INTRODUCTION Rice crop is affected by several biotic factors i.e., viruses, fungus, bacteria, nematodes, and insect pests. Many diseases produced by diverse phytopathogens, such as blast, sheath blight, sheath rot, stem rot, and bacterial leaf blight (BLB), have a significant impact on rice production. Among these diseases, rice sheath blight (ShB) caused by Rhizoctonia solani Kuhn, is a devastating disease that causes significant yield loss and quality degradation throughout the world (Eizenga et al., 2002; Nagarajkumar et al., 2004; Nirmalkar et al., 2017a). Management of diseases of crop plants is difficult due to absence of resistance in the host and unavailability of resistant cultivars of rice, therefore, chemical control is only effective methods for the management of diseases however, it also causes soil and water pollution. Despite the fact that pesticides are recommended for pathogen reduction, they are not considered to be long-term remedies because of concerns about cost, fungicide residues, exposure risks, toxicity to non-target organisms, and other health and environmental issues. Therefore, current efforts have been concentrated on producing environmentally safe & ecofriendly, long-lasting, and effective therapies against a wide range of plant diseases. Thus, the alternative ways for reducing the use of harmful agrochemicals have become a prominent emphasis in recent years (Boukaew et al., 2013). Biocontrol is a critical strategy for reducing the use of chemicals in disease management (Han et al., 2015; Nirmalkar et al., 2017a). The Bacillus spp. are classified in the order Bacillales and family Bacillaceae. Bacillus subtilis was first discovered by Ehrenberg in 1835. First known as Vibrio subtilis, it was renamed in 1872 by Cohn. Bacillus subtilis is a gram-positive, rod-shaped bacteria with peritrichous flagella (Nakano and Hulett 1997) and spore-forming bacterium that is widely distributed in the soils and environment, having the characteristics of high thermal tolerance, rapid growth in liquid culture, and readily form resistant spores. Several important plant pathogens, including Fusarium sp. (Cao et al., 2011), Rhizoctonia solani (Kumar et al., 2012), and Sclerotium rolfsii, can be suppressed by B. subtilis (De Curtis et al., 2010). Bacillus subtilis, which forms endospores, is one of the PGPRs that plays an important role in plant growth promotion and biocontrol of plant pathogens (Glick, 1995). Now a days, widely used of biological agent in reducing plant diseases has become more attractive and effective method due to their benefits and advantage of plant growth enhancing besides disease control. Such enhancement and benefits has been found in Bacillus subtilis. Therefore, in view of plant growth promotion and disease suppression ability of Bacillus subtilis the present study on antagonistic activity and bioefficacy of Bacillus subtilis against sheath blight of rice. MATERIALS AND METHODS Bacterial strains. Eleven isolates of Bacillus subtilis were used for present investigation. Six isolates of Bacillus subtilis were procured from State Bio Control Laboratory (SBCL), Chorbhatti, Bilaspur, Chhattisgarh and five isolates were obtained from the rhizosphere soil samples of different rice growing region of Chhattisgarh. Isolation of Bacillus subtilis. One g of soil sample was suspended in 9 ml sterile water and subjected to serial dilution (10-1-10-8). An aliquot of 0.1 ml/ 100µl of each dilution was spread on LB agar medium (Tryptone 10g, Yeast extract 5g, NaCl 10g, Agar agar 15g in 1 L distilled water) by pour plate method (Janisiewicz, 1988 and Roberts, 1990; Pramer and Schmidt 1965). The inoculated plates were incubated at 28° for 24 h. After incubation, the individual colonies were selected based on the their colour, shape, edges further subculture to obtained pure culture (Rangaswami and Mahadevan 2008). The isolated pure colonies were examined to their morphological characteristics and gram staining for the identification of isolates as Bacillus subtilis. Rhizospheric isolates designated as BS7 to BS11 and procured isolates designated as BS1 to BS6. Diseased plant sample collection. Diseased plant samples of sheath blight of rice caused by Rhizoctonia solani were used in the present studies was collected from rice field from Sendri, Bilaspur, Chhattisgarh. Isolation and identification of Rhizoctonia solani. For the isolation of R. solani, infected leaf sheath/ tillers were used and surface sterilize with 0.5% sodium hypo chlorite for 3 min and washed with distilled water in three changes. Sheath segments were blotted dry with blotting paper and cut into small bits using sterile blade and placed on Potato Dextrose Agar (PDA) medium with the help of forceps. Inoculated plates were incubated at 25±2oC for 7 days. Young active growth of fungal mycelial (5mm) was cut with the help of cork borer and sub-cultured on a new PDA medium to obtained pure culture. Fungal cultures were maintained on PDA slants