Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of
Macrophomina phaseolina (Tassi) Goid. 93
Discussion
Results of the present finding have been discussed under the following heads:
A. CHARACTERIZATION OF FUNGAL PATHOGEN AND ENDOPHYTIC
BACTERIA OF VIGNA MUNGO
In the present investigation the fungus isolated by blotter and water agar techniques
from Vigna mungo was identified as Macrophomina phaseolina on the basis of grayish
black to black coloured colony on PDA medium forming jet black microsclerotia of
irregular sizes (Fig. 2 A-B). A similar characteristic feature of this fungus has also been
reported earlier by Dhingra and Sinclair (1977). During parasitic phase M. phaseolina
attacks number of plants including Vigna mungo (Dhingra and Sinclair (1978) and
forms sclerotia that survives in soil, stem roots or seeds. Dubey and Upadhyay (2001)
have reviewed the survival mechanisms of M. phaseolina. Association of M. phaseolina
with charcoal rot disease has also earlier been reported by Deshwal et al. (2003).
Microsclerotia present in soil and the infected host tissues serve as primary
inoculum (Dhingra and Sinclair, 1977). Root exudates induce germination of
microsclerotia and root infection of hosts. The infective hyphae enter into the plant
through root epidermal cells or wounds. During the initial stages of pathogenesis, the
mycelium penetrates the root epidermis and is restricted primarily to the intercellular
spaces of the cortex of the primary roots. After onset of flowers the hyphae grow first
intercellularly in the cortex, then intracellularly through the xylem colonizing the
vascular tissue and form microsclerotia that plug the vessels. Once present within the
vascular tissue M. phaseolina spreads through the taproot and lower stem of the plant
producing microsclerotia that plug the vessels (Short et al., 1978; Mayek-Pérez et al.,
2002). The infected plants die as the result of necrosis of roots and stems, mechanical
plugging of xylem vessels by microsclerotia, and also by toxin production and
Discussion
Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of
Macrophomina phaseolina (Tassi) Goid. 94
enzymatic action (Jones and Wang, 1997). The role of toxin(s) produced by M.
phaseolina in disease initiation has recently been reported by Sett et al. (2000). They
found that the two avirulent mutants of M. phaseolina were able to initiate infection in
germinating Phaseolus mungo seeds only in the presence of phaseolinone. The
minimum dose of phaseolinone required for infection in 30% seedlings was 2·5 mg/ml.
The other substrate-specific enzymes viz., pectinase, cellulase, protease, amylases
and lipase have also been reported to be produced by M. phaseolina which are
associated with host-pathogenesis (Dubey and Dwivwdi, 1988; Ahmad, et al. 2006).
Jones and Wang (1997) have analyzed in planta β-1,4-endoglucanase production by M.
phaseolina by probing tissue blots with a peptide-specific antibody. Endoglucanase was
readily detected after inoculation to corn and tobacco stems. Enzyme production
continued along with growth of the fungus in stem tissue. Endoglucanase was rapidly
transported through the xylem resulting in distribution to distal portions of the plant.
Enzyme production at the site of infection was correlated with symptom expression that
suggested a role for endoglucanases in disease progression.
Phaseolinone is a nonspecific exotoxin which plays a key role in pathogenesis. This
toxin inhibits seed germination of a large number of plants. The concentration required
for complete inhibition of seed growth of Phaseolus mungo (blackgram) has been found
as 25 g/ml (Bhattacharya, 1987; 1992). It also causes wilting of seedlings and leaf
necrosis in several plants. These symptoms were similar to those produced by the
fungus itself.
A total of twenty endophytic bacterial isolates were screened on YEMA, CrYEMA
and Bacillus agar and KB medium. Among those sixteen isolates from VR1 to VR20
were chosen for further work in detail. The diverse endophytic bacteria have also been
isolated from root nodules of many leguminous plants including Hedysarum (Benhizia
et al., 2004), a tree Conzattia multiflora (Wang et al., 2006), Vigna radiata, and V.
unguiculata (Appunu et al., 2009).
Discussion
Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of
Macrophomina phaseolina (Tassi) Goid. 95
The tissues of healthy plants can be colonized internally by microorganisms. The
term ‘endophyte’ is commonly used to describe such microorganisms. The best-
characterized microbial endophytes are nonpathogenic fungi, for which much
compelling evidence of plant/microbe mutualism has been provided. The fungal
endophytes are thought to benefit from the comparatively nutrient rich, buffered
environment inside plants. However, endophytic fungi comprise only part of the
nonpathogenic microflora found naturally inside plant tissues. Bacterial populations,
exceeding 107 colony forming units g
-1 plant matter, have been reported within tissues
of various plant species. Much less is known about bacterial endophytes compared to
their fungal counterparts. Work with plant species of agricultural and horticultural
importance indicates that some endophytic bacterial strains stimulate host plant growth
by acting as biocontrol agents, either through direct antagonism of microbial pathogens
or by inducing systemic resistance to disease-causing organisms (Chanway, 1998).
The isolates VR1– VR10 from V. mungo were Gram-negative, rod shaped and
motile (Table 1). Appunu et al. (2009) also have reported that Bradyrhizobium
yuanmingense nodulated Vigna mungo, V. radiata, V. unguiculata plants grown at
different sites and in different agronomical-ecological-climatic regions of India. They
formed translucent, round, gummy, convex, highly EPS producing small sized (2 to 2.3
mm) colonies on YEMA medium. In present study it was found that some of the
isolates (VR1–VR10) were slow growing (in contrast to Rhizobium), and generation
times of VR1 and VR2 were recorded 7 and 7.5 hours, respectively. Saharan et al.
(2011) have also reported similar result that Bradyrhizobum take 6 to 8 hours to double
its population size and 3 to 5 days to create moderate turbidity in liquid broth media.
The physico-chemical characteristics of all the bacterial isolates were similar as
described by Holt et al. (1994).
Moreover, the isolates VR1–VR20 accumulated PHB and showed positive results
for catalase activity, esculin hydrolysis (except VR11, VR12, VR14 and B. subtilis
MTCC 441), and indole production (except VR12 and VR14). Kumar (2012) has also
reported the production of PHB, esculin hydrolysis, indole production and catalase
Discussion
Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of
Macrophomina phaseolina (Tassi) Goid. 96
activity by the some species of Rhizobium, Bacillus and Pseudomonas. Mandal et al.
(2007b) also reported the production and composition of extracellular polysaccharide
synthesized by a Rhizobium isolate of Vign amungo. Accumulation of PHB granules by
50 isolates of bean root nodule bacteria has also been reported by Rodriguez-Navarro et
al. (2000). Only few of them have capacity to utilize the citrate and tolerate 8% of
KNO3 solution.
On the basis of physical, and biochemical characteristics all Bradyrhizobium
isolates were compared by UPGMA analysis done by NTSYS-pc (Numerical
Taxonomy and Multivariate Analysis System) Version 2.02e software (Rohlf, 1997).
VR1 and VR2 showed 92% similarity, whereas Bradyrhizobium sp. NAIMCC-B-00262
and node of VR1 and VR2 and were 87.9 % identical. However, VR15 and VR16
showed 85.1 % similar, and VR3 and VR5 were 83% similary in biochemical
characteristics (Fig. 5). These similarities are due to the presence of some of the
common physico-chemical characteristics among them. Minamisawa and Fukai (1991)
also correlated the production of indole-3-acetic acid (IAA) by Bradyrhizobium
japonicum' and established genotype grouping and rhizobitoxine production.
All the bacterial isolates screened on King’s B medium were Gram–negative rods,
non-capsulated, non-endospore forming and motile bacteria (Table 3). Assessment of
phylogenetically relatedness among all the isolates was done on the basis of UPGMA
analysis done by NTSYS-pc (Numerical Taxonomy and Multivariate Analysis System)
Version 2.02e software (Rohlf, 1997) (Fig. 5; Appendix Table I). The KB medium
isolates VR15-VR16 and the standard culture Pseudomonas sp. MTCC-129 formed the
second group. The isolates VR15 and VR20 showed 85% similarity, whereas
Pseudomonas sp. MTCC-129 showed 79.1 % similarity with node of VR15-VR20.
VR16 and node 12 showed 54.6% similarity (Fig 5; Appendix Table I). On the basis of
these characters and comparison with the standard culture of Pseudomonas sp. MTCC-
129, the isolates VR15 to VR20 were identified as Pseudomonas spp. Pseudomonas
isolates are Gram-negative, non-capsulated, non-endospore forming motile (polar
flagella) rods with average generation time 1.4 h (Holt et al., 1994). Pseudomonas
Discussion
Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of
Macrophomina phaseolina (Tassi) Goid. 97
species are ubiquitous inhabitants of soil, water, and plant surfaces that belong to the
Gamma-proteobacteria and fall under the family Pseudomonadaceae and the most
common genera of PGPR (Kloepper, 1993).
