Book Environmental and Microbial Relantionships
Book Environmental and Microbial Relantionships
Karl Esser
THE MYCOTA
A Comprehensive Treatise on Fungi
as Experimental Systems for Basic and Applied Research
Environmental and
Microbial Relationships
IV
Third Edition
Irina S. Druzhinina
Christian P. Kubicek
Volume Editors
The Mycota
Edited by
K. Esser
The Mycota
Edited by K. Esser
IV
Volume Editors:
Environmental and Microbial
Relationships
3rd Edition
I.S. Druzhinina and C.P. Kubicek
Series Editor
Professor Dr. Dr. h.c. mult. Karl Esser
Allgemeine Botanik
Ruhr-Universität
44780 Bochum, Germany
Tel.: +49 (234)32-22211
Fax.: +49 (234)32-14211
e-mail: [email protected]
Volume Editors
Irina S. Druzhinina
(born 1974) obtained background education in
Mycology in Lomonosov’s Moscow State University
(Russia) and got her doctoral degree at the University
of Vienna (Austria). For the last decade, she runs the
research group on Microbiology in the Department of
Biotechnology and Microbiology at the Institute of
Chemical Engineering of the Vienna University of
Technology (Austria). The group is now focused on
ecological genomics, molecular evolution, and eco-
physiology of mycoparasitic and plant beneficial
fungi from the order Hypocreales. In a second line
of research, she is interested in microbial diversity in
tropical regions, fungal taxonomy, and development
of DNA barcoding tools for microbial identification
and for the development of new applications.
Christian P. Kubicek
(born 1951) is retired Professor for Biotechnology
and Microbiology at the Vienna University of Tech-
nology (Austria). His scientific work concentrated on
both basic and applied research on filamentous fungi
with most emphasis on those from the genus Tricho-
derma. These studies comprised enzymology and
protein chemistry, metabolic regulation, microbial
ecology, and functional gene characterization,
all “omics” approaches toward the improvement of
fungal strains used in industry and agriculture by
science-driven strategies. His work is documented
in more than 300 original papers in peer-reviewed
journals, as well as many patents and books, and was
distinguished by several international honors.
ThiS is a FM Blank Page
Series Preface
Mycology, the study of fungi, originated as a sub discipline of botany and was a
descriptive discipline, largely neglected as an experimental science until the early
years of this century. A seminal paper by Blakeslee in 1904 provided evidence for
self incompatibility, termed “heterothallism”, and stimulated interest in studies
related to the control of sexual reproduction in fungi by mating-type
specificities. Soon to follow was the demonstration that sexually reproducing
fungi exhibit Mendelian inheritance and that it was possible to conduct formal
genetic analysis with fungi. The names Burgeff, Kniep and Lindegren are all
associated with this early period of fungal genetics research.
These studies and the discovery of penicillin by Fleming, who shared a Nobel
Prize in 1945, provided further impetus for experimental research with fungi.
Thus began a period of interest in mutation induction and analysis of mutants
for biochemical traits. Such fundamental research, conducted largely with
Neurospora crassa, led to the one gene: one enzyme hypothesis and to a second
Nobel Prize for fungal research awarded to Beadle and Tatum in 1958.
Fundamental research in biochemical genetics was extended to other fungi,
especially to Saccharomyces cerevisiae, and by the mid-1960s fungal systems
were much favored for studies in eukaryotic molecular biology and were soon
able to compete with bacterial systems in the molecular arena.
The experimental achievements in research on the genetics and molecular
biology of fungi have benefited more generally studies in the related fields of
fungal biochemistry, plant pathology, medical mycology, and systematics. Today,
there is much interest in the genetic manipulation of fungi for applied research.
This current interest in biotechnical genetics has been augmented by the develop-
ment of DNA-mediated transformation systems in fungi and by an understanding
of gene expression and regulation at the molecular level. Applied research initia-
tives involving fungi extend broadly to areas of interest not only to industry but to
agricultural and environmental sciences as well.
It is this burgeoning interest in fungi as experimental systems for applied as
well as basic research that has prompted publication of this series of books
under the title The Mycota. This title knowingly relegates fungi into a separate
realm, distinct from that of either plants, animals, or protozoa. For consistency
throughout this Series of Volumes the names adopted for major groups of fungi
(representative genera in parentheses) areas follows:
Pseudomycota
Division: Oomycota (Achlya, Phytophthora, Pythium)
Division: Hyphochytriomycota
viii Series Preface
Eumycota
Division: Chytridiomycota (Allomyces)
Division: Zygomycota (Mucor, Phycomyces, Blakeslea)
Division: Dikaryomycota
Subdivision: Ascomycotina
Class: Saccharomycetes (Saccharomyces, Schizosaccharomyces)
Class: Ascomycetes (Neurospora, Podospora, Aspergillus)
Subdivision: Basidiomycotina
Class: Heterobasidiomycetes (Ustilago, Tremella)
Class: Homobasidiomycetes (Schizophyllum, Coprinus)
We have made the decision to exclude from The Mycota the slime molds which,
although they have traditional and strong ties to mycology, truly represent
nonfungal forms insofar as they ingest nutrients by phagocytosis, lack a cell
wall during the assimilative phase, and clearly show affinities with certain proto-
zoan taxa.
The Series throughout will address three basic questions: what are the fungi,
what do they do, and what is their relevance to human affairs? Such a focused and
comprehensive treatment of the fungi is long overdue in the opinion of the
editors.
A volume devoted to systematics would ordinarily have been the first to
appear in this Series. However, the scope of such a volume, coupled with the
need to give serious and sustained consideration to any reclassification of major
fungal groups, has delayed early publication. We wish, however, to provide a
preamble on the nature of fungi, to acquaint readers who are unfamiliar with
fungi with certain characteristics that are representative of these organisms and
which make them attractive subjects for experimentation.
The fungi represent a heterogeneous assemblage of eukaryotic microorgan-
isms. Fungal metabolism is characteristically heterotrophic or assimilative for
organic carbon and some nonelemental source of nitrogen. Fungal cells charac-
teristically imbibe or absorb, rather than ingest, nutrients and they have rigid cell
walls. The vast majority of fungi are haploid organisms reproducing either
sexually or asexually through spores. The spore forms and details on their
method of production have been used to delineate most fungal taxa. Although
there is a multitude of spore forms, fungal spores are basically only of two types:
(i) asexual spores are formed following mitosis (mitospores) and culminate
vegetative growth, and (ii) sexual spores are formed following meiosis (meios-
pores) and are borne in or upon specialized generative structures, the latter
frequently clustered in a fruit body. The vegetative forms of fungi are either
unicellular, yeasts are an example, or hyphal; the latter may be branched to
form an extensive mycelium.
Regardless of these details, it is the accessibility of spores, especially the direct
recovery of meiospores coupled with extended vegetative haploidy, that have
made fungi especially attractive as objects for experimental research.
Series Preface ix
The ability of fungi, especially the saprobic fungi, to absorb and grow on rather
simple and defined substrates and to convert these substances, not only into
essential metabolites but into important secondary metabolites, is also noteworthy.
The metabolic capacities of fungi have attracted much interest in natural products
chemistry and in the production of antibiotics and other bioactive compounds.
Fungi, especially yeasts, are important in fermentation processes. Other fungi are
important in the production of enzymes, citric acid and other organic compounds
as well as in the fermentation of foods.
Fungi have invaded every conceivable ecological niche. Saprobic forms
abound, especially in the decay of organic debris. Pathogenic forms exist with
both plant and animal hosts. Fungi even grow on other fungi. They are found in
aquatic as well as soil environments, and their spores may pollute the air. Some
are edible; others are poisonous. Many are variously associated with plants as
copartners in the formation of lichens and mycorrhizae, as symbiotic endophytes
or as overt pathogens. Association with animal systems varies; examples include
the predaceous fungi that trap nematodes, the microfungi that grow in the
anaerobic environment of the rumen, the many insect associated fungi and the
medically important pathogens afflicting humans. Yes, fungi are ubiquitous and
important. There are many fungi, conservative estimates are in the order of
100,000 species, and there are many ways to study them, from descriptive
accounts of organisms found in nature to laboratory experimentation at the
cellular and molecular level. All such studies expand our knowledge of fungi
and of fungal processes and improve our ability to utilize and to control fungi for
the benefit of humankind.
We have invited leading research specialists in the field of mycology to
contribute to this Series. We are especially indebted and grateful for the initiative
and leadership shown by the Volume Editors in selecting topics and assembling
the experts. We have all been a bit ambitious in producing these Volumes on a
timely basis and therein lies the possibility of mistakes and oversights in this first
edition. We encourage the readership to draw our attention to any error, omis-
sion or inconsistency in this Series in order that improvements can be made in
any subsequent edition.
Finally, we wish to acknowledge the willingness of Springer-Verlag to host
this project, which is envisioned to require more than 5 years of effort and the
publication of at least nine Volumes.
In their concept of The Mycota, Karl Esser and Paul Lemke (Series Editors)
determined that there was a need for a volume that emphasizes research on
fungal populations and communities and their biotic and abiotic interactions
with environments. Two volumes written in this line have so far been published.
We were invited to prepare a third edition of Volume IV that continues concen-
trating on fungal responses to physical environments, interactions with other
fungi, but also bacteria, plants, and invertebrates, and their role in ecosystem
processes.
The dramatic progresses in genetic manipulation of fungi, genome-wide
analytical tools, and bioinformatics have also revolutionized research in the
above-described area. Consequently, while several authors of the third edition
were asked to rework and update their chapters for this volume, we also felt the
need to also incorporate new chapters to emphasize important findings and
trends in this area.
The first section highlights two important aspects of the fungal population:
Sun and Heitman discuss the fundamental roles that recombination plays in
fungal evolution by generating novel allele combinations, as well as by increasing
the genetic diversity and facilitating natural selection. They thereby particularly
focus on the diverse mating systems and mating-type determination mechanisms
in fungi, which provide a rich resource to study the effects of recombination on
mechanisms of sex determination and sexual development, as well as the evolu-
tion of sex determination systems and sex chromosomes in general. Leavitt and
Lumbsch illuminate new insights into the geographical distributions of lichen-
forming fungi and the factors that shape these distributions. Besides discussing
general perspectives of biogeography of lichen-forming fungi, they focus on
major themes directly related to ecological biogeography, such as dispersal and
establishment of lichens, landscape genetics and gene flow, and the role of
photobionts in determining distributional ranges.
The second section is dedicated to the determinants of fungal communities.
Among these, sunlight is definitely one of the most important cues for all living
organisms on earth. Casas-Flores and Herrera-Estrella describe how light regu-
lates several physiological and developmental processes, including phototropism,
synthesis of pigments, circadian rhythms, sexual and asexual development, and
primary and secondary metabolism, among other processes. They also explain
the fungal components involved in sensing light and how this signal is transduced
downstream. Morris and coauthors deal with the impact of events that disrupts
ecosystem, community, or population structure such as changes in resources,
substrate availability, or the physical environment on fungal communities. They
xii Volume Preface
show that such disturbances that alter the fungal community have major con-
sequences for ecosystem dynamics by changing nutrient cycles and affect plant
diversity. However, also the type of fungal community that reestablishes on a
given site is affected by the reestablishing plant community structure and nutri-
ent dynamics across the landscape.
Fungi are capable of the degradation, utilization, and/or transformation of a
wide variety of organic and inorganic substances, including xenobiotics, metals,
radionuclides, and minerals. Fungal populations are therefore intimately
involved in element cycling at local and global scales and such processes have
major implications for living organisms, notably plant productivity and human
health. Geoffrey Gadd outlines in his chapter some important interactions of
fungi with organic and inorganic pollutants and highlights the interdisciplinary
approach that is necessary to further understand the important roles that fungi
play in pollutant transformations, the chemical and biological mechanisms that
are involved, and their environmental and applied significance.
Fungi are also the major organisms for the recycling of biomass on this planet
and thus essentially contribute to the global carbon cycle. Ramoni and Seiboth
illustrate how the plant cell wall that is composed of cellulose, different hemi-
celluloses, pectins, and the polymer lignin and represents material extremely
recalcitrant to degradation and decomposition can be used as a nutrient by
different fungi via different strategies and describe the enzymes involved in this
process.
The largest section of this volume is dedicated to the field of fungal interac-
tions which today is one of the hotspot of fungal research. Pawlowska starts this
section by describing the accumulated data about newly discovered bacterial–
fungal symbioses. Particularly, the heritable alliances formed by early divergent
lineages of Mucoromycotina and Glomeromycota with beta-proteobacteria and
Mollicutes yielded novel insights into the forms of evolutionary trajectories in
mutualisms and into mechanisms of symbiont genome evolution. Tarrka and
Deveau continue on this topic by discussing the interactions of fungi and bacte-
ria, which are frequent because they coexist in various environments and often
share niches. They consequently often undergo physical associations that have
beneficial effects for both partners. They highlight recent contributions to the
understanding of bacteria–fungi interactions and focus on the rapid methodo-
logical development in this area.
Fungal plant interactions have been known for a long while. “Mycorrhiza”—
the beneficial and mutualistic associations between plant roots and fungi—
thereby has a major impact on earth’s plant growth. Marmeisse and Girlanda
summarize the recent advances in the field of mycorrhizal ecology, particularly
prompted by “omic” approaches, which have offered insights into the genome
signatures of different fungal trophic strategies and the roles of mycorrhizal fungi
in the functioning of terrestrial ecosystems. In addition, root endophytes—while
not being mycorrhizal fungi—have only recently been recognized to have a
significant impact on plant nutrition. Yuan and coauthors describe the ecological
significances of fungal root endophytes, particularly those termed class 4 endo-
phytes which represent the main root associates. They particularly introduce a
new model system for studying this process—Harpophora oryzae—that is a root
endophyte of wild rice. Interestingly, H. oryzae is a close relative of the most
Volume Preface xiii
devastating pathogen of rice, Magnaporthe oryzae, and the authors highlight the
metabolomic and transcriptomic differences that have been found in the two
binary fungal-root systems which reveal details about the key elements leading to
either mutualistic or pathogenic interactions.
Moving on the further interactions performed by fungi, Chenthamara and
Druzhinina focus on mycotrophic fungi and—on the basis of genome-wide
investigations—reveal unique features in the intracellular mycoparasite Crypto-
mycota and outline similar and apparently convergent mechanisms employed by
a diversity of fungicolous Asco- and Basidiomycota. Herrera-Estrella and coau-
thors deal with the ancient and diverse group of nematophagous fungi that use
refined mycelial structures or their conidia to capture their preys. They review the
current state of knowledge of their biology and molecular physiology and partic-
ularly highlight the recent genomic insights into the virulence factors of nema-
tophagous fungi. Schigel illustrates the ecological complexity of fungus–beetle
interactions from European boreal forests. Larvae or adults of the fungivorous
species of the genus Coleoptera selectively feed on a primarily fungal diet, fruit
bodies, mycelia, and spores. He describes how the evolutionary success and
diversity of both fungi and the beetles result in complex patterns of co-occurrence
and interactions, culminating in diverse species assemblage patterns and varying
degrees of trophic specialization of beetles.
We hope that this volume will prove useful to both scientists who wish to
update themselves in any of the research areas outlined above and students for a
first overview before entering these areas in depth. We are grateful to all the
authors who took the time and effort to collaborate with us on the updating of this
volume and particularly that they all helped us getting this task finished within
the expected time schedule.
10 Mycorrhizal Fungi and the Soil Carbon and Nutrient Cycling . . . . . . . 189
ROLAND MARMEISSE, MARIANGELA GIRLANDA
xvi Contents
M.F. ALLEN
Center for Conservation Biology, University of California, Riverside, CA, USA
INES ASCHENBRENNER
Institute of Plant Sciences, Karl-Franzens-University, Graz, Austria
GABRIELE BERG
Institute of Environmental Biotechnology, Graz University of Technology, Graz,
Austria
SERGIO CASAS-FLORES
División de Biologı́a Molecular, IPICYT, San Luis Potosı́, SLP, México
TOMISLAV CERNAVA
Institute of Environmental Biotechnology, Graz University of Technology, Graz,
Austria
KOMAL CHENTHAMARA
Institute of Chemical Engineering, Microbiology Group, TU Wien, Vienna,
Austria
AURÉLIE DEVEAU
Interactions Arbres-Microorganismes, INRA, Champenoux, France
Interactions Arbres-Microorganismes, Université de Lorraine, Vandœuvre-lès-
Nancy, France
IRINA S. DRUZHININA
Institute of Chemical Engineering, Microbiology Group, TU Wien, Vienna,
Austria
C.F. FRIESE
Department of Biology, University of Dayton, Dayton, OH, USA
G.M. GADD
Geomicrobiology Group, College of Life Sciences, University of Dundee, Dundee,
UK
Laboratory of Environmental Pollution and Bioremediation, Xinjiang Institute of
Ecology and Geography, Chinese Academy of Sciences, Urumqi, People’s
Republic of China
xviii List of Contributors
MARIANGELA GIRLANDA
Dept. Scienze della Vita e Biologia dei Sistemi, University of Torino, Torino, Italy
MARTIN GRUBE
Institute of Plant Sciences, Karl-Franzens-University, Graz, Austria
JOSEPH HEITMAN
Department of Molecular Genetics and Microbiology, Duke University Medical
Center, Durham, NC, USA
ALFREDO HERRERA-ESTRELLA
Laboratorio Nacional de Genómica para la Biodiversidad, LANGEBIO, Irapuato,
Guanajuato, México
CHRISTIAN P. KUBICEK
Research Area Biotechnology and Microbiology, Institute of Chemical
Engineering, TU Wien, Wien, Austria
STEVEN D. LEAVITT
Science and Education, The Field Museum, Chicago, IL, USA
Committee on Evolutionary Biology, University of Chicago, Chicago, IL, USA
H. THORSTEN LUMBSCH
Science and Education, The Field Museum, Chicago, IL, USA
ROLAND MARMEISSE
Ecologie Microbienne, UMR CNRS 5557 – Université Lyon 1, Université de Lyon,
Villeurbanne, France
S. J. MORRIS
Biology Department, Bradley University, Peoria, IL, USA
TERESA E. PAWLOWSKA
Plant Pathology and Plant-Microbe Biology, School of Integrative Plant
Science, Cornell University, Ithaca, NY, USA
JONAS RAMONI
Molecular Biotechnology, RD Biotechnology and Microbiology, Institute of
Chemical Engineering, TU Wien, Vienna, Austria
DMITRY SCHIGEL
Metapopulation Research Centre and LUOMUS – Finnish Museum of Natural
History, University of Helsinki, Helsinki, Finland
BERNHARD SEIBOTH
Molecular Biotechnology, RD Biotechnology and Microbiology, Institute of
Chemical Engineering, TU Wien, Vienna, Austria
List of Contributors xix
ZHEN-ZHU SU
State Key Laboratory for Rice Biology, Institute of Biotechnology, Zhejiang
University, Hangzhou, People’s Republic of China
SHENG SUN
Department of Molecular Genetics and Microbiology, Duke University Medical
Center, Durham, NC, USA
MIKA TARKKA
Helmholz-Centre for Environmental Research, Department of Soil Ecology, UFZ,
Halle, Germany
ZHI-LIN YUAN
Institute of Subtropical Forestry, Chinese Academy of Forestry, Fuyang, People’s
Republic of China
CHU-LONG ZHANG
State Key Laboratory for Rice Biology, Institute of Biotechnology, Zhejiang
University, Hangzhou, People’s Republic of China
The Fungal Population
1 Running Hot and Cold: Recombination Around and Within
Mating-Type Loci of Fungi and Other Eukaryotes
molleri, the X and Y chromosomes are homo- HMR and HML, which carry epigenetically
morphic, and the Y chromosomes do not show silenced copies of MATa and MATa alleles,
extensive degeneration, which is likely due to respectively. Mating-type switching occurs dur-
occasional X-Y interchromosomal recombina- ing the G1 phase of the cell cycle in haploid cells
tion (which may occur in sex-reversed females) and starts with the excision and elimination of
that keeps homogenizing alleles from the same the allele at the active MAT locus by a
species, followed by selective sweeps by the specialized DNA endonuclease called HO and
newly arisen recombinant Y chromosomes exonucleases. The gap is subsequently filled in
that have reduced levels of deleterious muta- through gene conversion with genetic informa-
tions, thus maintaining Y chromosome integ- tion present at either the HML or the HMR
rity and preventing gradual degeneration locus. Typically, MATa cells repair the gap
(Stöck et al. 2011). using the MATa allele from the HML locus,
These studies illustrate that while inter- and vice versa, thus resulting in mating-type
chromosomal crossovers are highly repressed switching. Mating-type switching in S. pombe
between heteromophic highly rearranged sex shares similar features with S. cerevisiae,
chromosomes, recombination can still occur including the presence of one active MAT
and is likely at higher frequencies than previ- locus and two silent mating cassettes on the
ously appreciated in the regions outside of the same chromosome, as well as efficient direc-
highly rearranged key sex-determining regions tional switching initiated by a DNA lesion dur-
or even within these regions in the form of ing the mitotic cell cycle. However, the two
gene conversion. We discuss in the following systems evolved independently, and they differ
sections how these general processes regarding in the genes encoded within the MAT locus, as
recombination and sex determination regions well as the organization of the MAT locus and
also operate in many fungi and unicellular the silent cassettes. Additionally, the mechan-
eukaryotes (protists). Additionally, recombi- isms by which the silent cassettes are main-
nation plays important roles in fungal sex tained, and the means by which the DNA
determination, as well as in sexual develop- lesion that initiates switching is created, also
ment. differ between the two species. Instead of a
double-strand break induced by the HO endo-
nuclease, the switching in S. pombe is initiated
by a stall at the DNA replication fork that cre-
II. Mitotic Recombination in Mating- ates a lesion at the active MAT locus, which is
Type Determination subsequently repaired using the sequence from
a silent cassette, facilitated by various recombi-
A. Mating-Type Switching in Fungi national repair factors (Arcangioli and de
Lahondes 2000; Dalgaard and Klar 1999; Kay-
Mating-type switching was first discovered in kov and Arcangioli 2004; Vengrova and Dal-
the model yeasts Saccharomyces cerevisiae and gaard 2004, 2006). Thus, recombination,
later also in Schizosaccharomyces pombe. This specifically gene conversion, plays a central
involves one active mating-type cassette that role in mating-type switching in S. cerevisiae
can be rewritten by gene conversion from either and S. pombe. In S. cerevisiae, cells typically
of two distant mating-type cassettes that are switch their mating types every other genera-
otherwise maintained in a silent fashion by tion and only mother cells that have
specialized heterochromatin (Dalgaard and divided twice switch. Thus, the frequency of
Klar 1999; Egel 2005; Haber 2012; Heitman mating-type switching is likely considerably
et al. 2007; Klar 2007, 2010) (Fig. 1.1). Specifi- higher compared to typical mitotic recombina-
cally, in the genome of S. cerevisiae, the active tion frequencies in other chromosomal regions,
MAT locus defines a bipolar mating system which are typically 100- to 1000-fold less
with two alternative mating-type alleles, MATa common than meiotic recombination (Roeder
and MATa, and the two silent mating cassettes, and Stewart 1988).
Running Hot and Cold: Recombination Around and Within Mating-Type Loci of Fungi and Other. . . 5
Fig. 1.1 Recombination associated with the MAT loci have deleterious effects within the MAT locus. Gene
in different fungal species. (a) The three locus models conversion hot spot within the MAT locus of C. neofor-
of mating-type switching mediated by gene conversion mans could be the result of DSB repair through non-
in S. cerevisiae (I) and S. pombe (II) and the two locus crossover pathways. It could also function to maintain
models of mating-type switching mediated by inversion proper alignment of the two divergent MAT alleles
in H. polymorpha and P. pastoris (III) are illustrated. IR during meiosis. (c) Evolutionary transitions of the
inverted repeat. (b) Recombination hot spots flanking MAT locus, including transitions between heterothal-
the MAT locus C. neoformans break the linkage of MAT lism and homothallism (I), as well as between tetrapo-
and its original genetic background during meiosis and lar mating system and bipolar mating systems (II),
increase the efficiency of natural selection. They may could be the result of ectopic recombination mediated
also function to repress crossover events which could by repetitive sequences or transposable elements
6 S. Sun and J. Heitman
Remarkably, it has recently been shown et al. 2014) (Fig. 1.1). Additionally, while this
that another budding yeast, Kluyveromyces lac- simpler two-cassette inversion system differs
tis, also undergoes mating-type switching from the more canonical three MAT cassette
through a mechanism that is again distinct mating-type switching paradigms of both bud-
from that in S. cerevisiae (Barsoum et al. 2010; ding and fission yeasts, there are strong
Rusche and Rine 2010). Additionally, it has grounds to hypothesize that this was an ances-
been found that a specialized protein a3 is tral state from which the more complex switch-
essential for switching in K. lactis. a3 is homol- ing yeasts evolved. Models involving inversion
ogous to transposases, and the amino acids of just two mating-type loci were in fact
conserved among transposases are required advanced many years ago by Jim Hicks and
for successful mating-type switching in K. lac- Ira Herskowitz (Hicks and Herskowitz 1977)
tis, suggesting that similar to the HO gene in S. and others as one possible model for mating-
cerevisiae, a3 originated through domestication type switching. Given that there are other
of a transposase (Barsoum et al. 2010; Keeling invertible genetic switches in biology that
and Roger 1995; Rajaei et al. 2014). underlie flagellar phase variation in Salmonella
Recently, another novel mechanism for (Andrewes 1922; Haykinson et al. 1996; Silver-
mating-type switching has been discovered in man et al. 1979) and plasmid copy number
two closely related ascomycetous species, Han- amplification of the two-micron circle in S.
senula polymorpha (¼Ogataea angusta) and cerevisiae (Futcher 1986), it would not be
Pichia pastoris (¼Komagataella pastoris) (Han- surprising if other examples of similar
son et al. 2014; Maekawa and Kaneko 2014) inversion-mediated genetic switches remain to
(Fig. 1.1). In both species, there are just two be discovered.
mating-type loci, one active and one silenced, Additionally, mating-type switching has
and the mating-type switching involves an also been reported in one basidiomycetous spe-
inversion of the region that encompasses the cies, Agrocybe aegerita (Labarère and Noël
two, essentially interchanging the chromo- 1992), further demonstrating that this form of
somal position of active and silent MAT locus homothallism has evolved repeatedly and inde-
to enable switching between the two opposite pendently in different fungal lineages.
mating types, MATa and MATa. Specifically, in
H. polymorpha, the MATa and MATa loci are
located approximately 19 kb apart on the same B. Recombination-Mediated Sex
chromosome, and the chromosomal region Determination in Other Small Eukaryotes
encompassing the two MAT loci lies beside
the centromere. As a consequence, depending A recent study reports the exciting discovery of
on the orientation of this chromosomal region, the complex and fascinating mating-type locus
one of the two MAT loci will be silenced by the of the model ciliate Tetrahymena thermophila,
centromeric chromatin due to its proximity to solving an ~50-year-old mystery surrounding
the centromere, leaving the other MAT locus its unique mode of sex determination involving
active and capable of defining the mating type seven different sexes (Bloomfield 2014; Cer-
of the cell (Hanson et al. 2014; Maekawa and vantes et al. 2013). T. thermophila was among
Kaneko 2014). In P. pastoris, the two MAT loci the first microbial eukaryotes in which mating
are approximately 123 kb apart on the same types were discovered in the 1950s following
chromosome, and depending on the orienta- similar discoveries in the ciliate Paramecium
tion, one of the two MAT loci will be located and the yeast Saccharomyces. This opened a
close to the end of the chromosome and conse- new era of invaluable microbial models for
quently silenced due to its close proximity to research in genetics and evolution for species
the telomere. The inversion that results in with more than two sexes or mating types.
switching between the mating types occurs by While the yeast mating-type locus has become
recombination between inverted-repeat a textbook paradigm in genetics and molecular
sequences flanking the MAT loci (Hanson biology, the sex determination mechanisms in
Running Hot and Cold: Recombination Around and Within Mating-Type Loci of Fungi and Other. . . 7
the model ciliates languished in relative obscu- of multiple mating types a genetically tractable
rity due to their complex biology and genetics. research question. Discovery of the MAT locus,
Cervantes et al. took clues from the basic its genomic structure, and its differentiation in
biology of T. thermophila and ingeniously T. thermophila now provides biologists a foun-
applied RNA-seq to reveal the fascinating dation to obtain insights into the evolution of
nature of the MAT locus in this species (Cer- mating systems and further stresses the impor-
vantes et al. 2013). They found that the T. ther- tance of studying the unique and unconven-
mophila MAT locus contains an array of up to tional biology and genetics of ciliates.
seven tandem gene pairs, one pair
corresponding to each mating type, in the
germ line genome of the species. Only one of
these gene pairs is assembled into a functional III. Meiotic Crossover and Gene
dual protein-coding unit through homologous Conversion Related to the MAT
recombination during differentiation of the Locus
somatic genome, and all of the other MAT
modules are eliminated. Hence, different sib- A. Recombination Hot Spots Flanking and
ling progenies of the same parents may have Within the MAT Locus in Cryptococcus
different mating types depending upon which neoformans
gene pair is assembled in their soma. This is
astounding, especially because Tetrahymena Cryptococcus neoformans is a basidiomycetous
biology ensures that all siblings are exactly human fungal pathogen. It has a bipolar mating
genetically identical in their germ line genome, system that is determined by the presence of
yet somatic differentiation generates different one of two alternative alleles, a and a, at the
phenotypes (mating types). The discovery of unusually large MAT locus (>100 kb in size
the mating-type determination mechanism in with >20 genes) (Fraser et al. 2004; Lengeler
this unicellular eukaryote thus provides a et al. 2002; Loftus et al. 2005). Mating typically
gripping account of how phenotypic plasticity occurs between isolates of opposite mating
can be generated through developmental types (bisexual reproduction) (Kwon-Chung
genome rearrangements. In this context, the 1975, 1976a, b), although sexual reproduction
study salutes insights of the senior authors by can also occur between two MATa isolates
supporting their molecular model predicted (unisexual reproduction) (Lin et al. 2005,
~20 years ago based purely on classical genetic 2007, 2009). The C. neoformans MAT locus in
approaches. many ways mirrors the sex chromosomes of
Additionally, Cervantes et al. found that the other organisms. Specifically, extensive
two mating-type-specific proteins are large (161 sequence divergence and chromosomal rear-
and 194 kDa) and while they lack homology rangements, including translocations and
with any known proteins, they do contain a inversions, have accumulated between the a
few conserved furin-like repeats similar to and a MAT alleles, indicating meiotic recombi-
those observed in signaling receptors (Cer- nation is likely repressed within the MAT locus.
vantes et al. 2013). This suggests either rapid Additionally, studies have identified evolution-
evolution of ancestral proteins or an indepen- ary strata within the MAT locus, suggesting it
dent evolution of novel mating-type specifici- underwent gradual cessation of recombination
ties, in line with the recent discovery of novel between the two alleles during the evolution of
master sex determinants in fishes and novel the MAT locus (Fraser et al. 2004).
sex determinants found in other ciliates. It has been hypothesized that the extensive
Together with the recent discovery of the sequence divergence and chromosomal rear-
trisexual mating-type locus of the social rangements between the a and a alleles pose
amoeba Dictyostelium discoideum (Bloomfield physical barriers for crossing-over within the
et al. 2010), characterization of mating-type MAT locus during a-a bisexual reproduction in
genes in T. thermophila has made the evolution C. neoformans. Indeed, in a study in which
8 S. Sun and J. Heitman
more than 150 meiotic progeny from a-a bisex- intervariety hybridization (Marra et al. 2004;
ual reproduction of serotype D C. neoformans Sun et al. 2014; Sun and Xu 2007). One possible
were analyzed, no crossover was detected explanation could be that the two recombina-
within the MAT locus. However, gene conver- tion hot spots that flank the MAT locus repress
sion does occur within the MAT locus around a recombination within MAT through crossover
GC-rich intergenic region between the RPO41 interference, as these flanking hot spots have
and BSP2 genes, which have relatively high been shown to be active also during a-a unisex-
sequence similarity between the two alleles ual reproduction (Sun et al. 2014).
(Sun et al. 2012). Interestingly, the C. neofor-
mans MAT locus is also flanked by two GC-rich
regions, and it has been shown that both GC- B. Pseudobipolar Mating System and Its Effect
rich flanking regions contain recombination on Meiotic Recombination
hot spots that elevate crossover frequencies in
these regions during meiosis and crossover at Typically in fungi, the mating type is deter-
one side of the MAT is typically associated with mined by restricted regions in the genome
another crossover at the other side of the MAT called MAT loci, which usually contain genes
(Hsueh et al. 2006) (Fig. 1.1). It is possible that that encode pheromone/pheromone receptors
during meiosis, DNA double-strand breaks are and/or sexual development transcription fac-
induced similarly at high frequency in the MAT tors (Heitman et al. 2013), although in some
flanking recombination hot spots and the intra- species, such as Cryptococcus heveanensis,
MAT gene conversion hot spot. While repair in Cryptococcus amylolentus, and Cryptococcus
the flanking regions can result in crossovers, neoformans, the MAT loci apparently have
breaks within the MAT gene conversion hot undergone expansion by recruiting additional
spot are more likely repaired through mechan- genes into MAT (Findley et al. 2012; Fraser et al.
isms that result in gene conversion (e.g., 2004; Lengeler et al. 2002; Metin et al. 2010). In
synthesis-dependent strand annealing), or the basidiomycetes, the pheromone/pheromone
crossover products fail to survive, due to struc- receptors and sexual development transcrip-
tural or genetic constraints exerted by chromo- tion factors are typically located in two separate
somal rearrangements between MAT alleles. loci, called the PR locus and HD locus, respec-
Recent studies also suggest that sequence tively, that lie on different chromosomes, con-
divergence and structural variation between the stituting a tetrapolar mating system. However,
two mating-type alleles are not the only in some cases, such as C. neoformans, as well as
mechanisms that act to repress meiotic recom- Ustilago hordei, Malassezia species, and Micro-
bination within MAT. In a study of meiotic botryum species, the two MAT loci have
recombination in a-a unisexual reproduction, become fused, possibly through ectopic recom-
during which two MATa alleles align with each bination or nonhomologous end joining,
other in meiosis, and the physical constraints resulting in a bipolar mating system with linked
on recombination posed by sequence diver- PR and HD loci (Fig. 1.1). This structural tran-
gence and chromosomal rearrangements are sition can sometimes capture previously non-
no longer present, the authors found crossing- sexual autosomal genes between the two loci
over is still highly repressed, albeit not and subsequently represses recombination in
completely eliminated (Sun et al. 2014). Specif- these newly captured chromosome regions. In
ically, among 156 unisexual meiotic progeny some cases, this repression of recombination
analyzed, only one crossover event occurred can span the entire chromosome on which the
within the MAT locus, yielding a frequency of PR and HD loci now reside, resulting in mating-
crossover within the MAT that is considerably type chromosomes that share features with the
lower than expected given the size of MAT and sex chromosomes of animals (Gioti et al. 2013a;
the average recombination frequencies in other Giraud et al. 2008; Hood et al. 2013; Lee et al.
chromosomal regions during both a-a bisexual 1999; Petit et al. 2012; Votintseva and Filatov
and a-a unisexual reproduction, as well as the 2009; Whittle et al. 2015). In both Malassezia
Running Hot and Cold: Recombination Around and Within Mating-Type Loci of Fungi and Other. . . 9
and Microbotryum species, while the PR and HD and the a2 fungal sex chromosomes in Micro-
loci are linked, more than two mating types have botryum species. The ratio of nonsynonymous
been identified in the natural population, which to synonymous (dN/dS) substitutions in the
is different from canonical bipolar species NRR regions of the sex chromosomes com-
where there are only two mating types, and pared to non-MAT chromosomes was signifi-
thus, they have pseudobipolar mating systems cantly higher for 4 of 12 species examined
(Gioti et al. 2013a). Additionally, given the large based on a Codeml, PAML-based comparison,
size and the syntenic nature between the two whereas the dN/dS ratio was significantly
mating types in the region linking the PR and higher for 9 out of 12 species based on singleton
HD loci, crossover may still occur occasionally analysis that focused only on more recent
in this region, although this is hypothesized to events. These observations provide evidence
be detrimental as it breaks up the proper allele for a higher rate of deleterious mutations on
associations of the PR and HD loci. both fungal sex chromosomes, consistent with
Several recent studies on the dimorphic the genetics of the a1/a2 genotype only being
mating-type chromosomes of an unusual present in the zygote and heterokaryon stages
group of closely related fungal species in the of the life cycle, and the absence of an equiva-
genus Microbotryum that have expansions in lent to the homogametic state enabling differ-
the MAT loci reveal unique features of their ential sex chromosome repair in species with
evolutionary trajectory. Both mating-type chro- XY- and ZW-specific sex-determining systems.
mosomes (a1 and a2) were isolated and Taken together, these studies reveal not only
sequenced and also subjected to optical parallels in the evolutionary trajectory of sex
mapping to generate very high-quality assem- chromosomes in fungi, plants, and animals but
blies and sequences for the reference genome. also lineage-specific features giving rise to dis-
This scaffold then served to assemble data from tinct patterns of sex chromosome-linked gene
12 other related Microbotryum species. The mutation and decay in symmetric vs. asymmet-
mating-type chromosomes could be partitioned ric patterns.
into two types of regions termed recombining
regions (RR) and nonrecombining regions
(NRR), analogous to sex-specific and pseu-
doautosomal regions for animal and plant sex
IV. Consequences of Recombination
chromosomes. By comparing these regions, in the Evolution of the MAT
recombination suppression in these chromo- Locus
somes predates species divergence times and
thus is ancient. Evolutionary strata were not Recombination, or lack thereof, within and in
clearly apparent, in contrast to studies on XY the proximity of the mating-type locus can have
and ZW sex chromosomes and other studies of broad effects on the evolutionary dynamics and
fungal mating-type loci, suggesting one major trajectories of the MAT loci, as well as the
event may have driven this expansion to a fun- mating-type determination systems.
gal sex chromosome. Additionally, 11 examples First, recombination is required for efficient
of gene conversion events within the NRR natural selection. Therefore, chromosomal
regions were identified. regions in which recombination is repressed,
A key feature of XY and ZW sex chromo- such as the Y chromosome in animals, usually
somes is the degeneration of genes resident on degenerate due to accumulation of deleterious
one of the two sex chromosomes, namely, those mutations and transposable elements. Addi-
mutations that are sheltered on the Y or W tionally, chromosomal rearrangements, such
chromosome that is only present in the hetero- as translocations and inversions occurring
gametic sex and not in a homogametic fashion within the recombination-repressed region,
that would allow repair from a homolog. In also cannot be corrected efficiently through
contrast to this pattern, an evolutionary signa- recombination-mediated repair mechanisms.
ture of gene decay was observed for both the a1 These will in turn further compromise proper
10 S. Sun and J. Heitman
alignment of these regions during meiosis and Homothallism can also be due to the pres-
consequently reinforce the repression of recom- ence of MAT loci of opposite mating types in
bination within these regions. This is also true one genome. The two loci can be unlinked,
for fungal MAT loci that have undergone expan- such as in Aspergillus nidulans (Galagan
sion. For example, in C. neoformans, the two et al. 2005), or they can be linked, as found
mating-type alleles show elevated sequence in Neurospora pannonica and Neurospora ter-
divergence and extensive chromosomal rear- ricola (Gioti et al. 2012, 2013b; Pöggeler 1999),
rangements compared to other chromosomal Cochliobolus luttrellii (Lu et al. 2011; Yun et al.
regions. Additionally, repetitive sequences as 1999), as well as Pneumocystis species and
well as transposable elements have been found Taphrina deformans (Almeida et al. 2015; Tur-
within the MAT locus of C. neoformans, which geon and Inderbitizen 2015). In these species,
could have played roles in mediating the chro- it is likely that the linkage between the two
mosomal rearrangements within the MAT locus MAT loci originated from heterothallic ances-
through ectopic recombination (Fraser et al. tors through recombination at regions shared
2004; Lengeler et al. 2002). Additionally, natural between alleles from opposite mating types
C. neoformans populations have been found to (Fig. 1.1). For example, when the sequence
be largely clonal, suggesting recombination may from the MAT locus of the homothallic species
not occur at high frequency or be difficult to C. luttrellii is aligned with the MAT1-1 and
detect if recombination occurs in a clonal pop- MAT1-2 alleles from the heterothallic sister
ulation. Thus, the recombination hot spots species Cochliobolus heterostrophus, there is a
found flanking the MAT locus of C. neoformans stretch of eight nucleotides that is identical
could function to increase the effective popula- between the two species and lies at the fusion
tion size of MAT, thus enhancing the selection junctions between opposite mating-type alleles
efficiency on this functionally important locus. in C. luttrellii, which could have mediated the
Second, ectopic recombination likely ectopic recombination in the heterothallic
mediated the emergence of homothallism ancestor that resulted in the extant MAT con-
from ancestral heterothallism in many fungal figuration observed in C. luttrellii (Lu et al.
species. In heterothallic species, sexual repro- 2011; Turgeon and Inderbitizen 2015; Yun
duction only occurs between two individuals et al. 1999).
with different mating types. However, for Ectopic recombination mediated by repeti-
some fungal species, a single spore/cell can tive sequences or transposable elements could
initiate sexual reproduction without the pres- have also played critical roles in the transition
ence of another individual with a different mat- from an ancestral tetrapolar mating system to
ing type, and we call these species homothallic. the extant pseudobipolar and bipolar mating
The aforementioned species that can undergo systems observed in basidiomycetous species
mating-type switching are examples of homo- such as U. hordei, C. neoformans, as well as
thallism as the mother cell that underwent Malassezia and Microbotryum species (Findley
mating-type switching can mate with a daugh- et al. 2012; Fraser et al. 2004, 2007; Gioti et al.
ter cell and the two mating partners are identi- 2013a; Lee et al. 1999; Metin et al. 2010). Addi-
cal by descent (except at MAT). Homothallic tionally, in species such as C. neoformans,
sexual reproduction can also be achieved which have regional centromeres that are
through special configurations at the MAT enriched with common, shared transposable
locus. For example, in species such as Neuros- elements and their remnants, it is also possible
pora tetrasperma (Merino et al. 1996), Agrocybe that ectopic recombination that gave rise to the
semiorbicularis, Conocybe tenera for. bispora, initial linkage of the P/R and HD loci was
Coprinellus ephemerus (Raper 1966), and Agar- mediated by centromeres that brought the two
icus bisporus (Li et al. 2004), the meiotic spores MAT loci onto the same chromosome, and sim-
contain separate nuclei with compatible mating ilar events of chromosomal arm exchanges have
types and can germinate into heterokaryotic been observed in the evolution of the patho-
mycelia and proceed with sexual reproduction genic Cryptococcus species complex (Janbon
by themselves. et al. 2014).
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2 Ecological Biogeography of Lichen-Forming Fungi
also many other fungi present in lichen thalli, tions (Galloway 1988, 2008; Galloway and Apt-
including endolichenic and lichenicolous fungi. root 1995; Culberson 1972; Werth 2011; Du
Also bacteria are commonly found associated Rietz 1940). However, with the increasing avail-
with lichens. Lichens are ubiquitous components ability of molecular data, our understanding of
of most terrestrial ecosystems, playing important lichen phylogeography has dramatically
ecological roles, for example, as pioneers or as improved in recent years. This is due, in part,
stabilizers of soil. to the increased recognition of the importance
Specifically, we briefly discuss general per- of cryptic, or previously unrecognized, lineages
spectives of biogeography of lichen-forming in lichen-forming fungi (Lumbsch and Leavitt
fungi, followed by a synthesis of four major 2011; Crespo and Pérez-Ortega 2009; Crespo
themes directly related to ecological biogeogra- and Lumbsch 2010), large-scale collaborative
phy, including (1) dispersal and establishment projects (Lumbsch et al. 2011), and develop-
of lichens, (2) landscape genetics and gene flow, ments in analytical approaches (Yang and Ran-
(3) modeling lichen distributions, and (4) the nala 2006; Rabosky et al. 2014; Ree and Smith
role photobionts play in determining species 2008). A general perspective of lichen biogeog-
distributions. We conclude by discussing the raphy is presented by Galloway (2008), and
role of ecological biogeography in conservation Werth (2011) also provides a valuable review.
and climate change research. Lichens exhibit biogeography and/or eco-
Reflection on the broad scope of both past logical distribution patterns that in many cases
and current views of lichen biogeography can differ from other co-occurring biota (e.g., vascu-
improve our perspective into biogeographic pat- lar plants, animals, etc.; Galloway 2008; Culber-
terns and the underlying processes shaping spe- son 1972). For example, a disproportionately
cies distributions, in addition to directing future high number of lichen-forming fungal species
research. A comprehensive summary of the mul- have bipolar distributions, meaning that they
tifaceted and developing field of lichen biogeog- occur in polar regions of the Northern and
raphy is well beyond the scope of a single Southern Hemisphere (Wirtz et al. 2008; Fernán-
chapter. Rather, our emphasis is on summarizing dez-Mendoza and Printzen 2013; Myllys et al.
our current understanding of contemporary fac- 2003; Lindblom and Søchting 2008); other spe-
tors that influence ecological and spatial distri- cies occur in ecologically and geographically
butions of species (Monge-Nájera 2008). Our restricted regions (Lücking et al. 2014); and
hope is that this review will encourage a more many species occur across incredible ecological
comprehensive perspective of lichen biogeogra- and geographic distances (Leavitt et al. 2013b;
phy by promoting the consideration of a wide Printzen et al. 2013). Traditionally, it has been
variety of factors that potentially shape species thought that species with broad, intercontinen-
distributions, in addition to historical processes tal distributions have high dispersal abilities
(traditional phylogeographic perspective). This and general ecological preferences. Broad distri-
expanded focus of lichen biogeography should, butions have alternatively been explained as
in turn, promote creative biogeographic research older species of lichen-forming fungi that
incorporating the fields of ecology, landscape have had more time to disperse and reach exten-
genetics, species distribution modeling, symbi- sive distributions (reviewed in Werth 2011).
ont interactions in lichens, etc. However, increased interest in species delimita-
tion research and estimating divergence times
for groups of lichen-forming fungi have
challenged these traditional perspectives (Amo
II. Perspectives of Biogeography de Paz et al. 2011; Divakar et al. 2012; Leavitt
of Lichen-Forming Fungi et al. 2015a; Otálora et al. 2010). For example, in
the genus Melanohalea, many of the species-
There has been a tradition of studies attempting level lineages with more ancient diversification
to understand distributions of lichen-forming histories, including M. multispora s. lat. and M.
fungi and the factors that shape these distribu- ushuaiensis s. lat., have geographically restricted
Ecological Biogeography of Lichen-Forming Fungi 17
distributions in western North America and respond to distinct biogeographic regions, with
southern South America, respectively. In con- the exception of R. shushanii which is known
trast, species with more recent diversification exclusively from subalpine habitat on the
histories, including M. elegantula and M. exas- Aquarius Plateau in southern Utah, USA
peratula, generally have much broader geo- (Fig. 2.1; Leavitt et al. 2011, 2013b).
graphic distributions (Otte et al. 2005; Leavitt Our limited understanding of morphologi-
et al. 2013a). cal adaptations is also exemplified by the pres-
Iconic examples of phylogenetic structure ence of cryptic species that are only distantly
corresponding to major biogeographic regions related but occur under similar ecological con-
or major tectonic events are relatively scarce in ditions. For example, Parmelina quercina was
the groups of lichen-forming fungi that have believed to occur in areas with Mediterranean-
been investigated to date, although other type climate throughout the world, but molec-
striking biogeographic patterns have been ular studies have demonstrated that distinct
observed in a number of cases (Miadlikowska species occur in each continent, with the Aus-
et al. 2011; Lücking et al. 2013; Del-Prado et al. tralian taxon now being classified in a separate,
2013). Rather than biogeographic patterns unrelated genus, Austroparmelina (Argüello
driven by dispersal limitations or vicariance, it et al. 2007; Crespo et al. 2010a, b). Another
appears that major climatic shifts may have example is Physcia aipolia, which was believed
played the dominant role in the diversification to occur in temperate to subtropical regions of
and distributions of lichen-forming fungi in the the Northern Hemisphere but also in Australia
family Parmeliaceae (Amo de Paz et al. 2011, (Moberg 2001). However, the Australian sam-
2012; Kraichak et al. 2015). ples were shown by molecular data to represent
Ultimately, the interplay of climate-driven a group of three separate species, unrelated to
diversification, dispersal and establishment, the populations in the Northern Hemisphere
and vicariance results in biogeographic pat- (Elix et al. 2009).
terns that may be difficult to generalize. The Given our limited ability to make general-
Rhizoplaca melanophthalma group (Lecanora- izable inferences and predictions on species
ceae) provides an interesting example of the distributions within a historical biogeographic
challenges inherent to elucidating biogeo- framework, we emphasize that more effective
graphic patterns in lichen-forming fungi. Rhi- incorporation of an ecological biogeographic
zoplaca melanophthalma s. lat. occurs on all perspectives into biogeographic research of
continents, except Australia, in a broad range lichen-forming fungi will provide an improved
of habitats, from extremely arid continental understanding of the range of factors shaping
habitats to upper montane coniferous forests the geographical distributions of species.
and the lower portions of the alpine tundra
(Leavitt et al. 2013b). Although R. mela-
nophthalma s. lat. was traditionally assumed
to represent a single, cosmopolitan species, III. Assessing Dispersal Capacity
molecular sequence data support the conclu- of Lichen-Forming Fungi
sions that this nominal taxon is comprised of
multiple species-level lineages, three of which Accounting for differences in reproductive
occur across broad intercontinental distribu- strategies and dispersal capacities of lichen-
tions, and the remaining lineages are known forming fungal species is central to understand-
exclusively from western North America (Lea- ing and interpreting biogeographic patterns.
vitt et al. 2011, 2013b). All of the known species Lichen-forming fungi generally use two major
within the R. melanophthalma group can be reproductive strategies, sexual reproduction via
found within a limited geographic region in meiotically produced fungal spores and vegeta-
the southwest USA; species have not been tive reproduction using asexually produced
shown to have distinct ecological preferences diaspores. Sexual reproduction is restricted to
nor do distributions of species, otherwise cor- characteristic fungal fruiting bodies (ascomata)
18 S.D. Leavitt and H.T. Lumbsch
500 Km
B
R. melanophthalma s.l.
3400 m
R. melanophthalma
R. parilis
3270 m R. polymorpha
R. porterii
R. occulta
3175 m
R. shushanii
in
au
unta
Plate
3000 m
s Mo
ntain
2875 m
Lake
Mou
2725 m
sand
lder
Thou
2550 m
Bou
2400 m
2285 m
2220 m
Fig. 2.1 (a) Worldwide distribution of the Rhizoplaca endemic to subalpine habitats in the southern Utah,
melanophthalma group; (b) Distribution of species USA. Modified from Leavitt et al. (2011) and Leavitt
along an altitudinal gradient on the Aquarius Plateau et al. (2013b)
in southern Utah, USA; and habitat of R. shushanii,
that produce meiospores (ascospores). Ascos- usually require acquisition of the appropriate
pores are dispersed independently of the photobiont partner in order to reestablish the
photosynthesizing partner (photobiont) and lichenized condition. Ascospore ejection
Ecological Biogeography of Lichen-Forming Fungi 19
springtails, etc. (Pickup 1988; Stubbs 1989; Apt- IV. Ecological Biogeography
root and Berg 2004).
Endozoochory, diaspores carried within an In contrast to historical biogeography, the study
animal, plays an important role in seed plant of ecological biogeography attempts to elucidate
dispersal, and it appears that gastropods contemporary factors that influence ecological
grazing on lichen communities likely serve as and spatial distributions of species (Monge-
important vectors for lichen dispersal (Boch Nájera 2008). The study of contemporary biogeo-
et al. 2011; McCarthy and Healy 1978). Boch graphic relationships focuses on biotic interac-
et al. (2011) found that two lichens, Lobaria tions among organisms, environmental changes
pulmonaria (Fig. 2.2) and Physcia adscendens, that potentially impact a species distribution,
were able to regenerate from fecal pellets after and how landscape and environmental features
passing through their digestive tracts of com- influence gene flow and population structure.
mon snail species occurring in temperate Eur- While evolutionary patterns may not be explic-
ope. This gastropod–fungus–alga association itly of interest in ecological biogeography, there
represents another level of complexity in lichen is no distinct boundary between historical and
symbioses, and endozoochory provides a previ- ecological biogeography; and ecological biogeog-
ously overlooked mechanism for lichen dis- raphy can extend back in time to reconstruct
persal. Viable lichen fungal spores and demographic histories, ecological interactions,
photobionts have also been in fecal pellets environmental controls, and evolutionary rela-
from slugs (McCarthy and Healy 1978), and tionships (e.g., Richardson and Meyer 2012;
lichenivorous mites have also been shown to Chan et al. 2011; Lira-Noriega et al. 2015).
distribute lichens with their feces including via-
ble cells of both fungal and algal partners (Meier
et al. 2002). Rotifers have also been shown to
ingest ascospores of Xanthoria parietina and A. Ecological Biogeography and Lichens
deposit viable spores in their feces (Pyatt 1968).
Given the range of distribution patterns in The overall importance of ecology in determin-
lichen-forming fungal taxa, increased interest ing the distributions of lichens is well known
in understanding mechanisms for fungal dis- (Renhorn et al. 1996; Kantvilas and Minchin
persal and variation in dispersal capacity will 1989; Culberson and Culberson 1967). The dis-
likely provide novel insight into biogeographic tributions of some lichen-forming fungal spe-
patterns in lichens. Recent advancements in cies and overall diversity are commonly
sampling environmental DNA (eDNA) and determined by microclimatic differences
“next-generation” sequencing technologies (Palmqvist and Sundberg 2000; Renhorn et al.
provide promising avenues for more accurately 1996; Hauck et al. 2007; Ranius et al. 2008), and
characterizing dispersal capacities of lichen- a wide variety of contemporary factors poten-
forming fungi (Shokralla et al. 2012). Rather tially influence lichen distributions. For exam-
than relying exclusively on visual observations ple, Nelson et al. (2015) recently demonstrated
of lichen propagules, eDNA collected from that different combinations of lichen functional
migratory birds and other animals can be traits, including choice of photobiont, dispersal
used to determine if animals consistently capacity, microsite specificity, and water rela-
carry evidence supporting animal-mediated tions, peak along environmental and distur-
lichen dispersal. Similarly, eDNA can be used bance gradients.
to assess the presence of lichen-forming fungal The distribution and abundance of species
DNA from air and water samples. In the fore- can be explained by the combination of dis-
seeable future it is reasonable to assume that persal and environmental filtering. The occur-
source populations of eDNA samples could be rence of a particular species is the product of
accurately identified by using highly variable the probability of establishment (environmen-
DNA markers, affording an exciting avenue tal filtering), and the number of propagules
for future research. arriving at the site (dispersal) (Schei et al.
22 S.D. Leavitt and H.T. Lumbsch
2012). Schei et al. (2012) underscore that the In a saxicolous lichen community in coastal
relative importance of local dispersal and envi- Norway, vegetation cover, rather than radia-
ronmental filtering varied widely among sites in tion, maritime influence, and microhabitat
deciduous forests in southwest Norway, partic- variables, was the predominant factor explain-
ularly in terms of abundance patterns. How- ing variation in community composition (Bjel-
ever, environmental filtering by tree species land 2003). However, in this study over 90 % of
was more important than local dispersal overall the total variation in lichen community compo-
(Schei et al. 2012). Of course inferences from sition remained unexplained. A combination of
any study investigating the relative roles of a variety of factors potentially contribute to our
dispersal and environmental filtering are scale ability to more fully account for community
dependent (Jackson and Fahrig 2014). Other composition and species’ occurrences, includ-
studies of fine-scale epiphyte distribution pat- ing ignoring local historical factors affecting
terns have revealed somewhat conflicting views contemporary distributions, failing to account
of the overall importance of dispersal versus for influential environmental variables, sto-
environmental filtering. chasticity in species establishment, and/or
It has been found that traditional bio- lack of fit of data to response models.
geographical variables explain little of the vari- Cladonia species occurring in the Wiscon-
ance in lichen richness in the Antarctic sin Pine Barren ground-layer lichen-moss com-
Peninsula at local and regional scales (Casano- munity tend to occupy slightly different
vas et al. 2013). Interestingly, while the majority habitats (Lechowicz et al. 1974). Although
of variability in moss richness at a region scale there is some overlap in microdistributional
in the Antarctic Peninsula was explained by patterns, differences in net photosynthesis tem-
summer mean sea surface temperature, lichen perature responses appear to underlie the dis-
richness in the same region was not correlated tributions of C. arbuscula subsp. mitis, C.
with any of the variables investigated (Casano- carolinensis, C. rangiferina, and C. uncialis,
vas et al. 2013). These data suggest that site- highlighting the putative role of temperature
specific habitat characteristics that were not in structuring species distributions. While the
investigated (substrate, water availability, etc.) eco-physiological responses observed for these
likely play an important role in explaining var- Cladonia species provide some intuitive insight
iance in lichen richness. into their biogeography and microhabitat selec-
In biological soil crusts in western North tion in the Wisconsin Pine Barrens, the
America, lichen community composition is nuanced interactions remain largely unac-
strongly related to vascular plant species, soil counted for.
texture and pH, and climate variables (Root Assessing the combined biological effects of
and McCune 2012). While species-rich biotic light, temperature, and humidity on photosyn-
crust lichen sites were scattered throughout thetic activity of Usnea sphacelata in Antarc-
the region, areas impacted by physical distur- tica, Bölter et al. (1989) demonstrated that
bances, including grazing, fire, etc., had the although individual microhabitat conditions
lowest overall lichen richness (Root and (e.g., light, humidity, and temperature) show
McCune 2012). Strikingly, a third of the nearly long periods of favorable conditions for meta-
100 lichen-forming fungal species encountered bolic activity in U. sphacelata, the combined
in this study were only observed a single time, analysis of these variables considering thresh-
highlighting the fact that a substantial propor- old values for metabolism drastically shortens
tion of lichen diversity may not be found ubiq- favorable periods for growth. Experimental
uitously even across relatively similar habitats. manipulations revealed that the combination
Interspecific competition among lichens and of light with desiccation has caused photoinhi-
other biotic interactions have also been shown bitory damage in pendulous lichens that com-
to be the major drivers of lichen community monly occur in boreal forests and that the
structure in soil crust communities in central species-specific production of sunscreening
Spain (Maestre et al. 2008). fungal pigments plays a major role in the verti-
Ecological Biogeography of Lichen-Forming Fungi 23
cal canopy gradient of epiphytes (Färber et al. noting that dispersal does not equal to gene
2014). Specifically, high-light-tolerant Bryoria flow. Some lichen-forming fungi may be able
species producing melanin are more commonly to effectively disperse, but without successful
found in the upper canopy than light- reestablishment and reproduction, the dis-
susceptible species in the genera Alectoria and persal event has no impact. Only those dis-
Usnea (sunscreening pigment ¼ usnic acid) persal events that occur in suitable habitats,
(Färber et al. 2014). with compatible symbiotic partners, and at the
The interplay between the source of hydra- appropriate spatial and temporal scales will
tion and light availability plays a major role in have the potential to contribute to gene flow
structuring epiphytic lichen distributions and impact genetic structure.
(Gauslaa 2014). Lichens utilize a variety of Colonization rates of epiphytic lichens
sources for water, including rain, dew, and appear to be tied to a number of crucial factors,
humid air, facilitating active photosynthesis. including connectivity to occupied patches and
Sources of hydration vary on temporal and species traits (e.g., niche breadth and propagule
spatial scales (e.g., regional, landscape, stand, size, local and long-distance dispersal, and
tree); and distinct atmospheric hydration patch dynamics) (Johansson et al. 2012). The
sources influence and shape lichen diversity majority of studies of landscape genetics in
and distributions (Gauslaa 2014). lichen-forming fungi have focused on the
In short, the studies highlighted above model epiphyte genus Lobaria pulmonaria
demonstrate that a variety of environmental, (Fig. 2.2; Werth et al. 2006, 2007; Walser et al.
ecological, and historical factors have the 2005; Walser 2004; Widmer et al. 2012). Among
potential to significantly influence lichen distri- the first studies of landscape dynamics in L.
butions. However, identifying the specific fac- pulmonaria, Walser et al. (2005) used microsat-
tors and teasing apart their relative ellite loci to elucidate regional population dif-
contributions remains challenging. Similarly, ferentiation and isolation by distance in
determining the most appropriate scales for populations in western North America (British
sampling poses a significant challenge. The Columbia, Canada) and central Europe (Swit-
contribution of these factors in structuring zerland). The use of highly variable microsatel-
lichen distributions may vary dramatically lite markers revealed striking genetic
across different temporal and spatial scales, differentiation among populations of L. pulmo-
and extrapolations between studies can poten- naria, with Swiss populations being distinct
tially be misleading. from those occurring in British Columbia,
with additional differentiation between coastal
and mainland populations in western North
1. Landscape Genetics and Gene Flow America (Walser et al. 2005). Subsequent stud-
ies of L. pulmonaria suggest that its occurrence
From a landscape genetic perspective, research- may not be limited by dispersal capacities, but
ers attempt to understand how geographic and that ecological constraints at level of the sam-
environmental factors impact gene flow and pled tree stands result in establishment limita-
genetic structure in populations and indivi- tions (Werth et al. 2006). However, dispersal
duals. Two of the major objectives of modern characteristic of L. pulmonaria appears to be
landscape genetics are (1) to improve our at least as important as landscape configuration
understanding of how recent global change in determining the spatial scale of population
(e.g., climate change, land use, etc.) affects neu- connectivity (Wagner et al. 2006). In fragmen-
tral and adaptive genetic variation and (2) ted relict stands in the boreal rainforest in cen-
understand if species are likely to adapt to tral Norway, nearly all genetic variation found
ongoing global change on an ecological time in L. pulmonaria could be attributed to varia-
scale (Manel and Holderegger 2013). While tion within sites and spatial genetic structure
assessing gene flow is a central component in was absent or appeared on very small scales (5–
understanding landscape dynamics, it is worth 10 m; Hilmo et al. 2012). While this study high-
24 S.D. Leavitt and H.T. Lumbsch
lights that relict stands may contain high levels charismatic epiphyte Letharia vulpina (Parme-
of genetic diversity, disturbances (e.g., fire and liaceae) is known to occur in western North
intensive logging) may have long-lasting nega- America, Europe, the Caucasus, and Morocco,
tive consequences on adaptive potential and European populations are genetically depau-
reproduction in epiphytic lichens (Singh et al. perate, resulting from an apparent genetic bot-
2014). Overall, studies of landscape genetics in tleneck caused by limited long-distance
L. pulmonaria support the conclusion under- dispersal (Högberg et al. 2002; Arnerup et al.
scored by Werth et al. (2007) that dispersal in L. 2004).
pulmonaria is quite effective, but not spatially Similar patterns of effective gene flow have
unrestricted. also been observed at regional scales. In west-
Arctic and alpine ecosystems are facing sig- ern North America, the epiphytic lace lichen
nificant impacts from global climate change, (mycobiont ¼ Ramalina menziesii) occurs in
including ongoing increases in temperature inland foothills along the Pacific Coast, ranging
and altered precipitation patterns (Ernakovich from Baja California northward to Alaska. Sork
et al. 2014). Two species in Flavocetraria (Par- and Werth (2014) demonstrated that broad
meliaceae), F. cucullata and F. nivalis, have range of this epiphytic lichen has been shaped
widespread artic–alpine distributions in the not only by long-distance dispersal across suit-
Northern Hemisphere but also occur in a few able habitats but also by lineage formation and
disjunct populations in the Southern Hemi- persistence. While the majority of genetically
sphere. Geml et al. (2010) found high genetic distinct R. menziesii lineages tended to occur
diversity and effective long-distance dispersal in distinct ecoregions, high migration out of
capacity among populations of both F. cucul- populations in coastal and the Pacific North-
lata and F. nivalis occurring in the Northern west into inland California populations was
Hemisphere. Long-distance gene flow appears observed. In contrast, populations of R. men-
to have prevented pronounced genetic differen- ziesii in Baja California appear to be relatively
tiation among disjunct populations and sug- isolated. Previously, a complete lack of local
gests that these taxa are able to effectively genetic structure was observed in R. menziesii
track their potential niche during climatic shifts populations growing on distinct oaks species in
(Geml et al. 2010). an oak-savanna ecosystem in southern Califor-
Long-distance, intercontinental dispersal nia (Werth and Sork 2010). The low level of
and subsequent gene flow appear to be com- local genetic structure in some lineages within
mon in a number of other lichen-forming fun- R. menziesii is consistent with high effective
gal species. For example, in the rock-dwelling gene flow in other epiphytic lichen species.
lichen Porpidia flavicunda (Lecideaceae), a lack While population structure corresponding to
of fixed nucleotide polymorphisms and wide different host trees was not observed in R.
sharing of identical haplotypes between dis- menziesii, population differentiation was
junct geographical regions indicates recurrent observed in Xanthoria parietina populations
long-distance gene flow of propagules (Busch- occurring in different habitats (rock vs. bark)
bom 2007). Similarly, lichens of the genus but not in populations occurring in the same
Thamnolia (Icmadophilaceae) occur at high habitat (Lindblom and Ekman 2006). There-
altitudes/latitudes across incredibly broad geo- fore, in some cases habitat isolation, rather
graphic distances without any evidence of phy- than dispersal limitations, may play an impor-
logeographic structure (Nelsen and Gargas tant role affecting gene flow.
2009), although gene flow among disjunct Population structure corresponding to geo-
populations has not been explicitly tested. The graphic regions has been observed in some
broad distribution and lack of phylogeographic species of lichen-forming fungi with broad,
structure is particularly striking given that this intercontinental distributions. Fernández-Men-
taxon is thought to reproduce almost exclu- doza et al. (2011) demonstrated striking genetic
sively via vegetative fragmentation and asco- structure in Cetraria aculeata (Parmeliaceae)
mata are unknown in this genus. While the corresponding to distinct geographic regions.
Ecological Biogeography of Lichen-Forming Fungi 25
However, robust hypotheses of species bound- used habitat models to forecast the frequency of
aries in the C. aculeata group are lacking, and occurrence of epiphytic lichens under different
additional data will be required to accurately forest management strategies in northwestern
circumscribe species and adequately character- USA. These habitat models were able to success-
ize gene flow among disjunct populations fully estimate the occurrence of lichens modeled,
(Printzen et al. 2013). and forecast that forest management strategies
Ongoing research into landscape genetics reducing even-aged stands will increase the fre-
of model systems, such as Lobaria pulmonaria, quency of epiphytes associated with old-growth
will likely continue to provide novel insight forests. Logistic regression models have been
into how geographic and environmental factors shown to accurately predict the occurrence of
impact gene flow and genetic structure in six lichen epiphytes in Switzerland, including
lichens. Fortunately, many of the molecular two threatened or vulnerable lichens, by using
techniques that have previously only been the statistical relationships between response
available for model systems can now be and explanatory variables to predict the distribu-
incorporated into studies of non-model groups tion of lichens in previously unsampled geo-
using genomic data generated from high- graphic areas (Bollinger et al. 2007). Shrestha
throughput sequences (Ekblom and Galindo et al. (2012) used nonparametric multiplicative
2011). Incorporating non-model groups into regression analysis to model the potential distri-
landscape genetic studies of lichen-forming bution of the sensitive indicator species Usnea
fungi will ultimately provide a much more hirta (Parmeliaceae) in western North America.
nuanced perspective into how lichens respond Their predictive model suggested that average
to changing environments. monthly minimum and maximum temperatures,
precipitation, and solar radiation were the major
2. Modeling Distributions of Lichens macroclimatic factors influencing the distribution
of Usnea hirta, providing a useful ecological
Modeling species distributions has the potential niche-based approach to forecast the distribution
to play a pivotal role in biogeographic research. of air pollution-sensitive lichens based on macro-
While species distribution models have become climatic variables (Fig. 2.4). Similarly, ecological
commonplace in biogeographic research, only a niche modeling has also played an important role
limited number of studies of lichen-forming in improving survey design of rare epiphytic
fungi have incorporated modeling approaches macrolichens in the Pacific Northwest, USA
to elucidate biogeographic patterns. Modeling (Edwards et al. 2005).
distributions of species is most commonly Recently, Martellos et al. (2014) utilized
based on pattern-recognition approaches, ecological niche modeling to characterize dif-
where associations between records of a species ferences in the distribution of two varieties of
geographic occurrence and a suite of predictor the soil crust lichen Squamarina cartilaginea in
variables are explored to allow an estimation of Italy. Their study highlights not only the role
the species’ ecological requirements. Accurate the modeling plays in forecasting distributions
modeling depends, in part, on how the appro- but also the potential for using ecological niche
priate use of occurrence data and the adequacy modeling for species delimitation research. In
of the predictors used for model building fact, within an integrative species delimitation
(Anderson et al. 2003). framework, distribution modeling serves as an
Many lichens show significant relationships important independent line of evidence to cor-
with macroclimatic variables (Giordani and roborate lineage separation (Leavitt et al. 2015a;
Incerti 2008; Glavich et al. 2005), and overall it Pelletier et al. 2014). The study by Martellos
appears that there is general support for biocli- et al. (2014) provides the first implementation
matic modeling in lichens (Braidwood and Ellis of ecological niche modeling for resolving tax-
2012). Species distribution models have been used onomic issues in lichen-forming fungi.
in a number of ecological-, biogeographical-, and While distribution modeling can provide
conservation-related studies. McCune et al. (2003) important insight into species distributions
26 S.D. Leavitt and H.T. Lumbsch
Probability Class
0.0 - 0.118
0.119 - 0.224
0.225 - 0.348
0.349 - 0.412
0.412 - 0.498 N
0.499 - 0.588
0.589 - 0.655
0.656 - 0.725
0.726 - 0.796
0.797 - 1.0
10 5 0 10 20
kilometers
Fig. 2.4 Modeled distribution map for Usnea hirta in white hexagons represent sites with no U. hirta, and
the White River National Forest in central Colorado grey circles represent inaccessible sites. White areas
using nonparametric multiplicative regression analysis. represent no data value due to lower average neighbor-
Blue stars represent sites where U. hirta was recorded, hood size. Modified from Shrestha et al. (2012)
and biogeographic patterns, effective distribu- design, the results of which will impact all
tion modeling faces a number of general chal- downstream analyses (Edwards et al. 2006).
lenges, including: (1) clarification of the niche Even when occurrence data is relatively well
concept; (2) weaknesses in sampling ability and characterized, bioclimatic modeling may not
design; (3) variation in parameterization within provide results congruent with known species
each technique, potentially providing different occurrences. Distribution models for the rare
modeled distributions; (4) a need for improved lichen species Staurolemma omphalarioides,
model selection and predictor contribution; known for its disjunct distribution in the Medi-
and (5) a need for more robust model evalua- terranean region and central Norway, well out-
tion strategies (Araújo and Guisan 2006). side of the main range of the species, indicate
Among the most practical issues of distri- that either the species has not reached its
bution modeling is effective sample survey potential distribution or the models fail to
Ecological Biogeography of Lichen-Forming Fungi 27
accurately characterize the actual species distri- ally, lichen photobionts are considered the sub-
bution (Bendiksby et al. 2014). Furthermore, sidiary member of lichen associations;
while models can incorporate macroclimatic however, it now is clear that at least some
variables, as indicated above, lichens are inher- photobionts exhibit differential preference for
ently sensitive to microhabitat variation, and environmental factors (Peksa and Škaloud
measuring and incorporating this variation 2011). Algal preferences potentially limit the
remains challenging. As an example, microhab- ecological niches available to lichens, further
itat conditions, including proximity to water- supported the idea of habitat-specific lichen
courses and bark pH, play important roles in guilds, where lichen communities growing in
determining the occurrence Platismatia norve- similar habitats share the same photobionts
gica (Parmeliaceae) in suboceanic habitats at (Rikkinen et al. 2002). Factors such as photo-
the fringe of the species’ distribution (Lidén biont availability (Werth et al. 2006; Rikkinen
and Hilmo 2005). Similarly, the distribution of et al. 2002), fungal specificity in photobiont
some epiphytic lichens in different forest types choice (Yahr et al. 2004), ecological constraints
is strongly influenced by vertical position (Peksa and Škaloud 2011), and symbiont inter-
within the forest canopy (Coxson and Coyle actions (del Campo et al. 2013; Werth et al.
