Characterization of effective bio-control agent Bacillus sp. SRB 27 with high salt tolerance and thermostability isolated from forest soil sample

A potential antagonist Bacillus sp. SRB 27, against Fusarium oxysporum, Rhizoctonia solani and Alternaria solani was obtained from forest soil sample by carrying out in vitro and in vivo screening techniques. This study reports the identification and characterization of a Bacillus sp. SRB 27 that may be used as a bio-control agent against the plant diseases in crop plants. It was identified as Bacillus sp. SRB 27 based on 16S rDNA sequence analysis and biochemical tests. The isolate showed a wide range of antifungal activity in vitro against a number of phytopathogens such as F. oxysporum, Alternaria and Rhizoctonia in terms of percentage of growth inhibition which were 76.78, 78.57 and 77.55%, respectively. Strain SRB 27 was tolerant to high salt concentration up to 13%, was phosphate solubilizer, proteolytic and amylase positive and coagulase negative. It had compatibility with broad spectrum of fungicides with field recommended dose. Apart from the antagonistic activities, it showed a positive effect on the growth of the castor and cotton plants (both under seed and soil treatments) in comparison with control (non-inoculated). The growth parameters of the test plant surpassed the control in all the cases.


INTRODUCTION
Microorganisms can colonize the tissues of healthy plants. Such endophytic bacteria have been reported to prevent disease development by controlling the spread of plant pathogens or by enhancing plant resistance (Stein, 2005;Ryan et al., 2008). Therefore, there is a considerable potential for finding new and beneficial endophytic bacteria that can serve as bio-control agents. Bacillus sp. were considered as potential bio-control agents due to their high spore production ability, resistance and ability to survive desiccation, heat, ultraviolet (UV) irradiation and organic solvents (Romero et al., 2007). Bacillus sp. (Gram positive) form biofilms on *Corresponding author. E-mail: gouri1333@gmail.com.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License root surfaces, which are multicellular matrixes of bacteria surrounded by extra cellular polysaccharides called a glycocalyx. The glycocalyx acts as a physical barrier and is strongly anionic, thereby can protect the microcolony from external agents (Jeyasekaran and Karunasagar, 2000). Recent studies suggested that the biofilm mode is important for the bacteria's ability to act as bio-control agents (Bais et al., 2004).
According to Backman et al. (1997), the effectiveness of endophytes as biological control agents (BCAs) is dependent on many factors. These factors include: host specificity, the population dynamics and pattern of host colonization, the ability to move within host tissue, and the ability to induce systemic resistance. For example, Pseudomonas sp. strain PsJN, an onion endophyte, inhibited Botrytis cinera pers. Pseudomonas putida 89B-27 and Serratia marcescens 90-166 reduced cucumber mosaic virus in tomatoes and cucumbers (Raupach et al., 1996) as well as anthracnose and Fusarium wilt in cucumber (Liu et al., 1995). Jetiyanon (1994) established that cabbage colonized by endophytes in the green house had season-long reduced black rot in the field due to induction of defense mechanisms. Non-treated cabbage plants reached the economic threshold (symptoms of systemic disease) approximately 33 days after inoculation with Xanthomonas campestris pv. campestris.
The objective of present study was to investigate the antagonistic properties, salt and heat tolerance, phosphate solubilization, compatibility with broad spectrum of fungicides and growth promoting ability of Bacillus sp. SRB 27.

Isolation
The forest soil sample was collected in air tight polythene bags from Nallamala forest located at Munnanoor in Mahabubnagar district, Andhra Pradesh, India. The processed soil sample was serially diluted, spread plated on full strength nutrient agar and incubated at 28°C for 48 h. A total of 46 different colonies were isolated on nutrient agar (NA). Pure cultures were developed with repeated culturing which were maintained in 20% glycerol at -20°C. A potential isolate was screened and selected based on the antagonistic properties, phosphate solubilization and salt tolerance. Further, the isolate was identified based on phenotypic and 16S rDNA sequence.