and stored at 4oC in refrigerator for further use (Killani et al., 2011). Pathogenicity test: Before the experiment was carried out to test the virulence of R. solani. Artificial inoculation of rice seedlings sown in earthen pot using sclerotial inoculation method. In this method growing sclerotia was used and placed in between the leaf sheath of rice. After 3 days of inoculation, a typical symptoms of sheath blight were appeared on to the sheath with greyish, water soaked lesion, dark brown margin on the leaf sheath above water line. This confirmed that pathogen (R. solani) has ability to cause disease and expressed in the form of symptoms (Nagendran et al., 2019). To screen of the isolates of Bacillus subtilis for their antagonistic activity against Rhizoctonia solani under in vitro Dual culture method: The antagonistic activity of different isolates of Bacillus subtilis against Rhizoctonia solani and was investigated performed by dual culture technique under in vitro condition (Elkahoui et al., 2012). A mycelial disc (5mm), obtained from 5 days old culture of Rhizoctonia solani (test pathogen) was cut with the cork borer, and a loopful Bacillus subtilis of 24 h old cultures were placed on the Bacillus Potato Dextrose Agar medium (Rajkumar et al., 2018; Anillo et al., 2021) opposite to each other, equidistant from the periphery, and Petri dishes were incubated at 28±2oC. Three replications were maintained for each treatment. The Petri plate containing test pathogen alone served as control. After 7 days of incubation, radial growth of pathogen was recorded, and the percentage inhibition was calculated using following formula (Muthukumar and Venkatesh 2013). Percentage of mycelial growth inhibition was calculated by using the formula: I = (C-T/C×100) where, I is inhibition of radial mycelial growth, C is radial growth measurement of pathogen in control (mm) and T is radial growth measurement of pathogen (mm) in the presence of antagonists. After 7 days of incubation, number of sclerotia was counted from each treatment (Nagendran et al., 2019). In vivo evaluation of isolates of Bacillus subtilis against sheath blight of rice Mass culture of Bacillus subtilis. A single colony of 24 h old culture of test isolates, picked from the culture slants, was transferred and grown to a 250 ml capacity of conical flask containing 100ml LB broth medium and incubated at room temperature for 4 days at 200 rpm using arotatory shaker. The prepared culture being used as a mass culture. Ten % (10ml broth/100ml distilled water) bacterial suspension was used as spray material (Kumar et al., 2012; Khedher et al., 2015). Preparation of pathogen inoculums. Five mm diameter of mycelia disc were used from 48 hold active growth culture of R. solani was inoculated on PDA medium contained in 30cm diameter of plastic plate seal covered with sterile polythene. The inoculated plates were incubated at 25±2° in BOD incubator for 7 days. After incubation, fungal cultures were cut into rectangular sections (3 cm length× 1.5cm width) and placed into the sheath in between the tillers (Nagendran et al., 2019). In vivo studies: Twenty days old seedlings were used for transplanting. Five seedlings were transplanted into earthen pots of 9'' height with the diameter of 6.3'' having loam soil. After the establishment of seedlings and at maximum tillering stage, each pot was inoculated with mass culture of R. solani. Fungal inoculums grown on PDA was cut into rectangular bits and placed into the sheath in between the tillers. After inoculation, plants were covered with polythene bag to maintain humidity. After the appearance of first symptoms, liquid formulations (10ml/100ml distilled water) of different strains of B. subtilis were sprayed on host plant. Hexaconazole@0.1% was used as positive control while distilled water alone used as a negative control. Two sprays were done, first spray was done after the disease appearance and second spray was done after 10 days interval of first spray. Each treatment was maintained in three replications. Pot experiment were carried out in greenhouse conditions using CRD design for 45 days. The plants were observed critically for visual scoring of disease incidence and severity using the standard evaluation scale (0-9 scale) suggested by the International Rice Research Institute (IRRI), 1996. Visual scoring of sheath blight incidence rating scale given by International Rice Research Institute (IRRI), 1996. Per cent Disease Index (PDI) was calculated by using the formulas given by IRRI, (1996). PDI= (Sum of all individual rating)/(Total No.of plant observed)×100/(Maximum disease scale) Observations recorded. Percent Disease Index (PDI) based on 0-9 scale (Nagendran et al., 2019). Percent tillers mortality: Number of dead tillers/Total number of tillers ×100 Statistical analysis. All the experimental data were statistically analysed using appropriate design i.e., CRD with desired transformation as applicable. RESULTS AND DISCUSSION