Bio-efficacy of Bacillus subtilis isolates against soil borne pathogens

Introduction

Biological control using beneficial microorganisms particularly Bacillus subtilis has emerged as a promising strategy for managing soil borne pathogens (Kloepper et al., 2004; Compant et al., 2005) [1,2] . Bacillus subtilis is a Gram-positive, rod-shaped, aerobic and motile bacterium that belongs to the family Bacillaceae and the order Bacillales (Claus & Berkeley, 1986) [3] . This saprophytic bacterium is widely distributed in nature and can be isolated from soil, water, air and decaying plant materials (Earl et al., 2008; Chen et al., 2009) [4] . Its ability to produce a wide range of antimicrobial metabolites and promote plant growth makes it one of the most effective and well-studied biocontrol agents.

2.Materials and Methods

The present investigations entitled "Bio-efficacy of Bacillus subtilis isolates for management of soil borne pathogens" was conducted in Plant Pathology Laboratory, College of Agriculture, Nagpur during the year 2024-2025. Five isolates of Bacillus subtilis were obtained from different districts of Maharashtra (Amravati, Akola, Nagpur, Bhandara, and Buldhana) using the serial dilution method and confirmed by morphological and biochemical tests. Dual culture assays were conducted to evaluate the antagonistic potential of these Bacillus subtilis isolates against four soil-borne pathogens (Fusarium oxysporum f. sp. capsici, F. oxysporum f. sp. ciceri, Sclerotium rolfsii and Rhizoctonia bataticola).

Dual culture of Bacillus subtilis against four soil borne pathogens

The pathogens was inoculated at the center of a Petri plate containing a combined medium of Potato Dextrose Agar (PDA) and Nutrient Agar (NA). Bacillus isolates were streaked in a International Journal of Advanced Biochemistry Research 2025; SP-9(9): 1173-1178 square shape around the centrally placed pathogen. The plates were then incubated at 28 °C for three to seven days. A control plate, inoculated with the pathogen alone in the absence of any antagonistic bacteria was also maintained. All treatments, including the control, were performed in triplicates. After the incubation period, the radial growth of the fungal mycelium on each plate was measured. The percent inhibition of radial growth of the pathogen by Bacillus isolates, in comparison to the control was calculated using the formula described by Vincent (1927) [7] .

Where; I = Percent inhibition of mycelium C = Growth of fungal mycelium in control. T = Growth of fungal mycelium in treatment.

Results and Discussions

An experiment was conducted to identify efficient isolates of Bacillus subtilis, namely BS1 (T1), BS2 (T2), BS3 (T3), BS4 (T4) and BS5 (T5), against soil-borne plant pathogens using combined media (CM) plates through the dual culture technique.

Dual culture of Bacillus subtilis against F. oxysporum f. sp. capsici

The data presented in Table 1, Plate1 and Fig. 1 revealed that there was a significant difference among the five Bacillus subtilis isolates in their ability to inhibit the growth of Fusarium oxysporum f. sp. capsici. At 3 days after inoculation (DAI), the minimum mycelial growth of the pathogen was recorded in treatment T3 (8.25 mm), followed by T4 (8.75 mm) and T1 (11.50 mm). At 5 DAI, the lowest mycelial growth was again observed in T3 and T4 (12.25 mm each), followed by T1 (14.75 mm). By 7 DAI, treatment T4 exhibited the minimum mycelial growth (19.25 mm), followed by T3 (21.50 mm) and T2 (22.50 mm). In contrast, the maximum growth of F. oxysporum f. sp. capsici was consistently recorded in the control treatment. With respect to percent inhibition over control, isolate T4 exhibited the highest inhibition at 7 DAI (76.60%), closely followed by T3 (73.87%) and T1 (72.33%). Similar antagonistic potential of Bacillus strains against Fusarium wilt of sweet pepper was also reported by Abada and Ahmed (2014) [6] . The data presented in Table 2, Plate 2 and Fig. 2 indicated that there was a significant difference among the five Bacillus subtilis isolates in inhibiting the growth of Fusarium oxysporum f. sp. ciceri. At 3 days after inoculation (DAI), the minimum mycelial growth of the pathogen was recorded in treatments T2 and T3 (5.50 mm), followed by T4 (6.00 mm). At 5 DAI, treatments T3 and T4 again showed the lowest mycelial growth (8.75 mm for T3 and 10.50 mm for T1), with T3 exhibiting the highest percent inhibition (77.27%). At 7 DAI, the lowest mycelial growth was observed in T2 and T4 (10.50 mm), while the highest percent inhibition was also noted in T2 and T4 (82.13%). In contrast, the maximum mycelial growth of F. oxysporum f. sp. ciceri was consistently observed in the control treatment. All B. subtilis isolates significantly inhibited pathogen growth, with T2 and T4 showing the strongest antagonism. Similar reports highlight the efficacy of B. subtilis against Fusarium spp. through antibiotics, lipopeptides and secondary metabolites (Kumar et al., 2012) [8] .

