Appl Microbiol Biotechnol (2008) 80:199–209

DOI 10.1007/s00253-008-1567-2

MINI-REVIEW

Use of nitrogen-fixing bacteria as biofertiliser

for non-legumes: prospects and challenges
Rumpa Biswas Bhattacharjee & Aqbal Singh &
S. N. Mukhopadhyay

Received: 30 May 2008 / Accepted: 4 June 2008 / Published online: 4 July 2008
# Springer-Verlag 2008

Abstract The potential of nitrogen-fixing (NF) bacteria to on their potential to promote host plant growth and yield. In
form a symbiotic relationship with leguminous plants and addition, plant growth-promoting traits observed in these
fix atmospheric nitrogen has been exploited in the field to NF bacteria and their mode of interaction with the host
meet the nitrogen requirement of the latter. This phenom- plant have been described briefly.
enon provides an alternative to the use of the nitrogenous
fertiliser whose excessive and imbalanced use over the Keywords Biological nitrogen fixation (BNF) .
decades has contributed to green house emission (N2O) and Nitrogen-fixing (NF) bacteria . Endophyte . Rhizobium .
underground water leaching. Recently, it was observed that Non-legume . Growth promotion
non-leguminous plants like rice, sugarcane, wheat and
maize form an extended niche for various species of NF
bacteria. These bacteria thrive within the plant, successfully Introduction
colonizing roots, stems and leaves. During the association,
the invading bacteria benefit the acquired host with a Non-leguminous plants like rice, maize and wheat belong-
marked increase in plant growth, vigor and yield. With ing to the Poaceae family form staple food for the
increasing population, the demand of non-leguminous plant approximately 6.5 billion people around the world. An
products is growing. In this regard, the richness of NF flora exponential rise in world population indicates the need for
within non-leguminous plants and extent of their interaction increased crop production. According to the Food and
with the host definitely shows a ray of hope in developing Agriculture Organization of the United Nations, world
an ecofriendly alternative to the nitrogenous fertilisers. In cereal production in 2008 is forecast to increase 2.6% to a
this review, we have discussed the association of NF record 2,164 million tons. This rise in crop production has
bacteria with various non-leguminous plants emphasizing been a result of the indiscriminate use of chemical fertilisers
(N, P, K) in combination with advanced technology.
Nitrogen fertilisation of non-leguminous crops is one of
R. B. Bhattacharjee : S. N. Mukhopadhyay the most expensive inputs in agriculture. However, approx-
Department of Biochemical Engineering and Biotechnology,
Indian Institute of Technology Delhi, Hauz Khas,
imately 65% of the applied mineral nitrogen is lost from the
New Delhi 100 016, India plant–soil system through gaseous emissions, runoff,
erosion and leaching. Environmental impact of this loss
A. Singh (*) ranges from greenhouse effects, diminishing stratospheric
National Research Centre on Plant Biotechnology,
Indian Agricultural Research Institute, Pusa,
ozone and acid rain to changes in the global N cycle and
New Delhi 100 012, India nitrate pollution of surface and ground water (Rejesus and
e-mail: [email protected] Hornbaker 1999). With growing environment-related con-
cerns, various alternatives are being harnessed to reduce the
Present address:
R. B. Bhattacharjee
dependence on N fertiliser for plant nutrients. It is in this
Department of Molecular and Cell Biology, University of Guelph, context that the use of the nitrogen-fixing (NF) bacteria in
Guelph, Ontario, CanadaN1G 2W1 agricultural practices is gaining importance. A NF bacteri-
200 Appl Microbiol Biotechnol (2008) 80:199–209

um can exist freely or in symbiosis and in either case or mercuric chloride to remove contaminant surface
entraps atmospheric nitrogen and converts the unreactive bacteria. Reinhold-Hurek and Hurek (1998) have reviewed
N2 molecule to NH3, a form that is readily utilised by various surface sterilisation techniques and their efficiencies
plants. This process is termed as biological nitrogen in isolating endophytes from non-legumes. The extract from
fixation (BNF) and is catalysed by the oxygen-sensitive the surface-sterilised plant part is then plated on nitrogen-
enzyme nitrogenase, present within the bacteria, by the free media. The competence of the isolated bacteria as an
following reaction: endophyte is verified by re-inoculation to sterile rice
seedlings (Yanni et al. 1997; Prayitno et al. 1999). Based
N2 þ8H þ 8e
þ16ATP
Nitrogenase




! 2NH3 þH2 þ16ADP þ 16Pi
on the ability of the isolated endophyte to re-infect the host
plant and fulfill ‘Koch’s postulate,’ they are designated as
A rich flora of NF bacteria has been discovered within
‘true endophytes’ (Reinhold-Hurek and Hurek 1998).
and around non-leguminous plants. The NF bacterial
Koch’s postulate states the criteria to determine whether
population thriving within the plant without causing any
certain bacterium is the disease-causing agent. One of these
apparent disease is termed ‘endophytes,’ whereas those
criteria is to re-isolate the disease-causing bacterium from
isolated from the rhizosphere of the plant are referred to
the patient. In case of the endophyte–plant interaction, the
‘rhizospheric bacteria.’ It has been suggested that endo-
bacterium responsible for the growth promotion of a plant
phytes are placed in a more favourable environment than
after inoculation should be re-isolated from infected plants.
rhizospheric bacteria as they are less vulnerable to
More recent approaches of isolation involve direct analysis
competition from other soil bacteria and are shielded from
of bacterial gene sequences obtained from the source plant
various biotic and abiotic stresses (Reinhold-Hurek and
tissue harbouring the endophyte (Engelhard et al. 2000;
Hurek 1998). Additionally, endophytes enjoy direct provi-
Hurek et al. 2002). By this technique, Conn and Franco
sion of nutritional elements within the host and a low O2
(2004) established a larger diversity of endophytes in wheat
factor that assists optimal nitrogenase activity. In return,
as opposed to that obtained by culture-dependent method.
endophytes benefit the host plant’s growth and develop-
Culture-dependent methods confer selective advantage to a
ment through BNF and growth-promoting substances
certain group of bacteria and hence do not provide a
(Sevilla and Kennedy 2000). Apparently, this intimate
complete overview of the endophytic population. Knauth et
association led researchers to anticipate the use of these
al. (2005) developed a messenger ribonucleic acid
bacteria in developing a sustainable agriculture. In our
(mRNA)-based profiling of nitrogenase (nifH) genes and
review, based on developments in the last decade, we have
obtained a significant varietal difference in rice root
discussed natural tendencies of NF bacteria (endophytic/
associated nifH-expressing communities. Similarly, Zhang
rhizospheric) to coexist with non-host gramineous crops
et al. (2007) used a nifH gene-based short oligonucleotide
and benefit their growth and development. We have also
microarray to analyse NF diazotrophs in roots of wild rice
elaborated various growth-promoting activities of the
in Namibia. Their results demonstrated that a small
bacteria responsible for the growth promotion of the host
population of the total identified diazotroph was fixing
plant during the interaction.
nitrogen actively within the host.

