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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



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



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



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



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



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



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



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



(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



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



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



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



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



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



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



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



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



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



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



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



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



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



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



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



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



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



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



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



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



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



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



(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.

discussion - inflibnetshodhganga.inflibnet.ac.in/bitstream/10603/8437/10/10... · 2015-12-04 ·...

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