ORIGINAL PAPER

Multifarious plant growth promoting characteristics of chickpearhizosphere associated Bacilli help to suppress soil-bornepathogens

Rajesh Kumar Singh • D. Praveen Kumar • Pratiksha Singh •

Manoj Kumar Solanki • Supriya Srivastava • Prem Lal Kashyap •

Sudheer Kumar • Alok K. Srivastava • Pradeep K. Singhal •

Dilip K. Arora

Received: 6 April 2013 / Accepted: 26 October 2013

� Springer Science+Business Media Dordrecht 2013

Abstract Wilt and root rot are the major constraints in

chickpea production and very difficult to manage through

agrochemicals. Hence, for an ecofriendly and biological

management, 240 strains of Bacillus and Bacillus derived

genera were isolated from chickpea rhizosphere, further nar-

rowed down to 14 strains on the basis of in vitro production of

indole acetic acid, siderophore, phosphate solubilization,

hydrolytic enzymes and were evaluated for antagonism against

chickpea pathogens (Fusarium oxysporum f. sp. ciceri race 1,

F. solani and Macrophomina phaseolina). The strains were

identified on the basis of physiological characters and 16S

RNA gene sequencing. The genotypic comparisons of strains

were determined by BOX-polymerase chain reaction profiles

and amplified rDNA restriction analysis. These isolates were

evaluated in greenhouse assay in which B. subtilis (B-CM191,

B-CV235, B-CL-122) proved to be effective in reducing wilt

incidence and significant enhancement in growth (root and

shoot length) and dry matter of chickpea plants. PCR ampli-

fication of bacillomycin (bmyB) and b-glucanase genes sug-

gests that amplified genes from the Bacillus could have a role to

further define the diversity, ecology, and biocontrol activities

in the suppression of soil-borne pathogens.

Keywords Bacillus � Chickpea � Fusarium wilt �Genetic fingerprinting � Plant growth promoting

rhizobacteria

Introduction

The increasing global population creates significant con-

cern over the agricultural production to cope with

requirement of sufficient food for all. In this context,

management of diseases in cereal and pulse crops is vital to

alleviate food shortages and to improve efficiency in food

production. Among pulses, chickpea (Cicer arietinum L.)

is the world’s third most important crop and India con-

tributing approximately 75 % to world’s chickpea pro-

duction (Nikam et al. 2007). Chickpea productivity,

however, remained virtually stagnant from past few dec-

ades because of its susceptibility to diseases such as wilt

(Fusarium oxysporum f. sp. ciceri, FOC), black root rot (F.

solani, FS) and charcoal rot (Macrophomina phaseolina,

MP). The synergistic interaction of these pathogens can

cause more than 80 % yield losses, if proper and timely

measures are not taken to manage these pathogens (Pandey

et al. 2007). Moreover, farmers heavily rely on fungicides

to manage these soil-borne pathogens. Because of concerns

regarding both human health and environment, viable

alternatives to these chemicals are being sought (Franks

et al. 2006). It has been recognized that a large number of

naturally occurring rhizospheric bacteria are antagonistic

towards plant pathogens and as a result may offer a viable

substitute for abandoning the use of these chemicals to

enhance crop growth and yield.

Plant rhizosphere is a versatile and dynamic ecological

environment of intense plant–microbe interactions for

harnessing essential micro-and macro-nutrients from a

Electronic supplementary material The online version of thisarticle (doi:10.1007/s10725-013-9870-z) contains supplementarymaterial, which is available to authorized users.

R. K. Singh � D. P. Kumar � P. Singh � M. K. Solanki �S. Srivastava � P. L. Kashyap � S. Kumar (&) �A. K. Srivastava � D. K. Arora

