Evaluation of Antagonists Against Rhizoctonia solani Causing Foot Rot in Broccoli

dBroccoli (Brassica oleracea L. var. italica) is a nutritionally rich crop with increasing global demand, yet its productivity is severely constrained by soil-borne pathogens such as Rhizoctonia solani, the causal agent of foot and root rot. Chemical fungicides, while widely used, face limitations due to resistance development, environmental risks, and regulatory concerns, highlighting the need for sustainable alternatives. The present study evaluated the antagonistic potential of Trichoderma harzianum, T. viride, and Bacillus subtilis against seven isolates of R. solani under in vitro conditions. Antagonists were isolated from broccoli field soils and tested using dual culture and paper disc methods on Czapek’s sucrose nitrate agar medium. Results revealed significant variability in pathogen inhibition across isolates and antagonists. T. harzianum demonstrated the highest mean mycelial growth inhibition (63.04%), followed by T. viride (57.66%) and B. subtilis (49.78%). Maximum inhibition was recorded in R Isolate-006, where T. harzianum achieved 69.42% suppression and T. viride 67.28%. Conversely, the lowest inhibition was observed in R Isolate-005, with B. subtilis showing only 42.35% suppression. The findings confirm that T. harzianum consistently exhibited superior antagonistic activity, suggesting its potential as a promising biocontrol agent against R. solani. These results align with earlier reports of Trichoderma spp. exhibiting mechanisms such as mycoparasitism, antibiosis, and competition, thereby supporting their integration into eco-friendly disease management strategies. Further research on formulation development and field-level validation is essential to translate these laboratory findings into practical applications for sustainable broccoli production.

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Introduction

Broccoli (Brassica oleracea L. var. italica) has emerged as one of the most important vegetable crops worldwide due to its exceptional nutritional value and increasing market demand (Siomos et al., 2022) [15] . The crop is rich in vitamins A and C, minerals, fiber, and bioactive compounds, including glucosinolates and sulforaphane, which provide protective effects against cancer, diabetes, stroke, and myocardial infarction (Mittal et al., 2025) [12] . Broccoli is a high-value crop with the potential to double farmers' income through organic farming practices. The crop's by-products offer sustainable applications in developing agrochemicals, nanoparticles, and functional foods, supporting zero-waste solutions with economic benefits (Han et al., 2021) [5] . Chemical fungicides remain essential for crop disease control, but face significant limitations, including resistance development, environmental impacts, and regulatory challenges. Fungicide resistance can develop in virtually any pathogen through mechanisms such as target site alterations, efflux pump activation, and mutations (Janssen et al., 2024). This resistance poses a threat to effective crop protection across major fungicide classes, including benzimidazoles, sterol demethylation inhibitors, and succinate dehydrogenase inhibitors (Yin et al., 2023) [18] . The discovery of new fungicides faces mounting regulatory hurdles and increasing costs, though innovation continues, particularly in novel modes of action for oomycete control (Leadbeater, 2016) [9] . Resistance management strategies include rotating different modes of action, limiting applications of site-specific fungicides, and implementing Integrated Pest Management programs (Yin et al., 2023) [18] . Microbial antagonists play a crucial role in the biological control of plant pathogens, with Trichoderma, Pseudomonas, and Bacillus being the most prominent genera (Mahapatra et al., 2024) [11] .

Trichoderma species establish symbiotic relationships with plant roots and employ multiple mechanisms, including resource competition, production of antifungal metabolites, stimulation of plant defense mechanisms, and mycoparasitic interactions that directly attack pathogenic fungi (Elango et al., 2024) [3] . These biological control agents support sustainable disease management strategies and offer environmentally friendly alternatives to chemical pesticides, with proven effectiveness in laboratory, greenhouse, and field applications (Bansode, et al., 2022) [1] .

Materials and Methods

Isolation of Trichoderma and Bacillus spp.: Ten nonrhizosphere soil samples were collected from the agricultural fields of Chitali Putali village in the Jalna district to isolate Trichoderma and Bacillus spp. The soil samples were collected randomly and diagonally from a broccoli field and were used for the enumeration of fungi following the Soil Plate Method (Warcup 1950; Parkinson 1973) [13,17] on Martin's peptone dextrose agar medium. For the isolation of Bacillus bacteria, Thornton's standardized medium was utilized following the Soil Dilution and Plate Count Method (Waksman 1927) [16] . The Petri dishes were incubated at 28 ± 1°C for 5 days, and the recording of colonies for various microorganisms continued for 15 days or until no new colonies developed.