Extended niche for the nitrogen-fixing bacteria

Rhizobial inoculation enhances plant growth
The occurrences of Rhizobia (species of Rhizobium, and development
Mesorhizobium, Bradyrhizobium, Azorhizobium, Allorhi-
zobium and Sinorhizobium) as a natural endophyte in The importance of isolated endophyte in agribusiness
leguminous plants have been widely documented. However, depends on their performance in promoting host plant
the domain of endophytes is not restricted to the class growth and development in field. Various experiments
Leguminoceae. In the last decade, a large population of conducted under gnotobiotic as well as pot and field
endophytes ranging from 106 to 107 cells per gram of conditions illustrate the biofertlisation properties of some of
fresh tissue were isolated from various non-leguminous the isolated endophytes towards the acquired host (Yanni et
plants in different parts of the world (Yanni et al. 1997; al. 1997, 2001; Matthews et al. 2001; Muthukumaraswamy
Muthukumarswamy et al. 1999; Mirza et al. 2001). Unlike et al. 2005, 2007). Endophytes, used as inoculants for
the legume–Rhizobium interaction, endophytes are not plants (non-legume), proved to be an efficient source of N
restricted to a specific compartment within the non-legumes that can partly substitute urea N in the cultivation of rice
but occur in the mainstream plant roots, stems and leaves. and other cereals (Yanni et al. 1997; Baldani et al. 2000;
For isolating endophytes, plant surfaces (root, shoot) are Govindarajan et al. 2007). Plant growth promotion by
systematically surface-sterilised using sodium hypochloride rhizobial inoculation involved rise in plant biomass,
Appl Microbiol Biotechnol (2008) 80:199–209 201

nitrogen content, grain yield and carryover effect on straw Biswas et al. 2000a,b; Gutierrez-Zamora and Martinez-
and resulted in persistence of the inoculated strain in N- Romero 2001; Matthews et al. 2001; Riggs et al. 2001;
deficient and N-containing soil (Yanni et al. 1997, 2001; Feng et al. 2006). Unfortunately, it is not known which of
Biswas et al. 2000a, b; Peng et al. 2002). Table 1 shows the these techniques deliver the bacteria most efficiently to
relative ability of different NF bacteria to benefit their the plant for maximum output in terms of growth and
cereal host under gnotobiotic, green house and field production.
conditions. Previous investigations have now established that many
Yanni et al. (1997) isolated 11 strains of the clover- of the endophytic inoculations to commercially important
nodulating bacteria, Rhizobium leguminosarum bv. trifolii crops like rice, sugarcane and wheat can reduce N fertiliser
from rice roots in Egypt where rice has been grown in input in cultivation (Yanni at el. 1997, Baldani et al. 2000,
rotation with clover for generations. Inoculation of R. Matthews et al. 2001, Saleh et al. 2001; Govindarajan et al.
trifolii E11 and E12 to rice variety Giza significantly 2006, 2007). Yanni at el. (1997) used one third of the
increased its total N content (95% confidence) grain yield, recommended dose of N fertilisation in addition to R. trifolii,
grain N content and harvest index of rice (99% confidence) in a rice field to produce equivalent grain yield as obtained
under gnotobiotic and field conditions (Yanni et al. 1997, by the full-recommended dose of fertiliser (144 kg N ha−1).
2001; Biswas et al. 2000a,b). The overall yield and N Similarly, Burkholderia MG43 inoculation in sugarcane
accumulation of the plant in field went up by 3.6 t h−1 and resulted in an effect greater than increasing the fertiliser
19–28%, respectively (Yanni et al. 1997, 2001; Biswas et from half to the full recommended rate, saving the cost of
al. 2000a). It was observed that rhizobial inoculation ∼140 kg ha−1 N fertiliser (Govindarajan et al. 2006).
enhanced stomatal conductance, thereby increasing the Herbaspirillum is a broad-host-range endophyte, which
photosynthesis rates by 12% in rice varieties where 16% colonises sugarcane, rice, wheat, sorghum and other cereal.
grain yield increase was noted. This indicated a positive Baldani et al. (2000) tested eighty different strains of
correlation between increased grain yield and photosyn- H. seropedicae originally isolated from rice, maize and
thetic rate at zero N-level (Peng et al. 2002). Apparently, sorghum in order to select inocula for rice. They observed
they suggested that certain strains of rhizobia can promote that 12% of the tested strains led to a 100% increase in rice
rice growth and yield through the mechanism that improve fresh weight over control. In successive experiments, only a
single leaf net photosynthetic rate. Alternately, some of the few strains could maintain their performance. In a green-
Rhizobium isolates inhibited rice seedling growth and house, Herbaspirillum increased rice yield significantly (at
development (Prayitno et al. 1999; Perrine et al. 2005). 5% probability level) to 7.5 g per plant (Mirza et al. 2000). In
This inhibition occurred in presence of nitrate/nitrite addition, the N content of Al-tolerant rice varieties inocu-
supplied as the sole source of N in the media. Perrine- lated with H. seropedicae Z67 demonstrated a significant
Walker et al. (2005, 2007a) hypothesised that the inhibitory rise of 29–61% in roots and 37–85% in shoots (Gyaneshwar
effect observed in these strains were due to enzymes of et al. 2002). Similarly, Burkholderia sp. is another endo-
nitrate metabolism encoded by genes in the pSymA plasmid phyte, which has been widely studied in the field. Different
of the bacteria. The activity of these enzymes led to the forms of the bacteria (rhizospheric and endophytic) in field
reduction of nitrate to nitrite and subsequent accumulation increased rice grain yield by 0.5–0.8 t ha−1 and plant
of nitric oxide (NO), which is inhibitory to the plant biomass by 22 mg per plant (Baldani et al. 2000). This is
growth. Interestingly, plant growth-promoting rhizobacteria equivalent to a 69% increase over the uninoculated control
(PGPR) R. trifolii R4 is capable of further reducing NO to plant biomass. Burkholderia sp. strain PsJN required a gene
N2 due to the presence of additional reductases (nitrous/ similar to nadC to promote potato tube growth (Wang et al.
NO; Perrine et al. 2007a). 2006). The gene nadC encodes the enzyme quinolinate
In most of the trials, an initial inoculum density of 108– phosphoribosyltransferase (QAPRTase). The enzymatic
9
10 cells per millilitre was enough to obtain an optimum activity of QAPRTase catalyses the de novo formation of
growth response in the inoculated plant (Yanni et al. 1997, nicotinamide dinucleotide forming nicotinic acid mononu-
2001; Biswas et al. 2000a,b; Chaintruel et al. 2000). cleotide (NaMN) as the by-product. A nadC mutant was
However, different techniques were adopted to inoculate unable to synthesise the intermediary substrate NaMN and
the plant in green house or in fields. Commonly used failed to promote the growth of the host plant. However,
methods include: dipping seed or seedling roots in broth the growth-promoting activity of a PsJN mutant was
culture before sowing, application of bacterial suspension restored by the in vitro supplementation of commercial
directly to the soil, inoculating seedlings and setts of NaMN (10–100 μM) to the media. In addition to growth
sugarcane, seed coating with inoculum strains and foliar promotion, B. phytoformis PsJN also offered the inoculated
spraying with the bacterial suspension (Yanni et al. 1997, plant with cold tolerance compared to a non-bacterialized
2001; Muthukumarasamy et al. 1999; Baldani et al. 2000; control (Barka et al. 2006). Under chilling conditions,
202 Appl Microbiol Biotechnol (2008) 80:199–209