National Bureau of Agriculturally Important Microorganisms,

Mau 275101, Uttar Pradesh, India

e-mail: sudheer.nbaim@gmail.com

P. K. Singhal

Department of Biological Science, Rani Durgavati University,

Jabalpur 482001, Madhya Pradesh, India

123

Plant Growth Regul

DOI 10.1007/s10725-013-9870-z

limited nutrient pool (Solanki et al. 2012a). We focused on

bacterial genera that are often found in large populations in

soils with general disease suppression, such as gram posi-

tive spore-forming species belonging to Bacillus and

Bacillus derived genera. Bacillus has many characteristics

as an excellent biocontrol agent, including the production

of diverse antibiotics (Solanki et al. 2012b; Liu et al. 2006),

formation of viable spores (Cenci et al. 2006), promotion

of plant growth (Ryu et al. 2004), and ubiquitous presence

in soil (Gajbhiye et al. 2010). Some of the well documented

characteristics of Bacillus spp. related to soil fertility and

plant nutrition mobilization are the production of bacterial

phytohormones and solubilization of mineral phosphates

(Calvo et al. 2010), which allow them to inhabit diverse

niches in agro-ecosystems. Additionally, strains of Bacillus

have also paramount advantages over other biocontrol

bacteria in several manners such as they are mostly soil

inhabitants, have long shelf life and also impart phos-

phorus-solubilization, resulting in improved growth and

yield of crops (Quan et al. 2006). It has been previously

demonstrated that an effective antagonistic strain isolated

from one region may not perform as effectively in other

soils or with other plants (Kiely et al. 2006). For this rea-

son, it has become important to screen different environ-

ments to understand the role of crop rhizosphere in

selection for bacterial populations. Therefore, present site

specific explorative study of Bacillus spp. associated with

chickpea rhizosphere from different regions (Mau, Vara-

nasi, Lucknow and Kanpur) of Indo-Gangetic plains (IGP)

was carried out.

Considering the multiple applications of Bacillus spp., it

is essential to study their genetic and phenotypic assort-

ment and their PGPR activity (Huang et al. 2007), which is

useful in designing strategies to exploit them as bioinocu-

lants for sustainable and integrated disease management

without causing harm to the environment and farmers.

Moreover, there is a very limited knowledge regarding the

biological suppression of chickpea pathogens by the

application of PGPR in IGP regions, India. Thus, the major

objectives of present investigation were to (1) investigate

the antagonistic ability of Bacillus spp. isolated from IGP

regions against chickpea pathogens; (2) evaluate their PGP

activities in order to use them further as inoculant strains;

and (3) assess their genetic diversity through 16S rDNA-

RFLP and BOX-PCR.

Materials and methods

Site description and soil sampling

Chickpea rhizospheric soil samples were collected from

four different sites of IGP regions including Mau

(25�570N–25�570E), Varanasi (25�200 N–80�000 E), Luc-

know (26�550 N–80�590 E) and Kanpur (26�280 N–80�240

E), Uttar Pradesh, India. Five healthy plants were randomly

sampled from each site in a sterile specimen container and

immediately transported to the laboratory. The soil parti-

cles attached to roots were carefully collected after

uprooting plants, stored at 4 �C and processed within 24 h

of collection.

Isolation of bacterial strains

Root adhered soil (*10 g) was suspended in 90 ml of

sterile distilled water and heat treatment method described

by Solanki et al. (2012a) was used for the isolation of

Bacillus spp. to partially select spore-forming bacteria in

the samples. The suspensions from all samples were seri-

ally diluted (up to 10-6) and 100 ll of each dilution was

spread on nutrient agar medium plates. The plates were

incubated for 2–7 days at 30 ± 2 �C. The colonies of

distinct morphotypes arising on the plates were enumerated

and all the strains were purified and stored at -80 �C in

nutrient broth with 30 % glycerol.

Tests for antagonism

The pathogenic isolates of Fusarium oxysporum f. sp. ci-

ceri (FOC)—race 1 (NAIMCC-F-00856), Fusarium solani

(FS) (NAIMCC-F-01024) and Macrophomina phaseolnia

(MP) (NAIMCC-F-01263) were obtained from the

National Agriculturally Important Microorganisms Culture

Collection (NAIMCC), Uttar Pradesh, India. Bacillus

strains were evaluated for their in vitro antifungal activity

in dual culture assays on potato dextrose agar (PDA; Hi-

Media, India) with the plant pathogens FOC (race 1), FS

and MP. A fungal disk (FOC/FS/MP) of 5 mm diameter

taken from an actively growing culture and was placed at

centre on the surface of the PDA plate. Bacillus strains

grown in nutrient broth to a concentration of

*6 9 108 cell ml-1 were streaked in a straight line on the

edge of the NA ? PDA (1:1) plate (3 cm from the centre)

containing fungal disk, followed by incubation at

28 ± 2 �C for 5 days or till the fungal mycelia completely

covered the entire plate in control. Plate inoculated with

fungal disk alone was used as a control. The antifungal

activity was evaluated by measuring the growth inhibition

against test fungi. The Percentage of inhibition was cal-

culated as follows:

Percent inhibition ¼ C � Tð Þ=C½ � � 100

where, C is the radial growth of fungus in control, and T is

the radial growth of the fungus in the presence of test

organism. The strains exhibiting more than 50 % of

mycelial growth inhibition were considered as promising

Plant Growth Regul

123

antagonist. All assays were repeated thrice with five

replications.

