Isolation and maintenance of Bacillus sp.
Soil samples were collected from rhizosphere and non-rhizospheric soils in and around Odisha. Bacillus sp. was isolated by soil dilution method. Soil samples (10g) from rice rhizosphere transferred to 90 ml sterile distilled water and mixed thoroughly by shaking the flask on a rotatory shaker for 5 min. After serial dilution 0.1 ml suspension was spread over presterilized and cooled down nutrient agar plates in triplicates. The inoculated plates were incubated at 30 ± 2 ^æ%C for 2days. Rough and abundant colonies with waxy growth and irregular spreading edge were obtained and maintained on the Nutrient Agar Medium and kept at 4⁰C for further use.

Physiological and biochemical characterization of isolates were performed by using HiBacillus Identification Kit KB013 and Hi Carbohydrate TM Kit Hi25TM (Himedia) followed by as per the manufacture’s instruction. The culture strains were identified following the Bergey’s Manual of Determinative Bacteriology¹⁵.

Bacillus sp. inoculum preparation
The inoculum preparation was carried out according to the method described (Sethi and Adhikary, 2009a; Sethi and Adhikary, 2009b). Loopfuls of each isolated bacillus sp. from 2-3 days old cultures were transferred separately to 50 ml nutrient broth (NB) medium and incubated overnight at 28±2°C for 2-3 days. Viability was confirmed by taking loopful of cultures and restreaked on nutrient agar medium. This inoculum was prepared to be used in vitro for antifungal activities of the Bacillus isolates and also their biocontrol activities in vivo. The inoculums also used to net house experiments were prepared from a 24 h old shaken culture of each of the Bacillus isolate incubated at 28±2°C. Bacillus culture suspension containing 10⁷ cfu/ml was used as inoculant.

Isolation and preparation of phytopathogenic fungal sp.
Samples of diseased leaf stem, root parts and seeds were randomly collected and these fungal pathogens were isolated from infected plants and diseased seeds following water agar and blotter techniques. Besides some other standard fungal pathogens were also used in this study such as Rhizoctonia solani, Sclerotium rolfsii and Sclerotium oryzae obtained from different agricultural research institutes, India. Fungal strains and inoculum was prepared by culturing it on Potato dextrose agar (PDA) for 10 days in Petridishes. The microconidial suspension of these three pathogens was prepared by pouring 1 ml of sterile water in each petridish. The inoculum was then scrapped with the help of a sterile spatula from the surface of the Petridishes. The spores from the mycelia of the fungi to get a concentration of 10⁶ spore/ml concentration and applied in sterile soils as method described (Ajilogba et al. 2013; Adebay and Ekpo 2005).

Phosphate Solubilisation
Phosphate solubilization is an important and complex phenomenon for selectively screening the bacteria having P-solubilizing activity which have the ability to release inorganic phosphate from tricalcium phosphate. Pikovskaya’s medium, is a selective medium for phosphate solubilizing microorganisms (PSM) was used to which tricalcium phosphate (TCP) has been added as it will enhance formation of halo zones. The medium was sterilized and poured into sterile petridishes (Himedia). Isolates were streaked on the Petridishes and incubated for 3 days at 28±2°C. Bacillus isolates that were able to solubilize developed clear zones around colonies (Pikovskaya 1948).

Determination of Indole Acetic Acid Production (IAA)
Luria Bertani Broth (Himedia) was prepared and freshly grown cultures were inoculated into 10 ml LB broth in each test tube and incubated at 28±2°C for 24 h at 120rpm. Grown cultures removed from each test tube and centrifuged at 10,000 rpm for 15 min. An aliquot of 1 ml of supernatant was transferred into a fresh tube to which two drops of orthophosphoric acid were added. The mixture was incubated at room temperature for 25 minutes. The development of a pink colour indicated the presence of indole acetic acid (Gordon and Weber 1951.).

Detection of Hydrogen Cyanide (HCN) Production
Production of HCN was detected according to the method described (Lorck 1948). 24h freshly grown cells were spread on agar medium which contained 4.5 g/l glycine had been added and a sterilized filter paper saturated with 1% solution of picric acid and 2% sodium carbonate was placed in the upper lid of the Petridish, which was then sealed with parafilm and incubated at 28±2°C for 4 days, a change in colour of the filter paper from yellow to reddish brown was an index of cyanogenic activity while no colour change represent no cyanogenic activity.

Determination for the production of different cell wall degrading enzymes of Bacillus isolates Chitinase activity
The different isolates obtained were screened for chitinase production based on the halo produced on plates with minimal salts medium amended with chitin. The qualitative assay for chitinase production was performed following the method (Dunne et al. 1997) and modified method given by (Roberts and Selitrennikoff , 1988). Isolates were inoculated by spotting on the plates having chitin minimal medium (CMM) as sole source of carbon and incubated at 30±2°C for 10 days. These plates were examined for development of clear zones around colonies.

Preparation of colloidal chitin
5.0 g of chitin was added to 60 ml of concentrated HCl (acid hydrolysis) by constant stirring using a magnetic stirrer at 4°C and kept in refrigerator overnight. The resulting slurry was then added to 2000 ml. of ice-cold 95% ethanol and kept at 26°C for overnight (ethanol neutralization). Then it was centrifuged at 3000 rpm for 20 min at 4°C. The pellet was washed with sterile distilled H₂O. The final colloidal chitin was stored at 4°C until further use.