Table 1 Benefits of the association of NF bacteria and non-leguminous plants

Host plant Endophyte/diazotroph Colonisation Condition Percent increase Reference

inoculant of cultivation

BNFa
Rice Azoarcus Roots of grasses Gnotobiotic 16 (total dry weight) Reinhold- Hurek
and Hurek 1997;
Engelhard et al. 2000
Burkholderia Roots Greenhouse 68 (shoot biomass), Baldani et al. 2000
19 (seed biomass)
Photosynthetic Rhizosphere Gnotobiotic 20 (total plant biomass) Chaintruel et al. 2000
Bradyrhizobium (survives as endophyte)
Gluconacetobacter Stem, roots Gnotobiotic 30 (total dry weight) Muthukumarasamy
diazotrophicus et al. 2005, 2007
Herbaspirillum seropedicae Roots Gnotobiotic 38–54 (root biomass), Elbeltagy et al. 2001,
22–50 (shoot biomass), Gyaneshwar et al. 2002,
37.6 (plant dry weight), James et al. 2002,
52–112, 71 (fresh and dry Baldani et al. 2000
weight)
Serratia marcescens Roots, stem Gnotobiotic 23 (total dry weight) Gyaneshwar et al. 2001
Maize Burkholderia sp. Stems, roots, Greenhouse, Field 36–48, 5.9–6.3 (yield) Estrada et al. 2005,
rhizosphere Riggs et al. 2001
Azospirillum brasilense Roots, stems Greenhouse 13–25 (yield) Riggs et al. 2001
Sugarcane Gluconacetobacter Roots, stems Pot trial 18.83–49.86 (plant biomass) Suman et al. 2005, 2007
diazotrophicus
H. seropedicae, – Greenhouse 35 (dry matter) Oliveira et al. 2002
H. rubrisubalbicans
PGPRa
Rice Rhizobium leguminosarum Roots Greenhouse 15–22, 8–22 (grain yield) Yanni et al. 1997, 2001;
bv. trifolii and field Biswas et al. 2000a,b
B. vietnamiensis Rhizosphere Nursery pot trial, 23 and 59 (shoot/root weight), Trân Van et al. 2000
field 19 (yield), 13–22 (yield)
Wheat R. trifolii Roots Pot trial 24 (wheat shoot dry matter Hilali et al. 2001
and grain yield)
Cellulomonas sp. Rhizosphere Greenhouse, field 33 (root growth) Egamberdiyeva
and Höflich 2002
Maize R. trifolii Roots Greenhouse, field 34 (yield), 11 (yield) Riggs et al. 2001
Sinorhizobium sp. – Greenhouse 49–82 (yield) Riggs et al. 2001
A. brasilense Roots Pot, field 50–90, 33 (grain yield) Dobbelaere et al. 2001
R. etli bv. phaseoli Roots Gnotobiotic 20–45 (total biomass) Gutierrez-Zamora
and Martinez-Romero,
2001
H. seropedicae Roots Greenhouse, field 49–82 (yield), 19.5 (yield) Riggs et al. 2001
Pseudomonas sp. Roots Gnotobiotic 11.7 (total biomass) Shaharoona et al. 2006
Sugarcane G. diazotrophicus Micro-propagated Greenhouse 26 (plant dry weight) Muñoz-Rojas
and Caballero-
Mellado (2003)
BNF+PGPR
Rice Pantoea agglomerans Root and shoot tissue Gnotobiotic 63.5 (total biomass) Verma et al. 2001;
Feng et al. 2006
Combination – Pot, field 9.5, 23.6 Govindarajan et al. 2008
of G. diazotrophicus
LMG7603, H. seropedicae
LMG6513, A. lipoferum
4B LMG4348,
and B. vietnamiensis
LMG10929
B. vietnamiensis MGK3 Roots, shoots Pot, field 5.6–12.16 (yield) Govindarajan et al. 2008
Wheat H. seropedicae Seeds Greenhouse 49–82 (total biomass) Riggs et al. 2001
Appl Microbiol Biotechnol (2008) 80:199–209 203

Table 1 (continued)

Host plant Endophyte/diazotroph Colonisation Condition Percent increase Reference

inoculant of cultivation

Sugarcane B. vietnamiensis Rhizosphere, Field 19.5 (yield) Govindarajan et al. 2006

stem, roots
G. diazotrophicus Roots, stems Field 13–16 (yield) Govindarajan et al. 2006
H. seropedeceae Roots, stems Field 5–12 (yield) Govindarajan et al. 2006
Enterobacter Roots Gnotobiotic 55 and 70 Mirza et al. 2001
(root and shoot biomass)
Kleibsiella sp. GR9 Roots, stems Field 13–19.5 (biomass) Govindarajan et al. 2007
a
Mechanism of growth promotion: BNF refers to biological nitrogen fixation and PGPR refers to plant growth-promoting rhizobacterial activities

B. phytoformis PsJN characteristically improved the photo- Realizing the potential of endophyte in agribusiness,
synthetic activity and starch accumulation (P<0.05) in Vitis countries like Brazil have already adopted the practice of
vinifera when compared to the uninoculated plant. The use of plant growth-promoting bacteria in non-legume
increased resistance was a result of the rise in proline and cultivation. With the huge number of bacteria isolated and
phenolic content of the plant due to bacterial colonisation, identified to have a positive effect on the growth and
which plays an important role in the adaptation to stress development of non-legumes like rice, wheat, maize
(Barka et al. 2006). The elevation in phenolics is a kind of and sugarcane, the prospect of developing effective
stress response in the host plant due to bacterial invasion, microbial biofertilisers for these plants appears to be bright.
also observed in rice–endophyte interaction (Mishra et al. Nevertheless, the widespread adoption of this practice
2006). would require a critical analysis of the production variabil-
Endophytic bacteria can be used discretely or as a mixture ity that was observed at different sites and in different crop
for inoculating plants in pots or fields. A mixture of bacterial rotations.
isolates used as an inoculum gave a synergistic result in
terms of plant growth and development (Govindarajan
et al. 2008). Govindarajan et al. (2008) used a mixture of Plant growth promotion by endophytes: proposed
H. seropedicae LMG6513, Azospirillum lipoferum 4B mechanism
LMG4348, Gluconacetobacter diazotrophicus LMG7603
and B. vietnamiensis LMG10929 at a concentration of Beneficial effects savoured by the host plant in an
108 cfu ml−1 to inoculate 5-day-old uninfected rice seed- endophyte–plant interaction have been speculated to be
lings. Among all of the assays, the mixed inoculant the result of (1) BNF by the colonizing bacteria and (2)
performed best, producing an increase of 14.4% in yield plant growth-promoting substances produced by the rhizo-
compared to an average of 6.2% obtained from individual bacteria. In some cases, a cumulative participation of both
strains (Govindarajan et al. 2007). However, in using a the above mechanisms was observed. Table 1 summarises
mixed inoculum, the compatibility of each strain with the different mechanism by which endophytes has been
each other in the mixture determined the overall perfor- proposed to participate in the host plant growth promotion.
mance of the inoculum in enhancing plant growth. A
mixture of G. diazotrophicus LMG7603, A. amazonense Nitrogen accumulation
and Burkholderia sp. when applied to sugarcane gave a
comparatively lower yield than individual inoculation of Nitrogen is the most significant yield-limiting element in
B. vietnamiensis MG43 and G. diazotrophicus LMG7603 many agricultural production systems. It is known that in
(Oliveira et al. 2002; Govindarajan et al. 2006). However, legumes, BNF by symbiotic bacteria provides a substantial
a mixed inoculum of G. diazotrophicus LMG7603, amount of nitrogen required by the plant. When NF
B. vietnamiensis LMG10929, H. seropedicae LMG6513 bacterium co-exists as an endophyte within non-legumes,
and A. lipoferum 4B LMG4348 when compared to the above the plant’s total nitrogen content rises uniformly. Nitrogen
mixture performed with a higher growth response in rice accumulation in inoculated non-legumes may be the result
(Govindarajan et al. 2007). Although the performance of the of: BNF (Boddey et al. 1995; Elbeltagy et al. 2001; Oliveira
mixture used by Govindrajan et al. (2007) has not been et al. 2002) or the increase in nitrogen uptake from the soil
assessed in sugarcane, these studies emphasise the impor- (Yanni et al. 1997; Prayitno et al. 1999). Systematic study
tance of strain selection in a mixed inoculum for obtaining by various workers in Brazil over the years led to the
higher performance in the plant. observation that some sugarcane varieties grown for
204 Appl Microbiol Biotechnol (2008) 80:199–209

