Assignment and Lab Cover Page

Title: THE SCIENTIFIC METHOD AND EXPERIMENTAL DESIGN AND the ATTACK OF THE
KILLER FUNGUS EXPERIMENT
Date of the report submitted: April 1, 2022
Author’s Name: Saloni Ashesh Patel
Student ID number: 501122884
Course and section: BLG144-022
TA Full name: Morrigan Everatt
 Introduction

Nematophagous fungi are soil-dwelling organisms with a variety of worm-trapping methods. This
provides fungi with food, is beneficial to the environment's ecological balance, and gives agricultural
benefits. This review has gathered literature regarding nematophagous fungi and the regulation of
worm capture.

1. Arthrobotrys oligospora
Background Information
Fungi are eukaryotic microorganisms that are abundant across the globe and inhabit
many different types of niches. They have a variety of benefits, both for society and
the environment. These organisms are consumed for nutrition and used for food
preservation purposes, for example through the addition of specific microbes to
cheeses (11, 29). They also naturally produce antibacterial substances like penicillin
(3). In addition, fungi are essential members of the ecosystem. Mycorrhizal fungi
associate with plant roots, which increases the surface area of the roots, allowing
greater uptake of water and nutrients. They also protect plant roots from infection by
harmful pathogens. Saprotrophic fungi colonize dead material through hyphal
penetration of materials such as wood, leaves and manure, which are rich in
cellulose. As animals are incapable of metabolizing cellulose, it is essential that fungi
can break these items into simple compounds like sugars and carbon dioxide.
Fungi also have uses in the agricultural community as potential control agents for
parasites in farm animals (9). Although most species of fungi forage on decaying
matter, some species, which include the fungi of interest, are carnivorous and use
predatory techniques to capture their prey. Of the many predacious species currently
under study, Arthrobotrys oligospora holds great promise in the agricultural industry
for its potential as a pest control agent for both crops and animals.
A. oligospora belongs to the class Leotiomycetes, and the family Orbiliaceae. It is
mostly found on compost and decomposing wood, as well as animal excrements and
metal polluted soils (9, 12, 38). It is broadly dispersed across the globe, found in both
terrestrial as well as marine environments. Moreover, A. oligospora is considered the
most abundant predacious fungus in the environment. In nutritious environments, A.
 oligospora survives through a saprotrophic mechanism (12), by processing dead and
decaying matter. However, in low-nutrient environments, this species has an
increased potency for capturing prey, which leads to the formation of traps.