2003; Antoine and McCune 2004). Land use 2013) have all been shown to have a major
intensity has been shown to influence the local impact on the occurrence of lichens.
variation of lichen diversity in Mediterranean The spiny heath lichen (mycobiont ¼
ecosystems (Giordani et al. 2010). However, Cetraria aculeata; Fernández-Mendoza and
other lichen communities appear to be unaf- Printzen 2013; Pérez-Ortega et al. 2012; Fernán-
fected by human activity and are predomi- dez-Mendoza et al. 2011; Printzen et al. 2013),
nantly determined by macroclimatic factors lace lichen (mycobiont ¼ Ramalina menziesii;
(Werth et al. 2005). Werth and Sork 2010, 2014), and lung lichen
Going forward, species distribution model- (mycobiont ¼ Lobaria pulmonaria; Werth et al.
ing holds important promise in ecological bio- 2006; Dal Grande et al. 2012; Singh et al. 2014)
geographical research of lichens. Due to the fact have become model groups for understanding
that species distributions reflect the dynamic the dynamic roles that photobionts play in deter-
interplay of geographic and environmental pro- mining lichen distributions. Based on broad geo-
cesses with biotic factors, including species dis- graphic sampling of the spiny heath lichen
persal capabilities and interactions with other (mycobiont ¼ Cetraria aculeata), Fernández-
species, developing a stronger link between Mendoza et al. (2011) provide evidence that
ecological factors and modeling will be benefi- photobiont switches played an important role
cial for developing more approaches for distri- in increasing the geographical range and ecolog-
bution modeling. Advances in spatial data ical niche of lichen mycobionts by associating
technologies, geographic information system them with locally adapted photobionts in climat-
(GIS) data, and modeling approaches will con- ically distinct regions. In this specific case, the
tinue to facilitate ecological and evolutionary photobiont switch allows C. aculeata that is
insight and predict distributions of lichen- common in temperate and alpine habitats to
forming fungi. extend into semiarid regions in the Mediterra-
nean. In the lace lichen (mycobiont ¼ Ramalina
menziesii), ecological specialization of the
3. Role of Photobionts in Ecological photobiont (Trebouxia decolorans), geography,
Biogeography and climate shape the distribution of this lichen
(Werth and Sork 2014). Algal specialization on
Lichens represent iconic examples of sym- local environmental conditions, including
bioses, and therefore taking their symbiotic macroclimatic factors and substrate ecology,
partners into account in biogeographic allows R. menziesii to associate with locally
research is likely to provide valuable insight adapted photobiont strains. As discussed previ-
into elucidating biogeographic patterns. Gener- ously, dispersal limitations of the mycobiont
28 S.D. Leavitt and H.T. Lumbsch
Lobaria pulmonaria is not the most important species-level classification system for major
mechanism underlying differentiation among groups of lichen photobionts in order to
populations. Availability of the appropriate enhance communication about diversity, distri-
photobionts has been proposed as a potential butions, ecological patterns, and interactions in
mechanism generating population structure in lichen symbioses (Leavitt et al. 2015b).
L. pulmonaria (Werth et al. 2006, 2007).
While species richness of lichen-forming
fungi is associated with both climate and forest
structure variables, specific responses to these
IV. The Role of Biogeography in
different variables were dependent on the type Conservation and Climate
of photobiont. A study of patterns in lichen Change Research
richness across Italy highlights the photobiont-
dependent response of species richness to vari- Lichens are not easy targets for conservation
ous environmental factors (Marini et al. 2011). measures due to the fact that lichens represent
Mycobiont species paired with chlorococcoid the symbiotic phenotype of multiple interacting
green algae was correlated with increasing for- species (Scheidegger and Werth 2009). This
est cover. While species richness of cyanoli- challenge is further confounded by the fact
chens—lichen-forming fungi associating with that many lichens have specific habitat require-
photosynthetic cyanobacteria—was related to ments that are not generally shared with other
area and precipitation, lichens with Trentepoh- organisms. An ecological biogeographic per-
lia algae were enhanced by rainy and warm spective of lichen-forming fungi highlights the
climates (Marini et al. 2011). In some cases, importance of seeing distributions of species as
the mycobiont’s habitat preferences may be the result of dynamic interactions among sym-
determined by factors that are independent of bionts and ecological and environmental fac-
those of the photobiont at the landscape level tors. Scheidegger and Werth (2009) provide an
(Nadyeina et al. 2014). The differential invaluable perspective into lichen conservation
responses of lichens associating with different biology, including potential strategies to effec-
types of photobionts to ecological, climatic, and tively protect lichens and develop priorities for
other environmental factors demonstrate the conservation approaches. Here we only high-
challenges in predicting lichens distributions. light in brief a number of examples from lichen
These studies clearly indicate the potential conservation biology literature, emphasizing
importance of carefully taking the role of the the role of maintaining habitat quality, connec-
photobiont into consideration when consider- tivity, and size.
ing ecological biogeography. Currently, studies Relative to more charismatic species, con-
of species interactions in lichen symbioses are servation of lichens generally receives very lim-
limited by uncertainty in the circumscription ited attention from governmental organizations
photobiont species (Kroken and Taylor 2000; and other institutions. Conservation status of
Sadowska-Deś et al. 2014; Blaha et al. 2006; most lichens is unknown, and therefore, con-
Dahlkild et al. 2001; Tibell 2001). For example, servation approaches may fail to protect vul-
in spite of the fact that traditional morphology- nerable lichen and lichen communities. For
based species circumscriptions have consis- example, the Natura 2000 Program was estab-
tently been shown to be inadequate to charac- lished in the European Union as the main
terize species-level diversity in Trebouxia instrument for nature conservation, focusing
(Kroken and Taylor 2000; Sadowska-Deś et al. on protecting the most threatened habitats,
2014; Blaha et al. 2006; Dahlkild et al. 2001), only ensuring the long-term survival of species,
a limited number of studies have implemented and reducing the loss of biodiversity caused
objective methods for delimiting species-level by anthropogenic impact (http://www.natura.
lineages (Sadowska-Deś et al. 2014; Kroken org/). While Martı́nez et al. (2006) demonstrate
and Taylor 2000). We argue that there is a press- that the Natura 2000 network may include key
ing need to develop and adhere to a practical, habitat in conserved forests and mountain
Ecological Biogeography of Lichen-Forming Fungi 29
ranges for a number of lichens in Spain, the practices, changes in atmospheric pollution
effectiveness of the Natura 2000 network in levels, etc. Measurable responses of individual
protecting Mediterranean lichens is quite low lichen thalli (e.g., differential accumulation of
(Rubio-Salcedo et al. 2013). Natura 2000’s atmospheric pollutants) and lichen commu-
reserve network, based mainly on vascular nities (e.g., changes in community composition
plant data, may be ineffective for neglected and population density) can provide a means to
taxonomic groups, like lichens, and highlights quantitatively assess ecosystem health
the need to include “noncharismatic” species to (McCune 2000).
improve reserve design and other conservation Climate change is forecast to promote
strategies (Rubio-Salcedo et al. 2013). However, major ecological shifts worldwide (Parmesan
even when explicitly taking lichens into consid- and Yohe 2003; Araújo and Rahbek 2006). Spe-
eration when developing models to predict spe- cies that are unable to tolerate or adapt in situ
cies richness, it appears lichen surveys are to altered environmental conditions or migrate
critical for assessing species abundance, to suitable habitats face potential extinctions.
dynamics, and viability (Waser et al. 2007). Alternatively, climate change may lead to major
Ultimately, successful lichen conservation shifts in species distributions. Some compo-
is contingent on a wide variety of factors, nents of cryptogamic communities, including
including effective approaches for prioritizing lichens, have been shown to be particularly
the protection of vulnerable habitat and species sensitive to climatic shifts (Cornelissen et al.
(Bowker et al. 2008), promoting interdisciplin- 2001; Bjerke 2011). Although lichens are well-
ary collaborations (Campbell 2005), incorpor- known indicators of air quality (Leavitt and St.
ating population/landscape genetics into policy Clair 2015), recent studies indicate that they
decisions (Scheidegger and Werth 2009), and may also be useful in assessing ecological shifts
increasing the number of bio-inventory surveys related to climate change (Bjerke 2011; Ellis
which include lichens. Lättman et al. (2009) et al. 2007a, b; Cornelissen et al. 2001). Some
suggested that dispersal capability is likely to lichens, due to their sensitivity, may play an
be commonly underestimated for lichens important role in monitoring the potential
defined to habitat with long ecological continu- impacts of climate change.
ity, and this perspective was corroborated for In general, specific responses of most spe-
epiphytic lichens occurring in the boreal rain- cies, including lichen-forming fungi, to rapid
forest in central Norway (Hilmo et al. 2012). climate changes in vulnerable habitats remain
Therefore, successful conservation of some uncertain, and detailed, long-term monitoring
lichens may be possible even when a suitable will be essential to accurately assessing biologi-
habitat is highly fragmented. cally meaningful shifts in community composi-
Lichens are included among a suite of indi- tion and species distributions (Eaton and Ellis
cators of ecosystem health, which also includes 2012). On a global scale, macrolichens in cli-
bryophytes (Frego 2007; Pesch and Schroeder matically milder arctic ecosystems may decline
2006), vascular plants (Coulston et al. 2003), if and where global changes cause vascular
some terrestrial and aquatic invertebrates plants to increase in abundance (Cornelissen
(Hodkinson and Jackson 2005), and other sen- et al. 2001). In the UK, the southern elements
sitive species and/or communities (Leavitt and of Britain’ s lichen flora, and other lichen spe-
St. Clair 2015). Rather than abiotic metrics, bio- cies adapted to warmer climates, are projected
indicators are likened to canaries in a coalmine, to expand northward, while the montane spe-
serving as a direct surrogate for assessing dis- cies appear to be disproportionally threatened
turbances on biological communities. Lichens by climate change (Ellis et al. 2007b). Other
are particularly useful as bio-indicators due to data suggest that a warmer, humid climate in
the fact that many live and grow continuously Norway will likely be beneficial for the general-
for decades, or even hundreds of years, showing ist species H. physodes, but detrimental to the
cumulative responses to ecological changes, subalpine birch specialist Melanohalea olivacea
including climate change, land management (Evju and Bruteig 2013).
30 S.D. Leavitt and H.T. Lumbsch
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Ecological Biogeography of Lichen-Forming Fungi 37
Fungi, like other organisms, require infor- from night and alters their physiology accord-
mation about which way is up or down, how to ingly.
orient themselves, and clues to whether the Notably, the yeast Saccharomyces cerevisiae
fungus is in the soil, inside a host, or exposed is absent from the long list of photoresponsive
to air and other types of stress. In contrast with fungi. Although exposure of this yeast to
photosynthetic organisms, fungi use sunlight increasing illumination results in a reduction
not as a source of energy but to obtain infor- in growth rate, the effect of light is weak, and it
mation from their environment. The ability of a appears to occur only at low temperatures
fungus to respond to light is likely to contribute (Edmunds et al. 1978). Additionally, cell divi-
to its survival, its fitness, and its ability to com- sion and amino acid transport in S. cerevisiae
pete. Visible light, while not necessarily damag- are synchronized to light/dark (LD) cycles
ing in itself, can provide early warning of (Edmunds et al. 1979). The lack of robust light
impending UV damage (Gressel and Rau 1983; responses could mean that photoresponsive-
Berrocal-Tito et al. 1999). ness was selected out, at least in laboratory
Although fungi typically grow in damp, dark strains.
habitats, numerous studies have revealed that To respond to light fungi require photore-
exposure to light is accompanied by major ceptors (proteins or protein complexes) able
changes in their physiology. A response to light to perceive light that generate a signal, which
might be very rapid, simply changing the direc- is propagated into the cell to initiate a cellular
tion of growth, or might provide the initial cue response. Their light-absorbing cofactors,
to alter transcriptional profiles. These, in turn, known as pigments or chromophores, are typi-
provide the enzymes and metabolites needed for cally small molecules. Among these chromo-
protection from damage as well as programming phores, riboflavin (vitamin B12), flavin
the development of new structures. Develop- mononucleotide (FMN), flavin adenine dinu-
mental decisions triggered by light, depending cleotide (FAD), and porphyrins such as a
on the intensity, require from nanoseconds to heme group absorb light in the visible range.
minutes of exposure and amounts as low as Consequently, most—if not all—biological
1010 molm2 (Betina and Zajacova 1978; Hor- responses to visible light must be initiated by
witz et al. 1990; Corrochano 2007). Fungi display one of these pigments (Casas-Flores and
very rapid responses to light (e.g., phototro- Herrera-Estrella 2013).
pism), some of which were discovered more
than 150 years ago (Payen 1843). In fact, many
alterations in fungal morphology provoked by II. The Visual System of Fungi
light have been described in hundreds of fungi
(Marsh et al. 1959). However, it was Max Del- A. Flavin-Binding Photoreceptors
brück who in his pioneering work established
the basis for the development of fungal photobi- Light regulates growth and development in
ology in the 1960’s (Delbrück and Shropshire fungi, including sexual and asexual develop-
1960; Delbrück and Varju 1961). ment, phototropism, biological rhythms,
Rhythmic changes such as day and night, to metabolism, and gene transcription, among
which most organisms are well adapted, require others (Herrera-Estrella and Horwitz 2007;
anticipation of dusk and dawn and proper reac- Idnurm and Heitman 2005; Casas-Flores and
tion to the changing environment represent a Herrera-Estrella 2013; Fuller et al. 2014). As
major evolutionary adaptation. Fungi are no mentioned above, to respond to this environ-
exception and possess biological clocks, includ- mental cue, fungi require photoreceptors that
ing a ca 24 h clock (the circadian clock), which enable them to respond to different light quali-
uses light as one of the main cues to keep time. ties and quantities.
The machinery involved in the regulation of the Phycomyces was probably the first fungus in
circadian clock helps fungi distinguish day which light perception was analyzed. Max Del-
The Bright and Dark Sides of Fungal Life 43
brück concentrated in studying the capacity of PAS domains are involved in protein-protein
this fungus to “see.” He described in great detail interaction (Taylor and Zhulin 1999). These
the properties of the response to light of Phy- photoreceptors, in general, also contain GATA
comyces and the genetic zapping of the signal zinc finger DNA-binding domains, nuclear
transduction pathways which was initiated in localization signals (NLS), and activation
his laboratory but faced serious difficulties in domains in several cases (Corrochano 2011),
the molecular characterization of the first step but there are exceptions to the presence of the
of these pathways, photoperception (Delbrück latter (Fig. 3.2), which implies the participation
and Shropshire 1960; Bergman et al. 1973). of additional factors for the activation of tran-
Another fungus for which the search for ele- scription (Casas-Flores et al. 2004). The N.
ments involved in light perception was initiated crassa WC-1 protein dimerizes with its tran-
at about the same time was Trichoderma (Gres- scriptional partner, the WC-2 protein, which
sel and Hartmann 1968; Kumagai and Oda contains a PAS, a GATA zinc finger domain,
1969). In both cases it was concluded that the and an activation domain to form the White
most likely receptor for UV-blue light was a Collar Complex (WCC) (Fig. 3.2). Mutants in
flavoprotein. Accordingly, the operation of either wc-1 or wc-2 are affected in all known
cryptochromes was suggested—a hypothesis blue-light responses. In a similar way, mutants
corroborated many years later by the presence in other fungal models such as Trichoderma
of genes encoding potential flavoproteins that spp. and A. nidulans are also affected in blue-
could act as photoreceptors in several fungi light perception (Casas-Flores et al. 2004; Cas-
(Ballario et al. 1996; Casas-Flores et al. 2004; tellanos et al. 2010; Purschwitz et al. 2008).
Idnurm et al. 2006). In Mucor circinelloides, three white collar-1
Flavin-binding photoreceptors contain a genes (mcwc-1a, mcwc-1b, and mcwc-1c) ortho-
specialized PAS (Per-Arnt-Sim) domain called logous to the N. crassa WC-1 have been identi-
LOV (light-oxygen-voltage), which is a highly con- fied, and all three contain a LOV domain.
served domain among proteins that sense UV- mcwc-1a regulates phototropism, whereas
blue light in plants and fungi (Fig. 3.1a). LOV mcwc-1c regulates photocarotenogenesis (Silva
domains are about 110–120 aa (amino acids) in et al. 2006). The mcwc-1b product is also
length and present a highly conserved structure involved in carotenoid synthesis in some
from bacteria to plants, which adopt a conserved mutant background strains (Silva et al. 2008).
a/b-fold that binds FAD or FMN (Figs. 3.1b and Some fungi contain in their genomes a sec-
3.2). Upon exposure to blue light, a covalent ondary photoreceptor (Vivid/Envoy), which is
adduct between the flavin molecule and a con- entirely conformed by a LOV domain (Fig. 3.2)
served cysteine residue in the LOV domain is and binds either FAD or FMN as chromophore
formed. Once in the darkness, this adduct is (Zoltowski and Crane 2008; Lokhandwala et al.
dissociated and the photocycle is completed 2015). These photoreceptors are important for
(Salomon et al. 2000). photoadaptation under constant light and to
The founding member of blue-light fungal respond to changes in light intensity. Vivid
photoreceptors is the White Collar-1 (WC-1) fine-tunes these light responses by interacting
protein from Neurospora crassa; however, the with the WCC through the WC-1 LOV domain,
existence of this kind of light receptors was first to quench these responses (Vaidya et al. 2011).
suggested in Trichoderma spp. during the first Cryptochromes (Cry) are flavoprotein
half of the nineteenth century that was further photoreceptors with a structure closely related
supported by the cloning of the blue-light to that of DNA photolyases (Fig. 3.2), which
receptor 1 (Blr1), the WC-1 orthologue directly bind DNA or RNA, are sensitive to
(Casas-Flores et al. 2004). Fungal photorecep- blue and UV light, but display strongly reduced
tors belonging to the WC-1 family contain in or have lost DNA-repair activity (Daiyasu et al.
their structure three PAS domains, where the 2004; Lin and Todo 2005). Crys regulate growth
first one is a LOV domain, specialized in sens- and development in plants and circadian
ing blue light (He et al. 2002). The other two rhythms in plants and animals (Daiyasu et al.
44 S. Casas-Flores and A. Herrera-Estrella
Fig. 3.1 (a) Evolutionary relationships of fungal LOV the LOV1 and LOV2 domains from Arabidopsis thali-
domains taxa. The evolutionary history was inferred ana phototropin (GenBank accession no. AEE78073.1),
using the neighbor-joining method (Saitou and Nei Neurospora crassa WC-1 (GenBank accession no.
1987). The optimal tree with the sum of branch length Q01371), and T. atroviride ENV1 (GenBank accession
¼ 5.27804499 is shown. The tree is drawn to scale, with no. AAT40588.1), the blue-light photoreceptor from
branch lengths in the same units as those of the evolu- Bacillus subtilis (GenBank accession no. CEI58264.1).
tionary distances used to infer the phylogenetic tree. LOV domains representative of the different classes of
The evolutionary distances were computed using the fungi were also included—Agaricostilbum hyphaenes
Poisson correction method (Zuckerkandl and Pauling (ID: 234740; Pucciniomycotina), Ustilago maydis (Gen-
1965) and are in the units of the number of amino acid Bank accession no. XP_011389633.1; Ustilaginomyco-
substitutions per site. The analysis involved 18 LOV tina), Agaricus bisporus var. bisporus (ID: 186808;
domain amino acid sequences which are listed in (b). Agaricomycotina), Ascobolus immersus (ID: 242339;
All positions containing gaps and missing data were Pezizomycetes), Arthrobotrys oligospora (ID: 7017;
eliminated. There were a total of 93 positions in the Orbiliomycetes), Aspergillus nidulans (ID: 8655; Euro-
final dataset. Evolutionary analyses were conducted in tiomycetes), Cochliobolus carbonum (ID: 21495; Dothi-
MEGA6 (Tamura et al. 2013). (b) LOV-domain amino deomycetes), Yarrowia lipolytica (ID: 65092;
acid sequences alignment, which depicts the flavin- Saccharomycotina), Rhizophagus irregularis (ID:
interacting residues, and the forming adduct cysteine 88544; Glomeromycota), Mucor circinelloides (ID:
with the chromophore. The sequence analysis includes 117241; Mucoromycotina), Conidiobolus coronatus
The Bright and Dark Sides of Fungal Life 45
Fig. 3.2 Domain organization of the different classes of which uses folate as harvesting cofactor. They also
known and putative fungal photoreceptors and photo- contain a FAD-binding domain of DNA photolyase.
lyases. The flavin-binding photoreceptors contain a Opsins resemble the microbial opsin-related proteins,
specialized LOV domain, which is known to bind FAD which bind rhodopsin to perceive green light. The phy-
or FMN as pigment to harvest light (WC-1, VVD/ENV1, tochrome binds biliverdin as chromophore, and its
and CRY). The WC-1 orthologous also contains two structure includes a GAF small ligand-binding domain,
PAS domains, a GATA zinc finger DNA-binding a phytochrome region (PHY), a histidine kinase A
domain, a nuclear localization signal, and activation (phosphoacceptor) domain, one dimerization, one
domains. The WC-2 orthologous, the transcriptional phosphoacceptor domain (HisKA), a HisKA-like
partners of WC-1, contain one PAS domain, a GATA ATPase domain, an RRD, and a signal receiver domain
zinc finger domain, and activation domain. The DNA 6- that receives the signal from the sensor partner in a
4 and CPD photolyases and cryptochrome are related two-component system. It contains a phosphoacceptor
flavoproteins, containing a DNA photolyase domain, site that is phosphorylated by HisKA
2004; Lin and Todo 2005). Based on phyloge- chromes lack the latter domain (Fig. 3.2). A.
netic analysis, cryptochromes have been classi- nidulans possesses only one cryptochrome/
fied in plant, animal, and DASH (from photolyase-like photoreceptor, named CryA,
Drosophila, Arabidopsis, Synechocystis, which includes a PHR domain containing both
human) cryptochromes (Partch and Sancar DNA photolyase and FAD-binding domains.
2005). At the structural level, cryptochromes This protein shows sensory and regulatory
have an amino-terminal photolyase-related roles during A. nidulans development and
region (PHR) and a carboxy-terminal domain DNA-repair activity when expressed in Escher-
of variable length. However, DASH crypto- ichia coli (Bayram et al. 2008a).
⁄
Fig. 3.1 (continued) (ID: 58233; Entomophthoromyco- itation. LOV residues that interact with FMN are
tina), Ramicandelaber brevisporus (ID: 90268; Kickxel- marked with arrowheads. Colored residues in LOV
lomycotina), and Catenaria anguillulae (ID: 114754; domains are according to their physicochemical prop-
Blastocladiomycota)—but are not shown by space lim- erties
46 S. Casas-Flores and A. Herrera-Estrella
In the filamentous fungus N. crassa, the though when NOP-1 and opsin (OPS) show
CRY-DASH-type cryptochrome binds FAD high sequence similarity, their biochemical
and MTHF (methenyltetrahydrofolate). The characteristics suggest different roles in light
robust accumulation of cry and CRY upon a sensing and proton pumping, respectively
light pulse is wc-1 dependent. Purified CRY (Furutani et al. 2006). The differences between
protein binds to both single- and double- the photochemical behavior of LR and NOP-1
stranded molecules of DNA or RNA in vitro are due to the replacement of the cytoplasmic
(Froehlich et al. 2010). As described for N. proton donor Asp with Glu (Furutani et al.
crassa, the CRY-DASH cryptochrome tran- 2006).
script (cryD) from Fusarium fujikuroi is highly The filamentous fungus Fusarium fujikuroi
induced by light and is regulated by the WC-1 possesses two genes, which encode putative
orthologue. Recently, a member of the crypto- retinal-binding opsins called CarO and OpsA,
chrome/photolyase family, Cry1, was character- and a gene for an opsin-related protein (hspO).
ized in T. reesei. The cry1 transcript is positively OpsA was classified as NR-like rhodopsin by
regulated and modulated by the Blr and Env1 sequence similarity to that from N. crassa,
proteins. Heterologously expressed Cry1 binds which suggests also a very slow photocycle
to undamaged and 6-4PP damaged DNA and and lack of proton pump activity. On the con-
photorepairs it but does not repair CPD and trary, CarO was classified as an auxiliary ORP-
Dewar DNA lesions (Guzmán-Moreno et al. like rhodopsin that contains all conserved
2014). amino acids required to function as proton
pump and, together with LR-like rhodopsins,
shows fast photocycles. The carO and opsA
B. Opsins or Rhodopsins genes are light regulated, and their induction
is WC-1 (wcoA) dependent. Interestingly, carO
Opsins/rhodopsins comprise seven transmem- and opsA transcripts are not accumulated in
brane domain proteins that bind retinal via a carotenoid-overproducing mutants (Estrada
conserved lysine residue to form green-light- and Avalos 2009). Indeed, CarO is a green-
responsive ion pumps to transport ions (H+ or light-driven proton pump as demosntrated by
Cl–) across membranes or sensory receptors in patch-clamp electrophysiological experiments
microorganisms (reviewed by Spudich et al. with heterologously expressed CarO. Fusion of
2000; Spudich 2006). In fungi, the funding CarO with GFP led to determine that CarO is
member of this protein family is NOP-1 from strongly accumulated in conidia, upon expo-
N. crassa, which was isolated from an expressed sure of mycelia to light (Garcı́a-Martı́nez et al.
sequence tag from a mycelial library (Bieszke 2015).
et al. 1999). In the brown leaf spot fungus Bipolaris ory-
The nop-1 transcript is prominently zae two opsin-like genes, ops1 and ops2, were
expressed in conidia and sexual structures identified. The ops1 transcript is highly
under light conditions. However, the nop-1 expressed in mycelia under near-UV irradia-
transcript level was not affected in the WC-1- tion, but the ops2 transcript is constitutively
defficient mutant during conidiation (Bieszke expressed in mycelia under dark conditions
et al. 2007). NOP-1 binds retinal in vitro and and weakly induced after near-UV application.
undergoes a very slow photocycle, whereas pro- The expression of ops1 and ops2 is Blr1 (the
ton pump activity was not detected, which indi- orthologue of WC-1 in B. oryzae) dependent
cated a putative role in light sensing in N. crassa (Kihara et al. 2009).
(Bieszke et al. 1999). In Leptosphaeria macu- In Blastocladiella emersonii light percep-
lans, the opsin LR (Leptosphaeria rhodopsin) tion is accomplished by the function a novel
shows a fast bacteriorhodopsin-like photocycle type I (microbial) rhodopsin domain and gua-
and pumps protons light dependently. In this nylyl cyclase catalytic domain encoding gene
case the LR transcript is not induced by light, (BeGC1), which is highly expressed in late spor-
which contrasts with nop-1 from N. crassa. Even ulation cells, during zoospore biogenesis.
The Bright and Dark Sides of Fungal Life 47
Photobleaching of rhodopsin inhibits the accu- iliformis (ascomycetes), and Ustilago maydis
mulation of cGMP and phototaxis of zoospores and Cryptococcus neoformans (basidiomycetes)
when exposed to green light, whereas depletion but not in the yeasts Saccharomyces cerevisiae,
of guanylyl cyclase activity negatively impacts Schizosaccharomyces pombe, Candida albicans,
phototaxis. The BeGC1 protein was located in or Ashbya gossypii (Blumenstein et al. 2005).
the external surface of the zoospore eyespot Intriguingly, FphA forms a complex with LreA
positioned close to the base of the swimming and LreB, the orthologues of WC-1 and WC-2,
flagellum. Therefore, Blastocladiomycota fungi the central components of blue-light perception
have a cGMP signaling pathway involved in in N. crassa.
phototaxis similar to the vertebrate vision-
signaling cascade integrated in a novel gene
fusion encoding a protein composed of the dif-
ferent protein domains and of distant evolu- III. Growth and Primary Metabolism
tionary ancestry to type II rhodopsins of
animals (Avelar et al. 2014). Filamentous growth of a variety of fungi is
affected in diverse ways by light. Branching in
N. crassa hyphae increases in cultures grown in
the light, as compared to cultures grown in the
C. Phytochromes
dark, resulting in more compact colonies (Lau-
Phytochromes are a widespread family of pro- ter et al. 1998). This effect of light requires the
teins that covalently attach to bilin-type (or WC proteins and has also been observed in the
linear tetrapyrrole) chromophore and absorb truffle Tuber borchii (Lauter et al. 1998; Ambra
in the red/far-red region of the spectrum. The et al. 2004). The inhibition of mycelial growth
bilin-type chromophores enable photoconver- by light may promote the growth of Tuber
sion between red-absorbing (Pr) and far-red- underground (Ambra et al. 2004). A similar
absorbing (Pfr) forms of phytochromes (Rock- effect of light has been observed in another
well and Lagarias 2010). The most common ascomycete, Trichoderma atroviride, whose
structural architecture of bacterial, plant, and hyphal growth is inhibited by light but in a Blr
fungal phytochrome is composed of an N- (WC) independent fashion (Casas-Flores et al.
terminal photosensory core with three con- 2004). Promotion of hyphal branching has also
served PAS-GAF-PHY domains (Per/Arndt/ been documented for the plant pathogen Colle-
Sim-cGMP phosphodiesterase/adenylyl cyclase/ totrichum trifolii (Chen and Dickman 2002).
FhlA-phytochrome specific) and a C-terminal In the case of Neurospora, the Ser/Thr pro-
regulatory histidine kinase-related domain tein kinase Cot-1 is involved in the regulation of
(HKRD; Fig. 3.2) (Rockwell and Lagarias branching and colonial growth, and expression
2010). Phytochrome means “plant pigment” of the corresponding gene is regulated by light
and was first discovered in higher plants in through an as yet not fully understood mecha-
the mid-twentieth century, based on the ability nism (Yarden et al. 1992; Lauter et al. 1998;
of red and far-red light to control several pro- Gorovits et al. 1999). The C. trifolii homologue
cesses of plant growth and development (Quail of the Neurospora cot-1 gene (tb-3), like cot-1, is
2002; Smith 2000). A. nidulans possesses a phy- regulated by light, but TB3 may act as a tran-
tochrome (FphA) that is more closely related to scriptional regulator for hyphal branching, as
bacterial than to plant phytochromes. FphA suggested by its nuclear localization and the
binds biliverdin as chromophore and is located presence of putative transcriptional activation
in the cytoplasm, which indicates that red-light domains (Chen and Dickman 2002).
perception occurs in the cytoplasm (Blumen- As we have seen, light influences growth in
stein et al. 2005). Furthermore, FphA has kinase the fungi, which is very likely linked to the
activity and is likely to act as red-light sensor. major changes in metabolism that have been
Orthologues of FphA have also been found in detected when a fungus grows exposed to light
the genomes of A. fumigatus, Gibberella mon- or darkness.
48 S. Casas-Flores and A. Herrera-Estrella
Fig. 3.3 Schematic representation of the impact of bisphosphate (yellow oval), no alteration was
light on the glycolytic pathway and the consequent found, and in case of gray ovals, no data on the
effect on polysaccharide biosynthesis and utilization. levels of these metabolites in light are available (as
Green ovals indicate increased levels of the metabo- interpreted by Tisch and Schmoll 2010). Red rectan-
lite when a fungus is cultivated in light, and red gles indicate lower transcript and enzymatic activ-
ovals represent decreased levels. For fructose-1,6- ities of the indicated protein
The Bright and Dark Sides of Fungal Life 51
addition of dibutyryl cyclic AMP (dBcAMP), an not in nitrogen-rich media (Cetz-Chel et al.
analog of cAMP, to T. reesei cultures in the 2016).
presence of a potent cellulase inducer repressed
or activated the synthesis of endoglucanase
depending on the dBcAMP concentration. Con-
sistently, the addition of the phosphodiesterase IV. Development
inhibitor 3-isobutyl-1-methylxanthine (IBMX)
provoked an increase in intracellular cAMP Fungal development is often modified or trig-
levels and promoted the synthesis of endo- gered by the incidence of light. The main
glucanase. In this regard, it has been established developmental transitions in the fungal life
that light induces changes in intracellular cAMP cycle are spore germination, hyphal growth
levels (Gresik et al. 1988) and that IBMX stimu- and branching, and the formation of resting
lates photoconidiation in Trichoderma or reproductive structures. Fungi take advan-
(Berrocal-Tito et al. 2000; Sulová and Farkás tage of the presence of ambient light, and
1991). changes in its quality and quantity, to modu-
Another example of the regulation by light late several steps of their development. The
of extracellular enzymes is found in Aspergillus regulation by light of fungal development is
niger, which when exposed to blue or red light generally referred to as fungal photomorpho-
showed enhanced glucoamylase activity (Zhu genesis, and can be measured precisely, allow-
and Wang 2005). ing us to determine useful parameters such as
Regarding nitrogen metabolism, until action spectra and thresholds, and has
recently there were only some indications to attracted the attention of scientists worldwide.
its relation with light. However, there is Within the spectrum of visible light, blue is in
increasing evidence of the effect of light on the general most effective in fungal photomorpho-
abundance of amino acids. genesis but other wavelengths are also impor-
In A. ornatus, the uptake of many amino tant.
acids clearly decreases upon cultivation in The fact that light induces initiation and
light (Hill 1976), and in P. blakesleeanus, illumi- development of sclerotia—multicellular resting
nation of dark-adapted cultures decreased orni- structures, resistant to adverse conditions—in
thine decarboxylase activity (Lapointe and various filamentous fungi has been known for a
Cohen 1983). In the basidiomycete Polyporus long time (Chet and Henis 1975; Carlile 1956;
hispidus, phenylpropanoid metabolism and his- Heath and Eggins 1965; Rai et al. 1967; Rudolph
pidin biosynthesis are regulated by light (Nam- 1962; Tarurenko 1954). Back in the 1960s, Kai-
budiri et al. 1973). Further, in T. viride glutamic ser (1964) found that in Verticillium albo-
acid decarboxylase, catalyzing alpha- atrum, red light promoted microsclerotia pro-
decarboxylation of L-glutamic acid to produce duction while blue light inhibited it, and ultra-
gamma-aminobutyric acid (GABA) is also sti- violet radiation completely abolished
mulated by light (Pokorny et al. 2005; Strigacova microsclerotial formation in one isolate
et al. 2001), which is considered a pathway (Brandt 1964). Similarly, cultures of Sclerotium
required for conidial germination in N. crassa rolfsii produce considerably greater numbers of
(Schmit and Brody 1975). In addition, in N. sclerotia when exposed to light (Gruelach and
crassa several genes involved in nitrogen metab- Mohr 1947), and Trevethick and Cooke (1973)
olism are controlled by the circadian clock found that in Sclerotium rolfsii, Sclerotium del-
(Correa et al. 2003), and nitrate reductase activ- phinii, and Sclerotinia sclerotiorum, both num-
ity decreases (Klemm and Ninnemann 1979). ber and size of sclerotia could be determined by
Finally, a recent study on the role of the light- photoperiod length.
induced gene blu7, encoding a putative tran- Until very recently, the mechanism by
scription factor, revealed that it is involved in which light influenced sclerotial morphogene-
the expression of several amino acid transpor- sis was not understood at all. However, a puta-
ters, and deletion of the gene results in clearly tive histidine kinase (HK) gene Bcphy3 from B.
reduced growth in nitrogen-limited media but cinerea, containing a conserved GAF and a PHY
52 S. Casas-Flores and A. Herrera-Estrella
domain (putative phytochrome chromophore- 1990). Accordingly, the main blue-light recep-
binding sites), in which expression is induced tor involved in this process is the Blr complex
by light, was recently cloned. Mutants in which (Blr1/Blr2), which is also essential for the con-
Bcphy3 was disrupted were found to produce a trol of gene expression (Casas-Flores et al. 2004;
substantially reduced amount of sclerotia com- Rosales-Saavedra et al. 2006). Similarly,
pared to the wild-type strain (Hu et al. 2014). mutants in the Trichoderma reesei homologues
These results indicated that Bcphy3 might of the blr genes do not conidiate in response to
encode a highly divergent phytochrome-like light (Castellanos et al. 2010).
HK. The exact reason why Bcphy3-deleted Interestingly, overexpression of blr2
mutants lost the ability to produce sclerotia is resulted in enhanced sensitivity to light in T.
still unknown. Nevertheless, as in the case of V. atroviride (Esquivel-Naranjo and Herrera-
albo-atrum, sclerotia production in B. cinerea Estrella 2007). Nevertheless, recent biochemical
appears to be promoted by red and far-red light and molecular data suggest the participation of
(Tan and Epton 1973). Thus, it is possible that at least two light perception systems that regu-
the functional loss of the phytochrome-like HK late photoconidiation (Berrocal-Tito et al. 2000;
gene Bcphy3 may affect the sclerotia produc- Rocha-Ramirez et al. 2002; Rosales-Saavedra
tion process. et al. 2006; Casas-Flores et al. 2006). In this
As in the case of sclerotia formation, copi- sense, increases in cAMP-dependent protein
ous literature on the effects of light on asexual kinase (PKA) activity after a pulse of blue
reproduction in ascomycetes and the related light have been observed (Casas-Flores et al.
Fungi Imperfecti exists since the 1930s. Already 2006). However, such activation occurred even
in 1933 it was reported that light is required for in Dblr1 and Dblr2 mutant strains, confirming
the production of macroconidia in Sclerotinia the existence of an alternative system for light
fructigena (Hall 1933), whereas Sclerotinia fruc- perception linked to cAMP. The same authors
ticola requires darkness. showed that transformants expressing an anti-
Conidiation may be triggered by several fac- sense version of pkr-1, a gene encoding the
tors related to stress such as light, nutrient avail- regulatory subunit of PKA, with higher levels
ability, oxidant state, and mycelial injury (Aguirre of PKA activity did not produce conidia after
et al. 2005; Casas-Flores et al. 2004; Hernández- exposure to a pulse of blue light. Unfortunately,
Oñate et al. 2012; Horwitz et al. 1985). the light receptor responsible for the activation
The fact that continues darkness results in a of the cAMP pathway has not been identified.
delay or failure of asexual sporulation of Tri- Conidiation in Aspergillus involves a com-
choderma was reported in the 1950s, as well as plex regulatory network of gene expression and
the considerable differences between isolates in cell differentiation (reviewed by Adams et al.
their capacity to respond to illumination (Gut- 1998) and is induced by light, but other aspects
ter 1957). In contrast, abolishment of the con- of Aspergillus development are also influenced
idiation process in the phytopathogenic fungi by light. A. nidulans produces hyphae and sex-
B. cinerea and A. solani by blue light was also ual structures in the dark and mainly conidio-
described in the middle of the past century; phores and conidia in the light. Thus, the ratio
nevertheless, this process was reverted by expo- of sexual to asexual development is altered by
sure to red light (Lukens 1963; Tan 1973). light. In this case apparently only red light sti-
A good example of light-induced conidia- mulates conidiation, and the stimulatory effect
tion is T. atroviride, which grows as mycelium may be reversed by far-red light, which sug-
if nutrients are not limiting but upon exposure gested the participation of a phytochrome-like
to a brief pulse of blue light produces conidia in photoreceptor in Aspergillus (Mooney and
a nearly synchronous manner (Gressel and Yager 1990). In this regard, the discovery of
Galun 1967). Early photobiology studies carried fphA in the A. nidulans genome allowed Blu-
out using T. atroviride as a model indicated that menstein et al. (2005) to demonstrate that FphA
blue-light-activated conidiation was regulated acts as a red-light sensor and represses sexual
by a single photoreceptor (Horwitz et al. development under red-light conditions.
The Bright and Dark Sides of Fungal Life 53
for initiation in Polyporus arcularius (Gibson Interestingly, the threshold for this photo-
and Trapnell 1957) and speeds up the process response is unusually high for Phycomyces.
in Coprinus lagopus (Madelin 1956), while in The unique shape of the action spectrum with
Poria ambigua light is required for hymenium maximum efficiency at 350–410 nm indicates
development (Robbins and Hervey 1960). that the photosystems involved in the inhi-
Light is required throughout fruiting in bition of sexual development have special fea-
Sphaerobolus stellatus. Interestingly, in this tures not shared by other photoreceptor
case high intensities are needed early in devel- systems in Phycomyces.
opment, but very low intensities suffice at a later Studies in N. crassa reported that fruiting-
stage (Alasoadura 1963). These early observa- bodies are produced in the dark and located on
tions suggested that light may play important the surface of the growing medium even with-
roles at different stages of sexual development, out illumination. However, the perithecial
but no much information was provided in the necks are ever oriented to the light source,
study on the qualitative and quantitative para- whereas in the dark they point to any direction
meters or on the mechanisms involved. Later (Harding and Melles 1983). Additionally, the
on, more in-depth reports on the effect of light position of the neck on the perithecium is also
in sexual development started to appear in the light dependent (Oda and Hasunuma 1997).
literature, as in the case of the photoinduction of Moreover, the number of protoperithecia is
fruiting-bodies by light of defined wavelengths greatly increased upon a blue-light pulse (Inno-
in Schizophyllum commune. In this case, several centi et al. 1983). These processes completely
properties of the induction were established. depend on the wc-1 and wc-2 products, since
The exposure-response relationship for such responses were completely abolished in
induced fruiting was determined for light of Dwc-1 and Dwc-2 strains (Harding and Melles
448 nm. The Bunsen-Roscoe law of reciproc- 1983; Degli-Innocenti and Russo 1984; Oda and
ity—which establishes that a given quantity of Hasunuma 1997).
photons delivered in pulses of different dura- A rather interesting example of the influ-
tion should have the same final response—was ence of light in the control of fungal reproduc-
found to hold for the photoinduction of fruit- tion is that of Coprinus cinereus. The
ing-bodies for the interval from half a minute to basidiomycete C. cinereus forms dikaryons
half an hour. Although the photoreceptor after fusion of two homokaryon mycelia of
involved in this phenomenon is still unknown, compatible mating type and develops a fruiting
it is thought to be a flavoprotein because fruit- body where meiosis takes place to produce
ing-body formation is induced by UV- and basidiospores (reviewed in Kües 2000; Kamada
blue-light. Neither red light nor far-red light 2002). Additionally, C. cinereus develops differ-
induced fruiting bodies or affected the sensitiv- ent types of reproductive and specialized cells:
ity of the fungus to blue light (Perkins and haploid unicellular spores (oidia) develop on
Gordon 1969). oidiophores in the aerial mycelium, and large
In contrast with the effect of light on S. chlamydospores appear in submerged, old
commune, sexual development of P. blakeslea- mycelium. Hyphal knots can give rise to scler-
nus is inhibited by light. Effective wavelengths otia in old cultures or serve as primordia of
for this inhibition are shorter than 490 nm, but fruiting-bodies. As in most fungi, different
the shape of the action spectra and the most environmental conditions determine which
effective wavelength depend on the stage of developmental pathway will be followed, light
sexual development. Longer wavelengths are conditions being the major signal (reviewed in
more effective for the inhibition of the final Kües 2000; Fischer and Kues 2003).
stages of sexual development. Furthermore, Sclerotia are produced in the dark, and
biphasic fluence-response curves were initiation of the fruiting body occurs only in
observed using some wavelengths, providing the light. Fruiting-body development depends
additional support of the presence of a complex on dark/light cycles; hyphal knot formation is
photosensory system for photoinhibition of inhibited by light; the formation of fruiting-
sexual development (Yamazaki et al. 1996). body initials, maturation of primordia, and
The Bright and Dark Sides of Fungal Life 55
karyogamy are induced by light; and meiosis with natural isolates (Seibel et al. 2009; Chen
completion is inhibited by light (reviewed in et al. 2012). Mating of Denv1, Dblr1, and Dblr2
Kües 2000). The effect of light/dark cycles in (Seibel et al. 2009) with the wild-type strain was
meiosis, and possibly of the whole fruiting- fruitful, and discharge of ascospores was not
body development, seems to allow the matura- abated. These data indicated that deletion of
tion of fruiting-bodies for spore dispersal the photoreceptors does not affect male fertility
shortly after daybreak, regardless of night dura- (Seibel et al. 2012). Sexual reproduction in fully
tion (Lu 2000). Blind mutants, as well as other male and female wild-type background, bearing
mutants altered in different steps of fruiting- mutation in blr1 and blr2, showed altered
body development, have been isolated (Mura- fruiting-body formation compared to the wild
guchi and Kamada 2000). The blind mutants type with fewer, but larger, fruiting-bodies. The
are blocked in fruiting-body development, blr1 and blr2 products repress the formation of
forming only “dark stipes.” A gene affected in stromata under constant light and are neces-
one of these mutants was cloned, and its sary to speed up the formation of stromata
sequence was found to be similar to that of under 12 h light/12 h dark (LD) regime (Chen
the N. crassa wc-1, suggesting that this gene et al. 2012). In contrast, crosses of Mat1-1 and
plays a similar role in C. cinereus photo- Mat1-2 bearing a deletion in env1 provoked a
morphogenesis (Terashima et al. 2005). defect in sexual development characterized by
Cryptococcus neoformans is a heterothallic the lack of fruiting bodies, and Env1 was shown
yeast and a human pathogen (Hull and Heitman to be essential for female fertility. Recently, the
2002), in which blue light inhibits mating and participation in sexual development of the
haploid fruiting. As proposed for C. cinereus, pheromone precursor gene hpp1 and that of
mutations in any of the Cryptococcus wc genes the orthologous gene to the pheromone trans-
resulted in a blind-mating phenotype (Idnurm porter in yeast ste6 is regulated by the
and Heitman 2005; Lu et al. 2005), and their phosducin-like protein (PhLP1), and the G-
overexpression resulted in a stronger light- protein b (Gnb1) and g (Gng1) subunits were
dependent inhibition of mating (Lu et al. 2005). demonstrated. It has been proposed that
A. nidulans develops fruiting-bodies in the phosducin-like proteins mediate light trans-
dark, whereas upon exposure to far-red light, duction through a G-protein signal transduc-
their production is repressed (Mooney and tion pathway (Tisch et al. 2011). The
Yager 1990). As mentioned before, light control participation of the protein kinase A (PkaC1)
of development in A. nidulans is regulated by a and the adenylate cyclase (Acy1) in sexual
number of photoreceptors, including the phy- development in T. reesei was also recently eluci-
tochrome FphA, which senses red light, and it is dated. Deletion of pkac1 and acy1 provokes
responsible for repression of fruiting-body for- delayed fruiting-body formation but does not
mation, while it positively regulates asexual alter their development. Similar results were
spore development (Blumenstein et al. 2005). reported for Dcr-1 (adenylate cyclase) in N.
Interestingly, integration of blue- and red-light crassa (Ivey et al. 2002). Based on these results,
signaling affecting the developmental programs it can be concluded that the cAMP pathway is a
of this fungus was recently demonstrated. positive input in the regulation of sexual devel-
Mutants in lreA or lreB do not produce cleis- opment; however, it is not essential for this
tothecia under light conditions, which is largely process in this fungus (Schuster et al. 2012).
suppressed by deletion of fphA. Intriguingly, As mentioned before, light impacts in cAMP
double and triple mutants of lreA, lreB, and synthesis and protein phosphorylation (Gresik
fphA exposed to light produced similar quan- et al. 1988); however, the effect of light on
tity of cleistothecia when compared with dark mating using pkac1 and acy1 mutant back-
conditions. grounds was not investigated.
Sexual development of T. reesei requires Recently, the role of Velvet (Vel1) in light
light to take place, which has been shown to response, development, and secondary meta-
occur not only with laboratory strains but also bolism in T. reesei was described (Bazafkan et al.
56 S. Casas-Flores and A. Herrera-Estrella
2015). Lack of vel1 leads to defects in growth, human eye and is achieved through the action
conidiation, and mating in the dark, whereas in of two photosystems that operate at different
light the vel1 product was not necessary for light intensities (Galland and Lipson 1987). Tri-
female fertility. However, this protein was essen- fluoperazine affects both sporangiophore
tial for female fertility in both mating types. Addi- growth and phototropism, suggesting that
tionally, the absence of vel1 negatively affected microtubules and calmodulin are involved in
the transcription of hpr1, hpr2, hpp1, and ppg1, the response of the fungus to light (Valenzuela
genes that are part of pheromone system, in a and Ruiz-Herrera 1989). Additionally, the role
mating-type-dependent mode and depending on of calcium in dark adaptation of phototropism
the mating partner of a given strain. These mating of Phycomyces suggests that the corresponding
defects only occurred for the pheromone precur- signaling pathway may play a relevant role in
sor hpp1 and hpr2 and receptor genes associated phototropism (Sineshchekov and Lipson 1992).
with the Mat1-2 mating type and for the mating- A genetic screen for phototropic mutants,
type gene mat1-2-1. It has also been demon- conducted in the Delbrück’s lab, led to the
strated that a Dvel1 strain secretes different meta- isolation and physiological characterization of
bolites compared to the wild type; when the wild- mad mutants and the first draft of the signal
type strain was confronted with a compatible transduction pathway for Phycomyces (Berg-
partner, it produced a set of secondary metabo- man et al. 1973). The discovery of additional
lites, but the set changed if a Dvel1 strain was the mad mutants and detailed genetic characteriza-
partner. Therefore, this fungus uses both phero- tion allowed the identification of ten unlinked
mones and secondary metabolites to interact with mad genes (madA to madJ) (Alvarez et al. 1992;
mating partners, which is conducted in part by Orejas et al. 1987; Campuzano et al. 1995). The
Vel1 (Bazafkan et al. 2015). blue-light photoreceptor eliciting phototropism
in Phycomyces is a molecular complex, similar
to the N. crassa WCC constituted by the pro-
teins MadA and MadB (Idnurm et al. 2006; Sanz
V. Phototropism et al. 2009). The MadA-MadB complex acts as a
light-regulated transcription factor eliciting
As mentioned above, light may serve as a cue to phototropism and other responses pertaining
change the directionality of growth of fungi, to photodifferentiation. Further, madC is
which helps them “decide” on the positive or understood as an early phototropism mutant
negative consequences of approaching this sig- with a 106 higher threshold, and the corres-
nal (positive or negative phototropism). The ponding gene encodes a Ras GTPase-activating
best-studied phototropic response is that protein (Polaino-Orts et al. 2013).
observed in the sporangiophore of P. blake- Sporangiophores of another zygomycete,
sleeanus. The Phycomyces sporangiophore is a Mucor circinelloides, also respond to light, exhi-
single coenocytic cell of 2 mm length and biting positive phototropism. Analysis of the
100 mm diameter that elongates at an astonish- phototropic response of knockout mutants in
ing rate of 2–3 mm h1. Light perception, the three wc-1 homologues present in the M.
growth modulation, and phototropism all circinelloides genome revealed that while the
occur in the small growing zone extending 2– wild-type strain is responsive to white, blue,
3 mm below the sporangium (Bergman et al. and green light, the mcwc-1a mutant was
1969). When sporangiophores are exposed for unable to respond to light of any of these wave-
6–8 h to unilateral light, they bend in response lengths. In contrast, photocarotenogenesis is
to near-UV and blue light of fluence rates induced only by blue light and unaffected in
extending 10 orders of magnitude (109 and the mcwc-1a mutant (Silva et al. 2006). There-
102 Wm2) and transiently accelerate their fore, multiple photosystems are involved in
growth rate (Galland et al. 1985; Bergman et al. triggering photoresponses in this organism.
1969; Galland and Lipson 1987). This remark- Although not studied in as much detail as
able sensory dexterity resembles that of the in Zygomycetes, many other fungi display pho-
The Bright and Dark Sides of Fungal Life 57
totropic responses. Such is the case of A. gigan- responses. In these cases they have been
teus, in which conidiophores show positive shown to present negative phototropism upon
phototropism toward white light, restricted to repetitive exposure to UV-light, likely aimed at
the apical 240 mm. During the response, the rate evading its harmful effects (Brasch and Menz
of extension of the proximal wall (relative to the 1995).
direction of the light source) decreases by about
5 %, while that of the distal wall increases by
about 5 %; thus, there is no net change in the VI. Circadian Rhythms
rate of wall growth during phototropism. The
sign of the phototropic response is reversed The biological answer to the changing condi-
when conidiophores are unilaterally stimulated tions brought about by the rotation of earth are
with UV-light (280 nm) (Trinci and Banbury circadian rhythms. A requirement for photo-
1968). Young sporangiophores of Pilobolus periodism—the assessment of the light or the
kleinii also respond to unilateral illumination dark period—entails a timer, which in plants
by bending or growing toward near-UV and and animals is generally linked to the circadian
blue light and exhibit a negative phototropic system. As described above, the pattern of
response to UV light. The use of small beams spore release in many fungi, which discharge
of light and the behavior of sporangiophores their spores, is influenced by light. This is
submerged in mineral oil suggested that the clearly shown when, with periods of light and
photosensitive region is located in the tip of dark alternating, spore discharge may show a
the young sporangiophore (Page and Curry marked periodicity. Studies of circadian peri-
1966). odic discharge related to light were reported
Positive phototropism was also shown for back in the 1950s and 1960s for Pilobolus, Dal-
N. crassa perithecial beaks as beak bending in dinia and Sordaria, Sphaerobolus, Sporobolo-
maternal structures. This effect is induced by myces, and an extended range of
blue light and abolished by deletion of wc-1 and Pyrenomycetes (Callaghan 1969).
causes sexual spores to be ejected toward the Perhaps the first indication of endogenous
direction of light (Harding and Melles 1983; rhythms in the fungi derives from the dung-
Froehlich et al. 2010). In Sordaria fimicola, pho- loving fungus Pilobolus, which shoots spores
totropism of the perithecial necks has been as far as 2 m. Klein (1948) reported that when
observed, with wavelengths below 550 nm Pilobolus was submitted to various symmetrical
being more effective. These observations are in non-24-h LD cycles, the spore-shooting rhythm
agreement with early findings on the sensitivity was absent. His experiments probably repre-
of light-stimulated spore discharge in this fun- sent the first systematic investigation of the
gus (Ingold and Hadland 1959). influence of photoperiod on the sporulation
The germ tubes of the phytopathogen B. rhythm. Short (LD 4:20) and long (LD 20:4)
cinerea present negative phototropism to photoperiods appear to suppress rhythmicity,
near-UV and blue (300–520 nm) light followed while using LD 9:15 and 15:9, rhythmicity per-
by far red (700–810 nm), whereas red light sists with a low amplitude and with high ampli-
(600–700 nm) induces positive phototropism tude in LD 12:12. The first report providing
significantly. Near-UV and blue light, trigger- strong evidence for the endogenous nature of
ing negative phototropism, promoted this rhythmicity included experiments both in
infection-hyphae formation on both onion constant light (LL) and darkness (DD) (Schmi-
scale and broad bean (Vicia faba) leaf epider- dle 1951). When Pilobolus is maintained in a LD
mal strips. Conversely, red light that induces 12:12 cycle and released to constant light, the
positive phototropism suppressed the forma- rhythm rapidly dampens and continues when
tion of infection-hyphae (Islam et al. 1998). released to DD. Rhythmicity in DD also persists
Dermatophytes, such as Trichophyton after release from several days in LL.
rubrum, Trichophyton mentagrophytes, and Later, Uebelmesser (1954) reported
Microsporum canis, also show phototropic species-specific phase angles in LD cycles (P.
58 S. Casas-Flores and A. Herrera-Estrella
sphaerophorus peaks at ZT 03, P. crystallinus at the dark. Under continues light exposure, the
ZT 08). In contrast with Klein’s report, she banding pattern stops, a phenomenon observed
observed the rhythm in all photoperiods in many fungi. Light blocks the circadian system
(except LL) with a systematically different completely, which is supported by nonrhythmic,
phase angle. Uebelmesser also investigated the elevated clock gene (cg) expression in constant
ranges of entrainment and found that synchro- light (Crosthwaite et al. 1995), in addition to a set
nization persisted in 29 h cycles. She submitted phase relationship of the free-running circadian
Pilobolus to symmetrical LD cycles, ranging rhythm upon release to darkness.
from 18:18 down to 2:2. While in N. crassa Undoubtedly, the reproduction of fungi is
accumulation of conidia appears to be driven just as seasonal as in other organisms, especially
with a constant phase angle in reference to if they live far enough away from the equator.
lights off (Merrow et al. 1999), the phase angle Like in other organisms whose internal temper-
of the spore-shooting rhythm in Pilobolus ature varies considerably, fungal seasonality is
varied with varying cycle lengths, possibly controlled both by photoperiod and tempera-
reflecting circadian entrainment. However, a ture. Any collector or gourmet of mushrooms
more in-depth investigation revealed that the knows that fungal fruiting-bodies appear only
Pilobolus sporulation rhythm is also driven by in certain times of the year. The amount of
the LD cycle but by lights on. spores they produce also differs drastically
Sordaria fimicola and Daldinia also show over the course of a year. Studies carried out in
circadian rhythmicity in sexual spore shooting the Antarctic showed that all airborne fungal
in the dark, which are entrainable in light-dark spore types were most abundant in the summer
illumination cycles (Austin 1968; Ingold and months, except for chlamydospores, which were
Cox 1955). In Daldinia, circadian rhythmicity most numerous during the winter (Marshal
persists under continues light, damping after 1997). These annual rhythms have been directly
several days, while in Sordaria, exposure to correlated to environmental conditions such as
continuous light appears to suppress rhythmic nutrient availability, humidity, wind speed, day
spore release (Austin 1968). Interestingly, S. or night length, temperature, and other climatic
fimicola shares a functional frequency (frq) parameters, such as humidity and rainfall, or
orthologue with N. crassa (Merrow and Dunlap even triggered by other organisms in the form
1994), suggesting that the mechanics in the of food and, in the case of pathogens, host avail-
light input and the circadian programs of ability (Ingold 1971; Roenneberg and Merrow
these species are similar. Like Daldinia, spore 2001).
release of the basidiomycete Pellicularia fila- Similarly to many photoperiodic plants,
mentosa is circadian in either continues light some diurnal sporulators require light induc-
exposure or complete darkness and entrainable tion followed by a dark period to initiate their
by light-dark cycles (Carpenter 1949). There are seasonal reproduction (Durand 1982; Leach
also species that show some kind of rhythmicity 1967). Like with short-day plants, their matura-
in light-dark cycles, but this rhythmicity is lost tion process is highly light sensitive and is
in continues light or in the dark (e.g., Pyricu- inhibited when exposed to a short (seconds)
laria; see Barksdale and Asai 1961), indicating interrupting light pulse (Durand 1982). Timing
that sporulation, although rhythmic, is not of the light pulse and duration of the dark
always controlled by a circadian clock. period are crucial for the inhibitory effect.
Fungal circadian clocks can be exquisitely Despite the unquestionable seasonality in
light sensitive, in terms of fluence and duration. fungi, investigations into photoperiodic mecha-
Amazingly, the Pilobolus clock requires less than nisms and/or photoperiodic memory are
half a millisecond of light to be completely reset scarce. Due to their economic importance,
(Bruce et al. 1960), and conidial banding in many of the investigations into the influence
Neurospora is driven by light fluences as low as of photoperiod on fungal development and
moonlight levels (Merrow et al. 1999). In N. crassa, reproduction have focused on host-infecting
conidial banding occurs about once per 22 h in pathogens and more recently litter decompo-
The Bright and Dark Sides of Fungal Life 59
sers. When the entomopathogenic fungus A key component of the central oscillator in
Metarhizium anisopliae is grown in different N. crassa is frequency (FRQ); modifications of
temperatures (25, 28, and 30 C) and photoper- this protein result in altered periodicity or
iods (24, 16, 12, and 8 h), its colony size and the oscillation abolishment (Cha et al. 2011; Dieg-
number of conidia produced are maximal at mann et al. 2010). Nevertheless, clock-
28 C and a day length of 16 h (Alves et al. controlled genes which oscillate independently
1984). Mycelial growth of the phytopathogen of FRQ have been found (Correa et al. 2003),
Colletotrichum manihotis is greater in complete indicating the existence of an additional oscil-
darkness than under continues light (Makam- lator, the frequency-less oscillator (FLO)
bila 1984). When grown under light-dark (Heintzen and Liu 2007; Lakin-Thomas and
cycles, the inhibitory effect of light depends Brody 2004). In 2010 Brody and coworkers
on photoperiod length, as it is less in LD 12:12 (Brody et al. 2010) suggested that components
cycles, compared with longer and shorter regulating the conidiation process or part of the
photoperiods. The amplitude of the response RAS-cAMP-protein kinase pathway could con-
increases with light fluence and temperature tribute to the FLO. Recently, using genetic
and is not observed at low temperatures approaches, an oscillator mechanism that
(20 C). Chlamydospore formation of the corn drives rhythmic spore development in the
pathogen Exserohilum turcicum is also signifi- absence of the FRQ/WCC oscillator and in con-
cantly affected by photoperiod and temperature stant light was uncovered in N. crassa. While
(Levy 1995). Furthermore, day or night length this novel oscillator does not require the FRQ/
can also modify the development of sexual WCC oscillator for its activity, it requires the
spores, when seasonal changes in fungal abun- cryptochrome (CRY). The CRY-dependent
dance, activity, and community composition oscillator has a period of 1 day in constant
were investigated in the litter and organic and light and is temperature compensated (Nsa
upper mineral soils of a temperate Quercus et al. 2015). Another component involved in
petraea. The litter community was found to the regulation of the circadian clock is epi-
exhibit more profound seasonal changes than genetics, for which more and more evidence is
the community in the deeper horizons. In the being accumulated (Ripperger and Merrow
litter, saprotrophic genera reached their sea- 2011). Nevertheless, in fungi it is not clear if
sonal maxima in autumn, but during summer there is epigenetic control of the circadian
the highest abundance of ectomycorrhizal taxa clock, perhaps due to our poor understanding
was detected. Although the composition of the of epigenetic control in general.
litter community changed over the course of Research with Trichoderma spp., in con-
the year, the mineral soil showed changes in trast, did not yet provide any hints as to the
biomass (Vořı́šková et al. 2014). presence of circadian rhythms or their regula-
In fungi, the mechanisms by which circa- tion by homologues of WC-1, WC-2, and FRQ
dian rhythmicity is achieved are best studied at in these fungi. Nevertheless, given the ubiquity
the molecular level in N. crassa (Brunner and of circadian regulation in countless organisms,
Kaldi 2008; Heintzen and Liu 2007). As in other it would be surprising if this mechanism would
eukaryotes, the circadian clock consists of pos- not be operative in Trichoderma spp. Thus, it
itive and negative transcriptional/translational seems that robust circadian rhythms in conidi-
feedback loops, resulting in cycles of gene ation cannot be observed under the culture
expression covering various functions (Brun- conditions currently used in the laboratory.
ner and Kaldi 2008). Expression of most Since most Trichoderma species require
clock-controlled genes (ccgs) peaks just before light for conidiation, colonies growing in the
dawn, reflecting the preparation of the cell to dark were induced by LD cycles, determining
deal with exposure to DNA-damaging radia- that conidiation occurred, corresponding to the
tions, desiccation, and other stresses provoked interval of illumination. When Trichoderma is
by the imminent exposure to sunlight (Vitalini returned to the dark after a pulse of light,
et al. 2006). conidiation is observed only at what was the
60 S. Casas-Flores and A. Herrera-Estrella
Fig. 3.4 Model for the light-dependent regulation of indicate interactions fully supported by experimental
stress responses in T. atroviride, integrating compo- evidence and dotted lines indicate interactions sug-
nents (Pbs2 and Tmk3) of the SAPK pathway and the gested by these evidences
blue-light receptor complex (Blr1/Blr2). Solid lines
colony perimeter at the time of exposure to induced a ring of conidia, but it also delayed
light. Such result allowed to conclude that con- the reappearance of the dark banding pattern
idiation in T. viride is not rhythmic but can be (Deitzer et al. 1988). Recently, Steayert and
synchronized by a light pulse (Betina and Zaja- coworkers described that dark-grown cultures
cová 1978). Similarly, under continues light, of T. pluroticola form rings of green conidia at
conidiation is not rhythmic; however, banding interval ca. 24 h. Light, however, did not induce
patterns are formed under light/dark cycles. rhythmicity in conidiation (Steayert et al.
Interestingly, a Trichoderma mutant (B119) 2010). Nevertheless, similarly to what has been
that conidiates rhythmically in the dark has described in N. crassa, the frq gene of T. atro-
been described, and composition of the growth viride is regulated by light, and such regulation
medium influences the period length of coni- depends on functional blr1 and blr2 (Garcı́a-
diation. Medium containing sodium deoxy- Esquivel et al. 2015; Liu and Bell-Pedersen
cholate, an ionic detergent that delays the 2006). Orthologous genes to those described
growth of Trichoderma, increased the interval as involved in the regulation of circadian
between dark bands from 12 to 24 h. As in the rhythms (wc-1, wc-2, and frq) in several fungal
wild-type strains, the application of light species have been also found in all Trichoderma
The Bright and Dark Sides of Fungal Life 61
species, whose genomes have been sequenced. CryA, shown to control sexual development in
However, the role of their products in regu- the fungus, are examples of the dual functions
lation of circadian clocks has not been esta- that these types of proteins might play
blished (Casas-Flores and Herrera-Estrella (Berrocal-Tito et al. 1999; Berrocal-Tito et al.
2013). Further research on the molecular 2007; Bayram et al. 2008a). Other examples are
aspects of circadian rhythms in Trichoderma Phl1 from Cercospora zeae-maydis, which as a
should lead us to understanding this pheno- member of the cryptochrome/6-4 photolyase
menon in the genus. subfamily is involved in the repair of DNA
damage by UV, but is also involved in the regu-
lation of the CPD photolyase and participates in
the development of the fungus and secondary
VII. Light as Warning Signal for metabolism (Bluhm and Dunkle 2008); cry1
Abiotic Stress from Sclerotinia sclerotiorum is a DASH-type
cryptochrome encoding gene expressed in
As mentioned above, the sunlight that impinges response to UV-A light, which deletion results
on our planet comprises harmful wavelengths in diminished sclerotial biomass (Veluchamy
that can generate mutations and/or damaging and Rollins 2008). Yet another example is the
reactive oxygen species. Therefore, fungi must T. reesei cryptochrome/6-4photolyase (Cry1),
display a rapid reaction to exposure to UV whose transcript levels are induced by light in
light. Accordingly, blue light appears to be a a blr1-dependent manner and are repressed by
cue for fungi to start a rapid response to illumi- Env1 (Guzmán-Moreno et al. 2014). Similarly,
nation. the second-most highly light-induced gene in
Photolyases play a major role in protecting A. umigatus is phr1, a gene encoding a pre-
the cell from UV damage by repairing DNA dicted CPD photolyase (Fuller et al. 2013). Sur-
damage, through a process called photoreactiva- prisingly, and although induction of phr1 is lost
tion. The cryptochrome/photolyase family con- in a DlreA mutant, mutants in the photo-
sists of 55–70 kDa proteins that bind FAD, non- receptors lreA, fphA, and a double lreA-fphA
covalently, and an antenna chromophore of mutant are as resistant to UV treatment as the
pterin (MTHF) or flavine type (8-HDF, FMN, or WT in A. fumigatus, suggesting that additional
FAD) in the photolyase homology region (PHR) photoreceptors may be functioning to promote
(Sancar 2003; Chaves et al. 2011; Fig. 3.2). CPD UV resistance in response to light (Fuller et al.
photolyases repair cyclobutane pyrimidine 2013).
dimers (CPDs) and 6-4 photolyases repair the The molecular chaperones known as heat-
pyrimidine (6-4) pyrimidone photoproducts shock proteins (HSPs), together with their part-
(Sancar 2008; Weber 2005). In fungi genes ners, are critical components for the normal
encoding proteins similar to cryptochrome/ function of stress signaling pathways, parti-
photolyases have been found, and most, if not cularly high temperature (Nollen and Mori-
all of them, are transcriptionally activated upon moto 2002). In mammalian cells, it is known
exposure to blue light (Herrera-Estrella and Hor- that HSP expression can be induced by UV light
witz 2007; Corrochano 2007). Further, DNA- and is suggested to mediate protection from
damaging agents, including UV and ROS, can UV-induced cell death (Trautinger et al. 1996).
reset the clock (Gamsby et al. 2009). HSPs interact with the MAPK pathway and
Members of the cryptochrome/photolyase show mutual regulation in response to heat
family with functions, both as regulator shock (Dorion and Landry 2002). In N. crassa,
cryptochrome-photolyase type and photorepair a protein related to HSP30, belonging to the
enzyme, have been reported. Phr1, a protein group of LLRGs, was found to be responsive
with CPD photolyase activity that regulates its to light (Chen et al. 2009). Similarly, the
own expression, and modulates the expression P. blakesleeanus HSP encoding gene hspA is
of other light-responsive genes in T. atroviride, induced by heat shock and light (Rodriguez-
and its closest orthologue from A. nidulans, Romero and Corrochano 2004, 2006).
62 S. Casas-Flores and A. Herrera-Estrella
a stronger effect. Depending on the intensity, appears to be released in the wild type after
complete cessation of growth and/or inhibition blue-light exposure. Additionally, light induc-
of ochratoxin A biosynthesis was observed. tion of AOH formation was partially dependent
Interestingly, exposure to light has the opposite on LreA, which suggests also an activating
effect on ochratoxin A and citrinin production, function (Pruß et al. 2014). Similarly, in dele-
two mycotoxins, which can be produced simul- tion mutants in the white collar orthologues
taneously in P. verrucosum. Citrinin was pro- (Fgwc-1 and Fgwc-2) of Fusarium grami-
duced essentially under conditions that nearum, conidiation was derepressed in con-
inhibited ochratoxin A biosynthesis. Similarly, stant light and impaired early onset of
a derivative of ochratoxin b in A. carbonarius carotenogenesis, photoreactivation, and the
was produced in higher amounts under blue maturation of perithecia (Kim et al. 2014).
light, conditions in which the production of However, in contrast with A. alternata, it was
ochratoxin A ceased (Fanelli et al. 2012a; not derepressed in darkness, and individual
Schmidt-Heydt et al. 2011). mutants produced more aurofusarin and tri-
In Fusarium verticillioides, wavelengths chothecenes than the wild-type strain in both
across the visible spectrum, from red to blue, constant light and darkness (Kim et al. 2014).
stimulate growth and fumonisin production, by A significant breakthrough in fungal bio-
up to 150 % as compared to the levels observed logy was the discovery of the heterotrimeric
in the dark. However, high blue-light intensities VelB/VeA/LaeA transcriptional complex
reduced fumonisin biosynthesis. Pulses of (known as the Velvet complex) which connects
white light had no effect on growth but reduced secondary metabolism (SM) with light signal-
fumonisin production to half of that observed ing and development (Bayram et al. 2008a). The
in the dark. Analysis of gene expression showed founding member of this complex is velvet
a good correlation between the mRNA levels of (VeA), discovered in a veA mutant, which was
fum1, fum21, and Fvve1, which encode proteins described more than 40 years ago. VeA and the
involved in fumonisin biosynthesis (Fanelli other subunits of the complex are highly con-
et al. 2012b). served in several fungi (Myung et al. 2012). To
As described earlier in this chapter, fungal coordinate SM biosynthesis with fungal devel-
development is strongly controlled by light, and opment, different members of the velvet pro-
an important correlation has been found tein family partner with each other (reviewed in
between the developmental stage of a fungus Bayram and Braus 2012). However, the way in
and its capacity to produce secondary meta- which Velvet controls SM production is still not
bolites. Such is the case of Alternaria alternata, fully understood.
a phytopathogen that produces more than 60 Because many genes for the synthesis of
different secondary metabolites and causes SMs are found in clusters in fungal genomes
considerable loss of economically important (Keller and Hohn 1997), regulation of SM genes
crops for feed and food worldwide. Among the can be in part explained by transcriptional con-
most important mycotoxins produced by this trol through hierarchical levels of transcrip-
fungus are alternariol (AOH) and altertoxin tional regulatory elements including cluster-
(ATX). In this case, mycotoxin production specific regulatory elements, global regulators,
and spore formation are regulated by light in and transcriptional complexes like Velvet
opposite ways. Whereas spore formation is (reviewed in Yin and Keller 2011). In A. nidu-
greatly inhibited by blue light, the production lans, the methyltransferase-domain protein
of AOH is stimulated two- to threefold, and LaeA apparently links light regulation to chro-
ATX production is strictly dependent on it. matin modification. In the dark, VeA forms a
Deletion of the orthologue of the N. crassa heterotrimeric complex with VelB and the
white collar 1 (wc-1) gene (lreA) results in global regulator of secondary metabolism
derepression of spore formation and ATX for- LaeA (Bayram et al. 2008a; Bok and Keller
mation, regardless of the light conditions, sug- 2004). A third velvet protein (VosA) also inter-
gesting a repressing function of LreA, which acts in the dark with VelB, and it has been
64 S. Casas-Flores and A. Herrera-Estrella
proposed that this heterodimer can repress In several fungi, a regulatory connection
conidia formation as well as control spore mat- between sporulation and the production of
uration and trehalose biogenesis (Ni and Yu secondary metabolites has been reported
2007; Bayram et al. 2010). Shuffling of VelB (Calvo et al. 2002). The fact that in Tricho-
between the VelB-VosA and VeA-LaeA com- derma Dblr1 and Dblr2 strains do not conidi-
plexes is determined by the LaeA protein, ate in response to light allowed hypothesizing
which plays a fundamental role in the regula- that they are altered in secondary metabolism
tion of secondary metabolism and development and synthesis of peptaibols. Peptaibols are a
in A. nidulans (Bayram et al. 2010). The func- family of short peptides (20 residues),
tion of the fourth member of this family (VelC) synthesized by non-ribosomal peptide synthe-
is much less understood (Bayram and Braus tases (NRPS). Naturally occurring peptaibols
2012). The trimeric velvet complex might are isolated from soil fungi and often exhibit
directly perceive the light signal to coordinate antibacterial and antifungal activities. There
secondary metabolism and development. In are over 300 annotated sequences of non-
this sense, it has been proven that VeA can ribosomal peptides (Whitmore and Wallace
physically interact in the nucleus with the red- 2004), and the list is continuously increasing
light phytochrome receptor FphA, which is also since a single NRPS can produce up to three
associated with the blue-light receptors LreA distinct peptaibols. Neuhof et al. (2007) ana-
and LreB (Purschwitz et al. 2008; Bayram et al. lyzed 28 phylogenetically related Trichoderma
2010). strains, identifying 48 different classes of pep-
The A. nidulans laeA mutant does not taibols.
express AflR, the transcriptional activator that Recently, it was determined by mass
controls the secondary metabolite cluster for spectrometry that T. atroviride did not produce
the production of the aflatoxin precursor sterig- peptaibols during vegetative growth; however,
matocystin. LaeA is not only required for sterig- the production of peptaibols was evident when
matocystin and penicillin biosynthesis but also the fungus initiated conidiation. Therefore, dif-
for lovastatin, an important compound used to ferent stimuli that induce conidiation were
lower cholesterol to prevent cardiovascular dis- tested to determine if in all cases conidiation
eases (Bok and Keller 2004). Similarly, laeA and production of peptaibols were intimately
homologues of other fungi are involved in the correlated. Interestingly, conidiation induced
control of secondary metabolism. by light promoted high production of peptai-
A velvet complex was recently detected in bols, whereas in the dark it was observed in
the filamentous fungus Penicillium chryso- minimum quantities. Light-associated synthe-
genum, the main industrial producer of penicil- sis of peptaibols was dependent on blr1 and
lin. The core components of this complex are blr2 products. As expected, no conidiation was
PcVelA and PcLaeA, which in addition to sec- observed in the blr1 and blr2 mutants. On the
ondary metabolite production regulate hyphal other hand, carbon deprivation also induces the
morphology, conidiation, and pellet formation. production of both peptaibols and conidia,
As in A. nidulans, other subunits of the velvet independent of blr1 and 2, hence light indepen-
complex have been found, such as PcVelB, dent. These data are apparently contradictory,
PcVelC, and PcVosA. Functional analyses since it was reported that the carbon depri-
using single- and double-knockout strains vation signal is blr1 and 2 dependent; however,
served to determine that the velvet subunits such dependence is only observed when cul-
have opposing roles in the regulation of peni- tures are subject to sudden carbon deprivation
cillin biosynthesis. PcVelC, together with (Casas-Flores et al. 2006). Mechanical injury of
PcVelA and PcLaeA, activates penicillin biosyn- mycelia also triggered peptaibol production;
thesis, while PcVelB represses it (Kopke et al. however, it depended completely on light
2013). The phytopathogen F. fujikuroi also stimulation, and no peptaibol production was
requires LaeA for gibberellin production (Hoff observed in the absence of light, in spite of
et al. 2010; Wiemann et al. 2010). sporulation. These discoveries were also
The Bright and Dark Sides of Fungal Life 65
observed in blr mutants. Together these results different fungi will allow us to understand the
indicate the existence of a Blr-independent complexity in regulation by light in secondary
pathway for peptaibol production stimulated metabolism.
by light (Komon-Zelazowska et al. 2007).