The isolates VR11-VR14 were screened on Bacillus agar medium screened from the
nodules of V. mungo. Several other workers have isolated Bacillus isolates from
legumes and non-legumes such as cotton, common bean, soybean, pine, etc. and
reported as PGPR (Srinivasan et al., 1996; Singh et al., 2008b; Gajbhiye et al., 2010;
Wahyudi et al., 2011). Colonies of VR11-VR14 were circular, flat, off-white in colour,
small in size. They were Gram-positive rods, aerobic, motile and had ability of
endospore formation. Due to spore forming ability and adaptation it has been exploited
for commercial formulation and field application (Liu and Sinclair, 1993). All the
isolates were positive for urease and oxidase production, and nitrate reduction. Isolates
VR11 and VR 13 did not utilize starch. All the isolates were negative for 8% KNO3
tolerance, methyl red test, Voges Proskaur (except VR14) and citrate utilization (Table
3). The entire Bacillus agar isolates VR11-VR14 and the standard culture Bacillus
MTCC-441 formed a third group when analysed by using NTSYS-pc (Numerical
Taxonomy and Multivariate Analysis System) Version 2.02e software (Rohlf, 1997)
(Fig. 5; Appendix Table I). In this group the isolates VR11 and VR13 were 85%
similar, and Bacillus MTCC-441 showed 76.4% similarity with node 2 of VR11 and
VR13. The isolates VR12 and VR14 also showed 76.4% similarity between one another
(Fig. 5; Appendix - Table I). On the basis of these characters and comparison with the
standard culture of Bacillus MTCC-441, the isolates VR11 to VR14 were identified as
Bacillus spp.
Yüksekdağ et al. (2004) investigated poly-beta-hydroxybutyrate (PHB) production
by Bacillus subtilis 25 and Bacillus megaterium 12 strains in nutrient broth medium at
different incubation times (between 6 h and 48h). They recorded PHB productions of
0.101 g/L, 0.142 g/L after 45h; but there was a decrease in PHB yields after 48h.
Discussion
Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of
Macrophomina phaseolina (Tassi) Goid. 98
Kumar et al. (2012b) have also found the Bacillus isolates to form endospores and
secrete antibiotics. These features contribute to their survival under adverse
environmental conditions for extended periods of time. The physico-chemical
characteristics of Bacillus isolates are also similar to those as have been described by
Holt et al. (1994).
In the present study the minimum and maximum temperature range tolerated by all
the isolates was 10º C and 50º C, respectively. Only four isolates grew at 10ºC and three
isolates at 50ºC. Similarly Kulkarni et al. (2000) have also reported that 33.7 to 48.7ºC
was the maximum tolerated temperature by B. japonicum. The optimum temperature
regime recorded for all the isolates in present work was 28oC to 30ºC at which
substantial growth was observed (Kucuk et al., 2006; Baoling et al., 2007; Singh et al.,
2008a; Ali et al., 2009; El-Akhal et al., 2009). A similar finding for optimum
temperature for endophytic bacterial survival has also been reported by Kumar (2010)
and Kumar (2012). Marsh Lurline et al. (2006) also reported that 30ºC/20ºC was the
optimum temperature for Bradyrhizobium strains. Optimum pH range for the growth of
all isolates VR1-VR10 was 6 to 7. Similar result has also been reported by Rodrigues et
al. (2006) and Ali et al. (2009) for root nodulating bacteria. It was studied that optimum
pH for rhizobial population is neutral to slighty acidic. Studies of Taurian et al. (1998)
also show that acidic soil negatively affected the rhizobial population.
In the present study minimum and maximum tolerated pH values recorded were 4
and 10, respectively. Cordovilla et al. (1994) and Rao and Sharma (1995) stated that
salinity is hazardous to agricultures and one of the major problem of land. During
present study it was reported that all the isolates grew very well up to 3% salt
concentration and maximum tolerated salt concentration was 6% by only two isolates.
Growth of none of the bacterial isolates on more than 6% salt concentration has also
been recorded by Kumar (2012).
In the present study all the endophytic bacterial isolates were tested for utilization of
carbon sources (such as monosaccharides, pentose, hexoses, disaccharides,
Discussion
Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of
Macrophomina phaseolina (Tassi) Goid. 99
trisaccharides, polysaccharides, organic compounds and sugar alcohols). Such findings
on utilization of viz., xylose, rhamnose, glycerol and mannitol were utilized by all the
isolates. None of the isolate was able to utilize L-arabinose, D-arabinose, sorbose,
melezitose, sodium gluconate, salicin, glucosamine, α-methyl-D glucosidase, α-methyl-
D mannosidase and dulcitol, have also been reported by Kumar (2010) and Kumar
(2012). Only Pseudomonas isolates were able to utilize mannose and inulin was
consumed by only Bacillus isolates. Our findings are in conformity with those reported
earlier by Fang et al. (2001), Idriss et al. (2002) and Kumar et al. (2010). All the
isolates were compared on the basis of carbon sources utilization by UPGMA analysis
and grouped into sixteen clusters. VR11 and VR13 were 95% identical and showed 92.4
% similarity with VR14. VR16 and Pseudomonas MTCC-129 were 88.7 % and 83 %
similar with VR19, respectively. The isolate VR2 was 85.7 % similar with standard
strain Bradyrhizobium sp. NAIMCC-B-00262 and showed 80.6% similarity with VR1
which was 73 % identical with VR4. Similarity establishment among bacterial isolates
on the basis of carbon utilization has also been recently reported by Kumar (2012).
Metabolic fingerprinting of the bacterial isolates VR1-VR6 was done by using
Biolog GN2. Some of the isolates utilized the common bio-chemicals present in the
wells of Biolog Kit (Fig. 11). On the basis of morphological, biochemical and
physiological characteristics and metabolic fingerprinting the bacterial isolates were
identified as Bradyrhizobium sp. strains VR1 to VR6 (Fig. 12). These similarities are
only due to utilization of some of the common bio-chemicals present in the wells.
The isolate VR1 showed 100% sequence similarity with Bradyrhizobium japonicum
EU333382 and Bradyrhizobium sp. NR042177, and isolate VR2 showed 100% 16S
rRNA gene sequence similarity with Bradyrhizobium sp. AB681396 and
Bradyrhizobium elkanii AB672634 (Fig. 15). Therefore, on the basis of 100%
similarity of the 16S rRNA gene sequence of isolate VR1 with Bradyrhizobium
japonicum EU333382 and Bradyrhizobium sp. NR042177, the isolate may be
designated as below:
Discussion
Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of
Macrophomina phaseolina (Tassi) Goid. 100
VR1 = Bradyrhizobium japonicum strain VR1
` VR2 = Bradyrhizobium sp. (Vigna) strain VR2.
Bacterial classification can be based on phenotypic and/or genotypic features.
Phenotyping is based on morphological, physiological or biochemical aspects and, in
the case of the family Rhizobiaceae, also on symbiotic compatibility with legume host
plants. Genotyping can be done by various methods including DNA (rRNA) nucleotide
sequence analysis. The official classification of the genus Bradyrhizobium, as presented
in Bergey’s Manual of Systematic Bacteriology (Jordan, 1984), considers only
phenotypic features and mol% G+C. Later, genotypic features were also described
(Elkan and Bunn, 1992). High rDNA similarity is a prerequisite for more closeness
within the two bacterial species (Oyaizu et al., 1993).
Koppell and Parker (2012) carried out phylogenetic clustering of Bradyrhizobium
symbionts on legumes indigenous to North America spanning at 48.5° of latitude
(Alaska to Panama). Phylogenetic relationships for nifD conflicted with a tree inferred
for five housekeeping gene loci. Within-region permutation tests also showed that
bacteria clustered significantly on particular host plant clades at all levels in the
phylogeny of legumes (from genus up to subfamily). Nevertheless, some bacterial
groups were dispersed across multiple regions and were associated with diverse legume
host lineages. These results indicate that migration and horizontal gene transfer, and
host interactions have all influenced the geographical divergence of Bradyrhizobium
populations on a continental scale.
B. PLANT GROWTH PROMOTING PROPERTIES IN ENDOPHYTIC
BACTERIA
Plant growth promoting attributes of all the endophytic bacterial isolates VR1 to
VR20 associated with both direct and indirect growth promotion were studied in vitro.
In present work all the isolates were tested for HCN production and observed that none
of the isolate was able to produce HCN. Antoun et al. (1998) have isolated 266 strains
Discussion
Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of
Macrophomina phaseolina (Tassi) Goid. 101
(nodule inducing bacteria) and examined that only three percent were cyanogens (HCN)
producers.
Direct growth promotiom mechanism of PGPR also involves the various effects on
the plants such as phytohormones production such as IAA, gibberellins, cytokinins etc.
IAA (indole-3- acetic acid) is the most common phytohormone which positively affects
plant growth. In the present investigation IAA production by all the isolates of Bacillus,
Bradyrhizobium and Pseudomonas have been recorded (Table 10). Produced of IAA by
species of Bradyrhizobium (Boiero et al., 2007), Bacillus (Singh et al., 2008b) and
Pseudomonas in the presence and absence of tryptophan (precursor) has also been
reported that involve several pathways. Patten and Glick (1996, 2002) have reported
that 80 % of the all rhizospheric bacteria produced IAA and Antoun Hani et al. (1998)
screened 266 strains of nodule inducing bacteria and found that 58% of this produced
indole 3-acetic acid (IAA).