2. Caenorhabditis elegans

Background Information
C. elegans come from the genus Caenorhabditis, a line of nematodes that extends to
23 different species. C. elegans inhabit the soil and eat the microorganisms which
live there (16). As an adult, C. elegans follow the typical nematode body plan, which
is unsegmented, cylindrical, and tapered at both ends. The body is separated by a
pseudocoelom into two tubes: an outer tube that consists of the cuticle, hypodermis,
nervous system, muscle system, and excretory system and an inner tube that
includes the pharynx, intestine, and reproductive system. Homeostasis is maintained
through an internal hydrostatic pressure (2). Images of C. elegans anatomy can be
found in the Worm Atlas (2).
The two sexes of adult C. elegans are self-fertilizing hermaphrodites (XX) and males
(XO). Hermaphrodites possess 959 somatic cells while adult males have 1031 cells,
however, adult males tend to be slimmer and shorter. The major difference between
the two lies in the latter’s development of male-specific sexual organs in the posterior
half of the body. Hermaphrodites, in contrast, also possess an egg-laying apparatus
and undergo a 3-day reproductive life cycle. While self-fertilizing hermaphrodites can
produce about 300 progeny, those that mate with males’ breed 1200 to 1400
offspring (2).
The developmental stages of C. elegans are the embryonic stage, the four larval
stages L1 through L4, and adulthood, as illustrated in the Worm Atlas Introduction
Figure 6 (2). Egg-laying and subsequent embryogenesis and hatching occurs within
roughly nine hours. Development through the four larval stages is triggered by
feeding post-hatching. Molting occurs between each stage as C. elegans develop.
The time course of this process at 25C is as follows: beginning when the egg is laid,
L1/L2 molting occurs after approximately 18 hours, L2/L3 molting after 25.5 hours,
L3/L4 molting after 31 hours, and L4/adult molting after 39 hours. In the L1 larva
stage, five of the eight classes of motor neurons are developed, along with somatic
gonad precursors and the formation of the germ line. At this stage, males are already
distinguished. In the L2 larva stage, the nervous and reproductive systems continue
to develop. Of note, in the event of unfavorable environmental conditions such as
high temperatures, C. elegans at the end of the L2 stage can go into the dauer
stage, an arrested state, which is maintained until growth conditions are favorable
again. The L3 stage begins the more concrete formation of the parts of the
reproductive system in hermaphrodites and males, which is then completed in the L4
stage. The completion of these larval stages at 45 to 50 hours after hatching marks
the adult stage and the reproductive life cycle.
C. elegans is known as a colonizer of environments rich in microbes, such as
decomposing plants (10). While the natural habitat of C. elegans is unknown, this
organism is mostly found in human-made habitats: compost, mushroom beds, and
garden soil in Europe, North Africa, Asia, North America, Hawaii, and Australia (16).
Within its community, C. elegans shares their environment with arthropods,
mollusks, and other nematodes. C. elegans find their food source through a complex
olfactory chemosensory system that recognizes the by-products of microbes. As
such, potential predators include pathogenic microbes that use this system to their
advantage (26). Additionally, C. elegans are preyed upon by fungi that adhere to the
cuticles of nematodes or uses trapping devices to paralyze its movement and
puncture the organism (10).