Antagonistic ability
The in vitro antagonistic assay was performed using dual culture method on potatoes dextrose agar (PDA) medium. Seven days old culture agar discs (5 mm) of Fusarium oxysporum f.sp. ricini, Rhizoctonia solani and Alternaria solani were disposed at the center of Petri dishes and the bacterial strain was streaked in a square form around the agar disc at 4 cm distance. The antagonistic activity of the studied bacterial strain was estimated by the inhibition of the fungal growth monitored by measuring the diameter in millimeter of the colony until seven days at 28°C in biochemical oxygen demand (BOD) incubator. The experiment was replicated thrice. The percentage of growth inhibition of the fungus was calculated according to the formula given by Whipps (1987).
Per cent growth inhibition = ((R 1 -R 2 )/R 1 ) × 100 Where, R 1 is the farthest radial distance (measured in millimeter grown by the fungus after seven days of incubation in the direction of the antagonist (a control value). R 2 is the distance of fungal growth from the point of inoculation to the colony margin in the direction of the antagonist. All in vitro antagonism assays were replicated thrice. The bacterial strains that showed obvious inhibition to F. oxysporum, R. solani and A. solani were selected for further evaluation and stored in 20% glycerol at -20°C.

Fungicide susceptibility test by agar well diffusion method
Agar well diffusion assay is the key process used to evaluate the antifungal potential of fungicides. In the present study, six systemic fungicides; that is, carbendazim, tebuconazole + trifloxystrobin, hexaconazole, propiconazole, azoxystrobin and benomyl were used. Petri dishes (100 mm) containing 20 ml of Mueller Hinton agar (MHA) were seeded with approximately 100 µl inoculums of bacterial strains (inoculums size was adjusted so as to deliver a final inoculum of approximately 10 8 CFU/ml). After solidification of the media, wells of 6 mm diameter were cut into media using a sterilized cork borer. 100 µl of each fungicide was poured into respective well and the plates were incubated at 32°C overnight. The experiment was replicated thrice under strict aseptic conditions to ensure consistency of all findings. Fungicidal activity on inoculums was expressed in terms of the mean of diameter of zone of inhibition (in mm) produced at the end of the incubation period.

Phosphate solubilization
The isolate was screened for phosphate solubilization as per the methodology described by Gupta et al. (1994) on modified Pikovskaya agar (Glucose -10 g, Ca 3 (PO 4 ) 2 -5 g, KCl -0.2 g, MgSO 4 -0.1 g, MnSO 4 -trace, FeSO 4 -trace, Yeast extract 0.5 g, Agar -15 g, Distilled water-1 L, pH -7.0) and the plates were incubated at 30±1C for 48-96 h. Phosphate solubilization is indicated by the formation of a solubilization or a clear zone around the bacterial colony. A loop full of SRB 27 culture was placed on the center of agar plates and incubated at 30±1C for 5 days. The solubilization zone was determined by subtracting the diameter of bacterial colony from the diameter of total zone. Halo surrounding the colonies were measured and the solubilizing efficiency (SE) was calculated by the following formula: SE= (Solubilization diameter (S)/Growth diameter (G)) x 100.

Salt tolerance
The bacterial isolates were inoculated separately on specific agar medium containing NaCl at 4 to 13% concentration. Four replications of the plates for each isolate and concentration were maintained along with control. After 48 h of incubation, observations for survival and growth of inoculum started (Benson, 1990). The test was repeated for the promising isolates for the salt tolerance confirmation.

Temperature tolerance study
SRB 27 culture was streaked on nutrient agar plates and incubated at different temperature (40, 50 and 60°C) for 24 h with four replications. Control was also maintained for comparison. All the inoculated Petri plates were incubated in BOD incubator at 28±2°C up to 2 to 15 days (Benson, 1990). Observation was recorded for survival and growth of inoculum.