Experiment was conducted under in vitro conditions, the antagonist Bacillus subtilis exhibited a significant inhibition of the R. solani compared to the control (Table 1, Fig. 1). All the eleven B. subtilis isolates were tested and the maximum percent growth inhibition was recorded by B. subtilis isolate BS1 (88.73%) followed by BS3 (78.84%) which was statistically at par among themselves. Whereas, the other B. subtilis isolate showed (60.14%) per cent inhibition by B. subtilis isolate BS2, (59.83%) inhibition by B. subtilis isolate BS8 (59.83%) which were statistically at par among themselves, whereas, the least per cent inhibition (2.14%) of R. solani was recorded by B. subtilis isolate BS11 over control. The maximum number of sclerotia was recorded in B. subtilis isolate BS11 (52.33) followed by BS10 (48.66), BS9 (47.00), BS4 (45.33), BS5 (41.66), BS6 (33.33), BS2 (25.66), BS8 (21.33), BS7 (19.00) and BS3 (18.66), whereas, minimum number of sclerotia was recorded in B. subtilis BS1 (16.33). Severely, Huang et al. (2017) also reported such types of results and concluded their findings that in vitro antagonistic activity of Bacillus subtilis strain SL-44 showed significant antifungal activities with an inhibition rate of 42.3% against Rhizoctonia solani. Jamali et al. (2019) reported that the Bacillus subtilis strain RH5 exhibited significant antagonistic activity (84.41 %) against the fungal pathogen Rhizoctonia solani. Nagendran et al. (2019) reported that Bacillus subtilis strain Bs 7 showed maximum mycelial growth inhibition 51.1% over the control followed by Bs19 observed 49.4% mycelial growth reduction against Rhizoctonia solani causing sheath blight diseases. Ghazy and Nahrawy (2021) reported that the antifungal activity of Bacillus subtilis significantly inhibited with inhibition percent was 51.55% and reduced the growth of Cephalosporium maydis by 4.36 cm compared to control. In vivo evaluation of isolates of Bacillus subtilis against sheath blight of rice. Pot experiment was carried out in the in vivo greenhouse condition for disease controlling potential of different isolates of Bacillus subtilis against sheath blight of rice. Different isolates of B. subtilis were used as foliar spray@10% concentration in controlling sheath blight of rice. All the isolates of B. subtilis were found more effective in plant disease reduction over control. Results indicate (Table 2, Fig. 2) that the least per cent disease index was recorded in B. subtilis treatment T4 (33.29%) which showed maximum disease controlling potential on sheath blight and which was statistically at par with the other treatments i.e. T8 (36.28%) and T1 (40.70%), whereas, the isolates i.e. T9 (73.32%) was not effective in controlling sheath blight and other B. subtilis isolates i.e. T5 (64.40%) and T2 (62.29%) which was significantly effective in controlling sheath blight over control (80.73%) which was statistically at par with each other. The significantly less minimum per cent tillers mortality was recorded from B. subtilis T4 treatment (42.73%) and treatment T3 (42.80%) compared to control which was statistically at par among themselves which showed the less effective treatment on sheath blight over control (91.26%). Moreover, significant maximum per cent tillers mortality was recorded in B. subtilis treatment T9 (89.69%) followed by B. subtilis treatment T2 (75.19%) and B. subtilis treatment T10 (68.95%), T12 (68.86%), T5 (68.64%), T6 (67.11%) which were statistically at par with each other. Present results confirmed to the finding of Ali and Nadarajah (2013) also reported that the efficacy of Bacillus subtilis and Trichoderma isolates showed effective is ease reduction under greenhouse conditions against Rhizoctonia solani. Combine application of Bacillus subtilis and Trichoderma isolates T2+Bs and T7+Bs showed significant disease severity and disease incidence (4.33% and 11%), respectively, over the control (42.33% and 67.0%). B. subtilis showed 17.67% and 44.33% disease severity and disease incidence against R. solani alone. Similarly, Khedher et al. (2015) reported the efficacy of Bacillus subtilis V26 as a biological control agent against Rhizoctonia solani on potato under pot experiment. Here ported that the disease incidence on potato plant roots was significantly lower compared to control. StrainV26 (SPO2-CM) treatment resulted in low disease incidence on potato plant roots, with 63% of reduction. The application of V26 significantly reduced potato tuber caused by black scurf diseases and decrease disease incidence rate to 81% over the untreat done when using strain V26 (SPO2) treatment. Different bioagents i.e., T. harzianum, B. subtilis and P. fluorescens earlier proved as a potential bio-agent of soil borne plant pathogens (Nirmalkar et al., 2017) was found effective against different pathogens i.e., Rhizotonia solani, Fusarium oxysporum f. sp. Ciceris etc may be used alone or in combination with different bioagents as a seed and soil treatment.