In addition, Jangu et al. (2011) have found that various rhizospheric bacteria
improve the availability of nutrients and showed detrimental effect on plant pathogens
by producing hormones e.g. auxins and majority of the Pseudomonas mutants increased
the root growth of seedling in black gram (due to IAA production). Further they stated
that IAA produced by bacteria positively affected the plant growth and nodulation in
green gram (V. radiata) and black gram (V. mungo). IAA production in various strain of
B. japonicum has also been reported by Kiwamu et al. (1991) and Deshwal et al.
(2003). For the first time, Boiero et al. (2007) have reported IAA production by B.
japonicum in pure cultures using quantitative physicochemical methods. Kumar and
Dubey (2012) have also reviewed the plant growth promoting rhizobacteria for
biocontrol of phytopathogens and yield enhancement of Phaseolus vulgaris with special
reference to IAA production. Mandal et al. (2007a) have examined the influence of
endogenous root nodule’s phenolic acids (protocatechuic acid, 4-hydroxybenzaldehyde
and p-coumaric acid) on indole acetic acid (IAA) production by its symbiont
(Rhizobium) and reported that the phenolic acids present in the nodule might serve as a
stimulator for IAA production.
Discussion
Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of
Macrophomina phaseolina (Tassi) Goid. 102
Mishra and Kumar (2012) have investigated plant growth promoting and
phytostimulatory potential of Bacillus subtilis and B. amyloliquefaciens. They found
that malate followed by acetate was the most suitable sole carbon source for both the
IAA and siderophore production by the strains. Calvo et al. (2010) characterized
Bacillus isolates of potato rhizosphere for their potential PGPR characteristics and
found 81% of them as producer of auxin indole-3-acetic acid. Araujo et al. (2012)
studied the diversity and growth-promoting activities of Bacillus sp. in maize. They
found 40 isolates as auxin (IAA)-producers, phosphate solubilizers in vitro as well as
root colonizers, besides being as potential antagonists to plant pathogenic fungi.
Tryptophan-dependent production of indole-3-acetic acid (IAA) affecting level of
plant growth promotion by Bacillus amyloliquefaciens FZB42 has been reported by
Idris et al. (2007). They suggested that phytohormone-like acting compounds involved
in the phytostimulatory action are exerted by the plant-beneficial rhizobacterium B.
amyloliquefaciens FZB42. A five-fold increase in IAA secretion was registered in the
presence of 5 mM tryptophan. Prashanth and Mathivanan (2010) reported that B.
licheniformis MML2501 did not solubilise phosphate but produced indole acetic acid
(IAA) with a maximum of 23 μg/ml under optimised conditions such as pH 7.0,
temperature 35°C, tryphtophan at a concentration of 16 mM and at 200 rpm shaken
conditions.
Pseudomonas is the most abundant auxin producer microorganism. Growth
regulators especially IAA often affects the root systematic features such as root primary
growth, side-root formation and root hairs (Glick et al., 1995). Singh et al. (2010) have
found that ten strains of Pseudomonas aeruginosa (PN1 - PN10) isolated from
rhizosphere of chir-pine showed plant growth promontory properties in vitro, where P.
aeruginosa PN1 produced IAA. Khare and Arora (2010) have found that production of
indole-3-acetic acid (IAA) by rhizobacteria has been associated with plant growth
promotion, especially root initiation and elongation. They found the maximum
production of IAA by P. aeruginosa. Ahmad et al. (2005) have reported IAA
production by the indigenous isolates of Azotobacter and fluorescent Pseudomonas in
Discussion
Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of
Macrophomina phaseolina (Tassi) Goid. 103
the presence and absence of tryptophan. They quantitatively measured that production
of IAA by fluorescent Pseudomonas isolates increased with an increase in tryptophan
concentration from 1 to 5 mg/ml. In the presence of 5mg/ ml of tryptophan, they
recorded IAA production by 6 isolates in the range of 23.4 to 36.2 mg/ml. The
Rhizobium sp. isolated from the root nodules of common pulse plant Vigna mungo has
been found to provide the high levels of IAA to young and healthy root nodules
(Mandal et al., 2007a).
Pseudomonads are the most common genera of PGPR (Kloepper, 1993) which
control pathogens by production of antibiotics (Gutterson et al., 1988), HCN (Defago et
al., 1990), siderophores (Kloepper et al., 1980a), etc. and competition for space and
nutrients (Elad et al., 1987). The other endophytic bacteria such as Rhizobium,
Bradyrhizobium (Lalande et al., 1989, Deshwal et al., 2003, Mazen et al., 2008),
Bacillus etc. (Kumar et al., 2012b) have also been reported for PGP activities. These
bacteria carry out nitrogen fixation and provide several direct and indirect effects such
as phytohormone production, iron-chelation, phosphorous solubilization, hormone
production, HCN production, chitinase production, etc. (Deshwal et al., 2003).
Wahyudi et al. (2011) have also studied plant growth promoting activities of 118
isolates of Bacillus species from the rhizosphere of soybean plant. The principal
mechanisms of growth promotion include : production of growth stimulating
phytohormones, solubilization and mobilization of phosphate, siderophore production,
antibiosis (i.e., production of antibiotics), ethylene synthesis, and induction of plant
systemic resistance to pathogens (Gutierrez-Manero et al., 2001; Whipps 2001; Idris et
al., 2007; Richardson et al., 2009).
In the present work all the bacterial isolates (except VR10) showed phosphate
solubilisation property by forming clearing zone around the inoculation spot on the
Pikovskaya’s agar medium. Insoluble inorganic phosphate would have been solubilised
due to secretion of organic acids by all bacterial isolates resulting in formation of clear
zones (Fig. 3H).
Discussion
Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of
Macrophomina phaseolina (Tassi) Goid. 104
After nitrogen, phosphorous is a vital nutrient required by both plant as well as
microorganisms. Plant can take phosphorous from soil only in soluble form. Average
percentage of phosphorous in soil is about 0.05% (w/w), but only 0.1% of this is
available to plants (Scheffer and Schachtschabel, 1992; Illmer and Schinner, 1995).
Therefore, various chemical fertilizers containing phosphate are being used to
agricultural field due to non-availability of phosphorous to the plant. There are number
of endophytes which can make available this phosphorous to the plant by converting it
into the simple soluble forms. This improves and enhances the growth of both
leguminous and non-leguminous plants (Barea et al., 2005; Sridevi and Mallaiah,
2009). The most efficient phosphate solubilising microorganisms (PSM) belong to
genera Bacillus, Rhizobium, Bradyrhizobium and Pseudomonas amongst bacteria, and
Aspergillus and Penicillium amongst fungi (Kumar, 2012).
Wahyudi et al. (2011) isolated 118 isolates of Bacillus species from the rhizosphere
of soybean and examined the plant growth promoting activities. Among them 90
isolates (76.3%) positively produced the phytohormone, indole acetic acid (IAA). All
those 12 isolates produced siderophore and 11 isolates (91.7%) were able to solubilize
phosphate. Antoun et al. (1998) isolated 266 strains of nodule inducing bacteria and
stated that 54% were found to solubilise phosphorus. Idriss et al. (2002) have also
observed B. mucilaginous for its capacity in solubilizing phosphate. Thus PSM is good
inoculants for various crops of agricultural importance.
In the present investigation eight isolates (VR1, VR2, VR11, VR12, VR13, VR14,
VR15 and VR19) showed siderophore production (hydroxamate type) by forming
orange halo around the inoculation spot (Fig. 3E). Siderophore production by VR1,
VR2, VR11 and VR13 was quantitatively determined, and found that Bradyrhizobium
isolate VR2 produced the maximum quantity of siderophore (37 µg/ml) followed by
Bacillus isolates VR11, VR13 and B. japonicum strain VR1. Several workers have also
reported siderophore production by various endophytic bacteria such as Bradyrhizobium
sp. (Gupta et al., 2000; Deshwal et al., 2003), Bacillus sp. (Park et al., 2005; Wilson et
al., 2006; Kumar, 2012), Pseudomonas sp. (Bhatia et al., 2008; Kumar, 2012), Ensifer
Discussion
Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of
Macrophomina phaseolina (Tassi) Goid. 105
sinorhizobium (Dubey et al., 2010). Antoun et al. (1998) isolated 266 bacterial strains
out of which 83% of strains were found to produce siderophores.
Besides phosphorous, iron is also an essential element which is found in nature
copiously in the form of ferric iron (Fe III). It is soluble in nature and too low in
concentration to support microbial growth. Hence, to survive in such type of
environment microorganisms secretes Fe-binding ligands called ‘siderophores’, which
form complex with iron and made them available to plant root surfaces. Besides iron
uptake, proliferation of phytopathogens is also prevented, thereby facilitating the plant
growth (Kloepper et al., 1980b). Guerinot et al, (1990) have reported that levels of
hydroxamate-type siderophores in soil to be high (10 M) because majority of soil
microorganisms form siderophores containing hydroxamate ligands and this level
should be enough to support the growth of Bradyrhizobium sp. The ability to utilize
another organism's siderophores may grant a selective advantage in the rhizosphere
(Plessner et al., 1993).