To study antifungal activity of cell free crude extract of

selected screened bacteria against fungal pathogens, bac-

terial strains were grown in yeast extract-glucose (YEG)

medium for three days in an orbital incubator shaker with

80 rpm at 30 ± 2 �C. Culture filtrate was separated from

bacterial cells through centrifugation (10,000 rpm for

10 min at 20 �C) and filter sterilized (0.2 lm pore size),

and was stored at -20 �C until further use. The spores/

mycelium of pathogens were scraped and suspended in

sterile distilled water (10 ml). Diluted spore suspension

(0.1 ml, 105 cfu ml-1) of the test fungi were spread on

petri dishes (90 mm diameter) containing PDA. Wells of

5 mm diameter were punched into the agar medium and

filled with 150 ll of cell free culture filtrate. The plates

were incubated at 28 ± 2 �C for 6 days. Filter sterilized,

un-inoculated medium (150 ll) was taken as a control.

Characterization for plant growth promoting traits

The growth promotion traits of bacterial isolates were

evaluated by performing standard protocol for the estima-

tion of indole acetic acid (IAA), P-solubilization, sidero-

phore, hydrogen cyanide (HCN) and ammonia production

according to Glickmann and Dessaux (1995), Brick et al.

(1991), Schwyn and Neilands (1987), Lorck (1948) and

Dey et al. (2004), respectively. For P-solubilization, plates

containing Pikovskaya’s media amended with tri-calcium

phosphate were observed for clearing or solubilization

zones around the colonies. Similarly, siderophore produc-

tion was confirmed by observing clear halo zone formation

on Chrome Azurol S medium. Productions of hydrolytic

enzymes (chitinase, b-glucanase, and protease) were

determined according to Solanki et al. (2012a). All assays

were repeated thrice with five replications.

Scanning electron microscopy

The interaction of the plant pathogenic fungi (FOC race 1)

with selected bacterial strain (B-CM191) was studied by

scanning electron microscopy (SEM) using the mycelial

disc (5 mm) from the area of interaction zone and trans-

ferred on glass cover slips. Cover slip with mycelial disc

was washed in 0.1 M sodium cacodylate buffer (pH 7.4)

and fixed in 2.5 % glutaraldehyde in 0.1 M sodium caco-

dylate buffer for 4 h at 4 �C followed by post-fixation with

1 % OsO4 in 0.1 M sodium cacodylate buffer (pH 7.4) and

dried in a critical point dryer (EMITECH model K850

Hitachi). The preparations were mounted on stubs, sputter-

coated with 10 nm Au and observed by SEM (Hitachi

model S3400 at 15–30 kV, 2–5.00 lm). FOC (race 1)

fungus served as a control.

DNA extraction from isolates

Pure cultures of Bacillus strains were grown in nutrient

broth at 30 ± 2 �C for 24 h and pelleted cells from 1.5 ml

broth were resuspended in 0.5 ml SET buffer (75 mM

NaCl, 25 mM EDTA and 20 mM Tris) with 10 ll lyso-

zyme (10 mg ml-1). Genomic DNA was extracted as

described by Pospiech and Neumann (1995). Finally, the

washed DNA pellet was incubated at 37 �C for 25–30 min

to completely remove ethanol, and then resuspended in

50 ll TE buffer. The extracted DNA was checked on

agarose gel and stained with ethidium bromide.

Partial sequencing of the 16S rRNA gene

Amplification of 16S rRNA gene was performed from the

genomic DNA of strains using universal primers pA

(50-AGAGTTTGATCCTGGCTCAG-30) and pH (50-AAG

GAGGTGATCCAGCCGCA-30) (Solanki et al. 2012a).

PCR cocktail (25 ll) contained 10 pM of each primer,

50 ng of genomic DNA, 109 Taq DNA polymerase buffer,

1 U of Taq DNA polymerase (Banglore Genei, India) and

2.5 mM of each dNTP. Amplification was performed in a

thermo cycler (Bio-Rad Laboratories, CA, USA) at 95 �C

for 5 min, followed by 30 cycles of 1 min at 95 �C, 1 min

at 55 �C and 1 min at 72 �C with a final extension at 72 �C

for 5 min. A 5 ll aliquot of each amplified product was

electrophoresed on 1.2 % agarose gel along with 1 kb

DNA ladder as marker in 19 TAE buffer at 50 V for

45 min, stained with ethidium bromide and visualized with

a UV transilluminator (Bio-Rad Laboratories, CA, USA).

Phylogenetic tree analysis

PCR products were purified using PCR purification kit

(Promega, India) and sequenced directly with the Taq-

mediated Di-deoxy chain terminator cycle sequencing kit

(Applied Biosystem, India) in an ABI 3130xl automated

genetic analyser (Applied Biosystem, UK) according to

manufacturer’s instructions (Applied Biosystems). The

sequences were aligned by ClustalW and BlastN search

programme was used to compare the sequences deposited in

public databases and the phylogenetic tree was constructed

with the MEGA software version 4.1 (Saitou and Nei 1987).