Recently, it was shown that mycelia of T.
virens wild-type strain (Gv29.8) exposed to
light exhibited a slight increase in the expres-
IX. Impact on Pathogenicity
sion of veA transcript (Mukherjee and Kenerley
2010). Mutants in T. virens veA are defective in Although the effects of light on pathogenicity of
induction of genes that encode secondary some fungi have been known for quite some
metabolism enzymes and showed a null pheno- time, the molecular basis of this effect is still
type in the synthesis of gliotoxin. Lack of glio- only poorly understood. Light may directly or
toxin correlates with low expression levels of indirectly influence disease development
gliP, the NRPS encoding gene responsible through modulation of sporulation, motility,
for gliotoxin production in A. nidulans. In A. adhesion, toxin biosynthesis or activation, or
nidulans VeA interacts with the phytochrome- host defense responses (Idnurm and Crosson
white collar light regulator complex (FphA- 2009). An example of this is how the photo-
LreA-LreB) and bridges VelB and LaeA to period—comparable with the length of a
form the velvet complex (VelB/VeA/LaeA). day—influences the infectivity of the insect
The latter complex coordinates light signal pathogen P. fumosoroseus (Avery et al. 2004),
with fungal development and secondary meta- where increasing the duration of the light phase
bolism (Bayram et al. 2008b; Bayram et al. results in an increase in colonization of whitefly
2010). Furthermore, mutants in the wcoA nymphal hosts by blastospores of P. fumo-
gene, the blr1 orthologue in Fusarium fujikuroi, soroseus. Furthermore, the well-known effects
sustained carotenoid synthesis in response to of light on secondary metabolism in Aspergillus
light. On the contrary, production of fusarin spp. and many other fungi (Fox and Howlett
showed a drastic reduction in the light and 2008; Yu and Keller 2005) clearly indicate the
less synthesis of gibberellins and more bika- importance of light in pathogenicity.
verins when mycelia were growing under In F. oxysporum, knockout mutants lacking
nitrogen-limiting conditions. These results the putative photoreceptor Wc1 used in infec-
indicate that blr1 orthologous genes play piv- tion experiments with tomato plants and
otal roles in secondary metabolism in F. fuji- immunodepressed mice revealed that Wc1 is
kuroi. It is noteworthy that F. fujikuroi does not dispensable for pathogenicity on plants but
sporulate when exposed to light; on the con- required for full virulence on mammals (Ruiz-
trary, this stimulus seems to have a repressing Roldan et al. 2008). The injection of micro-
role on conidiation in this fungus. However, conidia of the wild-type or the Dwc1+wc1
DwcoA showed reduced levels of conidiation strain into immunodepressed mice resulted in
on minimal agar when compared with the 65 % and 80 % mortality, respectively. By con-
WT strain (Estrada and Avalos 2008). Together, trast, infection with the Dwc1 mutant produced
these results indicate an important role of only 20 % mortality rate, although during the
WcoA in nitrogen-regulated processes, as well infection of mice, the fungus remains in the
as for gibberellin and bikaverin synthesis in a dark. Similar results had previously been
light-independent pathway. reported for mutants in the orthologous gene
The available data indicate notable differ- (Bwc1) of the human pathogen C. neoformans,
ences in the use of VeA and Blr1 and its partner which, although not essential for virulence,
in secondary metabolism. Further studies on significantly contributes to disease severity
single, double, and triple mutants as well as (Idnurm and Heitman 2005).
the demonstration of the interactions between As mentioned above, the germ tubes of
the different components of VelB/VeA/LaeA B. cinerea show negative phototropism to
and phytochrome-white collar complexes in near-UV and blue light followed by far red,
66 S. Casas-Flores and A. Herrera-Estrella
whereas red light induces positive phototro- In addition to the possible direct involve-
pism. Exposure to near-UV and blue light also ment of photoreceptors in pathogenicity and
promotes the formation of infection-hyphae on the light control of secondary metabolism by
onion and broad bean, whereas red light sup- members of the velvet family, genes belonging
presses it, resulting in a high proportion of to the latter have been found to be important
germ tubes without infection hyphae. In broad for conversion of Histoplasma capsulatum from
bean leaf infection, the number of infection the filamentous form typically found in the soil
points and area of necrosis are higher under to the pathogenic yeast found inside macro-
near-UV-blue light than that of a dark control. phages, although it is not known whether the
Conversely, lower numbers of infection points function of Velvet in this fungus involves a
and very small necrotic lesions develop under response to light (Webster and Sil 2008).
red light (Islam et al. 1998). In this sense, Exposure of pepper plants to Colleto-
the role of the B. cinerea phytochrome like trichum acutatum, the causative agent of
(Bcphy3) in pathogenesis was recently anthracnose, under white, blue, red, and green
addressed on wounded capsicum, tomato, light resulted in larger lesion size than when
grape berry, cucumber, carrot, and intact let- incubated in the dark (Yu et al. 2013). On the
tuce. The wild-type strain caused obvious radial contrary, inoculation of rice plants with
lesions 2–6 days postinoculation. In contrast, Magnaporthe oryzae under light conditions
the invasion symptoms produced by the suppressed the infectious process, and such
corresponding mutant were drastically weak- suppression was clearly dependent on the
ened on different host plants, evidently suggest- WC-1 orthologs (Kim et al. 2011). Canessa
ing the involvement of Bcphy3 in pathogenicity et al. (2013) demonstrated that BcWCL1, the
(Hu et al. 2014). Nevertheless, the authors of the putative blue-light photoreceptor of B. cinerea,
mentioned report suggest that the disruption of plays an important role during plant infection
the gene may affect the growth rate and general in the presence of light. Arabidopsis thaliana
chitin synthesis of the cell wall, both of which in and Phaseolus vulgaris plants inoculated with a
turn increased the susceptibility of the mutant Dbcwcl1 mutant showed reduced lesion sizes,
to plant defense responses. with a more dramatic effect under constant
Recently, by a random mutagenesis light than in photocycles. Interestingly, it was
approach, a novel virulence-related gene found that excessive light impairs the growth of
encoding a GATA transcription factor the mutant due to the generation of ROS, indi-
(BcLTF1 for light-responsive TF1), which cating that the mutant is affected in its capacity
expression is strongly induced by light, was to cope with ROS produced by the plant within
identified (Schumacher et al. 2014). This tran- the extent of an oxidative burst, hampering the
scription factor has homologues previously ability of the fungus to colonize the plant tissue
characterized in A. nidulans (NsdD) and N. (Canessa et al. 2013).
crassa (SUB-1). By deletion and overexpres-
sion of bcltf1, it was confirmed that the tran-
scription factor plays a role in virulence. The
results obtained by the authors indicate that
X. Relevance in Biotechnological
BcLTF1 is required to cope with oxidative Applications
stress that is caused either by exposure to
light or arising during host infection. BcLTF1 Cellulose is the main component of plant bio-
is an important modulator of the transcrip- mass and it is associated mainly with hemi-
tional responses to light influencing the celluloses, lignin, and other components such
expression of most light-responsive genes in as proteins, fats, waxes, terpenes, phenols, alco-
B. cinerea. While deletion of bcltf1 mainly hols and alkanes, which together form the com-
affects advanced stages of infection, its over- plex and rigid structure of the cell walls. The
expression impairs penetration by germ tubes filamentous fungus T. reesei has been consi-
(Schumacher et al. 2014). dered one of the most prolific cellulase produ-
The Bright and Dark Sides of Fungal Life 67
cers in industry, since it could be used for effect was considerably enhanced in a mutant
conversion of cellulosic waste material into glu- lacking the cAMP-dependent protein kinase A
cose (Buchert et al. 1998; Galante et al. 1998). It (PkaC1) or the adenylate cyclase (Acy1) encod-
was thought that the cellulase enzyme complex ing genes. Acy1 exerts a positive effect on cellu-
of T. reesei consisted of at least three types of lase gene expression in light and darkness
enzymes to convert crystalline cellulose to glu- compared to the wild type. PkaC1 stimulates
cose (Lynd et al. 2002). However, the genome of the accumulation of mRNA of cellulase encod-
this fungus contains a lower number of cellulo- ing genes in light but exerts a negative regula-
lytic enzyme-encoding genes than any other tion in darkness. Transcriptional analysis of
sequenced fungus able to hydrolyze plant cell cellulase regulator genes showed that the regu-
wall polysaccharides, including cellulases, latory output of the cAMP pathway might be
hemicellulases, and pectinases (Martinez et al. settled by the regulation of Xyr1 abundance,
2008). The T. reesei genome encodes two cello- which is an essential transcription factor for
biohydrolases, four endo-b-1,4-glucanases, and cellulase gene expression. These data point to
several b-glucosidases, hemicellulases, and a role of adenylate cyclase and protein kinase A
accessory enzymes (Häkkinen et al. 2012). As in light-modulated cellulase gene expression in
briefly mentioned in the section on metabo- this fungus (Schuster et al. 2012). Additionally,
lism, cellulase gene expression in T. reesei is T. reesei strains lacking the env1 gene present
potentiated by light, and Env1 is involved in reduced intracellular levels of cAMP (Tisch
the light-dependent transcriptional regulation et al. 2011), which exert a considerable light-
of the cellobiohydrolase I-encoding gene dependent influence on transcription of the
(cbh1). Intriguingly, light does not lead to cel- major cellulase gene cbh1/cel7a. It was also
lulase gene expression in the absence of an demonstrated that Gna3 strongly regulates
inducer (Schmoll et al. 2005). cAMP levels, whereas Gna1 has only moderate
A positive role of the Blr1 and Blr2 proteins influence on them (Schmoll et al. 2009; Seibel
(the Trichoderma spp. putative photoreceptor et al. 2009). Therefore, Env1 links the light
complex) on cellulase gene transcription was response to carbon source signaling, probably
established, since mutants in blr genes showed through heterotrimeric G-protein cascades by
noninducible cellulase gene transcription in modulating the levels of gna1 and gna3 mes-
response to light (Castellanos et al. 2010). In sengers provoking effects on the cAMP levels.
T. reesei the induction of endoglucanase activ- In T. reesei the expression of cellulases and
ity by sophorose was stimulated by dBcAMP, as hemicellulases is dependent on the function of
well as by IBMX. On the other hand, the addi- the methyltransferase Lae1. Furthermore, it was
tion of exogenous dBcAMP or IBMX did not demonstrated that this regulation is dependent
induce endoglucanase activity (Šesták and Far- on xyr1 and that xyr1 overexpression does not
kaš 1993). The exposure of T. viride to a blue- rescue the lae1 deletion phenotype (Seiboth
light pulse led to a transient increase in the et al. 2012). On the other hand, a genome-
intracellular level of cAMP (Gresik et al. 1988). wide analysis of H3K4 and H3K9 methylation
Together, these results point to the participa- patterns in lae1 mutants did not show any
tion of Pka through phosphorylation of tran- methylation changes at the cellulase loci
scription factors in the synthesis of cellulases. (Karimi-Aghcheh et al. 2013), which contra-
In this regard, in T. reesei, cellulase gene tran- vene with the hypothesis that LaeA plays a
scription is not only exerted by cellulase but role removing the repressive chromatin
also by lactose. Contrasting with cellulase gene (Reyes-Dominguez et al. 2010). In T. reesei
transcription of T. reesei growing in cellulose, a vel1 mRNA expression is regulated by both,
considerable increase in transcript abundance light and darkness, and Vel1 is necessary for
of cellulase genes in darkness was determined cellulase gene expression. This is in agreement
compared to light upon growth on lactose. This with the hypothesis that the regulation of cellu-
68 S. Casas-Flores and A. Herrera-Estrella
lase gene expression by Lae1 takes place lulose degradation, which is most probably
through the Velvet complex (Karimi-Aghcheh also conserved in other ascomycete species
et al. 2014). (Schmoll et al. 2012).
Analysis of the cellulolytic activity of N.
crassa wild-type Dwc-1, Dwc-2, and Dvvd
mutants points to a role of WC-1 and WC-2 in
promoting cellulose utilization, given that the
XI. Concluding Remarks
Dwc-1 and Dwc-2 strains showed the lowest
activity among all tested strains. In contrast, Fungi are central to the global carbon cycle,
VVD appears to antagonize cellulose utiliza- constitute the major group of plant pathogens
tion, since a Dvvd mutant presented higher in managed and natural ecosystems, and serve
endoglucanase activity than the wild type. as symbionts with heterotrophic and auto-
Transcription analysis of the N. crassa cel7a trophic organisms alike. The ecological success
and cel6a homologues to T. reesei cbh1 and and importance of fungi to life can certainly be
cbh2, respectively, presented significantly attributed to the large diversity of enzymes and
lower expression levels in the Dwc-1 and Dwc- metabolites that they produce, which allows
2 mutants, whereas their expression levels in them to access a large diversity of carbon
Dvvd were comparable to those of the wild- sources and ecological niches. On the other
type strain. Transcriptomic analysis of wc hand, the influence of light on their growth,
mutants showed significant downregulation of reproduction, and metabolism has been
the major predicted cellulase and hemicellulase known for a long time. Thus, it is only logic to
genes in this fungus. On the other hand, the expect that such strong influence of light be
Dvvd strain showed an enrichment in carbohy- reflected in their ecological behavior, conse-
drate metabolism genes, which explains the quently impacting life on earth.
enhanced cellulose degradation by the Dvvd It is clear now that any defect in their ability
strain. The transcriptional analysis on pre- to perceive light represents an ecological dis-
dicted cellulolytic showed that in addition to advantage regardless of their habitat, as light is
cel7a and cel6a, six other cellulolytic genes a cue that alerts them of the conditions of an
were downregulated in the Dwc-2 mutant, ever-changing environment.
whereas the Dvvd strain had increased and During the last 20 years, but particularly in
extended expression levels of genes regulated the last decade, significant advances have been
by the WCC, including those encoding cellulo- made toward understanding light perception
lytic enzymes. Furthermore, all three photo- and the molecular events triggered by this envi-
receptors showed a negative effect on ronmental cue. Nevertheless, we are still far
transcription of the carbon metabolism regula- from elucidating the unexpectedly complex
tor encoding gene cre-1, which point to their network involved in all light responses
regulatory contribution to carbon catabolite observed. Most efforts have been focused on
repression (CCR) and not only subject to understanding blue-light responses through
photoadaptation. However, optimal expression what is perhaps the main blue-light perception
of genes encoding plant cell wall-degrading system.
enzymes and cellulolytic activity and release The advent of omics technologies (genomics,
from CCR strictly require the presence of transcriptomics, proteomics, metabolomics,
inducing molecules. These data point to a role etc.) has allowed us to discover new players in
of the WCC in regulating genes involved in the physiology of fungi and to construct meta-
plant cell wall degradation, probably through bolic and regulatory pathways when interacting
the activation of a WCC-dependent transcrip- with sunlight. Until today, over 100 fungal gen-
tion factor. Together, the data derived from the omes have been sequenced, including human
analysis on T. reesei and N. crassa indicate that and plant pathogens (https://www.broadinsti-
they use common mechanisms for the control tute.org/scientific-community/science/projects/
of enzymes involved in plant cell wall and cel- fungal-genome-initiative/fungal-genomics). In
The Bright and Dark Sides of Fungal Life 69
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metabolomes of several fungi under different ent photoperiods on the growth, infectivity and
colonization of Trinidadian strains of Paecilo-
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together will lead us to a better understanding Trialeurodes vaporariorum, using a glass slide bio-
of why and how sunlight provokes detrimental assay. J Insect Sci 4:38
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4 Disturbance in Natural Ecosystems: Scaling from Fungal
Diversity to Ecosystem Functioning
efficient nutrient capture and by expanding the pose large challenges for those characterizing
resources available to plants (Hart et al. 2003). As patho- fungal structure and function in response to
gens, fungi alter vigor and reproduction of other organ-
isms which are very important for maintaining
any environmental change. This is especially
biodiversity and mediating competition (Winder and problematic because differences in life history
Shamoun 2006; Hodge and Fitter 2013). As saprophytes, strategies among fungal species may mask their
fungi are the primary decomposers of substrates and are response to environmental change, altering the
essential for recycling nutrients to plants (van der Wal interpretation of presence-absence data
et al. 2013). Disturbance at any scale has the potential to
alter the biomass or reproductive activities of fungi, and
depending on the techniques employed (Brun-
can affect the diversity of fungi in ecosystems. Popula- drett and Ashwath 2013).
tion or community dynamics of the fungi involved in
these activities will be altered to differing degrees based Fungi, because of the size of individual hyphae or yeast
on physical location of organisms in the ecosystem and cells, and because they are predominantly studied in
type of disturbance (Snyder et al. 2002). With the differ- the laboratory under a microscope, are viewed as
ences in characteristics of these functional groups in microorganisms that primarily respond to minute
mind, we will attempt to discuss fungi in general with quantities of substrates in their environment. In mass
examples from each of these groups. Unfortunately, and spatial extent, fungi can be macroorganisms, often
there is greater information available about how distur- extending across large patches. These patches may be
bance alters some of these groups than others. This rather static in space, existing over long time periods,
constrains our conclusions here, but more importantly, such as the matforming ectomycorrhizal (ECM) fungi
limits our general understanding of the impacts of dis- or the Armillaria mycelial networks (Allen et al. 1995).
turbance on fungi. It certainly brings to the forefront the However, because these large organisms are still made
necessity to understand the response of these groups of up of microscopic hyphae with the potential to function
organisms to disturbance and to understand their independently, detailing their life-history characteris-
impacts on ecosystems. tics means understanding both the macroscopic and
microscopic aspects of their existence. As a result of
Fungi have unique physiology, morphol- this dichotomy in the structure of fungal “individuals,”
any disturbance, from an individual gopher mound to a
ogy, and reproductive biology. This makes volcano, has important ramifications to the types of
attempts to describe their dynamics difficult organisms and roles they play in ecosystem dynamics.
in the rather conventional terms used for For example, a large disturbance such as a volcanic
higher plants and animals. Further, Taylor eruption can destroy the entire fungal community,
et al. (2014) provide data that suggest our cur- requiring subsequent reinvasion and establishment
subject to classical models of succession (Allen et al.
rent estimates of 0.6–1.5 million species of 1992, 2005b). In contrast, disturbances such as mound
fungi may significantly underestimate the true building by gophers, or ants can disrupt mycelial net-
number. Their estimate is closer to six million works within multispecies patches and within “individ-
species of fungi. This would suggest that our ual” fungal types. These scenarios allow for some
current understanding of fungi in soil is woe- recolonization from existing species should the myce-
lial network rebuild from the remnant hyphal frag-
fully inadequate. The major limitations study- ments. Each of these disturbance extremes directly
ing fungi are their microscopic size and a and indirectly regulate the composition of the plant
marginal understanding of their life history community and, in turn, the animal community. It is
strategies. Recent uses of molecular techniques this range in the activity that we will describe. Rather
for evaluating fungal communities have than just providing examples, we will develop a set of
conceptual models that will allow us to begin to link our
allowed us to move a long way toward improv- understanding of fungal biodiversity and ecosystem
ing understanding of the needs and existence of functioning.
fungi in ecosystems, especially compared to
evaluation through direct observation using
culture techniques and microscopes. However,
we have not yet been able to determine aspects II. Ecosystem Disturbance:
of biology important for studying these organ- A Conceptual Framework
isms, such as cues involved in sporulation,
germination characteristics of spores, sensitiv- It has long been a goal of ecologists and mycol-
ity of hyphae vs. spores to physical or chemical ogists to understand the factors influencing
change, etc. These limitations in methodology the diversity, distribution, and abundance of
Disturbance in Natural Ecosystems: Scaling from Fungal Diversity to Ecosystem Functioning 81
organisms in a community. Disturbance can that scale from patch to ecosystem-level pro-
fundamentally alter these characteristics of fun- cesses (Fig. 4.1). Our overview includes ele-
gal communities; however, the mechanism by ments of these disturbance scales and the
which disturbance impacts any organism is a factors that affect the various states of the dis-
consequence of the intensity and frequency of turbance cycle. The conceptual model inte-
the disturbance. The effect of a disturbance on a grates processes operating at the level of the
community depends on how far the distur- patch disturbance, such as soil enrichment,
bance shifts the community from equilibrium with those at the ecosystem level, such as spatial
or how often it recurs compared to the life span variation in natural disturbance density (e.g.,
of the organisms. For example, disturbance fire, animal digging), soils, and microbial and
has been proposed to influence community plant community structure (Fig. 4.1). We
diversity and structure through a variety of believe that emphasis on the feedback between
mechanisms. Small-scale disturbances such as patch dynamics and local ecosystem processes
burrowing animals or insects can alter resource is an important key to increasing our under-
type or availability. A single disturbance type, standing of the role of natural disturbance in
e.g., gopher mounds, can enhance (Allen et al. fungal community structure and ecosystem
1984, 1992) or retard (Koide and Mooney 1987) function.
the rate of succession by altering soil resources
or species diversity. At even larger scales, such
as that of plowing, soil disturbance was found III. Fungal Community Dynamics:
to alter mycorrhizal community dynamics A Natural Disturbance Model
more than host plant identity (Schnoor et al. for Fungi
2011). Disturbances may also affect commu-
nities by creating new sites for colonization
(Platt 1975; Collins 1987) or creating new sub- Communities are characterized by all of the inter-
strates (Cooke and Rayner 1984; Gams 1992). acting species that occur within a given area.
Disturbances that decrease resources for one The area is determined by the extent of those
functional group of fungi may result in an interactions. Fungal communities can involve
increase of resources for other functional organisms that span many functional roles. To
groups of fungi (Lindahl et al. 2010; Xiong adequately describe the impacts of disturbance
et al. 2014). For example, disturbances that on fungal community dynamics, a disturbance
decrease photosynthate available for mycorrhi- model would need to integrate understandings
zae will provide increased substrate for sapro- of structural and functional impacts to fungi
phytes as hyphal networks become available for across spatial and temporal scales.
decomposition. Ultimately, the degree to which
any disturbance type modifies the quantity or
quality of a substrate on which a fungal com- A. Species Diversity
munity exists will determine the extent to A natural disturbance model that evaluates
which the community will change. These mod- fungi must examine species diversity in the
ifications can include alterations to substrate context of the concomitant impacts on ecosys-
chemistry (e.g., differing litter types) or to the tem dynamics. Diversity within a given system
environmental factors under which those sub- is characterized by richness and evenness.
strates can exist, such as changes in soil mois- However, it is virtually impossible to delineate
ture content with removal of the litter layer. If evenness due to the microscopic size of the
disturbances differentially affect substrate and individual organisms.
environment, then disturbances caused by a
variety of agents will alter fungal species diver-
Regardless of the disturbance size, fungal biomass, and
sity within a community in different ways. therefore evenness and diversity will be altered. More
We have developed a conceptual model of problematic is richness. The size of the organism mat-
natural disturbance that focuses on processes ters here. If a fungus occupies tens to hundreds of m2,
82 S.J. Morris et al.
• FUNGAL COMMUNITY
>1.0 µm to ? • PLANT DISTURBANCE
• ANIMAL DISTURBANCE
• SOIL NUTRIENT DYNAMICS
• FUNGAL POPULATION DYNAMICS
Fig. 4.1 Conceptual framework illustrating how ecolog- microbial functional groups, to larger-scale spatial pat-
ical concepts of hierarchy and scale can be used to terns and processes (e.g., ecosystem dynamics) (Friese
integrate and model the relationship of small-scale et al. 1997)
phenomenon, such as the impact of disturbance on
Disturbance in Natural Ecosystems: Scaling from Fungal Diversity to Ecosystem Functioning 83
then richness might not be affected by a small distur- B. Disturbance Types and Characteristics
bance. However, individual yeast and microfungi could
well be eliminated. How these diversity elements alter There are many types of disturbance that will
specific ecosystem characteristics is dependent on the affect a fungal community. In the fungal litera-
disturbance event itself, the substrates that exist follow-
ing the disturbance, the subsequent microclimate and ture, disturbances have been broken into two
its effects on substrate, and the surrounding organisms. main groups, enrichment and destructive dis-
A disturbance event directly alters the fungal commu- turbances (Cooke and Rayner 1984). For under-
nity by destroying hyphae and propagules of existing standing fungal community dynamics, these
species, although impacts of any disturbance will be classifications are often too broad because dis-
modified by the intensity of the disturbance and the
seasonality of the event (Gochenaur 1981). Understand- turbances at the scale that relates to the fungal
ing how these modifications regulate outcomes is community can never be entirely enriching or
important for predicting the impacts of disturbance entirely destructive. For example, Lindahl et al.
on communities. Many recent studies have identified (2010) found that the disruption of carbon to
legacy effects wherein the long-term effect of the dis- root systems from girdling in trees decreased
turbance is moderated by the specific circumstances of
the disturbances such that there is a unique response the ECM in the humus layers; however, it
from the community of interest. Legacy effects, defined increased opportunistic saprophytic fungi.
by Wurst and Ohgushi (2015) as “effects that persist Štursová et al. (2014) found similar results fol-
after the biotic interaction that caused the effect lowing a bark beetle-induced tree die back.
ceases”, occur when the disturbance differentially What was a destructive disturbance for the
removes or impacts the pre-disturbance biotic commu-
nity. Such was the case following the volcanic eruption mycorrhizal species was an enriching distur-
at Mt. St. Helens wherein the existence of snowpack bance for the saprophytic fungi.
protected some of the plant community from initial ash Disturbances that alter plant and animal
fall but not others areas causing significant differences community dynamics will resonate through the
in disturbance severity and differences in recovery tra- fungal communities. Wind storms strip leaves
jectories for plant and soil communities. (Crisafulli
et al. 2005) and blow down canopy trees in a forest. These
An additional dilemma in determining the impacts are usually greater near the edge which
effects of disturbance at the patch or ecosystem decreases habitat quality especially for small
scale is determining the extent of a fungal com- stands. These wind events provide substrate for
munity (Cooke and Rayner 1984). These effects saprophytic fungi; however, they decrease host
can be studied if the interpretation that com- density for mycorrhizal fungi. In complicated
munities are made of individuals with definable food webs, such as those that involve truffle-
sets of species interactions (MacMahon et al. eating flying squirrels, windthrow events can
1978) is used to define our concept of commu- decrease food resources and canopy connectiv-
nity. Fungal communities are comprised of ity, which can decrease squirrel populations
organisms with different functional roles (Carey 2000; Ransome et al. 2004) ultimately
(mutualist, saprophyte, pathogen), and these decreasing squirrel dispersal of the mycorrhizal
functional groups or guilds may have different fungal spores contained in the truffles. As fungi
responses to the same disturbance (Lindahl participate in many different relationships in
et al. 2010; Štursová et al. 2014; Xiong et al. ecosystems, a single disturbance, regardless of
2014). Large-scale disturbances can alter an size or enrichment/destructive capacity, has
entire landscape (Turner and Dale 1998), potential to impact fungi directly or indirectly
while small-scale disturbances may affect only through the organisms upon which fungi
a patch within the community. For this reason, depend. Ultimately, this will feed back to alter
the model developed here (Fig. 4.1) predicts the fungal community dynamics.
effects of disturbance within the patch, yet in
some circumstances, the patch may encompass C. Biotic and Abiotic Characteristics of the
an entire community. Following disturbance, Disturbance Model
the changes within the patch may affect the
community of which it was a part or may The greatest effect of disturbance on the fungal
become a community of its own. community will be through influences on key
84 S.J. Morris et al.
ECOSYSTEM
Plant Community Microbial Functional
Structure Dynamics
COMMUNITY
Overall Pattern of Fungal Species Diversity
Community & Distribution
Disturbance
PATCH
Size, Duration & Fungal Establishment
Timing of Individual
Disturbance Types
Disturbance Agent
(e.g., fire,
animal digging)
Biotic & Abiotic
Characteristics of
Individual
Disturbance Types
(Figure 3)
Fig. 4.2 Theoretical conceptual model depicting how larger-scale patterns and processes of community-
patch-level disturbance processes can affect microbial and ecosystem-level dynamics (Friese et al. 1997)
functional dynamics, which, in turn, can shape the
factors that impact the growth of fungal struc- ical environment within which organisms grow.
tures and germination of spores. These factors, For fungi in most terrestrial ecosystems, the
denoted as biotic and abiotic characteristics in complex system of decomposing wood and lit-
Figs. 4.2 and 4.3, include physical characteris- ter on the forest floor or simply litter in grass-
tics, resources, soil flora and fauna, and fungal land systems is an essential part of their
propagules or hyphal fragments (Boddy 1984; ecosystem. The litter and wood debris layer
Gams 1992). The physical components of the can provide substrate directly, or it can modify
fungal community affected by disturbance the soil quality below it.
include light, temperature, moisture, and pH
(Gentry and Stiritz 1972; Rogers and Lavigne Bässler et al. (2012) and Abrego and Salcedo (2013)
1974; Mandel and Sorenson 1982). The found fungal diversity linked to the diversity of decay-
resources include water, oxygen, organic mat- ing wood types or wood quality and structural hetero-
ter, and a variety of mineral nutrients for geneity rather than biomass of materials available.
growth and sporulation, for which some McGuire et al. (2010) investigating the response of
fungal taxa to specific substrates, identified patterns of
may be required in higher quantities than for response to substrate availability such community
vegetative growth (Moore-Landecker 1990). response to substrate availability could be predicted.
Another key resource included here is the phys- However, they also found difference in substrate use
Disturbance in Natural Ecosystems: Scaling from Fungal Diversity to Ecosystem Functioning 85
Fig. 4.3 Detailed model of the biotic and abiotic char- wide diversity of disturbance patches with unique
acteristics of individual disturbance types. This model biotic and abiotic characteristics. If each disturbance
is a subset of the complete conceptual model depicted type creates a unique set of biotic and abiotic charac-
in Fig. 4.2. The disturbance agent determines such teristics, then it is hypothesized that the fungal com-
aspects as the size, duration, and timing of an individ- munity will also be differentially affected within each of
ual disturbance event. All of these variables create a these disturbance types (Friese et al. 1997)
profiles among individual species suggesting that loss fungal biomass remained the same under elevated CO2,
of rare species could alter nutrient turnover. As such the total through-put actually increased. Fire can also
the forest floor composition and characteristics, and alter fungal-animal relationships. For example, the loss
impact of disturbances on the litter layer can have of the litter layer, a physical factor and/or substrate, by
severe consequences for the fungal biomass as well as environmental factors such as fire, results not only in
diversity in terrestrial systems. substrate loss, but also in increases in soil temperatures
and decreases water-holding capacity. As this happens,
soil organisms such as mites and collembolans will
Plants and animals also regulate the com- migrate more deeply into the soil to escape drought,
position of fungi in soils but often in a nonlin- reducing grazing on fungi but also reducing fungal
ear manner. Plants can provide both energy dispersal near the surface (Klironomos and Kendrick
and carbon for fungal growth, but they also 1995). Thus, changes in each of the above characteris-
provide many inhibitory substances. Many tics will be dependent on the type and intensity of the
disturbance and the interaction among the response
studies recently have documented impacts of variables.
plant species on belowground communities
through addition or removal of tissue (Bouasria
The spatial structure of systems, as a conse-
et al. 2012; Grove et al. 2012) or photosynthate
quence of environmental patterning or small-
and exudate release (Lindahl et al. 2010).
scale disturbance, has the potential to impact
Animals can remove (graze), disperse, provide
our ability to determine the effects of distur-
housing, or provide substrate (defecate or
bance on organisms. For example, the existence
die) for different members of the fungal com-
of an ant mound has potential to alter the impact
munity depending on fungal requirements.
of a disturbance such as fire on an ecosystem.
Additionally, changes in more than one of the
Alterations may be a consequence of different
parameters can act synergistically or antagonis-
physical structures, for example, the movement
tically, to further change the emergent fungal
of wind across the raised surface or decreased
community (Snyder et al. 2002).
burn heat as a consequence of increased mound
moisture. The difficulty is separating the
In one case, the addition of CO2 increased plant pro-
duction, and C inputs to soil fungi. However that input
impacts of fire on the system from the impacts
was matched by the increased grazing by soil animals of prefire small-scale disturbance. Failure to
(Allen et al. 2005c). So, although the standing crop of detect the specific impacts of fire may be the
86 S.J. Morris et al.
result of sampling a mound without incorporat- ited by lack of good techniques for identifying
ing the specific characteristics of a mound. Boer- either. While culture techniques allowed us to
ner et al. (2000) found that enzyme dynamics examine some species, the availability of molec-
related to bacterial and fungal activity in single- ular techniques has greatly increased our
tree influence circles (see Zinke 1962) were knowledge of fungi as they exist in their natural
altered by fire. Detecting changes in fungal environment. However, molecular identifica-
dynamics following disturbance requires under- tion does not always help with understanding
standing of pre-disturbance spatial dynamics the role of fungi in ecosystems, as within
and scale-appropriate post-disturbance sam- genera, closely related species can occupy
pling efforts. Failure to understand the impacts widely divergent niches (Taylor et al. 2014).
of patch-level disturbances and other agents Further, several studies examining arbuscular
capable of creating spatial structure in fungal mycorrhizal (AM) fungi have determined that
communities will decrease likelihood of detect- spore numbers, often used to predict existence
ing post-disturbance impacts. or survival of fungal species after disturbance,
are not always related to presence as mutualists
within roots, in the same way that seeds and
plant mass are not necessarily related. The rela-
IV. Scaling from Patch to Catastrophic
tionship between hyphal presence and spore
Disturbance production and cues for spore production
may be species specific, which would alter our
Disturbances impact fungi at a number of interpretation of the presence or importance of
levels. As discussed earlier, the smallest level specific taxa (Vargas et al. 2010; Brundrett and
reasonably available for description of distur- Ashwath 2013). As many studies carried out
bance impacts is the patch, and the largest is the before the use of molecular techniques were
ecosystem. While in theory large-scale distur- used to develop our understanding of patch
bances can influence entire landscapes and the dynamics and the role of individual, we are
last ice age certainly disturbed entire biomes, now gaining new knowledge of the complexity
our discussion focuses on individuals, commu- of these relationships. Many of the questions
nities, and impacts at the ecosystem level as generated regarding individuals and impacts of
these are the most common levels currently disturbance are just beginning to be addressed
studied. Landscapes can be viewed as large in greater detail; our discussion of individuals
mosaics of divergent ecosystems across a large of necessity depends on data derived from a
integrated spatial distance, or they can be dis- number of different approaches.
cussed as groupings of similar ecosystems at
different successional stages recovering from Patch level disturbances often have greatest impact at
similar disturbance regimes (Forman 1995). the individual level. Plants link with multiple fungi, and
This point is important as adjacent ecosystems individual fungi infect multiple individual plants.
house communities that provide propagules for Through interconnections, disturbances alter fungal
the reestablishment of similar communities. mycelial networks, disrupt the hyphal networks of indi-
vidual fungi, and alter environmental conditions for
While not addressing this directly, the avail- production and germination of fungal spores. Indivi-
ability of propagules similar to pre-disturbance duals will therefore change in response to patch level
communities is an essential component of rees- disturbances yet this may or may not impact fungal
tablishment which, over evolutionary time, communities. The loss of the species from the ecosys-
tem will only occur if the individual impacted is rare,
may have produced the mechanisms and com-
has poor sporulation capacity or mechanisms, poor
munity dynamics discussed below. dispersal ability, is unable to germinate under new
conditions or is not represented in another reasonably
close resource patch. Differences in these characteris-
tics among fungi and consequences for continued exis-
A. Individuals tence following disturbance have been explored. Often
patch dynamics are linked to fragmentation and con-
Our understanding of impacts of disturbance nectivity among patches. Nordén et al. (2013) found
on fungal individuals or species has been lim- that connectivity amongst patches was, not surpris-
Disturbance in Natural Ecosystems: Scaling from Fungal Diversity to Ecosystem Functioning 87
ingly, more important for highly specialized species, greatest impacts of these types of patch disturbances
compared to the generalist species. Given that the spe- were on individual species through changes in biomass
cies impacted by the lack of connectivity were more and organic matter and overall fungal community
likely to be red listed species, it is important to under- diversity across the site was impacted to a much lesser
stand impacts of connectivity at the species level for degree.
predicting the impact of patch level dynamics on the
survival of rare species.
Most recently, studies have begun to iden-
tify individual fungal species that are sensitive
Further research on the impacts of distur- or insensitive to a given disturbance allowing
bance, such as tillage, on agricultural soils sug- for predictions based on understanding the
gests that soil disturbance decreases fungal characteristics of survivor species. Duan et al.
biomass and alters soil structure (Denef et al. (2011) found, within the AM community, fungi
2001; Rillig and Mummey 2006). In natural that were insensitive to disturbance and could
systems, tillage was also found to alter mycor- compensate for those that were sensitive to dis-
rhizal community composition, with the distur- turbances, supporting the importance of diver-
bance increasing variability in the community sity within communities for resilience following
structure among disturbed plots (Schnoor et al. disturbance. Similarly, Gehring et al. (2014)
2011). While impacts from these types of dis- found that multiple stressors on a community
turbances often occur at the patch level in nat- of ECM decreased fungal diversity and moved
ural systems, the degree to which they impact communities to more generalist species; how-
individuals and species has been little studied. ever, there was some suggestion that those fungi
It is likely that the level of patch disturbance by may have been better mutualistic partners for
digging animals and insects alters species com- hosts under the prevailing disturbance regime.
position at the patch while maintaining fungal In contrast, Lekberg et al. (2012) failed to find
community composition at the ecosystem. impacts on the AM fungal community following
Studies that examine soil disturbances such as a disturbance involving plant removal suggest-
ant mounds have detected some of these pat- ing that resilience may be a community charac-
terns in arid systems. teristic that is highly influenced by patch
dynamics and disturbance scale.
Friese and Allen (1993) found biotic enrichment of
microorganisms in ant nests at the study sites in Color-
ado and Wyoming. In this case disturbance by har-
vester ant digging increased total number of AM B. Community-Level Effects
fungal propagules in the ant nests compared to adjacent
soil from blue grama grass (Bouteloua gracilis) at a site Change in the species composition within a
in Colorado and under shrubs (Artemisia tridentata) in patch may cause changes within the larger
a site in Wyoming. The assemblages and dominant ecosystem-level fungal community. The estab-
species of fungi also differed in mound vs. non lishment of fungi within the patch can affect the
mound sites, with more fungi (characteristic of mesic
sites) occurring in ant nests (Friese et al. 1997). Fungal
diversity and distribution of fungi within the
species richness was higher from mound associated community or it can cause the establishment of
material than from soil adjacent to mounds, or soil a new community that exists only within the
collected under shrubs at the Wyoming site, but not patch. The exact outcome will be determined by
from soil adjacent to the mounds at the Central Plains the characteristics of the disturbance, especially
Experimental Range location. It appeared that for
both locations, microenvironments selected a distinct
the size of the disturbance, but also by the
assemblage of dominant fungi with Fusarium spp. dom- heterogeneity of the landscape prior to distur-
inating the root material, and Aspergillus and Penicill- bance. A landscape matrix is established by a
lum species predominating in seed cache soil. However, series of disturbances of increasing scale.
Aspergillus fumigatus had high densities in off-mound The patches are nested within a habitat that
soil from the Colorado site. Mucoraceous taxa (i.e.,
Cunninghamella, Rhizopus, and Syncephalastrum)
can be relatively homogenous or heterogeneous
were isolated primarily from mound material, suggest- depending on the scale and intensity of other
ing that the ant mounds may represent refugia for these disturbances that occur across the landscape.
more mesic adapted fungi. These results suggest the From an area of no disturbance, the smaller-
88 S.J. Morris et al.
scale disturbances are overlaid by the increas- are highly artificial, and organisms exist along
ing intensity of the larger-scale disturbance. gradients of these extremes. For example, a
The impact of the smaller-scale disturbances common fungus of burned pine forests is
may alleviate the effects of the larger-scale dis- Morchella. Is that because it tolerates the fires
turbances by acting as islands of inoculum or and harsh conditions following fires (S), com-
dispersal agents. This was the case for the petes well with other residuals and immigrants
arbuscular mycorrhizal (AM) fungi at Mt. St. by growing hyphae fast and utilizing resources
Helens. Disturbances by gopher digging and elk in the early spring before other saprophytes are
droppings returned the AM fungal inoculum to getting started (C), produces a massive sporu-
the site that was completely decimated by the lating fungus that disperses spores by wind and
volcano blast (Allen et al. 1984, 1992, 2005b). animals when released from competition (R),
Initially, ECM fungi were predominantly dis- or (most probably) has some effective combi-
persed back onto the site by wind (Allen nation of all strategies?
1987). Thus, across the pyroclastic flow zone,
small AM fungal patches reformed along ani- Taylor and Bruns (1999) examined the ECM commu-
mal pathways, whereas the ECM plants initially nity structure in a mature pine forest. They detected
established at random locations. It was the rees- minimal overlap in the active mycorrhizal community
tablishment of these individuals or the small and the community present as resistant propagules.
This suggested that differences in colonization strate-
eclectic group of fungi that existed in the depos- gies such as the C, S, and R described above and
ited inoculum that led to recolonization follow- resource preferences, combined with resistant fungal
ing the most severe disturbance type. Similarly structures, allow diversity to be maintained in forest
Jumpponen (2003) found the fungal commu- communities so that organisms can respond to envi-
nity in the youngest soils adjacent to a receding ronmental cues and disturbances that have been histor-
ically part of the ecosystems. Peay et al. (2007) found
glacier to have dormant mycorrhizal fungi even that ECM communities were very strongly structured at
before the arrival of the plant community. As the tree level, with each tree acting as an island of
these fungi are biotrophs dependent on plant diversity rather than a homogenous mixture across a
hosts, airborne spore deposition preceded plant landscape. Given their findings they suggest that the
arrival providing symbionts for arriving seeds. trade-off between competition and dispersion strongly
determines composition of the island patches and the
The composition of the post-disturbance overall landscape community. The “island” structure
community is also dependent on the competi- of the community would protect diversity in patches
tive interactions among residuals and immi- that would be available for reestablishment following
grants. A good deal of work has been disturbance.
undertaken studying the interactions of com-
petitive (C), stress-tolerant (S), or ruderal (R) Other studies on mycorrhizal communities
strategies in saprophytes following disturbance suggest that community development is depen-
(Cooke and Rayner 1984). Immediately follow- dent on the type (enrichment vs. destructive),
ing a disturbance, the R-strategist is likely intensity, and frequency of disturbance. Lilles-
to predominate although, depending on the kov and Bruns (2003) found the timing of
disturbance, many S-strategists may remain root colonization by two ECM fungi was altered
as residuals. Presumably the C- and more by soil nutrient status. Rhizopogon occidentalis,
S-strategists will predominate later. Fungi an early successional species, colonized roots
such as Mucor and Rhizopus are presumed to early and then was replaced as a dominant
be ruderals because they exploit simple carbo- species by Tomentella sublilacina as the forest
hydrates rapidly (R-strategy). Alternatively, matured. Under high-nutrient conditions,
Phanerochyte grows slowly but can degrade however, this replacement was delayed suggest-
almost any type of substrate (S-strategy). ing that interspecific interactions between
Cephalosporium is an outstanding competitor the two species were mediated by soil nutrient
because it expends a large amount of resources content.
to produce antibiotics that restrict access to its Fungal pathogens have unique roles in dis-
own resource base. However, these separations turbances. Pathogen density and diversity can
Disturbance in Natural Ecosystems: Scaling from Fungal Diversity to Ecosystem Functioning 89
trial ecosystems have a great deal more com- differences in nutrient acquisition. The associa-
plexity than these two species model, it is likely tions that tie belowground community struc-
that changes induced by disturbance have dif- ture to aboveground dynamics are established,
ferential effects on the species present which maintained, and disrupted by disturbances that
will ultimately affect the composition of the alter fungal community dynamics.
post-disturbance community. Understanding the impacts of changes in
More complex ecosystem impacts and feed- communities of saprophytic fungi on ecosys-
back loops in understanding fungal community tem function is also essential for understanding
dynamics are being identified in studies exam- the overall impacts of a given disturbance type.
ining invasive species. Invasive species can be Modeling has been used recently to examine
understood as a disturbance to an ecosystem. linkages between communities and ecosystem
Further, many studies have identified that inva- dynamics. While these models have been
sive species more easily enter an ecosystem important for understanding global change sce-
following a disturbance or a change to a distur- narios, they have also allowed independent
bance regime (Moles et al. 2008) often resulting evaluation of the key components of below-
in a secondary or interactive impact on below- ground communities on ecosystem dynamics.
ground communities from the invasive inter-
acting with the disturbance impact. Studies on Hunt and Wall (2002) modeled effects of species loss on
the invasive species Alliaria petiolata (garlic biodiversity. They found that deletion of saprophytic
mustard), for example, have shown decreases fungi and bacteria caused changes to net primary pro-
ductivity. As they were modeling a large number of
in AM fungi (Rodgers et al. 2008) and ECM belowground groups and detected little change with
fungi (Wolfe et al. 2008) following invasion. deletions of other groups, these results emphasize the
Garlic mustard may, in large part, be an effec- importance of saprophytes as determinants of ecosys-
tive North American invader because in alter- tem characteristics. It also emphasizes that changes in
ing the mycorrhizal inoculum, it can shift the this group as a result of disturbance has potential to
alter larger scale functioning. Fisk et al. (2011) demon-
competitive advantage from the native species, strated that alterations to substrate through distur-
a feat it does not accomplish in its native range bance had significant impacts on the community of
(Callaway et al. 2008). However, Barto et al. fungi involved in decay. There is currently a great deal
(2010) were able to demonstrate that native of interest in tying community structure to function at
plants associated with AM fungi were able to the ecosystem level. The response of fungal commu-
nities to disturbance events might provide good sys-
withstand the invader suggesting that the tems to evaluate these linkages.
impact of the invasive species was temporally
mediated such that the allelochemicals released
by garlic mustard may only be effective against
the establishment of symbioses, rather than
established mutualisms. D. Disturbance and Species Diversity
Mycorrhizal fungi may provide the links
necessary to evaluate the impact of community Disturbances influence the composition and
structure and changes in community structure species richness of communities through a vari-
on ecosystem function (Read and Perez- ety of mechanisms. Some disturbances result in
Moreno 2003). The effects of individual fungal decreases in the fungal biodiversity, while
species on plant communities are expressed others are hypothesized to release ecosystems
through their impacts on aboveground produc- from unproductive states of “retrogression”
tivity and diversity. Fungal species can increase (Wardle 2006).
productivity through increased resource acqui-
sition. They can alter plant diversity directly by Within a short time scale, some disturbances affect the
presence or absence through mechanisms of entire community simultaneously, such as volcanic
eruptions (Andersen and MacMahon 1985; Allen et al.
specificity and by altering the outcome of 1984, 2005b; Allen 1988) and catastrophic winds (e.g.,
aboveground competition. They can cause Dunn et al. 1983), while other disturbances such as
changes to litter and tissue quality through animal diggings (Koide and Mooney 1987; Allen et al.
Disturbance in Natural Ecosystems: Scaling from Fungal Diversity to Ecosystem Functioning 91
1992; Friese and Allen 1993), defoliation (Williamson small-scale disturbances which have the capacity to
and Wardle 2007) or invasive species (Eviner and Cha- change the community composition of a patch. The
pin 2003; Bohlen 2006; Wolfe et al. 2008) influence only deposition of dung containing viable mycorrhizal
a relatively small portion of the community at a time. spores from areas adjacent to the blast zone on the
Others initially start small and expand over longer time tephra at Mt. St. Helens by elk, days following the
scales such as invasive species (van Mantgem et al. blast, allowed the return of fungal propagules to a
2004), N deposition and fertilization (Egerton- biotically sterile area (Allen 1987). Chronic small-scale
Warburton and Allen 2000; Miao-Yan et al. 2009), gla- disturbances can also be caused by large ungulates.
cial expansions and retreats (Helm et al. 1999; Jumppo- Serengeti ecosystems are heavily grazed, resulting in
nen et al. 2002). The scale and intensity of disturbances increased nitrogen and dung applications to the soil
can, in turn, significantly affect the response of organ- (Seagle et al. 1992). This increases nitrogen levels and
isms and resulting successional patterns (e.g., Bazzaz mineralizable carbon sources for the microorganisms.
1983; Mooney and Godron 1983; Sousa 1984; Pickett Studies of mycorrhizal distribution in this ecosystem
and White 1985; Pickett et al. 1989; McClendon and demonstrated an inverse relationship between soil fer-
Redente 1990; Egerton-Warburton and Allen 2000). tility and the presence of mycorrhizal fungi (McNaugh-
Further, the frequency of the disturbance may alter ton and Oesterheld 1990). This relationship is also
community composition by encouraging the growth associated with a smaller gradient of nutritional status
of rapid growing more generalist species, where in associated with the vegetation. The mycorrhizae allow
less frequent disturbance regimes may allow the estab- the plants to maintain a high nutritional status across a
lishment and continued growth of species that grow broad range of soil nutritional ranges, which ultimately
more slowly. (Koide et al. 2011; Gehring et al. 2014) results in better forage for the animals and a return of
nutrients to the soil in the form of urine and dung.
Small-scale disturbance can affect the
diversity of fungal communities in a variety of Large-scale disturbances affect the diversity
ways. These disturbances are often poorly char- of fungal communities in different ways. Volca-
acterized because they create a mosaic of het- noes, the most extreme of the large-scale dis-
erogeneous patches within the landscape that turbances, can completely destroy fungal
are often functionally and structurally different communities, leaving topsoil buried under ster-
from the landscape that surrounds them. ile tephra (Allen et al. 1984; Hendrix and Smith
Patches are defined ecologically as discrete spa- 1986) or lava (Gemma and Koske 1990) or even
tial patterns with easily identifiable boundaries by creating new lands (Henriksson and Hen-
(Pickett and White 1985). Disturbances such as riksson 1974). Fungal communities in the most
mound building by animals create or alter the severely damaged areas can be destroyed
patchiness of a landscape (Allen 1988; Friese completely. The recovery of these areas is
and Allen 1993; Snyder et al. 2002). These driven by wind or small animal vectors capable
patch disturbances disrupt existing external of bringing new propagules from surviving
soil mycelial networks such as those described areas, patches, or from source areas at differing
by Finlay and Read (1986) for ECM and Friese distances. Materials deposited as a consequence
and Allen (1991) for AM fungi (e.g., absorptive of the eruption, such as tephra or lava, can have
hyphal networks and hyphal bridges). Disrup- different characteristics, such as altered bulk
tions in the soil hyphal network will create density, chemistry, or pH, which leads to the
openings for the colonization and spread of creation of new communities. On the most
new fungi, thus increasing fungal biodiversity, devastated area of Mt. St. Helens following the
just as occurs for colonial animals (Connell blast of 1980, Ascomycotina were found to col-
1961) and higher plants (Allen and Forman onize the tephra within the first year followed
1976). Gophers and ants are examples of ani- by Basidiomycotina (Carpenter et al. 1987).
mals that are capable of overturning soil and After 10 years, AM and ECM were reestablished
moving mycorrhizal propagules within that soil on the site mediated by gophers and wind dis-
to new patches in the soil matrix. Additionally, persal (Allen et al. 1992), although the ECM
gophers trap spores within their fur and can were initially poorly developed.
transport these fungi to new areas. The rate at which reinvasion progresses on
the most severely disturbed sites depends on
Ingestion and excretion of viable propagules at new the availability of sources of fungal inoculum.
locations by large mammals are also considered With the existence of nearby or internal source
92 S.J. Morris et al.
patches of inoculum, fungal communities can profile (Pattinson et al. 1999; Bastias et al.
begin reinvading and establishing. 2006).
The rates of reinvasion of mycorrhizal fungi onto the Fire can have a number of effects on mycorrhizal
pumice plain of Mt. St. Helens, to Krakatau following spores, depending on the maximum ground tempera-
the 1883 eruption (Allen 1991) and from Hawaii ture reached while burning. Vilarino and Arines (1991)
(Gemma and Koske 1990) demonstrate that reinvasion found that, following fire, the number and viability of
of these fungi is dependent on location and type of AM spores decreased. They also determined that on at
inocula, and upon the reinvasion of vegetation. Mt. St. least one site the dominant species of mycorrhizal fun-
Helens, by having mycorrhizae in the most damaged gus changed from Acaulospora laevis to Acaulospora
areas as soon as 1 year after the eruption, recovered scrobiculata. Percent root colonization by AM
more quickly than Krakatau, where facultatively increased over the year following the burn but did not
mycorrhizal plant species were reported 3 years after reach the levels found before the fire. The depression in
the eruption. This is because the most severely dam- levels of soil colonization following fire in this site was
aged areas of Mt. St. Helens were surrounded by rem- detected for longer than in other similar research, such
nant vegetation patches rather than water, as in the as that of Dhillion et al. (1988) on prairie soils. The
island of Krakatau. On the Hawaiian Islands, there authors suggested that the temperatures reached on
was a rapid invasion of mycorrhizal species from adja- these soils with a shrub and tree vegetation were greater
cent kipukas, or isolated patches of vegetation that than the temperatures reached with a herbaceous vege-
remained untouched by disturbance (Gemma and tation producing the longer-lasting effects. Treseder
Koske 1990). Volcanic eruptions such as the ones et al. (2004) found that fire in Alaskan boreal forests
above occur relatively frequently and are considered had little impact on AM fungi however recolonization
predictable and therefore subject to selective evolution- by ECM appeared to be delayed up to 15 years following
ary pressure. Presumably, dispersal strategies and life the disturbance. Heat may impact structures in the
histories of the plants on these islands are adapted to fungal life cycle in different manners altering growth
these disturbances, and it is probable that the same is and reproduction patterns. Peay et al. (2009) found that
true for the fungi. ECM spores were differentially impacted by heat asso-
ciated with fires resulting in differences in recoloniza-
tion patterns following fires. Secondary forests in the
Fire presents another example of large- Yucatan Peninsula with a very shallow, highly organic
scale disturbance which has the ability to affect soil, a hot fire virtually eliminated all inoculum (Allen
fungal communities in different ways. Follow- et al. 2003) whereas following a cooler fire, where some
ing a severe fire, there is an initial drop in the organic matter persisted, the richness of fungi was
number of propagules (Wright and Bollen much higher. The two types of fires can cause signifi-
cantly different patterns of vegetation recovery (Allen
1961) and shift in the diversity of fungi present et al. 2003, 2005a). Therefore, the frequency and inten-
in an area (Wicklow 1973; Allen et al. 2003, sity of a fire, which is determined largely by the struc-
2005a; Bastias et al. 2006). Changes in soil pH ture of the plant community (e.g., forest vs. grassland),
and mineralization rates caused by fire regulate can determine both the spatial and temporal patterns of
the fungi that can initially establish in the area fungal community development. While, there is great
heterogeneity on the effect of fire on fungal commu-
(Gochenaur 1981). Following the initial nities, it is apparent that the fungal community is active
decrease in fungal propagules, a rapid increase after fire and have a role in soil stabilization and reme-
in fungal biomass occurs often to more than ten diation. (Claridge et al. 2009)
times the prefire value (Ahlgren and Ahlgren
1965; Wicklow 1973). These species, often At an even larger scale are the large-scale
referred to as pyrophilous fungi, are capable perturbations to fungal communities resulting
of taking advantage of the new resources from global climate change. While the planet
made available by the fire. has seen changes in temperature, moisture, and
Mycorrhizal species may decrease in num- atmospheric greenhouse gas concentrations
ber or diversity following fire (Vilarino and historically, these have taken place over much
Arines 1991; Allen et al. 2003, 2005a; Tuininga longer time frames. Current changes have taken
and Dighton 2004) or remain unaffected within place over much shorter time periods, that of
the plant root (Molina et al. 1992). However, hundreds of years, and have resulted in signifi-
some of these effects are relatively shallow and cant stochasticity and variability in weather
do not effect diversity or density lower in the patterns leading to increased ecosystem distur-
Disturbance in Natural Ecosystems: Scaling from Fungal Diversity to Ecosystem Functioning 93
bance (Stocker et al. 2013). The literature on the standing the roles of disturbance in fungal com-
impacts of elevated CO2 on fungi from natural munities and the feedbacks from the fungal
enrichment sites such as volcanoes suggests communities to ecosystem functioning is cru-
that some fungi are sensitive to CO2 certainly cial to understanding the results from these
more so than soil bacteria and archaea, and larger global concerns. Developing models for
fungi are differentially sensitive such that linking the range of scales that comprise distur-
wood decomposing fungi may have a greater bance dynamics depends on linking two dis-
tolerance to elevated CO2 than litter decompo- tinct types of studies. First, we must begin to
sers (McFarland et al. 2013). Mutualists showed build an array of case studies from particular
changes in community structure; however, it is ecosystems in which we know the natural his-
not possible to determine whether this is direct tory of the fungi and how these natural histories
response to CO2 or to changes in plant function. contribute to the existing community composi-
In terms of impacts from other disturbances tion and functioning. Second, we must develop
such as warming, Xiong et al. (2014) found a conceptual framework that integrates all of
significant impacts on fungi such that Basidio- these various case studies into a comprehensive
mycota increased, while other groups such as view of how communities work and what fac-
Ascomycota and some of the more rare species tors regulate them. Finally, we must continu-
showed variable responses. Overall, our capac- ously reevaluate those conceptual models to
ity to predict the outcomes for fungi from develop a quantitative model of the complex
changes to global climate change as a distur- roles of fungi within landscapes that are under-
bance is currently limited which impacts our going anthropogenic and natural change.
capacity to predict alterations to natural and While it would be easiest to evaluate distur-
agronomic systems. bance as a static entity, it is unfortunately a
moving target. As anthropogenic impacts alter
natural ecosystems, we are also altering distur-
V. Conclusions bance regimes and impacts of disturbance
events. Ecosystems contain organisms that
A. The Impact of Disturbance on the have interacted with their biotic and abiotic
Functional Role of Fungi environments over exceedingly long time
scales. The systems examined today are a con-
This chapter was designed to demonstrate that sequence of organisms responding to the stres-
disturbance may be the single most important ses experienced on predictable time scales. As
process regulating the structure and function- anthropogenic disturbances have escalated
ing of fungal communities. This is due to the only over the last 100 years, we cannot ade-
unique physiology, morphology, and reproduc- quately predict the consequences of a given
tive biology of fungi. While microbial ecologists disturbance based on historical data because
are beginning to describe the importance of we do not know if the system we are examining
individual disturbance events, we know far is similar to that which existed in the past.
less about the interactions of small- and large- Ecologists use the terms resistance and resil-
scale perturbations set in the larger landscape ience to evaluate the degree to which a system
of single or multiple plant communities. The resists change in the face of disturbance or the
patch dynamics that operate in systems in degree to which it returns to the pre-
advance of disturbance create a heterogeneous disturbance state. In theory, a system facing a
landscape that often precludes simple predic- predictable disturbance would respond in a
tions of outcomes. This distinction becomes of resistant or resilient fashion. However, with
even greater importance in the light of the added unpredictable stresses such as chronic
global dimensions of anthropogenic influences N deposition, herbicide use, global warming,
on ecosystems such as N fertilization, exotic and atmospheric pollution, systems may be
species migrations, habitat fragmentation, and less resistant or resilient to disturbances that
global climate change. We suggest that under- they may have easily recovered from in the past.
94 S.J. Morris et al.
Loss of the evolutionary history within ecosys- Future research on fungal community dynam-
tems not only decreases our ability to under- ics should also focus on linking the issues of
stand the complex effects of disturbance to fungal biodiversity and functionality with both
fungal communities, but it also has great poten- natural disturbance and anthropogenic change.
tial to irrevocably alter the ecosystems that cur- This research direction is critical for us to
rently exist within our biosphere. completely understand and explain the impor-
tance of fungi in ecosystem dynamics. Attempts
to explore and integrate all of the above factors
B. Future Research Directions are crucial if we are ever to gain a comprehen-
sive understanding of the functional role of
As a result of the widespread interest in anthro- fungi in diverse ecosystems and the biosphere
pogenic changes to the earth’s atmosphere and as a whole.
the effects that these changes may have on the
biosphere, it is important to develop a better
understanding of how small-scale microbial References
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5 Fungi and Industrial Pollutants
G.M. GADD1,2
CONTENTS I. Introduction
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
II. Predicted Effects of Pollutants on Fungal Fungi may be exposed to a wide variety
Populations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
III. Fungi and Xenobiotics . . . . . . . . . . . . . . . . . . . . . . . 101 of organic and inorganic pollutants in the envi-
IV. Effects of Acid Rain and Airborne Pollutants ronment. Since fungi play a major role in
on Fungal Populations . . . . . . . . . . . . . . . . . . . . . . 103 carbon, nitrogen, phosphorus and other bio-
V. Effects of Toxic Metals on Fungi . . . . . . . . . . . . 104 geochemical cycles (Wainwright 1988a, b;
A. Effects of Metals on Fungal Populations 104 Gadd 2006, 2007, 2008a, b, 2011), impairment
B. Morphological and Growth Responses
to Toxic Metals . . . . . . . . . . . . . . . . . . . . . . . . . . . 106 of fungal activity could have important conse-
C. Mycorrhizal Responses Towards quences for ecosystem function. It is obviously
Toxic Metals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 desirable that more is known about the impact
D. Metal and Metalloid Transformations of pollutants on these organisms. Unfortu-
by Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 nately, while it is easy to speculate on the likely
1. Metal Mobilisation . . . . . . . . . . . . . . . . . . . . . 108
2. Metal Immobilisation . . . . . . . . . . . . . . . . . . . 109
effects of pollutants on fungi, it is often far
3. Organometal(loid)s . . . . . . . . . . . . . . . . . . . . . 110 more difficult to demonstrate such effects.
E. Accumulation of Metals and Radionuclides Studies on pollutant effects on fungal popula-
by Macrofungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 tions are difficult, largely because of the inade-
F. Accumulation of Radiocaesium quacy of many of the techniques which are
by Macrofungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
G. Fungi as Bioindicators of Metal and
available to study fungi and the complexity of
Radionuclide Contamination . . . . . . . . . . . . . . 111 mixed microbial communities (Anders and
H. Bioremediation, Biotechnology and Domsch 1975; States 1981; Doelman 1985;
Bioprocessing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 Gadd et al. 2007). However, an appreciation of
1. Bioleaching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 the effects which pollutants can have on fungi
2. Biosorption and Bioaccumulation . . . . . . 113
3. Metalloid Bioremediation . . . . . . . . . . . . . . 114
can be obtained by a combination of the follow-
4. Mycoremediation and the ing measurements: (1) pollutant concentration,
Mycorrhizosphere . . . . . . . . . . . . . . . . . . . . . . 114 composition and distribution, (2) pollutant
5. Nanoparticle Formation and bioavailability, (3) pollutant concentrations
Nanobiotechnology . . . . . . . . . . . . . . . . . . . . . 115 that cause a toxic or physiological response
6. Soil Treatment Processes . . . . . . . . . . . . . . . 115
in vitro, (4) effects of the pollutant on fungal
VI. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 population/community size and composition
and (5) secondary changes resulting from pol-
1
lution effects on fungal populations, e.g. impact
Geomicrobiology Group, School of Life Sciences, University
on leaf litter decomposition. While pollutant
of Dundee, Dundee DD1 5EH, UK; e-mail: g.m.gadd@dundee.
ac.uk concentration and composition may be deter-
2
Laboratory of Environmental Pollution and mined using standard analytical techniques,
Bioremediation, Xinjiang Institute of Ecology and Geography, with varying degrees of difficulty depending
Chinese Academy of Sciences, Urumqi 830011, People’s on the pollutant and the environmental matrix,
Republic of China
the analysis of pollutant speciation and the bio- wright 1988b). Although toxic disturbance is
availability remain challenging problems. likely to predominate, instances will occur
The effect of pollutants on fungal popula- where both types of disturbance are found
tion/community size and composition is par- together. Toxic disturbance of fungal popula-
ticularly difficult to assess. Many earlier studies tions is likely to be particularly damaging to
used the dilution plate count or similar ecosystem function, while the rarer enrichment
approaches to assess changes in fungal commu- disturbance may occasionally produce beneficial
nity composition. The shortcomings of this effects on soil processes. Toxic disturbance is
technique have been criticised at length and likely to lead to a reduction in fungal numbers
are well known. To overcome problems relating and species diversity, as well as biomass and
to the use of plate counts, biomarkers such as activity changes which may detrimentally influ-
phospholipid fatty acid (PLFA) composition ence fundamentally important processes such as
and extraction and analysis of DNA are now litter decomposition (Freedman and Hutchin-
routinely used, though no methods are exempt son 1980; Hiroki 1992; Fritze and Baath 1993).
from problems (Gadd et al. 2007). Another The resultant degree of toxic disturbance will
problem is that it is unlikely that a meaningful depend upon both toxicant concentration and
picture of how fungi respond to pollutants in its availability to the fungal population, as well
the environment can be gained from determin- as to the susceptibility of the individuals
ing responses to pollutants added to solid or involved. Toxicants may be selective and affect
liquid growth media in laboratory experiments. only a few species, or they may have a more
The effects of toxic metals on soil fungi growing generalised effect. Selective inhibition may have
in vitro, for example, are markedly influenced less of an impact on overall soil fungal activity
by the composition of the medium used: metals than might be imagined, since susceptible spe-
are likely to be more toxic to fungi in low- cies can be replaced by more resistant fungi,
carbon media than in carbon-rich media some of which may be more active in a given
where the production of large amounts of extra- physiological process than the original popula-
cellular polysaccharides and chemical interac- tion. While concentration effects are generally
tions with the medium will tend to reduce metal emphasised, it is surprising how often the ques-
availability. Medium components may also tion of toxicant bioavailability is avoided in
complex or precipitate metals out of the solu- studies on the effects of pollutants on microor-
tion, making them unavailable (Gadd and Grif- ganisms. In soils, for example, bioavailability of
fiths 1978; Hughes and Poole 1991; Gadd 1992). a pollutant will generally depend upon factors
Finally, interactions between different pollu- such as (1) adsorption to organic and inorganic
tants and their breakdown products may have matter, (2) chemical speciation, (3) microbial
a major influence on the toxicity of a pollutant transformation and/or degradation and (4)
in the natural environment. This chapter will leaching. Another factor of importance in rela-
outline some of the main effects of organic and tion to the effects of toxicants on soil fungi con-
inorganic pollutants on fungi and will include cerns nutrient availability. Fungi are generally
the discussion of effects at cellular and commu- thought to be already stressed by the low levels
nity levels and their applied and environmental of available carbon present in most soils and
significance. other environments (Wainwright 1992). They
will grow slowly, if at all, under these conditions
and may be more susceptible to pollutants than
when growing in high-nutrient conditions.