decades or even a century do not show any decline in the colonised and invaded the plant upon inoculation contribut-
soil N reserve or yield despite the supply deficit of N ed the fixed nitrogen (Boddey et al. 1995; Oliveira et al.
(Boddey et al. 1995). In some varieties of sugarcane, grown 2002). Even in grasses, nitrogen fixation by colonizing
in well-irrigated and fertilised tank (with proper supply of bacteria was documented, although the amount of nitrogen
K and P) without N, yield increase was in the range of 170 fixed was lower compared to rice or sugarcane (Iniguez et
to 230 t ha−1 in the first year. In sugarcane varieties CB45-3, al. 2004). Plants inoculated with wild Azoarcus had higher
SP70-1143 and Krakatau, the trend of yield increase dry weights, lower N15 and 1.4 mg more N than plants
continued for 3 subsequent years. In these varieties, 60– inoculated with the nif K− mutant strain BHNKD4 (non-NF;
80% of the nitrogen accumulated was a result of BNF Hurek et al. 2002). They speculated using a N balance study
(Boddey et al. 1995). that the difference in N content was not due to N uptake
The ability of an endophyte to fix atmospheric nitrogen from potting media, as the soil in which the experiments
within a host has been proved using different approaches: were carried out was not fertilised with N for more than
acetylene reduction assay, 15N isotope dilution experiments, 8 months.
15
N2 reduction assays or 15N natural abundance assays. At the molecular level, BNF in host–endophyte interac-
Dalton and Kramer (2006) have discussed the experimental tion was ascertained using nif mutants of the non-legume-
details and shortcomings of these assays. These experi- colonizing endophyte (Iniguez et al. 2004; Hurek et al.
ments have conclusively shown that an increase in the 2002; de Campos et al 2006). Rice plants grown in
host-plant N content as high as 30–45 mg of N per plant nitrogen-deficient media and inoculated with non-NF
(6-week-old seedlings) in rice to 170 kg of N per hectare nifH mutant of Klebsiella pneumoniae 324 showed severe
per year in sugarcane was a result of BNF (Boddey et al. signs of nitrogen deficiency in contrast to the wild K.
1995; Iniguez et al. 2004). In the wild rice variety Oryza pneumoniae-inoculated batch (Iniguez et al. 2004). The
officinalis, acetylene reduction and 15N2 gas incorporation wild K. pneumoniae-inoculated plants assimilated 42% and
were deployed to determine the in planta nitrogen fixation 41% of the plant’s nitrogen from the atmosphere. Contrary
after inoculation with endophytic Herbaspirillum sp. strain to the previous case, rice plants inoculated with a mutant
B501. The percentage of 15 d N2 incorporation was 381 as strain of A. brasilense Sp7:: Tn5-33 with enhanced in vitro
compared to 0.4 of the uninoculated plant, which proved the nitrogen fixation accumulated 351 mg per plant dry matter
role of nitrogen fixation by Herbaspirillum sp. strain B501 in (de Campos et al. 2006). This accumulation is equivalent to
rice (Elbeltagy et al. 2001). Another instance is the growth- the control plant treated with an additional 5 mM NH4NO3.
promoting endophyte Burkholderia colonizing rice, where Observations from these workers highlighted a correlation
an estimated 31% (372 μg N per plant) of rice plant nitrogen between nitrogen accumulations in plants by the NF
were derived by BNF (Baldani et al. 2000). Commercially bacteria and growth promotion. Subsequently, Hurek et al.
important rice Basmati and Super Basmati are also known (2002) isolated 85-nucleotide-long nifH poly(A) mRNA
to benefit from inoculation with Herbaspirillum and from roots of inoculated Kallar grass, showing that
Azospirillum (Mirza et al. 2000). Under greenhouse trials, Azoarcus sp. BH72 was metabolically active in expressing
these rice varieties derived 19% and 47% of their nitrogen nitrogenase gene within the plant. From the same sample,
requirement from the atmosphere. In a separate experiment, isolated from roots of inoculated grass, nifH genes could be
Oliveira et al. (2002) inoculated micropropagated sugar- amplified, whereas nifH mRNA levels in control plants
cane with 2×105 cells per millilitre of five different strains were not sufficiently high to allow detection. The expres-
of NF bacteria (G. diazotrophicus, H. seropedicae, H. sion of BH72 nifH in test plants but not in control plants
rubrisubalbicans, A. amazonense and Burkholderia sp.) confirmed that the source of plant nitrogen in inoculated
originally isolated from sugarcane. These strains were used plants was from N2 fixation by Azoarcus. In spite of the fact
together in various combinations. After acclimatisation for that gramineous plants do not posses in vivo specialised
45 days in a greenhouse, plantlets were transferred to pots features that make a conducive environment for the
containing N15 for assessment of nitrogen fixation by the functioning of enzymes involved in the BNF pathway,
N15 isotope dilution technique. The bacterial inoculation endophytic bacteria successfully express nitrogen fixation
documented a maximum rise of 39% in total biomass (645 g structural nif genes within the host. The expression of the
per plant, 400 days after inoculation) over the uninoculated gene encoding iron protein of nitrogenase (nifH) was
control. In the process, the inoculated bacteria assimilated detected in epidermal cells, the intercellular region of root
30% nitrogen by BNF (Oliveira et al. 2002). Similarly, a cortex and vascular tissue of wheat, maize, sorghum and
phytohormone-producing diazotroph Enterobacter of sugar- rice roots (Hurek et al. 2002; Egener et al. 1999; Roncato-
cane inoculated to roots of micropropagated sugarcane Maccari et al. 2003). Indeed, in 7-day-old seedlings of
assimilated 29% of nitrogen by atmospheric fixation (Mirza O. officinalis, the nifH transcription of the colonizing
et al. 2001). In all the above cases, the bacteria that Herbaspirillum sp. B501gfp followed a circadian rhythm
Appl Microbiol Biotechnol (2008) 80:199–209 205