Gaps were treated by pairwise deletions and bootstrap ana-

lysis was done by using 1,000 pseudoreplications. The

nucleotide sequences of 16S rRNA gene were deposited in

NCBI GenBank to obtain the accession numbers.

BOX-PCR genomic fingerprints

The antagonistic Bacillus spp. were characterized by BOX-

A1R (50-CTACGGCAAGGCGACGCTGACG-30) primer

Plant Growth Regul

123

for genetic variability (Rademaker and de Bruijn 1997).

PCR reaction (25 ll) contained 5 ll 59 Gitschier buffer,

50 ng of genomic DNA, 1 ll BOX AIR primer

(0.3 lg ml-1), 2.5 ll DMSO (100 %), 1.5 ll dNTP mix

(100 mM), 0.2 ll BSA (20 mg ml-1), and 0.4 ll Taq DNA

polymerase (3 U ml-1) (GeNei, India) and reaction mix-

ture was balanced with nuclease free water. Amplification

was performed in thermal cycler (Bio-Rad Laboratories,

CA, USA) programmed with an initial denaturation at

94 �C for 1 min and 35 cycles at 94 �C for 30 s, 53 �C for

1 min, and 72 �C for 8 min, with an extension at 72 �C for

16 min. Amplicons were separated on 2.0 % agarose gel

(55 V/4 h) stained with ethidium bromide with a low range

DNA ladder (100 bp–3 kb) and photographed under UV

transillumination.

Detection of bacillomycin (bmyB) and b-glucanase

genes

Genomic DNA of Bacillus strains was extracted by cell

lysis method and PCR amplification of bacillomycin

(bmyB) and b-glucanase genes was performed using the

PCR primers: For bmyB gene, F-50-TGAAACAAA

GGCATATGCTC-30 and R-50-AAAAATGCATCTGCC

GTTCC-30 (Joshi and McSpadden Gardener 2006) and

b-glucanase gene, F-50-AATGGCGGTGTATTCCTT-

GACC-30 and R-50-GCGCGTAGTCACAGTCAAAGTT-30

(Solanki et al. 2012b). PCR cocktail (25 ll) contained

10 pM of each primer, 50 ng of genomic DNA, 109 Taq

DNA polymerase buffer, 1.0 U of Taq DNA polymerase

(Banglore GeNei, India) and 2.5 mM of each dNTP.

Amplification was performed in a thermo cycler (Bio-Rad

Laboratories, CA, USA) at 94 �C for 4 min; 35 cycles of

denaturation at 92 �C for 1 min, annealing at 54 �C for

bmyB and 55.5 �C for b-glucanase for 45 s, and extension

at 72 �C for 1 min each, followed by a 7 min extension at

72 �C. The PCR products were resolved on 1.2 % agarose

gel with 100 bp ladder.

Evaluation of biocontrol activity of isolates under green

house condition

The efficacy and disease suppression capability of all

screened potent antagonistic strains of Bacillus spp. was

studied in green house experiments to manage Fusarium

wilt of chickpea. Chickpea (cv. BGD-72) seeds were

obtained from Directorate of Seed Research, Mau, India.

For multiplication of FOC inoculum, a 5 mm disk of

actively growing culture was inoculated into 100 ml of

potato dextrose broth in 250 ml flasks and incubated for

8 days at 25 �C, entire contents of a flask was dilute with

sterile distilled water to get a final inoculum concentration

of 6.5 9 105 conidia/ml to be use as inoculum. Now, these

inoculums were used for soil infestation in plastic pots

(20 cm diameter) filled with sterilized soil. Pots with

sterilized soil alone served as healthy control and soil

infested with FOC served as pathogen control.

For seed inoculation, chickpea seeds were surface ster-

ilized in 2 % NaOCl, and rinsed with sterile double dis-

tilled water. Surface sterilized chickpea seeds were soaked

for 4 h in carboxy-methyl cellulose (1 %) suspension

containing bacterial cells (*108 CFU ml-1) and dried

overnight. Treated seeds were sown in the pots, five seeds

per pot and ten pots treatments were maintained in a

greenhouse at 10:14 h light/dark cycles. The relative

humidity in the greenhouse was maintained at around

70 %, and the temperature at 28 ± 2 �C. For comparing

the efficacy of various treatments, both healthy (without

pathogen inoculation) and pathogen-inoculated control

were included in the experiment. Disease incidence was

recorded 60 days after sowing and ten plants per treatment

were randomly removed for measuring root length (cm),

shoot length (cm), fresh and dry weight (mg plant-1). Pots

were irrigated at regular intervals and disease symptoms

were monitored during experiment on 0–4 scale was used

for severity progression: 0 is 0 %, 1 is 1–33 %, 2 is

34–66 %, 3 is 67–100 % and 4 is dead plant (Sharma et al.