3. Nematode-capturing Fungi
Importance of Nematode-capturing Fungi
Nematodes can infect and destroy plant roots, resulting in reduced nutrient uptake
and increased susceptibility to diseases. Use of pesticides is only a short-term
solution to nematode infestation of agricultural crops and has resulted in a surge of
resistant nematodes. Several nematode-trapping fungi have been identified and
studied for their potential role in controlling nematode growth. This would provide an
alternative to the use of toxic nematicides, which are potentially dangerous to our
food supply and the environment.
The three primary consumers in the soil food web are bacteria, fungi, and
nematodes, and all of these serve as food sources for other organisms (31). Some
nematodes, such as C. elegans, utilize bacteria as their food source, and
nematophagous fungi can consume these bacteria-eating nematodes. Bacteria and
fungi often co-inhabit areas, known as the bacteria-fungus interface. Interestingly, in
vitro experiments have shown that the presence of soil bacterial strains increases A.
oligospora trap induction (18). This suggests bacteria and fungi may share a
symbiotic relationship in controlling the nematode population.
There have been two models suggested for explaining the relationship between
nematophagous fungi and nematodes and the importance of trapping for these fungi
(31). The numerical response model suggests that nematode-trapping fungi are
obligate parasites that use nematodes as a carbon and nitrogen source. This plays
an important role in the ecological cycle because during the decomposition process,
the population of microorganisms increases. This increase in food would result in an
increased nematode population, but nematophagous fungi can control growth by
capturing these nematodes as their own energy source. On the other hand, the
supplemental nitrogen model suggests nematodes serve only as a nitrogen source
for the fungus. Thus, fungi are facultative parasites that degrade nematodes for
nitrogen to survive in a nitrogen-poor environment while they exploit other
organic matter as a carbon source and for energy.
Classification of nematode-trapping fungi
Nematode-preying fungi are classified as “nematophagous fungi” or “endophytic
fungi”. Endophytic fungi grow within plant tissue without causing diseases and are
important for preventing parasitic nematodes from growing on plant roots. On the
other hand, nematophagous fungi, a diverse group of fungi, colonize and parasitize
nematodes for exploitation of nutritious substances (21).
Nematophagous fungi are subcategorized into facultative parasites or obligate
parasites. Facultative parasitic fungi utilize trapping structures and secrete
antimicrobial and nematicidal compounds. They produce adhesive spores or develop
specialized hyphae to penetrate the nematode. Trapping structures are usually
complex three-dimensional nets with the branches covered with adhesive material.
The adhesive knobs are composed of adhesive polymers produced on the apex of a
slender hyphal stalk. The trapping structure also consists of constricting rings that
swell to trap the worm. Toxins are secreted to immobilize the nematodes before
penetration of the hyphae through the nematode cuticle. Scanning electron
micrographs of A. oligospora traps can be found in Nordbring-Hertz et al. (1986).
Obligate parasitic fungi release spores that are ingested or adhere to the nematodes.
Ingested spores germinate inside the intestine or adhere to the cuticle of the
nematode and sporulate on the surface, generating an infection on the nematode.
Zoospores are also used by parasitic fungi to infect nematodes that inhabit inside of
the surface of plant roots as cysts or root knots.
Classification of nematodes
Nematodes are generally classified as “free-living nematodes” or “plant-parasitic
nematodes”. Free-living nematodes can move freely through the soil and
rhizosphere and are captured by fungi that form trapping networks of constricting
rings or adhesive hyphae. Plant-parasitic nematodes are sedentary and
characterized by the formation of cysts (egg masses). They localize near the roots of
plants, form a root-knot and feed and reproduce permanently on the roots of the
infected plants. The larvae that grow from the egg masses can infect
the plant roots and draining the plant’s photosynthate and nutrients. Fungi that prey
on plant-parasitic nematodes colonize the rhizosphere and grow into cysts on the
nematodes (15). The C. elegans we are using are classified as free-living nematodes
that are preyed on by nematophagous fungi.
Actions of A. oligospora following worm capture
A schematic of the events described below can be found in Figure 18 of the article
by Veenhuis et al. (1986).
After contact with the nematode cuticle, A. oligospora utilizes a fibrillar matrix,
approximately 0.1μm thick, to attach the nematode to the hyphae (8). This adhesive
is not found on vegetative hyphae. The fibrillar matrix becomes reorganized in one
direction targeting the site of capture on the worm, and indentations of the nematode
cuticle begin forming (34). Then, the hyphae begin penetrating the nematode at the