Guerinot et al. (1990) found that out of 20 strains of B. japonicum, one strain (B.
japonicum 61A152), produced a siderophore which was determined to be citric acid. In
an experiment iron-deficient cells actively transported radiolabelled ferric citrate. These
results indicate a role for ferric citrate in the iron nutrition of this nitrogen-fixing
efficient strain on a variety of soybean cultivars.
Utilization of siderophores made by the other organisms is a sound strategy for iron
acquisition because siderophores are excreted into soil where they are freely available.
The majority of soil microorganisms form siderophores containing hydroxamate
ligands; levels of hydroxamate-type siderophores in soil have been reported to be as
high as 10M, which should be sufficient to support the growth of bradyrhizobia
(Guerinot, et al., 1990). Bradyrhizobium japonicum USDA 110 and 61A152 utilize the
hydroxamate-type siderophores ferrichrome and rhodotorulate, in addition to ferric
citrate, to overcome iron starvation. These strains can also utilize the pyoverdin-type
siderophore pseudobactin St3 (Plessner et al., 1993).
Discussion
Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of
Macrophomina phaseolina (Tassi) Goid. 106
Recently, Aeron et al. (2011) have shown the production of siderophore, IAA,
phosphate solubilization and biocontrol of M. phaseolina by Ensifer meliloti
RMP6Ery+Kan+
and Bradyrhizobium sp. BMP7Tet+Kan+
.
Iron is the fourth most abundant element in the earth’s crust but it is extremely
insoluble at neutral pH under aerobic conditions and is predominantly found as
precipitated, oxyhydroxide polymers. Several rhizobial and bradyrhizobial strains
release citric acid as a siderophore under iron-deficient growth conditions. They release
citric acid as a siderophore under conditions of iron-deficiency by rhizobial soybean
endosymbiont Bradyrhizobium japonicum strain 61A152 (Guerinot et al., 1990). The
ability to produce siderophores seems to be more widespread among rhizobial species
than among bradyrhizobial strains. Moreover, iron has been shown to be a pathogenicity
factor (Expert et al., 1996), so rhizobia must have mechanisms for accessing iron which
is generally unavailable to invading pathogens.
Among all the bacterial isolates only four isolates (VR1, VR2, VR11 and VR13)
produced a very important bacterial enzyme ACC deaminase in dual culture, resulting
in maximum growth inhibition by 50.5%, 71.5%, 78.6% and 60.2%, respectively.
Earlier Shah et al. (1997) have proposed that ACC deaminase-producing bacteria
increased root length by lowering the concentration of plant ethylene (Glick et al.,
1998). Many previous workers have also reported the production of ACC deaminase by
Rhizobium, Bradyrhizobium and other bacteria (Gupta et al. 2006, Kumar et al. 2010,
Dubey et al. 2012a), Pseudomonas putida GR 12-2 (Jacobson et al., 1994). Glick,
(2005) reported the significance of the role of ACC deaminase in the regulation of a
plant hormone, ethylene and enhancement of the growth and development of plants.
Remans et al. (2007) have also examined the potential of PGPR containing ACC
deaminase to enhance nodulation of common bean (P. vulgaris). Acinetobacter sp.,
Pseudomonas sp., Enterobacter sp., Micrococcus sp., and Bacillus sp. and some other
isolates have shown ACC deaminase activity (Kumar et al., 2012b).
Discussion
Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of
Macrophomina phaseolina (Tassi) Goid. 107
Govindasamy et al. (2011) have reviewed the ACC deaminase containing PGPR for
potential exploitation in agriculture. The enzyme ACC deaminase cleaves plant-
produced ACC, which is the immediate precursor of the stress hormone ethylene. ACC
deaminase containing PGPR act as a sink for ACC and protects the developing
seedlings from deleterious effects of stress ethylene that is synthesized during various
environmental stresses like phytopathogens, flooding, drought, salt, heavy metals,
organic contaminants, and high and low temperatures. ACC deaminase is a pyridoxyl 5′
phosphate-dependent enzyme and genes expressing this particular trait have been
isolated and characterized from a number of PGPRs of different genera. Several studies
have reported the potential exploitation of ACC deaminase containing PGPR in
improving the crop yields, improving shelf-life and quality of vegetables and
ornamental flowers, protecting crop plants against a range of abiotic and biotic stresses,
and phytoremediation of organic pollutants and heavy metal contamination in soils.
In the present study all the isolates were tested for chitinase activity; it was found
that only six isolates, two of Bradyrhizobium (VR1 and VR2), three of Bacillus (VR11,
VR12 and VR13) and one of Pseudomonas (VR15) produced chitinase on defined
medium and formed a clear zone around their respective colonies (Fig. 3 F; Table 10).
Chitinase is used to utilize the substrate chitin present in growth medium. Since fungal
mycelia also consist of chitin, chitinase is secreted by some of microorganisms to utilize
fungal chitin as substrate. Many previous workers have reported the production of
chitinase and β-1,3-glucanase by Bacillus cereus (Jian-Gang et al., 2008), Rhizobium,
Bradyrhizobium and other bacteria (Gupta et al. 2006, Mazen et al. 2008, Kumar et al.
2010, Dubey et al. 2012a). Chitinase production by Rhizobium leguminosarum isolates
TR1 and TR4 have also been recorded by Kumar et al. (2011b).
Development of several deformities in hyphae and sclerotia of M. phaseolina by
Bradyrhizobium strain VR2 in dual cultures such as fragmentation, shrinkage and lysis
of hyphae, cytoplasm vacuolation, loss of mycelial pigment, and inability of sclerotia
formation have recently been reported by Dubey et al. (2012a). Gupta et al. (2006) have
reported chitinase-mediated destructive antagonistic potential of Pseudomonas
Discussion
Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of
Macrophomina phaseolina (Tassi) Goid. 108
aeruginosa GRC1 against Sclerotinia sclerotiorum causing stem rot of peanut.
Morphological abnormalities, such as perforation, lysis and fragmentation of hyphae of
S. sclerotiorum caused by P. aeruginosa GRC1 were observed under scanning electron
microscopic (SEM) studies. This strain produced extracellular chitinase enzyme, the
role of which was clearly demonstrated through Tn5 mutagenesis. Morphological
deformity in mycelia of Sclerotinia sclerotiorum caused by Pseudomonas fluorescens
PS1as evident by hyphal perforation, fragmentation and lysis have also been observed
under scanning electron microscopy (Aeron et al., 2011).
Jian-Gang et al. (2008) have characterized chitinase secreted by Bacillus cereus
strain CH2 and evaluated its efficacy against Verticillium wilt of eggplant. The strain
secreted chitinase on chitin–Ayers (CA) medium. Germination of the fungal spores was
effectively suppressed by the bacterial suspension, supernatant from the suspension, and
0.005% solution of chitinase extracted from the strain CH2. Shali et al. (2010)
investigated the possible role of chitinase in in vitro growth inhibition of the wheat
pathogens Fusarium graminearum and Bipolaris sorokiniana by Bacillus pumilus SG2.
They found that a chitinolytic bacterium, B. pumilus SG2 produced two different
chitinases that had inhibitory activity against F. graminearum and B. sorokiniana.
Recently, Garg and Gupta (2010) have isolated and purified the M. phaseolina-
induced chitinase from moth beans (Phaseolus aconitifolius). They found that the
enzyme possibly generate the defense mechanism in non-host plants also. Chitinase was
purified by gel filtration chromatography in vitro and in vivo conditions.
C. BIOCONTROL POTENTIAL OF ENDOPHYTIC BACTERIAL ISOLATES
In the present investigation all the Bradyrhizobium isolates (VR1 to VR10) were
tested for their antagonistic activity and found that five isolates of Bradyrhizobium
(VR1, VR2, VR3, VR4 and VR5) showed antagonism and caused inhibition of fungal
growth (Fig. 18; Table 13). Among them Bradyrhizobium sp. (Vigna) strain VR2 was
more effective than B. japonicum strain VR1 which inhibited growth of the fungal
Discussion
Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of
Macrophomina phaseolina (Tassi) Goid. 109
pathogen by 71.5% and 50.5%, respectively. Many species of rhizobia promote plant
growth besides inhibiting the growth of certain pathogenic fungi (Lalande et al. 1989).
Antagonistic properties of many species of rhizobia against several pathogenic fungi,
such as M. phaseolina, Rhizoctonia solani, Fusarium oxysporum, Pythium spp., etc.
both in leguminous and nonleguminous plants have been reviewed by Dubey and
Upadhyaya (2001). In the present work metabolites of isolates B. japonicum VR1 and
Bradyrhizobium sp. strain VR2 was secreted in zone of interaction that caused several
deformities such as fragmentation, lysis, shrinkage, perforation and loss of pigment in
hyphae and sclerotia of M. phaseolina. Such abnormalities result in loss of fungal
viability. Similar post-interaction events have been reported in Fusarium udum caused
by Sinorhizobium fredii KCC5 (Kumar et al. 2010) and in M. phaseolina by
Bradyrhizobium AHR-2 (72% growth inhibition) (Deshwal et al., 2003). Strains of
Bradyrhizobium sp. and Rhizobium meliloti have been reported to be antagonistic
against M. phaseolina and to have plant growth promoting properties in urad (Dubey et
al., 2012a) and groundnut (Arora et al., 2001; Deshwal et al., 2003). Two fluorescent
pseudomonads strains PS1 and PS2 were isolated and selected for the antifungal activity
against M. phaseolina in vitro. Both the strains showed antagonistic activity against the
pathogen and inhibited its growth by 71% and 74%, respectively (Bhatia et al., 2008).