Dual culture of Bacillus subtilis against Sclerotium rolfsii

The data presented in Table 3 and Fig 3 indicated that there was a significant difference among the five Bacillus subtilis isolates in inhibiting the growth of Sclerotium rolfsii. At 3 days after inoculation (DAI), the minimum mycelial growth of the pathogen was recorded in T3 (10.50 mm), followed by T4 (11.50 mm) and T2 (13.25 mm). At 5 DAI, treatment T3 again exhibited the lowest mycelial growth (13.75 mm), followed by T4 (14.25 mm) and T5 (17.50 mm). By 7 DAI, T3 showed the minimum mycelial growth (17.50 mm), followed by T4 (18.00 mm) and T2 (18.50 mm). In contrast, the maximum mycelial growth of S. rolfsii was consistently recorded in the control treatment throughout all intervals. With respect to percent inhibition over control, treatment T3 recorded the highest inhibition at 7 DAI (79.94%), followed closely by T4 (80.52%) and T5 (76.50%). All B. subtilis isolates significantly inhibited the mycelial growth of S. rolfsii, with T3 and T4 being most effective. These results agree with earlier findings (Gopalakrishnan et al., 2014) [9] highlighting the antagonistic potential of B. subtilis through antifungal metabolites, competition and mycoparasitism, confirming its promise as a biocontrol agent against collar rot. isolates in inhibiting the growth of Rhizoctonia bataticola. At 3 days after inoculation (DAI), the minimum mycelial growth of the pathogen was recorded in T4 and T5 (5.00 mm each), followed by T3 (8.50 mm). At 5 DAI, the lowest mycelial growth was observed in T5 (7.50 mm), followed by T4 (8.50 mm) and T3 (10.50 mm). By 7 DAI, T5 exhibited the minimum mycelial growth (10.50 mm), followed by T4 (11.00 mm) and T3 (11.50 mm). In contrast, the maximum mycelial growth of R. bataticola was consistently observed in the control treatment across all observation periods. With respect to percent inhibition over control, treatment T5 demonstrated the highest inhibition at 7 DAI (88.25%), followed closely by T4 (87.69%) and T3 (86.84%). All B. subtilis isolates significantly inhibited the mycelial growth of R. bataticola, with T5 and T4 showing the strongest antagonism. These results are consistent with earlier reports (Meena et al., 2015) [10] highlighting the role of B. subtilis in suppressing dry root rot through rhizosphere colonization and production of antifungal metabolites.

Conclusion

All Bacillus subtilis isolates exhibited significant antagonism against major soil-borne pathogens, with specific isolates showing superior activity. T4 and T3 against F. oxysporum f. sp. capsici, T2 and T4 against F. oxysporum f. sp. ciceri, T3 and T4 against S. rolfsii, and T5 and T4 against R. bataticola. The consistent inhibition across pathogens confirms the strong biocontrol potential of B. subtilis, mediated through mechanisms such as antifungal metabolite production, competition, and rhizosphere colonization.

Acknowledgement

AcknowledgmentsI am thankful to the Head of the Plant Pathology Section, College of Agriculture, Nagpur for providing all the necessary facilities for conducting the research work.

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