(You et al. 2005). During the light phase, the transcription production is being deployed as tool for identification and
level of the nifH gene reached 100 times the level during screening of endophytes (Khalid et al. 2005; Shaharoona et
the dark phase. This is contradictory and goes against the so al. 2006).
called ‘oxygen paradox’ as the light phase generates an
aerobic condition (21% O2). At this stage, it is not known
how the enzyme activity is protected under such circum- Non legume–Rhizobium interaction
stances. However, You et al. (2005) suggested that this might
be an adaptation of the endophyte directed to derive Despite the widespread occurrence of endophyte in non-
maximum benefit of the photosynthate generated during leguminous plants, there is only limited data on the
the light phase. mechanism involved in the endophyte–host interaction.
Infection and colonisation of a non-legume by the NF bacteria
Other PGPR activities of endophytes differ significantly from Rhizobium–legume symbiosis.
Using various techniques like fluorescent-tagged endo-
Some workers observed that the overall growth promotion phytes, antibodies, fluorescent microscopy, scanning and
and nitrogen assimilation in a plant inoculated with bacteria transmission electron microscopy, the bacterial route of
is not solely due to BNF by the endophyte. During entry into the host plant has been tracked and scored in
extensive greenhouse and field experiments using non- many cases (Prayitno et al. 1999; Chaintreuil et al. 2000;
sterilised soils, Riggs et al. (2001) observed that when James et al. 2001; Verma et al. 2004; Perrie-Walker et al.
maize seeds are inoculated with H. seropedicae under 2007b). Within 90–120 min of inoculation of a non-legume,
greenhouse conditions, the yield increased by 49–82% with green fluorescent protein (GFP)-labelled Rhizobium
applied fertilizer N, whereas without fertilisation, the strains ANU843, E4 and R4 were observed on the main
increase was only 16%. This indicated the participation of root surface (Prayitno et al. 1999; Perrie-Walker et al.
factors other than BNF, which improved the maize plant’s 2007b). Nevertheless, a 24-h time period was required for
proficiency to use the available fertiliser N (Table 1). complete adherence of the bacteria to the root surface,
Similarly, Sevilla et al. (2001) also suggested the participa- which thereafter remained stable for a further 2 weeks
tion of other growth-promoting factors in addition to N (Prayitno et al. 1999; Gyaneshwar et al. 2001; Chi et al.
fixation as both wild and nifH − mutants of A. diazotrophi- 2005; Perrie-Walker et al. 2007b). Endophytic bacteria are a
cus promoted growth of sugarcane in the presence of more aggressive coloniser and are capable of outcompeting
nitrogen. Further, in the association of the NF R. trifolii or others in the surroundings (Verma et al. 2004). R. trifolii
Bradyrhizobia and rice, there was no evidence of in planta occurred at a density of ∼1.7×106 per gram of rhizosphere
nitrogen fixation by the bacteria (Yanni et al. 1997, 2001; soil surrounding rice roots (Yanni et al. 1997). In some
Chaintruel et al. 2000). regions of a protruding lateral root, curled root hairs
Most endophytes with plant growth-enhancing properties enclosing high numbers of GFP-labelled bacteria were
are producer of phytohormones: indolacetic acid, gibber- observed with R. trifolii R4-treated rice (Perrie-Walker et
ellins and cytokinins (Biswas et al. 2000a, b; Yanni et al. al. 2007b). In addition, infection thread-like structures were
2001, Verma et al. 2001), iron-sequestering siderophores also evident within inoculated plants. Subsequently, the
(Yanni et al. 2001; Verma et al. 2001), phosphate-solubilising bacteria are propagated to the next generation through seeds
enzymes (Verma et al. 2001) and 1-aminocyclopropane- or vegetative means (Verma et al. 2001). Successful entry
1-carboxylate (ACC) deaminase (Khalid et al. 2005). into the host plant by endophytes is made through: root tips,
Growth hormones produced by the bacteria enhanced the lateral root cracks at the point of emergence of lateral roots,
development of lateral roots, improving the plant’s nutrient injured sites on root epidermis and damaged stomata
uptake from the rhizosohere (Yanni et al. 2001). ACC (Chaintreuil et al. 2000; James et al 2002; Sevilla et al.
deaminase producing Pseudomonas spp. brought signifi- 2001; Chi et al. 2005; Perrine-Walker et al. 2007b). In
cant increases in plant height, root weight and total biomass Arabidopsis thaliana, 100% of the inoculated plants were
in the presence of nitrogen in maize plants (Shaharoona colonised at the point of emergence of lateral roots with
et al. 2006). ACC deaminase production by PGPR lowered H. seropedicae (James et al. 2002). After a successful
ACC, the immediate precursor of ethylene, thereby lower- infiltration, Rhizobium is disseminated throughout the host
ing the level of ethylene. Lower levels of ethylene in and plant interior without evoking an observable defense
around roots in turn promoted growth and elongation of reaction in the plant. The intercellular and cortex regions
roots (Glick 1995). Release of auxins and ACC deaminase of the root formed a preferred site of initial colonisation of
in vitro by the rhizobacteria was linearly correlated with the endophytic bacteria (Chaintruel et al. 2000; Verma et al.
the host plant growth promotion (Khalid et al. 2005). 2004). Colonisation further extended to the intercellular
Subsequently, indole-3-acetic acid and ACC deaminase space of the root cortex to the xylem vessels to further
206 Appl Microbiol Biotechnol (2008) 80:199–209

intercellular spaces in leaf mesophylls (Gyaneshwar et al. during an endophytic association. This is an exclusive
2002; Roncato-Maccari et al. 2003). For the mechanism of phenomenon observed in an endophytic interaction and
bacterial dissemination in aerial parts, many workers could not be seen in a pathogenic interaction. Although not
proposed the theory of ‘xylem translocation’ (James et al. well understood, the product of this kinase has a role to
2002; Chi et al. 2005). It was observed that a large play in the signal transduction process involved in the
population of G. diazotrophicus occurred in the xylem establishment of a successful endophytic interaction. An
vessel and parenchyma (possibly the phloem) of inoculated ISR developed in response to PGPR did not alter the
sugarcane plantlets and greenhouse-grown plants (Fuentes- establishment of an interaction but rather enhanced the
Ramirez et al. 1999; James et al. 2001). Further, Chi et al. invading plant’s growth and development (Mirza et al.
(2005) observed that following an endophytic colonisation 2000). It was observed that the specific tolerance of
by GFP-tagged Sinorhizobium meliloti and Azorhizobium sugarcane towards A. diazotrophicus was due to certain
caulinodans ORS57, the bacteria ascended into the stem glycoproteins of the host which binds to cells of A.
base, leaf sheaths and leaves reaching a population of diazotrophicus. The glycosidic moiety is composed of
9×1010 rhizobia per cubic centimetre of infected tissue. fructose units linked by β-(1-2) bonds and adheres more
Once the bacteria penetrate the plant, cell wall-hydrolysing effectively to A. diazotrophicus compared to Leuconostoc
enzymes, like CMCase, polygalacturonase, pectinolyase mesenteroides, an epiphytic bacterium which lives on
produced by R. leguminosarum bv. trifolii, cellulase and sugarcane leaf surfaces (Legaz et al. 2000). In a recent
pectinase produced by Pantoea agglomerans, H. seropedicae study by Ormeño-Orrillo et al. (2008), the role of rhizobial
Z67 and H. rubrisubalbicans assisted the process of lipopolysaccharide (LPS) in maize rhizosphere and root
invasion and dissemination of the bacteria within the host colonisation has been emphasised. They observed that three
(Yanni et al. 2001; Verma et al. 2001; James et al. 2002). transposon mutants of R. tropici defective in LPS biosyn-
The use of cell wall-degrading enzymes endogluconase thesis were significantly impaired in competitive root
and polygalacturonase in the infection of V. vinifera by colonisation with the parental strain when co-inoculated in
Burkholderia sp. was also emphasised in the work of a 1:1 ratio. In addition, LPS provided a protective coat
Compant et al. (2005). against many hydrophobic and lipophilic antimicrobial
An intriguing question in such interactions is how the compounds produced by plants as the mutants were more
plant identifies a beneficial microorganism. At present, not susceptible than the wild.
much is known about rhizobial factors that help the
endophyte to suppress or avoid host defence responses.
Nevertheless, it is likely that some kind of ‘quoram- Challenges and future perspectives
sensing’ mechanism as in Rhizobium–legume symbiosis
might also exist in this case which helps in the establish- In the last decade, numerous studies were undertaken to
ment of a successful relationship. Rhizobial inoculation to optimise conditions and reap maximum benefit from
rice plants is associated with an increased accumulation of various endophyte–non-legume interactions. However,
phenolics such as gallic, tannic, ferulic and cinnamic acids most of the experiments to test the performance of
in the plant leaves (Mirza et al. 2001). Such increases in endophytes were conducted under controlled conditions.
phenolic acid are a pathogenic stress-related phenomenon A general decrease in performance was observed when the
in plants (Pieterse et al. 2002). Defense reactions triggered pot-grown inoculated plants are shifted to the field (Riggs
in response to rhizobial invasion is termed as rhizobacteria- et al. 2001; Gyaneshwar et al. 2002). Some of the factors
mediated ‘induced systemic resistance’ (ISR). ISR is that may affect the performance of an endophyte are:
controlled by a signalling pathway in which jasmonic acid nitrogen content of the soil (Muthukumarasamy et al. 1999,
and ethylene play key roles; in contrast, pathogen-induced 2002), soil type (de Oliveira et al. 2006) and host plant age
‘systemic acquired response’ is regulated by salicylic acid and variety (Yanni et al. 1997; Muñoz-Rojas and Caballero-
(reviewed by Pieterse et al. 2002). Ethylene signalling is Mellado 2003; de Oliveira et al. 2006). In numerous
triggered by a family of receptors in sugarcane in response investigations, the use of NF bacteria in combination with
to G. diazotrophicus and Herbaspirillum sp. inoculation. In the N fertiliser subsequently reduced the amount of external
contrast to pathogenic interaction, putative ethylene recep- supply of fertiliser being applied to the plant (Yanni et al.
tors expression was up-regulated during invasion by 1997; Saleh et al. 2001). The challenge, however, lies in
beneficial bacteria (Cavalcante et al. 2007). They speculat- optimizing the amount of applied fertiliser to obtain a good
ed that the up-regulation of one such receptor SCER1 might survival rate of NF bacteria in the rhizosphere. High-
reduce ethylene sensitivity and therefore plant defence nitrogen fertilised soil (ammonia) reduced colonisation of
against the diazotrophic endophytes. Another receptor-like sugarcane by both G. diazotrophicus and H. seropedicae
kinase, SHR5, identified from sugarcane, was repressed (Fuentes-Ramırez et al. 1999; Bueno dos Reis Junior et al.
Appl Microbiol Biotechnol (2008) 80:199–209 207