Biological control involves destruction of the propagating units of plant pathogens,
prevention of formation of surviving inocula, weakening of pathogen in infested
residues, reduction of pathogen’s vigour by antagonistic microorganisms. The disease is
controlled by various modes of action of antagonism and induced resistance or plant
growth promotion due to production of antimicrobial substances, such as chitinolytic
enzymes, laminarinase, cellulose, HCN, antibiotics, siderophore and nutrient
competition. Bacterial antagonism responsible for biological control may operate by
antibiosis, competition and/or parasitism. Parasitism relies on lytic enzymes for the
degradation of cell walls of pathogenic fungi (Chet et al., 1990). Several studies have
shown that the interaction between plants and some endophytic bacteria was associated
Discussion
Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of
Macrophomina phaseolina (Tassi) Goid. 110
with beneficial effects such as plant growth promotion and biocontrol potential against
plant pathogens (Chen et al., 1995; Hallman et al., 1995; Pleban et al., 1995).
Kumar and Dubey (2012) have also reviewed the PGP rhizobacteria for biocontrol
of phytopathogens and yield enhancement of Phaseolus vulgaris with special reference
to IAA production, phosphate solubilization, organic acid production, solubilization of
zinc and potassium, production of ACC deaminase, HCN, siderophore(s), oxalate-
oxidase enzyme, lytic enzyme, and nitrogen fixation. PGPR have the potential to
contribute in sustainable agricultural systems by functioning in three different ways: (i)
synthesizing particular compounds for the plants, (ii) facilitating the uptake of certain
nutrients from the soil, and (iii) preventing the plants from diseases (Deshwal et al.,
2003; Singh et al., 2008b, 2010).
Cell-free culture filtrates of above given strains have also been observed for fungal
growth inhibition. Percentage growth inhibition by cell-free culture filtrates of VR1
(37.6%), VR2 (49.2), VR11 (54.5%) and VR13 (53.4%) was much less than that of dual
culture. Very recently, Dubey et al. (2012a) have also reported the inhibitory effect of
culture filtrates of plant growth promoting Bradyrhizobium sp. (Vigna) strains VR1 and
VR2 on growth and sclerotia germination of Macrophomina phaseolina in vitro. They
found the complete inhibition in mycelial dry weight and sclerotia germination of M.
phaseolina at 45% concentration of culture filtrate of strain VR2.The inhibitory effect
may be due to the presence of toxins and/or cell wall lytic enzymes, or some other
inhibitory factors produced by these isolates in culture filtrates. Chakraborty and
Purkayastha (1984) examined the presence of a toxic substance in culture filtrate of R.
japonicum which was identified as rhizobiotoxin, which was responsible for inhibition
of growth M. phaseolina. Similarly Kelemu et al. (1995) also observed that
Bradyrhizobium or their cell-free culture filtrate negatively affects the mycelia growth,
sclerotial formation and germination of Rhizoctonia solani AG-1. Cell-free culture
filtrates of three strains of Bradyrhizobium also had inhibitory effects on the growth of
the bacteria Eschenchia coli DH5a and Xanthomonas campestris pv. phaseoli CIAT
555.
Discussion
Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of
Macrophomina phaseolina (Tassi) Goid. 111
Mazen et al. (2008) also recorded the cultural filtrates of the three wild rhizobial
isolates M. L (R1), L. C (R2), T. S (R3), R. leguminosarum ICARDA 441 (R4) strain
and arbuscular mycorrhiza (AM) fungi as potential biocontrol agent for control of
damping-off and root rot diseases of faba bean plants, when applied in individual or
combined treatments, under naturally infested soil with pathogenic fungi, Rhizoctonia
solani, Fusarium spp. and F. solani.
All the Bacillus isolates (VR11, VR12, VR13 and VR14) showed antagonism
against the test pathogen M. phaseolina. Among them VR11 (78.6%) was the more
potential in growth inhibition than the other isolates followed by VR13 (60.2%). Post-
interaction events showed that M. phaseolina gradually lost pigment which led to
hyaline fungal hyphae. The other abnormalities observed in fugal hyphae, mycelia and
sclerotia were hyphal shrinkage, cytoplasmic vacuolation, fragmentation, discolouration
of mycelia and sclerotia, etc. Kumar (2012) tested the potential of several Bacillus
strains and reported that Bacillus strain BPR7 was one of the strains which control the
several phytopathogens such as M. phaseolina, Fusarium oxysporum, F. solani,
Sclerotinia sclerotiorum, Rhizoctonia solani, and Colletotricum sp. in vitro. Singh et al.
(2008b) have also reported the potential of biocontrol in B. subtilis BN1 against M.
phaseolina. Similar findings of biocontrol by Bacillus have also been reported by
several other workers (Chung et al., 2008; Gajbhiye et al., 2010).
Cell-free culture filtrates of all the Bacillus isolates (VR11, VR12, VR13 and VR14)
showed antagonistic activity against M. phaseolina. But Bacillus sp. VR11 showed the
maximum growth inhibition (54.5%) followed by Bacillus sp. VR13 (53.4%). Similarly,
Kumar et al. (2012b) has also found the inhibitory effect of cell-free culture filtrate of
Bacillus strain BPR7 against several phytopathogens.
Among all the Pseudomonas strains (VR15, VR16, VR19 and VR20) only one
isolate (VR15) positively showed antagonism against fungal pathogen. Similarly
Siddiqui et al. (2002) studied the potential of P. aeroginosa IE-6S+
, P. fluorescence
CHA0 and B. japonicum 569Smr
singly or in combinations for biocontrol against
Discussion
Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of
Macrophomina phaseolina (Tassi) Goid. 112
multiple tomato pathogens such as M. phaseolina, F. solani and Rhizoctonia solani AG
8 and root-knot nematodes e.g. Meloidogyne javanica. Because of root knot nematode
(Meloidogyne spp.), root infecting fungi viz., M. Phaseolina, R. solani and Fusarium
spp. causes various disease comples in uradbean resulting serious losses in crop
(Ehteshamul-Haque, 1994; Ghaffar, 1995). Rhizobia are also known to reduce the soil-
borne root infecting fungi (Ehteshamul-Haque and Ghaffar, 1993; Siddiqui et al., 1998).
Earlier, the role of chitinase (E.C.3.2.1.14) and β-1,3-glucanase (E.C.3.2.1.39) produced
by fluorescent pseudomonads inhibiting the growth of Fusarium oxysporum, M.
phaseolina and Sclerotinia sclerotiorum in vitro have been reported by Gupta et al.
(2002, 2006). Many workers have also reported the potential of Pseudomonas species
against several pathogens such as Sclerotinia sclerotiorum by P. aeruginosa GRC1
(Gupta et al. 2006), M. phaseolina by Pseudomonas strains PS1 and PS2 (Bhatia et al.,
2008), Fusarium udum by Pseudomonas fluorescens LPK2 (Kumar et al. 2010) and M.
phaseolina by Azotobacter chroococcum AZO2 (Dubey et al., 2012b).
In the present investigation mycelia dry weight of M. phaseolina was inhibited more
by Bradyrhizobium (Vigna) strain VR2 followed by B. japonicum strain VR1 than the
other Bradyrhizobium strains (Table 4). The inhibitory effect may be due to the
presence of toxins and/or cell wall lytic enzymes, rhizobiotoxin, and secondary
metabolites including antibiotics, toxins, etc. produced in culture filtrates (Chao, 1990).
Inhibition of myclelial dry weight in control sets may be explained to be due to dilution
of nutrient medium that affected dry weight. Inhibitory effect of CFCF of
Bradyrhizobium strains VR1 and VR2 on mycelia yield was significantly (P >0.1) low
at 15% concentration than 30% and 45% concentrations. At 45% both the strains
showed complete inhibition of mycelia yield of M. phaseolina. The presence of toxin(s)
in culture filtrate of Bradyrhizobium cannot be ruled out (Deshwal et al., 2003). The
inhibitory properties of rhizobial culture filtrate containing rhizobitoxin have also been
reported by Chakraborty and Purkayastha (1984). Rhizobitoxin is an important
compound involved in symbiosis between rhizobia and legumes that enhances
nodulation and competitiveness of Bradyrhizobium elkanii on a legume host (Yuhashi
Discussion
Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of
Macrophomina phaseolina (Tassi) Goid. 113
et al., 2000). Chitinolysis plays an important role in biological control of plant diseases
and has been substantiated with increased disease control by chitin supplemented
application of chitinolytic biocontrol agents. Kumar (2012) has also observed that cell-
free culture filtrates of R. leguminosarum RPN5 showed significant inhibition of M.
phaseolina, F. oxysporum, F. solani, S. sclerotiorum, R. solani, and Colletotrichum sp.
due to the production of siderophores (Loper and Buyer, 1991), antibiotics (Homma et
al., 1989), hydrolytic enzymes such as chitinases and β-1,3-glucanases (Fridlender et
al., 1993) and the other secondary metabolites like hydrocyanic acid (HCN) (Bagnasco
et al., 1998) and induced systemic resistance (Liu et al., 1995).