II. Predicted Effects of Pollutants Fungal populations are unlikely to remain
on Fungal Populations static when confronted with a toxic agent, and
resistant populations are likely to develop
Environmental pollution might be expected to which will be a major factor in determining
lead to both toxic (destructive) and enrichment population responses to the pollutant. On the
disturbances on fungal populations (Wain- other hand, a number of studies have shown
Fungi and Industrial Pollutants 101
ity to degrade complex mixtures of PAHs, such contain large populations of PAH-transforming
as those in creosote and coal tar, but actual bacteria (Johnsen et al. 2002) and fungi (April
bioremediation of contaminated soils using et al. 2000; Saraswathy and Hallberg 2002).
these fungi has met with varying success Combinations of several microorganisms are
(Canet et al. 2001; Cerniglia and Sutherland usually better able to degrade benzo[a]pyrene
2001; Pointing 2001; Hestbjerg et al. 2003). and other high-molecular-weight PAHs than
Non-ligninolytic fungi, including Cunningha- pure cultures (Kanaly et al. 2000).
mella, Mucor, Fusarium and Penicillium spp.,
have also been considered for PAH bioremedi-
ation (Colombo et al. 1996; Pinto and Moore
2000; Ravelet et al. 2001; Saraswathy and Hall-
IV. Effects of Acid Rain and Airborne
berg 2002). Pollutants on Fungal Populations
Biodegradation may require the presence of
mixed bacterial and fungal communities, Although acid rain is generally regarded as a
although less is known about the pathways of long-range pollution phenomenon, high con-
PAH degradation by co-cultures (Juhasz and centrations of mineral acids will pollute ecosys-
Naidu 2000). The evolution of 14CO2 from 14C- tems close to point source emissions (Helander
phenanthrene in soil was enhanced almost two- et al. 1993). Acid rain effects will also impinge
fold (from 19.5 % to 37.7 %) when P. chrysos- on the availability and effects of other pollu-
porium was added to the indigenous soil tants such as toxic metals, which may accom-
microflora (Brodkorb and Legge 1992). pany atmospheric dispersal and/or be released
Boonchan et al. (2000) combined Penicillium from soil components as a result of increased
janthinellum with either Stenotrophomonas acidity (Wainwright et al. 1982; Tabatabai 1985;
maltophilia or an unidentified bacterial consor- Francis 1986; Persson et al. 1989). Baath et al.
tium. The fungus could partially degrade pyr- (1984) showed that soil biological activity, as
ene and benzo[a]pyrene but could not use determined by respiration rate, was signifi-
either as a carbon source; S. maltophilia could cantly reduced following treatment with
use pyrene as a carbon source and co- simulated acid rain. Mycelial lengths (FDA
metabolise benzo[a]pyrene. The fungal–bacte- active) were also reduced by the treatment,
rial combinations grew on pyrene, chrysene, while plate counts showed no response. Fritze
benz[a]anthracene, benzo[a]pyrene and (1987), on the other hand, showed that urban
dibenz[ah]anthracene, converting 25 % of the air pollution had no effect on the total length of
benzo[a]pyrene to CO2 in 49 days. The white- fungal hyphae in the surface horizons of soils
rot fungus P. ostreatus and the brown-rot fun- supporting Norway spruce (Picea abies). Bew-
gus Antrodia vaillantii enhanced the degrada- ley and Parkinson (1985) showed that the con-
tion of fluorene, phenanthrene, pyrene and tribution which fungi make to the total
benz[a]anthracene in artificially contaminated respiration of a soil was reduced by acid rain,
soils (Andersson et al. 2003). Unlike P. ostrea- while, in contrast, Roberts et al. (1980) con-
tus, which inhibited the growth of indigenous cluded that the addition of acid rain to forest
soil microorganisms, A. vaillantii stimulated soils did not affect the normal 9:1 balance of
soil microbial activity. fungal to bacterial respiration. These studies
Ligninolytic fungi partially oxidise PAHs by clearly illustrate how difficult it is to generalise
reactions involving extracellular free radicals about the effects of atmospheric pollutants on
(Majcherczyk and Johannes 2000), making the soil microorganisms. Among higher fungi,
PAHs more water soluble so that they are able simulated acid rain has been shown to increase
to serve as substrates for bacterial degradation the dominance of some ectomycorrhizal fungi,
(Meulenberg et al. 1997). Partial oxidation while decreasing species diversity among sap-
increases PAH bioavailability in most contami- rophytic species (Sastad and Jensenn 1993).
nated sites (Mueller et al. 1996; Meulenberg Shaw et al. (1992) also showed that fumigation
et al. 1997), and PAH-contaminated soils may with sulphur dioxide or ozone had no effect on
104 G.M. Gadd
mycorrhizal populations. Acid treatments have and Pugh 1975; Baath 1991; Gadd 1992). ‘Resis-
been shown to impair the decomposition of tance’ is probably more appropriately defined as
both deciduous leaves and conifer needles the ability of an organism to survive metal tox-
(Baath et al. 1984; Prescott and Parkinson icity by means of a mechanism produced in
1985). Small-scale inhibitory effects were com- direct response to the metal species concerned,
mon, although stimulatory effects were also the synthesis of metallothionein and g-glutamyl
observed. Pollution in the form of alkaline peptides in response to Cu and Cd, respectively,
dust from iron and steel works was shown to providing perhaps the best examples (Mehra
lead to a doubling of the total length of fungal and Winge 1991). Metal tolerance may be
hyphae (Fritze 1987, 1991). defined as the ability of an organism to survive
The measurement of leaf litter and cellulose metal toxicity by means of intrinsic properties
decomposition provides a means of assessing and/or environmental modification of toxicity
the impact of atmospheric pollutants on soils. (Gadd 1992). Intrinsic properties that can deter-
However, in the absence of a means of parti- mine survival include possession of imperme-
tioning the relative impact of the toxicants on able pigmented cell walls, extracellular
fungi, bacteria and soil animals, such methods polysaccharide and metabolite excretion, espe-
provide only a measure of the effects of the cially where this leads to detoxification of the
pollutants on the total soil community. Atmo- metal species by binding or precipitation (Gadd
spheric pollutants from coking works can, for 1993a). However such distinctions are often dif-
example, reduce populations of soil microar- ficult to recognise because of the involvement in
thropods, a response which retards the rate of fungal survival in response to metal toxicity of
litter decomposition in deciduous woodland several direct and indirect physicochemical and
soils (Killham and Wainwright 1981). biological mechanisms. Biological mechanisms
Few examples of the effects of enrichment implicated in fungal survival (as distinct from
disturbance by air pollutants on fungal popula- environmental modification of toxicity) include
tions can be found in the literature. However, extracellular precipitation; complexation and
some fungi have been reported to utilise atmo- crystallisation; the transformation of metal spe-
spheric pollution deposits from coking works cies by, for example, oxidation, reduction,
as a nutrient source, as well as being able to methylation and dealkylation; biosorption to
oxidise the reduced sulphur which these parti- cell walls, pigments and extracellular polysac-
cles contain (Killham and Wainwright 1982, charide; decreased transport or impermeability
1984). and efflux; intracellular compartmentation;
and finally precipitation and/or sequestration
(Fig. 5.1) (Gadd and Griffiths 1978; Gadd 1990,
1992, 2007; Mehra and Winge 1991).
V. Effects of Toxic Metals on Fungi
The ability of fungi to survive in the presence of A. Effects of Metals on Fungal Populations
potentially toxic metals depends on a number of
biochemical and structural properties, includ- A range of fungi from all the major groups may
ing physiological and/or genetical adaptation, be found in metal-polluted habitats (Gadd
morphological changes and environmental 1993a, 2007, 2011). In general terms, toxic
modification of the metal in relation to the spe- metals may affect fungal populations by reduc-
ciation, availability and toxicity (Fig. 5.1) (Gadd ing abundance and species diversity and select-
and Griffiths 1978; Turnau 1991; Gadd 1992, ing for a resistant/tolerant population (Jordan
2007). Terms such as resistance and tolerance and Lechevalier 1975; Babich and Stotzky 1985;
are often used interchangeably in the literature, Arnebrant et al. 1987). However, the effect of
and may be arbitrarily based on the ability to toxic metals on microbial abundance in natural
grown on a certain metal concentration in labo- habitats varies with the metal species and the
ratory media (Tatsuyama et al. 1975; Williams organism present and also depends on a variety
Fungi and Industrial Pollutants 105
Aerial deposition
Fig. 5.1 Diagrammatic representation of the interac- mal and plant and microbial biomass is not shown but
tions of toxic metals and radionuclides with fungi in will be an important part of metal cycling. Fungal roles
the terrestrial environment. The dotted line shows in metal solubilisation from naturally occurring sub-
direct effects of metal species on fungi; this may some- strates and/or industrial materials are indicated (see
times occur and is more likely for metal species, such as Burgstaller and Schinner 1993; Gadd 2007). For more
Cs+, which are highly mobile. The release of metal/ detailed information regarding physiological and cellu-
radionuclide species from dead and decomposing ani- lar interactions, see Gadd (1993a, 2007, 2010)
of environmental factors making generalisa- and Zn and analysed after 6 months. PLFA
tions difficult (Gadd and Griffiths 1978). 18:2o6 is regarded as an indicator of fungal
General reductions in fungal ‘numbers’ (as biomass, and this increased with increasing
assessed by the dilution plate count in many metal contamination for all metals except Cu,
earlier studies) have often been noted in soils possibly reflecting the well-known mycotoxi-
polluted with Cu, Cd, Pb, As and Zn (Bewley city of Cu. However, in forest soils, such an
and Stotzky 1983; Babich and Stotzky 1985). increase in PLFA 18:2o6 was not observed
However, numerical estimates alone may pro- because of masking by identical PLFAs derived
vide little meaningful information unless possi- from plant material (Frostegard et al. 1993).
ble changes in fungal groups and species are Several studies have shown that microbial
considered, and the problems associated with population responses to toxic metals are char-
plate counting are in any case well known. acterised by shift from bacteria, including
Frostegard et al. (1993) analysed the phospho- streptomycetes, to fungi (Mineev et al. 1999;
lipid fatty acid (PLFA) composition of soil in Khan and Scullion 2002; Chander et al. 2001a,
order to detect changes in the overall composi- b; Kostov and Van Cleemput 2001; Olayinka
tion of the microbial community and provide and Babalola 2001). However, other studies
more reliable information on fungal popula- have shown a higher metal sensitivity of the
tions than can be produced using plate counts. fungal component of the microbial biomass
Two soils were amended with Cd, Cu, Ni, Pb (Pennanen et al. 1996). What seems clear is
106 G.M. Gadd
that all nutritional groups of fungi (sapro- Aureobasidium pullulans and Cladosporium
trophs, biotrophs and necrotrophs) can be species were the most numerous organisms
affected by toxic metals. Ruhling et al. (1984) (Bewley 1980). In fact, numbers of A. pullulans
found that the soil respiration rate, fluorescein showed a good positive correlation with lead,
diacetate (FDA) active mycelium and mycelial whether derived from industrial or vehicular
standing crop were all reduced with increasing sources, and this fungus was frequently the
copper concentration in soils proximal to a dominant microorganism present (Bewley and
brass mill. Nordgren et al. (1983, 1985) also Campbell 1980; Mowll and Gadd 1985).
showed that fungal biomass and soil respiration In conclusion, elevated concentrations of
decreased by ~75 % along an increasing con- toxic metals can affect both the qualitative and
centration gradient of metal pollution. A rela- quantitative compositions of fungal popula-
tive decrease in an indicator fatty acid for tions although it is often extremely difficult to
arbuscular mycorrhizal fungi and an increase separate their effects from those of other envi-
for other fungi have been reported for zinc- ronmental pollutants. It is apparent that certain
polluted soil (Kelly et al. 1999). Toxic metals fungi can exhibit considerable tolerance
(Cd, Cr, Cu, Ni, Pb and Zn) led to a decrease in towards toxic metals and can become dominant
the number of arbuscular mycorrhizal fungi microorganisms in some polluted habitats.
and low colonisation of plant roots and, as a However, while species diversity may be
result, changes in mycorrhizal species diversity reduced in certain cases, resistance/tolerance
(Del Val et al. 1999; Mozafar et al. 2002; Moy- can be exhibited by fungi from both polluted
nahan et al. 2002). Toxic metals also reduce and nonpolluted habitats. Physicochemical
plant root colonisation by ectomycorrhizal properties of the environment, including
fungi and ectomycorrhizal species composition changes associated with the metal pollution,
(Hartley et al. 1999; Markkola et al. 2002). The may also influence metal toxicity and thereby
most frequent soil saprotrophic microfungi affect species composition (Gadd 1984, 1992,
isolated from heavily metal-polluted habitats 1993a; Baath 1989).
in Argentina, the Czech Republic and Ukraine
were reported to be species of Penicillium,
Aspergillus, Trichoderma, Fusarium, Rhizopus B. Morphological and Growth Responses
and Mucor, as well as Paecilomyces lilacinus, to Toxic Metals
Nectria invertum, Cladosporium cladospor-
ioides, Alternaria alternata and Phoma fimeti Effects of toxic metals on fungal growth have
(Kubatova et al. 2002; Massaccesi et al. 2002; shown intra- and interspecific variability and
Fomina, Manichev, Kadoshnikov and Nako- dependence on the metal species present (Gadd
nechnaya, unpublished). Melanised fungi, 1993a; Plaza et al. 1998). For T. virens and
such as Cladosporium sp., Alternaria alternata Clonostachys rosea colonising spatially discrete
and Aureobasidium pullulans, were often toxic metal-containing domains, colonisation
isolated from soil containing high concentra- distance, hyphal extension rates and the effi-
tions of copper and mercury (Zhdanova et al. cacy of carbon substrate utilisation decreased
1986) and can be dominant members of the with increasing concentrations of copper and
mycoflora of metal-contaminated phylloplanes cadmium (Fomina et al. 2003). A decrease in
(Mowll and Gadd 1985). Dark septate endo- metal toxicity may be correlated with an
phytes were found to be dominant fungi increase in available carbon source (Ramsay
among isolates from roots of Erica herbacea L. et al. 1999; Fomina et al. 2003).
in Pb-, Cd-, and Zn-polluted soil (Cevnik et al. Several toxic metals can induce or acceler-
2000). ate melanin production in fungi, leading to
Metal pollution of plant surfaces is wide- blackening of colonies and chlamydospore
spread, but many filamentous and polymorphic development (Gadd and Griffiths 1980). Chla-
fungi appear to be little affected (Smith 1977; mydospores and other melanised forms have
Bewley 1979, 1980; Bewley and Campbell 1980; high biosorption capacities for metals, the
Mowll and Gadd 1985). On polluted oak leaves, majority of metal remaining within the wall
Fungi and Industrial Pollutants 107
(Gadd 1984, 2009; Gadd and Mowll 1985; Gadd ration between the conidia and the substrate
et al. 1987; Gadd and de Rome 1988). In rhizo- than in non-synnematal colonies, and this may
morphs of an Armillaria sp., the highest con- aid dispersal as well as ensuring conidia forma-
centrations of metals were located on the tion away from the substrate toxicants (Newby
melanised outer surface (Rizzo et al. 1992). and Gadd 1987).
Fungal morphology can be altered by toxic
metals, and changes in mycelial density and
morphology can occur (Darlington and Rauser C. Mycorrhizal Responses Towards
1988; Lilly et al. 1992; Jones and Muehlchen Toxic Metals
1994; Gabriel et al. 1996; Baldrian and Gabriel
1997; Gardea-Torresdey et al. 1997; Ramsay Mycorrhizal fungi are involved in phosphate
et al. 1999; Fomina et al. 2000, 2005b). Biomass solubilisation, proton-promoted and ligand-
distribution within Trichoderma viride colonies promoted metal mobilisation from mineral
was altered by toxic metals, with biomass con- sources, metal immobilisation within biomass
centrated in the periphery of the colonies in the and extracellular precipitation of mycogenic
presence of Cu and towards the interior of the metal oxalates (Fomina et al. 2004, 2005a; Fin-
colonies in the presence of Cd (Ramsay et al. lay et al. 2009; Gadd 2007, 2010, 2011). Plant
1999; Gadd et al. 2001). symbiotic mycorrhizal fungi can therefore
During growth of fungi in metal-containing accumulate metals from soil components, and
agar tiles simulating a spatially heterogeneous this may have consequences for metal nutrition
distribution of metal concentrations and avail- of the symbiosis as well as increased or
able nutritional resources, a range of morpho- decreased toxicity (Brown and Wilkins 1985a,
logical changes and growth responses occurred b; Jones and Hutchinson 1986, 1988a, b). Since
including negative chemotropism, cessation of plants growing on metalliferous soils are gener-
growth, swelling and lysis of hyphal tips ally mycorrhizal, an important ecological role
(Fomina et al. 2003). Penetration of hyphae for the fungus has frequently been postulated
into metal-containing domains was often fol- although such a role, e.g. phytoprotection, is
lowed by the formation of very dense mycelia often difficult to confirm (Meharg and Cairney
or mycelial ‘bushes’ (Fomina et al. 2003). Such 2000a, b). Ericaceous plants appear to be
hyphal aggregation could facilitate substrate entirely dependent on the presence of ericoid
colonisation and the production of high local mycorrhizas for protection against copper, the
concentrations of extracellular metabolites such fungus preventing metal translocation to plant
as complexing agents (e.g. organic acids, side- shoots (Bradley et al. 1981, 1982). Arbuscular
rophores, polyphenolic compounds), metal mycorrhizas (AMs) from metal-contaminated
precipitating agents (e.g. oxalate) and polysac- sites are often more metal tolerant to, for exam-
charides and pigments with metal-binding abil- ple, Cd and Zn, than other isolates, suggesting a
ities (Gadd 1993a; Dutton and Evans 1996; benefit to the plant via increased metal resis-
Baldrian 2003). Under poor nutritional condi- tance, nutrient uptake, etc., though in some
tions, fungi often produced long sparsely instances, AM plants do not necessarily require
branched or branchless hyphae in toxic metal- fungal colonisation for survival (Griffioen
containing domains representing an explorative 1994). It is often postulated that mycorrhizas
growth strategy (Fomina et al. 2003). Some provide a barrier to the uptake of potentially
fungi also exhibited multiple repeated ‘phase toxic metals (Bradley et al. 1981, 1982; Wilkins
shifts’ with a mixture of mycelial ‘bushes’ and 1991; Hetrick et al. 1994; Wilkinson and Dick-
long branchless explorative hyphae (Fomina inson 1995; Leyval et al. 1997; Meharg and
et al. 2003). Further, microfungi-penetrating Cairney 2000a, b) though this has not been
metal-contaminated domains may form myce- confirmed in every case. Further, in some
lial cords and synnema which may be atypical instances, AM may mediate enhanced accumu-
for these fungi under normal conditions. The lation of essential metals, which, unless regu-
production of synnema results in a wider sepa- lated, may lead to phytotoxicity (Killham and
108 G.M. Gadd
Firestone 1983). It is generally concluded that changes in metal speciation and mobility are
local conditions in metal-contaminated sites important survival determinants as well as
may determine the nature of the relationship components of biogeochemical cycles for
between the plant and the AM fungus, since metals and many other elements including car-
detrimental, neutral or beneficial interactions bon, nitrogen, sulphur and phosphorus (Gadd
have all been documented (Meharg and Cairney 1999, 2004b, 2007, 2008c).
2000a, b). For ericaceous mycorrhizas, clear Metals and their compounds interact with
host protection is observed for host plants, fungi in various ways depending on the metal
e.g. Calluna sp., Erica sp. and Vaccinium sp., species, organism and environment, while fun-
growing on polluted and/or naturally metallif- gal metabolism also influences metal speciation
erous soils (Bradley et al. 1981, 1982). Further, and mobility. Many metals are essential, e.g.
ericaceous plants are generally found on Na, K, Cu, Zn, Co, Ca, Mg, Mn and Fe, but all
nutrient-deficient soils, and it is likely the can exert toxicity when present above certain
mycorrhiza additionally benefits the plants by threshold concentrations (Gadd 1993a, b).
enhanced nutrient uptake (Smith and Read Other metals, e.g. Cs, Al, Cd, Hg and Pb, have
1997). A protective metal-binding effect of ecto- no known biological function, but all can be
mycorrhizal fungi (EcM) has been postulated accumulated by fungi (Gadd 1993b, 2001a, b).
frequently (Denny and Wilkins 1987; Leyval Metal toxicity is greatly affected by environ-
et al. 1997; Dixon and Buschena 1988; Colpaert mental conditions and the chemical behaviour
and Van Assche 1987, 1993). During growth, of the particular metal species in question.
mycorrhizal fungi often excrete low-molecu- Despite apparent toxicity, many fungi survive,
lar-weight carboxylic acids and siderophores grow and flourish in apparently metal-polluted
(Martino et al. 2003; Fomina et al. 2004). Eri- locations, and a variety of mechanisms, both
coid mycorrhizal and ectomycorrhizal fungi active and incidental, contribute to tolerance.
can dissolve a variety of cadmium, copper, Fungi have many properties which influence
zinc and lead-bearing minerals including metal toxicity including the production of
metal phosphates (Leyval and Joner 2001; Mar- metal-binding peptides, organic and inorganic
tino et al. 2003; Fomina et al. 2004, 2005b). precipitation, active transport and intracellular
compartmentalisation, while fungal cell walls
have significant metal-binding abilities (Gadd
D. Metal and Metalloid Transformations and Griffiths 1978; Gadd 1993b, 2007; Fomina
by Fungi and Gadd 2002). All the mechanisms by which
fungi (and other microorganisms) effect
Fungi can transform metals, metalloids (ele- changes in metal speciation and mobility are
ments with properties intermediate between survival determinants but also components of
those of metals and non-metals: the group biogeochemical cycles for metals and many
includes arsenic, selenium and tellurium) and other associated elements including carbon,
organometallic compounds by reduction, nitrogen, sulphur and phosphorus (Gadd
methylation and dealkylation (Gadd 1993b, 2004a, b, 2006, 2007, 2008a; Gadd et al. 2005,
2007). These are all processes of environmental 2007). These may be simply considered in
importance since the transformation of a metal terms of metal mobilisation or immobilisation
or metalloid may modify its mobility and toxic- mechanisms.
ity. For example, methylated selenium deriva-
tives are volatile and less toxic than inorganic
forms, while the reduction of metalloid oxya- 1. Metal Mobilisation
nions, such as selenite or tellurite to amorphous
elemental selenium or tellurium, respectively, Metal mobilisation from solids, e.g. rocks,
results in immobilisation and detoxification minerals, soil, ash, mine spoil and other sub-
(Thompson-Eagle and Frankenberger 1992; strates, can be achieved by chelation by
Morley et al. 1996). The mechanisms by which excreted metabolites and siderophores and
fungi (and other microorganisms) effect methylation which can result in volatilisation.
Fungi and Industrial Pollutants 109
moolooite (Cu(C2O4)·0.4H2O) (Fomina et al. applied to other metals and insoluble biomin-
2005a, 2007b) and lindbergite (MnC2O4·2H2O) erals for bioremediation or biorecovery of valu-
(Wei et al. 2012). A similar mechanism occurs able elements (Rhee et al. 2014a, b). Another
in lichens growing on copper–sulphide-bearing research has demonstrated that fungi can solu-
rocks, where precipitation of copper oxalate bilise uranium oxides and depleted uranium
occurs within the thallus (Purvis 1996). Oxalate and reprecipitate secondary uranium phos-
production by Aspergillus niger and Serpula phate minerals of the meta-autunite group,
himantioides has been shown to induce the uramphite and/or chernikovite, which can
dissolution and conversion of both rhodochro- encrust fungal hyphae to high accumulation
site and Mn oxides to Mn oxalate minerals values of 300–400 mg U g dry wt 1 (Fomina
(Sayer et al. 1997; Wei et al. 2012). Oxalate can et al. 2007c, 2008). Such minerals appear
act as a reductant of Mn(IV) oxides, and this can capable of long-term U retention (Fomina
result in mobilisation of Mn(II), which can then et al. 2008). The phosphate may arise from inor-
precipitate. Both A. niger and S. himantioides ganic sources in the environment or from
were capable of solubilising the insoluble man- phosphatase-mediated hydrolysis of organic P
ganese oxides MnO2 and Mn2O3, mycogenic sources, with the hyphal matrix serving to local-
manganese oxide (MnOx) and birnessite ise the resultant uranium minerals (Liang et al.
[(Na0.3Ca0.1K0.1)(Mn4+,Mn3+)2O4·1.5H2O]. Pre- 2015).
cipitation of insoluble manganese oxalate Many fungi precipitate reduced forms of
occurred and manganese oxalate trihydrate metals and metalloids in and around fungal
was detected after growth of S. himantioides hyphae, e.g. Ag(I) can be reduced to elemental
with birnessite which subsequently was trans- silver Ag(0), selenate [Se(VI)] and selenite [Se
formed to manganese oxalate dihydrate (Wei (IV)] to elemental selenium and tellurite [Te
et al. 2012). Several free-living and mycorrhizal (IV)] to elemental tellurium [Te(0)] (Gharieb
fungi can attack and transform pyromorphite et al. 1995, 1999).
(Pb5(PO4)3Cl) to lead oxalate (Sayer et al. 1999;
Fomina et al. 2004, 2005b). It has also been
shown that certain fungi (e.g. Paecilomyces 3. Organometal(loid)s
javanicus, Metarhizium anisopliae) were able
to mediate transformation of metallic lead into Organometals (compounds with at least one
pyromorphite, representing biomineralisation metal–carbon bond) can be attacked by fungi
of mobile lead species into a very stable form with the organic moieties being degraded and
(Rhee et al. 2012, 2014a, b). This might be an the metal compound undergoing changes in
important process occurring in lead-containing speciation. Degradation of organometallic com-
environments and of relevance to proposed pounds can be carried out by fungi, either by
bioremedial treatments (Rhee et al. 2012). It is direct biotic action (enzymes) or by facilitating
likely that acidolysis and complexation involv- abiotic degradation, for instance, by alteration
ing excreted organic acids play an important of pH and excretion of metabolites. Organotins,
role in mediating precipitation of pyromorphite such as tributyltin oxide and tributyltin
(Rhee et al. 2012, 2014a, b) and other metal naphthenate, may be degraded to mono- and
phosphates (Fomina et al. 2007c, 2008). Fungal dibutyltins by fungal action, inorganic Sn(II)
activity can also play an important role in the being the final degradation product. Organo-
biocorrosion and transformation of lead metal mercury compounds may be detoxified by con-
into pyromorphite in the aquatic environment. version to Hg(II) by fungal organomercury
The ability of fungi to immobilise mobile lead lyase, the Hg(II) being subsequently reduced
species in an insoluble form provides a further to Hg(0) by mercuric reductase, a system anal-
approach for the removal and detoxification of ogous to that found in mercury-resistant bacte-
lead from aqueous solution by bioprecipitation. ria (Gadd 1993b).
The principles of such a process could also be
Fungi and Industrial Pollutants 111
E. Accumulation of Metals and Radionuclides et al. 1987; Byrne 1988; Dighton and Horrill
by Macrofungi 1988; Haselwandter et al. 1988; Clint et al.
1991; Dighton et al. 1991; Muramatsu et al.
Elevated concentrations of toxic metals and 1991; Heinrich 1992); these organisms appear
radionuclides can occur in the fruiting bodies to have a slow turnover rate for Cs and com-
of higher fungi sampled from polluted environ- prise a major pool of radiocaesium in soil (Clint
ments. This phenomenon is of significance in et al. 1991). Mean activities of 25 Ukrainian, 6
relation to the use of macrofungi as bioindica- Swedish and 10 North American collections
tors of metal pollution and because of human were 4660, 9750 and 205 Bq (kg dry wt) 1,
toxicity resulting from the consumption of wild respectively (Smith et al. 1993). Deviations in
fungi. In general, levels of Pb, Cd, Zn and Hg the 137Cs:134Cs ratios attributable to Chernobyl
found in macrofungi from urban or industrial have revealed considerable accumulation of
areas are higher than from corresponding rural pre-Chernobyl Cs in macrofungi, probably as
areas, although there are wide differences in the result of weapon testing (Byrne 1988;
uptake abilities between different species and Dighton and Horrill 1988). It appeared that
different metals (Tyler 1980; Bressa et al. 1988; about 20 % of the 137Cs in Eastern Europe
Lepsova and Mejstrik 1989). Cadmium is accu- (Moscow area, Belarus, Ukraine) was of non-
mulated to quite high levels in macrofungi, Chernobyl origin (Smith et al. 1993). Radiocae-
averaging around 5 mg (kg dry wt) 1 although sium accumulation in basidiomycetes appears
levels of up to 40 mg (kg dry wt) 1 have also to be species dependent, with influences
been recorded (Byrne et al. 1976). Laccaria exerted by soil properties. Significantly higher
amethystina caps exhibited total As concentra- activities may be found in mycorrhizal species
tions of 100–200 mg (kg dry wt) 1 (Stijve and compared to saprotrophic and parasitic fungi
Porette 1990; Byrne et al. 1991). Accumulation (Smith et al. 1993). Smith et al. (1993) found
of 110Ag and 203Hg was studied in Agaricus that many prized edible mycorrhizal fungi may
bisporus, and concentration factors (metal con- contain unacceptably high levels of 137Cs, that
centration in mushroom, metal concentration is, at levels of greater than 1000 Bq (kg dry
in substrate) were found to be up to 40 and 3.7, wt) 1. It has also been demonstrated that the
respectively, with the highest Ag and Hg con- fungal component of soil can immobilise the
tents recorded being 167 and 75 mg (kg dry total Chernobyl radiocaesium fallout received
wt) 1, respectively (Byrne and Tusek-Znidaric in upland grasslands (Dighton et al. 1991)
1990). As well as fruiting bodies, rhizomorphs although grazing of fruiting bodies by animals
(e.g. of Armillaria species) can concentrate may lead to radiocaesium transfer along the
metals up to 100 times the level found in soil. food chain (Baaken and Olson 1990).
Concentrations of Al, Zn, Cu and Pb in rhizo-
morphs were 3440, 1930, 15 and 680 mg (kg dry
wt) 1, respectively, with the metals primarily G. Fungi as Bioindicators of Metal and
located in extracellular portions (Rizzo et al. Radionuclide Contamination
1992).
As mentioned above, higher fungi growing on
contaminated sites can show significantly ele-
F. Accumulation of Radiocaesium vated concentrations of metals in their fruiting
by Macrofungi bodies, and some experiments have demon-
strated a correlation between the quantities of
Following the Chernobyl accident in 1986, there metals in a growth substrate and the amounts
were several studies on radiocaesium (mainly subsequently found in the fruiting bodies
137
Cs) accumulation by fungi. Free-living and (Wondratschek and Roder 1993). The concept
mycorrhizal basidiomycetes can accumulate of bioindicators has been usually discussed in
radiocaesium (Haselwandter 1978; Elstener terms of reaction indicators and accumulation
112 G.M. Gadd
indicators. Reaction indicators may comprise Table 5.1 Higher fungi proposed as bioindicators for
individual organisms and/or communities metal pollution based on metal analyses of fruiting
bodies (see Mejstrik and Lepsova 1993; Wondratschek
which may decline or disappear (sensitive spe- and Roder 1993)
cies) or show increases (tolerant species). For
accumulation indicators, the indicator organ- Species Metal(s)
ism is analysed for the pollutant. Some organ- Agaricus arvensis Hg, Cd
isms, in theory, can therefore serve as both Agaricus campestris Hg, Cd
reaction and accumulation indicators. As Agaricus edulis Hg, Cd
described previously, alteration of macrofungal Agaricus haemorrhoidarius Hg
communities by metal pollution has frequently Agaricus xanthodermus Hg
Agaricus sp. Pb, Zn, Cu
been recorded. Ruhling et al. (1984) noted a Amanita rubescens Hg
decline from about 40 species per 100 m2 to Amanita strobiliformis Hg
about 15 species near the source of metal con- Coprinus comatus Hg
tamination (smelter emissions), with only Lac- Lycoperdon perlatum Hg
caria laccata increasing in frequency at more Lycoperdon sp. Pb, Zn, Cu
Marasmius oreades Hg
polluted locations. Other higher fungi which Mycena pura Hg, Cd
are apparently tolerant of high metal pollution
include Amanita muscaria and several species
of Boletus; some Russula species, on the other
hand, appear metal sensitive (Wondratschek A wide variation in Cd content has also
and Roder 1993). been recorded in macrofungi with ranges of
Fungi possess several advantages over reported values from <0.1–229 mg (kg dry
plants as metal accumulation indicators. The wt) 1 (Tyler 1980). However, there is frequently
fruiting bodies may accumulate greater a lack of correlation between the fungal Cd
amounts of metals than plants, while the large content and the Cd content of the soil (Won-
area of mycelium ensures contact with and dratschek and Roder 1993). Compared to other
translocation from a large area of soil. Further- common metal pollutants, lower concentra-
more, fruiting bodies may project above the tions of Pb tend to be found in macrofungi,
ground for only a short period, thereby mini- with much of the Pb content being derived
mising contamination from aerial or wet depo- from aerial sources. Levels of Pb around 0.4–
sition of metal pollutants. Sporophores are also 36 mg (kg dry wt) 1 have been reported in
easily harvested and amenable to rapid chemi- sporophores, with higher levels occurring in
cal analysis (Mejstrik and Lepsova 1993). How- urban areas (Tyler 1980). Zinc, an essential
ever, it is debatable whether a sufficiently clear metal for fungal growth and metabolism,
relationship exists between indicator species occurs at high concentrations within fungi,
and the metal pollution under consideration. 50–300 mg (kg dry wt) 1 (Tyler 1980), with a
For mercury, wide variations in metal content few genera apparently showing high affinities
of fruiting bodies occur in different species for the metal (Table 5.1). Copper may also be
sampled at the same site, ranging over as found at high levels (20–450 mg (kg dry wt) 1)
much as three orders of magnitude, with some in higher fungi (Tyler 1980). However, with
species showing extremely high Hg accumula- both Cu and Zn, there is a tendency for metal
tion values. Mercury concentrations in fungi concentrations in fruiting bodies to be inde-
generally occur in the range 0.03–21.6 mg (kg pendent of soil concentrations which reduces
dry wt) 1 although concentrations greater than their value as bioindicators (Gast et al. 1988).
100 mg (kg dry wt) 1 have been recorded from It is clear that many factors contribute to
polluted sites. Despite this, several macrofungi the wide variations in recorded metal contents
have been suggested as being suitable bioindi- of macrofungal fruiting bodies, even in the
cators of mercury pollution (see Mejstrik and same species sampled at the same site. Despite
Lepsova 1993; Wondratschek and Roder 1993) numerous studies, most investigations tend to
(Table 5.1). be contradictory and provide little useful infor-
Fungi and Industrial Pollutants 113
mation (Wondratschek and Roder 1993). Apart not compare with the established bacterial bio-
from organism-related factors, environmental leaching processes and may be more suited to
factors are of paramount importance in relation bioreactor applications. Metals can be solubi-
to metal accumulation by higher fungi and lised from fly ash (originating from municipal
include physicochemical soil properties like solid waste incineration), contaminated soil,
moisture and temperature, all of which influ- electronic scrap and other waste materials by
ence metal availability as well as the physiolog- fungal activity (Brandl 2001; Brandl and Fara-
ical activity of the fungus. It can be concluded, marzi 2006).
therefore, that a perfect fungal bioindicator
does not exist, although macrofungi may be
useful in determining the extent of a polluted 2. Biosorption and Bioaccumulation
or unpolluted area.
Biosorption is a physicochemical process,
simply defined as ‘the removal of substances
H. Bioremediation, Biotechnology and from solution by biological material’. It is a
Bioprocessing property of both living and dead organisms
(and their components) and has been proposed
Several fungal metal and mineral transforma- as a promising biotechnology for the removal
tions have potential for the treatment of envi- (and/or recovery) of metals, radionuclides
ronmental pollution (Gadd 2004a, 2005; and organic pollutants for many years because
Pumpel and Paknikar 2001). While several of its simplicity, analogous operation to con-
fungal-based systems have received interest in ventional ion exchange technology and appar-
the context of bioremediation of organic pollu- ent efficiency (Gadd 1986, 2001a, b, 2009;
tants, there has not been so much attention Volesky 1990; Garnham et al. 1992; Gadd and
given to metals. However, it should be stressed White 1990, 1993; Wang and Chen 2006, 2009).
that fungi will be components of the microbiota Modification of biomass has been attempted to
in any metal-polluted sites where their activities improve efficiency or selectivity of microbial
may contribute to natural attenuation of the biosorbents. Fungal–clay biomineral sorbents
pollutants and will also be involved in many combined the sorptive advantages of the
soil and waste treatment processes, revegeta- individual counterparts, i.e. the high density
tion strategies and effluent treatments. Fungi of metal-binding sites per unit area and
were clearly important in remediation of high sorption capacity of fungal biomass, high
selenium-contaminated soils (Thompson- sorption affinity and the high surface area per
Eagle and Frankenberger 1992). In addition, unit weight mechanical strength and efficient
fungal mineral-solubilising properties are sorption at high metal concentrations of the
important in plant nutrition and soil fertility clay minerals (Fomina and Gadd 2002). S. cer-
especially regarding phosphates. In addition evisiae mutants (pmr1D) hypersensitive to
to bioremediation, metal and mineral transfor- heavy metals due to increased metal uptake
mations have applications in other areas of have been investigated for the ability to remove
biotechnology and bioprocessing, including Mn2+, Cu2+, Co2+ or Cd2+ from synthetic
biosensors, biocatalysis, electricity generation effluents by a combination of biosorption
and nanotechnology. and intracellular uptake (Ruta et al. 2010).
Phytochelatins (PCs) are metal-binding
1. Bioleaching cysteine-rich peptides, enzymatically synthe-
sised in plants and certain fungi from glutathi-
Fungal solubilisation of metals from solid one in response to heavy metal stress.
minerals and metal and mineral wastes, includ- Overexpression of PC synthase in bacteria
ing contaminated soil, for metal recovery, recy- could be a means of improving the metal con-
cling and bioremediation purposes have all tent of organisms for bioremediation (Valls
been investigated, although fungal systems can- et al. 2000).
114 G.M. Gadd
plants by enhanced nutrient uptake (Smith and (Hennebel et al. 2009). The use of metal-
Read 1997). The development of stress-tolerant accumulating microbes for the production of
plant–mycorrhizal associations may therefore nanoparticles, and their assembly, may allow
be a promising strategy for phytoremediation control over size, morphology, composition
and soil amelioration (Schutzendubel and Polle and crystallographic orientation. The produc-
2002). Ericoid mycorrhizal fungal endophytes, tion of such biomimetic materials is relevant to
and sometimes their plant hosts, can evolve the production of new advanced materials, with
toxic metal resistance which enables ericoid applications in metal and radionuclide bioreme-
mycorrhizal plants to colonise polluted soil diation, antimicrobial treatments (e.g. nano-
(Perotto et al. 2002; Martino et al. 2003). This silver), solar energy and electrical battery appli-
seems to be a major factor in the success of cations and microelectronics (Dameron et al.
ericoid mycorrhizal taxa in a range of harsh 1989; Klaus-Joerger et al. 2001). Because of their
environments (Cairney and Meharg 2003). high specific surface area and high catalytic
The importance of mycorrhizas in plant properties, biogenic metal products also offer
phosphorus nutrition has been appreciated for potential for sorption and degradation of organic
a long time, and their ability to dissolve and contaminants, as well as a variety of other appli-
transform calcium-containing insoluble com- cations, e.g. electricity generation in fuel cells,
pounds and minerals (calcium phosphates, car- novel catalysts and sensors. Biogenic Mn oxides
bonate and sulphate) has been widely studied can sequester metals like Pb, Zn, Co, Ni, As and
(Callot et al. 1985a, b; Lapeyrie et al. 1990, 1991; Cr and also oxidise certain organic pollutants
Gharieb and Gadd 1999). However, toxic metal (Hennebel et al. 2009). In contrast to bacteria,
mineral solubilisation has received little atten- rather less attention has been given to fungal
tion, though this should be considered in any systems in this context although fungal reductive
revegetation, natural attenuation or phytore- transformations of metalloids and Ag and Au
mediation strategies. The ectomycorrhizal species to nano- or colloidal forms are well
fungi Suillus granulatus and Pisolithus tinctor- known, as well as metal-containing reactive crys-
ius can promote the release of cadmium and tallites (Dameron et al. 1989) and Mn oxides
phosphorus from rock phosphate (Leyval and (Miyata et al. 2004, 2007).
Joner 2001), while the ericoid mycorrhizal fun-
gus Oidiodendron maius can solubilise zinc
oxide and phosphate (Martino et al. 2003). 6. Soil Treatment Processes
Many ericoid mycorrhizal and ectomycorrhizal
fungi are able to solubilise zinc, cadmium, The application to soils of certain amendments
copper phosphates and lead chlorophosphate that immobilise metals, e.g., lime or phosphate
(pyromorphite) releasing phosphate and metals treatment, has demonstrated enhanced natural
(Fomina et al. 2004). Both non-mycorrhizal remediation resulting in improved vegetation
Pinus sylvestris and pines infected with the growth, increased microbial activity and diver-
ectomycorrhizal Paxillus involutus could sity and reduced off-site metal transport. How-
enhance zinc phosphate dissolution, withstand ever, while long-term stability of certain metal
metal toxicity and acquire the mobilised phos- complexes and compounds has been shown in
phorus (Fomina et al. 2006). model systems (Adriano et al. 2004), the influ-
ence of plant roots and its microbial and
mycorrhizal associations on such stability has
5. Nanoparticle Formation and often been neglected. For example, pyromor-
Nanobiotechnology phite (Pb5(PO4)3Cl), which can form in urban
and industrially contaminated soils, can be
Metal-containing micro-/nanoparticles have solubilised by phosphate-solubilising fungi,
applications as new ceramic–metal (cermet) with concomitant production of lead oxalate
or organic–metal (orgmet) composites or (Sayer et al. 1999; Fomina et al. 2004). The
structured materials for a variety of applications ability of free-living and mycorrhizal fungi to
116 G.M. Gadd
transform pyromorphite (and other toxic clear that because of the complexity of the fun-
metal-containing minerals) should be taken gal growth form and their multiplicity of
into account in risk assessments of the long- biological responses and interactions with pol-
term environmental consequences of in situ lutants, coupled with the complexity of the
chemical remediation techniques, revegetation terrestrial (and other) environments, a wealth
strategies or natural attenuation of contami- of knowledge still awaits discovery.
nated sites. The bioweathering potential of
fungi has been envisaged as a possible means Acknowledgements The author gratefully acknowl-
edges research support from the Biotechnology and
for the bioremediation of asbestos-rich soils. Biological Sciences Research Council, the Natural Envi-
Several fungi could extract iron from asbestos ronment Research Council and the British Nuclear
mineral fibres (e.g. 7.3 % from crocidolite and Fuels plc. G. M. Gadd also gratefully acknowledges an
33.6 % from chrysotile by a Verticillium sp.), award under the 1000 Talents Plan with the Xinjiang
thereby removing the reactive iron ions respon- Institute of Ecology and Geography, Chinese Academy
of Sciences, Urumqi, China.
sible for DNA damage (Daghino et al. 2006).
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6 Degradation of Plant Cell Wall Polymers by Fungi
selection programs have resulted in industrial strains of active proteins is mycoCLAP (https://mycoclap.
Aspergillus niger and T. reesei which can yield more fungalgenomics.ca/mycoCLAP/ Murphy et al.
than 100 g/l of amylases or cellulases (Durand et al.
1988; Berka et al. 1991; Cherry and Fidantsef 2003),
2011). Biochemical properties and functional
therefore making them also versatile production hosts annotations described in mycoCLAP are manu-
for other enzymes and recombinant proteins. Another ally curated, and the database covers fungal
advantage of these fungi is that they are easy and GHs, CEs, and PLs and enzymes with auxiliary
inexpensive to grow in large bioreactors and are suit- activities.
able for genetic recombination technologies. Indeed, it
was not surprising that these biotechnological applied
fungi were among the first filamentous fungi for which
a genome sequence became available (Martinez et al. II. Structure and Composition
2008; Pel et al. 2007). The recent advances in the of Plant Cell Walls
sequencing and annotation of fungal genomes give an
impression of the hidden enzymatic potential in these
organisms, and transcript and proteome analysis has Plant cells are enclosed by cell walls which
accelerated our current understanding of the different provide rigidity to the cell for structural and
genes expressed on various natural substrates. How- mechanical support, maintain and determine
ever, despite the advanced technologies used today,
the inability to efficiently convert the crystalline and cell shape, counterbalance osmotic pressure,
insoluble cellulose to fermentable sugars still poses a direct growth, and, ultimately, determine the
major barrier for the commercialization of biofuel pro- architecture and form of the plant. In addition,
duction (Himmel and Bayer 2009). the plant cell wall protects against environmen-
tal factors or pathogens. The plant cell walls
This chapter aims to summarize our current consist mainly of polysaccharides such as
knowledge on plant cell wall degradation, giving cellulose, hemicelluloses, pectins, and the aro-
the reader an overview about the structures of matic polymer lignin. Together, they form a
the substrates and the enzymatic strategies for complex and rigid structure termed lignocellu-
their degradation. As an example for the com- lose.
plex deconstruction of lignocellulose, the bio-
polymer degradation strategy of brown- and Typically, three regions are distinguished in plant cell
white-rot fungi is highlighted in more detail. A walls, which are the middle lamella, the primary, and
complete listing of all genes and their encoded the secondary cell wall. The middle lamella is the out-
enzymes involved in the breakdown of plant cell ermost layer, composed primarily of pectin and its
function is to cement the cell walls of adjacent cells
walls is beyond the scope of this chapter. Today, together. Primary walls are synthesized during growth,
a number of web-based databases are available whereas secondary walls are thickened structures con-
which accomplish these tasks. One example is taining lignin and surrounding specialized cells such as
the Carbohydrate-Active Enzymes Database vessel elements or fiber cells. All differentiated cells
(www.CAZy.org) which is a manually curated contain walls with distinct compositions, resulting in
a spectrum of specialized cell walls with primary and
list of enzyme classes and families known to act secondary walls as two extremes (Keegstra 2010). Pri-
on carbohydrates (Cantarel et al. 2009; Levas- mary walls are comprised of 15–40 % cellulose, 30–50 %
seur et al. 2013; Lombard et al. 2014). As a pectins, and 20–30 % xyloglucans and minor amounts
complement to CAZy, the CAZypedia (www. of arabinoxylans and proteins. The primary cell wall is
cazypedia.org) describes the different enzyme expanded inside the middle lamella and consists of
several interconnected matrices. In these matrices, cel-
classes and families in more detail. Initially lulose microfibrils are aligned at all angles and cross-
focusing on the glycoside hydrolase (GH) families, linked via hemicellulosic tethers to form the cellulose–
other CAZyme classes were introduced to the hemicellulose network (e.g., with xyloglucan and galac-
CAZy database within the last years, including toglucomannan). This network is embedded in the
polysaccharide lyases (PL), glycosyltransferases, gelatinous pectin matrix composed of homogalacturo-
nan, rhamnogalacturonan I, and rhamnogalacturonan
carbohydrate esterases (CE), as well as auxiliary II (O’Neill and York 2003). The pectins are particularly
redox enzymes and non-catalytic carbohydrate- important for wall hydration. Two main types of pri-
binding modules (CBMs). A database specialized mary cell walls are distinguished (Carpita and Gibeaut
on the biochemical properties of lignocellulose- 1993): type I is found in all dicotyledons, non-
graminaceous monocotyledons, and gymnosperms
Degradation of Plant Cell Wall Polymers by Fungi 129
and typically contains xyloglucan and/or glucomannan tent soil humus. The fungi found in these
and 20–35 % pectin. Type II is found in grasses, where decomposition sequences live in complex and
arabinoxylans constitute most of the matrix, and
mixed-linkage glucans transiently comprise 10–20 %
diverse communities and are often specialized
of wall mass (Carpita et al. 2001; Gibeaut et al. 2005). to degrade only certain types of polymers,
An exception are celery and sugar beet parenchyma reflecting their genomic capabilities. The effi-
which are rich in cellulose and pectin, but have little cient breakdown of the plant cell wall by fungi
hemicelluloses (Thimm et al. 2002; Zykwinska et al. is linked, on the one hand, to their hyphal
2007).
In addition to primary walls, many plants synthe-
growth, which provides penetrating power,
size a secondary wall, which is assembled between the and, on the other hand, to their highly
plant cell and the primary wall. These secondary walls specialized extracellular plant cell wall-
are found in plant tissues that have ceased growing. degrading enzyme systems. The enzymatic
They provide strength and rigidity and are comprised decomposition of plant cell walls is usually syn-
primarily of cellulose and lignin. In addition, the hemi-
celluloses xylan, and glucomannan replace xyloglucan
ergistic: individual, highly specialized enzymes
and pectins. Secondary cell walls are also less hydrated operate as components of multi-enzyme sys-
than primary cell walls. The cellulose microfibrils are tems to efficiently degrade the polymers. The
generally aligned in the same direction but, with each synthesis of these enzymes is governed by a
additional layer, their orientation changes slightly. The sophisticated signal transduction and gene reg-
secondary wall is altered during development by suc-
cessive encrustation and deposition of cellulose fibrils
ulation system, and a highly productive secre-
and other components. Nonstructural components of tory machinery is available for their cellular
the secondary wall represent generally less than 5 % of export. These characteristics enable fungi to
the dry weight of wood and include compounds such as successfully compete with other microorgan-
phenols, tannins, fats, sterols, proteins, and ashes. isms in their environment, and they are today
the main agents of decomposition in terrestrial
The structure of the polysaccharides found and aquatic ecosystems.
in the cell wall is highly diverse and comprises a Degradation occurs extracellularly, since
spectrum ranging from simple linear polymers the substrates are usually large polymers
composed of a single type of glycosyl residue which are insoluble or even crystalline. Two
(e.g., cellulose is composed of 1,4-linked b-glu- principal types of extracellular enzymatic sys-
cosyl residues), over polymers with a regular tems for the degradation of the polymeric frac-
branching pattern (e.g., xyloglucan and rham- tion have been developed: the hydrolytic
nogalacturonan II), to rhamnogalacturonan I, a system, which degrades the polysaccharides
polymer substituted with a diverse range of mainly by glycoside hydrolases, and a unique
arabinosyl and galactosyl-containing oligosac- oxidative ligninolytic system, which depoly-
charide side chains. Understanding the struc- merizes lignin. But even for the complete
tures of these polymers and determining their degradation of the chemically simple polysac-
primary structures remain a major challenge, charide cellulose, several enzymes are neces-
especially because their biosynthesis is not tem- sary and the number of enzymes is further
plate driven (O’Neill and York 2003). increased when the polysaccharides are substi-
tuted. Historically, three classes of enzymes can
be distinguished although recent advancements
III. Degradation of the Plant Cell Wall have revealed overlapping roles of some of the
enzymes originally classified into one of these
Due to the overall structural and chemical com- classes. These classes include (1) exo-acting
plexity of plant cell walls, a complete break- enzymes, which release mainly mono- and
down of the different components is brought dimers of the ends of the polymeric chain, (2)
about only by a wide range of organisms acting endo-acting enzymes, which cleave in the mid-
in a consortium. This degradation follows a dle of the sugar chain, and (3) enzymes (often
characteristic decomposition sequence, which exo-acting) which are specialized in cleaving
starts with the colonization of living plants the resulting oligosaccharides into their mono-
and ends with the production of highly persis- mers. The resulting low molecular break down
130 J. Ramoni and B. Seiboth
products are then readily taken up into the cell enzymes are necessary. These include cellobio-
and further degraded by a wide range of hydrolases (EC 3.2.1.91; 1,4-b-D-glucan cello-
specialized catabolic pathways. In contrast to biohydrolases), which attach to the cellulose
polysaccharides, the complex heteropolymer chains and act processively from their ends to
lignin lacks a stereochemical regularity and is generate mainly the glucose disaccharide cello-
therefore degraded by a nonspecific and non- biose. The cellobiohydrolases are classified in
stereoselective mechanism. two GH families with GH6 enzymes character-
ized by an inverting mechanism and cleavage
GHs are the main actors in polysaccharide degradation from the nonreducing cellulose end. In con-
and are represented by 135 GH families in the CAZy trast, GH7 family members cleave cellobiose
database. Additional families involved in plant cell wall from the reducing end by a retaining mecha-
degradation are found in the group of the polysaccha-
ride lyases which employ b-elimination reaction
nism. Endoglucanases (EC 3.2.1.4; 1,4-b-D-glu-
mechanisms to cleave pectins and carbohydrate can 4-glucanohydrolase) are capable of
esterases which catalyze the de-O or de-N-acylation of cleaving within the amorphous regions inside
substituted polysaccharides. To date, polysaccharide the cellulose chain, thereby generating addi-
lyases form 23 and carbohydrate esterases 16 charac- tional sites for the attack of the cellobiohydro-
terized families. “Auxiliary activities” or AAs cover
redox enzymes that are currently represented by 13
lases. b-Glucosidases (EC 3.2.1.21) degrade the
families, with the majority active in lignin degradation accumulating cellodextrines and cellobiose to
and with four families active on polysaccharides, D-glucose (Beguin 1990; Teeri 1997). Within
namely the lytic polysaccharide monooxygenases the last years, another main player in cellulose
(LPMOs). The catalytic modules or functional domains degradation was discovered with the lytic poly-
of the different enzymes are often associated with other
functions to improve their action on lignocellulose. The
saccharide monooxygenases (LPMOs). These
most prominent additional domain are carbohydrate- copper dependent oxidases originally classified
binding modules (CBMs), which are represented by 71 as fungal GH61 enzymes attack the highly
families in the CAZy database. crystalline regions of cellulose by an oxidative
cleavage. Studies on the T. reesei cellulase
CEL61A revealed that its addition to commer-
A. Cellulose Degradation cial enzyme mixes can significantly boost de-
gradation (Harris et al. 2010). These LPMOs act
Cellulose is the most abundant biopolymer on on crystalline cellulose by generating both oxi-
earth, with approximately 1010 metric tons of dized and non-oxidized chain ends. One group
CO2 and H2O getting transformed into this oxidizes the C1 of D-glucose, thereby releasing
polymer every year (Field et al. 1998). It con- lactones that are hydrolyzed to aldonic acids
sists of a linear chain of several 100 up to 12,000 (Beeson et al. 2011). Other LPMOs act on the
b-1,4-linked D-glucopyranose units. The glu- nonreducing end, producing ketoaldoses, or a
cose hydroxyl groups from one chain form combination thereof (Beeson et al. 2012).
hydrogen bonds with the oxygen atoms on the Therefore, these CEL61 enzymes were reclassi-
same, or on a neighboring chain, which leads to fied in auxiliary activity family 9 (AA9), which
the formation of cellulosic microfibrils. These exclusively contains eukaryotic LPMOs. Origi-
microfibrils provide high tensile strength in cell nally, these LPMOS were detected in the de-
walls and prevent the cell wall from stretching gradation of chitin (Hemsworth et al. 2014;
laterally. Cellulose crystallizes shortly after its Vaaje-Kolstad et al. 2010) before their action
biosynthesis, but less-ordered amorphous on cellulose or starch was discovered (Vu
regions can occur. A large number of fungi are et al. 2014; Levasseur et al. 2013; Lo Leggio
able to grow on amorphous cellulose and water- et al. 2015; Horn et al. 2012).
soluble derivates, but relatively few are able to
produce a complete enzyme system necessary By disrupting the crystalline structure of cellulose, they
to hydrolyze crystalline cellulose. For the com- facilitate the enzymatic cleavage of the classical cellu-
plete hydrolysis of cellulose (Fig. 6.1) to D-glu- lases and allow other enzymes like cellobiohydrolases
to attack the polymer at otherwise inaccessible sites
cose, a number of synergistically acting
Degradation of Plant Cell Wall Polymers by Fungi 131
Fig. 6.1 Enzymatic degradation of cellulose. Cellobio- oligomers and the dimer cellobiose are cleaved by b-
hydrolases (CBH) act on either the reducing or non- glucosidases (BGL) into D-glucose. The lytic poly-
reducing end of the glucose chain. Endoglucanases saccharide monooxygenases (LPMO) cleave within the
(EG) hydrolyze internal glycosidic bonds, thereby highly crystalline regions of cellulose in an oxidative
providing additional sites for the CBHs. Finally, smaller manner
(Horn et al. 2012). In this respect, LPMOs may be the the ability to swell or disrupt cellulose; the LPMOs
true C1 enzyme in the C1Cx model proposed by Elwyn might act like the C1 enzyme proposed by Reese.
T. Reese to explain cellulose degradation (Mandels and
Reese 1964; Reese et al. 1950). In this model, the non-
hydrolytic C1 enzyme is required for the initial attack
A structural comparison of the different
on crystalline cellulose and the Cx enzymes for the cellulases shows that these proteins comprise
hydrolysis of soluble cellulose. Later, in the 1990s, the besides the catalytic domain carbohydrate-
classical endo/exo model of cellulase degradation was binding modules. These modules (Várnai et al.
proposed (Wood and McCrae 1972) which consisted of 2014) can be found N- or C-terminally and are
EGs attacking amorphous cellulose regions of the
microfibrils, thereby generating new sites for exo-
about 40 aa in size. They were originally
glucanases. The exoglucanases attack the free cellulose described as cellulose-binding domains due to
ends. Finally, b-glucosidases convert cellodextrines and their presence in cellobiohydrolases but were
cellobiose to glucose. Here, the dominant EG act only later also found in other carbohydrate-
on amorphous cellulose and functions as the Cx activity degrading enzymes and therefore renamed
whereas the C1 is represented by the exoglucanases,
i.e., cellobiohydrolases. But Reese suggested that C1
to carbohydrate-binding modules (CBMs).
was a decrystallizing protein factor which possesses Removal of this domain leads to enzymes
which are still able to cleave glycosyl linkages
132 J. Ramoni and B. Seiboth
from smaller oligosaccharides, but their bind- cellulose fibers more accessible for the cellu-
ing to cellulose and, therefore, the action on lases to act upon (Saloheimo et al. 2002).
crystalline cellulose is impaired. CBMs are Although SWO1 has cellulose disrupting activ-
structurally similar, and their carbohydrate- ity (Andberg et al. 2015; Jäger et al. 2011), its
binding capacity can be attributed to several actual role seems to be in aiding degradation of
amino acids constituting the hydrophobic sur- hemicelluloses, as synergistic effects with endo-
face. The CBM domain is usually spatially sepa- xylanases rather than endo-cellulases or cello-
rated from the catalytic core domain by a biohydrolases have been reported (Gourlay
flexible linker region (Sammond et al. 2012). et al. 2013).
This linker region is rich in prolines, serines, Numerous genes encoding cellulases have
and threonines, and the latter two amino acids been isolated and the respective enzymes were
are highly O-glycosylated to impair proteolytic studied in great detail. They are found in
degradation (Langsford et al. 1987; Srisodsuk GH families 5, 6, 7, 12, and 45. One of the
et al. 1993). Recent studies on this connector best studied cellulolytic fungi is T. reesei,
have shown that their length and sequences are which was discovered during World War II on
optimized towards the type of GH and CBM the Solomon Islands as a severe degrader of
domain, which indicates that also the linker is cellulosic material of the US Army (Reese
of importance for the activity of the enzyme 1976) and is today the most commonly used
(Sammond et al. 2012; Payne et al. 2013). organism for commercial production of cellu-
lases (Gupta et al. 2014). Detailed analysis of the
The catalytic domain of the cellobiohydrolase II enzymes and gene regulation is available for,
(CEL6A) of T. reesei was the first cellulase crystal struc- e.g., A. niger, T. reesei, and P. chrysogenum
ture resolved at the atomic level (Rouvinen et al. 1990). (Aro et al. 2005; de Vries 2003; Louise Glass
Its structure demonstrates why the cellobiohydrolases
are only able to attack cellulose chain ends. The crystal
et al. 2013; Kubicek et al. 2009).
structure shows a tunnel-shaped active site which is so
tight that it can incorporate only one cellulose chain.
Despite a similar overall structure, endoglucanases pos-
sess a more open active site which allows the enzyme to B. Hemicellulose Degradation
attack the cellulose chains in the middle. The active site
topology of these polymer-degrading enzymes shows Hemicelluloses are the second most abundant
that these enzymes have extended active sites which polysaccharide in plant cell walls and represent
provide binding places for a number of sugar units. a heterogeneous group of polymers with a
These subsites position the substrate tightly and cor- backbone of 1,4-linked b-D-pyranosyl residues,
rectly with respect to the catalytic amino acids.
with the exception of arabinogalactan (O’Neill
and York 2003; O’Neill and Selvendran 1985).
In addition to the classical glycoside hydro- The backbone can consist of xylosyl-, glucosyl-,
lases and the new LPMOs, other types of pro- galactosyl-, arabinosyl-, or mannosyl residues
teins have been described to be involved in and, depending on the dominant sugar, they
cellulose degradation. One of them is the are, e.g., named xylans or arabinogalactans, if
T. reesei swollenin SWO1 which shows similar- both sugars occur in near-equal amounts. This
ity to plant expansins (Saloheimo et al. 2002; chemical diversity of hemicellulosic structures
Sampedro and Cosgrove 2005). Expansins requires a larger set of enzymes which attack
induce the extension of isolated cell walls by a the main chain or side chains. The main chain
non-hydrolytic activity on different cell wall is cleaved by (mainly) endo-acting enzymes
polymers, e.g., pectins and xyloglucans, which whereas exo-acting enzymes liberate the
are tightly bound to the cellulose microfibrills respective monomers. Only a few exo-acting
(McQueen-Mason and Cosgrove 1995). A pro- enzymes are known which attack the main
posed model is, that swollenins are able to dis- chain (Tenkanen et al. 2013). Accessory
rupt the cellulose microfibrils without any enzymes are activities necessary to cleave off
hydrolytic activity and thereby they make the the side chains, leading to the release of various
Degradation of Plant Cell Wall Polymers by Fungi 133
Fig. 6.2 Enzymatic degradation of hemicelluloses. The dase (GLU), feruloyl esterase (FES), acetyl xylan ester-
main chain of xylan is degraded by endo-1,4-b-xyla- ase (AXE), 1,4-galactosidases (BGA), and a-L-
nases (XYN). Accessory enzymes necessary for side arabinofuranosidase (ABF)
group removal are b-xylosidase (XYL), a-glucuroni-
mono- and disaccharides. In this way, the main sidases (1,4-b-D-xylan xylohydrolase; EC
chain also becomes more accessible for the 3.2.1.37). Exo-1,4-b-xylanase which cleaves
other group of enzymes. Most extensively stud- xylan from the ends has received less attention
ied is the enzymatic degradation of xylan, but, e.g., XYN4 from T. reesei shows both exo-
which involves exoxylanases, endoxylanases, and endo-xylanase activity (Tenkanen et al.
b-xylosidases, and accessory enzymes for the 2013). Endoxylanases cleave the main sugar
side chains (Fig. 6.2). chain depending on the type of xylan, the
degree of branching, and the presence of differ-
ent substituents (Polizeli et al. 2005). The main
1. Xylans hydrolysis products are substituted or non-
Xylans are characterized by a b-1,4-linked b-D- substituted oligomers which are further con-
xylosepyranose backbone substituted by differ- verted by b-xylosidases into tri-, di-, and
ent side chains. They are the major hemicellu- monomers. Endoxylanases can be classified
lose in the cell walls of cereals and in hardwood according to their end product into debranch-
and represent a minor component of the walls ing and non-debranching enzymes, based on
of dicotyledons and non-graminaceous mono- their ability to release L-arabinose from
cotyledons. arabinoxylan (Wong et al. 1988). Some
enzymes cut randomly between unsubstituted
D-xylose residues whereas the cleavage site of
Xylans found in cereals are highly substituted with
single residues or short side chains of a-1,2- or a-1,3- some endoxylanases is dependent on the neigh-
linked L-arabinofuranose residues and are therefore boring substituents of the side chains. b-Xylo-
referred to as arabinoxylans. Glucuronoxylans are typ- sidases can be classified according to their
ical hardwood xylans and contain large amounts of a- relative affinities for xylobiose or larger xylo-
1,2- and a-1,3-linked 4-O-methyl-a-D-glucuronic acid
and acetyl groups at O-2 or O-3. Glucuronoarabinoxy-
oligosaccharides and release b-D-xylopyranose
lans are found in softwood and are substituted with a by a retaining mechanism from the nonreduc-
higher content of a-1,2-linked 4-O-methyl-a-D-glu- ing end. b-Xylosidases are in general highly
curonic acid compared to hardwood and, in addition, specific for small unsubstituted D-xylose oligo-
contain a-L-arabinosefuranose but no acetyl groups. saccharides, and the activity decreases with
The L-arabinose residues may be esterified at O-5 with
feruloyl or p-coumaroyl residues, and a number of
increasing polymerization of the substrates.
other minor residues have been detected, too (Darvill Accumulation of the short oligosaccharides
et al. 1980; Ebringerova and Hienze 2000; Izydorczyk would inhibit the action of the endoxylanases,
and Biliaderis 1995). but the hydrolysis of these products by b-xylo-
sidases removes this possible cause of inhi-
The hydrolysis of the xylan backbone bition, thereby increasing the efficiency of
involves endo-1,4-b-xylanases (endo-1,4-b-D- xylan hydrolysis (Andrade et al. 2004).
xylan xylanohydrolases; EC 3.2.1.8) and b-xylo- Similar to cellulases, most of the genes encod-
134 J. Ramoni and B. Seiboth
ing endoxylanases and b-xylosidases have been mannan backbone randomly distributed b-
characterized in different Aspergillus spp., 1,4-linked b-D-glucopyranose and b-D-manno-
T. reesei, and Penicillium spp. as well as in pyranose are found. Further substituents
Agaricus bisporus and Magnaporthe grisea. include a-1,6-linked a-D-galactopyranose resi-
dues which can be substituted further by a-1,2-
linked a-D-galactopyranose. These poly-
2. Xyloglucan saccharides are usually referred to as galacto-
Xyloglucan is the predominant hemicellulosic mannans and galactoglucomannans (Brett and
polysaccharide of dicotyledons and non- Waldren 1996) which are the major hemicellu-
graminaceous monocotyledons, constituting lose structures of softwoods whereas gluco-
up to 20 % of the plant cell wall. mannan dominates in hardwood (Stephen
1982; Aspinall 1980). The D-glucose or D-man-
nose residues are partially substituted with ace-
Xyloglucans cross link cellulose microfibrills and sup-
port in this way the structural integrity of the cell wall tyl residues linked to O-2 or O-3. The backbone
(Hayashi and Kaida 2011). The backbone is composed is degraded by endo-1,4-b-mannanases (Man-
of 1,4-linked b-D-glucopyranose residues which are nan endo-1,4-b-mannosidase, EC 3.2.1.78) and
substituted at O-6 by D-xylopyranose via an a-1,6-link- b-mannosidases (EC 3.2.1.25). The ability of the
age. Depending on the number of D-glucopyranose endo-1,4-b-mannanases to degrade these poly-
residues attached to the main chain, they are classified
as XXXG or XXGG type. In the XXXG type, three mers depends on the number and position of
consecutive D-glucopyranose residues are substituted the side chain substituents. The enzymes
with D-xylopyranose followed by a fourth unbranched releasing glucose (b-glucosidase, EC 2.1.21)
D-glucopyranose residue. Additional sugars found and galactose (a-galactosidase, EC 3.2.1.22)
substituted to the D-xylopyranose include a-1,2-L-fuco- residues act in synergism with endo-1,4-b-
pyranose, b-1,2-D-galactopyranose, a-1,2-L-galacto-
pyranose, or a-1,2-L-arabinose residues. Some of these mannanases and b-mannosidases. b-Mannosi-
residues can also contain O-linked acetyl groups dases split off the b-D-mannose residue from
(O’Neill and York 2003). XXXG-type glucans are pres- the nonreducing end of the manno-
ent in numerous plants whereas the XXGG type occurs oligosaccharides and are characterized by a
in solanaceous plants. retaining mechanism.
linear chains of a-1,4-linked D-galacturonic acid resi- To efficiently degrade this complex pectin
dues which can carry methyl esters at the terminal structure, fungi have developed a broad spec-
carboxyl group and acetyl esters at the O-2 or O-3
position. Homogalacturonan with a high degree of
trum of pectinolytic enzymes. Enzymatic de-
methyl esterification is referred to as pectin whereas polymerization of pectin weakens the cell wall
pectic acid (pectate) has a low degree of esterification. and exposes the other cell wall polymers to
The esterification of the uronic acid group results in the degradation by other plant cell wall-degrading
elimination of the negative charge, which is of great enzymes. Therefore, pectin degradation is
significance for the gelling process of pectin, since the
complexes between the carboxyl groups and Ca2+ ions
important for plant pathogenic fungi (Kubicek
are involved in this (Vincken et al. 2003). Additionally, et al. 2014), while other often fungi show a
the number of methyl- and acetyl esters has a strong reduced set of these enzymes.
influence on the susceptibility to cleavage by the differ- Pectinases can be classified in terms of their
ent pectinases. Rhamnogalacturonan I+II and xyloga- reaction mechanism into hydrolases or lyases
lacturonan (XGA) are responsible for the “hairy”
regions of the pectin due to their abundant and often
and further according to their substrate speci-
branched side chains. The backbone of rhamnogalac- ficity into, e.g., polygalacturonases or rhamno-
turonan I consists of D-galacturonic acid and L-rham- galacturonases. Beside glycoside hydrolases,
nose in a [1!2)-a-L-Rha-(1!4)-a-D-GalA-(1!]n different lyases such as pectin- (EC 4.2.2.10),
linkage. Whereas D-galacturonic acid can be substituted pectate- (EC 4.2.2.2), and rhamnogalacturonan
with either methyl- or acetyl esters similar to those in
HG, 20–80 % of the L-rhamnose residues are substituted
lyases (EC 4.2.2.-) cleave polysaccharide chains
at the O-4 position by L-arabinose and D-galactose with via a b-elimination mechanism resulting in the
varying size from monomers up to branched, heteroge- formation of a D-4,5-unsaturated bond at the
neous oligomers. They can be terminated with a-L- newly formed nonreducing end.
fucose and (4-O-methyl)-b-D-glucuronic acid. Arabi- Most pectinases are found in GH family 28
nan chains are formed of a-1,5-linked L-arabinoses
which can be further substituted with a-1,3-linked L-
as they share a common conserved structure
arabinose residues. In addition, two types of arabino- which is in contrast to the cellulases and hemi-
galactan side chains are present: Type I consists of a cellulases, which are characterized by a high
chain of b-1,4-linked D-galactopyranose whereas type II diversity of protein structures. Although the
contains a backbone of b-1,3-linked D-galactopyranose overall sequence similarity is low, pectinases
residues which can be substituted with b-1,6-linked D-
galactopyranose residues. Both are occasionally substi-
share a central core consisting of parallel
tuted with L-arabinose at O-3. In addition, ferulic acid b-strands forming a large, right-handed helix
and p-coumaric acids have been identified in the pectic defined as parallel b-helix (Jenkins and Pick-
hairy regions attached to O-2 of L-arabinose and O-6 of ersgill 2001). Even though hydrolases and
D-galactose. Rhamnogalacturonan II consists of a short lyases differ in their catalytic mechanism, the
backbone of a-1,4-linked D-galacturonic acid which is
substituted either at the O-2 or O-3 position (Vidal et al.
substrate-binding sites are all found in a similar
2000). Its side chains have been found to be either location within a cleft formed on the exterior of
dimers or branched oligomers and to contain rare the parallel b-helix. This structure facilitates the
sugars such as D-apiose and L-fucose in addition to L- binding and cleaving of the buried pectin poly-
arabinose, D-galactose, and L-rhamnose. The backbone mers in the undamaged cell wall. The parallel
of another substructure found in hairy regions, the
xylogalacturonans, is similar to that of the HGs, but a
b-helix fold confers the stability needed by
major part of the D-galacturonic residues carry b-D- these pectinases for efficient aggressive action
xylose substituents at the O-3 position. It has been in a variety of hostile extracellular environ-
found in reproductive tissues, including soybean seed, ments. An exception to this rule is the rhamno-
apple fruit, and pine pollen (Schols et al. 1995). galacturonan lyase from A. aculeatus, which
Although the composition and structure of the individ-
ual subunits are well established, the manner in which
displays a unique arrangement of three distinct
they make up the pectin polymer is still under investi- modular domains (McDonough et al. 2004).
gation. For a long time, pectin was thought to consist of The pectinolytic system has been studied in
linear chains of homogalacturonan interspersed with great detail in Aspergillus spp. including A.
hairy regions. In another model (Vincken et al. 2003), niger (de Vries and Visser 2001). About 60
rhamnogalacturonan I forms the backbone, substituted
with homogalacturonan and the abovementioned ara-
genes of A. niger are predicted to encode pecti-
binan and galactan side chains. nases for which the expression of 46 was
detected (Martens-Uzunova and Schaap 2009)
136 J. Ramoni and B. Seiboth
and a high number was identified by mass The pectinolytic enzyme system of A. niger
spectrometry (Tsang et al. 2009). serves as an example of how a microorganism
Polygalacturonases (PGAs) are the most can degrade a pectin molecule. A. niger pro-
extensively studied class of pectinases and duces seven PGAs, two of them (PgaA and
include endopolygalacturonases (1,4-a-D-galac- PgaB) constitutively. These two are most active
turonan glycanohydrolase; EC 3.2.1.15) which on pectins containing 22 % methyl esters (Par-
catalyze the hydrolytic cleavage of a-1,4 enicova et al. 2000), thus making them suitable
D-galacturonic bonds within the chain and exo- for an initial attack on the native substrate.
polygalacturonases (galacturan 1,4-a-galactur- Enzymes subsequently induced at this early
onidase; EC 3.2.1.67) which cleave from the stage during growth on pectin are pectin
nonreducing end. Both endo- and exoPGAs methyl esterase PmeA, an exopolygalacturo-
belong to glycoside hydrolase family 28 and nase PgxA, and pectin lyases (PelA and PelD;
have similar reaction mechanisms and sub- (de Vries et al. 2002). The action of the
strate specificities, but their level of sequence methyl esterase renders the substrate accessible
identity is surprisingly low (Biely et al. 1996; to PGAs, which are expressed at a later stage
Markovic and Janecek 2001; Henrissat and after removal of the methyl esters, while the
Bairoch 1993). Among the endoPGAs, some pectin lyases contribute to the breakdown of
enzymes cleave only once per chain (single the still esterified polymer. The exoPGAs
attack or non-processive) whereas others attack cleave D-galacturonic acid monomers from
multiple times (processive behavior). Single- the homogalacturonan poly- and oligomers,
attack PGAs generally produce longer frag- which may serve as inducers for the other
ments, which are only gradually degraded into pectinases. Similar to homogalacturonans, the
dimers, trimers, or short oligomers, providing degradation of the rhamnogalacturonan I back-
possible sites for exoPGAs. bone is catalyzed by hydrolases and lyases
(Fig. 6.3). Endorhamnogalacturonases have
A factor which significantly influences the activity of been isolated from A. acculeatus and A. niger
PGAs is the number (and distribution) of methyl- and (de Vries and Visser 2001) and hydrolyze the a-
acetyl ester groups on the substrate. In general, most 1,4 glycosidic bonds in saponified hairy
endo- and exoPGAs prefer substrates with a low degree
of esterification, although some exceptions exist (Par-
regions. The resulting fragments were tetra-
enicova et al. 2000). In most cases, the activity of a and hexamers of the backbone, which partly
methyl/acetyl esterase is required to prepare the pectin were still substituted with D-galactose. This sug-
molecule for PGA digestion. Pectin-degrading fungi gests that—similar to some exoPGAs ability to
often produce multiple isozymes with a wide range of cleave XGA (van den Broek et al. 1996)—
enzymatic properties, substrate specificities, and pH
optima, which may reflect the complexity of the pectin
endorhamnogalacturonases are tolerant
molecule in plant cell walls and the need for enzymes towards monomeric substituents. Depending
capable of cleaving the homogalacturonan backbone in on the monosaccharide cleaved from the non-
a variety of structural contexts. Another structural fea- reducing end of the rhamnogalacturonan, two
ture which may determine the functional diversification kinds of exorhamnogalacturonases have been
of these enzymes is the presence or absence and type of
N-terminal extension, which has been suggested to
described: rhamnogalacturonan a-D-galactosy-
influence their substrate specificity and to play a role luron-hydrolase and rhamnogalacturonan a-L-
in their interaction with particular regions of the pectin rhamnohydrolase. All rhamnogalacturonan
polymer (Gotesson et al. 2002; Parenicova et al. 2000). hydrolases were classified as members of GH
The degree of esterification is also important for the family 28. Rhamnogalacturonan lyases cleave
functional classification of lyases. Pectate lyases prefer
substrates with a low degree of methyl esterification,
the rhamnogalacturonan backbone via b-elimi-
which therefore have a more acidic character, and are nation. Unlike the hydrolases, they act on the
strictly dependent on Ca2+ for catalysis. Pectin lyases, Rha-(1!4)-a-D-GalA bond, resulting in the
on the other hand, favor highly methyl-esterified sub- formation of D4,5-unsaturated D-galacturonic
strates and do not require Ca2+ ions (Jurnak et al. 1996). acid residues at the nonreducing end.