site where the fungal cells have the greatest adhesion to the cuticle, which usually
occur within 2 to 4 hours after capture. This begins by the accumulation and
association of small vesicles at the cellular membrane of fungal trapping cells.
A new cell wall begins forming on the cytoplasmic side of the fungal cell, and the
original nematode cell wall begins to degrade. This leads to the release of small
vesicles into the fibrillar matrix and emergence of the newly formed cell wall, creating
a penetration tube that indents the nematode cuticle. The cuticle begins thinning and
eventually, the fungal hyphae penetrate the nematode creating an infection bulb with
large numbers of electron- dense microbodies. New trophic hyphae then develop
from the infection bulb inside of the nematode within a few hours of capturing (36).
Mycelium growth outside of the nematode body is not observed until 10-24 hours
later. This mycelium only develops from trapping cells that had captured the
nematode. Trophic hyphae are rarely seen growing and developing outside of the
nematode, so this indicates that trophic hyphae are mainly utilized for the digestion
of nematodes to support the growth of vegetative mycelium. Approximately 6
hours after penetration, low levels of lipid droplets and microbodies accumulate in
the trophic hyphae (35). Lipid droplets are used for storage of nutrients derived from
13
the nematode. In the later stage of infection, lipid droplets begin increasing in
number and fusing into larger droplets while microbodies also develop and undergo
fission. Microbodies were found to contain catalase and thiolase and are often
associated with the lipid droplets. Eventually, the lipid droplets begin disappearing
while growth of vegetative mycelium accelerates. Veenhuis et al. suggest that the
catalase and thiolase from the microbodies provide enzymes for the β-oxidation
pathway of fatty acid metabolism to provide the carbon source that supports the
mycelium growth (35). These organelles eventually disappear, and only the
nematode cuticle, filled with trophic hyphae, remains after digestion.
IV. Regulation of Nematode Capture by Nematophagous Fungi
Below are results accumulated from a variety of published articles that examined
fungus-dependent worm capture. This is by no means an exhaustive list of the
available literature.
Time dependence of trap formation
After introducing the nematodes to the fungal plates, researchers have attempted to
determine the timescale of trap formation. The traps were observed at three-hour
intervals over a 27-hour period in a study by Nansen et al. (24). Another approach
was to observe the traps at 17 hours after worm addition (37). A third study reported
that no traps were observed until 24 hours, and after that, trap formation ceased
within 5 days (28). Thus, there seems to be variability on the observed timing of trap
formation. This may be due to differing factors such as media used, strain of the
fungus, or temperature of incubation. Furthermore, the age of the traps seemed to
have no impact on their ability to capture the worms, as young developing traps had
the same efficacy as already existing traps (35).
Larval density
There is a correlation between increasing concentrations of nematode population
and the number of traps formed (28). Researchers added 50, 100, and 200 worms to
fungal plates, and observed a much higher number of spores and rings in the first
day in the 200-nematode condition. This was also seen in another study testing
suspensions containing 50 to 3200 L3 nematodes, as increased nematode trapping
was seen at higher larval densities (22).
14
Temperature
Incubating the fungal plates with nematodes at multiple temperatures from 7 to 37C
elucidated the optimum temperature required for trap formation with the nematode
Heligmosomoides polygyrus. A. oligospora was found to have a peak growth rate
between 20 and 25°C. Predacity of the fungus was highest between 25 and 28°C.
The fungus was found to be significantly slower in capturing nematodes at much
lower temperatures (22).
Media conditions
Morgan et al. tested the effect of using corn meal agar diluted with water. Increased
trapping occurred on plates with a lower CMA concentration implying that nutrient
poor conditions forced the fungus to obtain more of its nutrients from the worms.
Scholler and Rubner analyzed carbon and nitrogen sources and concluded that the
fungus is more predacious when either is lacking (27). Additionally, they found that a
specific concentration of carbon and nitrogen was sufficient to prevent the fungus
from forming traps at all. It has been concluded that nematodes may not even be
necessary for trap formation and that a combination of a low nutrient medium and
small peptides will be sufficient to induce traps to form (25).
Learning Objectives:
1. Perform dilution calculations
2. Use micro pipettors with confidence
3. List benefits of fungi in nature
4. Describe how fungi and C. elegans are maintained in the lab
5. Develop a testable hypothesis
6. Design an experiment to test hypothesis
7. Data analysis