2005). Plant growth-promoting activity of Bacillus spp.

was assessed based on the seedling vigour index (SVI)

calculated by using the formula as described by Solanki

et al. (2011):

SVI = Percent healthy survivals

� mean shoot lengthþmean root length½ �

Greenhouse experiment was repeated thrice with ten rep-

lications and pooled data was used for analysis.

Statistical analysis

Experimental data was analyzed using standard analysis of

variance (ANOVA) followed by Duncan’s multiple range

test (DMRT). Standard errors were calculated for all mean

values. Differences were considered significant at the

p B 0.05 level. XLSTAT (2013) software was used for the

principle component analysis (PCA).

Results

Preliminary screening of strains and tests

for antagonism

A total of 240 bacilli were selectively isolated based on

heat treatment from the chickpea rhizosphere of IGP

regions of Uttar Pradesh, India. Details of sampling sites,

soil properties and number of total strains obtained from

Plant Growth Regul

123

each site are compiled in Table 1. All the isolates were

tested for in vitro screening against chickpea pathogens are

appended in Table 1. Out of them, only fourteen bacterial

(B-WM11, B-MM26, B-MV43, B-PM64, B-WK95,

B-PV116, B-CL122, B-CK148, B-WV169, B-PL180,

B-CM191, B-PK212, B-WL223 and B-CV235) isolates

displayed strong antagonistic activity and showed signifi-

cant inhibitory effect on mycelial growth (C15 mm

diameter) against all the three plant pathogens (Fig. 1).

Strain B-CM191, B-CV235 and B-CL122 were showed the

most efficient antagonistic activity and displayed 38.33,

37.03 and 36.80 mm inhibition zones against FOC (race 1),

respectively. Similar trends were observed with other two

pathogens.

Hydrolytic enzymes and plant growth promoting traits

Principal component analysis (Fig. 1) of all the 14 strains

for the assessment of chitinase, b-1,3-glucanase and pro-

tease in culture filtrates was performed. All the bacterial

strains isolated from chickpea rhizosphere were clustered

together and able to produce significant amount of cell wall

degrading enzymes i.e., chitinase, b-1,3-glucanase and

protease in culture filtrates. PCA plots revealed that the

first component explained 98.26 % of the variation with

chitinase, b-1,3-glucanase and protease, while explained

81.22 % of the variation with FOC (race 1), FS and MP. It

was observed that B-CM191, B-CV235 and B-CL122 were

able to produce higher amount of chitinase enzyme fol-

lowed by B-WM11 and B-PV116. Maximum production of

protease resulted in the B-CM191 as compared to other

strains (Fig. 1). The highest amount of cell wall degrading

enzymes was shown by B-CM191 in comparison to other

strains.

The bacterial antagonists were also evaluated for pro-

duction of IAA and it was varied from 11.08 to

42.59 lg ml-1 in the tryptophan supplemented medium

and a higher production was recorded in medium without

tryptophan (Table 2). B-CM191, B-CV235 and B-CL122

strains were able to produce maximum amount of IAA in

comparison to other strains. Solubilisation of tricalcium

phosphate and halo zone formation indicated that

B-WM11, B-PV116, B-MM26, B-MV43, B-PL180,

B-CL122, B-CM191, B-CV235 and B-CK148 were able to

solubilise phosphorous (Table 2). In vitro siderophore

production assay revealed that 62.28 % of the potent

antagonistic strains were able to produce siderophores. Out

of fourteen distinct bacterial strains, only 50 % strains were

able to produce hydrogen cyanide (Table 2). It was

observed that B-WM11, B-PM64, B-MV43, B-CK148,

B-CM191, B-CV235 and B-WL223 were able to produce

ammonia (Table 2). On the basis of these results we

selected strain B-CM191 to study the direct interaction

between Fusarium oxysporum f. sp. ciceri through SEM.

Examination of direct interaction between antagonist

and plant pathogen

Antagonistic interaction between FOC (race 1) and B.

subtilis was investigated by SEM which revealed the

antagonistic effect of B. subtilis B-CM191 on FOC

exhibiting reduced apical growth, curling of hyphal tips

and distortion of the fungal mycelia (Fig. 2).

Molecular characterization, accession numbers

and phylogenetic analysis

Based on 16S rRNA gene partial sequencing, similarity

values C97 % suggested that all strains belongs to genus

Bacillus and Bacillus derived genera, viz. Lysinibacillus

fusiformis (B-WM11, B-PM64 and B-PV116), Lysiniba-

cillus spp. (B-MM26, B-WV169, and B-PK212), B. cereus

(B-MV43 and B-PL180), B. subtillus (B-CL122, B-CM191

and B-CV235), B. thuringiensis (B-WL223) and Bacillus

spp. (B-WK95, B-CK148). Partial 16S rRNA gene

sequences of the strains were submitted to NCBI GenBank

under accessions numbers HM588141-154.