Degradation of Plant Cell Wall Polymers by Fungi 137
Fig. 6.3 Enzymatic degradation of rhamnogalacturonan Terminal monosaccharides are removed by a-L-arabino-
I and homogalacturonan. The main chain of rhamono- furanosidases (ABF) and b-galactosidases (BGA).
galacturonan I is degraded by rhamnogalacturonan The main chain of homogalacturonan is degraded by
hydrolase (RGA) and rhamnogalacturonan lyase (RGL). endopolygalacturonases (PGA), exopolygalacturonases
The side chains are degraded by rhamnogalacturonan (PGX), pectin lyases (PLY), and pectate lyases (PEL).
acetyl esterase (RGAE), endoarabinase (ABN), endo-b- Pectin methyl esterase (PME) and pectin acetyl esterase
1,6-galactanases (GAL), and exogalactanases (GAX). (PAE) act on the side groups
D. Accessory Enzymes for Plant Cell Wall some of the major enzymes are listed here
Degradation (Figs. 6.2 and 6.3).
Side groups in xylans are generally small
Most plant cell wall-degrading enzymes (mono-, di-, and trimers) but can consist of
described above act on the backbone of the several different sugars and acids (e.g., acetic
respective polysaccharide, but often their activ- acid, L-arabinose, ferulic acid, D-galactose,
ity is impaired by monomeric substituents or D-glucuronic acid), and, consequently, multiple
larger side chains present in hemicelluloses and enzymes are required to make the backbone
pectins. To ensure an efficient and complete fully accessible for the xylanases. a-L-Arabino-
breakdown of such polysaccharides, these sub- furanosidases (EC 3.2.1.55) remove terminal
stituents have to be removed and degraded by L-arabinose residues but differences in the
accessory enzymes. These accessory enzymes specificity towards a-1,2-, a-1,3-, or a-1,5-ara-
work in synergism with the enzymes attacking binosidic bounds and towards the substrates
the main chain and often depend on each other themselves have been observed. Whereas sev-
for an efficient breakdown of the whole sub- eral representatives are also able to release
strate to monomeric sugars. The substrate L-arabinose from pectins and xylans, arabino-
specificity of these enzymes varies; some of xylan arabinofuranohydrolases (EC 3.2.1.99)
the enzymes can hydrolyze the intact polymer are strictly specific for L-arabinose bound to
whereas others show maximum activity only in xylan. In addition, some a-L-arabinofurano-
the presence of shorter breakdown products sidases are inhibited by the presence of D-glu-
(Puls and Schuseil 1993; Tenkanen and Siika- curonic acid residues adjacent to the targeted L-
aho 2000). A detailed list of all the enzymes arabinose. a-L-arabinofuranosidases contain
involved in the degradation of hemicellulose also CBMs which support their binding to cel-
and pectinase side chains and their mode of lulose, xylan, or arabinofuranose side chain.
action has been reviewed for e.g. Aspergillus The D-glucuronic acid and its 4-O-methyl
spp. by de Vries and Visser (2001), and only ethers are removed by a-glucuronidases (EC
138 J. Ramoni and B. Seiboth
compounds. Besides these fungi, brown-rot sisting of oxidized lignin, which cracks into
fungi are also able to degrade wood extensively. characteristic brick-like pieces. Representatives
Analysis of the genomes of different Basidio- of brown-rot basidiomycetes comprise Schizo-
mycota suggests that the categorization into phyllum commune, Fomes fomentarius, Serpula
white rot or brown rot is an oversimplification lacrimans, Postia placenta, Piptoporus betuli-
because of gradations both in the expression of nus, and Gloeophyllum trabeum. They are also
metabolites and the resulting patterns of decay the major cause of decay of woods in commer-
(Riley et al. 2014; Arantes et al. 2012). Some cial use and have an important role in conifer-
ascomycetes also colonize wood in contact ous ecosystems through their contribution to
with soil but alter the lignin component only humus formation. These fungi grow mainly in
slightly. Their action leads to decrease in the the cell lumen of the woody cells, and the deg-
mechanical properties of wood, giving rise to radation is not localized to the fungal hyphae
so-called soft rot, a process which often but found at greater distances from these. The
involves bacteria. Soft-rot fungi can degrade extracellular enzymes formed are too large to
wood under extreme environmental conditions penetrate healthy cell walls and therefore—as
(extreme wetness or frequent dryness) which noted above—degradation of cellulose by
prohibit the activity of other wood-degrading brown-rot fungi must involve diffusible low-
fungi. They are relatively unspecialized (hemi-) molecular agents. They employ small molecule
cellulolytic ascomycetes in the genera Chaeto- reactive species to depolymerize lignin, cleave
mium, Ceratocystis, and Phialophora, and some propyl side chain, and also demethoxylate the
basidiomycetes can also cause a soft rot-type of ring structures before repolymerizing the mate-
decay pattern. In soft rot, decay by fungi is rial elsewhere as a means of freeing the cellu-
closely associated with penetration by the fun- losic components and generating greater access
gal hyphae, because the enzymes cannot cross for deconstruction (Arantes and Goodell 2014).
the plant cell wall. Two distinct types of soft rot Brown rot fungi have evolved multiple times
are currently recognized. Type 1 is character- from the predecessors of current white rot
ized by longitudinal cavities formed within the fungi. During this evolution, the typical ligno-
secondary wall of wood cells and type 2 by an lytic enzyme systems of white rots and crucial
erosion of the entire secondary wall (Martinez types of cellulases have been lost (Floudas et al.
et al. 2005). Although many white rots and 2012).
brown rots secrete oxidative and hydrolytic Although some brown rots possess cello-
enzymes, it is generally recognized that their biohydrolases, they generally lack the ability
enzymes are unable to diffuse through healthy to hydrolyze crystalline cellulose enzymatically.
wood and that smaller, non-proteinaceous Nonenzymatic deconstruction of cellulose uses
molecules are involved in the initiation of iron-dependent Fenton chemistries (Arantes
decay. and Goodell 2014). Crystalline cellulose can
efficiently be degraded by a combination of
classical endoglucanases and an oxidative de-
gradation system such as extracellular reactive
A. Brown-Rot Fungi
oxygen species (ROS). These include hydroxyl
Brown-rot fungi degrade mainly cellulose and radicals ( OH) and the less reactive peroxyl
l
hemicellulose of coniferous softwoods and par- (ROO ) and hydroperoxyl ( OOH) radicals
l l
tially modify the lignin mainly by demethyla- (Hammel et al. 2002). There is a well-
tion but not by oxidation (Eriksson et al. 1990). established pathway for the generation of
Brown-rot fungi attack cellulose in wood, which these radicals via the Fenton reaction (H2O2 +
promotes rapid loss of mechanical strength Fe2+ +H+ !H2O+Fe3+ + OH). In order not to
l
leaving modified lignin behind. The term destroy the fungal hyphae and to act in the
“brown rot” refers to the characteristics of this lignified parts of the secondary cell wall, the
decayed wood: a reddish-brown material con- l
OH production has to occur at a distance
140 J. Ramoni and B. Seiboth
from the hyphae, and the fungal reductants Most brown rots secrete oxalic acid, which
should be stable enough to diffuse before they is a strong chelator of Fe3+ and Fe2+ but also
react to reduce Fe3+ and O2 to Fe+2 and H2O2. reduces the pH. The pH of wood itself is gener-
The production of OH radicals can take place
l
ally in the range 3–6 and is lowered to pH
in several ways including secreted hydroqui- values between 2.5 and 1.7. The reduction of
nones, cellobiose dehydrogenases, low pH is important for the function of the extra-
molecular-weight glycopetides, and phenolate cellular enzymes and has been identified as a
chelators. A chelator-mediated Fenton (CMF) key factor in several hypotheses related to
system has evolved in different brown rot fungi molecular weight degradation systems, as dis-
(Gloeophyllales, Polyporales, and Boletales), cussed in the reviews listed above.
providing an efficient mechanism for depo-
lymerization and modification of lignocellulo-
lytic biomass (Arantes and Goodell 2014; B. White-Rot Fungi
Eastwood et al. 2011). The CMF system is
unique as it is based on oxygen radical chemis- White rots are the most frequently found wood-
try that permits nonenzymatic deconstruction rotting organisms and are mainly basidio-
at a considerable distance from the organism. mycetes, but also some ascomycetes (Diatrypa-
The efficiency of the CMF system is thought to ceae and Xylariaceae) are able to cause white
provide brown rot fungi advantages in exploit- rot. They are characterized by their ability to
ing ecological niches, and, for example, these completely degrade lignin, hemicelluloses, and
fungi have displaced white rot predecessors in cellulose, thereby giving rise to the name-giving
the degradation of conifer wood. cellulose-enriched white colored wood mate-
rial. A typical white rot degradation is primarily
The principle of the quinone redox cycling for OH l
enzymatic, and the attack of the wood cell wall
production is that the fungus reduces the quinone proceeds only from lignocellulose surfaces
extracellularly to its hydroquinone which then reacts because degradative enzymes are too large to
with Fe3+ to Fe2+ and a semiquinone radical. The semi-
quinone reduces O2 to OOH, which is a source for
l
penetrate the intact cell wall. The enzymes
H2O2, and is in this way recycled to quinone. Gloeo- employed by the white rot fungi include a com-
phyllum trabeum produces extracellular quinones plete suite of cellulases, with some fungi pos-
including 2,5-dimethoxy-1,4-benzoquinone and 4,5- sessing a large number of LPMO encoding
dimethoxy-1,2-benzoquinone which can reduce Fe3+ genes (Busk and Lange 2015) and enzymes
and O2 rapidly under physiological conditions, thereby
generating both Fe2+ and H2O2. Moreover, the fungus that can oxidize lignin components, including
was shown to reduce the resulting dimethoxyquinones lignin, manganese, and versatile peroxidase or
back to hydroquinones, possibly by the action of an laccases (Pollegioni et al. 2015). Two different
intracellular quinone reductase. Another nonenzymatic white-rot patterns have been described which
system includes phenolate or catechole chelators are simultaneous and selective delignification.
(Goodell 2003). They have a high affinity for the bind-
ing of iron and have the ability to reduce Fe3+ to Fe2+. Simultaneous or nonselective delignification
The two compounds 4,5-dimethoxy-1,2-benzenediol acts mainly on hardwood and degrades cellu-
and 2,5-dimethoxy-1,4-benzenediol were identified in lose, lignin, and hemicellulose simultaneously.
the Gt chelator fraction and also their oxidized benzo- The cell wall is attacked progressively from the
quinone forms (see above). OH radicals can also be
l
cell lumen towards the middle lamella. De-
produced by the extracellular flavohaemoprotein cello-
biose dehydrogenase (CDH). CDH production has been gradation is associated with the fungal hyphae
reported for all types of wood-rotting fungi (Zamocky and substantial amounts of undecayed wood
et al. 2006), and CDH can act as cellobiose oxidase by remains. Basidiomycetes (e.g., Trametes versi-
reducing O2 to H2O2. However, Fe3+ is a better electron color, Irpex lacteus, P. chrysosporium, Hetero-
acceptor than O2 and, thus, CDHs are actually Fe3+ basidion annosum, and Phlebia radiata) and
reductases. Glycopeptides, implicated in wood de-
gradation, have been isolated from G. trabeum and some ascomycetes (e.g., Xylaria hypoxylon)
Tyromyces palustris. They reduce Fe3+ to Fe2+ and perform this type of degradation. Selective
bind Fe2+ (Goodell 2003; Enoki et al. 2003). In the delignification, or sequential decay, is found
presence of H2O2, the glycopeptide generates one- in hardwood and softwood. The initial attack
electron oxidation and possesses the ability to oxidize is selective for lignin and hemicellulose, and the
NADH in the presence of oxygen and thereby produces
H2O2. cellulose is attacked later. Lignin is degraded in
Degradation of Plant Cell Wall Polymers by Fungi 141
the middle lamella and in the secondary wall. plant cell, direct degradation is carried out only in
This type of degradation is performed exclu- exposed regions of the cell lumen (simultaneous
delignification). But microscopic studies of selective
sively by basidiomycetes (e.g., Ganoderma aus- lignin biodegradation revealed that white-rot fungi
trale, Phlebia tremellosa, C. subvermispora, remove the polymer from inside the cell wall, which
Pleurotus spp., and Phellinus pini). Many can be performed only by indirect oxidation mediated
white-rot fungi cause both types of rot, and by low molecular-weight diffusible compounds capable
the amount of simultaneous or selective of penetrating the cell wall.
decayed wood depends on the substrate and
varies even among different strains of the MnPs are closely related to LiPs and have
same species (Martinez et al. 2005; Eriksson an iron protoporphyrin IX (heme) prosthetic
et al. 1990). To date, P. chrysosporium is the group. They have the same catalytic cycle
most intensively studied white-rot fungus involving a two-electron oxidation of the
(Martinez et al. 2004; Cullen and Kersten 2004). heme by H2O2, followed by two subsequent
White-rot fungi degrade lignin via an one-electron reductions. But instead of veratryl
oxidative process involving peroxidases and alcohol, MnPs oxidize Mn+2 to Mn+3, which is
laccases (phenol oxidases) which act nonspecif- stabilized by organic acids such as oxalate,
ically by generating highly reactive, nonspecific fumarate, and malate.
free radicals that attack lignin causing sponta-
neous cleavage reactions (Hammel and Cullen Chelated Mn+3 ions can act as diffusible oxidizer on
2008). phenolic substrates and oxidize non-phenolic sub-
strates via lipid peroxidation reactions (Martinez et al.
Heme peroxidases, such as the lignin per- 2005; Hofrichter et al. 2010). The reaction is similar to
oxidase (LiP; EC 1.11.1.14), manganese peroxi- LiPs and initiated by the enzyme and H2O2 to form
dase (MnP; EC 1.11.1.13), and the versatile MnP compound I, a Fe4+-oxo-porphyrin-radical com-
peroxidase (VP; EC 1.11.1.16), have been plex. A monochelated Mn+2 ion transfers one electron
described as true ligninases due to their high to the porphyrin intermediate to form compound II
and is oxidized to Mn3+ before the resting state is
redox potential which enables them to oxidize reached by one electron transfer to form Mn+3 by an
non-phenolic aromatic substrates constituting electron transfer from Mn+2. The more recently discov-
up to 90 % of the lignin structure. Usually, a ered VP combines the enzymatic properties of both LiP
number of isoenzymes are produced which is and MnP and oxidizes Mn+2 and veratryl alcohol
either due to posttranslational modifications or (Hofrichter et al. 2010). A VP was firstly described for
Pleurotus eryngii (Martinez et al. 1996). The catalytic
the presence of multiple genes in the genome. cycle of VP combines both LiP and MnP, but this cycle
Peroxidases require the presence of H2O2 as differs from the classical MnPs by catalyzing the Mn+2-
oxidizing substrate and oxidize phenolic and independent oxidation of simple amines and phenolic
non-phenolic compounds (Hatakka 1994; Ker- monomers (Perez-Boada et al. 2005). The catalytic ver-
sten 1990; Hammel et al. 1986; Eggert et al. satility of VP permits its application in Mn+3-mediated
or Mn-independent reactions in both low- and high-
1997). LiP and MnPs were firstly described for redox potential aromatic substrates. Although VP from
P. chrysosporium (Glenn and Gold 1985; Kirk P. eryngii catalyzes the oxidation of Mn+2 to Mn+3 with
and Farrell 1987; Paszczyński et al. 1985). H2O2, it differs from classical MnPs in its manganese-
independent activity, thereby enabling it to oxidize
substituted phenols and the veratryl alcohol (Camarero
The catalytic, oxidative cycle of LiP is similar to those of et al. 1999).
other peroxidases. A heme group (Fe3+) in the active The crystal structures have been resolved for both
center oxidizes H2O2 while forming an intermediate LiP and MnP (Piontek et al. 1993; Poulos et al. 1993;
compound I known as LiP oxyferryl. Compound I is Sundaramoorthy et al. 1994). The prosthetic group
reduced by veratryl alcohol via one-electron transfer (iron protoporphyrin IX) of LiPs is accessible only
forming compound II before it is reduced to its peroxi- through a narrow pore (Piontek et al. 2001). Although
dase resting state via a second one-electron transfer its catalytic cycle is common to peroxidases, it is noted
(Hofrichter et al. 2010; Liu et al. 2003). Veratryl alcohol that the position of the iron-binding histidine residue
is oxidized to short-lived cation radicals which oxidize in ligninolytic peroxidases is located further away from
lignin directly or pass on the charge to other, more the heme iron which increases the redox potential. Also
stable carriers which can act as diffusible mediators. the existence of specific binding sites for substrate
Since enzymes such as LiPs are too large to enter the oxidation are unique (Martinez 2002). The substrate-
142 J. Ramoni and B. Seiboth
binding sites have been identified for LiP, MnP, and VP, hemicellulose and lignin, and (3) a cooperation
and explain the dual catalytic properties of VP. Mn2+ with the manganese peroxidases to make the
oxidation occurs at a binding site near the cofactor
which enables direct electron transfer. By contrast, ver-
abundant non-phenolic components of lignin
atryl alcohol is oxidized at the surface of the protein by accessible for MnP and laccases. The role of
a long-range electron transfer mechanism. The ratio- ROS in the initial attack of lignin has also
nale of the existence of this electron transfer mecha- been discussed (Hammel et al. 2002) and was
nism is related to the fact that many of the aromatic reviewed already in the brown-rot section.
substrates cannot penetrate inside the LiP/VP and,
therefore, these substrata are oxidized at the enzyme
surface, and electrons are transferred to the heme
(Doyle et al. 1998; Gold et al. 2000; Sundaramoorthy
et al. 1997).
V. Conclusions
Laccases (EC 1.10.3.2) are multicopper phe- The recycling of polymers by fungi is an essen-
noloxidases and generally larger than peroxi- tial process for life on earth which is today
dases. They directly oxidize phenols and exploited in the biotech industry to produce
aromatic amines and catalyze a one-electron fermentable sugars for different biorefinery
oxidation combined with a four electron reduc- products including bioethanol. Fungi produce
tion of O2 to H2O. Laccase catalysis requires a plethora of plant cell wall-degrading enzymes
four copper atoms, which are held in place at and efficiently degrade this highly complex car-
the catalytic center by four histidine-rich, bon source. Although most of these enzymes
copper-binding regions (Claus 2004). The phe- are known for decades, the recent discovery of a
nolic nucleus is oxidized by removal of one new group, the lytic polysaccharide monooxy-
electron, generating phenoxy-free-radical pro- genases, as main actors in polysaccharide de-
ducts, which can lead to polymer cleavage. Due gradation shows that our understanding of
to their low redox potential, non-phenolic sub- polysaccharide and lignocellulose deconstruc-
strates have to be oxidized by other mediators. tion is still limited. Comparative genomics,
Metabolites such as 3-hydroxyanthranilate can transcriptomics, and proteomics have provided
mediate oxidation in Pycnoporus cinnabarinus us over the past years with a number of poten-
(Eggert et al. 1997), and also lignin degradation tially interesting enzymes and accessory pro-
products can act as redox charge transfer mole- teins, which will reveal new insights into how
cules (ten Have and Teunissen 2001). fungi use plant cell walls for their metabolism.
H2O2-generating enzymes are essential for Still, a major challenge for future research is to
the function of the peroxidases and include understand the complex multienzyme process
glyoxal oxidase, glucose 1-oxidase, methanol- of lignocellulose decomposition and to transfer
oxidase, aryl-alcohol oxidase, and oxalate this knowledge for the development of novel
decarboxylase oxidases which reduce O2 to enzyme formulations to increase the efficiency
H2O2 (Zhao and Janse 1996). Flavin is often of plant cell wall saccharification in the bio-
used as cofactor, with the exception of, e.g., based economy.
copper-containing glyoxal oxidase from P.
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Biotic Interactions of Fungi
7 Evolution in Heritable Bacterial–Fungal Endosymbioses
TERESA E. PAWLOWSKA1
mycotina) with Burkholderia rhizoxinica and these associations have already generated and
B. endofungorum (beta-proteobacteria), the will continue to provide important insights into
symbionts reside directly in the host cytoplasm the evolution of heritable endosymbioses,
(Partida-Martinez and Hertweck 2005). The including: (A) the role of symbiont vertical
bacteria control fungal ability to reproduce transmission in evolution of mutualisms from
asexually (Partida-Martinez et al. 2007b) and antagonisms, (B) the form of evolutionary
provision the host with potent toxins, rhizoxin trajectories in mutualisms, and (C) the
and rhizonin, which may be important for fun- mechanisms of genome evolution in heritable
gal pathogenesis of plants (Partida-Martinez symbionts.
and Hertweck 2005; Partida-Martinez et al.
2007a). Despite fungal dependence on bacteria
for reproduction, both partners can be sepa- II. Evolution in Bacterial–Fungal
rated and cultivated independently of each Symbioses
other (Partida-Martinez and Hertweck 2005).
In contrast, in the symbiosis formed by the A. The Role of Symbiont Vertical
representatives of the Gigasporaceae family of Transmission in Evolution of Mutualisms
Glomeromycota, also known as arbuscular from Antagonisms
mycorrhizal fungi (AMF), with Candidatus
Glomeribacter gigasporarum (a beta- Vertical transmission allows for coupling of
proteobacterium hereafter referred to as Glo- partner reproductive interests and, over time,
meribacter gigasporarum), the endobacteria is expected to lead to a transformation of antag-
are contained in vesicles (Bianciotto et al. onistic interspecific interactions into mutual-
1996). The symbionts improve fungal ability isms (Yamamura 1993). Evolutionary theory
to elongate germ tubes emerging from asexual suggests that a symbiotic system will transition
spores (Lumini et al. 2007). This feature may from antagonism to mutualism if a parasite is
facilitate AMF colonization of plant roots able to dominate the co-evolutionary race with
important for the completion of the fungal the host and achieve a rate of vertical transmis-
host life cycle, as Glomeromycota are obligate sion that enables efficient reciprocal selection
biotrophs reliant on plants for assimilated car- between the partners (Yamamura 1993)
bon (Lumini et al. 2007). While for AMF the (Fig. 7.1). In other words, when an interaction
association with Glomeribacter is facultative is dominated by the host, resulting in low rates
(Lumini et al. 2007; Mondo et al. 2012), the of parasite vertical transmission, it will continue
endobacteria are uncultivable (Jargeat et al. to function as an antagonism. However, once
2004) and metabolically dependent on the fun- the parasite dominates the interaction and is
gus (Ghignone et al. 2012). Finally, in the asso- able to increase the rate of vertical transmission,
ciation between Glomeromycota and the its reproductive success will eventually become
recently discovered novel lineage of Mollicutes, bound to the host reproductive success. If the
referred to mycoplasma-related endobacteria increase in the rate of symbiont vertical trans-
(MRE), the endobacteria reside directly in the mission is accompanied by the development of
host cytoplasm (Naumann et al. 2010). Unlike host abilities to complement its metabolism
Glomeribacter, whose distribution is limited to with symbiont metabolites, a mutualism is
the Gigasporaceae family of AMF, MRE were expected to evolve (Yamamura 1993).
detected not only in all major lineages of Glo- While the model that explains the evolution
meromycota (Naumann et al. 2010) but also in of mutualisms from antagonisms through
some Endogone representatives of Mucoromy- changes in the rates of symbiont transmission
cotina (Desirò et al. 2014a). The role of MRE in is rather straightforward (Yamamura 1993), the
the biology of their fungal hosts is unknown. actual mechanisms that permit symbiont verti-
Despite the differences among symbioses cal transmission remain elusive as nearly all
formed by early diverging fungi with bacteria known heritable endosymbionts are uncultiva-
and uncertainties surrounding their biology, ble (Moran et al. 2008), and many hosts are
Evolution in Heritable Bacterial–Fungal Endosymbioses 153
Fig. 7.1 Evolutionary theory predictions concerning endosymbionts are shown as green dots. Relative host
the role of vertical transmission in evolution of mutu- fitness is reflected by the size of ovals
alism from antagonism. Hosts are depicted as red ovals;
Fig. 7.2 Hypothetical evolutionary trajectories in heri- expected to arrest an association at the facultative
table mutualisms. Hosts are depicted as red ovals; dependence stage. If conditions remain unfavorable
endosymbionts are shown as green dots. Relative host for prolonged periods of time, host populations would
fitness is reflected by the size of ovals. (a) Evolutionary be expected to completely lose endosymbionts. Image
trajectory leading to obligate reciprocal partner depen- modified from Mondo et al. (2012)
dence. (b) Shifting environmental conditions are
partners combined with evidence of phyloge- the null hypothesis that symbiotic partners
netic codivergence between the partners. evolve independently of each other and, in
However, evolutionary histories of facultative addition to calculating a test statistic for the
endosymbioses are difficult to reconstruct entire dataset, allows for examining individual
because phylogenies of the partners are often partner pairs to detect evidence of codiver-
incongruent with each other (Bright and Bul- gence. In the case of the Gigasporaceae–Glo-
gheresi 2010). Consequently, the apparent meribacter symbiosis, the ParaFit test rejected
shortage of old facultative endosymbioses may the hypothesis of independent partner evolu-
be related to impediments in estimating the age tion and identified partner pairs that contribu-
of these associations rather than to their ted to the overall significant signal of
ephemeral nature. codivergence (Fig. 7.3). These partner pairs
Because fossil record is available for many included representatives of several populations
fungal lineages, fungi offer excellent models for of Gigaspora margarita, Cetraspora pellucida,
studying coevolution in symbiotic associations Racocetra castanea, and R. verrucosa. Based
(Taylor et al. 2015). In particular, the hypothe- on this pattern of codivergence, the origin of
sis of the ephemeral nature of facultative sym- the Gigasporaceae–Glomeribacter symbiosis
bioses was tested using the Gigasporaceae– appears to predate the divergence of Gigaspora
Glomeribacter symbiosis (Mondo et al. 2012). from Cetraspora and Racocetra (former Scutel-
To achieve this goal, the phylogenetic histories lospora). The existence of Scutellospora fossil
of both the Gigasporaceae hosts and their record from the Rhynie chert dated to 396
Glomeribacter symbionts were reconstructed 12 mya (Dotzler et al. 2006) allows ascribing
and compared using a statistical tool ParaFit the origin of the Gigasporaceae–Glomeribacter
(Legendre et al. 2002) (Fig. 7.3). ParaFit tests symbiosis to at least 400 million years ago.
Fig. 7.3 Patterns of coevolution between the Gigaspor- gigasporarum bacterial endosymbionts (right). The
aceae fungal hosts (left) and the Ca. Glomeribacter fungal phylogeny was reconstructed using 18S rRNA,
156 T.E. Pawlowska
Older than 400 million years, the facultative the host (McCutcheon and Moran 2012). In
mutualism between Gigasporaceae and Glomer- maternally transmitted essential mutualists of
ibacter predates several obligate nutritional insects, genome reduction is a consequence of a
mutualisms of insects (Moran et al. 2008). The degenerative process. Endosymbionts are pro-
existence of an ancient facultative mutualism pagated exclusively through host reproductive
suggests that not all facultative mutualisms are structures, leading to transmission bottlenecks
transitional stages along the evolutionary tra- in every host generation (Mira and Moran
jectory leading to reciprocal partner depen- 2002). In addition, endosymbiont populations
dence (Fig. 7.2b). A discovery that facultative associated with individual host lineages are
mutualisms can be ancient and stable in their reproductively isolated from each other, result-
facultative state rises a question concerning the ing in endosymbiont clonality and population
mechanisms that prevent the host from evol- subdivision. All these phenomena reduce endo-
ving toward obligate dependence on the endo- symbiont effective population size and magnify
symbiont. While other heritable nonessential the impact of genetic drift relative to natural
mutualisms, such as defensive symbioses of selection (Charlesworth 2009). As a conse-
insects (Oliver et al. 2003; Scarborough et al. quence, slightly deleterious mutations undergo
2005), might also be arrested in a facultative rapid fixation (Ohta 1973), leading to the loss of
state, the Gigasporaceae–Glomeribacter symbi- gene functions. Due to the strong deletional
osis is uniquely suited to addressing this ques- bias prevailing in the bacterial genomes (Mira
tion. Because AMF depend on plant-assimilated et al. 2001; Kuo and Ochman 2009), mutation-
carbon, their interaction with Glomeribacter is compromised genes are eliminated, resulting in
likely to be tightly regulated to avoid unneces- genome contraction (Moran et al. 2009).
sary costs of supporting symbionts under con- Another consequence of rapid fixation of
ditions when they become a burden to the host slightly deleterious mutations is acceleration
fitness. Since the Gigasporaceae hosts can be of molecular evolution rate relative to free-
readily sampled from nature, as they are com- living taxa (Ohta 1973). While genome size
monly found in dune habitats (Bergen and reduction is apparent in several bacterial endo-
Koske 1984; Koske 1987; Gemma et al. 1989), symbionts of fungi examined thus far, includ-
the distribution of Glomeribacter across the ing (1) Burkholderia rhizoxinica (Lackner et al.
AMF hosts could be correlated with environ- 2011b), (2) Glomeribacter gigasporarum
mental conditions to reveal factors responsible (Ghignone et al. 2012), and (3) MRE (Naito
for the facultative nature of the Gigasporaceae– et al. 2015), none of these genomes appears to
Glomeribacter symbiosis. be a product of exclusively degenerative evolu-
tion (Castillo and Pawlowska 2010; Naito et al.
2015). Consequently, examination of genome
C. Mechanisms of Genome Evolution evolution in bacterial endosymbionts of fungi
in Heritable Endosymbionts is likely to reveal novel mechanisms responsi-
ble for genome contraction.
Genome reduction is one of the hallmarks of
endosymbiont reproductive dependence on
Fig. 7.3 (continued) 28S rRNA, and beta-tubulin gene codivergence after recombination was accounted for
sequences; the bacterial phylogeny is based on 16S in the bacterial dataset. Dashed orange lines indicate
rRNA, 23S rRNA, ftsZ, and pstA genes. Bayesian poste- partner pairs with no evidence of codivergence, likely
rior probabilities greater than 0.80 are shown above due to host switch events. Fungal isolates harboring
branches. Branches with Maximum Likelihood boot- endobacteria are colored red; individuals with no endo-
strap support over 70 % are thickened. Solid blue bacteria are shown in green. The node likely associated
lines connecting host and symbiont pairs indicate sig- with the Scutellospora Rhynie chert fossil record (Dot-
nificant evidence of codivergence detected by ParaFit. zler et al. 2006) is marked by an asterisk. Image mod-
Dashed blue lines link partners showing evidence of ified from Mondo et al. (2012)
Evolution in Heritable Bacterial–Fungal Endosymbioses 157
reciprocal selection and coevolution in bacte- Gemma JN, Koske RE, Carreiro M (1989) Seasonal
rial–fungal symbioses is expected to be a criti- dynamics of selected species of VA mycorrhizal
fungi in a sand dune. Mycol Res 92:317–321
cal source of novel findings needed to advance Ghignone S, Salvioli A, Anca I, Lumini E, Ortu G, Petiti
evolutionary theory. L, Cruveiller S, Bianciotto V, Piffanelli P, Lan-
franco L, Bonfante P (2012) The genome of the
Acknowledgments I thank Jonathan Gonzalez and Olga obligate endobacterium of an AM fungus reveals
Lastovetsky for comments on the manuscript. This an interphylum network of nutritional interac-
work was supported by the National Science Founda- tions. ISME J 6:136–145
tion grant IOS-1261004. Jargeat P, Cosseau C, Ola’h B, Jauneau A, Bonfante P,
Batut J, Bécard G (2004) Isolation, free-living capa-
cities, and genome structure of “Candidatus Glo-
meribacter gigasporarum,” the endocellular
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8 An Emerging Interdisciplinary Field: Fungal–Bacterial
Interactions
fungi and bacteria leads to changes in pH and tions to the understanding of bacterium–
to enrichment of certain metabolites, which fungus interactions and focuses on the rapid
induces the production of some but suppresses methodological development in this area. On
that of other compounds (reviewed in Tarkka the basis of this introductory review, we pro-
et al. 2009; Scherlach et al. 2013). Beneficial pose that the reader subsequently consults the
effects of microbial cocultures for food produc- review of Pawlowska (chapter “Evolution in
tion include cheese (Corsetti et al. 2001) and Heritable Bacterial-Fungal Endosymbioses”),
vinegar (Valera et al. 2015) production or sup- to learn more about the fascinating bacterium
pression of mold growth and mycotoxin pro- endosymbiosis in the hyphae of the oldest fun-
duction in foodstuffs (Dalie et al. 2010), but FBI gal mutualists, the Glomeromycota.
may also cause food spoilage by toxin produc-
tion (Garcia et al. 1999). The activation of
“silent” secondary metabolite gene clusters by II. FBI Theater Takes Place Anytime,
bacteria during FBI (Schroeckh et al. 2009) has Anywhere, Every Way
emerged as a novel strategy to produce thera-
peutical agents, but the FBI may either suppress The following section focuses on fungus–bacte-
or activate secondary metabolite production rium interactions taking place in simple in vitro
(Scherlach et al. 2013). cultures and different environments. Mediation
Pathogenicity or nutritional influence of of the FBI by metabolites, enzymes, and physi-
plant and animal symbionts may be affected cal associations is described; examples from
by FBI. Bacteria may interfere with fungi by a simple two-partner model systems to more
multitude of mechanisms, including competi- complex dynamic consortia are included; and
tive tissue colonization; synthesis of antibiotics, implications for medicine, biotechnology, agri-
organic acids, and hydrogen peroxide; produc- culture, and forestry are mentioned.
tion of lytic enzymes; detoxification of toxins;
and degradation of virulence factors (reviewed
in Compant et al. 2005; Harriott and Noverr A. Simple Models Drive the Analysis of FBI
2011). Stimulatory interaction exists between Evolutionary Ecology
nitrogen-fixing and phosphate-solubilizing
bacteria and plant beneficial mycorrhizal fungi Evolution and ecology of FBI has been targeted
(Requena et al. 1997). Some bacteria stimulate perhaps most elegantly with the simple yeast–
the virulence of a plant pathogen by and Hert- bacterium interaction. For instance, by using
weck 2005 producing virulence factors (Par- an artificial community composed of baker’s
tida-Martinez and Hertweck 2005), promote yeast Saccharomyces cerevisiae and nitrogen-
the growth of the pathogen (Xu et al. 2008) or fixing bacterium Rhizobium etli, Andrade-
anchor the mycelia of a fungal pathogen to the Domı́nguez et al. (2014) investigated what
target tissue, and stimulate the formation of happens when two species with no known his-
mixed FB biofilms (Silverman et al. 2010), tory of previous interaction meet for the first
which are less responsive to antimicrobials time. At the beginning of the interaction, yeast
than free-living cells. produced growth inhibitor orotic acid (OA)
The yearly growing number of studies pub- and growth-promoting C4-dicarboxylates, and
lished on the FBI points to an exciting and since the dicarboxylates dominated over the
emerging area of research with increasing num- impact of OA, the early interaction was com-
bers of researchers involved (Table 8.1). Niches mensal, bacterium favoring. When the growth
for fungal–bacterial networks are just begin- promoter OA levels later became depleted, the
ning to be described, and in the following interaction developed to a harmful one for the
review, we will focus on how the targeting of bacterium, and as a result, OA-resistant bacte-
the “fungus microbiome” in more detail will rial variants arose that allowed bacterial
help us understand fundamental biological pro- growth. The growth of the bacterium created a
cesses. The review highlights recent contribu- stage of nutrient competition with the yeast,
An Emerging Interdisciplinary Field: Fungal–Bacterial Interactions 163
Table 8.1 Same functional properties occur in FBI-based processes taking place in different niches
and as an ecological consequence of this diver- fungus-specific bacterial groups could be dis-
sification of bacteria, a new change in the com- tinguished, and among the Pseudomonas com-
munity appeared: the interaction became munities, there was an increase in diversity in
antagonistic. This was due to bacterium-caused most of the fruiting bodies tested. The selection
alkalinization of pH and consequent starvation of the bacteria was strongly related to their
of yeast cells. It led to the extinction of the yeast capacities to use characteristically fungus-
partner. enriched carbohydrates, arabitol, mannitol,
Elsewhere, Romano and Kolter (2005) dis- and trehalose.
covered a beneficial interaction, reminiscent to
the commensal phase of the last report, between
Saccharomyces cerevisiae and Pseudomonas
putida. When the bacteria were incubated B. Coevolution in Ant Fungus Gardens:
alone glucose-rich environment, they lost via- Antagonistic Warfare Keeps the Balance
bility rapidly during stationary phase: the bac- on the Side of Good Fungi
teria seemed unable to control the activity of
their glucose dehydrogenase, and the conver- The mutualism between “fungus-gardening”
sion of glucose to gluconate led to a pH drop insects and their fungal associates is an excel-
and bacterial growth arrest. The pseudomo- lent example of specialized networks of bacteria
nad’s stationary phase survival improved dra- and fungi, focused on insect nutrition. Most
matically by the presence of the yeast, since attention has been given to tripartite animal–
yeast rapidly consumed part of the glucose, bacterium–fungus interaction between fungus-
practically eliminating the pH drop. The growing attine ants, their bacterial compa-
interactions between bacteria and fungi in the nions, and fungal associates. The ants are
extramatrical mycelium may also express dependent on their fungal associates, since the
mutualistic features. For instance, the mycor- cultivars serve as the sole food source for the
rhizal fungus Morchella crassipes can be termed ant larvae and queen. The fungus gardens are
“bacterial farmer,” since the bacteria can under attack from parasites, in particular from
migrate on the fungal mycelium and consume the ascomycete genus Escovopsis, and the ants
nutrition from fungal exudates. The fungus keep their gardens free of microbial pathogens,
then harvests and translocates bacterial carbon thanks in part to beneficial bacteria (Currie
(Pion et al. 2013). Warmink et al. (2009) et al. 1999, 2006). These Actinobacteria sym-
showed that there is a decrease of the diversity bionts of the leaf-cutting ants, often belonging
of the total bacterial communities in the soil to Pseudonocardia and Streptomyces genera,
from beneath fruiting bodies, which also argues support the ants in defending their fungus gar-
for selection or “farming” of bacteria by the dens against infections by providing antimicro-
host fungal mycelium. Both universal and bial and antifungal compounds.
164 M. Tarkka and A. Deveau
Antibiotic assays suggest that despite gardens will give us further insight to this
Escovopsis being generally susceptible to inhi- fascinating FBI network.
bition by diverse Actinobacteria, the ant-
derived Pseudonocardia isolates inhibit the
parasite more strongly than they inhibit other C. Polymicrobial Biofilms as Virulence Factors
fungi and are better at inhibiting this pathogen
than most environmental strains, indicating Traditional microbiological research empha-
that fungus-growing ants may maintain sized the study of single isolates of microorgan-
specialized symbionts that help with garden isms in liquid culture, but the past decades have
defense (Cafaro et al. 2011). Some antibiotics witnessed a growing interest on the structure and
produced by Actinobacteria associated with function of the dominant mode of microbial life,
attine ants selectively target Escovopsis but not consortia between different species, and strains
the fungal garden, such as candicidin and den- of microorganisms. Wherever the submerged
tigerumycin (Oh et al. 2009; Haeder et al. 2009), microbial cells encounter surfaces, they can
and some of the secondary metabolites express switch from a planktonic lifestyle to form sessile
strong synergistic effects against Escovopsis, communities that are enclosed by a slimy
but some of the antifungal substances are not matrix termed biofilms (Battin et al. 2007).
only active against pathogenic fungi but also The biofilms are heterogeneous communities
the garden fungus itself, e.g., the antifungal encased in an extracellular matrix and from
antimycins (Schoenian et al. 2011). In conclu- their form often complex three-dimensional
sion, secondary metabolites of microbial sym- architectures (Xiao et al. 2012; Ramı́rez Granillo
bionts of leaf-cutting ants contribute to shaping et al. 2015). The chemical properties of biofilms
the microbial communities within the nests of are distinct from the planktonic cultures, as these
leaf-cutting ants. Parasitic yeast affects the ant tightly packed cells share resources, compete, or
fungus gardens as well. Phialophora, a genus of cooperate in order to survive.
ascomycete black yeasts, also grow on the ants’ The biofilms are complex and dynamic sys-
cuticle and appear to be localized to sites where tems, and as they grow and reproduce, they
mutualistic, Escovopsis-killing bacteria occur. may possess novel or amplified biochemical
The basis for this localization is unknown, but activities, as has been, e.g., suggested for
the yeasts appear to obtain nutrients from the petroleum-degrading microorganisms (White
bacteria and have a negative effect on their et al. 1995; Charlesworth et al. 2012). For path-
growth, which reduces their antibiotic effects ogenic ones, the protection of fungi and bacte-
on Escovopsis (Little and Currie 2008). In con- ria by biofilm formation is often manifested
clusion, the microbial symbionts of leaf-cutting through increased tolerance against host
ants contribute to shaping the microbial defenses and high-level resistance to antimicro-
communities within the nests of leaf-cutting bial drugs. There is considerable evidence to
ants. As the common patterns of garden fungus suggest that choosing antibiotics based on bio-
and parasites occur in the fungal gardens of film rather than conventional antimicrobial
ants, beetles, and termites, all insects that have susceptibility testing could potentially improve
independently evolved nutritional symbioses response to antimicrobial treatments. For these
with fungi, it is an important question whether reasons, Candida albicans and Aspergillus
the gardens harbor similar microbiomes. fumigatus, common fungal participants of
Aylwards et al. (2014) showed that this is pathogen polymicrobial biofilms in mammals,
indeed the case; the microbiota, their fungal have centered a wealth of pioneering FBI
gardens, are remarkably similar, comprising research, which has been extensively reviewed
primarily of the bacterial genera Enterobacter, in recent years (e.g., Wargo and Hogan 2006;
Rahnella, and Pseudomonas. Future work Harriott and Noverr 2011). For this reason, the
assessing the roles of these bacteria in fungal following includes just a few examples of inter-
An Emerging Interdisciplinary Field: Fungal–Bacterial Interactions 165
kingdom signaling and recent microbiome (Mowat et al. 2010). Whereas A. fumigatus
approaches. monoculture biofilms show organized struc-
C. albicans is a resident of mucosal tures with extensive hyphal growth and extra-
surfaces. It can overgrow under favorable cellular matrices, P. aeruginosa antibiosis leads
conditions, particularly when the immune sys- to disorganized fungal structures and limited
tem is compromised, leading to a wide range of hyphal growth (Ramı́rez Granillo et al. 2015).
diseases, referred to as candidiasis. C. albicans P. aeruginosa inhibits A. fumigatus, just like
infections often involve more than one cellular C albicans, by the production of phenazines,
morphology; yeast, pseudohyphal, and hyphal and at higher dozes, these antibiotics block
growth forms have all been associated with fungal growth. But lower concentrations of phe-
virulence (Wargo and Hogan 2006). Numerous nazines allow slow extension of mycelium and
studies have described its interactions with thereby affect the development of A. fumigatus
bacteria, and C. albicans–bacterium interac- by means of oxidative stress regulation (Zheng
tions can promote or prevent human diseases et al. 2015). Wrinkled colony biofilms with
(Morales and Hogan 2010). C. albicans infec- higher oxygen concentrations develop (Morales
tions are associated with the formation of et al. 2013) and fungal yeast-to-filament transi-
mixed biofilms, where C. albicans and bacteria tion is inhibited, but also asexual sporulation is
adhere to one another through the action of stimulated and an iron-uptake system is
surface adhesins and extracellular polymers. induced (Briard et al. 2015; Zheng et al. 2015).
Another fungal pathogen, Aspergillus fumiga- These data give insight to the interkingdom
tus, causes severe lung diseases such as asper- signaling and show that antifungal substances
gillosis in immunocompromised patients, and can be assessed not only as growth inhibitors
biofilm formation by A. fumigatus is an impor- but also as interspecies signaling compounds.
tant virulence factor in these diseases. Human serum induces hyphal development
C. albicans interactions with bacteria are of Candida albicans, and given the abundance
modulated by farnesol, a fungal quorum- of bacterial cell wall fragments in the intestine,
sensing molecule (Hogan et al. 2004). Farnesol a natural habitat and invasion site for C. albi-
represses hyphal growth and early biofilm for- cans, one would expect that the fungus
mation and is required for virulence during responds to bacterial cell wall fragments. Xu
scattered infection and affects bacterial patho- et al. (2008) showed that this is the case:
gens, inhibiting biofilm formation and bacterial C. albicans virulence is induced by the percep-
virulence. For instance, farnesol production by tion of bacterial cell wall fragments, muramyl
C. albicans leads to decreased antibiosis by dipeptides that are present in low concentra-
P. aeruginosa, and this takes place by decreased tions in the human serum. And C. albicans and
production of P. aeruginosa quinolone signal. bacteria may promote coaggregation and for-
The bacterial quinolone signal acts in concert mation of mixed-species biofilms.
with other bacterial quorum-sensing regulators Co-occurring in the oral cavity, C. albicans
and controls the production of P. aeruginosa and the caries-promoting bacterium Strepto-
extracellular proteases, hydrogen cyanine, and coccus mutans express a synergistic interaction
phenazines (Cugini et al. 2007). Farnesol usage and a most aggressive onset of caries. Mixed
may be a strategy of C. albicans growing in biofilms reach higher biomass and cell numbers
biofilms to compete under conditions of stress, than mono-species biofilms. The resulting
and due to the inhibition of sessile biofilm three-dimensional biofilm architecture displays
formation, it may also lead to dispersal of bac- small S. mutans colonies surrounded by fungal
teria from the polymicrobial biofilm (Harriott cells, which are embedded in a dense extracel-
and Noverr 2011). P. aeruginosa also sup- lular polysaccharide matrix (Falsetta et al.
presses Aspergillus fumigatus biofilm forma- 2014). Coexistence with C. albicans induces
tion in vitro, perhaps leading to comparably the expression of virulence genes in S. mutans,
low mortality in the lungs of cystic fibrosis and C. albicans achieves this by including the
patients with P. aeruginosa as an inhabitant complete quorum-sensing system of S. mutans
166 M. Tarkka and A. Deveau
(Sztajer et al. 2014). C. albicans forms biofilm other, to the mutual success of the interaction
communities also with Streptococcus gordonii, a (Frey-Klett et al. 2011). Plant-associated fungal
ubiquitous oral bacterium. The molecular systems have become complementary models
nature of bacterial-fungal contact has been of endobacterial symbioses. Hoffmann and
examined in this association, and the formation Arnold (2010) recovered phylogenetically
of dual-species S. gordonii–C. albicans biofilm diverse endohyphal bacteria occur in living
communities involves interaction of the bacte- hyphae of diverse foliar endophytes, but the
rial cell wall-anchored SspB protein and hyphal presence of intracellular bacteria is particularly
filament surface Als3 protein (Bamford et al. well established among the species of mycor-
2009; Silverman et al. 2010). The attachment is rhizal fungi (Bianciotto et al. 2004; Bertaux et al.
mediated by O-mannosylation in Candida albi- 2003). Reviewed in the paragraph X of this
cans surface (Dutton et al. 2014). volume, the endobacteria of Glomeromycota
In addition to cross-kingdom adhesion and were already discovered in the early 1970s on
quorum sensing, metabolic changes in the envi- the basis of electron microscope observations
ronment (pH, metabolic substrate production, (Mosse 1970).
inhibitor production, nutrient sensing/seques- The bacterial endosymbionts of the
tering, etc.) and indirect activity on the host Glomeromycota fungi provide ammonium and
response are mechanisms by which Candida thiamine for the fungus (Minerdi et al. 2001;
and other fungi interact with bacteria in the Ghignone et al. 2012) and stimulate spore for-
microbiome to regulate fungal levels and host mation and growth of germinating mycelium
responses to fungal colonization (Wargo and (Lumini et al. 2007). But another in-detail
Hogan 2006). These changes lead to enrichment investigated model of an intracellular symbiosis
of certain bacteria. When Fox et al. (2014) in fungi (Lackner et al. 2011) exists between the
investigated mixed biofilms composed of the plant pathogen, rice seedling blight fungus Rhi-
major fungal species of the gut microbiome, zopus microsporus, and an endosymbiont bac-
C. albicans, five prevalent bacterial gastrointes- terium, Burkholderia rhizoxinica. The key
tinal inhabitants, Bacteroides fragilis, Clostrid- virulence factor of rice seedling blight is the
ium perfringens, Escherichia coli, Klebsiella phytotoxin rhizoxin, a potent cell cycle inhibi-
pneumoniae, and Enterococcus faecalis, they tor that kills plant and animal cells. Rhizoxin is
observed that biofilms formed by C. albicans not a fungal metabolite but in fact produced by
provided a hypoxic microenvironment that B. rhizoxinica (Partida-Martinez and Hertweck
supported the growth of anaerobic bacteria, 2005). Endobacteria are often very difficult to
even when cultured in ambient toxic conditions get in culture, but Partida-Martinez and Hert-
that are normally toxic to the bacteria. In sus- weck (2005) were not only able to culture the
pension cultures, C. perfringens induced aggre- endosymbiont in vitro but also to produce
gation of C. albicans into “mini-biofilms,” bacterium-free Rhizopus mycelium. This
which allowed C. perfringens cells to survive in enabled them to examine the roles of the
a normally toxic environment. partners of this intriguing relationship. A
specialized mechanism has evolved during evo-
lution that guarantees the persistence of the
D. An Intracellular Bacterial Symbiont Rhizopus-Burkholderia endosymbiosis. The
in a Plant Pathogenic Fungus bacteria invade the Rhizopus cells by excreting
fungal cell wall-degrading chitinolytic enzymes
Beyond the most commonly observed FB cell- and chitin-binding proteins through type
to-cell interactions, there is a growing number 2 secretion system (T2SS). The T2SS is required
of known endosymbioses and endobacteria for the formation of the endosymbiosis
living inside the fungal cells. In these intracel- (Moebius et al. 2014). The presence of Burkhol-
lular associations, bacteria grow and multiply deria endosymbiont is necessary for vegetative
within fungal hyphae, and the two partners may reproduction of Rhizopus, in particular
have developed strategies to interact with each for spore production (Partida-Martinez and
An Emerging Interdisciplinary Field: Fungal–Bacterial Interactions 167
Hertweck 2007), and this control measure extramatrical mycelium, bacteria, fungal
against the host fungus takes place by another, saprophytes, and protists. In the same way the
Hrp T3SS. Interestingly, Lackner et al. (2011) rhizospheres exert a pressure on microbial
showed that mutants defective in T3SS not only populations (Barea et al. 2005), the mycorrhizal
failed to elicit sporulation of the host but also roots and hyphae of mycorrhizal fungi (MF)
showed reduced intracellular survival of the shape the bacterial species composition due to
bacterium, underlining the intimacy and inter- root and hyphal exudation and turnover. Bac-
dependency of this endosymbiotic association. teria have been visualized inside mycorrhizas,
General importance of the secretion systems as as colonies on soil-colonizing fungal hyphae
delivery systems of effectors in FBI is hinted by and inside the fungal mycelium (Bertaux et al.
the role of T3SS in mycorrhiza symbiosis stim- 2003). Mycorrhizal fungi and bacteria undergo
ulation expressed by the mycorrhiza helper complex interactions that influence the biology
bacterium Pseudomonas fluorescens BBc6R8 of the fungus and the nutrition of the plant
(Deveau et al. 2010). The respective bacterial (Frey-Klett et al. 2007), with the plant partner
effectors warrant further examination. selecting for bacterial strains beneficial for the
symbiosis and for the plant (Frey-Klett et al.
2005). Although the mycorrhizal fungi interact
E. Soil and Wood as Arenas of FBI Affecting with bacterial strains which can have beneficial,
Nutrient Cycling and Microbe–Plant neutral, or harmful effects on fungal physiol-
Interactions ogy, this “mycorrhizosphere effect” may lead to
improved plant nutrition, growth, and disease
Soil represents a very heterogeneous environ- resistance (Frey-Klett et al. 2005). There is a
ment for the available organic and mineral body of evidence that points to the influence
nutrients, for water, and for its microbiota. of fungi on bacterial community structure at
Both fungi and bacteria are involved in biogeo- both the taxonomic and functional levels, nota-
chemical cycling processes of the soil, and a bly in the mycorrhizosphere (Frey-Klett et al.
main energy source driving the soil system is 2011). Determining the functional significance
formed by plants through the provision of of the mycorrhizosphere organisms for plant
assimilated carbon as rhizodeposits. The productivity presents a major challenge for sus-
microorganisms, for their part, mobilize soil tainable agriculture (Artursson et al. 2006). The
nitrogen and phosphorus and transfer them intimate relationship between the oldest of all
from the soil back to the plant, with intensive symbioses, arbuscular mycorrhiza, and fungus
fungus–bacterium interactions (Nazir et al. endophytic bacteria is described in chapter
2010), which can be in many instances seen as “Mycorrhizal Fungi and the Soil Carbon and
a cooperative system, with fungi, bacteria, and Nutrient Cycling.” In the following we focus
plants interacting with each other (Barea et al. on another level of FBI with importance to
2005; de Boer et al. 2005). mycorrhizal symbiosis, bacteria which stimu-
In the carbon-limited soil, the emergence of late mycorrhiza formation.
plant roots and the formation of fungus-root The presence of bacteria that are directly
mutualism termed mycorrhiza create nutri- involved in mycorrhiza formation was first
tional hot spots for soil bacteria providing an indicated by the studies of Bowen and Theo-
extensive and enduring energy source. The dorou (1979), which showed that some bacte-
majority of plants form mycorrhizas, and the rial isolates promoted and others inhibited the
mycorrhizal symbiosis improves plant nutrient colonization of Pinus radiata roots by Rhizopo-
and water uptake, while the fungal partner gon luteolus. Confirmed in other mycorrhizal
gains carbohydrates from its host plant. The forms (Garbaye and Bowen 1987; Meyer and
mycorrhiza-associated organisms are known Linderman 1986), the bacteria able to promote
to influence each other, the outcome of which mycorrhizal development have been collec-
is described as the “mycorrhizosphere” (Foster tively named as MHB: mycorrhiza helper bac-
and Marks 1967), comprising mycorrhizas, teria (Garbaye 1994). The presence of MHB as
168 M. Tarkka and A. Deveau
an ubiquitous group of microorganisms and the aggregation of bacteria during the phagocy-
important for mycorrhizal symbiosis is sug- tosis process (Huh et al. 1998). In L. bicolor, the
gested by the following findings: (1) MHB tectonin orthologue could thus play a role in
have been found whenever they have been cell recognition and/or fungal cell interaction
looked for, (2) they are present in very different with P. fluorescens BBc6R8.
habitats, (3) many of these bacteria seem to be Second central MHB model is Streptomyces
closely associated with MF, and (4) MHB can be sp. AcH 505, which promotes the extension of
found from taxonomically diverse bacterial fungal mycelium and mycorrhiza formation by
groups (Frey-Klett et al. 2007). There exists a the symbiotic fungus fly agaric Amanita mus-
multitude of interaction mechanisms promot- caria and Norway spruce (Maier et al. 2004).
ing mycorrhiza formation, and some directly Streptomyces–Amanita interaction has a strong
affect fungal growth. influence on the growth pattern of fungal
Mechanisms by which ectomycorrhizal hyphae. The hyphal diameter and the mycelial
fungi perceive and react to surrounding bacte- density decreased in cocultures with AcH 505.
ria have been explored in ectomycorrhizal The dense and polarized actin cap in hyphal
(mycorrhiza of forest trees and shrubs) model tips of pure culture A. muscaria changed to a
systems. The most studied one concerns loosened and dispersed structures in coculture
Laccaria bicolor S238N, the first genome- (Schrey et al. 2007). Apart from changes in cell
sequenced mycorrhizal fungus (Martin et al. biology, the interaction with AcH 505 has a
2008), which is commercially used in France strong impact on gene expression levels in
for controlled mycorrhization of Douglas fir A. muscaria, suggesting that the fungal physiol-
seedlings, because it significantly improves ogy is strongly altered due to the contact with
plant growth and survival. As the bacterium, the bacterium (Schrey et al. 2005). Whereas
the MHB P. fluorescens strain BBc6R8, signifi- Streptomyces AcH 505 promotes the extension
cantly improves the pre-symbiotic survival and of A. muscaria, it inhibits the ectomycorrhizal
growth of L. bicolor in the soil (Brule et al. 2001) fungus Hebeloma cylindrosporum. Riedlinger
as well as in vitro (Deveau et al. 2007). et al. (2006) found that AcH 505 produced the
P. fluorescens is chemoattracted by fungal fungal growth-promoter auxofuran, and two
extracts but also by trehalose, a disaccharide antifungal substances, WS-5995 B and
in L. bicolor mycelium. Conversely, BBc6R8 C. Fungi that were sensitive against WS-5995
secretes thiamine which promotes fungal B, were found to be inhibited in cocultures with
growth (Deveau et al. 2010). Thiamine (vitamin AcH 505 (Lehr et al. 2007). A. muscaria induced
B1) is an essential cofactor of several enzymes conditions that are favorable to fungal growth,
of the central carbon metabolism, and it has promoted auxofuran, and suppressed WS-5995
been previously associated with growth promo- B production by AcH 505 (Riedlinger et al.
tion of the yeasts Debaryomyces vanrijiae by 2006). Lehr et al. (2007) characterized 11 WS-
Bacillus sp. TB-1 (Rikhvanov et al. 1999). 5995 B-sensitive and 1 WS-5995 B-tolerant
Coculture with BBc6R8 led to altered growth strains among a collection of fungal plant path-
pattern of L. bicolor mycelium; angles and ogen Heterobasidion abietinum isolates. Sensi-
numbers of hyphal branches, as well as num- tivity against the antibiotic is reflected by
bers of hyphal apices, changed. Simultaneously, decreased expression levels of cell stress-related
pleiotropic alterations in fungal transcriptome genes (Lehr et al. 2007). Mycorrhizal fungus
were observed, which varied in time (Deveau enhanced the growth of AcH 505 (Kurth et al.
et al. 2007). An early-stage BBc6R8-responsive 2013), and AcH 505 affected the structure of
fungal gene sharing 54 % similarity at the pro- microbial community and improved plant
tein level to tectonin II of Physarum polycepha- resistance against nematode colonization
lum was identified, and later shown to be (Caravaca et al. 2015). Furthermore, AcH 505
induced when L. bicolor interacts with other elicited a systemic defense response in oak,
bacteria, as well (Deveau et al. 2015). In P. which was associated in reduced powdery mil-
polycephalum, tectonins may be involved in dew symptoms in leaves, indicative of priming
An Emerging Interdisciplinary Field: Fungal–Bacterial Interactions 169
of plant defenses by the bacterium (Kurth et al. taxa, as the numbers of cultivable wood-
2014). The defense elicitation process was atte- inhabiting bacteria were considerably lower in
nuated by Piloderma croceum (Kurth et al. wood blocks that became colonized by the
2015), presumably to faciliate root colonisation white-rot fungi Hypholoma fasciculare and
by the mycorrhizal fungus. Resinicium bicolor than control blocks. Analy-
FBI take place also at the degradation of sis of the bacterial community structure in soil
recalcitrant organic matter, e.g., lignocellulose adhering to the mycelial cords of the fungi
in litter or wood (de Boer et al. 2005). Wood revealed fungal species-specific effects on bac-
decomposition is an important process for the terial community composition. Bacteria can
forest biogeochemical cycle that is driven by cause changes in wood chemistry, permeability,
microorganisms such as white-rot fungi which and structure, which may favor subsequent
naturally coexist with bacteria. Soils are usually succession by fungi (Clausen 1996). Nitrogen
not saturated in water, but in order to find is limited in wood, and associations with N-
water-saturated microaggregates and to fixing bacteria may enable wood-decaying
degrade organic compounds, the bacteria may fungi to meet their nitrogen requirements for
have to move larger distances. Wick et al. have vegetative and generative growth (Hoppe et al.
identified a role of mycelia for the translocation 2014; Herve et al. 2014). Nitrogen-fixing bacte-
of organic compounds and microorganisms. ria have been isolated from mycorrhizal
Briefly, contaminant biodegradation in soil is (including truffle) fungi and in the fungus
frequently limited by hindered physical access gardens of leaf cutter ants, further suggesting
of bacteria to the contaminants, and, e.g., positive inputs of bacteria to fungal nutrition
Schamfuß et al. (2013) showed that water- (Minerdi et al. 2001; Pinto-Tomas et al. 2009).
insoluble hydrophobic organic compounds, Bacteria may also provide nutritional benefits
polyaromatic hydrocarbons, were transported in the surrounding soil while assisting fungal
on the hyphae of mycelial oomycete Pythium degradation of preserved woods through the
ultimum along air-water interfaces. Mycelia- sequestration or detoxification of preserving
dispersed hydrocarbons were degraded by the agents (Wallace et al. 2008).
bacterium Burkholderia sartisoli and the bacte- The description of these associations
rium enriched on hyphal surfaces. This sug- demonstrates the functional diversity of bacte-
gests that both bacteria and their substrates rium–fungus interactions. Treatment of the
can migrate and confront each other on fungal literature implies that there are yet-to-be dis-
hyphae and that this interaction is beneficial for covered potentials to unravel among the FBI,
the bacterium. This could in particular stimu- and the following paragraphs shall devote to the
late the degradation of organic compounds methodologies which allow us to dig even dee-
with patchy distribution and low solubility in per to this challenging subject.
water (Schamfuß et al. 2013). The ability of
fungi to colonize and decompose woody
resources may be strongly influenced by III. How to Study the FBIs?
wood-inhabiting bacteria that grow on easily
utilizable compounds released by the fungal A wide spectrum of techniques can be applied
enzymes. Since fungi acidify the substrate at to explore FBIs. They mainly fall into two
decomposition, coexisting bacteria have to categories: cultivation-dependent and
also be acid tolerant (Valásková et al. 2009; cultivation-independent approaches (Fig. 8.1).
Frey-Klett et al. 2011). Decay of wood by Cultivation-dependent approaches have been
white-rot fungi can be either inhibited or pro- for long time the main way used to describe
moted by associated bacteria, depending on the and analyze FBIs, but the recent fast expansion
relative timing of the fungal and bacterial of -omics techniques and their relatively easy
inoculations (Murray and Woodward 2003). use allow now to get inventories of microorgan-
Indicated by the study of Folman et al. (2008), isms colonizing almost any environment and to
fungal presence may suppress certain bacterial list potential functions that they could detain.
170 M. Tarkka and A. Deveau
Fig. 8.1 Methods which have been applied to explore fungal–bacterial interactions
Thus, these new tools offer great opportunities isolate microorganisms will strongly influence
to better describe the world of FBIs. The two the type of microorganisms that will be recov-
approaches can bring complementary informa- ered. Combination of different media and/or
tion and both have important pitfalls, and the creation of media that mimic the environment
best means is probably to combine them. from which the microorganisms are isolated
can help broadening the diversity of microor-
ganisms that are recovered (Vieira and Nahas
A. Cultivation-Dependent Approaches 2005).
Such process was used by Frey-Klett et al.
One of the methods the most frequently used by (2005) to isolate 220 bacterial strains from ecto-
microbial ecologists consists in isolating micro- mycorrhizae of Laccaria bicolor, Douglas fir
organisms from specific environments. Collec- and surrounding soil, and permitted to show
tions of pure microorganisms are obtained by that ectomycorrhizosphere—the zone of influ-
plating on selective or nonselective media. ence of ectomycorrhizae—selects for specific
Microorganisms are then most often identified Pseudomonas fluorescens strains that are poten-
by the sequencing of specific DNA regions such tially beneficial for the symbiosis and the plant.
as 16S ribosomal DNA region for bacteria and Using similar plate media isolation methods,
internal transcribed spacer (ITS) or 18S rRNA Warmink and van Elsas 2008 also demon-
for fungi. Microbial collections can further be strated that the mycosphere of Laccaria prox-
screened for numerous phenotypes, metabolic ima offered a niche to specific bacterial
properties, and functions, thus generating communities and that it was particularly
hypotheses that can be further tested in vitro enriched in bacterial strains possessing type
or in situ. It is important to take into account III secretion systems (T3SS). This observation
that the choice of medium composition used to led them to hypothesize that T3SS could have
An Emerging Interdisciplinary Field: Fungal–Bacterial Interactions 171
new unknown functions and play a role in the BBc6R8 stimulates the growth of the ectomy-
interaction of those bacteria with their fungal corrhizal fungus Laccaria bicolor and promotes
host. the establishment of symbiosis between the
One of the main drawbacks of cultivation- fungus and tree roots. We first developed an
based analyses is due to the fact that a very in vitro assay in which the bacterium stimulates
small percentage of microorganisms can be the fungal growth and then analyzed the
cultivated. Therefore one shall keep in mind transcriptomic responses of the fungus and
that cultivable approaches will provide a very the bacterial strain during their interaction
biased view of the microbial community com- (Deveau et al. 2007, 2014). These responses
position. Thus it is most relevant to analyze, in were compared to those induce by and in
parallel, the community composition using fin- other bacterial strains to determine the degree
gerprinting or bar coding methods. A second of specificity of the interaction. In parallel,
limitation of cultivation is that functional capa- we acquired the genome sequence of strain
cities obtained by in vitro bioassays only pro- BBc6R8. Genome mining and comparative
vide an image of the functional potential of the genomics to other P. fluorescens strains pointed
microorganisms. Whether these functionalities to a peculiarity of strain BBc6R8: while being a
are expressed in the natural environment is nonpathogenic bacterium, it possesses a com-
hypothetical. Despite these drawbacks, cultiva- plete cluster encoding for a potential type III
tion approach has the immense advantage to secretion system (Cusano et al. 2010). These
generate hypotheses which can be tested in systems serve as molecular syringe to inject
controlled environments since the microorgan- effectors in eukaryotic host cells, but their func-
isms of interest can be grown and tracked. tion is unknown in commensal bacteria. How-
Specific microorganisms can then be used as ever, bacteria harboring T3SS-encoding genes
model systems to decipher molecular mechan- are enriched in the vicinity of some soil fungi
isms of interactions. Almost any molecular (Warmink and van Elsas 2008; Viollet et al.
biology tool can be applied to study FBIs and 2011), suggesting a potential role in the interac-
a large number of them have been successfully tion with fungi. We thus hypothesized that
used to decipher mechanisms of FBIs (Fig. 8.1). T3SS could be involved in the helper effect of
This requires the design of a bioassay setup in strain BBc6R8 and we carried out a targeted
which the interaction can be tracked. mutagenesis of the T3SS genes. We further
Depending on the microorganisms studied, showed that T3SS was essential to the helper
liquid cultures (Hogan and Kolter 2002; Bal- ability of strain BBc6R8 as T3SS mutants were
bontı́n et al. 2014), Petri dish agar-based bioas- not anymore able to promote symbiosis. The
say (Schrey et al. 2005; Morales et al. 2010; Mela creation of a library of random inserted trans-
et al. 2011; Pion et al. 2013), or more complex poson mutants of strain BBc6R8 and its screen
systems containing natural substrates such as allowed us to identify four additional mutants
soil (Nazir et al. 2014; Aspray et al. 2006; Kurth that lost their ability to promote symbiosis
et al. 2013) or wood (Hervé et al. 2014) or (Deveau et al. unpublished). Further analyses
including hosts (Peleg et al. 2008; Nash et al. are on going to identify the role of the mutated
2014) have been designed. Microfluidic systems gene products in the interaction. This combi-
that allow one to probe FBIs at the single cell nation of descriptive transcriptomic (Deveau
level were also recently developed (Stanley et al. et al. 2007, 2014; Mela et al. 2011), proteomic
2014). These devices provide a platform to fol- (Melin et al. 2002), or metabolomic (Phelan
low in vivo interactions by microscopy and to et al. 2014) methods together with targeted
perform time-lapse analyses that are otherwise analyses permit to obtain an overview of the
particularly challenging to obtain. As an exam- interaction and to identify mechanisms. Since
ple of the different tools that can be combined all these experiments are performed in vitro,
to analyze FBIs, we describe here the process we one may ask how much they tell about what is
used to study the mechanisms by which the really happening in natural settings. An inter-
soil-born bacteria Pseudomonas fluorescens esting approach consists in going back to
172 M. Tarkka and A. Deveau
(semi)complex environments using synthetic tion of the main microorganisms from DNA
communities in controlled environments. It fragments can be implemented through gel
requires the development of ways to track extraction followed by sequencing.
down the microorganisms such as quantitative However, one needs to keep in mind that
PCR, fluorescent labeling of microorganisms, the fingerprinting methods can only provide a
or fluorescent in situ hybridization. In this pur- partial image containing the major representa-
pose, a system based on gamma sterilized soil- tives of a microbial community. Nevertheless,
perlite substrate and amended with a defined they represent powerful tools to getting an
microbial starting community, using peduncu- overall profile of microbial communities, to
late oak as the plant host, was developed (www. compare community patterns and to analyze
trophinoak.de; Tarkka et al. 2013). In this sys- very large sets of samples for a reasonable
tem they could co-inoculate MHB bacteria, cost. For example, DNA fingerprinting was
ectomycorrhizal fungi, soil bacterial commu- used to test whether diversity of microorgan-
nities, and host plant and follow the impact isms and macroorganisms are driven by similar
plant and bacterial native communities on the processes – dispersal and selection (Ranjard
MHB-ectomycorrhizal fungi interaction (Kurth et al. 2013). The comparison of the bacterial
et al. 2013). This type of top-bottom bottom- diversity between 2085 soil samples covering
top approach has the advantage of providing 5.3105 km2 of French landscape by DNA fin-
experimental settings that are closer to the real- gerprinting demonstrated that the turnover
ity of complex communities while allowing one rate of bacterial diversity in soils on a wide
to test mechanistic hypotheses. scale is very high and strongly correlated to
the turnover rate of soil habitats.