Different concentrations of CFCF (15%, 30% and 45%) of Bacillus sp. strains
VR11, VR12, VR13 and VR14 inhibited the mycelia yield of M. phaseolina. Among
them CFCF of Bacillus sp. VR11 was found to be most effective for inhibition of
mycelia yield followed by VR13. At 45% concentration of CFCF of Bacillus sp. VR11
and VR13 the complete inhibition of growth of mycelia yield was recorded. Podile and
Dubey (1985) have reported the effect of concentrated cell-free culture filtrate of
Bacillus subtilis on growth of vascular wilt fungi such as Verticillium albo-atrum, V.
dahlia, Fusarium udum, F. oxysporum f. sp. lycopersicae, F. oxysporum f. sp.
vasinfectum and Ceratocyctis ulmi. They found the growth inhibition of all test fungi at
> 10% concentration of CFCF. Moreover, no fungal pathogen could growth im 5-fold
concentrated extract at 40% concentration. Singh et al. (2008b) also reported the
deleterious effect of cell-free culture filtrate of B. subtilis BN1 on the growth of M.
phaseolina in pine seedlings. Kumar (2012) has also observed that cell free culture
filtrates (CFCF) of Bacillus strain BPR7 showed significant inhibition of M. phaseolina.
Mishra et al. (2011) have reported that 4 to 6 days old culture filtrate of Bacillus subtilis
isolate MA-2 completely inhibited the growth of Alternaria alternata and Curvularia
andropogonis causing leaf blight of geranium (Pelargonium graveolans) and that of
Java citronella (Cymbopogon winterianus), respectively.
Different concentrations of culture filtrates of Bradyrhizobium strains VR1, VR2,
VR3, VR4, VR5 and VR6 were tested for their inhibitor effect on sclerotia germination
Discussion
Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of
Macrophomina phaseolina (Tassi) Goid. 114
and hyphal development (Tables 16-17). It was observed that Bradyrhizobium strains
VR1 and VR2 effectively inhibited sclerotia germination of M. phaseolina and caused
complete inhibition at 45% up to 48 h. This effect has been explained to be due to the
presence of inhibitory factors (toxins and/or cell wall lytic enzymes) in culture filtrates
that would have caused inhibition of sclerotia germination (Dubey et al., 2012a).
Microsclerotia are made up of mycelia network, the cells of which are tightly cemented.
Individual cell acts as a unit and all of them show germination; this is why a sclerotium
produces many hyphae emerging from it (Fig. 27 A-D)). Only the viable cells of a
sclerotium germinate and immediately produce secondary sclerotia required for its
survival (B). This is why number of hypha emerging from a sclerotium was more in
control (A) than culture filtrate-treated sclerotia (C). Such results on sclerotia
germination producing varying number of hyphae have recently been reported by
Dubey et al. (2012a).
Kelemu et al. (1995) have found the inhibitory effects of Bradyrhizobium strains or
their cell-free culture filtrates on mycelial growth, sclerotial formation, and sclerotial
germination of Rhizoctonia solani AG-1, a pathogen of tropical forage legumes.
Besides, cell-free culture filtrates of three Bradyrhizobium strains had inhibitory effects
on the growth of the other bacteria such as Escherichia coli DH5α and Xanthomonas
campestris pv. phaseoli CIAT 555. Das et al. (2008) have reported that 20%
concentration of the cell-free culture filtrates of fluorescent pseudomonads strains
significantly reduced the formation and germination of microsclerotia of M. phaseolina.
Role of chitinase in control of Sclerotium rolfsii and Rhizoctonia solani by Serratia
marcescens (Chet et al. 1990), and Sclerotinia sclerotiorum by Pseudomonas
aeruginosa GRC1 (Gupta et al. 2006) associated with certain plant diseases has been
reported. Chet et al. (1990) have found that S. marcescens releases N-acetyl D-
glucosamine from cell walls of S. rolfsii due to presence its chitinolytic activity. Use of
antagonistic Bradyrhizobium strains has dual advantage as compared to the other
biocontrol agents as the former assimilate atmospheric nitrogen besides killing
deleterious phytopathogens. The presence of inhibitory properties in culture filtrates of
Discussion
Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of
Macrophomina phaseolina (Tassi) Goid. 115
Bradyrhizobium strains helps to act as potential biocontrol agent for control of M.
phaseolina (Deshwal et al., 2003).
Similarly, the cell-free culture filtrates of Bacillus strains VR11, VR12, VR13 and
VR14 were tested for their effect on sclerotia germination and hyphal development
(Tables 18-19). It has been observed that Bacillus strains VR11 and VR13 effectively
inhibited sclerotia germination of M. phaseolina and showed complete inhibition at
45% concentration. Inhibition in scelrotia germination may be explained to be due to
presence of inhibitory factors such as toxins and/or cell wall lytic enzymes in culture
filtrates. Therefore, in control the numbers of sclerotia producing >7 hyphae were more
than the culture filtrate-treated sclerotia. Complete inhibition of hyphal development
was recorded at 45% concentration of culture filtrate.
Ahmed et al. (2009) evaluating the effect of different concentrations of Bacillus
subtilis culture filtrates on the linear growth and spore germination of Fusarium
oxysporum. They found that 50% concentration of filtrates of all the Bacillus isolates
No1 to No. 4 completely inhabited spore germination of F. oxysporum. Culture filtrates
of Bacillus subtilis No.2 and Bacillus spp. No.2 also were more effective in reducing
the mycelial growth of F. oxysporum by 80.74 and 80.37 %, respectively. On the other
hand Bacillus subtilis No.1 and Bacillus spp. No. 4 made lysis to mycelia of F.
oxysporum. Generally linear growth and spore germination were decreased by
increasing the concentrations of culture filtrate from 10% to 50%.
D. EFFECT OF BACTERIAL CONSORTIUM ON GROWTH AND YIELD OF
Vigna mungo IN POT TRIALS
In the present study all the sixteen bacterial isolates (VR1 to VR20) were tested for
synergistic or antagonistic effect between each other (Table 20). It was found that
Bradyrhizobium (Vigna) strains VR1 and VR2, and Bacillus strains VR11 and VR13
displayed synergistic interaction. Pseudomonas strains VR19 and VR20 also showed
synergism with each other. Some similarities such as common physiochemical
Discussion
Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of
Macrophomina phaseolina (Tassi) Goid. 116
properties, carbon utilization, etc. were the reasons of synergistic interaction among
them. They would have been sharing some of identical features of PGP properties such
as IAA production, absence of HCN production, hydroxamate type of siderophore,
chitinase production, etc. Similarly, Positive interaction between Rhizobium and
Pseudomonas sp. LG or Bacillus sp. has been obtained by Stajkovic et al. (2011).
Kumar (2012) have also reported the positive interaction among R. leguminosarum
RPN5, B. subtilis BPR7 and Pseudomonas sp. PPR8, and stated that they were
successfully grown as mixed cultures.
In the present work it was observed that growth of Bacillus sp. VR11 increased after
amending the CFCF of Bradyrhizobium sp. (Vigna) strain VR2 as compared to control
(Table 21). This shows that the culture filtrate of Bradyrhizobium sp. (Vigna) strain
VR2 synergistically affected the growth of Bacillus sp. VR11. Samavat et al. (2011)
conducted a greenhouse experiment to evaluate the potential of the two Pseudomonas
fluorescens isolates UTPF68 and UTPF109 in the biocontrol of bean damping-off
caused by Rhizoctonia solani (AG-4), when applied individually or in combination with
the culture filtrates of five rhizobia isolates (RH3 to RH7). They found that certain
rhizobia had a capacity to interact synergistically with P. fluorescens isolates having
potential biocontrol activity. Moreover, El-Batanony et al. (2007) found that the cultural
filtrates of Rhizobium leguminosarum showed potential synergetic activity with
arbuscular mycorrhizal (AM) fungi in the biocontrol of R. solani, Fusarium solani, and
F. oxysporum of faba bean.
Bacillus licheniformis MML2501 isolated from groundnut rhizosphere soil showed
the increased populations on spermozphere colonisation and significantly increased the
seed germination and other growth parameters in groundnut under in vitro conditions.
B. licheniformis MML2501 produced 23 μg/ml IAA under optimised conditions, such
as 7.0 pH, 35°C temperature, 16 mM tryphtophan and at 200 rpm shaken conditions.