Two major clusters and two minor clusters under each

major cluster were formed based on NJ method with 1,000

bootstrap sampling (Fig. 3). Isolates B-MV43, B-WK95,

Table 1 Details of sampling sites, soil properties and number of antagonistic strains obtained from chickpea rhizosphere soil samples (0–10 cm)

Site(s) Soil

type

pH EC

(dSm-1)

No. of

samples

Total N (%) Organic C

(%)

Strain

(s) obtained

Assessment of antagonistic bacteria using

in vitro dual culture

FOC (race 1) FS MP

Mau Sandy 8.1 ± 1.23 4.1 4 0.17 ± 0.14 0.87 ± 0.05 70 24 (34.28 %)a 24 (34.28 %) 22 (31.42 %)

Varanasi Loam 7.3 ± 1.14 3.9 4 0.12 ± 0.09 0.74 ± 0.12 70 22 (31.43 %) 24 (34.29 %) 24 (34.29 %)

Lucknow Sandy 7.8 ± 1.22 4.2 4 0.16 ± 0.12 0.95 ± 0.18 50 16 (32.00 %) 18 (36.00 %) 16 (32.00 %)

Kanpur Sandy 7.4 ± 0.86 4.0 4 0.14 ± 0.07 0.75 ± 0.08 50 17 (34.00 %) 18 (36.00 %) 15 (30.00 %)

a Number of antanositic bacteria; Values in the parenthesis are % antagonists of total strains obtained from a particular site

Values are mean ± standard error of three independent experiments. EC electrical conductivity, N nitrogen, C carbon

Plant Growth Regul

123

B-PM64, B-WM11, B-MM26, B-WV169 and B-PK212

showed similarity with L. fusiformis, Lysinibacillus sp.,

Bacillus sp. and B. cereus were grouped under cluster

I. Cluster II contained isolates B-PV116 and B-CK148

(L. fusiformis and Bacillus sp.). Isolates B-WL223 and

B-PL180 showing similarity to B. cereus and B. thuringi-

ensis in cluster III and cluster IV represents B-CL122,

B-CM191, and B-CV235 to B. subtilis.

BOX-PCR genomic fingerprints

Genotypic diversity of antagonistic strains was further

assessed using BOX-PCR fingerprints from genomic DNA.

The number of polymorphic bands ranging between 200

and about 3 kb were used for analysis. BOX-polymerase

chain reaction fingerprints of all the Bacillus strains

revealed a high genotypic diversity (Fig. 4). BOX-PCR

results ascertained the distinctness of fourteen Bacillus and

Bacillus derived genera.

Detection of antibiotic genes

Detection of amplified genetic markers (bacillomycin and

b-glucanase) in antagonistic Bacillus strains was shown in

Table 2. Bacillomycin (bmyB) gene amplification showed

one specific band at around 395 bp length in all Bacillus

spp. and amplification of b-glucanase gene was also spe-

cific on the 415 bp in all the fourteen strains.

Plant protection bioassay

A greenhouse experiment was performed based on the

results of in vitro inhibition and PGP traits to assess

antagonistic efficacy of all fourteen Bacillus isolates

against FOC (race 1). Fig. 5 shows the scatter diagram of

the scores of the first and second PCs obtained by the

combined PCA. Two principal components were extracted

accounting for 100 % of total variance in the analysis of

strains with disease index, vigour index, shoot and root

length, fresh weight and dry weight. All the fourteen

Bacillus isolates strains were tightly grouped and well

separated from control. These strains significantly

(P B 0.05) enhanced the growth (root and shoot length),

fresh weight (root and shoot), and dry weight (root and

shoot) as compared to control. Maximum protection

against FOC (race 1) was recorded with B. subtilis

B-CM191 followed by B. subtilis B-CV235 and B. subtilis

B-CL122. The seedling vigour index (SVI) calculated for

each treatment revealed all the strains of Bacillus isolates

to be efficient in plant growth promotion and B. subtilis

B-CM191 being the most effective antagonist (Fig. 5).

Discussion

In this site specific explorative study, we used Bacillus and

Bacillus derived genera to characterize the in vitro antag-

onistic potential towards chickpea soil-borne pathogens

(FOC race 1, FS and MP) and PGP traits of Bacillus

spp. obtained from four different sites (Mau, Varanasi,

Lucknow and Kanpur) of Indo-Gangetic plains, India.

The strains were taxonomically described as L. fusiformis

(B-WM11, B-PM64, B-PV116), Lysinibacillus spp.