DNA bar code sequencing of short genetic
B. Methods Which Are Independent of markers such as bacterial 16S ribosomal RNA
Cultivation or fungal internal transcribed spacer between
the 5.8S and 18S rRNA sequences, often
Research has focused for a long time on very referred to as monogenic rRNA metagenomic
specific organisms that have been identified to studies, are a powerful alternative to finger-
be either the good or the bad guys. The concept printing. Similarly to fingerprinting, they per-
of microbiome is very ancient and we know mit to measure the richness of a community—
since decades that microbes live and behave alpha diversity—and to compare community
within and as complex communities. However, profiles, but they also give access to the taxo-
most of the tools that allow to dissect and to nomic composition of community members.
question deeply these community behaviors The level of precision in the taxonomic identi-
were developed only very recently. The con- fication depends on the marker used and on the
cepts of keystone species, functional redundan- class of microorganisms studied. For example,
cies, community resilience, etc. that have been fungal ITS and rRNA 18S, and bacterial 16S
intensely analyzed for macroorganisms remain markers generally permit to assign sequences
understudied in microbial ecology, particularly up to genera but will not allow to distinguish
in the field of FBIs. Three different approaches between species (Bruno et al. 2015). These tools
are commonly used to address these questions: can be applied to describe bacterial commu-
taxonomic analyses of FBIs communities, func- nities associated to fungi or to analyze the
tional analyses, and spatial organization of response of mixed fungal–bacterial commu-
the FBIs in communities. A first glance of the nities to environmental changes.
diversity of a microbial community can be They have been applied to many different
given by fingerprinting techniques. Finger- types of ecosystems as, for example, cystic
printing methods (Zhou et al. 2015) differenti- fibrosis lung patients (Willger et al. 2014),
ate groups of microorganisms based on unique cheeses (Wolfe et al. 2014), decomposing
characteristics of biomolecular markers such as wood (Hervé et al. 2014), and truffle mush-
DNA sequence (DGGE, TGGE, ARISA, etc.) or rooms (Antony-Babu et al. 2014). Bar code
phospholipid (PLFA) composition. Identifica- sequencing has fantastically enhanced our
An Emerging Interdisciplinary Field: Fungal–Bacterial Interactions 173
capacity to describe the composition of micro- nities. They now need to be applied to the FBI
bial community and to analyze how they can be field.
impacted by biotic and abiotic factors and how
much these microbial communities influence
their host physiologies. Large catalogs of the IV. Perspectives
microorganisms that colonize many environ-
ments are now available. Despite this knowl- To date, the evidence for the FBI argues for
edge, most environments colonized by fungi common themes governing the physical and
and bacteria remain a black box because we biochemical characteristics of these associa-
have a poor understanding of the actual activ- tions, whatever the ecological niches investi-
ities performed by these microorganisms in gated. The development of bacterial-fungal
situ. In addition, little is known on how model systems (Hogan et al. 2004; Deveau
organized these communities are. Indeed, et al. 2007: Partida-Martinez and Hertweck
most microorganisms behave differently when 2005) has been crucial for unraveling the phys-
they are growing alone or in interaction with ical interactions and communication patterns
others (Frey-Klett et al. 2011; Scherlach et al. between the hyphae and bacterial cells. The
2013). Therefore the spatial organization of observation of growth substrate as well as bac-
complex microbial communities most likely terial migration on hyphal surfaces has revolu-
influences the activity of each organism. Net- tionized our idea on the migration of bacteria
work analyses of metagenomic datasets can and mobility of substrates over air gaps.
provide a first glimpse of this structure by Increased number of model systems investi-
inferring cooccurrence between microorgan- gated by functional genomics, microscopy,
isms (Faust and Raes 2012; Hoppe et al. 2014). and metabolite analyses, in a way that has
FBI community structures can also be assessed been implemented for the Burkholderia–Rhizo-
by microscopy using fluorescence in situ hybri- pus endosymbiosis, is necessary for a deeper
dization (FISH). Recent progresses made in understanding of the FBI. Confrontation stud-
confocal microscopy and the expansion of fluo- ies have been implemented mostly in simple,
rescent probes now allow to visualize simulta- artificial systems which are suitable for the
neously up to 15 different microbial phylotypes identification of interaction mechanisms
in a single sample (Valm et al. 2011). Addition- (Andrade-Domı́nguez et al. 2014), but the use
ally, visualization of 3D spatial distributions of of more complex substrates (Kurth et al. 2013)
microbial communities can now be obtained as is necessary when the relevance of the in vitro
illustrated by Erlacher et al. (2015) with lichen- data is questioned. Bacterial enrichment by
associated microbial communities. The next fungi (Fox et al. 2014) and fungal enrichment
question one will ask is what are the activities by bacteria (Caravaca et al. 2015) are emerging
these microorganisms are expressing in situ themes and clearly worthy of further consider-
and how are these activities altered by biotic ation as potential targets for investigation are
and abiotic parameters. This is probably the microbial community-based questions. Started
most important but also the most challenging by in situ analysis of microbial community
question to answer. Zhou et al. (2015) recently structure, subsequent reconstruction of artifi-
proposed an extended review of both open and cial microbial communities by adding different
close format technologies to analyze the com- combinations of widely distributed and cultiva-
plex microbial communities at the functional ble genera of bacteria and fungi on seminatural
level. “Close format techniques” refer to tech- substrate, and monitoring the establishment
nologies using known biomarkers such as Geo- and FBI (Wolfe et al. 2014), surely is a powerful
Chip (He et al. 2010) by contrast to the “open way to address FBI in a community context. A
format” ones which do not require prior knowl- growing number of examples suggest that the
edge of the community. Until now, these tech- FBI serve as a framework or even facilitates
niques have been mainly applied to study fungal symbioses with animals and plants
separately either bacterial or fungal commu- (Frey-Klett et al. 2007, 2011; Morales and
174 M. Tarkka and A. Deveau
Hogan 2010), and the future research should Bianciotto V, Genre A, Jargeat P, Lumini E, Bécard G,
dissect the direct FBI effect from direct impacts Bonfante P (2004) Vertical transmission of endo-
bacteria in the arbuscular mycorrhizal fungus
of bacteria and fungi on the host immunity or Gigaspora margarita through generation of vege-
nutrition. The data reviewed here strongly sup- tative spores. Appl Environ Microbiol 70:3600–
port the postulate (Frey-Klett et al. 2011) that 3608
fungus–bacterium interactions are an emerging Boer W, Folman LB, Summerbell RC, Boddy L (2005)
research theme which binds different scientific Living in a fungal world: impact of fungi on soil
bacterial niche development. FEMS Microbiol Rev
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Bowen GD, Theodorou C (1979) Interactions between
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9 Lichen–Bacterial Interactions
provide unique niches for bacteria, suggesting phylogenetically diverse bacteria colonize
that bacteria are an integral component of lichens and that the isolated bacteria also may
lichen symbioses. The bacterial communities contribute auxiliary functions to the symbiosis.
are host specific and reach densities of up to Although the cultivable fraction in most of
1010 bacteria per gram of lichen dry weight. the cases represents a very low proportion of
Moreover, they are able to form biofilm-like the whole microbiome, antagonistic bacteria
communities on the thallus surfaces (Grube are now well presented in the culture collec-
et al. 2009; Cardinale et al. 2012b). The associa- tions (Grube et al. 2009; Cernava et al. 2015).
tions of lichens with bacteria seem to be very Altogether, 24.5 % of all isolates were shown
old, as they were demonstrated in fossilized to display antagonistic properties against
lichens already from the lower Devonian period plant and lichen pathogens in vitro. Cultured
(Honegger et al. 2013). isolates are amenable to further experimental
The present data, which mainly come from approaches, which may show their interaction
lichens with green algal photobionts, reveal a with fungal hyphae in greater detail (Senevir-
predominance of Alphaproteobacteria as well as atne and Indrasena 2006). Cultivation-
presence of other bacterial phyla, such as Acid- independent methods, originally accomplished
obacteria or Actinobacteria. Many of these by DNA community fingerprints and clone
likely represent novel species (Selbmann et al. libraries, were used to gain more inside in the
2010; Lee et al. 2014; Sigurbjörnsdóttir et al. diversity of the total bacterial community or
2014), but only few of the lichen-associated of specific groups, such as Actinobacteria.
strains were so far described as new taxa (Li For example, Gonzáles et al. (2005) showed
et al. 2007; Lang et al. 2007; An et al. 2008, 2009; differences of actinobacterial associations with
Hamada et al. 2012; Yamamura et al. 2011a, b; lichens between tropical and cold climatic
Cardinale et al. 2011). Since only a minor frac- regions using fingerprinting methods.
tion of lichen-associated bacteria is culturable Grube et al. (2009) provided first evidence
and because fungal symbionts grow too slow for host specificity of bacterial communities.
for efficient experimental resynthesis of bacte- They compared DNA fingerprints of bacterial
rial associations, the analysis of interactions is a communities from three lichen species occur-
challenging topic. In the first section, we will ring in the same subalpine habitat. Host speci-
therefore present an outline of the different ficity was confirmed by subsequent studies,
methods that have so far been used to study either using gene clone library sequencing or
these hardly culturable symbioses. amplicon sequencing (Bjelland et al. 2011; Bates
et al. 2012). Amplicon sequencing meanwhile
became the gold standard to assess microbial
community structure, and this technique also
II. Methodological Approaches helped in exploration of ecological influence on
bacterial community structure (Cardinale et al.
Lichen-associated bacteria have been studied 2012a, b; Grube et al. 2012).
using a range of techniques. Culture-dependent In parallel with sequencing approaches,
studies date back to the first half of the last lichen-associated bacteria were also studied by
century and were used to verify the presence advanced microscopic approaches. While pre-
of nitrogen-fixing bacteria (e.g., Henckel and vious analyses with light or electron micros-
Yuzhakova 1936; Iskina 1938). While Cardinale copy revealed the presence of bacteria, it is
et al. (2006) confirmed the growth of isolated only possible with fluorescence in situ hybridi-
lichen-associated bacteria on N-free media, N- zation (FISH) to study the structure of bacterial
free enrichment media were used to selectively colonization in more detail. For this purpose,
sample nitrogen-fixing bacteria from tropical Cardinale et al. (2008) introduced a method to
green algal lichens (Liba et al. 2006). Molecular directly make all steps of FISH using fragments
data in these studies already revealed that of lichens instead of fixed sections. It was then
Lichen–Bacterial Interactions 181
Fig. 9.1 Confocal laser scanning microscopy (CLSM) of maximum projection; (f) single optical slice. In (b)
bacteria associated with L. pulmonaria. Bacteria are white box indicates the region shown in (d). Arrow in
stained by fluorescence in situ hybridization (FISH). (f) indicates an endophytic bacterial cell. Scale bars, (a–c)
Green, algae; blue/purple, fungi; yellow, Rhizobiales; red, 30 mm, (d–f) 5 mm. (Reproduced from Erlacher et al. 2015;
other bacteria. (a, b, d) Three-dimensional models made #Frontiers Media S.A.)
of isosurfaces and spheres; (c) volume rendering; (e)
182 M. Grube et al.
sent different species or their chemical variation naria, and most of the Rhizobiales belonged to
is driven by other factors. In addition, one new the families Methylobacteriaceae, Bradyrhizo-
compound, cyaneodimycin, an acrylate deriva- biaceae, and Rhizobiaceae. Erlacher et al.
tive, was isolated from the lichen-inhabiting (2015) studied this order in more detail using
S. cyanofuscatus. Six other known compounds the available metagenomic dataset. About 20 %
were also characterized (diketopiperazines, of our metagenomic assignments could not be
actinomycin derivatives, and indole deriva- placed in any of the Rhizobiales lineages, which
tives). Total EtOAc extract of S. cyanofuscatus indicates the incomplete knowledge of this
showed antibacterial activities against Staphylo- order. Focused on Rhizobiales, the SEED-
coccus epidermidis and antiproliferative proper- based functional analysis revealed again func-
ties against B16 and HaCaT cell lines (IC50 tions supporting the symbiosis, including auxin
0.33 mg/ml and 0.25 mg/ml, respectively). More- and vitamin production, nitrogen fixation, and
over, cynomycin exhibited antiproliferative stress protection.
properties against Jurkat cell lines after 72 h of Similar results were found meanwhile also
incubation with IC50 value of 18.5 mM. in other lichens, e.g., in an analysis of metagen-
omically derived 454 sequences from Peltigera
membranacea after subtraction of sequences
attributed to the primary fungal and cyanobac-
IV. Omics Technologies as Indicators terial symbionts (Sigurbjörnsdóttir et al. 2015).
of Interactions The dominant groups in P. membranacea are
Proteobacteria (Alphaproteobacteria 59 %,
Grube et al. (2015) explored the metabolic Betaproteobacteria 29 %), while Actinobacteria
potentials of the bacterial microbiome of the and Bacteroidetes represent minor fractions.
lung lichen Lobaria pulmonaria. Metagenomic This metagenomic data agrees largely with
and proteomic data were compared and visua- microscopic and amplicon sequencing studies
lized by Voronoi treemaps. The study was fur- (e.g., Cardinale et al. 2008; Mushegian et al.
ther complemented by molecular, microscopic, 2011; Hodkinson et al. 2012). Also P. membra-
and physiological assays. It was found that nacea hosts bacteria capable of synthesizing
more than 800 bacterial species grow on the indole acetic acid, albeit in a small number
lung lichen. This diverse collective may con- according to BLASTX hits to indoleacetimide
tribute multiple aspects to the symbiotic sys- hydrolase (most similar to those from Actino-
tem, including essential functions such as (1) bacteria and Betaproteobacteria). The few hits
nutrient supply, especially nitrogen, phospho- for chitinase A were nearly exclusively actino-
rous, and sulfur, (2) resistance against biotic bacterial. This is in accordance with observa-
stress factors (i.e., pathogen defense), (3) resis- tions that Actinobacteria are particularly
tance against abiotic factors, (4) support of associated with senescing thalli (Cardinale
photosynthesis by provision of vitamin B12, et al. 2012a). Several further glycosyl hydrolases
(5) fungal and algal growth support by provi- were representative for other bacterial classes,
sion of hormones, (6) detoxification of metabo- including Alphaproteobacteria. The use of
lites, and (7) degradation of older parts of the AppA phytase and AcpA acid phosphatase
lichen thallus. These findings showed the con- genes as query sequences for Peltigera meta-
siderable potential of lichen-associated bacteria genome data yielded diverse hits, with alpha-
to interact with the fungal as well as algal part- proteobacterial appA and betaproteobacterial
ner to support health, growth, and fitness of acpA homologs being particularly prominent.
their hosts. This supports the hypothesis that inorganic
Contributing to one third (32.2 %) of phosphate solubilization may be among the
the overall bacterial community, Rhizobiales roles of these abundant members (Sigurbjörns-
(Alphaproteobacteria) are the most common dóttir et al. 2015; Grube and Berg 2009; Grube
partners in the symbiosis of Lobaria pulmo- et al. 2015).
184 M. Grube et al.
Fig. 9.2 Functional model of the lichen symbiosis, partners, either in the algal layer or in cephalodia, is
including bacteria as third interacting partner on fun- not considered here; (2) the green algal photobionts
gal surfaces. Bacterial functions, light brown; algal form an apoplastic continuum with the mycobiont
functions, green; fungal functions, blue. Notes: (1) due to a surface layer with hydrophobins (Honegger
nitrogen fixation of lichens with cyanobacterial 1998)
VIII. Conclusions and Outlook Berg G (2015) Beyond borders: investigating micro-
biome interactivity and diversity for advanced bio-
control technologies. Microb Biotechnol 8(1):5–7
In this chapter, we characterized lichens as a Bjelland T, Grube M, Hoem S, Jorgensen SL, Daae FL,
more complex system than previously known, Thorseth IH, Øvreås L (2011) Microbial metacom-
by involving their relations with bacteria. These munities in the lichen–rock habitat. Environ
Microbiol Rep 3:434–442
new insights may stimulate further research,
Boustie J, Grube M (2005) Lichens—a promising
especially to find out what regulative mechan- source of bioactive secondary metabolites. Plant
isms help to keep the entire symbiotic systems Genet Resour 3:273–287
in balance and regulate the partnering physiol- Boustie J, Tomasi S, Grube M (2011) Bioactive lichen
ogies. Lichen–bacterial interactions are appar- metabolites: alpine habitats as an untapped
ently as old as the lichen lifestyle and eventually source. Phytochem Rev 10:287–307
Burkholder PR, Evans AW, McVeig I, Thornton HK
have also contributed to the fungal genome (1944) Antibiotic activity of lichens. Proc Natl
evolution. There are already indications for Acad Sci U S A 30:250–255
horizontal gene transfer events from bacterial Cardinale M, Puglia AM, Grube M (2006) Molecular
to fungal genomes, for example, in the diverse analysis of lichen-associated bacterial commu-
polyketide synthase genes (Schmitt and nities. FEMS Microbiol Ecol 57:484–495
Cardinale M, Vieira de Castro J Jr, Müller H, Berg G,
Lumbsch 2009). Yet, there are still many other Grube M (2008) In situ analysis of the bacterial
new questions for research. For example, little community associated with the reindeer lichen
is known so far about the diversity of lichen- Cladonia arbuscula reveals predominance of
associated Archaea. Lichens are a highly inter- Alphaproteobacteria. FEMS Microbiol Ecol
66:63–71
esting case of interkingdom interactions (Berg
Cardinale M, Grube M, Berg G (2011) Frondihabitans
2015), and they are specifically designed for cladoniiphilus sp. nov., a novel actinobacterium of
persistence and longevity under diverse ecolog- the family Microbacteriaceae isolated from the
ical conditions. We think this symbiosis reindeer lichen Cladonia arbuscula (Wallr.)
deserves more study in the future, since under- Rabenh. in the Austrian Alps. Int J Syst Environ
standing and exploiting of the functional prin- Microbiol 61:3033–3038
Cardinale M, Steinova J, Rabensteiner J, Berg G, Grube
ciples may be of considerable interest for M (2012a) Age, sun, and substrate: triggers of
biotechnological applications. bacterial communities in lichens. Environ Micro-
biol Rep 4:23–28
Acknowledgments MG and GB are grateful to the Aus- Cardinale M, Grube M, De Castro JV Jr, Müller H, Berg
trian Science Foundation FWF for the financial support G (2012b) Bacterial taxa associated with the lung
(P19098, I799, I882) and to Kathrin Riedel for the coop- lichen Lobaria pulmonaria are differentially
eration in the framework of a joint DFG-FWF project shaped by geography and habitat. FEMS Microbiol
(I882). Lett 329:111–115
Cardinale M (2014) Scanning a microhabitat: plant-
microbe interactions revealed by confocal laser
microscopy. Front Microbiol 5:94
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188 M. Grube et al.
2014; Chagnon et al. 2014; Schmitz and Harrison number of studies dedicated to the molecular
2014; Wyatt et al. 2014). diversity of AM fungi (i.e., Glomeromycota) in a
wide range of geographically distant “undis-
turbed” and disturbed soils (Öpik et al. 2013).
Current estimates of global species numbers
II. Mycorrhizae, a Diversity range from ca. 300 to 1600 (van der Heijden
of Plant-Fungal Associations et al. 2015). Such values reflect the fact that
many individual Glomeromycota species seem
The four main types that will be discussed in this to have a large, potentially global, geographic dis-
chapter are the so-called ectomycorrhizae (EcM), tribution; support a wide range of environmental
arbuscular mycorrhizae (AM), ericoid mycorrhi- conditions; and can associate to numerous, taxo-
zae (ErM), and orchid mycorrhizae (OrM). nomically unrelated, plants (Öpik et al. 2013).
According to an abandoned classification
The AM symbiosis can be well defined by the fungal scheme, AM, ErM, and OrM were all qualified
partners, which all belong to a single monophyletic fun- as “endomycorrhizae” as in all three cases the
gal lineage, the Glomeromycota clade (Schübler et al.
2001). With only one known exception (Geosiphon pyr-
fungal hyphae grow intracellularly in living host
iformis associated to Nostoc cyanobacteria), all Glomer- plant cells. Besides this similarity, these mycor-
omycota species are obligate biotrophs that cannot be rhizae are evolutionary and potentially function-
grown in vitro in the absence of a host plant. Both fossil ally distinct plant-fungal associations. First,
records (Redecker et al. 2000; Strullu-Derrien et al. 2014) hyper-ramified arbuscules are absent from ErM
and molecular phylogeny (Wang et al. 2010) suggest that
the AM symbiosis could be the most ancient mycorrhizal
and OrM where intracellular hyphae form so-
association, dating back to the Devonian period (at least called pelotons. Secondly, as suggested by their
400 Myrs ago), where it could have been instrumental in names, ErM and OrM concern a restricted num-
the colonization of nonaquatic terrestrial habitats by ber of plant species belonging, respectively, to the
green plants (Bonfante and Selosse 2010). As a matter families Ericaceae and Orchidaceae (Smith and
of fact, the AM symbiosis is found in most Embryophyta
lineages including early diverging ones such as liverworts
Read 2008; Brundrett 2009). Finally, ErM and
and hornworts (Selosse et al. 2015). A majority of present OrM also differ from each other with respect to
seed plant families, including families of agricultural their fungal partners. ErM involve a restricted
importance (e.g., the Fabaceae, Poaceae, and Solanaceae), number of fungal taxa (ca. 150 according to van
are concerned by the AM symbiosis, and absence of AM der Heijden et al. 2015, commonly assigned to the
associations in specific families (e.g., the Brassicaceae,
Caryophyllaceae, Chenopodiaceae, Juncaceae, Polygona-
Rhizoscyphus ericae (syn. Hymenoscyphus ericae)
ceae, and Proteaceae) should be seen as a secondary loss, aggregate (Helotiales, Ascomycota), Oidioden-
not as an ancestral character (Bonfante and Selosse 2010). dron species (Onygenales, Ascomycota), or the
The name arbuscular mycorrhiza derives from the arbus- Sebacinales (Basidiomycota) (Smith and Read
cule, a highly branched and metabolically active fungal 2008). However, there is evidence that the spec-
hypha that grows within a living plant rhizodermal or
cortical root cell. Despite growing intracellularly, the
trum of mycobionts detected in the Ericaceae
arbuscule does not disrupt the host plant cell integrity. may be considerably larger (Walker et al. 2011;
The arbuscule is indeed entirely surrounded by an inva- Gorzelak et al. 2012; Vohnik et al. 2012; Bruzone
ginated plant cell plasmalemma, and most of the apo- et al. 2015). As for OrM, they are mostly formed
plastic space between the fungal and plant plasmalemma by fungal species belonging to different clades in
is filled by a matrix of both plant and fungal origin (Genre
and Bonfante 2012; Gutjahr and Parniske 2013; Balestrini
the Agaricomycetes and Basidiomycota (such as
and Bonfante 2014). This AM symbiosis-specific struc- fungi in the “rhizoctonia” complex, encompass-
ture is thought to be the place where the bidirectional ing Tulasnellaceae, Ceratobasidiaceae, and Seba-
exchanges of nutrients between the plant and the fungus cinaceae), although Ascomycota in the
take place (Smith and Read 2008). Tuberaceae and Pezizaceae can also be involved
(Dearnaley 2007; Dearnaley et al. 2012).
Regarding the AM symbiosis, the large num-
ber and diversity of host plants contrasts with the Both ErM and OrM hosts can associate with fungi simul-
“relatively low” number of fungal species taneously forming EcM on neighboring trees (Villarreal-
described thus far, despite the ever-increasing Ruiz et al. 2004; Selosse and Roy 2009; Grelet et al. 2009,
Mycorrhizal Fungi and the Soil Carbon and Nutrient Cycling 191
2010; Hynson and Bruns 2010; Dearnaley et al. 2012). alpine, and arctic biomes are dominated by EcM
Although ErM and OrM could be regarded, at first forest trees, while this symbiosis is less frequent,
sight, as marginal cases of plant-fungal associations,
they have been the focus of numerous research works
but not absent in the tropics, with EcM trees in,
for two distinct reasons. Regarding ErM, a significant e.g., families Fabaceae, Caesalpiniaceae, or Dip-
fraction of land at high latitudes and altitudes and in terocarpaceae. From a fungal perspective, EcM
specific geographic zones (such as in the south of the fungi belong essentially to the Pezizomycotina
African continent) is covered by a vegetation dominated (Ascomycota) and the Basidiomycota (Smith
by Ericaceae plants (e.g., heathland), where it is sus-
pected that ericoid mycorrhizal symbionts play essential
and Read 2008). EcM fungi are “numerous” with
roles in plant nutrient acquisition (Smith and Read 2008). an estimated number of species exceeding 20,000
Orchids feature a peculiar mycorrhizal relationship, (van der Heijden et al. 2015) but do not share a
since association with symbiotic fungi providing organic common ancestor. Indeed, phylogenetic studies
carbon is essential for germination of the minute, reser- demonstrate that the “aptitude” at forming EcM
veless seeds and the development and survival of the
heterotrophic protocorm lifestage. At adulthood, achlor-
has appeared at least 80 times in different fungal
ophyllous orchids or orchids with reduced photosyn- lineages (Hibbett et al. 2000; James et al. 2006;
thetic efficiency (such as forest species, growing in Tedersoo and Smith 2013). Although EcM could
deeply shaded habitats) still depend on mycorrhizal be dated back to 100–200 M years ago (Brundrett
fungi for organic carbon supply. By contrast, green orch- 2002), transition from saprotrophy to symbiosis
ids thriving in exposed, grassland habitats are believed to
emancipate themselves from fungal-derived organic car-
could still be an ongoing process in some fungal
bon as soon as they form fully developed leaves, although lineages as suggested by the occurrence of both
they are thought to be still heavily reliant on their fungal saprotrophic and EcM species in single fungal
symbionts for mineral nutrition (Waterman and Bidar- genera such as Amanita (Wolfe et al. 2012).
tondo 2008; Selosse and Roy 2009; Rasmussen and Ras- Categorization of mycorrhizal associations
mussen 2014; Selosse 2014).
does not however reflect the diversity of these
associations under natural conditions. For
The last type of mycorrhizal association that example, many EcM plants can also host AM
will be discussed in this chapter is the ectomycor- symbionts (Smith and Read 2008) and most if
rhizal (EcM) one. As suggested by its name and as not all plant individuals simultaneously accom-
opposed to “endomycorrhizae,” in ectomycorrhi- modate different mycorrhizal symbiotic fungal
zae, the fungal hyphae do not penetrate plant cells species and genotypes on a single root system.
but rather entirely surround rhizodermal and/or For instance, Bahram and coworkers (2011)
external root cortical cells to form the so-called could identify up to 122 different EcM fungal
Hartig net where the nutrient exchanges between operational taxonomic units (OTUs, potentially
the plant and fungal partners take place. as many different species) and tens of genets of
Additionally, the fungal hyphae can entirely sur- the species Cenococcum geophilum on the root
round the plant absorbing rootlets to form a system of a single Populus tremula plant.
plectenchyma-like tissue called fungal mantle or
fungal sheath that “isolates” the plant root from
the surrounding soil (Smith and Read 2008). In
plants, the evolutionary origin of the EcM symbi- III. Mycorrhizae, a Hot Spot
osis is not fully resolved as it concerns essentially of Nutrient Exchange Between
woody plants (shrubs and trees) belonging to the Plants and Fungi
Gymnosperms (most of the Pinaceae) and the
Angiosperms. While most, if not all, species in, A classical and unified vision of mycorrhizae
e.g., the Fagaceae (oak, beech, chestnut), Betula- describes this association as a “balanced” plant-
ceae (birch, alder, hazelnut), Salicaceae (salix, fungus partnership where each of the two associ-
poplar), or Myrtaceae (eucalyptus) are ectomy- ates provides essential nutrients to the other one
corrhizal, a few woody species in otherwise over- at the plant-fungal interface (either arbuscules,
whelmingly AM plant families can also form EcM pelotons, or Hartig net). In a classical model,
(Smith and Read 2008; Brundrett 2009). From a plants allocate to their fungal partners
geographic perspective, forests in the temperate, photosynthesis-derived simple sugars (mono-
192 R. Marmeisse and M. Girlanda
and/or disaccharides) in exchange for soil- Nutrient versatility has essentially been
derived macronutrients (e.g., nitrogen, phospho- tested for EcM and ErM fungi, which can be
rus, potassium) provided by the fungi. Therefore, grown in vitro on controlled N and P sources. It
this model implicitly envisions the extraradical is now well established that, for example, EM
mycelium, which explores the soil matrix, as a fungal species can efficiently use proteins as sole
substitute to the root system itself for the func- N sources, thanks to secreted proteases (Abuzina-
tion of primary nutrient acquisition from the soil dah and Read 1986a, b; Bajwa et al. 1985). As a
solution (Girlanda et al. 2007). This model result, these fungi can provide N derived from
hypothesizes that the “beneficial effects” of the these macromolecules to their host plants (Abu-
mycorrhizal association recurrently observed on zinadah and Read 1986a, b). Thus, at the ecosys-
host plants’ growth and nutrition result from a tem scale, the mycorrhizal association “shortcuts”
combination of at least two main fungal traits. the N and P mineralization process of N- and/or
The first trait is the capacity of the extraradical P-containing organic molecules, normally neces-
mycelium to efficiently explore a larger volume of sary to the release of inorganic forms of P (PO42)
soil than the root system itself in the quest of low or N (NH4+ or NO3) that can be assimilated by
concentration and/or poorly mobile (e.g., phos- nonsymbiotic plants. “Unexpected” soil constitu-
phate ions) soil nutrients. The second trait is that ents have thus been demonstrated to represent
fungi display a wider metabolic versatility than direct and significant sources of P and/or N for
plants and can access soil nutrient sources not EcM plants. This is the case of pollen grains
readily available for plant direct uptake such as (Perez-Moreno and Read 2001a) or of the soil
the organic forms of nitrogen and phosphorus. animal necromass (Perez-Moreno and Read
2001b; Klironomos and Hart 2001).
Historically, the main predictions of this global model of
functioning of the mycorrhizal symbiosis have received
experimental support in a series of classical studies per-
formed on different plant-fungus associations. For exam- IV. Fungal “Omics,” Global
ple, labeled 14CO2 applied to plant leaves has been used to Approaches to Probe the Mycorrhizal
visualize the transfer of 14C from plant leaves to roots and
then to mycorrhizae and to the soil-colonizing extraradi- Symbiosis Lifestyle
cal mycelium (Ho and Trappe 1973; Bending and Read
1995). Conversely, the use of 32P-enriched poorly mobile Most of earlier studies on the mycorrhizal sym-
phosphorus sources added to soil compartments colo- biosis have addressed specific aspects of this
nized by fungal hyphae, but from which associated plant
roots were excluded, allowed visualizing the transfer of association such as the contribution of a particu-
phosphorus from mycorrhizal hyphae to the host plant lar fungal metabolic pathway to the plant mineral
(Finlay and Read 1986). More recently, the use of nonra- nutrition. Understanding this complex and
dioactive 13C and 15N sources fed to the plant and/or diverse plant-fungus interaction may benefit
fungal hyphae has also been used to visualize at the from a more holistic approach as provided by
cellular level by NanoSIMS the transfer of carbon and
nitrogen at the fungal-plant interface in both AM (Kaiser the so-called “omics” research field (genomics,
et al. 2015) and OrM (Kuga et al. 2014). In the AM transcriptomics, proteomics, but also environ-
symbiosis, the bidirectional transfer of nutrients at the mental genomics). Besides cataloging genes and
plant-fungus interface has also received direct molecular proteins from organisms, through their func-
support by the characterization of essential plasma- tional annotation and cross-species compari-
membrane nutrient transporters. From the plant side,
this is the case of a phosphate transporter (MtPT4 in the sons, “omic” approaches are also susceptible to
legume Medicago truncatula), present in most studied decipher the ecological roles of microbial species.
plant species, which is specifically expressed in
arbuscule-containing root cells and whose suppression
leads to premature arbuscule degeneration and a drop in
P transfer from the fungus to the plant (Harrison et al. A. The Saprotrophic-Mycorrhizal Continuum
2002; Javot et al. 2007; Breuillin-Sessoms et al. 2015). and Debate
From the fungal side, Glomus monosaccharide trans-
porter genes expressed in arbuscules were also shown to One of the most debated issue in the field of
be necessary to arbuscule formation (Helber et al. 2011). mycorrhizal research and on the roles of mycor-
Mycorrhizal Fungi and the Soil Carbon and Nutrient Cycling 193
rhizal fungi in soil nutrient cycling concerns In most cases, results from enzyme assays performed on
certainly the putative role of these species as environmental samples (root tips, soil) must however be
interpreted cautiously as the substrate used in these assay
soil saprotrophs (Baldrian 2009; Cullings and measures the activities of so-called exoenzymes hydrolyz-
Courty 2009; Lindahl and Tunlid 2015). Consid- ing either the end of plant cell wall (PCW) polysaccharides
ering that soil is one of the largest reservoirs of (e.g., cellobiohydrolase, xylosidase), disaccharides, or tri-
organic carbon on earth (Lal 2004) and that the saccharides (e.g., b-glucosidase). None of these enzymes is
mycelia of EcM fungal species can represent up probably susceptible to significantly degrade a long poly-
saccharide in the absence of cooccurring endoenzymes
to one third of the microbial biomass in some attacking the internal links between monomers and
forest soils (Högberg and Högberg 2002), the whose activities are seldom assayed in field studies.
debate over saprotrophy and mutualism is not
anecdotic. Indeed any significant contribution of Arguments against saprotrophy include the
mycorrhizal fungi to soil organic matter degra- theoretical one that the secretion of large
dation could in this context significantly affect amounts of plant cell wall (PCW)-degrading
the whole carbon budget of the entire ecosystem. enzymes by a mycorrhizal species would be
This debate over saprotrophy may have been damageable for the integrity of its host plant.
partially promoted by different interpretations Among factual observations, no study convinc-
of the word itself. In this chapter, we define a ingly reported the use of major PCW polymers
fungal saprotroph as a fungal species capable at (e.g., cellulose or hemicelluloses) as sole C
living autonomously in its natural habitat by sources by EcM fungal species. EcM fungal spe-
deriving all of its carbon and other elements cies may however use “minor” PCW polymers
needed for its metabolism and growth from such as pectins as in the case of Laccaria bicolor
organic carbon and other nutrients present in (Veneault-Fourrey et al. 2014) or polysacchar-
dead organic matter. According to this defini- ides absent from the PCW such as starch or
tion, all Glomeromycota AM species, which b-1,3 glucans as in the case of Hebeloma cylin-
have thus far failed to be maintained in pure drosporum (Doré et al. 2015). Along this line, a
culture in the absence of a host plant, are obligate selected set of 15 EcM species produced several
symbionts and not saprotrophs. Still according orders of magnitude less hydrolytic enzymes
to this definition, one of the many EcM fungal (exoenzymes) active on PCW polysaccharides
species whose mycelium can be grown in vitro on than saprotrophic species when inoculated to
a defined medium is a symbiotic species with sterilized leaf litter (Talbot et al. 2015). Oxida-
potential saprotrophic capacity. In this latter tive enzymes (e.g., peroxidases) did not follow
case, it remains however to demonstrate that this rule as they can be produced in similar
this species is indeed capable of surviving auton- amounts by EcM and saprotrophs. Current bio-
omously in soil, outcompeting soil saprotrophs chemical models (see below) suggest however
for the use of complex soil carbon sources. that, in EcM fungi, these latter enzymes do not
directly participate to fungal carbon provision,
In this context, arguments in favor of saprotrophy essen- but rather to the chemical modification of lig-
tially include the observation that ectomycorrhizae
formed by specific fungal species are often found in close
nin and other plant phenolics. Another argu-
association with coarse woody debris and that numerous ment used against saprotrophy is the
EcM root tips secrete hydrolytic enzymes (e.g., laccase, observation that sporocarps from EcM fungi
cellobiohydrolase, xylosidase) susceptible to attack con- display a significantly lower 13C natural abun-
stituents of the plant cell wall (Courty et al. 2005). As dance (d13C) by comparison to cooccurring
estimated by Talbot et al. (2013), enzymes secreted by
mycorrhizal root tips may however account for a small
sporocarps from saprotrophic species (Mayor
fraction of the total bulk soil enzyme activity. However, by et al. 2009). In this respect, d13C values of EcM
using an experimental setup allowing the specific coloni- sporocarps are closer to the d13C of the sur-
zation of an organic soil compartment by hyphae from rounding fully autotrophic plants suggesting
EcM fungal species, Phillips and colleagues (2014) showed that they derive most of their carbon from
that this soil compartment presented levels of hydrolytic
activities as similar to those assayed in control soils colo-
recently fixed atmospheric CO2 provided by
nized by hyphae from both EcM and saprotrophic species. their host plants (Högberg et al. 1999).
194 R. Marmeisse and M. Girlanda
Finally, in undisturbed forest soils, there is rot” wood-degrading basidiomycete species. White-rot
usually a strong physical separation between species, as exemplified by Phanerochaete chrysospor-
ium, initially fully hydrolyse lignin, thanks to a com-
the distribution of saprotrophic fungal species plex repertoire of class II peroxidases (16 gene copies)
present in the uppermost litter layer where and peroxide-producing enzymes. Exposed polysac-
most of the litter mass loss takes place and the charides, freed of lignin protection, are then fully
lower soil layers where EcM fungi predominate degraded by a complex array of GH enzymes active on
(Lindahl et al. 2007; Vořı́škov et al. 2014). cellulose and hemicelluloses (Martinez et al. 2004; East-
wood et al. 2011). By contrast, brown-rot species as
exemplified by Postia placenta that presumably evolved
from white-rot ancestors leave lignin almost
B. “Omics” and the Saprotrophic-Mycorrhizal untouched, as reflected by a lack or reduced number
of both class II peroxidases and peroxide-producing
Continuum enzymes encoding genes. To access lignin-protected
polysaccharides, brown-rot fungal species have devel-
Genome sequencing of several saprotrophic oped a specific strategy involving the production of
fungal species (degrading soil litter or produc- highly diffusible reactive oxygen species that perform
ing white or brown rot on wood) demonstrated the chemical attack of PCW polysaccharides (Martinez
that almost all of them can produce a wide et al. 2009; Eastwood et al. 2011).
range of hydrolytic enzymes susceptible to
completely hydrolyze most of the polymers In this context, the sequencing of the gen-
constitutive of the PCW (e.g., Martinez et al. omes of different mycorrhizal fungal species set
2004, 2009; Stajich et al. 2010; Eastwood et al. as a major objective the inventory of PCW-
2011; Floudas et al. 2012). Based on sequence degrading enzymes in their genomes to address
homology, these enzymes fall into different both the evolutionary transition from sapro-
protein families indexed in specialized data- trophism to mutualism and the potential impli-
bases such as CAZy (Lombard et al. 2014). cation of these enzymes in symbiosis
According to this database, these enzymes can establishment and functioning. Genome
be, for example, glycoside hydrolases (GH sequences are now available for at least one spe-
families), polysaccharide lyases (PL families), cies forming AM (Lin et al. 2014; Tisserant et al.
or auxiliary enzymes (AA families). Most auxil- 2014), EcM (Martin et al. 2008, 2010; Kohler et al.
iary enzymes are not directly involved in poly- 2015), ErM (Kohler et al. 2015) and OrM (Kohler
saccharide hydrolysis, but rather participate to et al. 2015). Both AM and EcM genomes share
the removal of lignin whose presence in PCW one relevant genomic feature, which is a strik-
represents an “obstacle” to the actual enzy- ingly low number or absence of genes encoding
matic hydrolysis of PCW polysaccharides. Lac- PCW-degrading enzymes. For example, the GH6
cases, fungal “Class II peroxidases,” and other family encoding endo-cellulases is absent in all
enzymes responsible for the production of AM and EcM genomes sequenced thus far. The
H2O2 necessary for peroxidase activity repre- GH7 family encoding either exo- or endo-
sent different auxiliary enzymes. cellulases is also absent from all species with the
exception of the basidiomycetes Piloderma cro-
In most saprotrophic fungal genomes, each PCW- ceum and Hebeloma cylindrosporum (Kohler
degrading enzyme family is also often represented by et al. 2015). Regarding this latter species, it can-
several active copies. For example, the wood-degrading not use cellulose as only C source in vitro and its
oyster mushroom (Pleurotus ostreatus) has 16 copies unique GH7 gene copy is transcribed at very low
and the coprophilic Coprinopsis cinerea has 6 copies of
glycoside hydrolase 7 (GH7)-encoding genes involved levels in both free-living mycelia and mycorrhi-
in cellulose degradation. Similarly, these two species zae (Doré et al. 2015). Sequencing of both AM
have also, respectively, 3 and 5 copies of GH10- and EcM species, which originated indepen-
encoding genes involved in xylan degradation (Rytioja dently from unrelated fungal lineages, thus dem-
et al. 2014). Saprotrophic fungal species do not however onstrate that mutualism is associated with the
represent a homogeneous functional group with respect
to their capacity at producing PCW-degrading parallel loss of unrelated genes involved in the
enzymes. For example, striking differences have been hydrolysis of structural PCW constituents.
observed between so-called “white-rot” and “brown-
Mycorrhizal Fungi and the Soil Carbon and Nutrient Cycling 195
Partial or total loss of PCW-degrading enzymes in AM of unicity of the different forms of mycorrhizal
and EcM genomes does not however seem to be part of a associations and plead for further research to
global reduction in the proteome repertoire of these spe-
cies as reported for the proteomes of several obligate
understand how these symbionts associate to a
pathogenic or symbiotic bacteria (McCutcheon and host plant without damaging its tissues.
Moran 2011). Indeed the gene content of the genomes of Besides a specific loss in the capacity of using
all mycorrhizal species sequenced thus far is almost as soil carbon sources, a strict dependency toward a
diversified and of the same order of magnitude to the gene host plant could also be explained by a partial or
content of phylogenetically related saprotrophic species
(Kohler et al. 2015). Gene loss seemed to have specifically
total loss of essential metabolic pathways leading
affected few gene families, among which those encoding to the production of key metabolites that would
enzymes active on structural constituents of the PCW. As need to be provided by the host plant. Such loss
already mentioned, the EcM fungus H. cylindrosporum, has not been reported for any of the EcM fungal
for example, retained a full repertoire of enzymes that genomes. In the case of the AM fungus Rhizopha-
allows it to grow on starch or b,1-3 glucans absent from
PCW. As for some of the putative PCW-degrading genes
gus irregularis, the absence from its genome of
that remained in the genomes of several EcM species, it genes encoding thiamin biosynthesis enzymes
can be hypothesized that they have gone through a pro- could be one of the reasons of its seemingly
cess of neo-functionalization and/or that they are the last obligate symbiotic status (Tisserant et al. 2014).
witnesses of a, not fully achieved, evolutionary transition
from saprotrophy to mutualism. Regarding the first
hypothesis, in a global transcriptomic analysis of L.
bicolor CAZyme genes, Veneault-Fourrey et al. (2014) C. Saprotrophy and the Soil Organic Nitrogen
showed that several of the few remaining PCW active Lead
genes in the genome of this species were upregulated at
either early of late stages of EcM formation, thus suggest- In natural unmanaged ecosystems, a significant
ing a role in host plant cell wall remodeling during this
ontogenic process. Regarding the second hypothesis, fraction of nitrogen enters the soil as complex
Wolfe et al. (2012) showed that in the genus Amanita organic N forms (e.g., proteins, nucleic acids)
in which coexists saprotrophic and EM species, all sym- trapped in plant litter. In the global N budget of
biotic species had lost both the ability to grow on ster- a forest ecosystem, the total N that returns back
ilized litter and genes encoding GH6 and GH7 exo- annually to the soil in litter can represent two
(¼cellobiohydrolases) and endo-cellulases active on cel-
lulose polymer. Many, but not all, of these symbiotic thirds of the N assimilated by the plants the year
Amanita species have however retained b-glucosidases before (Ranger and Bonneau 1984). According to
active on cellobiose but not on the original cellulose a classical scheme of N cycling in terrestrial habi-
polymer. The specific conservation of the final metabolic tats, complex N forms are first hydrolyzed as
gene in the pathway of cellulose degradation was also oligopeptides, amino acids, and nucleotides by
observed in L. bicolor (Veneault-Fourrey et al. 2014) and
H. cylindrosporum, a species which retained a seemingly microbial proteases and nucleases and further
inactive GH7 endo-cellulase gene (Doré et al. 2015). mineralized by microbes into ammonium and/
or nitrate, which can be assimilated back by
While the genome sequences of all AM and plants. As mentioned before, this classical view
EcM published so far are consistent with a loss has been challenged and it has been proposed
of saprotrophic capacities and a strong depen- that mycorrhizal (at least EcM and ErM) plants
dency to a host plant for carbon supply and shortcut this mineralization process and directly
survival in soil, this is not the case for both tap, via the associated fungus, into the organic N
ErM and OrM symbionts. Both Oidiodendron pool containing oligopeptides, amino acids, or
maius (Ascomycota, ErM symbiont) and Tulas- nucleotides (Read and Perez-Moreno 2003). Pref-
nella calospora (Basidiomycota, OrM symbi- erential acquisition of amino acids (up to 80 % of
ont) possess in their genome a diversified set N supply) over ammonium and nitrate by plants
of PCW-degrading genes (Kohler et al. 2015) in boreal soils has indeed been demonstrated in
consistent with earlier studies demonstrating situ using a nondestructive microdialysis tech-
their capacity to use plant-derived substrates nique (Inselsbacher and Näsholm 2012).
as C sources (Smith and Read 2008). These
observations illustrate the diversity and lack Most of the N present in dead and partially decom-
posed plant biomass becomes complexed with polyphe-
196 R. Marmeisse and M. Girlanda
nolic compounds that partially or totally protect it from pounds. Indirect correlative evidences suggest that
enzymatic hydrolysis. A series of studies proposed that several Basidiomycota EM fungi could indeed use man-
EcM mycorrhizal fungi would have retained different ganese (Mn)-dependent class II peroxidases to obtain
pathways, originally used by their saprotrophic ances- nitrogen bound to phenolic compounds in soil. First of
tors to modify organic matter including lignin, in order all, several species in the genera Hebeloma, Laccaria,
to specifically access this complexed N pool. Piloderma (Kohler et al. 2015), and Cortinarius (Böde-
ker et al. 2014) possess one or several copies of genes
encoding secreted class II peroxidases. Secondly, in a
Using the Boletales Paxillus involutus, that field study, Bödeker et al. (2014) observed a positive
may have evolved from a brown-rot sapro- correlation between the abundance of Cortinarius ITS
trophic ancestor (Kohler et al. 2015), and a rDNA amplicons from Swedish pine and birch forest
diverse set of analytical approaches, including soil samples and the level of peroxidase activity
spectroscopy, Rineau et al. (2012) demonstrated measured in these samples. Talbot et al. (2013) also
reported a similar positive correlation between EcM
that in pure culture P. involutus could partially fungi, but not saprotrophic fungi, community richness
degrade and/or modify the chemical structure in Californian pine forests and soil peroxidase and
of polysaccharides and phenolic compounds protease activity levels. Finally Bödeker et al. (2014)
present in the organic matter extract. The also recorded lower levels of soil peroxidase activities
observed chemical modifications are reminis- in ammonium-amended forest soils, a readily assimi-
lated N source for both plants and fungi which become
cent of the modifications performed by sapro- less dependent upon soil protein hydrolysis.
trophic brown-rot fungi on wood. These
modifications involved the Fenton reaction,
All these different genomic, laboratory, and
which leads to the production of hydroxyl
field observations suggest that while most, if
(HO ) radicals through the interaction between
l
tion of soil respiration and metabolic activity 2015) to a few 3–4 days for a 1–4 m tall tree
which cannot be attributable to saprotrophs (Högberg et al. 2008, 2010; Warren et al. 2012;
living on dead plant litter. The second one is Albarracı́n et al. 2013). Detection of labeled C in
that mycorrhizae, and more specifically EcM, roots is immediately (within less than 1 day) fol-
could represent essential drivers of long-term lowed by its detection in CO2 resulting from “soil
carbon storage in soils. Altogether, these two respiration” which combines the respiration of
hypotheses plead for incorporation of mycor- tree roots and of all other components of the soil
rhizae in models of terrestrial carbon cycling, biota, including soil fungi and bacteria. By com-
especially in a context of global change. bining NanoSIMS imaging to follow the fate of 13C
at the cellular level in mycorrhizal Triticum roots
and the 13C enrichment of bacterial- or fungal-
A. Fast Nutrient Transfer Between Plant and specific fatty acids extracted from the rhizosphere,
Fungal Partners Kaiser et al. (2015) concluded that a significant
fraction of recently fixed C entered the soil com-
The proportion of fixed carbon transferred by a partment through extraradical AM hyphae. In a
host plant to its mycorrhizal partners varies high-resolution follow-up of 13C in the soil biota
widely according to the type of association, following pulse labeling of Pinus foliage, Högberg
identity of the host plant, and type of study. et al. (2010) monitored a rapid (maximum at 14
However, values of 10–20 % and 20 %(-50 %) days postlabeling) labeling of fungal-specific fatty
been repeatedly reported for the AM and EcM acids as well as of newly formed sporocarps from
associations, respectively (van der Heijden et al. EM species, thus demonstrating that not only
2015). Similarly, it has also been estimated that basal metabolism, but also developmental pro-
a large proportion of mineral nutrients such as cesses of these fungi are sustained by immediate
phosphorus (up to 90 % for AM plants) assimi- host plant photosynthetic activity. Considering
lated by a mycorrhizal plant has been trans- that mycelia from EcM species can represent ca
ferred from the mycorrhizal fungi to the plant one third of forest soil microbial biomass in
(van der Heijden et al. 2015). Although these boreal forests (Högberg et al. 2001; Högberg and
figures have been reported for some time, the Högberg 2002), their belowground mycelial net-
consequences of these massive transfers of pri- works are therefore likely to represent, from a
mary nutrients between symbiotic partners on quantitative point of view, one of the main entry
the global functioning of the soil ecosystem point of recently fixed carbon in soil. This is
have long remained elusive. indeed supported by a large-scale girdling experi-
ment of Pinus sylvestris in Northern Sweden,
Two main experimental approaches are used to follow which resulted in a sharp and immediate drop
the fate and redistribution of assimilated nutrients in (within days) in soil respiration which reached
plants. The first one is the use of isotopically labeled ca 50 % after 2 months (Högberg et al. 2001),
nutrients such as 14C or 13C-labeled CO2 supplied to the
leaves of 15N-labeled NO3 of NH4 supplied to the roots.
thus suggesting that, in this case, the global soil
Although initially used in laboratory-grown plants, this biological activity was largely driven by recently
approach is now also being used in the field including fixed carbon and its translocation to EcM fungi.
on trees. In the case of trees, the impact of recently Dependency toward immediate carbon
assimilated CO2 on the soil microbiota can also be supply from the plant may however vary
appreciated by either root severing or tree girdling
(the annular sectioning of the tree bark and associated
between EcM species as indicated by Högberg
phloem tissues), which both result in the termination of et al. (2008) and Albarracı́n et al. (2013) who
the descending flow of phloem sap from the plant aerial both observed significant differences in the
parts to the root tips and the mycorrhizal symbionts. levels of 13C enrichment in EcM root tips
formed by different fungal species. Heterogene-
Experiments measuring the fate of labeled C ity between EcM fungal species was also
supplied to the leaves consistently show that its observed by Pena et al. (2010) who showed
transfer to fine roots is fast, occurring within that girdling led to the selective disappearance
hours for an herbaceous plant (Kaiser et al. of the less frequent EcM fungal species on
198 R. Marmeisse and M. Girlanda
beech tree root systems. In term of fungal com- the uppermost litter and fermentation soil hor-
munity structure, Lindahl et al. (2010) showed izons colonized by mainly saprotrophic fungal
that, as expected, local root severing resulted in species (characterized by high PCW degrading
a rapid decline in EM species abundance and enzymatic activities) and the horizons under-
diversity mirrored by an increase in abundance neath where mycorrhizal fungi predominate
of opportunistic saprotrophic species, which in (Lindahl et al. 2007; Vořı́šková et al. 2014).
the short term may use senescing hyphae from Besides this physical separation between these
EcM fungi as nutrient source. two functional groups, which limits direct com-
petition between them, it has also been recorded
While field measurements suggest that increased plant that litter degradation by saprotrophic species
photosynthetic carbon fixation results in increased car- results in an increase of the C/N ratio, which
bon allocation belowground and to associated mycorrhi- rather decreases in the lower soil horizons, pre-
zal symbionts (Druebert et al. 2009; Näsholm et al. 2013),
this does not always results in an anticipated increase in N
sumably as a result from N foraging activity by
allocation from the fungi to their host plants. Albarracı́n mycorrhizal fungi and subsequent N transfer to
et al. (2013) reported that while N fertilization led to host plants (Lindahl et al. 2007).
increased C flow belowground and to increased N con- By using 14C atomic bomb modeling, Clem-
centration in Pinus EcM mycorrhizae, most of this assimi- mensen et al. (2013) suggested that a large frac-
lated N became blocked in the symbiotic tissues and was
not transferred to the aerial parts of the plant. A similar N
tion of the recently stored soil carbon in late
retention in Pinus ectomycorrhizae was independently successional boreal forests could actually
reported by Näsholm et al. (2013) who concluded that in directly derive from root and EcM fungal bio-
N-limited soils the ectomycorrhizal association could mass instead of aboveground litter input. From
increase the N-starvation status of their associated plants. a mechanistic point of view, Clemmensen et al.
This “cheating” behavior of mycorrhizal fungi, which
derive plant carbohydrates for the acquisition of soil N
(2015) observed that in the course of boreal
to their exclusive profit, could be canceled by external N forest aging, the mycorrhizal fungal community
fertilization that led to effective N transfer from the fun- shifted from a predominance of “cord-form-
gus to the plant (Näsholm et al. 2013). ing” fungal species in the earlier stages to a
predominance of ericoid fungi producing mel-
anized hyphae. Melanized cell walls may
become recalcitrant to degradation (Fernandez
B. Mycorrhizal Fungi and Soil Carbon Storage and Koide 2014) and therefore contribute to the
long-term C storage in these forests.
Besides being the recipient of recently fixed
atmospheric CO2, EcM and ErM fungi could
also drive directly or indirectly the long-term
carbon storage in soil which represents one of
VI. Conclusions and Future
the major sink of C on earth (Lal 2004). In a Perspectives
global meta-analysis of soil carbon pools across
biomes, Averill et al. (2014) showed that EcM and In this chapter, we have summarized recent
ericoid-dominated ecosystems stored 70 % more advances in the field of mycorrhizal ecology
carbon per unit nitrogen than soils in ecosystems prompted by “omic” approaches, which have
dominated by AM-associated plants. Using a offered insights in the genome signatures of
model-based approach, Orwin et al. (2011) sug- different fungal trophic strategies and the
gested that increased C storage in EcM and roles of mycorrhizal fungi in the functioning
ericoid-dominated soils could result from either of terrestrial ecosystems.
higher C input to soils and/or from slower litter While the latter paragraphs clearly demon-
decomposition rates due to strong competition strate the importance of the mycorrhizal associ-
for N between saprotrophs and mycorrhizal ation in terrestrial carbon cycling, it remains
fungi. The latter hypothesis may marginally however to be seen if several of the hypotheses
apply for well-stratified forest soils in which formulated in recent studies can be generalized
there is a strong physical separation between or on the contrary modulated according to the
Mycorrhizal Fungi and the Soil Carbon and Nutrient Cycling 199
type of ecosystem. Indeed, many studies on car- Baldrian P (2009) Ectomycorrhizal fungi and their
bon cycling refer to forests, in boreal zones, with enzymes in soils: is there enough evidence for
their role as facultative soil saprotrophs? Oecolo-
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11 Understanding the Biodiversity and Functions of Root
Fungal Endophytes: The Ascomycete Harpophora oryzae
as a Model Case
ting sequences of the internal transcribed In this chapter, we will provide an overview
spacer (ITS), small-subunit (SSU), and large of current progress, future directions, and chal-
subunit (LSU) of the rRNA genes of a newly lenges regarding the diversity, binary root–fun-
found endophyte to BLAST analysis at public gal interaction, and field applications of root
databases sequences often leads to no hits, indi- endophytes. To provide a thorough under-
cating their potentially taxonomic novelty. In standing of the molecular basis of root–fungal
addition, frequently neither sexual nor asexual symbiosis, we will specially focus on a novel
sporulation can be seen, precluding the possi- DSE, Harpophora oryzae, and highlight its
bility of morphological descriptions. Recently, importance as a model organism to study fun-
a new class (Xolonomyectes) and a new order gal biology and evolution.
(Phaeomoniellales) within the Ascomycota
were established during a survey of tree fungal
endophytes (Gazis et al. 2012; Chen et al. 2015). II. An Overview of Diversity and
These findings further highlight that fungal Function of Root Fungal
endophytes comprise the important compo- Endophytes
nent of fungal biodiversity on a global scale.
From an evolutionary perspective, the abil-
A. Diversity of Root Fungal Endophytes
ity of filamentous fungi to colonize roots guar-
anteed the viability of earliest land plants as Due to the ancient coevolutionary processes
evidenced by fossil records (Bidartondo et al. that occurred between plant roots and fungi, it
2011). Root infecting fungi are usually detected is not surprising that today a diverse array of
by staining methods, traditional culture-based fungi are able to penetrate root tissues and
methods, and contemporary molecular tools finally establish a symbiotic relationship with
(such as pyrosequencing-based community hosts. Based on the pattern of symbiosis (e.g.,
profiling; cf. Lindahl et al. 2013; Toju et al. host range, colonization pattern, transmission
2013). On the other hand, detection of non- model, and in planta biodiversity), Rodriguez
mycorrhizal fungal root symbionts received et al. (2009) distinguished four classes of fungal
also strong attention in the past decades, endophytes, among which Class 2 and Class 4
because (a) individual plant roots generally endophytes are the primary root associates.
harbor a highly diverse array of fungal root Taxonomically, most of them belong to the
endophytic species. Among them, dark septate Ascomycota, with a minority belonging to Basi-
endophytes (DSEs) are frequently found in a diomycota.
wide range of host species and seem even Class 2 endophytes appear to show less
more predominant than mycorrhizal fungi at diversity compared to the other three groups,
least in some cases (Green et al. 2008; Mandyam but they exclusively express an extensive colo-
and Jumpponen 2015); and (b) some root endo- nization pattern within the whole plant and
phytes confer multiple beneficial services to are not only limited in roots. One of the most
plants, including substantial stimulation of striking characters observed in Class 2 endo-
plant biomass accumulation and improving phytes is their habitat-adapted benefits (see
stress tolerance. A novel microbial inoculant below).
called BioEnsure (formulated a Class 2 endo- Among the class 4 endophytes, DSEs are the
phytic Fusarium culmorum FcRed1) has representative members. It is important to bear
entered commercial applications for achieving in mind that some root endophytes do not fall
a sustainable agriculture (www.adaptivesym- strictly into either category, but their roles
biotictechnologies.com, see more details in the should not be neglected.
section “An Overview of Diversity and Function
of Root Fungal Endophytes”), exemplifying the For instance, non-mycorrhizal root endophytes in
importance of root endophyte-mediated plant orchids are often emphasized in recent studies (Oliveira
ecophysiology. et al. 2014): Fusarium semitectum isolated from an
Understanding the Biodiversity and Functions of Root Fungal Endophytes: The Ascomycete. . . 207
orchid (Cypripedium reginae) was proven to be and Narisawa 2013; Diene et al. 2013). Chlori-
effective in enhancing seed germination and seedling dium paucisporum, Leptodontidium orchidi-
establishment (Vujanovic and Vujanovic 2007). Nema-
tophagous and entomopathogenic fungi infecting
cola, Phialocephala dimorphosphora,
nematodes and insects also endophytically colonize Phialocephala fortinii, Phialophora finlandia,
roots (Lopez-Llorca et al. 2006). The entomopathogenic and Harpophora oryzae are the well-known
fungal endophyte–plant–insect tripartite interaction classic representatives of DSEs species (Jump-
model sheds light on the soil nitrogen cycling (Behie ponen and Trappe 1998; Rodriguez et al. 2009).
et al. 2012); these fungi are found to have the ability to
colonize roots and transfer the insect-derived nitrogen
The current knowledge of biodiversity of
to plants. Some soil-borne Trichoderma species have DSEs remains largely incomplete. In a semiarid
also been described as endophytic root symbionts ecosystem, 60 % of root associated fungal iso-
(Harman et al. 2004). lates were identified as DSEs using in vitro
The traditional way of detecting root infecting inoculation system (Knapp et al. 2012). The
fungi makes use of both staining and culturing
approaches: a large-scale survey of root fungal endo-
authors hypothesized that plants of (semi)arid
phytes in Mediterranean environments was performed grasslands share common DSE communities. It
by Maciá-Vicente et al. (2008) using a culture- becomes more and more clear that a large pro-
dependent method. Maciá-Vicente et al. (2012) further portion of DSEs fall into the order Pleospor-
examined the culturable root endophytes in halophytic ales,. and even many more taxa new to science
and non-halophytic plants. With the advent of ribo-
somal DNA-based methodology, molecular profiling
still await discovery (Knapp et al. 2015). The
of the fungal communities gained extensive popularity. endophytic pleosporalean genera are often
In a pioneering example, Porras-Alfaro et al. (2008) recorded from roots in stressful (arid, saline)
adopted a cloning library method to identify a novel environments (Porras-Alfaro et al. 2008; Maciá-
group of DSEs within the order Pleosporales. With the Vicente et al. 2012; Knapp et al. 2015).
advent of reasonable priced genome sequencing meth-
ods, however, e.g., the pyrosequencing technology
enables a holistic view of biodiversity of root micro-
biota. Gottel et al. (2011) examined the fungal commu- C. Ecological Functions of Class 2 and Class 4
nity in endosphere of Populus deltoids using the Endophytes
barcoded pyrosequencing targeting D1/D2 region for
fungal 28S rRNA genes. They found that 25 % LSU Based on an exhaustive characterization of root
sequences remained unclassified even at phylum level, fungal colonization pattern in different adverse
indicating the existence of potentially novel fungal
lineages contributing to the plants physiology. Simi- environments and related functions, Rodriguez
larly, Toju et al. (2013) used a nested-PCR targeting and coworkers introduced the concept of “hab-
internal transcribed spacer 2 (ITS2) to construct the itat-adapted symbiosis” for characterizing the
amplicon libraries of root samples of oak species (Quer- ecological significances of Class 2 endophytes
cus serrata), which showed that diverse ascomycetous (Rodriguez et al. 2008).
fungi, in particular DSEs, dominated the roots.
their low quantity in roots or need of special nutrient HM detoxification in E. pisciphila (Ban et al.
(Riess et al. 2014). In addition, even in the same loca- 2012; Shen et al. 2015). Beyond this, some
tion, a single plant species would host different Class
2 endophytes along a salinity gradient (Rodriguez RJ
work provides some microscopic evidence
and Redman RS, personal communication). Therefore, that DSEs have branched mucilaginous hyphae
selection of the plant part and methods for isolation containing lipid vesicles and finally form bio-
(e.g., surface sterilization and medium) have a signifi- films on the surface of roots, which prevent
cant impact on finding these cryptic microorganisms. direct exposure of roots to the adverse environ-
ments. This is thought to be a primary interpre-
Positive effects of DSEs on plant growth tation for DSEs conferring drought tolerance
and nutrition acquisition are also illustrated (Barrow 2003; Barrow et al. 2004).
by a meta-analysis approach (Newsham 2011).
Under controlled conditions, most DSEs signif-
icantly increase plant biomass, particularly III. Harpophora oryzae, a Novel DSE
when organic nitrogen sources were present
(Mahmoud and Narisawa 2013). There is grow-
Recovered from Wild Rice Roots
ing evidence that DSEs are capable of synthe- and Model System
sizing proteolytic enzymes like gelatinase
guaranteeing the mineralization of organic N- A. Morphology of H. oryzae
containing compounds in the rhizosphere
(Caldwell et al. 2000; Mandyam et al. 2010). H. oryzae has originally been isolated from wild
Organic nitrogen uptake enhancement in plants rice roots in Yunnan province, China (Yuan
achieved by colonizing them with DSEs may et al. 2010b), during isolation of DSE fungi in
highlight a larger role of DSEs in soil nitrogen 2007 and 2008.
cycling or nitrogen-related ecological process
than previously thought (Zijlstra et al. 2005). It shows a moderate growth rate, with a frequent pro-
duction of rope-like strands around the colony edge. It
In addition, the ability of phosphorus solubili- sporulates readily on artificial media and produces a
zation in a DSE fungus (Aspergillus ustus) has mass of sticky, single-celled, and hyaline conidia
been verified as, e.g., A. ustus improves plant (microconidia) from phialides (Yuan et al. 2010a), and
biomass when using unavailable P (rock phos- most conidia are very falcate or strongly curved.
phate and tricalcium phosphate) source Recently, it has been shown that H. oryzae also produces
macroconidia (Fig. 11.1). Size and shape of macroconi-
(Barrow and Osuna 2002). Thus, it is evident dia is variable (unpublished data), often fusiform, ellip-
that DSEs actively participate in host nutrient soidal, or amygdaliform and slightly inflated at either
acquisition from organic and recalcitrant end. Only macroconidia are able to germinate. In fact,
sources (Mandyam and Jumpponen 2005). two types of conidia produced by its close relatives have
With regard to DSEs-mediated abiotic stress been observed previously (Deacon 1974; Sivasitham-
param 1975; Wong and Walker 1975; Zhang et al.
response, it is often hypothesized that the high 2014). It is necessary to bear in mind that both micro-
melanin levels in hyphae is responsible for alle- conidia and macroconidia can germinate and are infec-
viating host abiotic stresses, despite experimen- tious in Magnaporthe oryzae (Zhang et al. 2014).
tal data is relatively limited. It is now well
recognized that melanin plays an important
role in the ability of melanized fungi to survive B. Molecular Phylogeny of H. oryzae and
under stressful environments. Li et al. (2011) Related Close Members
showed that a DSE fungus (Exophiala pisci-
phila) improved the tolerance of maize to Gams (2000) erected the genus Harpophora for
heavy metals (HM). The authors provided evi- anamorphs of Gaeumannomyces and Magna-
dence that E. pisciphila restricted the transloca- porthe within the Magnaporthaceae. In addi-
tion of HM from roots to shoots. In essence, E. tion to H. oryzae, four species including
pisciphila is a HM tolerant strain. Glutathione H. radicicola, H. maydis, H. graminicola, and
S-transferases (GSTs) gene family and high H. zeicola have so far been described, H. radi-
melanin content are of great importance for cicola thereby serving as the type species.
Understanding the Biodiversity and Functions of Root Fungal Endophytes: The Ascomycete. . . 209
Fig. 11.1 Non-germinating phialospores (microconida) H. oryzae. (a) Conventional light microscopy; (b) fluo-
and germinating phialidic conidia (macroconidia) pro- rescence microscopy. Bar¼10 mm
duced by GFP (green fluorescent protein)-tagged
H. graminicola also plays as a beneficial DSE cially in shoots (Fig. 11.2). The mechanisms
in grass roots (Newsham 1999), while the have not yet been addressed in detail. A com-
remaining members are thought to be potential plete genome analysis of H. oryzae may yield
pathogens. A phylogenetic tree inferred from important clues as genes involved in the syn-
ITS-5.8S rRNA gene regions reveals that thesis of auxin and gibberellin are highly
H. oryzae forms a distinctive subclade within enriched in H. oryzae (Xu et al. 2014). In addi-
the genus Harpophora and is not genetically tion, H. oryzae can protect rice roots from inva-
close to other species of Harpophora, although sion by its pathogen Magnaporthe oryzae,
it shows phylogenetic affinity to two unidenti- which makes it an attractive candidate for bio-
fied Harpophora sp. (Yuan et al. 2010a). control (Su et al. 2013). H. oryzae causes local
resistance by forming reactive oxygen species
(ROS) and highly antioxidative compounds and
IV. Effects of H. oryzae Colonization on also induces the OsWRKY45-dependent sal-
icylic acid (SA)-mediated systemic resistance
Plant Growth and Disease
against this disease (Su et al. 2013).
Resistance
Fig. 11.2 Harpophora oryzae colonization confers growth-promoting activity to tobacco (left), rice (upper right),
and Arabidopsis (bottom right) seedlings
Fig. 11.3 The differentiation and evolution of mutualistic or pathogenic interactions between rice and endophytic
fungus Harpophora oryzae or pathogenic fungus Magnaporthe oryzae
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tion for applying microbial inoculants. Thus, and uptake by dark septate fungi in four wing
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this paper was financially supported by the National logically little-differentiated anamorphs of diver-
Natural Science Foundation of China (No. 31370704, gent ascomycetes. Stud Mycol 45:187–199
31401687, and 30970097). Non-profit Sector Special Gazis R, Miadlikowska J, Lutzoni F, Arnold AE, Cha-
Research Fund of Chinese Academy of Forestry (No. verri P (2012) Culture-based study of endophytes
RISF2013005). associated with rubber trees in Peru reveals a new
class of Pezizomycotina: Xylonomycetes. Mol Phy-
logenet Evol 65:294–304
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12 Ecological Genomics of Mycotrophic Fungi
CONTENTS I. Introduction
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
II. Obligate Intracellular Mycoparasitism in A key feature of fungal communities—their
Cryptomycota . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216 interdependence with other organisms—is
III. Diversity of Interactions Between High explained by their inability of primary produc-
Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
tion (heterotrophy). In consequence, fungi can-
A. Fungi that “Stick Together” . . . . . . . . . . . . 217
B. Types of Hostile Interactions Between
not form separate self-sustaining communities,
Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218 and their occurrence is irrevocably linked
IV. Mycotrophic Hypocrealean Fungi . . . . . . . . . 220 with that of organisms on which they depend
A. Escovopsis: The Devastating Pest in for their nutrition (Hawksworth and Mueller
Gardens of Leaf-Cutting Ants . . . . . . . . . . . 220 2005). Contemporary interactions of fungi
B. Versatile Mycoparasites from the Genus with plants derived from initially saprotrophic
Trichoderma . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222 living of early fungi on dead algal material in
C. Clonostachys rosea Demonstrates an periodic dry, limnetic ecosystems. It is conceiv-
Alternative Toolkit for Successful able that some of these fungi may have formed
Mycoparasitism . . . . . . . . . . . . . . . . . . . . . . . . 230
mutualistic associations with early terrestrial
D. Further Candidates for Whole Genome
Sequencing of Mycoparasitic Fungi . . . . . 232 algae, which later gave rise to complex sym-
V. First Transcriptomic Insight into bioses between high fungi and vascular plant
Mycoparasitism of Ampelomyces modern algae. A phylogenomic study of pecti-
quisqualis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233 nase gene expansions demonstrated that the
VI. Genomic Properties of Pseudozyma early group of true fungi Chytridiomycota
flocculosa, a Mycotrophic Basidiomycete diverged from its sister clade and thus leading
That Evolved from an Advanced Plant to the high fungi Dikarya only after pectin
Pathogenic Ancestor . . . . . . . . . . . . . . . . . . . . . . 234
evolved in plant cell walls that happened not
VII. Conclusive Remarks on the Use of
Mycotrophic Fungi in Agriculture . . . . . . . . 235 earlier than 750 million years ago (Mya) (Chang
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238 et al. 2015).