Seed treatment of B. licheniformis MML2501 in groundnut showed a significant
increase in seed germination, other growth parameters and yield parameters under
potted plant experiments (Prashanth and Mathivanan, 2010).
Discussion
Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of
Macrophomina phaseolina (Tassi) Goid. 117
Wahyudi et al. (2011) found that the 12 isolates (13.3% of total) of Bacillus
species isolated from the rhizosphere of soybean plant significantly enhanced seed
germinatin, root length, shoot length and the number of lateral root of the seedling.
Furthermore, 3 isolates (25%) among them were able to inhibit the growth of Fusarium
oxysporum, 9 isolates (75%) inhibited the growth of Rhizoctonia solani, and 1 isolate
(8.3 %) of Bacillus sp. inhibited the growth of Sclerotium rolfsii.
Similarly, Srinivasan et al. (1997) reported that co-inoculation of Bacillus sp. with
Rhizobium etli led to increase in nodulation in common bean. Many other researchers
have also reported the effect of co-inoculation of two bacteria upon one another. Co-
inoculation of Bradyrhizobium with P. striata resulted in an enhanced biological
nitrogen fixation in soybean (Dubey, 1996); moreover, co-inoculation of
Bradyrhizobium with Bacillus enhanced soybean growth and nodulation (Bai et al.,
2003).
The intrinsic antibiotic resistance markers bacteria were used to monitor root
colonization of V. mungo. Significantly, this method is used due to simplicity,
uncomplicated, non-pricey and time-saving (Howieson et al., 2000; Dey et al., 2004;
Spriggs and Dakora, 2009; Yasmin et al., 2009). In the present study B. japonicum
strain VR1, Bradyrhizobium sp. (Vigna) strain VR2 and Bacillus sp. VR11 were
screened for antibiotic sensitivity against number of antibiotics (Table 22; Fig. 32). It
was found that B. japonicum strain VR1, Bradyrhizobium sp. (Vigna) VR2, Bacillus sp.
VR11 and Bacillus sp. VR13 showed resistance against number of antibiotics.
Bradyrhizobium sp. (Vigna) strain VR2 was used to develop the highest level of
tolerance (100 µg ml-1
) to the nalidixic acid. Further this marker strain was used for
study of seed bacterisation and root colonisation at different intervals. Use of antibiotic
marker strains for monitoring their propagation, population increase, and root
colonisation has also been done by several researchers (Gaur, 2001; Gupta et al., 2002;
Obaton et al., 2002; Bhatia et al., 2005).
Discussion
Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of
Macrophomina phaseolina (Tassi) Goid. 118
Obaton et al. (2002) found that the rhizobial marker strains remained resistant to
antibiotics even after twenty years of inoculation. Similarly, several other workers have
also reported the development of antibiotic resistant marker strains against number of
antibiotics and their use in root colonization study (Deshwal et al., 2003; Bhatia et al.,
2008; Kumar, 2010; Kumar, 2012).
Seeds bacterised with B. japonicum strain VR1, Bradyrhizobium sp. (Vigna) strain
VR2 and Bacillus sp. VR11, either singly, twin or three bacterial consortia with or
without M. phaseolina enhanced the seed germination and other vegetative parameters
such as root and shoot length, their dry weight and nodule numbers 30 DAS as
compared to control (Table 25). Bacterial consortium resulted in enhanced seed
germination and seedling emergence in all the treatments after 30 DAS as compared to
control. In T5 (VR1 + VR2+ VR11 + M. phaseolina) increased root length, shoot length
and nodule number were recorded as compared to control and M. phaseolina-infested
soil, 30 and 60 DAS. The growth promoting effect of the strain VR2 was followed by
the strains VR11 and VR1 (Tables 25-26; Figs. 35-36). T5 [consortium A- (VR1+
VR2+ VR11) + M. phaseolina] was the best in providing the satisfactory response 30
and 60 DAS. Thus Bradyrhizobium sp. (Vigna) strain VR2 (singly and in consortia)
enhanced the maximum seed germination, plant growth and yield, and nodule number.
Vigour index was calculated by multiplying the germination percentage with total
length of plant which increased with seed viability. Maximum vigour index was shown
by T5 (Consortium A) (36.5) followed by T2 (VR2) (33.3), T4 (VR11) (32.2) and T8
(Consortium D) (31.7) (Tables 24 -25). Similar findings have also been reported by
Deshwal et al. (2003). They found that seed bacterisation with Bradyrhizobium strains
positively affected seed germination, seedling biomass, nodule number and weight
compared to control. Many other workers have also used seed bacterization to improve
growth factors and biological control of pathogen (Gupta et al., 2006; Singh et al.
2008b; Samavat et al., 2011).
Xiao et al. (1990) isolated pseudomonads from cotton plants and found their
inhibitory effect on seedling diseases. Bhatia et al. (2008) reported that the fluorescent
Discussion
Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of
Macrophomina phaseolina (Tassi) Goid. 119
pseudomonads PS1 and PS2 used as bioinoculants increased crop yield by 66% and
77%, respectively and enhanced seed germination up to 15% and 30% with prevention
of fungal pathogen M. phaseolina causing charcoal rot disease. Seed treatment with two
isolates of Pseudomonas fluorescence (singly or in combination), mainly RH4
+UTPF107 and RH6+UTPF68 improved growth factors (root, shoot dry/fresh weights)
of bean (Samavat et al., 2011).
The cooperative interactions between rhizobia and other plant root colonizing
bacteria play a role in the improvement in nodulation and N2 fixation in legume plants
(Barea et al., 2005); other such examples include when rhizobia are co-inoculated with
Rhizobium leguminosarum bv trifolii and either B. insolitus or B. brevis (Sturz et al.,
1997), and with Bacillus spp. and the soybean endosymbiont Bradyrhizobium
japonicum (Liu et al., 1993; Bai et al., 2003). Geetha et al. (2008) reported that co-
inoculation enhanced growth and nodulation of the pigeon pea with Bacillus strains and
Rhizobium spp. Likewise, Selvakumar et al. (2008) have shown that the non-rhizobial
plant growth promoting bacteria Bacillus thuringiensis KR-1 from the nodules of
Kudzu promoted growth and positively influenced nutrient uptake in wheat seedlings.
Therefore, this report extends similar observations to another legume-rhizobium system
that of Sophora alopecuroides.
Deshwal et al. (2003) have found that the population of Bradyrhizobium strains
AHR-2, AHR-5 and AHR-6 increased the nodule weight seven-fold higher than the
control, and seeds coated with Bradyrhizobium strains AHR-2, AHR-5 and AHR-6 also
enhanced nodule fresh weight more than six-fold when sown in M. phaseolina- infested
soil. Moreover, presence of the microbial antagonists (Bradyrhizobium sp.) has shown a
significant positive effect on plant growth by reducing the colonization of sunflower
and mungbean roots by Sclerotium rolfsii. Use of biocontrol agents in S. rolfsii-infested
soil have shown a significant reduction in Root Colonization Index (RCI) accompanied
by increase in plant growth (Yaqub and Shahzad, 2011).
Discussion
Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of
Macrophomina phaseolina (Tassi) Goid. 120
Egamberdiyeva et al. (2004) studied the effect of inoculation of B. japonicum
S2492 on soybean growth, nodulation and yield in nitrogen-deficient soil of Uzbekistan.
They found 48% higher yield of soybean varieties in inoculated than un-inoculated
plants. The application of Bradyrhizobiurn is found to be very useful in soybean
cultivation for producing high yield as well as for keeping the soil fertile for the
succeeding crops. Several different ways of application of Bradyrhizobiurn for
enhancing the production and productivity of soybean crop had been work out.
Inoculation of seeds with Bradyrhizobiurn culture gave significantly taller plants with
more nodules, pods/plants, grains/pod and seed weight than untreated seeds; yield of
soybean increased due to inoculation (Singh, 2005).
Bhuiyan et al. (2008) found that the application of Bradyrhizobium inoculant
produced significant effect on seed and stover yields in both trials conducted in two
consecutive years. Seed inoculation significantly increased seed and stover yields of
mungbean as compared to uninoculated control. Bradyrhizobium inoculation also
significantly increased pods/plant, seeds/pod and seed weight. Inoculated variety BARI
Mung-2 produced the highest seed and stover yields as well as yields attribute, such as
pods/plant and seeds/pod. Application of B. japonicum strain USDA110 and
Pseudomonas sp. strain P18 liquid inoculants on soybean seed before sowing plus 20 kg
N/ha has been found to enhance the nodule number, fresh weight, dry weight of
nodules, yield components and grain yield in comparison to conventional farmers’
fertilizer level Son et al. (2007).
The maximum disease reduction was recorded in T5 having consortium
VR1fur+
+VR2nal+
+ VR11nor+
followed by T8 (consortium VR2+VR11), T6
(VR1fur+
+VR2 nal+
) and T7 (VR1fur+
+ VR11nor+
) 30 and 60 DAS when compared with
control. In T5, 68.7% disease reduction was recorded 60 DAS; but in T8, 51.2%
reduction 60 DAS (Table 26; Fig. 37).