2000; Muthukumarasamy et al. 1999, 2002). Even the one step closer to developing an ecofriendly nutrient source
presence of any concentration of Ca2+ and (PO4)3− above for cereal crops. Despite the recent advances, commercial-
50 mM in the media had a derogatory effect on the rate of isation of this technology demands extensive optimisation
Azospirillum adsorption on wheat root surface (Pinheiro et and comprehensive study of the aftereffects of the applica-
al. 2002). Consequently, Alfisol soil type (low soil fertility) tion. The prospects of this technology is far reaching
supported a better performance of the endophytic inoculant keeping in consideration the rising cost and declining
in terms of BNF contribution and stem yield without N reserves of fossil fuels. Given that the research succeeds,
fertilisation for 3 consecutive years (de Oliviera et al. the prodigious ramifications would mitigate environmental
2006). Muthukumaraswamy et al. (2002) speculated that concerns arising from the use of nitrogenous fertiliser and
high concentration of nitrogen sources especially ammonia its costs to poor farmers.
(25 mM NH4NO3) in media led to morphological changes
in the bacteria which might play a negative role in their
survival. Nevertheless, the use of compost as the nitrogen Acknowledgments One of the authors, RBB, is thankful to Council
of Scientific and Industrial Research, India, for the Ph.D. fellowship.
source was found to counteract the derogatory effect of N
We are thankful to Sujay Bhattacharjee for his editorial help.
fertiliser on bacterial colonisation and boost the number of
colonizing bacteria (Muthukumaraswamy et al. 2007).
Selection of plant genotype and age also influences the
References
consistency of performance of the bacteria to contribute to
host plant growth enhancement (Muñoz-Rojas and Caballero- Baldani VLD, Baldani JI, Döbereiner J (2000) Inoculation of rice
Mellado 2003; de Oliveira et al. 2006). Muñoz-Rojas and plants with the endophytic diazotrophs Herbaspirillum seropedicae
Caballero-Mellado (2003) observed a drastic decrease in and Burkholderia spp.. Biol Fert Soils 30:485–491
the G. diazotrophicus population with the age of the plant Barka EA, Nowak J, Clément C (2006) Enhancement of chilling
resistance of inoculated grapevine plantlets with a plant growth-
and the genotype. In some sugarcane varieties, apparently,
promoting rhizobacterium, Burkholderia phytofirmans strain
the persistence of the endophyte was for a longer period PsJN. Appl Environ Microbiol 70:7246–7252
and in higher numbers. In addition, environmental factors Biswas JC, Ladha JK, Dazzo FB, Yanni YG, Rolfe BG (2000a)
like the soil hydric stress and seasonal changes also Rhizobial inoculation influences seedling vigor and yield of rice.
Agronomy J 92:880–886
contribute to the observed variation in diazotrophic bacteria
Biswas JC, Ladha JK, Dazzo FB (2000b) Rhizobia inoculation
number (Bueno dos Reis Junior et al. 2000). More field improves nutrient uptake and growth of lowland rice. Soil Sci
trials are therefore required to optimise these parameters Soc Am J 64:1644–1650
including time and way of application of the inoculant and Boddey RM, Oliveira OCD, Urquiaga S, Reis VM, Olivares FLD,
Baldani VLD, Döbereiner J (1995) Biological nitrogen fixation
environmental factors (de Oliveira et al. 2006).
associated with sugarcane and rice: contributions and prospects
In this study, the documented ability of Rhizobium to for improvement. Plant Soil 174:195–209
interact naturally with rice or other gramineous plants Bueno dos Reis Junior F, Massena Reis V, Urquiaga S, Döbereiner J (2000)
flashes light on the use endophytic/rhizospheric bacteria for Influence of nitrogen fertilisation on the population of diazotrophic
bacteria Herbaspirillum spp. and Acetobacter diazotrophicus in
improving plant performance. However, to maximise
sugar cane (Saccharum spp.). Plant Soil 219:153–159
benefit from these endophytes either through BNF or plant Cavalcante J, Vargas C, Nogueira E, Vinagre E, Schwarcz K, Baldani J,
growth-promoting activities, a better understanding of Ferreira P, Hemerly A (2007) Members of the ethylene signalling
endophyte ecology and mechanism of interaction at pathway are regulated in sugarcane during the association with
nitrogen-fixing endophytic bacteria. J Exp Bot 58:673–686
molecular level will be required. With complete genome
Chaintreuil C, Giraud E, Prin Y, Lorquin J, Ba A, Gillis M, de Lajudie P,
sequencing of various endophytes and using transcriptomics/ Dreyfus B (2000) Photosynthetic bradyrhizobia are natural
proteomics, various genes that are induced or repressed endophytes of the African wild rice Oryza breviligulata. Appl
during colonisation can be identified. This insight in the Environ Microbiol 66:5437–5447
Chi F, Shen S-H, Cheng H-P, Jing J-X, Yanni YG, Dazzo FB (2005)
mechanism will be promising in developing a more efficient
Ascending migration of endophytic rhizobia, from roots to
plant–bacteria interaction to promote sustainable production leaves, inside rice plants and assessment of benefits to rice
of biomass in the field. growth physiology. Appl Environ Microbiol 71:7271–7278
Compant S, Reiter B, Sessitsch A, Nowak J, Clément C, Barka EA
(2005) Endophytic colonization of Vitis vinifera L. by plant
growth-promoting bacterium Burkholderia sp. strain PsJN. Appl
Conclusions Environ Microbiol 71:1685–1693
Conn VM, Franco CMM (2004) The effect of microbial inoculants on the
From the numerous literature available today, it is clear that indigenous actinobacterial endophyte population present in the roots
of wheat as determined by terminal restriction fragment length
the “NF bacteria–non-legume” interaction is a natural
polymorphism (T-RFLP). Appl Environ Microbiol 70:6407–6413
phenomenon. With the progressive understanding of the Dalton DA, Kramer S (2006) Plant-associated bacteria. Springer, The
benefits conferred to the host by this association, we are Netherlands, pp 105–130
208 Appl Microbiol Biotechnol (2008) 80:199–209