The establishment of interactions between
fungi and other opisthokonts (nucleariids,
other fungi and animals) is definitely more
ancient than their relationships with plants
and dates back to the origin of fungi as an entire
monophyletic group. The divergence of the
plant–animal–fungal lineages occurred likely
820–1200 Mya. Recent recalibrations of the
most important fungal fossils and the construc-
1
Institute of Chemical Engineering, Microbiology Group, TU tion of molecular clock phylogenetic trees
Wien, Gumpendorferstrasse 1a, 1060 Vienna, Austria; allowed to put fungal evolution on a right
e-mail: [email protected]
ilar findings were also made in microsporidia, in which using the better established botanical (and
the capacity to retrieve ATP from their hosts by the rarely zoological) terminology that creates con-
HGT-derived bacterial NTTs is linked with a severe
degeneration of their mitochondrion to a vestigial,
siderable confusion. The review of several
genome-less organelle called a mitosome (Williams inherent problems and ambiguities associated
et al. 2002). Analysis of Rozella’s proteome and secre- with terminologies used in general ecology to
tome, respectively, mainly revealed adaptations to describe fungal interactions is made by Tui-
endoparasitism that are convergent to those in other ninga (2005). Based on the way how fungi
lineages of single-celled eukaryotes with a similar life-
style. As expected for an obligate intracellular patho-
receive nutrients, she proposed to divide inter-
gen, the Rozella proteome is missing key components fungal interactions in nutritive and nonnutri-
of primary metabolism. Overall, the portion of the tive. So-called nutritive fungal interactions can
proteome responsible for primary metabolism of then be further be differentiated into biotrophy
Rozella is more similar to that of the apicomplexan (deriving nutrients from the cytoplasm of a
parasites, Plasmodium and Toxoplasma, than that of
microsporidia or other fungi. On the other hand, the
living host) and necrotrophy or predation, i.e.,
amino acid metabolism of R. allomycis is more similar rapid utilization of nutrients from an organism
to that of Metazoa and Amoebozoa, perhaps suggestive after killing it (Jeffries and Young 1994;
of a phagotrophic mode of protein consumption and Dighton et al. 2005; Atanasova et al. 2013).
amino acid extraction. However, proteins involved in While necrotrophy is ultimately beneficial for
protein–protein interactions (e.g., signal transduction,
protein folding, protein kinases, and proteins with
one partner only (the host or the predator),
WD40 domains) are all enriched in the R. allomycis biotrophic interactions may vary in their
proteome. James et al. (2013) hypothesized that some importance for the two fungi from mutualism
of the protein–protein interaction domains are actually (hypothetically assumed but almost not docu-
involved in the direct manipulation of host signaling or mented) to commensalism and classical para-
recycling of host proteins. In support of this argument,
they identified 22 genes of the Crinkler family of effec-
sitism.
tor proteins. Crinkler proteins are found in many sym-
biotic, microbial eukaryotes but are best known in
oomycete plant pathogens as secreted proteins that A. Fungi that “Stick Together”
translocate into the host cytoplasm or nucleus to
induce plant cell death. Thus, these new and most
advanced genomic studies clearly demonstrate the
To the best of our knowledge, cases of inter-
ancient nature of intimate interactions between fungal fungal mutualism are not well documented.
lineages. The existence of mainly obligate and endocel- Commensalism between fungi has been demon-
lular parasites in Cryptomycota sensu lato and the strated in vitro although explanations for such
obvious lack of less specialized facultative associations observations are still insufficient. Deacon
between organisms from early fungal lineages is proba-
bly best explained by the long evolutionary history in
(2005), working with thermophilic fungi Chae-
aquatic ecosystem. Consequently, such interactions are tomium (Sordariales, Ascomycota) and Ther-
rare among high fungi, which, however, interact in a momyces (Eurotiales, Ascomycota), showed
great diversity of ways. that the latter non-cellulolytic fungus clearly
benefited from the ability of Chaetomium to
degrade cellulose in the compost. When inocu-
lated together, the two fungi could degrade
III. Diversity of Interactions Between more of the cellulose filter paper sample com-
High Fungi pared to Chaetomium alone. The advantages of
Chaetomium from the presence of Thermo-
Even before the inclusion of Cryptomycota in myces remain to be explained. Beneficial inter-
true fungi, this kingdom was considered as one actions between fungi were also shown by
of the most diverse members of the eukaryotic Friedl and Druzhinina (2012), studying infra-
domain being probably only second after generic communities of Trichoderma (Hypo-
Arthropoda (Animalia). Consequently the ecol- creales, Ascomycota) in vertical profiles of the
ogy of these organisms in general and the struc- two undisturbed soils in the Danube valley.
tures of fungal communities in particular are They detected up to a dozen of Trichoderma
very complex. Unfortunately fungal ecosystems species to coexist in a soil sample of not more
and interactions are frequently described by than 200 mg.
218 K. Chenthamara and I.S. Druzhinina
Pairwise in vitro modeling of Trichoderma commu- grazing on fungal hyphae (e.g., by mites or
nities by cultivating one species on the culture filtrate ants) or fruiting bodies (e.g., by deer or
of the other species and measuring the resulting fitness
(growth rate and conidiation efficiency) revealed that
humans), (2) to various biotrophic interac-
many of such interactions provided a benefit, but cases tions with fungi ranging from mutualism
of no effect or even inhibition of growth and/or con- through commensalism and parasitism to
idiation were observed too. Our studies of Trichoderma predation, and (3) to saprotrophic nutrition
molecular evolution and diversity in different habitats on all types of dead fungal biomass. Fungiv-
demonstrate frequent cases of sympatric speciation and
cohabitation of sibling species that remains to be
ory nutrition is known for fungi, bacteria,
explained (Atanasova et al. 2010; Friedl and Druzhinina plants, vertebrates (particularly for birds
2012; Hoyos-Carvajal et al. 2009; López-Quintero et al. and mammals), invertebrates (gastropods,
2013; Migheli et al. 2009). Besides these few examples, nematodes, and insects), and protozoans
the absolute majority of described nutritional interac- including fungi-like oomycetes and amoeba.
tions between fungi are neither mutualistic nor based
on commensalism. The aggressive behavior of fungi
Thus, when Pythium (Pythiales, Oomycota)
against each other is widely used in agriculture to attacks a fungus, this interaction may be
suppress plant pathogenic fungi, but it may also cause referred as mycophagy (or fungivory or
adverse effects on mushroom farms and on fungal mycotrophy); in contrast, when a fungus
bioeffectors used for plant growth promotion such as attacks Pythium, another term should be
arbuscular mycorrhizal fungi. We therefore first
describe the types of such hostile interactions between
used (e.g., parasitism).
fungi and then focus on several best studied cases.
l Mycoparasitism—the case of mycophagy
when one fungus feeds on another fungus. It
includes the true cases of parasitism when
parasite does not kill its host. Such interactions
are biotrophic are beneficial for a parasite (or a
B. Types of Hostile Interactions Between Fungi pathogen) and are harmful for the host.
Necrotrophic mycoparasitism—the case of
“The term mycoparasitism applies strictly to mycophagy that is best described as preda-
those relationships in which one living fungus tion when the feeding fungus aims to kill its
[underlined by the authors] acts as a nutrient prey and then feed on its dead biomass. Some
source for another.” This definition by Peter authors prefer to use “prey” and “predator,”
Jeffries (Jeffries 1995) that limits the term to respectively, for simplicity and clarity (Ata-
the fungal kingdom is only one of numerous nasova et al. 2013; Barnett and Binder 1973;
similar clear statements commonly present in Druzhinina et al. 2011; Seidl et al. 2009).
books and articles (Barnett 1963; Deacon 2005; Necrotrophic mycoparasites tend to be more
Gupta et al. 2014). Ideally, the strictness of the aggressive and unspecialized (Chet and
definition should limit the use of a term to Viterbo 2007). Biotrophic mycoparasites, on
appropriate cases. Unfortunately it is not the other hand, are usually restricted to a
always the case in fungal ecology as numerous certain host range and may also develop
interactions between fungi and fungi-like pro- specialized structures to adsorb nutrients
tozoans (e.g., Oomycota) are also referred as from their hosts. Some fungi may behave as
mycoparasitic (Ait Barka and Clément 2008; biotrophic mycoparasites of some hosts,
Benhamou et al. 1999; Gaderer et al. 2015; Rey while in interactions with others they behave
et al. 2005; Vallance et al. 2009) due to the rather as predators (Zhang et al. 2015).
similar impact made by these and the true l Hyperparasitism—parasitism on a parasite.
mycoparasitic interactions to plant pathology. The term is not limited to fungi and may be
Below we describe terms related to non- used for any group of organisms. Different
mutualistic interactions between fungi: cases of mycophagy including mycoparasit-
l Fungivory, mycophagy, or mycotrophy—the ism may belong to this category. For example,
use of fungi for food. All three terms are when the host of a mycoparasitic fungus is a
synonymous and may be applied (1) to plant pathogen, mycoparasite may be consid-
Ecological Genomics of Mycotrophic Fungi 219
Fig. 12.2 Nutritive and nonnutritive interactions reesei and A. rolfsii. Trichoderma (left) is overgrown by
between fungi. (a) Mycoparasitism of Trichoderma har- aerial hyphae of A. rolfsii (right) that does not parasit-
zianum (left) on Athelia rolfsii. (b) Agonism between T. ize on Trichoderma
ered as hyperparasite. It is important to note rolfsii from this behavior is the reduced com-
that for any hyperparasitic interactions at petition pressure for space and resources
least two hosts and two parasites should be (Fig. 12.2). Tuininga (2005) notes that the
present. The use of this term in the absence of term “coantagonism” is preferable to the fre-
the primary host is not correct. For example, quently used term “competition,” because the
Trichoderma may be considered as a hyper- latter term describes only one possible mech-
parasite when it grows on sclerotia of Athelia anism of antagonistic interactions.
rolfsii (Agaricales, Basidiomycota) that are
Other theoretically possible and nonnutritive
formed on tomato plants. In this case, both
interfungal interactions are bilaterally neutral
Trichoderma and Athelia are parasites (and
cohabitation, neutral/beneficial commensalism,
pathogens), respectively, while Athelia and
and mutualism, but they are very rare in fungi
tomato are the hosts, respectively. When the
(vide supra) because of the usually present
same Trichoderma is in vitro confronted with
antagonism.
the same Athelia and it is performed in the
absence of tomato, the term pathogen (or
High fungi from many taxonomic groups that are able
parasite) is applicable to Trichoderma, not to either parasitize on plant pathogenic fungi or to
to Athelia, and no organisms may be called antagonize them have been proposed for use in plant
as a hyperparasite. protection. For example, in the late 1970s, Teratos-
l Antagonism—a type of nonnutritive interac- perma sclerotivorum (syn. Sporidesmium scleroti-
tions where one fungus inhibits the growth of vorum, Ascomycota) that is an obligate pathogen on
sclerotia of Sclerotinia spp. (Helotiales, Ascomycota)
other fungi, while continuing to grow unin- was suggested for use in biological control of the latter
hibited itself. Similar interactions include plant pathogen (see Fravel 2006 for the review). How-
coantagonism with negative outcome for ever, this technology likely did not get commercialized
both fungi and agonism when one fungus is as the recent literature on the topic is limited: public
harmed and the other receives benefit. The databases contain no gene sequences for this hyperpar-
asitic fungus (NCBI, November 22, 2015), and there
latter interaction is similar to mycoparasit- is also no recent descriptions of the mechanisms of
ism, but it should not be confused with it as respective mycoparasitic interactions. Most of the
the benefitting fungus does not feed on the modern antifungal biocontrol formulations use myco-
one that is harmed. An example for such trophic fungi from the order Hypocreales (Sordariomy-
interaction is A. rolfsii that is capable of over- cetes, Pezizomycotina, Dikarya) that are also best
studied at molecular biological, ecological, and taxo-
growing some strains of Trichoderma but nomic levels, respectively.
does not feed on them. The benefit for A.
220 K. Chenthamara and I.S. Druzhinina
Fig. 12.3 Dual confrontations of Escovopsis weberi (left) with other fungi. The white line indicates the growth of
E. weberi as detectable from the back side of the plate
of Leucoagaricus and was also proposed as a tán et al. 2015). Our own results suggest that parasitism
potential bioeffector against these ants (Rey- of E. weberi is specialized on the attack of Leucoagar-
icus spp. (K. Chenthamara and I.S. Druzhinina, unpub-
nolds and Currie 2004). According to the lished data) We investigated the antifungal potential of
above explained terminology, E. weberi should E. weberi in dual confrontation assays with a standard
not be assigned as a hyperparasite because range of plant pathogenic fungi that are used to esti-
Leucoagaricus—its host—is not a parasite but mate the biocontrol potential of Trichoderma and Clo-
a saprotroph. nostachys (Fig. 12.3). We thereby found that E. weberi is
generally not an aggressive fungus as it is hardly able to
attack Fusarium oxysporum (Hypocreales, Ascomy-
Until recently it remained unclear whether the primary cota) and is completely agonized by Thanatephorus
nutrient source for E. weberi was the mushroom itself sp. (Rhizoctonia solani, Cantharellales, Basidiomycota)
or the vegetative substrate placed on the gardens by and A. rolfsii (Agaricales, Basidiomycota). The interac-
ants, in other words whether the interaction was nutri- tion of E. weberi with wood-rotting fungus Lentinula
tive or rather nonnutritive. Reynolds and Currie (2004) edodes (shiitake, Agaricales, Basidiomycota) was more
demonstrated the true mycoparasitic nature of E. complex as growth of the latter one was somewhat
weberi by showing its rapid growth on pure culture of stimulated by the presence of E. weberi (data not
Leucoagaricus and negligible development on sterilized shown). Our attempts to cultivate E. weberi on plates
leaf fragments. Consequently, these authors described that were pre-colonized by such fungi as Trichoderma
E. weberi as a necrotrophic mycoparasite of Leucoagar- atroviride, Alternaria alternata (Pleosporales, Ascomy-
icus (Reynolds and Currie 2004). More recently, Marfe- cota), A. rolfsii, and L. edodes failed, which suggested
tán et al. (2015) based on the microscopic analysis of that E. weberi is not able to parasitize on them (data not
interactions between E. weberi and Leucoagaricus spp. shown).
revealed hooklike structures and the penetration of the
host hyphae and thus described E. weberi as a true
mycoparasite. Furthermore, the most virulent E. weberi Despite becoming a model system for the
isolates were those which developed hooks involved in study of coevolution and host–parasite dynamics
capturing Leucoagaricus sp. (Marfetán et al. 2015). The (Currie et al. 2003, 2006; Gerardo et al. 2004; Little
formation of these structures and growth rates posi- and Currie 2008; Mendes et al. 2012; Reynolds
tively correlated with virulence of individual E. weberi
isolates, while the formation of hyphal traps did not
and Currie 2004; Rodrigues et al. 2008; Seifert
show any correlation with virulence. Traps formed by et al. 1995; Taerum et al. 2007, 2010), little atten-
E. weberi were also not able to generate pressure over tion has been paid to the taxonomy of Escovopsis
their target nor degrade the Leucoagaricus sp. hyphae until recently. In the 1990s, when the genus Escov-
(Marfetán et al. 2015). Mycoparasitism of E. weberi is opsis was proposed, only two species were
accompanied by secretion of enzymes and chemotro-
pism toward Leucoagaricus (Marfetán et al. 2015; Rey-
known: E. weberi (Muchovej and Della Lucia
nolds and Currie 2004). Moreover water-soluble 1990) and E. aspergilloides (Seifert et al. 1995).
metabolites secreted by the latter fungus stimulate In 2013, three additional Escovopsis species—
growth of E. weberi and induce its conidiation (Marfe- E. microspora, E. moelleri, and E. lentecres-
222 K. Chenthamara and I.S. Druzhinina
cens—were described, and a new genus, Escov- (Kubicek et al. 2011; Martinez et al. 2008),
opsioides, was proposed (Augustin et al. 2013). which emphasizes the specialized nature of the
Later on, Meirelles et al. (2015) performed a sur- interaction between Escovopsis and ant agricul-
vey for Escovopsis species in gardens of the lower ture. While genes for primary metabolism have
attine ant Mycetophylax morschi in Brazil and been retained, the E. weberi genome is depleted
found four strains belonging to the pink-colored in carbohydrate-active enzymes, which may
Escovopsis clade. The examination of these strains represent a reliance on a host capable to per-
revealed significant morphological differences form these functions. E. weberi has also lost
when compared to previously described species genes necessary for sexual reproduction. Con-
of Escovopsis and related Escovopsioides. Based trasting these losses, the genome encodes
on sympodial type of conidiogenesis, percurrent unique secondary metabolite biosynthesis clus-
morphology of conidiogenous cells and non- ters, some of which exhibit upregulated expres-
vesiculated conidiophores, Meirelles et al. (2015) sion during host attack. The availability of the
described the four new strains as a new species whole genome sequences of E. weberi and sev-
E. kreiselii. Phylogenetic analyses using three eral species of Trichoderma makes the detailed
nuclear markers (28S and ITS1 and 2 or the comparison of ecophysiology of these fungi a
rRNA operon and the partial sequence of the challenging task.
translation elongation factor 1-alpha, tef1) from
the new strains and sequences retrieved from
public databases confirmed that all known fungi B. Versatile Mycoparasites from the Genus
infecting attine ant gardens comprise a mono- Trichoderma
phyletic group within the Hypocreaceae family.
Specifically, E. kreiselii is likely associated with Of all mycoparasites and/or mycotrophs, the
gardens of lower attine ants, but the mode of its hypocrealean genus Trichoderma is probably
pathogenicity remains uncertain. Even more the best studied and the most frequently
interestingly, a further new species of Escovopsis, applied bioeffector with the widest host/prey
E. trichodermoides, isolated from a fungus garden range (Atanasova et al. 2013; Baek et al. 1999;
of the lower attine ant Mycocepurus goeldii, Brunner et al. 2005; Druzhinina et al. 2011; Elad
which has highly branched, Trichoderma-like et al. 1980; Kotasthane et al. 2015; Kubicek et al.
conidiophores lacking swollen vesicles, with 2011; Mukherjee et al. 2013; Studholme et al.
reduced conidiogenous cells and distinctive con- 2013; Zhang et al. 2015). One of the many
idia morphology, was described by Masiulionis important qualities that makes Trichoderma
et al. (2015). We compared tef1 sequences of the outstanding as a biological control agent for
two almost simultaneously described and there- plant pathogenic fungi (biocontrol; see below)
fore not compared Escovopsis species and found is its high opportunistic potential (Jaklitsch
that they are only 90 % similar (see NCBI acces- 2011; Jaklitsch 2009) and adaptability to vari-
sion numbers KF033128 and KJ808766 for ous ecological niches (Atanasova 2014). It has
E. trichodermoides and E. kreiselii, respectively). been well documented that Trichoderma spp.
Thus, in November 2015, there are seven species used for biocontrol can act through a diversity
of Escovopsis recorded in the Index Fungorum of mechanisms and combinations of them.
database (http://www.indexfungorum.org/): Despite of the fact that these fungi are myco-
E. aspergilloides, E. kreiselii, E. lentecrescens, E. parasites, necrotrophic mycoparasites, and
microspore, E. moelleri, E. trichodermoides, and nonspecific mycotrophs (Kubicek et al. 2011;
the oldest E. weberi. All these taxa are only known see also Druzhinina and Kubicek 2013, for
from gardens of leaf-cutting ants. more references), they can establish themselves
The genome of E. weberi was sequenced by in the rhizosphere and stimulate plant growth
de Man et al. (2015) and shown to have a sig- and thus elicit a general plant defense reactions
nificantly reduced size and gene content com- against pathogens (Druzhinina et al. 2011; Gal-
pared to closely related but less specialized letti et al. 2015; Harman 2011; Kotasthane et al.
mycotrophic fungi from the genus Trichoderma 2015). Some Trichoderma spp. have been also
Ecological Genomics of Mycotrophic Fungi 223
isolated as endophytes too (Bae et al. 2009; various proteases, and small secreted cysteine-
Bongiorno et al. 2015; Chaverri et al. 2015; rich proteins. T. virens, on the other hand,
Gazis and Chaverri 2010; Rosmana et al. expressed mainly the genes for biosynthesis of
2015). All of these characteristics make Tricho- gliotoxin, respective precursors, and also gluta-
derma a genus of particular interest for appli- thione, which is necessary for gliotoxin biosyn-
cation in agriculture as biofungicide and thesis. In contrast, T. reesei increased the
biofertilizer. expression of genes encoding cellulases and
hemicellulases and of the genes involved in
The genomic properties of Trichoderma spp. that add solute transport. The majority of differentially
to their ability for biocontrol have been discussed regulated genes were orthologs present in all
(Martinez et al. 2008; Kubicek et al. 2011). In general three species or both in T. atroviride and
these properties can be divided into such related to
interactions with other fungi (Fig. 12.4) and such
T. virens, indicating that the regulation of
related to the interactions with plants and nonfungal expression of these genes is different in the
pathogens of plants (nematodes, bacteria). As the latter three Trichoderma spp. The genes expressed
topic is behind the scope of this review, the following in all three fungi exhibited a nonrandom geno-
description will only consider interfungal interactions mic distribution, indicating a possibility for
with participation of Trichoderma. Druzhinina et al.
(2011) and Druzhinina and Kubicek (2013) provided
their regulation via chromatin modification.
detailed reviews of Trichoderma’s ability to interact The authors concluded that the initial Tricho-
with living fungi as both mycoparasites and predators derma mycotrophy demonstrated earlier by
(necrotrophic mycoparasites) and also to their ability Kubicek et al. (2011) has differentiated into
to saprotrophically feed on dead fungal biomass. The several alternative ecological strategies. In the
targeted biotrophic interaction of Trichoderma with
other fungi includes such steps as sensing the presence
context of their study, when T. cucumeris was
of the host and optional coiling around their hyphae. used as an opponent for Trichoderma, the
host cell wall degradation and penetration of the host interactions ranged from parasitism of T. atro-
hyphae, repair of damages caused by hosts, and pro- viride to predation of T. virens and competitive
duction of toxic secondary metabolites that may even- cohabitation of T. reesei. The neutral response
tually kill the host and thus transforming it to a prey.
In this chapter we will focus on those studies that
of the latter species is best explained by the fact
functionally characterized genes involved in the inter- that the exclusively tropical T. reesei has never
actions between Trichoderma and other fungi been isolated from soil so far and is not able to
(Table 12.1). Most of them are involved in signal trans- recognize temperate soil-borne T. cucumeris as
duction during mycoparasitism, in fungal cell wall deg- its host or prey (Druzhinina et al. 2010). But it
radation, and in the production of antifungal secondary
metabolites. Fewer studies focused on general and spe-
is important to note here that the assumption
cific regulator genes such as nox1, noxR, laeA, vel1, and that T. reesei is merely a saprotrophic fungus
xyr1 and the role of proteases. that is not capable to mycotrophy is contra-
dicted by numerous studies that demonstrated
Table 12.1 demonstrates that the absolute the ability of this fungus to attack a variety of
majority of functional genetic investigations fungi (Druzhinina et al. 2010; Atanasova et al.
were performed on two species of Trichoderma 2013), as also shown in Fig. 12.4.
only, i.e. T. atroviride and T. virens. Atanasova
et al. (2013) used DNA microarrays to compare The other conclusion that can be drawn from Table 12.1
the transcriptional response of the latter two is that the majority of the studies made were based on
only a limited number of opponent fungi. In most
species in comparison to T. reesei to the pres- studies either T. cucumeris, A. rolfsii, or Botrytis cinerea
ence of Thanatephorus cucumeris (Rhizoctonia (Helotiales, Ascomycota) was used for confrontations
solani). They found that the three Trichoderma with Trichoderma. As most Trichoderma species are
spp. exhibited a strikingly different transcrip- capable of biotrophic and necrotrophic types of myco-
tomic response already before physical contact parasitism and may also efficiently feed on dead fungal
biomass, the conclusions of these studies therefore
with alien hyphae. T. atroviride expressed an demonstrated only partial reduction of either one or
array of genes involved in the production of another mycotrophic strategy employed by the respec-
secondary metabolites, GH16 b-glucanases, tive Trichoderma species in given interactions. The
224 K. Chenthamara and I.S. Druzhinina
Fig. 12.4 In vitro interactions between Trichoderma at 25 and 12 h cyclic illumination. Cases of non-
(left) and other fungi in dual confrontation assays as mycoparasitic interactions are marked by dark red
observed after 10 days of incubation on PDA medium (antagonism) and blue (agonism) background
recent study by Zhang et al. (2015) demonstrated a (Chaverri et al. 2015; Li et al. 2012)] on F. oxysporum, A.
clear role of nmp1 gene encoding a secreted neutral rolfsii, and A. alternata. However, NMP1 was also
deuterolysin metallopeptidase in the predation by found to be involved in mycoparasitism on B. cinerea
T. guizhouense [former T. harzianum species complex and S. sclerotiorum and did not have any role in the
Table 12.1 Genes of Trichoderma that have been studied for their role in fungal–fungal interactions
Mycoparasitism-related Phenotype due to the deletion or Phenotype caused by the gene
process Gene General function Predator/parasite Prey/host silencing overexpression Study Year
Signal transduction tga1 G-protein a-subunit; takes T. atroviride Thanatephorus cucumeris Light-independent hyper- An increase of coiling; inhibited Reithner et al. 2005
part in G-protein- (Rhizoctonia solani) sporulation; retarded sporulation; increased
mediated signaling mycoparasitic-related coiling mycoparasitism on R. solani
pathway; involved in against R. solani; loss of
conidiation, sexual GTPase activity, which is
reproduction; stimulated by the peptide
important for sensing toxin, Mas-7
mating partners/preys/
hosts
Signal transduction tga3 G-protein a-subunit; takes T. atroviride T. cucumeris (R. solani); Reduced growth; defect in n.a. Zeilinger et al. 2005
part in G-protein- Botrytis cinerea chitinase secretion; loss of
mediated signaling infection structure
pathway; involved in formation; avirulence against
conidiation, sexual R. solani and Botrytis cinerea
reproduction;
important for sensing
mating partners/preys/
hosts
Signal transduction tac1 Adenylate cyclase, a signal T. virens Athelia rolfsii (Sclerotium Retarded morphology; retained n.a. Mukherjee et al. 2007
regulator in cAMP rolfsii); T. cucumeris only 5–6 % of the wild-type
signaling pathway (R. solani); Pythium sp.a growth rate on agar; lowered
intracellular cAMP levels
below the detection limit;
loss of virulence against
Athelia rolfsii, R. solani, and
Pythium sp.; negatively
affected production of
secondary metabolites
Signal transduction tmkA Mitogen-activated protein T. virens, T. atroviride A. rolfsii (S. rolfsii); Reduced ability to parasitize n.a. Mukherjee et al. 2003
kinase (MAPK) T. cucumeris (R. solani) R. solani and S. rolfsii and the Viterbo et al. 2005
signaling pathway gene, ability to induce systemic Mendoza-Mendoza et al. 2003
involved in transmitting resistance in plant; in Reithner et al. 2007
signals for mating, another strain of T. virens,
filamentous growth, cell improved mycoparasitism
integrity, response to against R. solani; in T.
osmotic stress, and atroviride, it resulted in
Ecological Genomics of Mycotrophic Fungi
(continued)
225
Table 12.1 (continued)
226
Cell wall degradation ech42 (chi18-5) Endochitinase, secreted T. atroviride, A. rolfsii (S. rolfsii); In T. harzianum: unaffected T. atroviride: improved Carsolio et al. 1999
T. harzianum, T. T. cucumeris (R. solani) mycoparasitism of A. rolfsii mycoparasitism Deng et al. 2007
virens and T. cucumeris. In Baek et al. 1999
T. virens: reduced
mycoparasitism of
T. cucumeris
Cell wall degradation nag1 N-acetyl-b-D- T. atroviride, T. cucumeris In T. atroviride: resulted in n.a. Brunner et al. 2003
glucosaminidase, T. harzianum (R. solani); B. cinerea reduced ability to protect Dubey et al. 2012
secreted bean seedlings against R.
solani. In T. harzianum:
resulted in reduced
antagonistic activity against
B. cinerea
Cell wall degradation bgn3 b-1,6-glucanase, secreted T. virens Rhizopus oryzae (Mucorales, No effect on growth and Improved antagonism against Djonović et al. 2006b
Mucoromycotina); development; reduced ability P. ultimum, Rhizopus oryzae,
T. cucumeris (R. solani); to inhibit growth of and T. cucumeris
P. ultimuma P. ultimum
Cell wall degradation thpg1 Endopolygalacturonase, T. harzianum T. cucumeris (R. solani); Reduced PG activity, ability to n.a. Morán-Diez et al. 2009
secreted B. cinerea; P. ultimuma grow on pectin medium, and
ability to colonize Solanum
lycopersicum (tomato)
(Solanales, Streptophyta)
roots
Regulator nox1 NADPH oxidase T. atroviride, P. ultimuma Severely affected ability to form In T. harzianum: resulted in Montero-Barrientos et al. 2011
T. harzianum conidia in response to injury; improved biocontrol Hernández-Oñate et al. 2012
uncompromised hyphal potential against P. ultimum
regeneration; loss in ability
to produce reactive oxygen
species (ROS) in response to
injury
Regulator noxR Regulator of NADPH T. atroviride n.a. Affected ability to form conidia in n.a. Hernández-Oñate et al. 2012
oxidases involved in response to injury; hyphal
reactive oxygen species regeneration was not
formation compromised
K. Chenthamara and I.S. Druzhinina
Regulator laeA Methyltransferase. Global T. atroviride Alternaria alternata; Decrease in conidiation by 50 % Increased conidiation by 30–50 % Aghcheh et al. 2013
regulator that affects the T. cucumeris (R. solani); in light; no conidiation in in light; enhanced
expression of secondary B. cinerea darkness; abolishment of mycoparasitic vigor;
metabolite gene clusters sporulation in response to resistance to oxidative stress
and controls sexual and injury; increased sensitivity (H2O2, 5 mM); increased
asexual development to oxidative stress; affected production of a known
expression of genes encoding antifungal metabolite, 6PP
several proteases, GH16 b- (6-pentyl-2H-pyran-2-one)
glucanases, PKSes, and
SSCPs; decrease in
antagonism against A.
alternata, T. cucumeris, and
B. cinerea; decrease in
production of known
antifungal metabolites
including 6PP (6-pentyl-2H-
pyran-2-one)
General regulator xyr1 Regulator protein for T. atroviride Phytophthora capsici Reduced transcript levels of axe1 n.a. Reithner et al. 2014
cellulase and (Peronosporales)a; and swo1, which encode
hemicellulase gene B. cinerea; T. cucumeris accessory cell wall-degrading
expression in (R. solani) enzymes; delayed response
Trichoderma of Arabidopsis thaliana
during Trichoderma–
Arabidopsis interaction;
upregulation of prb1
expression; overall enhanced
competition with studied
plant pathogens probably
due to overexpression of
prb1
Secondary metabolites tri4 Cytochrome P450 T. arundinaceum, B. cinerea; T. cucumeris In T. arundinaceum: reduced In T. harzianum: reduced ability Malmierca et al. 2012
monooxygenase that T. harzianum (R. solani) antifungal activity against B. to colonize roots; Cardoza et al. 2015
oxygenates trichodiene cinerea and T. cucumeris; downregulation of defense-
to give rise to reduced ability to induce the related genes in S.
isotrichodiol expression of S. lycopersicum lycopersicum
defense-related genes
belonging to the salicylic
acid (SA) and jasmonate (JA)
when attacked by B. cinerea
Secondary metabolites tri5 Terpene synthase T. arundinaceum, B. cinerea, T. cucumeris In T. arundinaceum: no In T. brevicompactum: increase of Malmierca et al. 2013
T. brevicompactum (R. solani) production of HA (a non- the trichodermin; increase in Tijerino et al. 2011
phytotoxic trichothecene), the antibiotic activity against
which has role in a large panel of yeasts;
antagonistic activity against affected S. lycopersicum
fungal plant pathogens and growth and the lesions
induction of plant genes caused by B. cinerea
involved in defense
responses; altered the
expression of other tri genes
involved in HA biosynthesis;
altered the expression of
hmgR, dpp1, erg9, erg1, and
erg7, all genes involved in
terpene biosynthetic
pathways
Secondary metabolites gliP Involved in the production of T. virens Sclerotinia sclerotiorum; Abolition of gliotoxin production; n.a. Vargas et al. 2014
gliotoxin, one of the T. cucumeris (R. solani); reduced growth; dispersed
most studied secondary P. ultimuma; Galleria and less dense mycelium; less
Ecological Genomics of Mycotrophic Fungi
(continued)
227
Table 12.1 (continued)
228
phytoalexins in Gossypium
(Malvales, Streptophyta)
seedlings
Protease nmp1 Neutral deuterolysin T. guizhouense A. rolfsii; T. cucumeris; B. Reduced mycoparasitism; no Increased mycoparasitism; self- Zhang et al. 2015
metallopeptidase, cinerea; S. sclerotiorum; coiling around hyphae of F. toxicity
secreted A. alternata; Fusarium oxysporum f. sp. cubense 4;
oxysporum; F. fujikuroi reduced ability to produce
antifungal secondary
metabolites; reduced ability
to defend against other fungi
Accessory proteins sm1 Cerato-platanin, SSCPs T. virens n.a. Unaffected growth or Unaffected growth or Djonović et al. 2007
development, conidial development, conidial Djonović et al. (2006b) 2006
germination, production of germination, production of Salas-Marina et al. 2015
gliotoxin, hyphal coiling, gliotoxin, hyphal coiling,
hydrophobicity, or the hydrophobicity, or the
ability to colonize Zea mays ability to colonize Z. mays
roots; same levels of systemic roots; enhanced levels of
protection as in plants that protection against plant
have not been treated with diseases
Trichoderma; reduced level
of protection in plants
against diseases
(Colletotrichum graminicola
was used as a plant
pathogen)
Accessory proteins sm2 Cerato-platanin, SSCPs T. virens n.a. Dramatic decrease (more in n.a. Gaderer et al. 2015
compared to sm1) in the
ability of the fungus to
induce resistance against
disease caused by
Cochliobolus heterostrophus
in Zea mays (maize) (Poales,
Streptophyta)
Accessory proteins epl1 Cerato-platanin, SSCPs T. atroviride, T. virens Alternaria solani; B. cinerea In T. atroviride: resulted in An increase in disease resistance Salas-Marina et al. 2015
diminished systemic against all tested pathogens
protection of tomato plants
(S. lycopersicum) against A.
solani and B. cinerea,
whereas in T. virens was less
effective in protecting
tomato against Pseudomonas
syringae pv. tomato
(Pseudomonadales,
Proteobacteria) and B.
cinerea
a, b
Indicate nonfungal hosts from Oomycota and Insecta, respectively
c
The strain IMI 206040 was initially published as T. harzianum
Ecological Genomics of Mycotrophic Fungi
229
230 K. Chenthamara and I.S. Druzhinina
efficient attack of this fungus on T. cucumeris at all. antagonism of C. rosea BAFC3874 against Scler-
Moreover, the secretion of the protein was induced otinia sclerotiorum (Helotiales, Ascomycota) in
when the fungus was confronted with itself on dead
fungal biomass as the carbon source and was not acti-
pot-grown lettuce (Lactuca sativa) and soybean
vated when T. guizhouense was grown on glucose or (Glycine max) plants and established that the
potato dextrose agar. Besides the role of the exact pro- strain produced antifungal compounds. They
tease that is definitely only one of the numerous other comprised a microheterogeneous mixture of
proteases that likely act synergistically in different Tri- peptaibols. These are short linear peptides that
choderma species (Druzhinina et al. 2012), this study
demonstrates the diversity of types of interaction that
are rich in a-aminoisobutyric acid and bear an
may be formed by one individual Trichoderma strain acetylated N-terminus and an amino alcohol at
against a broad range of opponent fungi. It is thus the C-terminus (Kubicek et al. 2007). They form
impossible to assign Trichoderma to either exclusively helices that are inserted into the plasma mem-
biotrophic mycoparasitic fungi or describe them as brane of the host causing alterations in the
necrotrophic mycoparasites or saprotrophs. Figure 12.4
illustrates the diversity of interactions between eight
osmotic balance of the cell (Degenkolb et al.
Trichoderma strains from eight species representing 2006) and inhibit membrane-bound enzymes
the three major infrageneric clades and six opponent such as cell wall polysaccharide synthases (Lor-
fungi including three closely related strains of Fusarium ito et al. 1996). Such effects may explain some of
oxysporum and three unrelated Basidiomycota fungi. the changes observed in the mycelium of the
For this reason, Druzhinina et al. (2011) proposed the
more general term mycotroph as the best ecological
pathogen, including cell lysis of the hyphae and
identifier for Trichoderma spp. Results of Zhang et al. melanization (Rodrı́guez et al. 2011).
(2015) also demonstrate the need to study the role of
individual genes in at least several possible interactions Recently, Karlsson et al. (2015) sequenced the whole
including at least parasitism and predation. genome of C. rosea IK726. A comparative phylogenetic
analysis between C. rosea, Trichoderma spp., and
Fusarium spp. suggested that C. rosea are sister taxa
to Fusarium spp. (frequent plant pathogens), which
belongs to family Bionectriaceae. In their study Tricho-
C. Clonostachys rosea Demonstrates an derma spp., which belongs to family Hypocreaceae,
Alternative Toolkit for Successful appeared in basal position to C. rosea. A comparative
analysis of gene family evolution under the hypothesis
Mycoparasitism that evolution of mycoparasitism in Bionectriaceae and
Hypocreaceae results in selection for converging inter-
The mechanisms of interfungal interactions action mechanisms, revealed several differences
with the participation of still another hypocrea- between the studied mycoparasites. In comparison to
lean mycotrophic fungus—Clonostachys Trichoderma spp., C. rosea showed expansion in several
rosea—have only recently attracted research- gene families such as those involved in plant cell wall
degradation (polysaccharide lyase family 1 (pectin
ers’ interest. Schroers et al. (1999) classified lyase), auxiliary activity family 3 (glucose–methanol–
the mycoparasite Gliocladium roseum as Clo- choline oxidoreductases), and auxiliary activity family
nostachys rosea because it differed from the 9 (lytic polysaccharide monooxygenase)); secondary
type species of Gliocladium, G. penicillioides, metabolite synthesis (PKS, cytochrome P450 monoox-
in morphology, ecology, teleomorph, and ygenases, PKS, and NRPS genes), likely attributing to
production of antifungal components; ABC transporter
DNA sequence data. Jensen et al. (2004) used and major facility superfamily membrane transporters,
an ecological approach to present C. rosea as an attributing to the fungi’s high tolerance to toxins like
effective mycoparasite against Alternaria dauci boscalid, ZEN, and other microbial metabolites; and
(Pleosporales, Ascomycota) and A. radicina on several ankyrin repeat proteins. In contrast, the
carrots (Daucus carota subsp. sativus). C. rosea genome of C. rosea contains significantly fewer
carbohydrate-binding family 18 (CBM18, chitin bind-
showed a similar efficiency against these patho- ing) module containing genes (only two B group GH18
gens as the fungicide iprodione. A C. rosea chitinases, only two C group GH18 chitinases, eight A
strain, C. rosea IK726, was transformed with group GH18 chitinases), suggesting that cell wall deg-
GFP (green fluorescent protein) and was used radation of the fungal prey may not be a prominent
in biopriming of carrot seeds. Microscopy after strategy for interactions of C. rosea with other fungi.
7 days of this biopriming showed seeds covered
with a fine web of sporulating mycelium of C. Table 12.2 summarizes the results of func-
rosea. Rodrı́guez et al. (2011) demonstrated the tional characterization of C. rosea genes
Table 12.2 Genes of Clonostachys rosea studied for their role in fungal–fungal interactions
Mycoparasitism- Phenotype due to the deletion or
related process Genes General function Prey/host silencing Expression analysis Study Year
Secondary metabolites zhd101 Zearalenone hydrolase F. graminearum Lowered in vitro ability to inhibit n.a. Kosawang et al. 2014
growth of the ZEA-producing F.
graminearum. Failed to protect
wheat seedlings against foot rot
caused by the ZEA-producing
F. graminearum
Accessory proteins hyd1, hyd2, hyd3 Hydrophobins F. graminearum; B. cinerea; Higher growth rate of Dhyd1Dhyd3 in Repressed expression of Dubey et al. 2014
T. cucumeris (R. solani) high salinity. Faster overgrowth of hyd1, hyd2, and hyd3 in
Dhyd1Dhyd3, Dhyd1, and Dhyd3 on interactions with
prey/hosts. Improved protection of B. cinerea and F.
plants against fungal pathogens. graminearum.
Increased colonization ability by Upregulation of hyd1,
Dhyd1Dhyd3 on Arabidopsis hyd2, and hyd3 during
thaliana roots self-interaction. High
expression of hyd1 in
germinating conidia
Transporter abcG29 ATP-binding cassette (ABC) F. graminearum, F. oxysporum Delay in conidial germination and n.a. Dubey et al. 2015
transporter, induced by f. sp. radicis-lycopersici; subsequent reduction in total germ
zearalenone and the fungicides B. cinerea tube length when subjected to H2O2;
Cantus, Chipco Green, and increase in necrotic lesion area
Apron caused by B. cinerea measured on A.
thaliana leaves pre-inoculated with
DabcG29 strain spores compared to
the necrotic lesion area on leaves
pre-inoculated with C. rosea WT
spores; increase of F. graminearum
foot rot disease severity in barley
seedlings
Transporter abcG5 ATP-binding cassette (ABC) F. graminearum Reduced antagonism toward F. n.a Dubey et al. (2014b) 2014b
transporter, induced by graminearum; reduced biocontrol
zearalenone, secondary efficiency to protect barley
metabolites secreted by F. seedlings from foot rot disease
graminearum and different caused by F. graminearum;
Ecological Genomics of Mycotrophic Fungi
required for its interaction with other fungi. ents and essential growth factors from living
The availability of the genome sequence and host cells (Vujanovic and Goh 2009).
its comparative analysis now provides the
basis for studying of those gene families that Vujanovic and Goh (2009) demonstrated that S. quad-
are overrepresented in the genome of this fun- rangularis produces hook-shaped structures within four
gus (vide supra). days of subjection with F. avenaceum. Although S. quad-
rangularis also produces clamp-like or other contact
structures, hook-shaped contact cells are more promi-
nent. It is interesting that the diameter of hyphae para-
sitizing F. avenaceum is much smaller compared to its
D. Further Candidates for Whole Genome saprotrophic hyphae. S. quadrangularis also shows host
Sequencing of Mycoparasitic Fungi specificity, as it did not form any specialized attachment
structures when confronted with other Fusarium species.
A number of other hypocrealean fungi are myco- S. mycoparasitica, another biotrophic mycoparasite from
trophic with different degrees of specialization. the same genus, has however proven to be effective
However, most of them remain poorly investi- against a broad range of Fusarium species, showing
gated. These are such genera as, for example, positive effect on wheat (Triticum spp.) seed germination
and seedlings growth (Vujanovic and Goh 2012). This
Hypomyces (Põldmaa et al. 1997), Cosmospora, fungus is not affected by the mycotoxins produced by F.
and Verticillium, but none of them have been graminearum such as deoxynivalenol (DON), trichothe-
investigated on the levels of genes and/or gen- cene, and its acetyl derivatives 3-acetyldeoxynivalenol
omes. Hypomyces, with about 50 species recog- (3-ADON), 15-acetyldeoxynivalenol (ADON), and zear-
nized in recent studies (Poldmaa 1996; Rogerson alenone (ZEA) (Shinha and Bhatnagar 1998), probably
because of the presence of similar defense genes as found
and Samuels 1985, 1989, 1993, 1994), among in C. rosea (Karlsson et al. (2015).
which more than 30 are listed in NCBI taxonomy
browser (November 2015), is the largest genus of An interesting further target for more
almost exclusively fungicolous fungi. detailed investigations may be Calcarisporium
arbuscula (Watson 1955) that is only putatively
Hypomyces species occur mainly on discomycetes, related to Hypocreales based on the similarity of
boletes, agarics, or polypores. The polyporicolous
Hypomyces are more numerous than any other group, its nucleotide sequence encoding rpb2 gene for
with 19 species accepted by Rogerson and Samuels the RNA polymerase II large subunit 2 (NCBI
(1993). The genome of Hypomyces chrysospermus CBS accession number LN714633). Although the
394.52, a bolete mold that grows on Boletus (Polypor- morphology of interaction of these fungi with
ales, Basidiomycota) mushrooms, turning the host a their hosts that belong to Xylariales (cf. Physa-
whitish, golden yellow, or tan color and making it not
edible, is currently sequenced by JGI DOE in collabora- lospora) has been interpreted by Barnett (1958)
tion with Joe Spatafora (https://gold.jgi.doe.gov/proj- as intimate balanced mycoparasitism, no
ect?id¼36363). The same group has sequenced the advanced recent studies on this fungus have
whole genome sequence of Tolypocladium inflatum been performed. The low attention to Calcaris-
(Bushley et al. 2013), which is a pathogen of beetle porium is demonstrated by only 18 nucleotide
larvae but is closely related to fungicolous species of
Elaphocordyceps. The comparative analysis of H. chry- sequences deposited in public databases for the
sospermus with already sequenced fungicolous hypo- entire genus (NCBI, November 2015), none of
crealean fungi and T. inflatum may give insights in which were obtained in relation to studies of
convergent evolution of this lifestyle. interfungal interactions.
Numerous other fungicolous fungi are only
Sphaerodes quadrangularis (Ceratostoma- investigated on the level of their taxonomy that
taceae, Hypocreales) is another example of a also frequently yields unexpected results. For
facultative (i.e., able to grow saprotrophically example, Hawksworth et al. (2010) studied Rosel-
in vitro, even when the host is absent) contact liniella, a pyrenocarpous fungi growing on lichens
biotrophic mycoparasite. It establishes an inti- and forming single-celled brown ascospores and
mate relationship with its host Fusarium ave- persistent interascal filaments that were previ-
naceum (Nectriaceae, Hypocreales) by ously assigned to Sordariales. The molecular phy-
producing hook-shaped and clamp-like attach- logeny showed them to belong to Hypocreales.
ment structures that appeared to derive nutri- Jaklitsch and Voglmayr (2014) investigated and
Ecological Genomics of Mycotrophic Fungi 233
reinstated the fungicolous genus Thyronectria as whose chemical structure is yet unknown (Gu and Ko
also belonging to Hypocreales. 1997; Sundheim 1982). Penetration of the host hyphae
is made through either mechanical (Sundheim and
Krekling 1982) or enzymatic processes. Rotem et al.
(1999) reported the isolation of an exo-b-1,3-glucanase
from A. quisqualis, and in vitro production of lytic
V. First Transcriptomic Insight into enzymes has been reported for different isolates of A.
Mycoparasitism of Ampelomyces quisqualis (Angeli et al. 2012). Siozios et al. (2015),
quisqualis using a high-throughput sequencing approach, estab-
lished a catalog of transcripts that are formed by A.
quisqualis during mycoparasitic interactions with
Mycoparasitic fungi from other groups than Podosphaera xanthii (Erysiphales, Ascomycota). This
Hypocreales are studied less intensively. One catalog was then used to manufacture oligonucleotide
exception is the mycotrophic fungus Ampelo- microarrays for large-scale genome-wide analysis of
transcriptional changes that occur during the early
myces quisqualis (Pleosporales, Ascomycota) germination phase of A. quisqualis. They retrieved
that is a hyperparasite of Erysiphe, Podo- 1536 putative genes showing significant changes in
sphaera, Sphaerotheca, Uncinula, and others transcription during the germination of A. quisqualis,
that all belong to the order Erysiphales (Asco- documenting an extensive transcriptional reprogram-
mycota) and cause powdery mildew disease of ming of A. quisqualis induced by the presence of the
host. Genes encoding secreted proteases, virulence fac-
wine grapes, cucumber, carrots, mango, and tors, and enzymes related to toxin biosynthesis were
other plants (Sztejnberg et al. 1989; Takamatsu fund to be upregulated and interpreted as putative
2004). In total Ampelomyces has been described mycoparasitism related. They also found that a rapid
to be associated with more than 60 species from activation of the transcription and translation machin-
eight different genera of the order Erysiphales ery in the early stages of conidial germination is crucial
for the successful transition from a dormant state to
and is thus the most widespread and oldest vegetative growth of A. quisqualis. The later phase of
known natural enemy of powdery mildews hyphal germination is hallmarked by upregulation of
(Kiss 2008). It is therefore frequently used for the genes involved in proteasomal and vacuolar protein
biological control of this disease (Kiss 2003, degradation, protein secretion, transport, and localiza-
2008; Kiss et al. 2004; Sundheim 1982). tion, and genes related to the Snf7 family of proteins,
which is involved in protein sorting and transport to
The biology and life cycle of A. quisqualis lysosomal compartments (Peck et al. 2004). An involve-
has been extensively studied (Kiss et al. 2004; ment of these proteolytic genes in mycoparasitism has
Kiss 2008). Conidia of A. quisqualis are pro- also been suggested for other fungi (Grinyer et al. 2005;
duced in pycnidia, which develop intracellu- Monod et al. 2002; Muthumeenakshi et al. 2007;
larly in the parasitized mycelia of the powdery Olmedo-Monfil et al. 2002; Zhang et al. 2015). Further-
more, the authors detected homologues of secreted
mildew host. In the presence of water, conidia proteases such as dipeptidyl-peptidase 5 and the
become released and form hyphae that then tripeptidyl-peptidase SED3 and two putative genes
penetrate the nearby hyphae of powdery mil- with homology to the M6 family of metalloprotease
dew. A. quisqualis can withstand cold periods domain-containing proteins which all may facilitate
in the form of pycnidia that are saprotrophi- the penetration of the host mycelium. They also identi-
fied a small secreted protein related to the cerato-
cally produced in the killed plant tissues, but platanin family (Chen et al. 2013; Gaderer et al. 2014;
the fungus is not an efficient saprotroph. A. Skinner et al. 2001). They are widespread among fungi
quisqualis is able to infect and form pycnidia and believed to be involved in fungus–host interaction
within powdery mildew hyphae, conidiophores, phytotoxicity in different plant pathogens (Jeong et al.
and chasmothecia that causes reduced growth 2007; Pazzagli et al. 1999) or elicitors of the plant
defense response in mycoparasitic Trichoderma spp.
and death of the parasitic mildew (Kiss 2008). (Djonović et al. 2006a; Seidl et al. 2006). The actual
role of this protein in the mycoparasitic action of A.
Although ecological aspects of the mycoparasitic activ- quisqualis remains therefore to be determined.
ity in A. quisqualis have been widely investigated
(Angeli et al. 2011, 2012; Hashioka and Nakai 1980;
Kiss 2008; Kiss et al. 2004), its molecular physiology
Siozios et al. (2015) identified genes encod-
remained largely unstudied. It is known that conidia of ing proteins involved in toxin biosynthesis
A. quisqualis poorly germinate in water or in the pres- among the upregulated genes: a homologue of a
ence of glucose but their germination is stimulated by trichodiene oxygenase, which has a key role in
the presence of a water-soluble substance from the host
234 K. Chenthamara and I.S. Druzhinina
the trichothecene biosynthesis pathway (Car- the comparative genomics of P. flocculosa and
doza et al. 2011), and a homologue of the ster- plant pathogenic smut fungi U. maydis (Kaem-
igmatocystin biosynthesis P450 monooxygenase. per et al. 2006), U. hordei (Laurie et al. 2012),
Finally, two of the upregulated genes encoded and Sporisorium reilianum (Schirawski et al.
multidrug transporters and the major facilitator 2010) (all from Ustilaginales). Several Ustilagi-
superfamily to that resembled in C. rosea (Karls- nomycetes smut fungi share common features
son et al. 2015). that are essential for pathogenicity. U. maydis
Several genes reported for their role in interaction with maize (Zea mays) became the
mycoparasitism have been found in dormant model system in phytopathology for investiga-
conidia of A. quisqualis. These were cell wall- tion of factors essential for the establishment of
degrading enzymes, including different glyco- the biotrophic parasitism. The genome
syl hydrolases and homologues of MAPK 1 such sequence of U. maydis has revealed previously
as Pmk1 of Magnaporthe grisea (Magna- unknown genes that play key roles during such
porthales, Ascomycota) and the Tmk1 of T. pathogenicity (Kaemper et al. 2006). Among
atroviride. In fungi, MAPK signaling pathways these was a distinctive set of genes that coded
are involved in the transduction of a wide vari- for small secreted proteins referred to as effec-
ety of extracellular signals and play an impor- tor proteins (or effectors), of which many had
tant role in the regulation of different unknown functions. However, some were
developmental processes, including those essential for infection and several counteracted
related to pathogenicity (Table 12.1). The plant defense responses, thus facilitating infec-
authors also noted two lectin-related proteins tion by the smut fungus (Brefort et al. 2009;
that are well known for carbohydrate-binding Doehlemann et al. 2011).
properties and are widely distributed in ani-
mals, plants, and microorganisms (Lam and In the case of U. maydis, the secreted effectors were
Ng 2010). A. quisqualis-related lectins could found to be arranged in clusters and were upregulated
potentially be involved in the mycoparasitic upon recognition of the host plant, upon invasion, and
in developing tumor tissue. Cluster deletion analysis
process by recognizing the powdery mildew proved their importance in pathogenicity (Kämper
host and facilitating penetration. This study et al. 2006; Schirawski et al. 2010). The P. flocculosa
revealed several convergent strategies deployed genome comprises 6877 predicted protein coding genes
by mycoparasites from different taxonomic and exhibited genomic features, including hallmarks of
groups. Future studies, including the sequenc- plant pathogenicity, that were very similar to the plant
pathogens U. maydis, Sporisorium reilianum, and Usti-
ing of the A. quisqualis genome, could aid our lago hordei (Lefebvre et al. 2013). These findings and
understanding of the biology and evolution of phylogenomic analysis suggested that P. flocculosa
the mycoparasitic lifestyle in general. diverged from a plant pathogenic ancestor. Interest-
ingly, however, Lefebvre et al. (2013) observed a loss
of a specific subset of the secreted effector proteins
(CSEP) reported to influence virulence in U. maydis.
VI. Genomic Properties of Pseudozyma Although 345 CSEP-encoding genes were encoded by
flocculosa, a Mycotrophic the P. flocculosa genome, which is a similar number as
those found in the plant pathogenic Ustilaginales,
Basidiomycete That Evolved from orthologs for 51 out of 55 genes encoding secreted
an Advanced Plant Pathogenic proteins that influence plant pathogenicity and viru-
Ancestor lence were absent in P. flocculosa. Since otherwise P.
flocculosa has a high level of conservation of all other
pathogenicity-related genes, e.g., encoding for enzymes
Another hyperparasitic fungus that may be in cell wall degradation and biosynthesis of secondary
used to control powdery mildews is Pseudo- metabolites, this suggests that the loss of above
zyma flocculosa (Ustilaginales, Ustilaginomy- described effectors represents the crucial factor which
explains the not plant pathogenic lifestyle of P. floccu-
cotina, Basidiomycota) that is closely related losa.
to the model plant pathogen Ustilago maydis
yet not capable to attack plants (Kemen and
Jones 2012). Lefebvre et al. (2013) presented
Ecological Genomics of Mycotrophic Fungi 235
Yet the interaction between P. flocculosa in the biocontrol activity of P. flocculosa (Bélanger et al.
and its fungal host might be dictated by other 2012), these finding suggests that a feature is shared
with the mycoparasites, which requires further investi-
effector proteins. For example, the secretome of gation.
P. flocculosa includes two NPP1-containing
proteins that are absent from plant pathogenic
Ustilaginales (Kämper et al. 2006; Schirawski
et al. 2010; Laurie et al. 2012) and also from
other basidiomycetes and which are involved in VII. Conclusive Remarks on the Use of
the formation of necrosis and ethylene. They Mycotrophic Fungi in Agriculture
have so far only been identified in Moni-
liophthora perniciosa (Agaricales), the causal The biological control of plant diseases, or
agent of witches’ broom disease of Theobroma biocontrol, is an agricultural technique that
cacao (Meinhardt et al. 2008). Interestingly, the is based on the use of natural hyperparasites
NPP1-containing proteins exhibit structural and/or antagonists of plant pathogenic organ-
similarities to actinoporins, which form trans- isms to prevent or combat disease; in a broad
membrane pores (Ottmann et al. 2009), which sense, biocontrol may also include the appli-
fits well to previous observations that the col- cation of plant stimulating (micro)organisms
lapse of powdery mildew colonies caused by P. that help crops sustain abiotic stresses such as
flocculosa could be due to alteration of the drought or salinity. It is very important to note
plasma membrane and cytoplasmic leaking that not all organisms but only humans1 are
(Hajlaoui and Belanger 1991; Hajlaou et al. capable to do biocontrol. The success of bio-
1994; Mimee et al. 2009). Thus, NPP1- control is best defined by its result—reduced
containing proteins could be key elements disease index for crops, but not by the mecha-
explaining the antagonism of P. flocculosa nism of action and the type of interactions
toward powdery mildews. involved. Thus, efficient bioeffectors (organ-
Other species-specific genes also provided isms used in biocontrol) may (1) stimulate
further insights into how P. flocculosa acquired plants to induce their resistance, (2) compete
its potential to antagonize powdery mildews. For with plant pathogens, (3) antagonize plant
instance, two divergent GDSL lipases/esterases pathogens by means of secondary metabolite
(Akoh et al. 2004) that contain a CE16 carbohy- production, or (4) directly attack such patho-
drate esterase motif that is exclusive to P. floccu- gens as parasites or predators. Figure 12.5
losa have been identified that may be of relevance gives an overview of biocontrol relevant inter-
to its activity as an epiphytic competitor. fungal interactions. Nonnutritive antagonistic
interactions are depicted in pane a, while b–d
Another interesting observation differentiating P. floc- demonstrate cases of parasitism among which
culosa from the plant pathogens was the identification
of a gene encoding a subgroup C GH18 chitinase adja-
b and c are beneficial for the plant as the
cent to another gene encoding a chitin-binding LysM “good” fungus or bioeffector attacks either
protein. The same genomic arrangement has also been plant pathogenic nematodes (b) or plant path-
found in mycoparasitic Trichoderma species (Kubicek ogenic and therefore “bad” fungi. The nature
et al. 2011). Interestingly, the LysM protein TAL6 of T. of the interaction showed in e is disputable
atroviride inhibited its own spore germination, while it
had no effect on Aspergillus niger or Neurospora crassa
and may be considered as either nonnutritive
(Ascomycota, Sordariales) (Seidl-Seiboth et al. 2013), mutualism (plant gets stimulated while myco-
suggesting a self-regulatory role in fungal growth and parasitic fungus may find a greater diversity of
development. TAL6 could also act to protect the fungus host organisms) or commensalism when only
against self-degradation by its other chitinases during plant benefits.
mycoparasitism. Such a protective function for LysM
chitinases against wheat (Triticum aestivum) was
described during infection by for Mycosphaerella gra-
minicola (Capnodiales, Ascomycota) (Marshall et al.
1
2011). While there is no evidence for a role of chitinases The cases of natural agriculture as that of leaf-cutting ants are
briefly discussed above.
236 K. Chenthamara and I.S. Druzhinina
Fig. 12.5 A simplified overview of interfungal interac- also be accompanied by nonnutritive interactions
tions that are relevant for biological control of plant such as antagonism or agonism. Due to the potential
pathogenic fungi and nematodes. Exclusively nonnutri- applications of mycoparasitic fungi for crop protection,
tive antagonistic interactions are depicted on pane (a), the so-called “bad” fungi are frequently labeled as
while (b–d) demonstrate cases of parasitism among pathogens even in the absence of their host plants
which (b) and (c) are beneficial for the plant as the when solely fungal–fungal interactions are investigated.
“good” fungus or bioeffector attacks either plant path- However, in such studies, these “pathogens” serve as
ogenic nematodes (b) or plant pathogenic and there- hosts for “good” fungi that parasitize on them; there-
fore “bad” fungi (c). However, the cases of nutritive fore, the latter ones—the “good” fungi—should rather
mycoparasitism (biotrophic and necrotrophic) may be named as pathogens
A “good” label for a bioeffector organism is conditional the first place” that illustrates the future trend toward
and may only be applied in respect of exact interactions reduced use of chemical pesticides under the need to
and an exact crop plant (Fig. 12.6). The application of increase crop production for the growing population.
mycoparasitic and antagonistic fungi for biocontrol However, despite the generally accepted low risk, the
allows to reduce the use of chemical pesticides which release of bioeffectors in the environment may also
is usually strongly supported by the general public, and have adverse effects on both agricultural and natural
therefore respective research will likely attract more ecosystems. It appears to be conceivable that intro-
attention and funding. The Directive 2009/128/EC of duced biocontrol fungi in case of either importation
the European Parliament and of the Council of 21 or augmentation practices will increase competition
October 2009 “establishing a framework for Commu- pressure for naturally present plant-beneficial micro-
nity action to achieve the sustainable use of pesticides” organisms including other fungi and bacteria. For
(http://eur-lex.europa.eu/legal-content/EN/TXT/? instance, the most prominent and widely accepted as
uri¼CELEX%3A32009L0128) contains the respective “good” fungus Trichoderma may parasitize on arbus-
statement: “Appropriate risk management measures cular mycorrhizal fungi Gigaspora (Diversisporales,
shall be taken and the use of low-risk plant protection Glomeromycota) that are used to enhance plant nutri-
products as defined in Regulation (EC) No 1107/2009 tion and stress resistance (Lace et al. 2015) or even
and biological control measures shall be considered in affect the plant as demonstrated by the colonization of
Ecological Genomics of Mycotrophic Fungi 237
Fig. 12.6 Positive and negative arguments for the use of mycoparasitic fungi in biological control of plant pathogenic
fungi
broad areas of the root epidermis of Medicago trunca- The extensive colonization of harvested apples by T.
tula (Fabaceae, Angiosperms, Plantae) by T. atroviride minor and T. pallescens may diminish the prospects for
leading to localized death. However, reports on direct their commercial application as biocontrol agents, as
adverse effects of biocontrol fungi on plants are rare: registration as a biocontrol agent will become more
T. viride was diagnosed as a causative agent of dieback complicated (Baric et al. 2010).
of Pinus nigra (Pinales, Plantae) seedling in Italy
(Li Destri Nicosia et al. 2015) and different species of
Tilletiopsis (Entylomatales, Basidiomycota) that are
Several studies also document the adverse
well-known antagonists of powdery mildews caused effect of fungal hyperparasites on fungi used to
by Erysiphales fungi (Hijwegen 1986, 1989; Hoch and control insect pests. It has been shown that the
Provvidenti 1979; Klecan 1990; Knudsen and Skou mycoparasitic Syspastospora parasitica (Hypo-
1993; Urquhart 1994). Smut fungi belonging to genus creales, Ascomycota) attacks Beauveria bassi-
Tilletiopsis were demonstrated to cause “white haze” on
the apple surface by Boekhout et al. (2006), in particu-
ana (Hypocreales, Ascomycota) growing on a
lar under conditions of ultralow oxygen storage. Clearly Colorado potato beetle (Leptinotarsa decemli-
these fungi are able to reduce the growth of other fungi neata) cadaver (Klinger et al. 2006). Our own
that contributes to their success as apple colonizers. data indicate that this action that may also be
238 K. Chenthamara and I.S. Druzhinina
performed by almost any Trichoderma species Interestingly, to the best of our knowledge,
(Druzhinina, Atanasova, unpublished) and thus up to now there are no reports published on
the application of Trichoderma may counteract adverse effects of Clonostachys rosea on
the positive role of B. bassiana on the control of humans, cultivated mushroom, or biocontrol
the disease. Similar to this, the chytrid fungus insects. It could be possible that the mycopar-
Gaertneriomyces semiglobifer (Spizellomyce- asitic ability derived from herbivorous ances-
tales, Chytridiomycota) is capable to parasitism tors may possess fewer number of possible
of entomophthoralean gypsy moth Lymantria adverse effects compared to mycoparasites
dispar pathogen Entomophaga maimaiga (Ento- that evolved from an entomopathogenic-like
mophthorales, Entomophthoromycota) in soil organisms. No detailed ecological risk assess-
(Hajek et al. 2013). The authors propose that ment analyses on the use of mycotrophic fungi
mycoparasitism, whether by G. semiglobifer or have been performed. However, the newest
other mycoparasitic fungi, might be partially genome-wide mechanistic and evolutionary
responsible for declines in azygospore reser- studies would provide sufficient background
voirs, especially under wet conditions where for such research.
the motile zoospores of chytrids would have
better access to susceptible fungal host spores. Acknowledgments The work on this review was sup-
ported by Austrian Science Fund (FWF): project num-
Besides the direct impact on plants and ber 25613 B20 to ISD. The authors are thankful to
plant-interacting microorganisms, fungi used Mohammad Rahimi (TU Wien) for the gift of his
in biocontrol may also have adverse effects on image used on Fig. 12.2b and to Christian P. Kubicek
mushroom production (Castle et al. 1998; (TU Wien) for critical reading of the manuscript.
Hajek et al. 2013; Hermosa et al. 1999; Kim
et al. 2012; Komon-Zelazowska et al. 2007; Kre-
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13 Nematophagous Fungi
the Orbiliomycetes, which consists of a single recombination at the same geographic areas (Zhang
order (Orbiliales) and one family (Orbiliaceae). et al. 2012).
To investigate the genetic variation over the geo-
Phylogenetic analysis placed Orbiliomycetes as graphic and ecological contexts, two virulence asso-
a basal branch among the Pezizomycotina ciated genes and two housekeeping gene fragments of
(James et al. 2006), the largest subphylum of 80 natural Paecilomyces lilacinus strains were analyzed.
the fungi that includes the vast majority of Using this approach, it was found that 32 and 19 multi-
filamentous growing and fruiting-body-pro- locus genotypes were represented by a single isolate.
Several multilocus genotypes were shared by multiple
ducing species. The time of divergence of isolates, all of them from different geographical loca-
nematode-trapping fungi from the other Pezi- tions. Various degrees of polymorphisms and haplo-
zomycotina species is not completely clear: types were determined among the six genes analyzed.
Yang et al. (2007a, b, 2012) estimated 400– The analysis showed that P. lilacinus has a clonal repro-
520 Mya, whereas Meerupati et al. (2013) duction mode in natural populations. These results
suggest that these fungal isolates have limited geo-
arrived at a much more recent time frame graphic distribution and might have undergone strong
(198–208 Mya). The reason for this difference genetic differentiation during their adaptation to envir-
could be that the latter authors used 9632 onments in different geographic regions (Li et al. 2013).
orthologous genes (in contrast to five used by
Yang et al. 2012). Also the calibration of the
time scale differs: Meerupati et al. (2013) used
the split between ascomycetes and basidiomy-
III. Biology of Trap Formation and
cetes (500–650 Mya; Lücking et al. 2009), Nematode Infection
whereas Yang et al. (2007a, b) used two fossil
records of carnivorous fungi, dated to 100 Mya There are distinct differences in the mechanism
and 24 Mya, respectively, which however is of trapping with adhesive cells and the con-
complicated by the uncertainties in the identi- stricting ring. Fungi that feature adhesive
fication of the trap structures and the assign- traps capture nematodes by secretion of extra-
ment of the taxa in fossils (Meerupati et al. cellular polymers that accumulate at the site of
2013). Constricting rings are probably the infection (Tunlid et al. 1992), whereas those
most ancient device for nematode trapping with constricting rings ensnare the nematode
because the fungi displaying this type of struc- by rapid swelling of a ring formed by three cells
ture form a basal branch in the tree of (Meerupati et al. 2013). When the nematode
nematode-trapping fungi (Ahrén et al. 1998). enters the ring, the cells inflate and the nema-
tode is trapped. This closure occurs very rap-
Genotypification of 228 isolates from the nematode- idly (0.1 s) and is triggered by pressure of the
trapping fungus A. oligospora from different ecologi- nematode on the constricting-ring cells (Hig-
cal niches and geographical locations, by means of 12 gins and Pramer 1967). Ultrastructural exami-
single nucleotide polymorphic loci located at eight nations revealed that the cell wall of the
random DNA fragments, showed that ecological
niche separation contributed significantly, whilst geo-
constricting-ring cells is folded; when the cells
graphic separation contributed relatively little to inflate, the folded cell wall balloons out and
genetic variation. The differences found between forms the new cell wall (Heintz and Pramer
strains isolated from polluted zones versus those 1972; Liu et al. 2012). Interestingly, nematodes
from non-contaminated locations suggested that may still be able to escape trap formation: the
environmental stress might have contributed to eco-
logical divergence for populations. Thus, these data
constricting rings of Drechslerella doedycoides
confirm the relevance of local adaptation and ecologi- catch early larval stages with a diameter, which
cal niche specialization. Additionally, the lower level is similar to the trap opening. Yet there is a
of differentiation among geographical populations short delay between the ring entry and ring
suggested a long-distance dispersal and frequent closure, which allows the animal to withdraw
gene flow because of the predominant clonal repro-
duction between populations. It was also found that
from the trap before being caught. Mutants that
those strains isolated from stressful niches presented fail to suppress head movements in response to
more variability, an unambiguous evidence for touch are caught more efficiently than the wild-
Nematophagous Fungi 251
Fig. 13.3 Chemical structure of the ascaroside hormones from nematodes, according to Hsueh et al. (2013)
type. This demonstrates that the coordination After the nematode is killed, the fungus grows inside
of motor programs allows C. elegans to and feeds on it (Nordbring-Hertz et al. 1995). Interest-
ingly, Li et al. (2011) showed that attachment of the soil
smoothly retract from a fungal noose and bacterium Chryseobacterium spp. to the hyphae of A.
evade capture (Maguire et al. 2011). oligospora also induced trap formation in the absence
In contrast, the adhesive trap is surrounded of a nematode host. However, the mechanism has not
by a layer of fibrillar, extracellular polymers, been determined.
which becomes reorganized during the attach-
ment of the traps to the nematode cuticle (Tun- Andersson et al. (2014) used comparative
lid et al. 1992). Following the trapping of transcriptomics to investigate the molecular
nematodes, the infection mechanisms appear events during the infection process of two
to be rather similar in the species with con- nematodes (the root-knot nematode M. hapla
stricting rings and adhesive traps: the fungus and the sugar beet cyst nematode H. schachtii)
forms a penetration tube that pierces the nem- and of three nematode-trapping fungi that dis-
atode cuticle and paralyzes the nematode. Sub- play different trapping mechanisms, i.e., adhe-
sequently, the internal tissues are rapidly sive nets (A. oligospora), adhesive branches (M.
colonized and digested by fungal hyphae cionopagum), and constricting rings (A. dacty-
(Nordbring-Hertz et al. 1995). loides). They studied the phases of adhesion,
penetration, and digestion stages. Their data
One striking feature of the nematode-trapping fungi is showed that the divergence in interspecific
that they can sense the presence of their prey and only
then form the traps. Earlier work demonstrated that
gene expression between the three fungi was
nematodes secrete a morphogenic substance that significantly larger than that inferred by the
induces trap formation in the fungi (Pramer and Stoll nematode host used. The core set of genes,
1959). Hsueh et al. (2013) have recently shown that identified by the Pfam domains of the encoded
these substances are in fact ascarosides, nematode proteins, that was significantly expressed by all
pheromones that are composed of the dideoxy-sugar
ascarylose linked to a fatty acid-like side chain
three fungi included serine endoproteases
(Fig. 13.3). More than 100 different ascarosides have belonging to the subtilisin family, aspartyl pep-
meanwhile been identified from nematodes and func- tidases, proteins containing a CFEM domain (a
tion as inter-organismal signals that play a central role fungal-specific cysteine-rich domain that is
in regulating nematode development and behavior found in some proteins with proposed roles in
(Butcher et al. 2007; Srinivasan et al. 2008, 2012). In
both adhesive and constricting-ring types, the cuticles
fungal pathogenesis), proteins involved in fun-
of the captured nematodes are then penetrated and gal stress response, cell signaling, organization
finally an infection bulb is formed inside the nematode. of the cytoskeleton, vesicular transport and
252 A. Herrera-Estrella et al.
membrane transport, as well as several families biosynthesis GCN4 are induced, suggesting
of calcium-binding proteins and transcription that nematodes induce autophagy probably by
factors. triggering intracellular amino acid starvation.