(B-MM26, B-WV169 and B-PK212), Bacillus cereus

(B-MV43 and B-PL180), B. subtilis (B-CL122, B-CM191

and B-PM235), Bacillus spp. (B-WK95 and B-CK148),

and B. thuringiensis (B-WL223) on the basis of 16S rRNA

gene sequencing and subsequent molecular phylogeny

analysis. Phenotypic as well as genotypic (BOX-PCR)

analysis revealed a high degree of diversity among Bacillus

strains. These results were in accordance with Solanki et al.

(2012a), where they reported the antagonistic potential of

Bacillus megaterium, B. subtilis, B. amyloliquefaciens and

Fig. 1 Principle component analysis (PCA) of Bacillus strains on the

basis of a antagonistic reactions against FS, MP and FOC (race 1)

and; b hydrolytic enzymes

Plant Growth Regul

123

Bacillus spp. associated to the rhizosphere of healthy

tomato plants from Indo-gangetic plains of India.

Plant growth promoting rhizobacteria dwelling in the

rhizosphere have drawn much attention in recent years, as

they have the ability for root colonization and offensive

mechanisms have been proposed to explain the inhibition

of fungal pathogens by Bacillus spp., including production

of siderophore, antimicrobial compounds, secretion of

hydrolytic enzymes, competition for nutrients, or a com-

bination of mechanisms (Singh et al. 2012; Berg et al.

2005). All strains of Bacillus spp. screening with dual

culture and extra-cellular metabolite efficacy test showed

that they had antagonistic potential against chickpea

pathogens, FOC (race 1), FS and MP. Strains of B. subtilis

(B-CM191, B-CV235 and B-CL122) were most efficient

towards several pathogens that could be isolated from all

soils independent from a previous history of fungal disease

suppressiveness. Therefore, it is concluded that dual cul-

ture and extra-cellular metabolite efficacy assay can be

used as a standard test for the selection of biocontrol agents

and shows a cumulative effect of all mechanisms under-

going for biocontrol. This finding supports earlier obser-

vations that, virtually all natural and agricultural soils

studied so far possessed some ability to suppress the

activity of soil-borne plant pathogens due to the presence

and activity of soil microorganisms, generally referred as

soil suppressions (Postma et al. 2008).

An important role of hydrolytic enzymes has been well

documented as a variety of microorganisms that exhibit

hyper-parasitic activity, attacking pathogens by excreting

these enzymes (Chernin and Chet 2002). The mode of

action following antagonism by B. subtilis B-CM191was

studied by SEM, which demonstrated gradual destruction

of mycelia leading to the death due to cytoplasmic extru-

sion validating potency to inhibit FOC (race 1). The broad

range of antifungal activity of the antagonistic bacteria

demonstrates the multiple mechanisms of action (Tariq

et al. 2010) and hence may involve more than one anti-

fungal metabolite. These strains also possessed beneficial

PGP traits. Microbial-mediated solubilization of insoluble

phosphates through release of organic acid is often com-

bined with production of other metabolites like sidero-

phores, phytohormones and lytic enzymes, which take part

in biological control against soil-borne pathogens (Vassilev

et al. 2006). Our result showed the potential of P-solubi-

lizing B. subtilis B-CM191, B-CV235 and B-CL122,

Bacillus spp. B-CK148, and L. fusiformis B-WM11 for the

simultaneous synthesis of IAA and release of other path-

ogen suppressing metabolites. In addition, PCR amplifi-

cation was confirmed the presence of the bacillomycin

Table 2 In vitro characterization of antagonistic Bacillus spp. on the plant growth promotion traits and presence of antibiotic gene (bmyB and b-

1,3-glucanase)

Strain (s) Plant growth promoting traits Amplification of antibiotic gene(s)

IAA (lg ml-1) P-solubilisation Production of

(?) Tryptophan (-) Tryptophan Siderophore HCN NH3 bmyB b-1,3-glucanase

B-WM11 27.37cd 16.23bc ? - ? ? ? ?

B-MM26 18.92fg 10.63d–g ? - ? - ? ?

B-MV43 17.52fgh 9.54d–g ? - - ? ? ?

B-PM64 24.05de 13.56cd - ? ? ? ? ?

B-WK95 21.63ef 12.52cde - ? ? - ? ?

B-PV116 23.57de 11.63def ? ? - - ? ?

B-CL122 35.89b 19.66ab ? ? ? - ? ?

B-CK148 31.28c 16.36bc ? ? - ? ? ?

B-WV169 15.52ghi 8.41efg - - ? - ? ?

B-PL180 13.76hij 7.39fg ? ? - - ? ?

B-CM191 42.59a 23.47a ? ? ? ? ? ?