Bhatia et al. (2008) also found reduction in plant disease by Pseudomonas strains
PSI and PSII in peanut. Siddiqui and Husain (1992) examined the effect of Meloidogyne
Discussion
Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of
Macrophomina phaseolina (Tassi) Goid. 121
ineognica race 3, M. phaseolina and Bradyrhizobium sp. on root-rot disease complex of
chickpea (Cicer arietinum). They found that inoculation of Bradyrhizobium 10 days
prior to pathogens resulted in reduced damage. Inoculation of pathogens prior to
Bradyrhizobium resulted in more damage than prior or simultaneous inoculation of
Bradyrhizobium.
Inoculation of seeds with P. fluorescens strains demonstrated a drastic decline in
charcoal rot incidence in Vigna radiata (mungbean) by 84.7% (treatment Burkholderia
cepacia BAM-12+ P. fluorescens BAM-4) in M. phaseolina-infested soil, and 99% in
non-infested soil as compared to non-bacterized seeds raised in M. phaseolina-infested
soil where the disease was 97.25%. Maximum suppression of disease incidence (84.7%
over control) was achieved upon application of two bacterial strains together (Minaxi
and Saxena, 2010).
E. ROOT COLONISATION BY MARKER STRAINS VR1 VR2 nal+
Root colonization was first used by Kloepper et al. (1980b) to describe the recovery
of antibiotic resistant PGPR strains from external surfaces of potato roots following
application to seed pieces. Kloepper et al. (1992) reviewed the issues related to
measuring colonization of plant roots by bacteria. They suggested that the root
colonization should be done in competitive conditions, i.e. natural field soils by
applying different methods including spontaneous antibiotic resistance marking
systems. They defined that root colonization is an active process involving growth of
the introduced bacteria on or around roots, and is not simply a passive chance encounter
of a soil bacterium with a passing root. In the present study Bradyrhizobium sp. (Vigna)
strain VR2nal+
effectively colonised the uradbean root either present singly or in the
form of consortia (Table 27, Fig. 38). Successful root colonisation by Bradyrhizobium
sp. (Vigna) strain VR2nal+
or any bacteria is the first requirement for the enhanced plant
growth and yield and protection from soil-borne diseases by PGPR-mediated
siderophore production; the HCN, lytic enzymes and antibiotics suppressed fungal
Discussion
Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of
Macrophomina phaseolina (Tassi) Goid. 122
pathogens. A population of 6.87 log cfu and 7.4 log cfu g-1
of Bradyrhizobium VR2nal+
was recorded in T5 when co-inoculated with strain VR1fur+
+ Bacillus sp. VR11nor+
and
M. phaseolina. Similarly, 6.57 and 7.14 log cfu g-1
of Bacillus sp. VR11nor+
was
recorded 30 and 60 DAS, respectively. The optimum level of root colonisation should
be attained to reach about 105-10
6 cfu g
-1 of root for the protection of the soil-borne
diseases (Bull et al., 1991; Raaijmakers et al., 1999; Haas and Keel, 2003; Zaidi et al.,
2005). Root colonization by rhizobacteria has been found to be enhanced due to the
secretion of root exudates (Chandra et al., 2007) leading to excess siderophore
production and other compounds involved in biocontrol of phytopathogens (Bais et al.,
2006).
Population of B. japonicum strain VR1fur+
, Bradyrhizobium sp. (Vigna) strain
VR2nal+
and Bacillus sp. VR11
nor+ was always increasing continuously from 30 to 60
DAS but the population of strain VR2 was higher that of the other two strains,
VR1fur+
and VR11nor+
. Root colonisation by the strain VR2nal+
even in the presence of M.
phaseolina showed as a good root coloniser and aggressive biocontrol agent resulting in
plant growth enhancement and plant protection. Bradyrhizobium sp. (Vigna) strain
VR2nal+
would have inhibited the growth of M. phaseolina possibly through the
production of antifungal metabolites and the other lytic enzymes. There are several
workers who have reported the successful root colonisation by endophytic bacteria.
Siddiqui et al. (2002) found the effective root colonisation of tomato plant by
Pseudomonas fluorescens strain IE-6S+
than CHA0 and Bradyrhizobium japonicum
569Smr
. Pseudomonas fluorescens strain IE-6S+
successfully colonised the root when
used singly or with either IE-6S+
and/or 569Smr
. IE-6S+
was the only bacterium that
colonized inner root tissues of tomato plants.
Bacterization of peanut seeds with Pseudomonas aeruginosa GRC1 resulted in
increased seed germination and reduced stem-rot of peanut by 97% in S. sclerotiorum-
infested soil. Other vegetative and yield plant parameters such as nodules per plant,
pods and grain yield per plant were enhanced with a statistical significance in
comparison to control. Neomycin resistant (GRC1neo+
) bacterium was a good root
Discussion
Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of
Macrophomina phaseolina (Tassi) Goid. 123
colonizer and frequently isolated from rhizosphere of peanut plants. These findings
showed P. aeruginosa GRC1 as a potential biocontrol agent against S. sclerotiorum
(Gupta et al., 2006).
Singh et al. (2008b) proved B. subtilis BN1 as a good root coloniser and potential
biocontrol agent. Similarly Bhatia et al. (2008) also reported the fluorescent
Pseudomonas strains PS1 and PS2 as a good root coloniser and potential biocontrol
agent. The present work in in agreement with those of Singh et al. (2010) who found P.
aeruginosa strain PN1rif+strep+
as a good colonizer of chir-pine roots either singly or
combination with M. phaseolina.
Arora et al. (2001) have found that both Rhizobium meliloti isolates RMP3 and
RMP5 maintained high cfu g–1
root segments up to 60 days in the presence of M.
phaseolina, which was marginally lower than the population of both the isolates in soil
without fungal infestation. The population density of RMP5 was slightly higher than
that of RMP3 both in infested and non-infested soils. Both the strains strongly inhibited
the M. phaseolina population in the rhizosphere. The population of the pathogenic
fungus declined after 60 days due to bacterization by RMP3 and RMP5. Besides, co-
inoculation with Bacillus cereus MQ23+MQ23II has been found to have a more
significant effect on Sophora alopecuroides than alone inoculation in vitro for most of
the positive actions suggesting that they have a cooperative interaction. Results of plant
inoculation with endophytes indicated that the growth indexes of co-inoculated
MQ23+MQ23II were higher than those of inoculated alone (p < 0.05) (except root fresh
weight) when compared to negative control. Moreover, Bacillus cereus MQ23 was
shown to be able to produce siderophores, IAA, and certain antifungal activity to plant
pathogenic fungi (Zhao et al. (2011).
It is interesting to note that the living fungal propagules such as mycelia, conidia
sclerotia, etc. act as attractants for motile bacteria in soil, because the motile bacteria
consume fungal exudate as nutrients, and thus their propagules offer a niche for these
bacteria in soil. Singh and Arora (2001) found that Pseudomonas fluorescens strains
Discussion
Characterization of endophytic rhizobacteria from Vigna mungo (L.) Hepper and their role in biocontrol of
Macrophomina phaseolina (Tassi) Goid. 124
(LAM1-hydrophilic) and (LAM2-hydrophobic) showed positive chemotaxis towards
attractants (sugars, amino acids, polyols and organic acids) present in the exudate of M.
phaseolina. The varied response of motility traits such as speed, rate of change in
direction and net to gross displacement ratio (NGDR) was observed for different
chemo-attractants. Swimming speed of the strains was highest in 10-fold diluted
exudate or 100–1000 μM strength of different attractants, but further dilutions
significantly decreased the swimming speed. The results suggest that M. phaseolina
exudate contains chemical attractants that serve as signal for flagellar motility of P.
fluorescens. Motile P. fluorescens strains thus may consume fungal exudate as
nutrients. Bacterial attraction towards substances exuded by sclerotia of Macrophomina
phaseolina by Erwinia herbicola, Pseudomonas fluorescens, and P. putida in vitro has
been reported by Arora et al. (1983).
These studies lend supports that the bacterial marker strains besides colonizing the
plant roots also colonise the surfaces of fungal propagules and antagonize them
resulting in their lysis and death.
In the present study the IAA-producing, phosphate-solubilizing, siderophore-, ACC
deminase- and chitinase- producing Bradyrhizobium sp. strains VR1 and VR2, and
Bacillus sp. VR11 are not only a good and aggressive root colonizer but also have a
strong antagonistic activity against M. phaseolina, resulting in increased seed
germination, vegetative growth and nodulation. The use of antagonistic Bradyrhizobia
strains (VR1 and VR2) and Bacillus sp. VR11 has dual advantage when compared to
the other biocontrol agents as they assimilate atmospheric nitrogen besides killing the
deleterious pathogens. Based on the results of present investigation it may be concluded
that these strains are not only found to be a good root colonizers but also possess a
strong antagonistic activity against M. phaseolina and enhanced vegetative parameters
of V. mungo. The presence of inhibitory properties in culture filtrates of B. japonicum
strain VR1, Bradyrhizobium sp. (Vigna) strain VR2 and Bacillus sp. VR11 help to act
as potential biocontrol agent for control of M. phaseolina causing charcoal rot of V.
mungo.