Among the species-specific transcripts were those A transcriptomic analysis of Drechslerella stenobrocha,
encoding proteins of metallopeptidase families M1 which mechanically traps nematodes using a constrict-
and M24, lectins, tyrosinase, as well as some transcrip- ing ring also shed some light on the signal transduction
tion factors and cell-signaling components, and pro- cascade that is involved: like in entomopathogenic
teins containing the WSC (cell wall integrity and stress fungi during insect infection (Zheng et al. 2011), trap
response component) domain and the DUF3129 formation may involve the protein kinase C (PKC)
domain. The latter is a domain of unknown function pathway, as suggested by the strong upregulation of
that is found in the GAS1 protein of Magnaporthe gri- the pkc1 gene during trap formation. The authors pro-
sea, which participates in appressorial penetration and posed that one of the Ga-proteins (DRE_07451) could
lesion formation (Xue et al. 2002). The DUF3129 be the first step in this PKC pathway, which could be
domain protein-encoding gene was highly expressed activated by a signal from nematodes (see above). The
during infection among the species that forms adhesive authors also detected several putative transcriptional
branches and adhesive knobs. As for lectins, a fruiting- regulators of the fungal-specific Zn(2)Cys(6) type that
body lectin and a D-mannose-binding lectin were only could regulate the downstream genetic responses.
highly expressed in A. oligospora and not in the other Finally, they found that the transcription of genes
two fungi. Also, the WSC domain proteins, which are encoding a cell division protein and a cyclin peaked
one of those gene families that are expanded in during the phase of trap formation, suggesting that the
nematode-trapping fungi, are highly—yet differ- formation of constricting ring would involve cell divi-
ently—expressed during pathogenesis in different sion processes. In a similar approach, global patterns of
nematode-trapping fungi, suggesting that they contrib- gene expression in traps and mycelium of the fungus
ute to the specialization of the trapping mechanisms. Monacrosporium haptotylum were compared. In this
Two DUF3129-domain proteins, whose orthologs in case, the trap is a unicellular spherical structure called
other fungi were proven as virulence genes, were differ- the knob, which develops on the apex of a hyphal
ently expressed by the three fungi (Andersson et al. branch. Substantial differences in the patterns of
2014). Finally, a hydrophobin-like protein genes expressed in the two cell types were found, with
(AOL_s00006g570) was upregulated more than 12-fold about 23 % of the putative genes preferentially
in A. oligospora. Hydrophobins are able to assemble expressed in knobs. Various differentially expressed
spontaneously into amphipathic monolayers at hydro- genes were similar to genes known to be involved in
phobic–hydrophilic interfaces (Linder et al. 2005). In regulating morphogenesis and cell polarity in other
Beauveria bassiana, a nonspecific hydrophobic interac- fungi. This set of differentially expressed genes
tion between the fungal spore coat hydrophobin and included several putative small GTPases, such as rho1,
the insect epicuticle was found to be essential for the rac1, and ras1, and a rho GDP dissociation inhibitor
pathogenicity of the fungus (Zheng et al. 2011). (rdi1). Genes involved in stress responses, protein syn-
thesis and protein degradation, transcription, and car-
Trap formation has been shown to be bon metabolism were also among this set. A number of
favored under poor nutrient conditions. Chen the differentially expressed genes are also differentially
regulated during infection structure formation in plant-
et al. (2013) showed that the presence of the pathogenic fungi. Interestingly, gks1 a homologue of
nematodes induces autophagy—a process the Magnaporthe grisea (gas1/mas3) gene, which is
characterized by the degradation of unneces- specifically expressed in appressoria, was found
sary or dysfunctional cellular components that (Ahrén et al. 2005).
are involved in morphogenesis and morphol-
ogy in fungi—by nematodes during the early The initial phase of penetration is believed
stage of trap formation in A. oligospora. This is to be associated with recognition mediated by a
illustrated by the high expression of the atg8 lectin–carbohydrate interaction (Nordbring-
gene, which encodes an essential protein in the Hertz 1983). These lectins are located on fungal
autophagic pathway (Nakatogawa et al. 2007). traps or adhesive conidia that can specifically
Disruption of a homolog of this gene in A. bind a carbohydrate on the nematode cuticle. It
oligospora leads to reduce trap formation is has been suggested that after the recognition
(Chen et al. 2013). During the early stage of event, the fungus immobilizes the nematode
trap formation, the expression of genes encod- and secretes extracellular enzymes at the point
ing enzymes involved in amino acid biosynthe- of contact that allow the posterior parasitism
sis and the general regulator of amino acid (Tunlid et al. 1994). The nematode cuticle con-
Nematophagous Fungi 253
sists mainly of proteins, including collagens parative genomics indeed showed that some
(Cox 1992), and the nematode eggshell contains families of the metalloproteases (particularly
chitin fibrils embedded in a protein matrix, those that show collagenase activity) and of
with the chitin complex as a major barrier the subtilisin-type of the serine proteases are
against fungal infections (Warton 1980). Extra- strongly enriched in the nematophagous fungi
cellular enzymes that are capable of digesting (Meerupati et al. 2013; Lai et al. 2014; Fig. 13.4).
the main chemical constituents of the nema-
tode cuticle and eggshell (protein, chitin, and
lipids) have been isolated and identified in var- 1. Collagenases
ious nematophagous fungi (e.g., Lopez-Llorca The ascarid cuticle is a three-layered, fibrous
1990; Tunlid et al. 1994; Yang et al. 2005a, b; for structure, which contains nematode-specific
review see Yang et al. 2013a, b). When the types of collagen and keratin (Bird 1971). Col-
hyphae from the endoparasitic fungi Drech- lagens are among the most complex of proteins
meria coniospora and Hirsutella rhossiliensis and are slowly degraded in natural soils and
reach the eggshell, they form appressoria from waters (Weiss 1976). During the infection of
which these extracellular enzymes are then nematodes, nematophagous fungi must penetrate
secreted (Lopez-Llorca and Robertson 1992). the nematode cuticle, and particularly collage-
This formation of appressoria depends on the nases have been considered important enzymes
recognition of the host surface, and surface involved in the pathogenicity of nematophagous
hydrophobicity is considered an important rec- fungi (Dackman et al. 1992; Tunlid et al. 1994).
ognition factor in this process (Lopez-Llorca Meerupati et al. (2013) showed that peptidases
et al. 2002). belonging to the MEROPS family M10, which
display strong collagenolytic activity, are strongly
enriched in A. oligospora and M. haptotylum,
V. Virulence Factors whereas these genes are almost absent from insect
pathogenic fungi. This is in agreement with earlier
Virulence factors are molecules produced by studies that nematode-trapping fungi produced
pathogens, which are essential for major con- collagenase in the growth media of all tested spe-
tribution to their pathogenicity, by enabling cies (Schenck et al. 1980).
them to attach to the host, escape the defense The M10 metallopeptidases belong to a
mechanisms, and finally feed on it. The previ- group of peptidases known as the “metzincins”
ous chapter on the molecular mechanisms that due to a conserved methionine C-terminal to
take place during attack of the nematode by the the zinc ligands. Both subfamilies of M10 con-
fungus has already pointed to potential candi- tain mosaic proteins, which contain, e.g.,
dates for virulence factors. In this chapter, we glycine-rich C-terminal domains that can bind
will discuss those components for which calcium ions or domains homologous to hemo-
detailed scientific information is already avail- pexin and vitronectin that aid in binding to the
able. extracellular matrix. The gelatinases have
acquired three domains homologous to type II
segments of fibronectin nested within the pep-
tidase unit. Details about the structure of the
A. Proteases
fungal M10 metalloproteases, as well as genetic
Being saprophytes, most Pezizomycota are proof for their action as virulence factors, are
known to possess a rich arsenal of proteolytic however yet lacking.
enzymes. According to the MEROPS database,
the main groups found are aspartyl proteases, 2. Subtilisins
cysteine proteases, metalloproteases, and serine
proteases, the latter two making up for the bulk Almost all the proteases from entomopatho-
of secreted proteases (cf. Lai et al. 2014). Com- genic and nematophagous fungi that were iden-
254 A. Herrera-Estrella et al.
400
300
Number of Genes
200
100
0
AspP CysP MeP M10 SerP S8 Total
Fig. 13.4 Number of selected protease genes in the OPS family M10 metalloproteases within MeP, SerP
genomes of nematophagous and insect pathogenic serine proteases, S8 MEROPS family S8 subtilisins
fungi. Fungal species are indicated by color bars: blue: within SerP, Total all protease genes encoded in the
A. oligospora, red: M. haptotylum, grey: D. stenobrocha, respective genome. Data taken from Yang et al. (2011a,
yellow: H. minnesotensis, black: M. anisopliae, green: B. b), Meerupati et al. (2013), Liu et al. (2014), Lai et al.
bassiana. Abbreviations: AspP aspartyl proteases, CysP (2014), Gao et al. (2011), and Xiao et al. (2012)
cysteine proteases, MeP metalloproteases, M10 MER-
tified and shown to have nematicidal activity in Yang et al. 2011a, b). The A. oligospora subtili-
the pre-genomic era belonged to the large sub- sin PII immobilizes the active stages of Pana-
tilisin family of endopeptidases MEROPS M8 grellus redivivus and hydrolyzes its cuticle
found only in fungi and bacteria. This family (Tunlid et al. 1994). The enzyme is expressed
is enriched in nematophagous fungi, but also in under starvation conditions (Åhman et al.
insect pathogens (Meerupati et al. 2013; Lai 1996). Another subtilisin from A. oligospora
et al. 2014). The role of serine proteases as (Aoz1), whose amino acid sequence is 97 %
virulence factors was first demonstrated in similar to that of PII, also produced dramatic
invertebrate pathogenesis by the entomopatho- structural changes in the nematode cuticle
genic fungi Metarhizium anisopliae (St. Leger (Minglian et al. 2004). Deletion of the PII gene
et al. 1987) and B. bassiana (Bidochka and had only a limited effect on pathogenicity
Khachatourians 1987), where a 30 kDa serine (decreased adhesion and immobilization of
protease (Pr1) was found to play an important nematodes and formation of less traps), proba-
role in the infectious process (Morton et al. bly due to the presence of aoz1. Overexpression
2004). Proteases that share characteristics with of the PII-encoding gene, however, resulted in a
Pr1, such as size, sensitivity to inhibitors, and higher capacity to kill nematodes and forma-
substrate pattern (thus called Pr1-like), were tion of more traps (Åhman et al. 2002).
first purified and characterized from the oppor-
tunistic nematophagous fungi Paecilomyces More recently, a genome-wide transcriptional analysis
lilacinus (Bonants et al. 1995), P. rubescens of A. oligospora revealed that in fact the gene encoding
(Lopez-Llorca 1990), and P. chlamydospora yet another subtilisin (P186) was more than 40-fold
upregulated, while that encoding PII was even down-
(Segers et al. 1994). regulated, suggesting that P186 may be the main prote-
In A. oligospora, subtilisins appear to play a ase required for penetration of the nematode cuticle. In
key role in the early stages of infection, includ- fact, a protease belonging to the same subfamily in S8 as
ing immobilization of the captured nematode P186 was also found upregulated in the knob proteome
(Tunlid and Jansson 1991; Åhman et al. 2002; of M. haptotylum (Andersson et al. 2013).
Nematophagous Fungi 255
To better understand the cellular functions of lin membrane from eggs of Meloidogyne incog-
adhesive traps, the proteome and transcriptome of nita (Segers et al. 1994). Subsequent infections
trap cells versus mycelia of the fungus Monacrosporium
haptotylum were assessed. The comparison of protein
of these eggs by P. chlamydosporia degraded
expression between mycelia and knobs revealed that 54 extensively the eggshell to the degree of gener-
out of 336 detected proteins were highly expressed in ating large holes in the structure, with no evi-
the knobs compared with mycelia. Secreted proteins dent formation of appressoria while this was
were overrepresented: secretion signals were predicted not the case when eggs of G. pallida were trea-
in 26 sequences (48 % of de proteins identified), includ-
ing Small secreted cysteine-rich proteins (SSCRPs),
ted with VCP1, which points to the importance
peptidases and carbohydrate-binding proteins contain- of the different composition of nematode egg-
ing WSC and GLEYA domains, and proteins involved shells (Morton et al. 2004).
in stress response. WSC is a cysteine-rich domain with
eight conserved cysteine residues that are required for Sequence analysis of the vcp1 upstream region from 30
its function (Heinisch et al. 2010; Dupres et al. 2011). different isolates of Pochonia chlamydospora revealed
All the upregulated WSC domain proteins belong to a that this region is highly conserved, ranging from 91 to
large expanded cluster of paralogs in M. haptotylum. 97 % identity, and contains putative regulatory motifs
Various peptidases and homologs of experimentally for carbon (CREA and CREB) and nitrogen repression
verified proteins in other pathogenic fungi were also (GATA), and pH regulation (PacC). Indeed, the pres-
upregulated in the knob proteome. The expression of ence of glucose, ammonium, and changes in pH
only six of the upregulated knob genes was reflected in affected the expression of this gene. For instance, addi-
increased protein levels. These proteins included a tion of glucose to the growth medium significantly
putative surface protein of the PA14_2/GLEYA family, repressed VCP1 enzyme and mRNA levels, whereas
a glutathion S-transferase, an alcohol dehydrogenase, the presence of M. incognita eggs did not downregulate
and two hypothetical proteins with predicted secretion neither the VCP1 enzyme nor the mRNA. Furthermore,
signals. In agreement with the upregulated proteome, the presence of ammonium chloride significantly
the upregulated transcriptome was also enriched in reduced the VCP1 mRNA and proteins levels; however,
sequences predicted to have a signal peptide (20 %). at longer times (24 h), the enzyme and the mRNA levels
Therefore, the traps of M. haptotylum seem to have the were considerably upregulated (Ward et al. 2012).
necessary proteins for the early stages of infection Cryo-scanning electron microscopy revealed that
(Andersson et al. 2013). VCP1 production occurred only when the fungus and
P. chlamydospora eggs are in close contact. These
Subtilisins also appear to play dominant results indicated that the presence of preferable carbon
roles in the infection by non-trap forming sources and unfavorable pH in the rhizosphere/egg-
mass environment might negatively affect the nema-
nematophagous fungi: infection of nematode tode parasitism by P. chlamydosporia. On the contrary,
eggs by D. coniospora was blocked by the addi- the presence of ammonium nitrate may favor the bio-
tion of the serine protease inhibitor, chymosta- control of this nematode by the fungus at longer times
tin, indicating the possible role of serine (Ward et al. 2012).
proteases in the infection process (Jansson The regulation of the expression of subtilisins has
also been studied in the case of prC in Clonostachys rosea,
and Friman 1999). Addition of serine protease whose encoded protein immobilized nematodes and
inhibitors reduced egg penetration by the fungi hydrolysed proteins of the nematode cuticle (Li et al.
Lecanicillium lecanni and P. chlamydosporia, 2006): the presence of putative transcription control
further supporting the relevance of proteases sites in the promoter for nitrogen regulation (50 -
at the early stages of the infection process GATA), carbon regulation (50 -SYGGRG), pH regulation
(50 -GCCARG), and stress response element (STRE) (50 -
(Lopez-Llorca et al. 2002). A subtilisin named AGGGG) suggested that the expression of prC may be
P32 was immunolocalizated in appressoria of regulated by nitrogen sources, environmental pH, and/or
the fungus P. rubescens that infects eggs of the other stress conditions (Zou et al. 2010a, b). To study the
beet cyst nematode Hetrodera schachtii (Lopez- effect of pH, the C. rosea orthologue of the pH transcrip-
Llorca and Robertson 1992). tional regulator PacC was deleted. The expression of prC
was downregulated in DpacC mutants, and the prC tran-
The opportunistic fungus P. chlamydos- script levels were significantly higher under alkaline
poria produces an alkaline subtilisin (VCP1) growth conditions than under acidic growth conditions.
during the infection of nematode eggs. The Induction of prC expression by nematode cuticles was
incubation of nematodes eggs with purified significantly suppressed by glutamine, ammonia, and
VCP1 resulted in the removal of the outer vitel- serine protease inhibitors (Zou et al. 2010c).
256 A. Herrera-Estrella et al.
Aside from pH and nematode cuticle-induced trapping devices, they likely rely mainly on the
changes of gene expression, the expression of prC was extracellular enzymes as virulence factors to
also upregulated by oxidants (H2O2 or menadione) and
heat shock, probably through a stress response path-
help them penetrate and digest nematode cuti-
way. Interestingly, the addition of nematode cuticle cles (Huang et al. 2004; Yang et al. 2007a). It is
significantly attenuated the production of reactive oxy- likely that their subtilisins evolved towards
gen species induced by oxidants and heat shock in the increased activity and broad substrate specific-
wild-type strain but not in the DprC mutants (Zou et al. ity. In contrast, the nematode-trapping fungi
2010b). This suggests that PrC is not only involved in
the degradation of nematode cuticles but also plays a
degrade the trap-captured nematode without
role in the adaptation to environmental stresses. time constraints because it is already paralyzed
by the trap. This interpretation is also sup-
The serine protease Ver112 from the nema- ported by the fact that the subtilisin-like serine
tophagous fungus Lecanicillium psalliotae is proteases of the nematode-trapping fungi were
capable of degrading the nematode cuticle and found to be under positive selection, suggesting
killing nematodes effectively (Yang et al. 2005a, co-evolution of trapping structures and proteo-
b). The Ver112 gene was used to genetically lytic enzymes (Li et al. 2010).
transform P. lilacinus. Protease activity of the
transformants was higher than in the wild-type B. Chitinases
and correlated with a stronger ability to immo-
bilize, infect, and degrade the nematode Pana- Nematophagous fungi that parasitize nematode
grellus redivivus and Caenorhabditis elegans eggs must penetrate the eggshell during the
than the wild-type. The crude protein extract infection (Lopez-Llorca and Duncan 1988;
of the transformants showed enhanced nemati- Lýsek and Krajcı́ 1987). As mentioned above,
cidal activity compared to the wild-type (Yang the structure of the eggshell is formed by sev-
et al. 2011a, b). eral layers, including one formed by chitin
The evolution of subtilisin-like serine pro- (Warton 1980), which is the thickest and prob-
teases in Pezizomycotina has been analyzed (Li ably the major barrier for infection (Bird and
et al. 2010): molecular phylogeny divided the Bird 1991).
serine proteases from nematophagous fungi Most fungi are able to degrade chitin, and
into two clades with neutral proteases from two main enzyme classes cooperate in its deg-
nematode-trapping fungi clustering in clade A radation: chitinases (belonging to the glycoside
and the alkaline ones from nematode-parasitic hydrolase family GH18) and N-acetyl-b-D-glu-
and entomopathogenic fungi clustering in clade cosaminidases (belonging to GH20). The action
B. Both share a high degree of sequence iden- of the former leads to soluble chitooligosac-
tity, have very similar molecular structure, and charides with a chain length of at least two
play a similar role in degrading host cuticle amino sugar units, which are subsequently fur-
during fungal infection of nematodes. However, ther hydrolysed to NAcGln by the N-acetyl-b-D-
their structure reveals interesting differences in glucosaminidases. Furthermore, chitin can be
the 3D structure of the substrate-binding deacetylated by chitin deacetylases (EC
regions and some neighboring loops and turns 3.5.1.31) found in carbohydrate esterase family
(Liang et al. 2010; Yang et al. 2010a, b): disulfide 4 (CE 4) in the Carbohydrate Active Enzymes
bridges, which contribute to the stabilization of database (CAZy) classification (Hartl et al.
the local/global structures and enhance the 2012).
structural flexibility of two of the substrate
sites, were only present in the alkaline, but not Fungal chitinases can be further divided into three
in the neutral protease. This may explain why different subgroups, A, B, and C, based on the amino
the alkaline proteases have higher substrate acid sequences of their GH18 modules. These sub-
affinity and catalytic activity than neutral pro- groups differ in the architectures of their substrate-
binding cleft and thus their enzymatic activities (exo
teases. Since nematode-parasitic fungi, which vs. endo), and those from subgroups B and C contain
contain only alkaline proteases, do not produce different carbohydrate-binding modules (CBM 18 and
Nematophagous Fungi 257
CBM50; see also www.cazy.org; Gruber and Seidl- gests a role of A. oligospora chitinases in bio-
Seiboth 2011; Seidl 2008). Class A chitinases comprise control. The expression patterns of A.
some of the most frequently described fungal chiti-
nases. They typically have a Mr (Molecular weight
oligospora chitinases suggest that they play dif-
range) between 40 and 60 kDa, their active center is ferent roles in growth, differentiation, and
located in a deep cleft, and they are exo-acting. Class B infection (Yang et al. 2013a, b).
enzymes are usually somewhat smaller (30–50 kDa), When five Trichoderma species (T. harzia-
endo-acting, their active center is located close to the num, T. virens, T. atroviride, T. rossicum, and T.
proteins surface, and they typically contain a
carbohydrate-binding domain at their C-terminus (fre-
tomentosum) were tested against C. elegans, T.
quently of the cellulose-binding CBM1 type). Class C harzianum was more successful at parasitizing
chitinases, in contrast, are large proteins (120–200 kDa) eggs. During egg parasitism, the expression
that act in an exo-type with an active center in a deep levels of chi18-12 and chi18-5 were significantly
and narrow cleft. Most typical for them is the presence higher than controls, which suggest a role of
of a CBM18 chitin-binding or CBM50 peptidoglycan-
binding (LysM) domain. The presence of CBMs in class
these endochitinases in the infection process
B or C chitinases enables them to bind more tightly to (Szabó et al. 2012).
insoluble substrates (Eijsink et al. 2008). Bacterial pro-
teins with LysM domains have been reported to be
involved in specific recognition events between nitro-
gen fixing bacteria and their plant hosts (Knogge and C. Lectins
Scheel 2006). Besides nutritional purposes, subgroup B
chitinases appear also to be involved in mycoparasitic It has for a long time been assumed that the
and entomopathogenic functions. adhesion of nematophagous fungi to their host
might be mediated by the interaction between
The first report for chitinase activity in lectins on the surface of trapping devices or
nematophagous fungi was in Verticillium spp., adhesive spores and carbohydrate ligands on
isolated from infected nematode eggs, both in the nematode cuticle (Nordbringhertz and
screening on solid media with colloidal chitin Mattiasson 1979). Lectins are carbohydrate-
and in liquid media. Several chitinases have binding proteins that are present in all organ-
also been identified from egg-parasitic fungi, isms. A comparison of the genomic inventory
which were found to serve as a nematicidal of trap-forming fungi and nematode patho-
factor in infecting nematode eggs (Tikhonov genic or insect pathogenic fungi revealed that
et al. 2002; Khan et al. 2004; Gan et al. 2007). the trap-forming species indeed had a much
On the basis of their properties, most of them higher number of lectin-encoding genes than
seem to belong to the A subgroup. other fungi. A detailed analysis showed that
The Arthrobotrys oligospora genome con- the most abundant lectin family—like in other
tains 16 Open Reading Frames encoding puta- fungi—are the concanavalin A-like lectins that
tive chitinases that belong to the glycoside bind a-D-glucose and a-D-mannose (Fig. 13.5).
hydrolases (GH) family 18. These chitinases However, the ricin B-type lectins (PF14200), the
vary considerably in their functional domains, H-type lectin (binding to N-acetyl-b-D-galac-
size, and pI. Based on the phylogenetic relation- tosamine), the fucose-specific lectin, and the
ship, these were grouped into four clades: I, II, bulb-type lectin were all significantly more
III, and IV, that include an A. oligospora-spe- abundant in the trap-forming species A. oligos-
cific subclade (Clade IV-B), which includes pora, D. stenobrocha, and M. haptotylum (Lai
chitinases 100 kDa. Most of the A. oligospora et al. 2014).
chitinases genes are downregulated in absence
of carbon; conversely nitrogen starvation upre- During trap formation and infection, all trap-forming
gulates all chitinase-encoding genes. Nonethe- fungi expressed transcripts encoding RicinB_lectins,
less, chitinase AO-190 was upregulated in both, which are ribosome-inactivating proteins (RIPs) con-
carbon and/or nitrogen starvation. Further- sisting of a catalytic A-chain and a sugar-binding B-
chain (Michiels et al. 2010). The effects of these lectins
more, chitinases AO-59, AO-190, and AO-801 from trap-forming fungi on the nematode have not yet
increased their transcription in the presence of been studied, but a RicinB_lectin_2 domain-containing
colloidal chitin or R. solani cell wall. This sug- protein (MOA) of the basidiomycete Marasmius
258 A. Herrera-Estrella et al.
80
Number of Genes 60
40
20
0
AOL MHA DRS HIM MAN BBA
Fig. 13.5 Number of selected lectin-encoding genes in lum, DST D. stenobrocha, HMI H. minnesotensis,
the genomes of nematophagous and insect pathogenic MAN M. anisopliae, BBA B. bassiana. Data taken from
fungi. Blue Concanavalin A lectin; red ricin B-like lec- Yang et al. (2011a, b), Meerupati et al. (2013), Liu et al.
tin; grey H-type lectin; yellow fucose-specific lectin; (2014), Lai et al. (2014), Gao et al. (2011) and Xiao et al.
black bulb-type lectin; black total number of lectins. (2012)
Abbreviations: AOL A. oligospora, MHA M. haptoty-
oreades displayed nematotoxic activity against C. ele- acids long and containing four or more cysteine
gans (Wohlschlager et al. 2011). This nematotoxicity residues. Among them, hydrophobins,
was dependent on the cysteine protease activity of
MOA and the binding of its lectin domain to glyco-
hydrophobin-like proteins, and elicitor-like
sphingolipids in the worm intestine. A Sclerotinia scler- proteins make up for a major part, but many
otiorum agglutinin (SSA) also contains a RicinB- others are found for which no function has
lectin_2 domain and shows insecticidal properties been predicted. In Trichoderma spp., some
when fed to the pea aphid Acyrthosiphon pisum (Ham- members of this cluster contain CFEM domains
shou et al. 2010). Most recently, a ricin B-like single-
domain lectin (MpL) has been isolated and character-
or consensus sequences for glycosylphosphati-
ized from the parasol mushroom Macrolepiota procera dylinositol (GPI anchors), suggesting that they
(Žurga et al. 2014). MpL exhibits highest specificity for could be cell surface proteins with important
terminal N-acetyllactosamine and related b-D-galacto- roles in the interaction with other organisms, as
sides and contains a second putative carbohydrate- in C. albicans (Druzhinina et al. 2012). Meeru-
binding site with a low affinity for D-galactose. MpL
was shown to be toxic to C. elegans. Summarizing,
pati et al. (2013) detected that “SSPs” make up
there is now accumulating evidence that the RicinB- for a large amplified group of orphan genes of
lectins exhibit toxicity to nematodes, and they could the “knob-forming” fungus M. haptotylum—
well be the agent that paralyzes the host after forming but not in the “net-forming” A. oligospora.
the trap. This is a challenging topic of further work with 27.6 % of them were actually clustered in the
the trap-forming fungi.
genome, of which 34 genes that were >10-fold
upregulated in M. haptotylum during early
infection were located in clusters. This suggests
D. Small Secreted Cysteine-rich Proteins that the SSPs could play an important role in
knob formation in this species.
One of the largest groups of proteins secreted Proof for a function of SSPs has been
by mycoparasitic fungi like Trichoderma spp. obtained in the case of cerato-platanins, a
are the so-called “small secreted cysteine-rich group of small, secreted, cysteine-rich proteins
proteins (SSCPs).” They were identified by the that have been implicated in virulence of cer-
criteria that their Mr should be 300 amino tain plant pathogenic fungi and also shown to
Nematophagous Fungi 259
stimulate plant defense against pathogenic phomalactone, was isolated from P. chlamydosporia
fungi (Djonovic et al. 2007; Salas-Marina et al. (Hellwig et al. 2003); and culture filtrates from several
fungi grown in malt extract broth were toxic to infective
2015; Pazzagli et al. 2014). juveniles and eggs (Chen et al. 2000). Several secondary
The nematophagous fungus Dactylellina metabolites have been isolated from the nematode egg-
cionopaga, which is a known parasite of the parasite P. chlamydosporia, including radicicol
nematode plant pathogens Meloidogyne java- (¼monorden; a resorcylic acid lactone), tetrahydromo-
nica and Heterodera schachtii, develops adhe- norden, pseurotin A, pochonins A to J (Hellwig et al.
2003 and Shinonaga et al. 2009; Zhou et al. 2010), and
sive columns and two-dimensional networks various aurovertin-type metabolites (Niu et al. 2010).
(Khan et al. 2006; Jaffee and Muldoon 1995). Radicicol biosynthesis has been studied in detail, as
The cerato-platanin family of proteins plays usually found in fungal genomes, the genes encoding
roles in parasitism, recognition, adhesion, cell- the corresponding biosynthetic pathways are clustered
wall morphogenesis, fungal growth and devel- (rdc1–rdc5; Zhou et al. 2010) and encode two fungal
iterative polyketide synthases (PKS). Rdc5, the highly
opment, and induction of the systemic resis- reducing IPKS, and Rdc1, the nonreducing IPKS, are
tance to pathogens in plants (Djonovic et al. required for the biosynthesis of radicicol. The biochem-
2007; Salas-Marina et al. 2015). The transcrip- ical pathway, by which Rdc1 and Rdc5 catalyze the
tion levels of D. cionopaga snodprot increased biosynthesis of radicicol, and how the remaining
in the presence of nematodes and was induced genes of the cluster contribute, has been elucidated
(Zhou et al. 2010). During endophytic root coloniza-
during the development of traps and conidia. tion, P. chlamydosporia expressed 56 % of the second-
The recombinant protein changed the chemo- ary metabolism pathway genes found, including seven
taxis and increased the body-bend frequency of of the radicicol cluster.
C. elegans, but did not induce immunity in
plants. In agreement with the parasitism So far, 179 nematicidal compounds belong-
mechanisms of nematophagous fungi, chemo- ing to diverse chemical groups have been iden-
taxis, and locomotion mechanism of C. elegans, tified from nematophagous fungi, of which only
the possible targets of snodprot could be ASE three (oligosporon, 40 ,50 -dihydro-oligosporon,
and ASI neurons, which are involved in the and linoleic acid) were from A. oligospora (Li
process of chemotaxis to NaCl, the response to et al. 2007). A genomic comparison of PKS,
serotonin, and the locomotion of C. elegans (Yu nonribosomal peptide synthases, and terpene
et al. 2012). Together these results indicated synthases (TPS) revealed that the trap-forming
that snodprot is a novel parasitism-related pro- fungi contained the lowest number of these
tein of nematophagous fungi with a non- genes (Fig. 13.6), whereas the nematode endo-
described activity. parasite H. minnesotensis exhibited the highest
number of secondary metabolite synthases,
even in comparison with insect pathogens, par-
E. Secondary Metabolites ticularly of the PKS-1, NRPS, and TPS class (Lai
et al. 2014). Six NRPS and two TPS genes were
In order to antagonize or kill their competitors, unique to the nematode endoparasitic fungus,
many microorganisms produce toxic metabo- suggesting lineage-specific expansion of these
lites, such as antibiotics. Toxins are particularly families in the H. minnesotensis genome. Thus,
important for parasitic microorganisms, while trap-forming fungi do not seem to make
because they facilitate infection by debilitating strong use of secondary metabolites (possibly
the host (Morton et al. 2004). In addition, P. because the lectins already paralyzed the nema-
lilacinus produces acetic acid to paralyze juve- todes; vide supra), the endoparasites heavily
nile nematodes (Djian et al. 1991). rely on these metabolites to kill the host.
Trichoderma spp. are biocontrol agents
So far, the majority of nematicidal secondary metabo- widely used in plant protection due to their
lites characterized are those produced by opportunistic capacity to antagonize phytopathogenic fungi.
fungi. Fusarium equiseti produces compounds that
reduce hatch of root-knot nematode eggs and immobi- Nevertheless, it is known that some Tricho-
lize infective juveniles (Nitao et al. 1999); a metabolite derma spp. produce secondary metabolites
with nematicidal activity against infective juvenile, with nematicidal activity, including trichoder-
260 A. Herrera-Estrella et al.
100
Number of Genes
80
60
40
20
0
AOL DRS MHA HIM MAN BBA
Fig. 13.6 Number of secondary metabolite synthases in pora, MHA M. haptotylum, DST D. stenobrocha, HMI
the genomes of nematophagous and insect pathogenic H. minnesotensis, MAN M. anisopliae, BBA B. bassiana.
fungi. Red: polyketide synthases (PKS), green: non- Data taken from Yang et al. (2011a, b), Meerupati et al.
ribosomal peptide synthases (NRPS), grey: hybrid (2013), Liu et al. (2014), Lai et al. (2014), Gao et al.
PKS-NRPS, yellow: terpene synthases, blue: total num- (2011), and Xiao et al. (2012)
ber of synthases lectins. Abbreviations: AOL A. oligos-
min (Yang et al. 2010a, b), acetic acid (Djian penetrating the cell wall of epidermis and cor-
et al. 1991), gliotoxin (Watanabe et al. 2004; tex cells, from tomato and barley. However,
Anitha and Murugesan 2005), and the peptide these fungi never penetrate the vascular tissue
cyclosporin A. Recently, the volatile organic (Lopez-Llorca et al. 2002; Bordallo et al. 2002).
compound 6-pentyl-2H-pyran-2-one from Tri- Plants endophytically colonized by nematopha-
choderma spp. was shown to kill >85 % of gous fungi show enhanced defense responses
Panagrellus redivivus, Bursaphelenchus xylo- and biomass gaining (Maciá-Vicente et al.
philus, and C. elegans in 48 h at 200 mg/l 2009a, b). LopezLlorca and coworkers detected
(Yang et al. 2012). the production of serine protease P32, VCP1,
and SCP1 from a nematophagous fungus in
roots colonized endophytically by this microor-
ganism. As mentioned above, these proteases
VI. Plant Endophytism by are produce by nematophagous fungi on their
Nematophagous Fungi nematode host. Thus, the expression of these
proteins in the absence of their host would
Several nematophagous fungi may be found as imply that plants colonized by these fungi
endophytes of plant roots. A plant endophyte is could be protected from nematode attack
a plant microbial endosymbiont, which lives before contact (Lopez-Llorca et al. 2010).
part of its live cycle in the plant, without Lopez-Llorca and coworkers showed that
provoking negative effects to its host. On the fungi other than A. oligospora and P. chlamy-
contrary, the presence of endophytes frequently dosporia grow endophytically in roots (Lopez-
results in positive effects in plant growth and Llorca et al. 2006). For instance, the endopara-
development. Nematophagous fungi such as P. sitic basidiomycete Nematocnus robustus,
chlamydosporia and A. oligospora can present which infects nematodes through adhesive con-
endophytic life style for both monocots (Lopez- idia, penetrated and colonized barley roots and
Llorca et al. 2002) and dicots (Bordallo et al. formed clamp connections, whereas N. pachy-
2002). Both fungi were capable of growth inter- sporus did not colonize the root system, but
and intracellularly and form appresoria when colonized the root surface (Lopez-Llorca et al.
Nematophagous Fungi 261
2006). The nematode endoparasitic fungus Hir- related to detoxification were also detected. Genes
sutella rhossiliensis has a similar behavior since involved in cell wall biosynthesis and modification,
including chitin synthesis activators, chitin synthases,
it only colonizes the root surface. Furthermore, lipopolysaccharide modifying proteins, and hydropho-
fungi belonging to the basidiomycota, such as bins, were also found expressed under endophytism.
Pleurotus djamor that immobilizes nematodes Additionally, the P. chlamydospora genome encodes
with a toxin (Kwok et al. 1992) prior to infec- 409 putative transcription factors (TFs), grouped in
tion and digestion of its prey, also colonizes six families, of which the Zn2Cys6 fungal-specific type
TF contains the highest number of genes expressed
and penetrates barley roots (Lopez-Llorca under endophytism. In addition, the P. chlamydospora
et al. 2006). The nematode-trapping fungus genome encodes G-protein subunits (8), putatively
Arthrobotrys dactyloides is also a root colonizer involved in vegetative growth, conidiation conidium
that penetrates the epidermal cells and forms attachment, appresorium formation, mating, and path-
coiling structures in barley root cells (Lopez- ogenicity, from which six were expressed in endophyt-
ism. The genome of P. chlamydospora also contains 54
Llorca et al. 2006). homologues to Pth11-like G protein-coupled receptors
(GPCR), 27 small GTPase regulators, and 12 Rab
To better understand the endophytic process, the P. GTPase activators, all of which presumably regulate
chlamydospora genome was recently sequenced and its endophytic behavior. Similarly to entomopathogens
consists of 41.2 Mb, from which 12,122 gene models or plant pathogens, the P. chlamydospora genome con-
were predicted. Under the endophytic relationship with tains 153 genes for protein kinases involved in the
barley roots, 63 % (7586 genes) of the genome was regulation of cell and metabolic processes, from which
expressed. From the 1432 predicted secreted proteins, 75 had matches in the PHI database. Together with PKs,
57 % were expressed under this condition. 663 pre- genes coding for histidine kinases (HKs) were identi-
dicted genes did not exhibit any homologue in the fied, nearly all which had homologous in the PHI data-
NCBI database and almost a half of them (277) were base. From these 14 were expressed during
expressed during P. chlamydospora endophytic life- endophytism (Larriba et al. 2014). All together these
style. Phylogenetic analysis of genome-encoded ortho- results provide information for understanding the
logous showed that P. chlamydospora is most closely molecular mechanism involved in the multitrophic life-
related to Metarhizium anisopliae and M. acridium. A style of P. chlamydospora.
search for pathogenesis-related genes in the Pathogen–
Host Interaction (PHI) database that collects
pathogenesis-related genes of fungi, bacteria, and
Recently, a fusion PCR-based deletion
oomycetes (Winnenburg et al. 2008) showed that 1981 method was developed for P. chlamydosporia,
genes (16 %) shared homology with genes included in using the split marker strategy and PEG-
the PHI database, 24 % (468 genes) of which encoded mediated protoplast transformation. The
putatively secreted proteins. The majority of genes authors were capable of generating three stable
putatively associated with pathogenesis and endophyt-
ism encode hydrolytic enzymes and signal transduction
deletion mutants resistant to neomycin (G418
proteins. The hydrolases found in PHI included metal- sulfate) of genes induced during infection of
loproteases and chitinases, whilst those expressed nematode eggs by P. chlamydosporia: one chit-
under endophytism include serine and rhomboid pro- inase (VFPPC_01099) and two protease genes
tease families and a protein phosphatase. The P. chla- (VFPPC_10088 and VFPPC_06535) (Shen et al.
mydospora genome contains a wide set of genes
encoding hydrolytic enzymes, from which almost a
2014). After screening ~100 mutant candidates
half were expressed during endophytism. In addition by PCR, the average rate of gene knockout was
it contains 15 PKS and 12 putative non-ribosomal pep- 13 %. This method resulted in an efficient
tide synthases (PRPS), together with a number of PKS- homologous gene knockout strategy for P. chla-
and NRPS-like proteins, and 4 NRPS–PKS hybrid mydosporia, which together with the availabil-
genes. A radicicol gene cluster was also found. P. chla-
mydospora expressed 56 of the secondary metabolism
ity of the genome sequences opens the
pathway genes identified. From these seven genes opportunity for high-throughput genetic analy-
belonging to the radicicol cluster were expressed. The sis in this fungus (Shen et al. 2014).
P. chlamydospora genome contains 290 transporters of
the major facilitator superfamily (MFS), 58 ATP-
binding cassette (ABC) transporters, and 113 general
transporters, most of which exhibited homologs VII. Concluding Remarks
included in PHI database. P. chlamydospora expressed
drug resistance, sugar/inositol, oligopeptide, and
amino acid transporters during endophytism. Further- The last decade has resulted in a burst of scien-
more, a number of genes encoding oxidoreductases tific research on nematophagous fungi, fueled
262 A. Herrera-Estrella et al.
by the advance in techniques for genome gene expression in trap cells and vegetative hyphae
sequencing and transcriptome analysis. Thus, of the nematophagous fungus Monacrosporium
haptotylum. Microbiology 151:789–803
the knowledge that has previously accumulated Andersson KM, Meerupati T, Levander F, Friman E,
in relation to the structural characteristics of Ahrén D, Tunlid A (2013) Proteome of the
the specialized structures produced by nematode-trapping cells of the fungus Monacros-
nematode-trapping fungi during the interac- porium haptotylum. Appl Environ Microbiol
tion with their hosts and even in the life cycle 79:4993–5004
Andersson KM, Kumar D, Bentzer J, Friman E, Ahrén
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obtained complement by “-omics” data on sev- gene expression patterns in nematode-trapping
eral species, renders the nematophagous fungi fungi. BMC Genomics 15:968
an attractive subject to investigate their molec- Anitha R, Murugesan K (2005) Production of gliotoxin
ular physiology in detail. Despite all the above- on natural substrates by Trichoderma virens. J
Basic Microbiol 45:12–19
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14 Beetles versus Fungi: Trophic Interactions in Boreal Forests
DMITRY SCHIGEL1
Fungus–insect interactions can be com- Coleoptera is one of the most important, in numbers of
pared with plant–herbivore systems (Shaw species and the diversity of associations, orders of fun-
givorous insects (Hammond and Lawrence 1989). Other
1992). Herbivore invertebrates often specialise insect orders that share compact but structured fruit
on genera or genus groups, but certain guilds bodies with beetles are Diptera (Wertheim et al. 2000;
are monophagous. Noteworthy that higher Yamashita and Hijii 2007b), Hymenoptera, Hemiptera,
diversity of herbivores in tropics is mostly Lepidoptera and Thysanoptera. Ambrosia bark beetles,
explained by higher diversity of plants, while attine ants and termites are well known for their abilities
to cultivate fungal gardens. Noninsect invertebrate
there is no clear latitudinal trend in numbers of groups (Takahashi et al. 2005), such as mites (Makarova
consumers per host, consumer specialisation 2004), millipedes, molluscs, oligochaetes, nematodes,
and beta diversity (Lewinsohn and Roslin woodlice and springtails, are also tied into the complex
2008). There are much fewer studies on tropical network of ecological interactions.
(Yamashita et al. 2015), subtropical or Mediter-
ranean (Quinto et al. 2015) saproxylic and fun- The number of insect species associated
givorous food webs than on boreal and with fungi in the UK was estimated by
temperate systems, and a meta-analysis or a Paviour-Smith (1960) as 600 species. There are
gradient study would be needed to explore the estimated 300–400 fungivorous beetle species
parallels between plant–herbivore and fun- in Finland (Schigel 2011b) and potentially 500–
gus–insect host use systems. As in plant–her- 1000 species from at least 20 families in Europe.
bivore systems, interactions between fungi and Adults of truly fungivorous beetles are attracted
insects include grazing, defence (Rohlfs et al. by the fungal odours to the fruit bodies, they
2007) and dispersal interactions. meet and mate there, and their larvae exploit
fruit bodies until pupation, which takes place
inside the fruit body or in the soil. Certain
species of Ciidae are able to exploit the fungal
II. Species Diversity fruit body for several generations, until nothing
is left. Similarly to colonisation of trees by
European temperate and especially boreal for- fungi, the visible traces of beetle colonisation
ests harbour relatively low numbers of native of fungal fruit bodies are mostly apparent when
tree species compared to subtropical and tropi- fruit bodies are dead. There are certain excep-
cal forests. Only recently we came closer to tions of this general rule—numerous beetles
realise the huge number of species supported breed in the living fruit bodies of Polyporus,
by wood (Stokland et al. 2012). Of around 7000 Laetiporus and Inonotus, and fewer beetle spe-
of saproxylic (dead-wood dependent) species cies and individuals are found in the dead rem-
in North Europe, 2000 species are fungi (Stok- nants of their fruit bodies.
land et al. 2006). At least 1000 species of ligni-
colous fungi in boreal Europe are voluminous The smallest beetles, Ptiliidae, especially Nanosellinae
enough to host fungivorous insects in the fruit have fungivorous forms (Fogel and Peck 1975; Polilov
body interiors or at their surfaces. In combina- 2008), including the smallest beetle in Europe Bara-
tion with soil saprotrophs and mycorrhiza spe- nowskiella ehnstromi. Fungivory of Staphylinidae
cies, as well as with macroscopic non- (Lawrence 1989) is one of the least studied trophic
specialisations in this family. Saprophagous beetles
lichenized Ascomycetes, boreal forests harbour such as Scarabaeidae, Silphidae and Hydrophilidae
several thousand fungal species that are poten- can be found in decaying fungi. Clambidae, Cryptopha-
tially colonised or visited by insects. Through gidae and Corylophidae frequently visit sporulating
physical and chemical barriers, only a fraction fruit bodies and anamorphic fungi on decaying plant
of this fungal diversity is utilised by insects, and debris. In European fauna, key polypore destructors
are found among Anobiidae, Trogossitidae, Erotylidae,
various temporal and spatial factors contribute Endomychidae, Tenebrionidae, Mycetophagidae,
to population equilibrium of fungi and fungi- Tetratomidae, Ciidae and Melandryidae (Schigel 2009).
vores.
Beetles versus Fungi: Trophic Interactions in Boreal Forests 271
III. Functional and Life-Form (McGonigle 1995), while fruit bodies are less than 1 %
of fungal biomass (Frankland 1982). Most of reviewed
Diversity studies concern the laboratory experiments in con-
trolled environments. Grazing is reported to affect spe-
Fungal fruit bodies vary in size, shape, archi- cies richness by elimination or of fungal species,
relative abundances and community diversity, commi-
tecture and consistency, from flat, soft and
nution of substrate and fungal dispersal of fungi. In
ephemeral annual, through various intermedi- mycelial fungivory, Annelida, Nematoda and Arthro-
ate forms, to massive, hard and perennial fruit poda are identified as key taxa; within the latter phy-
bodies which may last sporulating for decades lum, Acari and Collembola are the best studied groups
(Niemelä 2005). For beetles, fungal fruit bodies through direct observation, examination of gut con-
tents and monitoring enumeration of hyphae. Grazing
offer the rich feeding and breeding substrate.
causes a variety of responses of single species of grazed
Fungi transform chemically challenging fungi and of community effects: reducing roles of dom-
organic molecules of wood. Fungal fruit bodies, inant and not dominant fungi, species replacements,
unlike mycelium, form the compact, protected selectivity and intensity of grazing, enhanced diversity
and relatively stable habitat and food source or polarisation of fungal communities. McGonigle
(2007) concludes that three factors control impact of
exploited by invertebrates. The kaleidoscope of
grazing: selectivity, intensity and fungal responses.
differences in longevity and seasonality and
chemistry and structure of fungal fruit bodies
support a variety of associated insect species, in
particularly of beetles. Perennial polypores have
more persistent fruit bodies than annual poly- IV. Ecology of Fungus – Beetle
pores, which are longer living than those of Relationships and Interaction
boletes and of agarics, with the diversity – Types
stability hypothesis is a likely explanation of
species richness patterns among fungivores
Ecology of beetle fungivory is based on fungal
(Hanski 1989).
fruit bodies, mycelia and their spores providing
Different stages of fungal life cycle attract
beetles with patchy and unpredictable habitats
various beetle colonisers and consumers: for
and food sources. The variability of these habi-
instance, in European taiga larvae of Melandryi-
tats is influenced by the speed of hyphal growth
dae occur mostly in living fruit bodies of poly-
and decomposition, yearly fluctuations in fruit
pores, Ciidae—in dead ones, Latridiidae on
body production, architecture of fruit bodies
sporulating fruit bodies. The roles of fungivor-
(Ryvarden 1991) and sporulation patterns.
ous beetles as spore vectors are rarely documen-
Fungi and beetles are involved in a variety
ted (but see Shaw 1992). The direct negative
of interaction types, including commensal,
(destructive) impact of the fungivores on the
mutualistic and combative interactions with
fungal community by, for example, reducing
fungi killing insects and insects grazing on
spore productivity has been shown in a number
fungi, insects vectoring fungal propagules and
of cases (Shaw 1992; Guevara et al. 2000a). Shaw
fungal symbionts of insects. The Biodiversity in
(1992) observes that few studies investigate
Dead Wood book (Stokland et al. 2012) chal-
mycelial grazing, most focusing on dipteran
lenges the classic pyramidal schemas of trophic
pests of commercial mushroom cultivation or
saproxylic food webs and suggests understand-
explore laboratory microcosms. Boddy and
ing the complexity through interaction web
Jones (2008) review in detail direct positive
where various wood-dependent taxa are
and negative effects of Basidiomycota on inver-
interconnected through a dense network of
tebrates, as well as role of invertebrates in dis-
relationships, which include tropic, nesting
persal, colonisation, modification of
and habitat uses.
environment and metabolism of fungi.
Six types of fungivory can be identified
since two life stages of beetles, larvae and adults
McGonigle (2007) reviews impact that fungivore
grazing on fungi in soil and litter, suggesting that it can be interacting with fungal spores, mycelia
should be high in natural condition based on estimates or fruit bodies. Moreover, fungivorous insects
that up to 75 % of the soil fauna biomass are fungivores can be divided into consumers of living and of
272 D. Schigel
dead fungi (Yakovlev 1995), though this border extreme of trophic specialisation some poly-
is fuzzy. Lawrence (1989) separates micro- and pores “escape” by producing fruit bodies
macrophagy of fungivorous beetles based on unpalatable for insect consumers (Schigel
mouthpart morphology and the type of feeding 2011a, 2012a).
and illustrates dramatic differences between the
mouthpart adaptations of larvae feeding on Trophic interactions between fungi and their beetles
loose food particles, such as spores and surface could be visualised using food web diagrams (Schigel
mycelia, and those adapted for dealing with 2011a). Frequencies of fungal fruit bodies in forest
compartments were used to measure host abundance
dense hyphal masses. and frequencies of occurrence of beetle larvae in these
fungi to measure consumer abundances in seven pro-
The life cycles of fungivorous beetles are synchronised tected boreal forests in northern, eastern and southern
with those of fungi. Beetles emerging from pupae seek Finland. Twenty-four out of 37 commonest polypore
mating and disperse to colonise new fungal habitats or species proved to be colonised by 32 beetle species,
continue using the parental fruit body. Most species of resulting in 87 fungus–beetle links (Fig. 14.1).
fungivorous beetles consume recently dead fungal fruit
bodies soon after final (or, in case of annuals, the only)
sporulation period. Spatial complexity of perennial
Fungivorous beetles demonstrate at least
fruit bodies allows accommodation and niche isolation three types of host–consumer interactions
of beetle larvae of different species. Fungivorous bee- and exploitation of host polypores: wide gener-
tles, visitors and colonisers come in waves during the alists, specialist (strict and not) and beetles
warm season, but succession also takes place during the forming species-rich communities which
whole life of a fruit body. Structural similarity, decom-
position stage and phylogenetic relations serve as a
jointly share a host fungus. With more links to
basis for attraction of certain fungivorous guilds. discover and more species of fungi to explore,
Many beetles feed on fungi only as adults, while those both the number of fungivorous species devel-
most closely associated have larvae with fungal diet. In oping in polypores and the assumed level of
some cases (e.g. many Ciidae) the entire life cycle of a beetle generality are likely to increase in accor-
beetle takes place within a fruit body. Most of fungal
fruit bodies can be called ephemeral substrates, but
dance with Martinez (1992).
perennial fruit bodies of some polypores, such as Phel-
linus spp., may stay on trees for decades (Niemelä In boreal forests, and most likely in other wooded land-
2005). scapes, diversity of fungivorous insects seems to posi-
tively correlate with volume of fully grown fungal fruit
body, with gigantic Polyporus squamosus (Klimas-
Significant species diversity of fungivorous zewski and Peck 1987), Laetiporus sulphureus (Benick
beetles from distantly related phylogenetic 1952), large Piptoporus betulinus and Fomes fomentar-
clades indicates an important role of fungi in ius (Thunes 1994), Fomitopsis pinicola and Fomes
the evolution of this insect order. Saprophagy fomentarius (Hågvar 1999; Økland 2002) hosting tens
and fungivory are ancestral types of feeding in of beetle species. Soft and voluminous polypores, such
as Polyporus squamosus and Grifola frondosa, as well as
Coleoptera (Lawrence 1989; Leschen 2000), and pleurotoid fungi, are also likely to host more species
fungivory has evolved several times indepen- and individuals of parasitoids than the dry and tough
dently in this order of insects (Crowson 1981). polypores with perennial or annual hibernating fruit
Palaeontology shed little light on history of bodies. Commonness, architectural complexity, sea-
insect fungivory; however, massive fruit bodies sonal and age changes and longevity of fungal fruit
body also increase the number of niches and number
of Basidiomycetes are known since Early Creta- of associated species, though fewer species at a time can
ceous, and there are evidences that fungi have be recorded or reared from a sample from a perennial
been consumed by insects for at least 100 mil- than from a soft large annual fruit body.
lion years (Schmidt et al. 2010).
A gradient of trophic specialisation types is Seasonality plays an important role in fun-
demonstrated by fungivorous beetles, from gus–insect interactions. While perennial fruit
mono- and oligophagy to the most common bodies are available all year round, certain
polyphagy. At one extreme, Cis bilamellatus is fungi produce ephemeral fruit bodies for only
an example of a very broad host range in north- a few weeks. Adult beetles hatch from pupae in
ern Europe (Orledge et al. 2010); at another advance, feed on various sources and travel to
Beetles versus Fungi: Trophic Interactions in Boreal Forests 273
Fomes fomentarius
Fomitopsis pinicola
Phellinus igniarius
Inonotus obliquus
Phellinus viticola
Cis bidentatus
Antrodia serialis
Gloeophyllum sepiarium
Fomitopsis rosea
Antrodia xantha
Cis punctulatus
Trametes ochracea
Phellinus nigrolimitatus
Cis glabratus
Cerrena unicolor
Antrodia sinuosa
Abdera affinis
Phellinus tremulae
Dorcatoma dresdensis
Oligoporus sericeomollis
Fig. 14.1 Fungus–beetle food web in Finnish boreal than 10 % fruit body frequency scores in forest com-
forests (Schigel 2011a). Bars on the left represent fre- partments were included. Names of fungi which hosted
quencies of fruit body occurrence of the host polypores no beetle larvae are set in bold face. Bars on the right
from the commonest (top left) to least frequent in seven represent the gradient of sums of beetle larvae frequen-
sites in Finland (bottom left). Only species with more cies in the selected fungi, from the most frequent in all
274 D. Schigel
breeding sites, when those become available. humidity and safety concerns. Entomopatho-
Various sporulating fungi are regularly visited genic fungi are found in most fungal clades
by adults of beetles breeding elsewhere. For except for the higher Basidiomycota; fungal
instance, sporulating fruit bodies of Fomes pathogens include aggressive species such as
fomentarius attract tens of beetle species during Metarhizium anisopliae and opportunists like
such period in spring, while fruit bodies appear Mucor hiemalis. Some of those fungi demon-
nearly deserted for the rest of the year. Among strate complicated life cycles changing host
fungal spore feeders, Leiodidae, Latridiidae and organisms, other cause mummification of
Sphingidae are frequently present in North insects. The major biological non-taxonomic
Europe, whose larvae require even more unpre- division of entomopathogenic fungi is into
dictable substrate—the myxomycetes. Adult destructively pathogenic and biotrophic fungi.
visitors feeding on the polypore spores, or on Both systems display a wide range of infection
the fungal fruit bodies, are often different spe- and response strategies, resulting in a complex-
cies from those breeding in the fungus as lar- ity of population biology, including epizootic,
vae, or adult visitors and coloniser may co- interactions (Charnley and Collins 2007).
occur. After being exposed to spore masses,
these visitors ramble at various feeding and
breeding sites and very likely contribute to V. Fungivory Studies
directional transport of fungal spores. Closer
to the end of decomposition of fungal fruit Great variety of fungus–insect interaction types
body and of the succession of beetles species, and different aspects of fungus–insect interac-
secondary anamorphic fungi may grow over the tion ecology are comprehensively covered in at
wet remnants of the fungus. These “moulds” least four books (Wheeler and Blackwell 1984;
are many and diverse, though poorly studied, Wilding et al. 1989; Vega and Blackwell 2005;
their species diversity may become less myste- Boddy et al. 2008) and in a number of book
rious with availability of DNA methods and chapters (Crowson 1981; Shaw 1992; Ehnström
they attract yet another guild of fungal feeders, and Axelsson 2002; Stokland et al. 2012), plus at
such as Cryptophagidae. least 600 research and review articles. Since
In reverse interactions, fungal abilities to 1990s, at least 70 studies of saproxylic, includ-
kill insects can be exploited for human needs. ing fungivorous, beetles and their fungal hosts,
Use of fungal insecticides has comparatively were carried out in North Europe. Additional
little impact on pest control, but is seen as a references can be found in the bibliography
promising direction for biocontrol develop- reviews by Fogel (1973a, b), Jonsell (1999) and
ment, despite constraints by the environment Schigel (2012b). The megadiverse order Cole-
Fig. 14.1 (continued) fungi (top right) to the least fre- chaptum species, pioneer decomposers of dead conif-
quent (bottom right). Thickness of the interaction cline erous trees. The heterogeneous set of beetle species
reflects the relative contribution of the fungal species to least frequently reared from the commonest polypores
diet of the beetle larvae. Generalist beetles, one-to- is found in the right-bottom corner of the diagram and
many interactions (upper right corner): a few generalist is recognised by the up-pointed interaction clines,
species utilise a wide range of host fungi; some beetle forming many-to-one group. Many of the host fungi
species are narrowly dependent on one (or two closely supporting beetles in many-to-one interactions have
related) host fungus, while other beetles are members perennial or semi-perennial (hibernating) fruit bodies.
of speciose communities inhabiting a certain host fun- Extended durability of the fruit bodies improves
gus only. Generalist beetles demonstrate ecological chances to discover the fungus individual both for a
flexibility with larvae developing in a variety of unre- beetle looking for habitat and for a researcher
lated and structurally diverse host fungi. Such flexibil- surveying the forest for polypores. These beetles toler-
ity is, however, not infinite: a broad preference may be ate competition with each other in the structured envi-
identified within the diverse host species range. Spe- ronment of relatively voluminous fruit bodies of
cialist beetles (one-to-one type of host–consumer perennial and hibernating fungi and form multispecific
interactions) are found in the middle part of the food communities
web diagram, e.g. Cis punctulatus is confined to Tri-
Beetles versus Fungi: Trophic Interactions in Boreal Forests 275
optera is one of the key players in the saproxylic Most of beetle fungivory research has focused on asso-
game, and fungivory, selective consumption of ciations with polypores, agaricoids and boletes, with
numerous fungal and beetle taxa left overlooked by
fungal spores, mycelia and fruit bodies, is fungivory explorers. Links of Coleoptera with Ascomy-
among the most apparent interaction types. cetes (Lawrence 1977) and other fungi (Leschen and
Carlton 1996; Stribling and Seymour 1988) and most
A number of Nordic studies approach fungus–beetle importantly with microscopic and soil fungi are in need
relationships from ecological, population biology and of further studies. Species and geographic coverage in
conservation directions (Økland 1995; Thunes et al. the fungus–beetle studies needs to be expanded; more
2000; Jonsell and Nordlander 2004). Habitat preference research is needed on ecological factors and processes
is measured by aggregation and frequency of occur- affecting fungus–insect systems (Hanski 1989; Heard
rence (Jonsell and Nordlander 1995; Jonsson et al. 1998; Hilszczajski et al 2005; Yorozuya 2006), including
1997). According to Jonsell et al. (1998), presence of competition (Yamashita and Hijii 2007a), acoustic
wood-decaying fungi determines the fauna of communication (Gilbert and Arrow 1924), subsocial
saproxylic beetles. Habitat loss and fragmentation behaviour (Ashe 1986; Leschen 1994) and predation.
pose a threat to the stability of the fungus–insect sys-
tems: Jonsson and Nordlander (2006) prove that dis- To build quantitative food webs, abun-
tance from an old-growth forest reserve affects
colonisation rates of fungivores.
dances of hosts and consumers need to be
measured. As a proxy to actual abundance of
fungal hosts, frequencies in forest biotopes can
The parallels between trophic associations
be used. A similar compromise is available for
and phylogeny and systematic position of bee-
the ranking beetle species: instead of the sam-
tle and fungal taxa were identified by Paviour-
pled frequency, contributions of individual
Smith (1960), Lawrence (1973), Kompantsev
beetle species into the whole spectrum of spe-
(1984), and Jonsell and Nordlander (2004).
cies interacting with a given fungal species are
Orledge and Reynolds (2005), based on original
obtainable. In traditional food web analysis,
and literature data, identified beetle fungal host
abundances of hosts, consumers and interac-
groups among 167 species of Ciidae.
tion frequencies are measured from the same
sampling. This approach uses the abundances
of all interacting species and numbers of inter-
VI. Discussion and Future Prospects actions per unit volume or area (Lewis et al.
2002); the alternative food web approach may
A typical research project on trophic interac- use presence-only frequency measures, such as
tions would either select a model system and contributions of individual consumer species
explore it in high detail in the controlled into the whole number of interactions with a
experiment or repeated field study or would host. Such methodology (Schigel 2011a) lacks
document species diversity and ecology using higher precision (Polis 1991) and may be used
heterogeneous best available data, including in an analysis of gradually growing datasets,
museum, digital, own, colleagues’ and litera- such as those coming from reserve surveys,
ture sources. Both accuracy and trust of citizen science initiatives, old project and
experimental/model systems and coverage museum data, and datasets on poorly known
and realism of field-based studies require and rare species. Beetles grazing on rare fungi
sample size of tens of fruit bodies to detect are slow to collect and rear in statistically ana-
most of the beetles for each host species. The lysable quantities and such studies are further
main methods in beetle fungivory studies are complicated by identification difficulties. Build-
field collections and rearing beetle larvae into ing molecular food webs is possible using high-
adults, with more than 40 publications based throughput sequencing and biomass measure-
on each approach (Schigel 2009), followed by ments for fungi and using molecular identifica-
less widespread methods such are trapping, tion and DNA barcodes for beetles; isotope
laboratory and field experiments (Komonen studies help to clarify the nature of interspecific
2008; Faticov et al. 2015). relationships: these are promising methodolog-
ical improvements in food web research.
276 D. Schigel
Chemical signals, volatile compounds, play The elements of this rich and secret world are
a key role in directing adult beetles to and from nearby, in the parks, in the managed and in the
certain fungi in certain conditions or life stages. protected forests, planted and natural. Many of
A large fraction of Finnish polypore species saproxylic species are small, inconspicuous and
appeared never visited nor colonised by beetles; hidden from eyes of an observer. Dead wood
chemical repellents are the very likely reasons harbours saproxylic organisms which support
for the mycelial (Shaw 1992) and fruit body boreal forests exist as stable habitats.
(Schigel 2012a) rejection. Chemical ecology
offers a range of powerful spectrometry meth-
ods to solve the mechanisms of attraction and
rejection. Host odours and volatiles attract bee-
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Biosystematic Index
A
Acaulospora, 92 Armillaria sp., 80, 107, 111
Acidobacteria, 180 Arthrobotrys, 248
Acremonium, 101 A. dactyloides, 251
Acromyrmex, 220 A. oligospora, 251, 252, 253, 254, 257
Actinobacteria, 180 A. tortor, 249
Acyrthosiphon pisum, 258 Ascobolus
Agaricomycetes, 190 A. immersus, 44
Agaricomycotina, 44 A. magnificus, 53
Agaricostilbum hyphaenes, 44 Ascomycetes, 47
Agaricus sp., 112 Ascomycota, 190
A. arvensis, 112 Ascomycotina, 91
A. bisporus, 10, 134 Ashbya gossypii, 47
A. bisporus var. bisporus, 44 Aspergillus spp., 52, 53, 62, 65, 87, 89, 101, 106,
A. campestris, 112 127, 134, 135, 137
A. edulis, 112 A. aculeatus, 135
A. haemorrhoidarius, 112 A. carbonarius, 62, 63
A. xanthodermus, 112 A. fumigatus, 47, 61, 62, 164, 165
Agrocybe A. giganteus, 48, 57
A. aegerita, 6 A. glaucus, 53
A. semiorbicularis, 10 A. nidulans, 10, 43, 44, 45, 47, 52, 53, 55, 61,
Alectoria, 23 62, 63, 64, 65, 66, 69, 262
Alliaria petiolata (garlic mustard), 90 A. niger, 51, 62, 110, 128, 135
Allomyces, 216 A. ornatus, 48, 51
Alphaproteobacteria, 180 A. steynii, 62
Alternaria Athelia rolfsii, 219, 221, 223, 224, 225, 226, 228
A. alternata, 63, 106, 185, 215, 226 Atta, 220
A. dauci, 230 Attini, 220
A. radicina, 230 Aureobasidium pullulans, 101, 106
A. solani, 52 Austroparmelina, 17
Amanita, 191
A. muscaria, 112, 168
B
A. rubescens, 112
Bacillus subtilis, 44
A. strobiliformis, 112
Bacteroides fragilis, 166
Ampelomyces, 233
Baranowskiella ehnstromi, 270
A. quisqualis, 233–234
Basidiomycetes, 47
Anobiidae, 270
Basidiomycota, 190
Antrodia vaillantii, 103
G I
Gaertneriomyces semiglobifer, 238 Icmadophilaceae, 24
Gaeumannomyces, 208, 209 Inonotus, 270
Ganoderma australe, 141 Irpex, 102
Gibberella moniliformis, 47 I. lacteus, 140
Gigaspora, 236
Gigasporaceae, 152
Gigaspora margarita, 154 K
Gliocladium, 230 Kickxellomycotina, 45
G. penicillioides, 230 Klebsiella pneumoniae, 166
G. roseum, 230 Kluyveromyces, 101
G. semiglobifer, 238 K. lactis, 6
Gloeophyllum trabeum, 139, 140 Komagataella pastoris, 6
282 Biosystematic Index
Rhizopogon Suillus
R. luteolus, 167 S. granulatus, 115
R. occidentalis, 88 S. luteus, 114
Rhizopus spp., 87, 88, 101, 106 Syncephalastrum, 87
R. microsporus, 151, 166 Syspastospora parasitica, 237
Roselliniella, 232
Rozella allomycis, 216, 217
T
Russula, 112
Taphrina deformans, 10
Tenebrionidae, 270
Teratosperma sclerotivorum, 219
S
Tetrahymena, 7
Saccharomyces, 6, 101
T. thermophila, 6
S. cerevisiae, 4, 42, 162
Tetratomidae, 270
Saccharomycotina, 44
Thamnolia, 20, 24
Scarabaeidae, 270
Thanatephorus, 221
Schizophyllum commune, 54, 139
T. cucumeris, 223, 225, 226, 227, 228, 230, 231
Schizosaccharomyces pombe, 4, 47
Thermomyces, 217
Sclerotinia
Thyronectria, 233
S. fructicola, 52
Thysanoptera, 270
S. fructigena, 52
Tolypocladium inflatum, 232
S. sclerotiorum, 51, 61, 224, 227, 228,
Tomentella sublilacina, 88
230, 258
Trametes
Sclerotium
T. versicolor, 140
S. delphinii, 51
Trametes spp., 102
S. rolfsii, 51
Trebouxia decolorans, 27
Sebacinaceae, 190
Trentepohlia, 28
Sebacinales, 190
Trichoderma spp., 43, 51, 52, 59, 60, 61, 64, 67,
Serpula himantioides, 110
106, 238, 249, 257, 258
S. lacrimans, 139
T. asperellum, 238
Silphidae, 270
T. atroviride, 44, 47, 52, 60, 61, 62,
Sordaria, 57, 58
64, 221, 223, 225, 226, 227,
S. fimicola, 57, 58
228, 229, 234, 235, 237,
Sphaerobolus, 57
249, 257
S. stellatus, 54
T. gamsi, 238
Sphaerodes
T. guizhouense, 224, 228, 230
S. mycoparasitica, 232
T. harzianum, 219, 224, 225, 226, 227, 238,
S. quadrangularis, 232
249, 257
Sphaerotheca, 233
T. koningiopsis, 238
Sporidesmium sclerotivorum, 219
T. longibrachiatum, 238, 249
Sporobolomyces, 57
T. pluroticola, 60
Squamarina cartilaginea, 25
T. reesei, 46, 48, 49, 51, 52, 55, 61, 66,
Staphylinidae, 270
67, 68, 127, 128, 132, 133, 134, 223, 228
Staurolemma omphalarioides, 26
T. rossicum, 249, 257
Stenotrophomonas maltophilia, 103
T. tomentosum, 249, 257
Streptococcus
T. virens, 65, 106, 223, 225, 226, 227, 228, 229,
S. gordonii, 166
249, 257
S. mutans, 165
Trichophyton
Streptomyces, 163, 168
T. mentagrophytes, 57
S. cyanofuscatus, 182
T. rubrum, 57
S. uncialis, 182
Biosystematic Index 285
A
Acid rain, 103–104 Biotechnological applications
Adenylate cyclase, 225 cellulases, 67
Adhesive networks, 248, 249 cellulolytic enzymes-encoding genes, 67
Agonism, 219, 224, 236 cellulose, 66
Airborne pollutants, 103–104 cell wall, 66
Alternative food web, 275 celulase producer, 66–67
Aminocoumarins, 182 glucose, 67
Angucyclines, 182 hemicellulases, 67
Antagonisms, 152, 153, 219, 224, 226, 230, 231, lignin, 66
235, 236 pectinases, 67
Ant mound disturbance, 85 polysaccharides, 67
Appressoria, 210 Biotechnology, 113–116
Arabinogalactans, 132 Biotrophic mycoparasitism, 218, 230, 232
Arbuscular mycorrhizae (AM), 86–88, 91, 92, Bioweathering, 116
106, 107, 114, 152, 190 Bisexual reproduction, 7
Arbuscule, 190, 191 Blue-light fungal photoreceptor
Aspartyl peptidases, 251 light-oxygen-voltage (LOV), 43
Atmospheric greenhouse gas, 92 Per-Arnt-Sim (PAS), 43
Atmospheric pollutants, 104 white collar-1 (WC-1), 43
ATP-binding cassette, 231 MadA, 56
MadB, 56
negative phototropic response, 57
B
photocarotenogenesis, 56
Bioavailability, 100, 103
photoresponse, 56
Biocontrol, 219, 221–223, 226, 231, 235–238,
photosystem, 56
248, 249
phototrophic response, 56–57
Biocorrosion, 110
phototropic response, 56
Biodegradation, 103
phototropism, 56
Biofertilizers, 223
positive phototropism, 57
Biofilm, 164–166
WCC, 56
Biofungicides, 223
Brown-Rot Fungi, 139–140
Biogeographic patterns, 17
Bioindicators, 111–113
Bioleaching, 113 C
Biorecovery, 110 Carbohydrate-active enzymes, 128
Bioremediation, 102, 103, 110, 113–116 Carbohydrate-binding modules, 128, 131
Biosorption, 106, 109, 113 Carbon cycling, 197
K N
Keratin, 253 N-acetyl-b-D-glucosaminidase, 226
Knob, 252, 255, 258, 262 Nanobiotechnology, 115
Necrotrophic mycoparasitism, 218,
221–223, 230
L
Nematodes, 247, 249–262
Laccase, 193
Neutral deuterolysin metallopeptidase,
Landscape genetics, 16, 23–25
224, 228
Large-scale disturbance, 86
Novobiocin, 182
Lead-bearing minerals, 108
Nutrition disturbance, 91
Lectin, 252, 257, 258
Legacy effects, 83
Lichen O
biogeography, 15, 16 Oligosporon, 259
distribution, 30 Opsin/rodopsin
symbioses, 21 CarO, 46
thallus, 19, 29, 179 green-light responsive, 46
Lichenicolous, 179 NOP-1, 46
Lignin, 138–139, 194 OpsA, 46
Lignocellulose, 127–130 phototaxis, 47
Organomercury, 110
Organometal(loid)s, 110
M
Organotins, 110
Mating-type, 7
Oxalates, 109
chromosomes, 8
switching, 4
MAT locus, 4, 7 P
Melanin, 106, 109, 208 Parvome, 182
Metal immobilisation, 109–110 Pectin, 129
Metalloids, 108–110, 114, 116 homogalacturonan, 135
Metallopeptidase, 252 rhamnogalacturonan, 135, 136
Metal mobilisation, 108–109 xylogalacturonan, 135
Metals, 100, 101, 103, 104, 108, 116 Pelotons, 190, 191
Metal toxicity, 104, 108 6-pentyl-2H-pyran-2-one, 260
Methyltransferase, 226 Peroxidases, 193
Metzincin, 253 PHA biopolymers, 185
Microbiome, 162, 166, 172 Phylloplane, 106
Microsclerotia, 207, 210 Phytochrome
Mineralization, 195 bilin-type chromophore, 47
Mitogen activated protein kinase (MAPK), biliverdin, 47
225, 234 C-terminal regulatory histidine kinase-
Modeling lichen distributions, 16, 25–27 related domain (HKRD), 47
Mollicutes, 151–152 Far-red-ansorbing (Pfr), 47
Mycoparasitism, 216–238 FphA, 47
Mycophagy, 218, 269 PAS-GAF-PHY domains, 47
Mycoplasma-related endobacteria (MRE), 152 red-absorbing (Pr), 47
Mycoremediation, 114–115 red/far-red, 47
Mycorrhiza helper bacteria (MHB), 167, 168 Phytoremediation, 114, 115
Mycorrhizosphere, 114–115 Plant cell walls, 129–130, 193
Mycotrophy, 218, 223 structure and composition, 128–129
Subject Index 291
Q T
Quantitative food web, 275 Telomere, 6
Termites, 270
Terpene synthase, 227
R
Tillage disturbance, 87
Radicicol, 259
Tramblaya princeps, 157
Radionuclides, 111–113, 116
Traps, 248, 250–252, 254, 255, 257–259
Reactive oxygen species, 209
Trichodiene, 227, 233
Rhizosphere, 220, 222
Tripartite, 179
Root-rot nematodes, 247
Trisexual, 7
Trophic specialization, 272
S
Saprotrophy, 191, 193, 215, 220, 221, 223, 230,
U
232, 233
Uncialamycin, 182
Saproxylic, 270
Unisexual reproduction, 7
Secondary metabolites, 62–64, 162, 164, 222,
Usnic acid, 182
223, 225–228, 231, 235, 259–260
siderophores, 109
Selenium, 108, 110, 114 V
Serine protease, 228, 253–256, 260 Velum formation protein 1, 228
Sex chromosomes, 3, 8 Virulence, 221, 225, 233, 234, 252–254, 256,
Sexual development 258, 262
ascospores, 55 Vitamin B12, 183
blr1, 55 Volatile organic compounds, 260, 276
blr2, 55 Volcano disturbance, 80, 83, 87, 90–92
conidiation, 56 Voronoi treemaps, 183
development, 55
env1, 55
W
female fertility, 55
White-rot fungi, 101, 102, 140–142
growth, 56
Wind disturbance, 89–91
Mat1-1, 55
Mat1-2, 55
mating, 55 X
pheromone transporter, 55 Xenobiotics, 101–103
secondary metabolism, 55
stromata, 55
Z
velvet, 55
Zearalenone hydrolase, 231
Silent mating cassettes, 4