B-PK212 11.08j 6.74g - - - - ? ?

B-WL223 12.99ij 7.76fg - ? - ? ? ?

B-CV235 39.12ab 22.54a ? ? - ? ? ?

SEM 1.40 1.39

CD (P B 0.05) 4.07 4.03

(?), positive; (-), negative; values are mean ± standard error of three independent experiments. Means followed by same letter within a row are

not significantly different (P B 0.05) according to Duncan’s Multiple Range Test (DMRT). SEM standard error of the difference between means,

CD critical difference

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123

(bmyB) and b-glucanase genes. Similarly, bmyB and

b-glucanase genes have been reported from different

Bacillus strains using PCR techniques (Joshi and McSp-

adden Gardener 2006; Solanki et al. 2012b). This suggests

that amplified genes from the Bacillus could also have a

role to further define the diversity, ecology, and biocontrol

activities of B. subtilis in the suppression of soil-borne

pathogens.

In general, strains that work well in one environment

may fail to elicit any plant response in others due to con-

ditions prevailing in new environment. Screening of natural

population of Bacillus spp. from various sampling sites

revealed that Mau has more population and species rich-

ness of effective antagonistic Bacillus isolates compared to

Varanasi, Kanpur and Lucknow. This conformed with the

objectives of the studies on diversity within a targeted

bacterial population, to screen bacterial antagonists that are

best adapted to particular environment stress or ecological

habitat (Solanki et al. 2012b; Achouak et al. 2000).

It is always difficult to extrapolate the biocontrol

activity of a given strain from the laboratory to natural

environments (Hermosa et al. 2000) However, the results

of biocontrol study under greenhouse condition suggest the

efficiency of Bacillus strains that showed aggressive

in vitro antagonism in controlling chickpea wilt caused by

FOC (race 1). Thus, the efficacy of the antifungal strains of

Bacillus in soil may be predicted from their in vitro per-

formance against fungal plant pathogens. The dual culture

and well diffusion assay can definitely serve as the selec-

tion criteria for the screening of BCAs and their perfor-

mance in soil.

The mechanism by which Bacillus spp. influenced

plant growth promotion were further evaluated under

greenhouse with the strains showed significant in vitro

antagonistic activity and colonized the chickpea rhizo-

sphere effectively. B. subtilis B-CM191 from chickpea

rhizosphere maintained and sustained higher level of

antagonistic population. This is in concurrence with the

results of others researchers (Al-Jedabi 2009; El-Hassan

and Gowen 2006), who reported that a culture based

approach can be an important tool to evaluate root

colonizing bacterial communities. The results of

Fig. 2 Scanning electron micrographs showing morphological

changes with fungal mycelia during the interaction FOC (race 1)

with B. subtilis B-CM191. a Healthy F. oxysporum f. sp. ciceri

mycelia (control); b Bacilli proliferation on the mycelia of F.

oxysporum f. sp. ciceri (race 1); c, d Hyphal destruction by

antagonistic B. subtilis B-CM191. Arrows indicates mycophagy

action of Bacillus cells over the intact mycelia and hyphal distortion

Plant Growth Regul

123

greenhouse biocontrol experiment were also in accor-

dance to that of dual culture assay and extra-cellular

metabolite efficacy test. Under greenhouse experiment,

B. subtilis B-CM191, B-CV235 and B-CL122 were

found to be most effective, showed more than 85–90 %

biocontrol efficacy.

Fig. 3 NJ phylogenetic tree of

full 16S rRNA gene sequences

from selected isolates. The

sequence data for several

closely related Bacillus cultures

were recovered from GenBank

and included in the tree. The

boot strap values from 1,000

pseudoreplications are shown at

each of the branch points on the

tree. Bar indicates % similarity

Fig. 4 BOX-PCR fingerprints generated from genomic DNA of antagonistic strains of Bacillus spp. M, molecular size marker (100 bp–3 kb),

low range DNA ruler (GeNeiTM)

Plant Growth Regul

123

Overall, the present study is successful in selecting

effective strains of Bacillus from chickpea rhizosphere to

manage soil-borne pathogens and we are reporting B.

subtilis B-CM191, B-CV235 and B-CL122 as an effective

biocontrol agent of chickpea wilt with the production of

diffusible and volatile antibiotics, siderophore, IAA and

phosphate solubilization. Hence, they can be used under

field conditions as an effective biocontrol agent promoting

plant growth with reduced disease incidence. They could

be incorporated within an integrated disease management

package including moderately resistant cultivars, limited

fungicide application and effective cultural practices.

Acknowledgments This work has been carried out under the net-

work project ‘Application of Microorganisms in Agricultural and

Allied Sectors’ and financially supported by Indian Council of

Agricultural Research (ICAR), New Delhi, India.

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