Amplification, sequencing and phylogentic analysis of 16S rRNA gene
The cells of isolate SRB 27 were harvested after incubation in liquid Luria Bertani (LB) medium for 24 h. After centrifugation at 10,000 rpm for 5 min, cells were collected and washed several times by Tris-HCI-EDTA (TE) (pH 8.0) buffer. Genomic DNA was extracted by X-pert bacterial genomic DNA extraction teaching kit (solution based Hi-media kit). Amplification of 16S rRNA gene of the isolate was carried out in PCR cycler using universal primers FD1 (5′-AGA-GTT-TGA-TCC-TGG-CTC-AG-3′) and RP2 (5′-ACG-GCT-ACC-TTG-TTA-CGA-CTT-3′). Amplification reactions were performed in a total volume of 50 µl of 10 X PCR buffer, 5 µl of 25 mmol/l, MgCl 2 , 5 µl of 2 mmole/l dNTPs mixture, 2 µl of primer FD1, and 2 µl of RP2, 1 µl of Taq DNA polymerase (5 U/µl) and 2 µl of template DNA. Thermo cycling procedure was as follow: an initial keeping at 94°C for 45 s, 55°C for 45 s and 72°C for 90 s, then an extension in the last cycle at 72°C for 5 min. The PCR product was purified using X-Pert gel extraction teaching kit (solution based Hi-media gel extraction kit). The PCR product was sequenced at Bioserve Pvt. Ltd, Hyderabad.

Coagulase test (method for tube test)
The plasma was diluted 1 in 10 in physiological saline (mix 0.2 ml of plasma with 1.8 ml of saline). In this test, three small test tubes were labeled as follows T = test organisms, Pos = positive control (Staphylococcus aureus broth culture), neg = negative control (sterile broth). To each tube, 0.5 ml of the diluted plasma was added and later five drops (about 0.1 ml) of the test organism culture was added to the tube labeled 'T'. To the tube labeled 'pos', five drops of the S. aureus culture was added. Later, five drops of sterile broth was added to the tube labeled 'neg' after mixing gently, after which the three tubes were incubated at 35-37°C. The test tubes were examined for clotting after 1 h and if no clotting occurred, examined at 30 min intervals for up to 6 h; each tube was gently tilted upon observing the clotting.

Protease production
Bacterial isolates were tested for production of protease by growing them on skim milk agar (SKM) (Chantawannkul et al., 2002). An ability to clear the skim milk suspension in the agar was taken as evidence for the secretion of protease. Petri plates with no bacterial inoculation were used as the control.

Growth promotion efficacy
Castor and cotton seeds were treated with Bacillus sp. SRB 27. For control treatments, seeds were not treated. Treated and controlled seeds were sown in pots. Root length, shoot length, root dry weight and shoot dry weight were measured 30 days after sowing. In another experiment, soil was treated with the test bacteria.

RESULTS AND DISCUSSION
In the present investigation, an attempt was made to isolate the potential bio-control agent with high salt tolerance and thermo-stability properties for which the bacteria was isolated. The isolate was characterized and identified using morphological, biochemical and 16S rDNA sequence analyses. Further, its effect on growth parameters of test plants under green house conditions was evaluated.

Assessment of in vitro antifungal activity
The isolate showing bio-control property was identified as Bacillus sp. SRB 27 by preliminary morphological and biochemical observations. The data presented in Table 1 explicates the antifungal activity shown by the Bacillus sp. SRB 27 against the fungi F. oxysporum, R. solani, A. solani (Figure 1). The Bacillus sp. SRB 27 showed the antagonistic activity in terms of growth inhibition in the     trifloxystrobin, hexaconazole, propiconazole, azoxystrobin and benomyl at 3000 and 4000 ppm.