Final chitinase detection medium
The final chitinase detection medium per litre comprises of 4.5 g colloidal chitin, 0.3 g magnesium sulphate, 3.0 g ammonium sulphate, 2.0 g potassium dihydrogen phosphate, 1.0 g citric acid monohydrate, 15 g agar, 0.15 g bromocresol purple and 200 µl of tween-80. The pH of the media was maintained at 4.7 and autoclaved at 121°C for 15 min. The fresh culture of Bacillus isolates to be tested for chitinase activity were inoculated into the sterile plates containing chitinase detection medium and incubated at 28±2°C for seven days and observed for the colored zone formation. Chitinase activity was identified due to the formation of purple colored zone. Color intensity and diameter of the purple coloured zone were taken as the criteria to determine the chitinase activity after three days of incubation.

Protease activity
The production of protease, a fungal cell wall degrading enzymes which is an important mechanism of fungal growth inhibition was detected for all selected isolates. Protease activity was determined according to the modified method described (Berg et al. 2002). Skim milk agar medium (5.15/100ml) was used for detection of protease activity, 48h old freshly grown cultures were inoculated on skim milk agar medium and incubated at 28±2°C for two to three days. Bacillus sp. which shows the protease activity gave a clearance zone indicating the production of protease enzyme. The degree of protease activities were measured based on the diameter of clearance zone.

Carbon Utilization study
Different carbon utilization of Bacillus sp. was studied by the HiCarbo Utilization kit (Himedia). Hi carbokit was used to study the biochemical profile of organisms is a standardized colorimetric identification system utilizing 35 carbohydrate utilization test. The test was based on the principle of change of pH and substrate utilization by selected bacteria. On incubation or organism undergo metabolic changes which are indicated by a spontaneous colour change in the media. A combination of 35 tests for utilization of carbohydrate tests. Kit contains Part A, Part B each having 12 carbohydrates utilization tests and Part C containing 11 sugars and 1 control. The organism was pure cultured and culture inoculums turbidity was 0.5 OD at 620 nm, then inoculate each well with 50µl of the above inoculums by surface inoculation methods and incubate at 28±2⁰C for 24 hr and data was interpreted as per result interpretation chart.

Antagonistic activity of Bacillus sp.
The test was carried out to see the effect of the Bacillus isolates on the three important rice phytopathogens in vitro. Pathogen inhibition test was performed by dual culture plate assay One 5-mm disc of a pure culture of the pathogen was placed at the corner of a Petri dish containing NA+PDA (Nutrient agar with Potato dextrose agar). A loopful of Bacillus culture was streaked over the surface of NA+PDA plate at opposing corners. Plates were incubated for 10 days at 28±2⁰C, and examined for inhibition of fungus growth by the Bacillus isolates. The growth diameter of the pathogen was measured and compared to control growth, where the bacterial suspension was replaced by sterile distilled water. Each experiment was used a single pathogen isolate was run in triplicate. The Results were expressed as the mean value with standard error deviation in inhibition distance between the growths of the fungus isolate and the presence of bacillus isolate tested. Percent inhibition was calculated using the following formula: % inhibition = [1-(Fungal growth/Control growth)] x100

Control experiment with only growth of Bacillus isolates and each pathogen in each separate Petridishes of three replicates were observed.

Pot experiment for Efficacy of Bacillus on biomass and yield
Healthy seeds of rice were treated with different treatments of Bacillus isolates and directly sown into 24cm-diameter pots. A loopful of bacterial isolate (starter culture) was transferred to a 500 ml flask containing sterile liquid medium and grown aerobically in a rotary shaker at 120-160 rpm for 48h at 28±2⁰C (Lamsal et al. 2013; Sethi and Adhikary 2009a,b; Sethi and Adhikary 2014; Sethi and Adhikary 2015). The bacterial culture suspension was diluted to become 10⁹ CFU/ml as per method described²³. Seeds were surface sterilized for 30 min in 20% sodium hypochloride, followed by rinse with 70% ethanol before sown in earthen pot (4 cm diameter with hole).  Each pot contained sterile garden soil. The outlined of the experimental design using 10 replicates (each replicates comprised of 3 plants) for each of the Bacillus isolates, a negative control with no fungus and a positive control with inoculation of fungus. Seeds were treated with bacterial suspension (containing 10⁸ CFU/ml) of each Bacillus sp. in the sterile trays 24h before showing in nursery bed. Seedlings were transplanted in to 30cm diameter pots each pots contain sterile soil. The different treatments with Bacillus isolates were applied as outlined in the experimental design using seven trials where each trial composed with ten replicates and a negative control with no pathogens. Plants watered daily in the net house at for 8 weeks. At the end of 8 weeks, 10 plants (one plant of each replicates) were harvested to assess the effect of the various Bacillus isolates on the different vegetative growth parameters. Significant difference was assessed from the mean of each of the different treatments. The experiment was repeated twice. The growth parameters assessed include: length of shoot and root length, biomass and randomly selected seedlings were used to determine each parameter per treatment. Cholorophyll content was also measured with a chlorophyll meter (SPAD meter, USA)

Statistical analysis
All experiments were performed twice. Analysis of Variance and Duncan’s multiple range test (DMRT) was employed to test for significant differences between treatments at p = 0.05 (Gomez and Gomez, 1984).