Hypothesis
The addition of Pseudomonas influences the trap creation of A. oligospora and the survival of
C. elegans.
 Results

Variable 1 Variable 2
25 26
13 42
45 22
12 28
20 37
19 44
18 29
11 33
30 21
15 19
28 23
31 41
41 34
15 27
24 36
t-Test: Two-Sample Assuming Unequal
Variances

  Variable 1 Variable 2
Mean 23.13333333 30.8
Variance 106.6952381 64.74285714
Observations 15 15
Hypothesized Mean Difference 0
df 26
t Stat -2.267767591
P(T<=t) one-tail 0.015942945
t Critical one-tail 1.70561792
P(T<=t) two-tail 0.031885891
t Critical two-tail 2.055529439  

 P(T<=t) two-tail is 0.031885891 which is smaller than 0.05.

 Control - 6 hrs - 16 degrees - 6 hrs - 27 degrees -
ratio of surviving ratio of surviving 6 hrs - ratio
worms on fungus worms on fungus of surviving
plate/worms on plate/worms on non- worms on
non-fungus plate fungus plate fungus
plate/worm
s on non-
fungus
plate
Plate 1 1.158 0.931 0.756
Plate 2 0.961 1.185 0.904
Plate 3 1.032 0.818 0.263
Plate 4 1.132 1.042 0.877
Plate 5 1.126 1.365 0.931