de Campos SB, Roesch LFW, Zanettini MHB, Passaglia LMP (2006) Hurek T, Handley LL, Reinhold-Hurek B, Piché Y (2002) Azoarcus
Relationship between in vitro enhanced nitrogenase activity of an grass endophytes contribute fixed nitrogen to the plant in an
Azospirillum brasilense Sp7 mutant and its growth-promoting unculturable state. Mol Plant–Microbe Interact 15:233–242
activities in situ. Curr Microbiol 53:43–47 Iniguez AL, Dong Y, Triplett EW (2004) Nitrogen fixation in wheat
de Oliveira ALM, de Canuto EL, Urquiaga S, Reis VM, Baldani JI provided by Klebsiella pneumoniae 342. Mol Plant–Microbe
(2006) Yield of micropropagated sugarcane varieties in different Interact 17:1078–1085
soil types following inoculation with diazotrophic bacteria. Plant James EK, Olivares FL, de Oliveira ALM, dos Reis FB Jr, da Silva LG,
Soil 284:23–32 Reis VM (2001) Further observations on the interaction between
Dobbelaere S, Croonenborghs A, Thys A, Ptacek D, Vanderleyden J, sugar cane and Gluconacetobacter diazotrophicus under labora-
Dutto P, Labandera-Gonzalez C, Caballero-Mellado J, Aguirre JF, tory and greenhouse conditions. J Exp Bot 52:747–760
Kapulnik Y, Brener S, Burdman S, Kadouri D, Sarig S, Okon Y James EK, Gyaneshwar P, Mathan N, Barraquio WL, Reddy PM,
(2001) Responses of agronomically important crops to inoculation Iannetta PPM, Olivares FL, Ladha JK (2002) Infection and
with Azospirillum. Aust J Plant Physiol 28:871–879 colonization of rice seedlings by the plant growth-promoting
Egamberdiyeva D, Höflich G (2002) Root colonization and growth bacterium Herbaspirillum seropedicae Z67. Mol Plant–Microbe
promotion of winter wheat and pea by Cellulomonas spp. at Interact 15:894–906
different temperatures. Plant Growth Regulation 38:219–224 Khalid A, Arshad M, Zahir Z (2005) Screening plant growth-
Egener T, Hurek T, Reinhold-Hurek B (1999) Endophytic expression promoting rhizobacteria for improving growth and yield of
of nif genes of Azoarcus sp. strain BH72 in rice roots. Mol Plant– wheat. J Appl Microbiol 96:473–480
Microbe Interact 12:813–819 Knauth S, Hurek T, Brar D, Reinhold-Hurek B (2005) Influence of
Elbeltagy AK, Sato NT, Suzuki H, Ye B, Hamada T, Isawa T, Mitsui H, different Oryza cultivars on expression of nifH gene pools in
Minamisawa K (2001) Endophytic colonization and in planta roots of rice. Environmental Microbiol 7:1725–1733
nitrogen fixation by a Herbaspirillum sp. isolated from wild rice Legaz M-E, de Armas R, Barriguete E, Vicente C (2000) Binding of
species. Appl Environ Microbiol 67:5285–5293 soluble glycoproteins from sugarcane juice to cells of Acetobacter
Engelhard M, Hurek T, Reinhold-Hurek B (2000) Preferential diazotrophicus. Int Microbiol 3:177–182
occurrence of diazotrophic endophyte, Azoarcus sp. in wild rice Matthews SS, Sparkes DL, Bullard MJ (2001) The response of wheat
species and land races of Oryza sativa in comparison with to inoculation with the diazotroph Azorhizobium caulinodans.
modern races. Environ Microbiol 2:131–141 Aspects Appl Biol 63:35–42
Estrada P, Mavingui P, Cournoyer B, Fontaine F, Balandreau J, Mishra RPN, Singh RK, Jaiswal HK, Kumar V Maurya S (2006)
Caballero-Mellado J (2005) A N2-fixing endophytic Burkholderia Rhizobium-mediated induction of phenolics and plant growth
sp. associated with maize plants cultivated in Mexico. Int J Syst promotion in rice (Oryza sativa L.). Curr Microbiol 52:383–389
Evol Microbiol 55:1233–1237 Mirza MS, Rasul G, Mehnaz S, Ladha JK, So RB, Ali S, Malik KA (2000)
Feng Y, Shen D, Song W (2006) Rice endophyte Pantoea agglomerans Beneficial effects of inoculated nitrogen-fixing bacteria on rice. In:
YS19 promotes host plant growth and affects allocations of host Ladha JK, Reddy PM (eds) The quest for nitrogen fixation in rice.
photosynthates. J Appl Microbiol 100:938–945 International Rice Research Institute, Los Baños, pp 191–204
Fuentes-Ramírez LE, Caballero-Mellado J, Sepúlveda J, Martinez- Mirza MS, Ahmad W, Latif F, Haurat J, Bally R, Normand P, Malik KA
Romero E (1999) Colonization of sugarcane by Acetobacter (2001) Isolation, partial characterization, and effect of plant
diazotrophicus is inhibited by high N-fertilization. FEMS Micro- growth-promoting bacteria (PGPB) on micro-propagated sugar-
biol Ecol 29:117–128 cane in vitro. Plant Soil 237:47–54
Glick BR (1995) The enhancement of plant growth by free-living Muñoz-Rojas J, Caballero-Mellado J (2003) Population dynamics of
bacteria. Can J Microbiol 41:109–117 Gluconacetobacter diazotrophicus in sugarcane cultivars and its
Govindarajan M, Balandreau J, Muthukumarasamy R, Revathi G, effect on plant growth. Microb Ecol 46:454–464
Lakshminarasimhan C (2006) Improved yield of micropropagated Muthukumarasamy R, Revathi G, Lakshminarasimhan C (1999)
sugarcane following inoculation by endophytic Burkholderia Diazotrophic associations in sugar cane cultivation in South
vietnamiensis. Plant Soil 280:239–252 India. Trop Agric 76:171–178
Govindarajan M, Kwon S-W, Weon H-Y (2007) Isolation, molecular Muthukumarasamy R, Revathi G, Loganathan P (2002) Effect of
characterization and growth-promoting activities of endophytic inorganic N on the population, in vitro colonization and
sugarcane diazotroph Klebsiella sp. World J Microbiol 23:997–1006 morphology of Acetobacter diazotrophicus (syn Gluconacetobacter
Govindarajan M, Balandreau J, Muthukumarasamy R, Kwon S-W, diazotrophicus). Plant Soil 243:91–102
Weon H-Y, Lakshminarasimhan C (2008) Effects of the Muthukumarasamy R, Cleenwerck I, Revathi G, Vadivelu M, Janssens
inoculation of Burkholderia vietnamiensis and related endophytic D, Hoste B, Ui Gum K, Park K, Son CY, Sa T, Caballero-
diazotrophic bacteria on grain yield of rice. Microbial Ecol Mellado J (2005) Natural association of Gluconacetobacter
55:21–37 diazotrophicus and diazotrophic Acetobacter peroxydans with
Gutierrez-Zamora ML, Martinez-Romero E (2001) Natural endophytic wetland rice. Syst Appl Microbiol 28:277–286
association between Rhizobium etli and maize (Zea mays L.). J Muthukumarasamy R, Kang UG, Park KD, Jeon W-T, Park CY,
Biotechnol 91:117–126 Cho YS, Kwon S-W, Song J, Roh D-H, Revathi G (2007)
Gyaneshwar P, James EK, Mathan N, Reddy PM, Reinhold-Hurek B, Enumeration, isolation and identification of diazotrophs from
Ladha JK (2001) Endophytic colonization of rice by a diazo- Korean wetland rice varieties grown with long-term application
trophic strain of Serretia marcessens. J Bacteriol 183:2634– of N and compost and their short-term inoculation effect on rice
2635 plants. J Appl Microbiol 102:981–991
Gyaneshwar P, James EK, Reddy PM, Ladha JK (2002) Herbaspirillum Oliveira ALM, Urquiaga S, Döbereiner J, Baldani JI (2002) The effect
colonization increases growth and nitrogen accumulation in of inoculating endophytic N2-fixing bacteria on micropropagated
aluminium-tolerant rice varieties. New Phytol 154:131–145 sugarcane plants. Plant Soil 242:205–215
Hilali A, Prévost D, Broughton WJ, Antoun H (2001) Effets de Ormeño-Orrillo E, Rosenblueth M, Luyten E, Vanderleyden J, Martínez-
l’inoculation avec des souches de Rhizobium leguminosarum Romero E (2008) Mutations in lipopolysaccharide biosynthetic
biovar trifolii sur la croissance du blé dans deux sols du Maroc. genes impair maize rhizosphere and root colonization of Rhizobium
Can J Microbiol 47:590–59 tropici CIAT899. Environ Microbiol 10:1271–1284
Appl Microbiol Biotechnol (2008) 80:199–209 209

