Isolation of rhizospheric Bacillus sp.
Chilli rhizosphere soils were collected from in and around Bangalore and Bacillus sp. were isolated by soil dilution method. The dilutions were heat treated at 80 °C for 20 min to ensure that only heat resistant strains remained. The different isolates obtained were screened for chitinase production based on the halo produced on plates with minimal salts medium amended with chitin (1 % chitin) (Cook 1985). The Bacillus sp., thus, obtained were maintained on Nutrient agar (NA) amended with chitin (1 %).
Morphological and phenotypic characterization of Bacillus sp.
This strain was characterised morphologically and biochemically by following Bergey’s Manual of Systematic Bacteriology (Sneath 1986) and was found to be a Bacillus sp. It was grown and maintained on NA at 30 °C. Polymerase chain reaction (PCR) was performed to amplify a partial 16S rRNA gene of the bacteria, and partial 16S rDNA sequencing was used to assist in the identification of the isolate. Isolation of genomic DNA, PCR amplification and sequencing of PCR product for analysis of 16S rRNA were conducted according to Marchesi et al. (1998). A similarity search for the nucleotide sequence of 16S rRNA of the test isolate was carried out using a blast search at NCBI (Altschul et al. 1990).
Preparation of colloidal chitin
Colloidal Chitin was prepared from crab shell chitin powder (Roberts and Selitrennikoff 1988). A few modifications were made as described: 10 g of chitin powder was added slowly into 100 mL of concentrated HCl under vigorous stirring for 2 h. The mixture was added to 1,000 mL of ice-cold 95 % ethanol with rapid stirring and kept overnight at 25 °C and then stored at −20 °C until use. When needed, the filtrate was collected and washed with 0.1 M phosphate buffer (pH 7) until the colloidal chitin became neutral (pH 7) and used for further applications.
Phytopathogens and chilli seeds
Colletotrichum gloeosporioides (OGC1) and chilli seeds (Arka Shweta variety) were obtained as a kind gift from IIHR, Hessarghatta, Bangalore.
Dual plate assay
The fungal growth inhibition capacity of Bacillus sp. strains was determined (Huang and Hoes 1976). A few modifications were made to suit the need. One 5-mm disc of a pure culture of the pathogen was placed at the centre of a Petri dish containing PDA. The Bacillus sp. was inoculated at two opposing corners. Plates were incubated for 72 h, at 28 °C, and growth diameter of the pathogen was measured and compared to control growth, where the bacterial suspension was replaced by sterile distilled water. Each experiment using a single pathogen isolate was run in triplicate. Results were expressed as the means of the percentage of inhibition of growth of the corresponding pathogen isolate in the presence of any of the strain of Bacillus sp.
Percent inhibition was calculated using the following formula:
Detection of extracellular hydrolytic activity
Plates with minimum salts medium (MSM) containing (1 % w/v) carboxy methyl cellulose (CMC) was prepared. The Bacillus sp. was spot inoculated in the centre of the plate. After an appropriate incubation period at 30 °C for 48 h, the agar medium was flooded with an aqueous solution of Congo red for 15 min. The Congo red solution was then poured off and plates containing CMC were visualised for zones of hydrolysis (Shanmugaiah et al. 2008; Moataza 2006; Teather and Wood 1982) detecting β-1,4 cellulase. Yeast Glucan containing plates (Chen et al. 1995) was used to detect β-1,3 cellulase activity. MSM with (1 % v/v) yeast cell glucan was prepared and spot inoculated with the isolate. Development of a clear zone surrounding the colony indicated enzyme production.
Assay of hydrolytic enzymes
Chitinase enzyme assay
Chitinase activity was measured with colloidal chitin as a substrate. The culture broth was centrifuged and enzyme solution of 1 ml was added to 1.0 ml of substrate solution, which was made by suspending 1 % of colloidal chitin in Phosphate buffer (pH 7.0). The mixture was incubated at 50 °C for 30 min. 1 ml of DNS was added and incubated at 100 °C in boiling water bath. The amount of reducing sugar produced in the supernatant was determined by dinitrosalicylic acid method (DNS) (Miller 1959). One unit of chitinase activity was defined as the amount of enzyme that produced 1 μmol of reducing sugars per min (Annamalai et al. 2008).
Cellulase β-1,3 assay
The specific activity of β-1,3-cellulase was determined by measuring the amount of reducing sugars liberated using dinitrosalicylic acid solution (DNS) (Annamalai et al. 2008). The culture broth was centrifuged and enzyme solution of 1 ml was added to 1.0 ml of substrate solution which contained 1 ml of yeast cell wall extract (YCW 1 % v/v). The mixture was incubated in a water bath at 50 °C for 30 min and the reaction was terminated by adding 1 ml of DNS solution and incubated in boiling water bath for 10–15 min till the development of the colour of the end product. Reducing sugar concentration was determined by optical density at 540 nm (Gadelhak et al. 2005).
Cellulase β-1,4 assay
The specific activity of β-1,4-glucanase was determined by measuring the amount of reducing sugars liberated using dinitrosalicylic acid solution (DNS) (Annamalai et al. 2008). The culture broth was centrifuged and enzyme solution of 1 ml was added to 1.0 ml of substrate solution which contained 1 ml of carboxy methyl cellulose solution (CMC 1 % v/v). The mixture was incubated in a water bath at 50 °C for 30 min and the reaction was terminated by adding 1 ml of DNS solution and incubated in boiling water bath for 10–15 min till the development of the colour of the end product. Reducing sugar concentration was determined by optical density at 540 nm (Moataza 2006).