Control - 24 hrs - 16 degrees - 24 hrs - 27 degrees -

ratio of surviving ratio of surviving 24 hrs -
worms on fungus worms on fungus ratio of
plate/worms on plate/worms on non- surviving
non-fungus plate fungus plate worms on
fungus
plate/worm
s on non-
fungus
plate
Plate 1 0.112 0.357 0.076
Plate 2 0.198 0.387 0.057
Plate 3 0.324 0.327 0.078
Plate 4 0.214 0.421 0.065
Plate 5 0.351 0.435 0.06
 50% CMA - 6 hrs - ratio of 2% Mannose - 6 hrs - ratio of P. aeruginosa - 6 hrs - ratio of Dark - 6 hrs - ratio of
surviving worms on fungus surviving worms on fungus surviving worms on fungus surviving worms on f
plate/worms on non-fungus plate/worms on non-fungus plate/worms on non-fungus plate/worms on non
plate plate plate plate
Plate
1
1.099 1.577 2.217 0.597
Plate
2
1.114 0.873 2.411 0.809
Plate
3
1.348 1.483 2.319 1.561
Plate
4
0.601 0.804 0.952 1.689
Plate
5
0.855 0.637 1.493 0.091

50% CMA - 24 hrs - ratio of 2% Mannose - 24 hrs - ratio P. aeruginosa - 24 hrs - ratio Dark - 24 hrs - ratio o
surviving worms on fungus of surviving worms on fungus of surviving worms on fungus surviving worms on f
plate/worms on non-fungus plate/worms on non-fungus plate/worms on non-fungus plate/worms on non
plate plate plate plate
Plate
1
0.397 0.782 1.47 0.096
Plate
2
0.755 0.605 0.544 0.132
Plate
3
0.537 1.102 0.375 0.154
Plate
4
0.378 0.853 0.954 0.099
Plate
5
0.29 0.959 1.404 0.066
 Class data exploring the influence of several variables on C. elegans capture by A. oligospora.
One week before the experiment, A. oligospora was struck on CMA plates and cultured at room
temperature.   The worms were cleaned off the CMA plates at t = 6 and t = 24 hours after worm
addition and computed % survival. The data is provided as a percentage survival ratio in the
variable condition versus the control condition. For each variable, data from at least 5 groups
were averaged, and the standard error of the mean (SEM) was calculated.

Discussion

The protocol involves the addition of C. elegans to plates with and without fungus. The “no fungus”
plate is a control condition to take into consideration worms that die naturally during the experiment.
At set times, worms are rinsed off the control and fungus plates and are counted. From these values, the
percent of worms that survive the fungus is calculated.
Percent Survival = Worms on Fungus Plate/ Worms on No Fungus Control Plate
Prior to adding the worms to the fungus plate at the beginning of the experiment, a rough count is taken
to approximate how many C. elegans are added to each plate. Equal numbers will be added to the
fungus and control plates. This experiment supports the initially mentioned hypothesis that is “The
addition of Pseudomonas influences the trap creation of A. oligospora and the survival of C. elegans”.

Conclusion

The purpose of the experiment is to measure the fungus dependent capture of C. elegans after 48 hours
of co-incubation and how a specific variable alters the ability of A. oligospora to capture C. elegans.
Percent survival is calculated as worms on fungus plate divided by worms on no fungus plate. This
value is calculated with both the control and variable conditions.
Independent variable is presence of pseudomonas. Dependent variable is survival of C.elegans.
Controlled variable is temperature and lighting. Reference/control is A. oligospora with/without
pseudomonas added.
References

BLG 144 LAB FORMAL EXPERIMENT

THE SCIENTIFIC METHOD AND EXPERIMENTAL DESIGN
and the ATTACK OF THE KILLER FUNGUS EXPERIMENT
(Experiment created by Brian K. Sato, Department of Molecular Biology and
Biochemistry, University of California, Irvine, CA 92697 found in the JOURNAL OF
MICROBIOLOGY & BIOLOGY EDUCATION, December 2013, p. 230-237 DOI:
http://dx.doi.org/10.1128/jmbe.v14i2.612)