Peng SB, Biswas JC, Ladha JK, Yaneshwar PG, Chen Y (2002) inoculation of sterile plants with Acetobacter diazotrophicus
Influence of rhizobial inoculation on photosynthesis and grain wild-type and nif− mutant strains. Mol Plant–Microbe Interact
yield of rice. Agron J 94:925–929 14:358–366
Perrine-Walker FM, Hocart CH, Hynes MF, Rolfe BG (2005) Plasmid- Shaharoona B, Arshad M, Zahir ZA (2006) Effect of plant growth
associated genes in the model micro-symbiont Sinorhizobium promoting rhizobacteria containing ACC-deaminase on maize
meliloti 1021 affect the growth and development of young rice (Zea mays L.) growth under axenic conditions and on nodulation
seedlings. Environ Microbiol 7:1826–1838 in mung bean (Vigna radiata L.). Lett Appl Microbiol 42:155–59
Perrine-Walker FM, Gartner E, Hocart CH, Becker A, Rolfe BG (2007a) Suman A, Gaur A, Shrivastava AK, Yadav RL (2005) Improving sugarcane
Rhizobium-initiated rice growth inhibition caused by nitric oxide growth and nutrient uptake by inoculating Gluconacetobacter
accumulation. Mol Microbe Plant Interact 20:283–292 diazotrophicus. Plant Growth Regul 47:155–162
Perrine-Walker FM, Prayitno J, Rolfe BG, Weinman JJ, Hocart CH Suman A, Gaur A, Shrivastava AK, Gaur A, Singh P, Singh J, Yadav RL
(2007b) Infection process and the interaction of rice roots with (2007) Nitrogen use efficiency of sugarcane in relation to its BNF
rhizobia. J Exp Bot 58:3343–50 potential and population of endophytic diazotrophs at different N
Pieterse CMJ, Van Wees SCM, Ton J, Van Pelt JA, van Loon LC levels. Plant Growth Regul 54:1–11
(2002) Signalling in rhizobacteria-induced systemic resistance in Trân Van V, Berge O, Kê SN, Balandreau J, Heulin T (2000) Repeated
Arabidopsis thaliana. Plant Biol (Stuttgart) 4:535–544 beneficial effects of rice inoculation with a strain of Burkholderia
Pinheiro RD, Boddey LH, James EK, Sprent JI, Boddey RM (2002) vietnamiensis on early and late yield components in low fertility
Adsorption and anchoring of Azospirillum strains to roots of sulphate acid soils of Vietnam. Plant Soil 218:273–284
wheat seedlings. Plant Soil 246:151–166 Verma SC, Ladha JK, Tripathi AK (2001) Evaluation of plant growth
Prayitno J, Stefaniak J, McIver J, Weinmen JJ, Dazzo FB, Ladha JK, promoting and colonization ability of endophytic diazotrophs
Barraquio W, Yanni YG, Rolfe BG (1999) Interactions of rice from deep water rice. J Biotechnol 91:127–41
seedlings with bacteria isolated from rice roots. Aust J Plant Verma SC, Singh A, Paul Chowdhury S, Tripathi AK (2004)
Physiol 26:521–535 Endophytic colonization ability of two deep-water rice endo-
Reinhold-Hurek B, Hurek T (1997) Azoarcus spp. and their phytes, Pantoea sp. and Ochrobactrum sp. using green fluores-
interactions with grass roots. Plant Soil 194:57–64 cent protein reporter. Biotechnol Lett 26:425–429
Reinhold-Hurek B, Hurek T (1998) Life in grasses: diazotrophic Wang K, Conn K, Lazarovits G (2006) Involvement of quinolinate
endophytes. Trends Microbiol 6:139–144 phosphoribosyl transferase in promotion of potato growth by a
Rejesus RM, Hornbaker RH (1999) Economic and environmental Burkholderia strain. Appl Environ Microbiol 72:760–768
evaluation of alternative pollution-reducing nitrogen management Yanni YG, Rizk RY, Corich V, Squartini A, Ninke K, Philip-
practices in central Illinois. Agric Ecosyst Environ 75:41–53 Hollingsworth S, Orgambide G, de Bruinj F, Stoltzfus J, Buckley
Riggs PJ, Chelius MK, Iniguez AL, Kaeppler SM, Triplett EW (2001) D, Schmidt TM, Mateos PF, Ladha JK, Dazzo FB (1997) Natural
Enhanced maize productivity by inoculation with diazotrophic endophytic association between Rhizobium leguminosarum bv.
bacteria. Aust J Plant Physiol 28:829–836 trifolii and rice roots and assessment of its potential to promote
Roncato-Maccari LDB, Ramos HJO, Pedrosa FO, Alquini Y, rice growth. Plant Soil 194:99–114
Chubatsu LS, Yates MG, Rigo LU, Steffens MBR, Souza EM Yanni YG, Rizk RY, El-Fattah FKA, Squartini A, Corich V, Giacomini A,
(2003) Endophytic Herbaspirillum seropedicae expresses nif de Bruijn F, Rademaker J, Maya-Flores J, Ostrom P,
genes in gramineous plants. FEMS Microbiol Ecol 45:39–47 Vega-Hernandez M, Hollingsworth RI, Martinez-Molina E,
Saleh SA, Mekhemar GAA, El-Soud AAA, Ragab AA, Mikhaeel Mateos P, Velazquez E, Wopereis J, Triplett E, Umali-Garcia M,
FT (2001) Survival of Azorhizobium and Azospirillum in Anarna JA, Rolfe BG, Ladha JK, Hill J, Mujoo R, Ng PK, Dazzo FB
different carrier materials: inoculation of wheat and Sesbania (2001) The beneficial plant growth-promoting association of
rostrata. Bulletin of Faculty of Agriculture, Cairo University Rhizobium leguminosarum bv. trifolii with rice roots. Aust J Plant
52:319–338 Physiol 28:845–870
Sevilla M, Kennedy C (2000) Genetic analysis of nitrogen fixation You M, Nishiguchi T, Saito A, Isawa T, Mitsui H, Minamisawa K
and plant-growth stimulating properties of Acetobacter diazotro- (2005) Expression of the nifH gene of a Herbaspirillum
phicus, an endophyte of sugarcane. In: Triplett EW (ed) endophyte in wild rice species: daily rhythm during the light–
Prokaryotic nitrogen fixation: a model system for analysis of a dark cycle. Appl Environ Microbiol 71:8183–8190
biological process. Horizon Scientific, Norwich, UK, pp 737–760 Zhang L, Hurek T, Reinhold-Hurek B (2007) A nifH-based oligonu-
Sevilla M, Gunapala N, Burris RH, Kennedy C (2001) Comparison of cleotide microarray for functional diagnostics of nitrogen-fixing
benefit to sugarcane plant growth and 15N2 incorporation following microorganisms. Microbiol Ecol 53:456–470