Evaluation of NaCl and temperature tolerance shown by Bacillus sp. SRB 27
The data pertaining to Tables 3 and 4 describes tolerance levels shown by Bacillus sp. SRB 27 towards high NaCl concentrations and temperatures, respectively. The tolerance range was designated in connection with growth of the bacteria (in terms of optical density) in the broth at various concentrations. The isolate was tolerant for a wide range of NaCl concentrations (4 to 13%). High tolerance (+++) was shown at 4 and 5% whereas moderate tolerance (++) at 6 to 8% and low tolerance at 9 to 13% ( Table 3). The test organism tolerated a wide range of temperatures (45 to 60°C) and exhibited high tolerance at 45°C (+++); whereas moderate tolerance (++) at 50°C and low tolerance at 55 and 60°C (Table 4).

Phenotypic and biochemical characterization
In light of the results obtained for the assessment of fungicide susceptibility, high NaCl concentrations and temperatures tolerance shown by the test organism, the phenotypic and biochemical characterization of the bacteria was carried out and the results given in Tables 5  and 6. The isolate was found to be Gram positive, aerobic and motile aerobic rod that produced a big size colony with mucoid surface in white colour with irregular margin. The bacteria produce endospores (sub terminal) and form a pellicle in liquid medium. It grows better at temperature 40°C and pH 7.
The results pertaining to the biochemical tests performed are presented in Table 6 which explicates that, the Bacillus sp. SRB 27 utilized ornithine as the nitrogen source and did not utilized ONPG, lysine and urea. It showed positive response for nitrate reduction whereas negative for phenylalanine deamination and H 2 S production. Response for citrate, voges proskauer's and indole tests was positive for the bacteria. The Bacillius sp. SRB 27 utilized some carbon sources (arabinose, cellobiose, saccharose, trehalose, glucose and lactose) and rejected the other (xylose, adonitol, rhamnose, melibiose and raffinose). The test organism showed positive activity for oxidase, amylase, protease, lipase and gelatinase whereas negative for catalase, coagulase and HCN production.

Phylogentic analysis of 16S rRNA gene and deposition in NCBI
The PCR amplified product was gel electrophoresed and showed the band with 1.5 kb on the gel (Figure 2). The 16S rDNA gene sequencing of the isolate was amplified analysed (NCBI Accession no. JX276739). The data pertaining to the basic local alignment search tool (BLAST) analysis is shown in Table 7. Neighbor-joining phylogenetic tree based on the alignment of the nearly complete 16S rDNA gene sequence of the Bacillus sp. SRB 27 with the 16S rDNA sequences of the seven described Bacillus type strains available in GenBank and NCBI databases is shown in Figure 3. BLAST analysis and a neighbor-joining dendrogram constructed using MEGA revealed that the bacterium belongs to the genus Bacillus and as closely clustered together with Bacillus amyloliquefaciens. The amplified 16S rRNA gene sequence of isolate Bacillus sp. SRB 27 was most closely related to that of Bacillus sp. HNR03 (GenBank accession number, EU373340.1) and showed 99% identity with the sequence from Bacillus sp. HNR03. The sequence showed 99% identity with the sequences obtained from most of the B. amyloliquefaciens strains and Bacillus subtilis strains. On the basis of the results of the classical bacteriological tests and the analysis of the 16S rRNA gene, it was concluded that the isolate was a strain of B. amyloliquefaciens and Bacillus sp. SRB 27.

Phosphate solubilization
The Bacillius sp. SRB 27 has the ability of phosphate solubilization (Table 8) the solubilizing efficiency (SE) of the bacteria was found to be 163.63%.