References for the Background Information

1. Ahman, J., T. Johansson, M. Olsson, P. J. Punt, C. A. van den Hondel,
and A. Tunlid. 2002. Improving the pathogenicity of a nematode-trapping
fungus by genetic engineering of a subtilisin with nematotoxic activity. Applied
and environmental microbiology 68:3408-3415.
2. Altun, Z.F. and Hall, D.H. 2012. Handbook of C. elegans Anatomy. In
WormAtlas.
3. Anke, H., Stadler, M., Mayer, A., Sterner, O. 1995. Secondary metabolites
with nematicidal and antimicrobial activity from nematophagous fungi and
Ascomycetes. Canadian Journal of Botany S1:S932- S939.
4. Ankeny, R. A. 2001. The natural history of Caenorhabditis elegans research.
Nat Rev Genet 2:474-479.
5. Ashafari, K. 2007. Obesity and the regulation of fat metabolism. Worm Book.
6. Bird, D. 2005. Model systems in agriculture: Lessons from worms. . Annals of
Applied Biology 146:147- 154.
7. Blaxter, M. 2011. Nematodes: the worm and its relatives. PLoS Biol
9:e1001050.
8. den Belder, E., Jansen, E., Donkers, J. 1996. Adhesive hyphae of
Arthrobotrys oligospora: an ultrastructural study. European Journal of Plant
Pathology 102:471-478.
9. Drechsler, C. 1937. Some Hyphomycetes That Prey on Free-Living
Terricolous Nematodes. Mycologia 29:447-552.
10.Felix, M. A., and C. Braendle. 2010. The natural history of Caenorhabditis
elegans. Current biology : CB 20:R965-969.
11.Goltapeh, E. M. S.-B., M.; Pakdaman, B. S. 2008. Sensitivity of the
nematophagous fungus Arthrobotrys oligospora to fungicides, insecticides
and crop supplements used in the commercial cultivation of Agaricus
bisporus. Journal of Agricultural Science and Technology 10:383-389.
12.Haard, K. 1968. Taxonomic studies on the genus Arthrobotrys corda.
Mycologia 60:1140-1159.
13.Hulme, S. E., and G. M. Whitesides. 2011. Chemistry and the worm:
Caenorhabditis elegans as a platform for integrating chemical and biological
research. Angew Chem Int Ed Engl 50:4774-4807.
14.Jensen, C., Neumeister-Kemp, H., Lysek, G. 1998. Fluorescence
microscopy for the observation of nematophagous fungi inside soil. Mycologist
12:107-111.
15.Karakas, M. 2007. Biological control of plant parasitic nematodes. Anadolu
University Journal of Science and Technology 8:1-12.
16.Kiontke, K., Sudhaus, W. 2006. Ecology of Caenorhabditis species. Worm
Book.
17.Larsen, M. 2000. Prospects for controlling animal parasitic nematodes by
predacious micro fungi. Parasitology 120 Suppl:S121-131.
18.Li, L., M. Ma, Y. Liu, J. Zhou, Q. Qu, K. Lu, D. Fu, and K. Zhang. 2011.
Induction of trap formation in nematode-trapping fungi by a bacterium. FEMS
microbiology letters 322:157-165.
19.Mello, C. C., and D. Conte, Jr. 2004. Revealing the world of RNA
interference. Nature 431:338-342.
24
20.Mendoza De Gives, P., Davies, K.G., Clark, S.J., Behnke, J.M. 1999.
Predatory behaviour of trapping fungi against srf mutants of Caenorhabditis
elegans and different plant and animal parasitic nematodes. Parasitology
119:95-104.
21.Moosavi, M., Zare, R. 2012. Fungi as biological control agents of plant-
parasitic nematodes. Plant Defense: Biological Control 12:67-107.
22.Morgan, M., J. M. Behnke, J. A. Lucas, and J. F. Peberdy. 1997. In vitro
assessment of the influence of nutrition, temperature and larval density on
trapping of the infective larvae of Heligmosomoides polygyrus by Arthrobotrys
oligospora, Duddingtonia flagrans and Monacrosporium megalosporum.
Parasitology 115 ( Pt 3):303-310.
23.Morgan, P., Kayser E., Sedensky, M. 2007. C. elegans and volatile
anesthetics. Worm Book.
24.Nansen, P., J. Gronvold, S. A. Henriksen, and J. Wolstrup. 1988.
Interactions between the predacious fungus Arthrobotrys oligospora and third-
stage larvae of a series of animal-parasitic nematodes. Veterinary
parasitology 26:329-337.
25.Nordbring-Hertz, B. 2004. Morphogenesis in the nematode-trapping fungus
Arthrobotrys oligospora - an extensive plasticity of infection structures.
Mycologist 18:125-133.
26.Rumbaugh, K. P. 2010. Fatal attraction: bacterial bait lures worms to their
death. Proc Natl Acad Sci U S A 107:16411-16412.
27.Scholler, M., and A. Rubner. 1994. Predacious activity of the nematode-
destroying fungus Arthrobotrys oligospora in dependence of the medium
composition. Microbiological research 149:145-149.
28.Simon, A. a. S. 2011. Effect of different level of inoculums of root knot
nematode on predacity of Arthrobotrys oligospora. Archives of Phytopathology
and Plant Protection 44:1483-1489.
29.Simon, S. 2011. Management of Root Knot Disease in Rice Caused by
Meloidogyne graminicola through Nematophagous Fungi. Journal of
Agricultural Science 3:122-126.
30.Soto-Barrientos, N., De Oliveira, J. 2011. In-vitro Predatory Activity of
Nematophagous Fungi from Costa Rica with Potential Use for Controlling
Sheep and Goat Parasitic Nematodes. Revista de biologia tropical 59:37-52.
31.Stirling, G. 2011. Biological Control of Plant-Parasitic Nematodes: An
Ecological Perspective, a Review of Progress and Opportunities for Further
Research. Progress in Biological Control 11:1-38.
32.Tunlid, A., J. Ahman, and R. P. Oliver. 1999. Transformation of the
nematode-trapping fungus Arthrobotrys oligospora. FEMS microbiology letters
173:111-116.
33.Tunlid, A., T. Johansson, and B. Nordbring-Hertz. 1991. Surface polymers
of the nematode-trapping fungus Arthrobotrys oligospora. Journal of general
microbiology 137:1231-1240.
34.Veenhuis, M., Nordbring-Hertz, B., Harder, W. 1985. An electron-
microscopial analysis of capture and initial stages of penetration of
nematodes by Arthrobotrys oligospora. Antonie van Leeuwenhoek 51:385-
398.
35.Veenhuis, M. H., W., Nordbring-Hertz, B. 1989. Occurance and metabolic
significance of microbodies in trophic hyphae of the nematophagous fungus
Arthrobotrys oligospora. Antonie van Leeuwenhoek 56:241-249.
25
36.Veenhuis, M. S., K., Nordbring-Hertz, B., Harder, W. 1989. An improved
method for light and electron microscopial studies of nematode/fungal
interactions. Antonie van Leeuwenhoek 55:361-368.
37.Xu, L. L., Y. L. Lai, L. Wang, and X. Z. Liu. 2011. Effects of abscisic acid
and nitric oxide on trap formation and trapping of nematodes by the fungus
Drechslerella stenobrocha AS6.1. Fungal biology 115:97-101.
38.Yang,J.,L.Wang,X.Ji,Y.Feng,X.Li,C.Zou,J.Xu,Y.Ren,Q.Mi,J.Wu,S.Liu,Y.Liu
,X. Huang, H. Wang, X. Niu, J. Li, L. Liang, Y. Luo, K. Ji, W. Zhou, Z. Yu,
G. Li, Y. Liu, L. Li, M. Qiao, L. Feng, and K.-Q. Zhang. 2011. Genomic and
Proteomic Analyses of the Fungus Arthrobotrys oligospora Provide Insights
into Nematode-Trap Formation. PLoS pathogens 7:e1002179.
39.Zhuang, J. J., and C. P. Hunter. 2012. RNA interference in Caenorhabditis
elegans: uptake, mechanism, and regulation. Parasitolog