Induction with fungal mycelium
The Bacillus sp. was grown on Nutrient broth supplemented with dead fungal mycelium (Colletotrichum gloeosporioides OGC1) as inducer for enzymes production at a concentration of 1.0 % and dispensed in Erlenmeyer flasks (250 ml), each flask containing 50 ml of medium. The flasks were autoclaved and inoculated with 1.0 ml of a pre-cultured Bacillus sp. culture. The culture was incubated in a shaker (120 rpm.), at 28 ± 2 °C. Aliquots from the flask were analyzed daily for chitinase and cellulases (β-1,3 and β-1,4) for a period of 5 days (Moataza 2006).
Preparation of hyphal wall
The pathogenic fungal culture (C. gloeosporioides) was inoculated into 50 ml of PDB broth and incubated at 30 °C for 5 days under shaking conditions. After incubation, the mycelia were collected by filtration. The mycelia were thoroughly washed with autoclaved distilled water and homogenised on ice, with a homogenizer for 5 min. The mycelia suspension was centrifuged at 10,000 rpm for 20 min at 4 °C (Remi: C 24). The pellet was resuspended in sodium phosphate buffer (0.1 M, pH 7.0). This preparation was used as substrate for the hydrolytic assay (Moataza 2006).
Hydrolytic activity of B. subtilis (wild type and mutants) culture filtrate
For assessing the hydrolytic activity, the reaction mixture (1 ml) containing 1 mg/ml of fungal mycelia with 1.0 ml of crude enzyme (from B. subtilis grown on NB + CMC) was incubated at 30 °C for 24 h. The released total reducing sugars (Miller 1959) in control and treated fungal cell wall was estimated using the DNS method.
Dual culture method
The differences in dry weights between the fungal cultures grown with B. subtilis strain or the control culture grown without any bacterium were recorded according to Broekaert et al. 1990. For this, 48 h grown dual cultures were passed through the pre-weighed Whatman No 1 filter paper. It was dried for 24 h at 70 °C and weights were measured (Saleem and Kandasamy 2002).
The fungal culture grown on NA agar plate without any bacterial culture served as control. The damage caused by the bacterium to the fungal mycelium by dual plate assay was studied microscopically. The mycelium along with the agar disc present in the inhibition zone and control mycelium was taken, stained with lactophenol cotton blue and observed under a Nikon Trinocular microscope (Saleem and Kandasamy 2002).
To characterise the antagonistic mechanism by this bacterium, a mutant of this bacterium was developed, which lost its antagonistic activity. UV mutagenesis of B. subtilis was carried out following the procedure of Miller (1992). During UV mutagenesis, the log phase culture was exposed to short wavelength UV light (280 nm, Philips TUV 30 W, G3018, Holland) from a distance of 30 cm for various time intervals (for 0.1 % UV survivors). From the serial dilutions of the mutagenized culture, 0.1 ml was plated on NA plates for isolated mutant colonies. The isolated colonies were further screened for the loss of antifungal activity using dual plate assay. The mutants were also screened for mycolytic enzyme activity and hydrolytic activity with the pathogen mycelium.
Sensitivity of the culture supernatant of wild type B. subtilis to proteolytic enzymes, TCA and heat
The crude supernatant (1 mL) was subjected to treatments for 1 h at 37 °C (for enzymes) or room temperature (for TCA). The proteolytic enzymes (Sigma) were used at a final concentration of 1 mg ml−1 in 10 mmol−1 potassium phosphate buffer, pH 7.0. The crude supernatant in buffer without enzymes as well as the enzyme solutions was exposed to the same conditions. For the heat treatment, the preparations were incubated at 70, 80 and 90 °C and autoclaving for 20 min. Antifungal activities were checked before and after all treatments on a test plate made with C. gloeosporioides as mentioned earlier (Tendulkar et al. 2007).
Germination efficiency and antagonism of the Bacillus sp. against C. gloeosporioides was checked on chilli seeds in vitro. The water agar plates were seeded with the following: Set 1-Seeds control-plain seeds coated with carboxy methyl cellulose (CMC); Set 2- Seeds coated with CMC and C. gloeosporioides spores; Set 3- Seeds coated with CMC and Bacillus sp. culture and Set 4- Seeds coated with CMC and both C. gloeosporioides spores and Bacillus sp. culture. Chilli seeds were surface sterilised successively with sterile distilled water and 0.1 % HgCl2. To remove the residual HgCl2, the seeds were washed with sterile distilled water. The isolate was inoculated into NB medium and incubated for 24 h at 30 °C (Moataza 2006). C. gloeosporioides was inoculated onto PDA plates and incubated at 28 °C for 3–4 days. The above three sets of treated seeds were seeded onto 1 % water agar plates. Plain CMC coated seeds on water agar were used as control. The four sets were monitored regularly for germination and growth. After 1 week, the sets were observed for germination and biocontrol against C. gloeosporioides coated seeds by the isolate.
All the experiments were done in triplicates and the values represented statistically are in ANOVA form.