Effect of the isolate on growth of castor and cotton plants in soil and seed treatments under green house conditions
The results pertaining to the effect of the Bacillus sp. SRB 27 for growth promotion of castor and cotton when treated to soil and seed under green house condition are mentioned in Table 9. The test organisms was able to influence the growth of castor and cotton in terms of the shoot length (LS), root length (LR), dry weight of shoot (DWS) and dry weight of root in connection with the seed and soil treatment when compared with control at 30 days after sowing. The shoot length was recorded as 25.5±0.2 and 28.4±0.6 cm for castor and cotton,     Romero et al. (2007) in which, the species of the genus Bacillus, particularly B. amyloliquefaciens and B. subtilis, have been shown to produce a range of antimicrobial dipeptides or cyclic lipopeptides. Some of the metabolites are strain-specific and may be associated with certain species and subspecies of B. amyloliquefaciens and B. subtilis. In the studies reported by Earl et al. (2008) and Rosas-Garcia (2009), it was revealed that the in vitro prescreening test of dual culture selected four B. subtilis strains, PLC1605, PCL1608, PCL1610 and PCL1612 with noticeable antifungal activity against R. necatrix and other soil-borne phytopathogenic fungi. B. subtilis possess several characteristics that enhance its survival in the rhizosphere and thus its effectiveness as a biopesticide. Compant et al. (2005) noticed that many root associated bacteria have a direct positive influence on plant growth and can indirectly stimulate plant health. Plant bacterial endophytic populations correlate to a certain extent with plant growth performance (Sessitsch et al., 2004). Studies conducted by Yun et al. (2011) revealed that B. amyloliquefaciens PEBA20 showed antimicrobial activity against a wide range of fungi and bacteria. The efficacy of the bacterial suspension in inhibiting fungal growth in vitro ranged from 38.44 to 89.37%, while that of the fermentation filtrate ranged from 19.2 to 82.62%. The diameter of the inhibition zone after treatment with bacterial suspension and fermentation filtrate of PEBA20 ranged from 9.97 to 19.14 mm and from 5.1 (very weak effect) to 19.05 mm. Nitrogen-fixing Bacillus sp. isolated by Jadhav et al. (2010) has 15% salt-tolerance, indicating they are new novel strains qualified by adaptation to environment and thereby acquiring additional traits. B. subtilis subsp. Subtilis NCIB 3610T and Bacillus sonorensis NRRL B-23154T isolated by Jadhav et al. (2010) tolerated very high salt concentration (10 and 15%, respectively). The inhibitory potential and antibiotic production of B. subtilis was investigated in vitro against phytopathogen fungus and bacteria (Földes et al., 2000). Touré et al. (2004) was attributed to lipopeptides produced by one isolate of Bacillus subtilis, the antagonistic action over mycelia growth, which they obtained over many phytopatogenic fungi from soil, as Fusarium, Pythium ultimum, Rhizoctonia solani and Rhizopus. Mariana et al. (2009) observed the differences in susceptibility for each fungal isolate, in relation to the antagonist action of Bacillus E164 which may be related to variations in the production of inhibitory metabolites by the bacterial strains. The phosphate solubilization of Bacillus cereus [0.32+0.05] and B. subtilis [0.38+0.01] were analyzed using various parameters such as pH [7 to 9], temperature (30 to 45°C], and nutrient supplementation. The role of phosphorus in increasing the yield and improving the quality of Bacillus sp. is well known. Phosphorous, next to nitrogen is a vital nutrient for plant and microorganisms.
In conclusion, the isolated bacterial species was identified as Bacillus sp. based on the phenotypic, biochemical and phylogenetic characterization. The Bacillus sp. SRB 27 was found to be tolerant to high temperatures and high salt conditions which is a positive aspect of the strain to grow better for the crops that grow under high saline conditions and withstand high temperatures. Apart from that, the Bacillus sp. SRB 27 has the superior quality of antagonistic activity against phytopathogenic fungal genera like Fusarium sp., Rhizoctonia sp. and Alternaria sp. The phosphate solubilization efficiency was found to be very high which helps in better uptake of the phosphorous under field conditions. Moreover, Bacillus sp. SRB 27 has shown proven effect on the growth of castor and cotton in specific and many plant species in general.