Test for Antagonism

Trichoderma spp. and Bacillus sp. isolated from infested soil were assessed for their antagonistic properties against seven isolates of R. solani on Czapek's dox medium in Petri dishes. A 5mm diameter piece of R. solani was transferred to the center of a Czapek's dox medium Petri dish, and circular pieces of T. harzianum and T. viride, grown on PDA, were placed at equidistant points near the periphery of the dishes. The Petri dishes were incubated at 28 ± 1°C for 7 days, and observations were made to record the growth inhibition of R. solani. For the Bacillus sp. paper disc plate method (Loo et al. 1945) [10] was implemented as follows. Circular discs (5 mm in diameter) made from Whatman filter paper (No. 42) were cut and dipped in the filtrate of Bacillus sp. These discs were then placed 2 cm inward from the periphery of Petri dishes at three equidistant points, with the inoculum of the pathogen (R. solani) positioned at the center. The inoculated dishes were kept in an incubator at a temperature of 28±1°C for 7 days, during which observations were made to measure the inhibition zone of R. solani.

Result and Discussion

Isolation of Trichoderma and Bacillus spp. A notable population of T. harzianum, T. viride, and various other fungal colonies was observed, alongside a similarly significant population of B. subtilis and other bacterial colonies. The results presented in Table 1 indicate that all three antagonists effectively inhibited the mycelial growth of the seven isolates of Rhizoctonia solani. Among these antagonists, Trichoderma harzianum demonstrated the highest mean inhibition rate at 63.04%, followed by T. viride at 57.66% and Bacillus subtilis at 49.78%. This suggests that T. harzianum was the most effective antagonist against the tested isolates. At the isolate level, the highest inhibition was measured in R-Isolate-006, with 69.42% inhibition by T. harzianum and 67.28% by T. viride. Conversely, the lowest inhibition was recorded in R Isolate-005, with only 42.35% inhibition by B. subtilis. Overall, the mean values indicate that while B. subtilis was comparatively less effective, but it still exhibited moderate suppression of the pathogen. Several studies have demonstrated the effectiveness of Trichoderma and Bacillus species as biocontrol agents against Rhizoctonia solani. Atef (2008) [1] found that combining T. harzianum and B. subtilis was more effective than using each agent alone for controlling wheat root rot, with the combination improving biochemical parameters in treated seedlings. Seema & Devaki (2012) [14] and Bansode et al (2022) [2] evaluated multiple biocontrol agents against R. solani, reporting that T. viride achieved 70% growth inhibition, T. harzianum 67%, and B. subtilis 50% in dual culture assays. The antagonistic effect was attributed to hyphal interactions causing pathogen lysis. Grosch et al. (2006) [4] selected six Trichoderma isolates, identified as T. reesei and T. viride, which significantly reduced Rhizoctonia symptoms in potato sprouts and controlled dry mass loss in lettuce under both controlled and field conditions. Interestingly, Kim et al. (2008) [2] demonstrated that certain Bacillus isolates could antagonize Trichoderma species, with isolates M27 and RM29 strongly inhibiting mycelial growth of T. harzianum, T. koningii, and T. viridescens.

R. solani isolate	Mycelial growth inhibition (%) of R. solani by antagonists	—	—	Mean
	T. harzianum	T. viride	B. subtilis sp.
R Isolate-001	58.45 (49.90)	53.62 (46.21)	54.28 (43.90)	55.45 (46.67)
R Isolate-002	62.13 (51.70)	56.08 (48.33)	52.47 (45.10)	56.89 (48.38)
R Isolate-003	67.58 (55.40)	61.32 (50.92)	47.20 (41.56)	58.70 (49.29)
R Isolate-004	61.05 (51.20)	55.22 (47.95)	45.10 (40.80)	53.79 (46.65)
R Isolate-005	57.92 (48.67)	46.95 (41.70)	42.35 (39.22)	49.07 (43.19)
R Isolate-006	69.42 (56.93)	67.28 (55.16)	51.25 (44.60)	62.65 (52.23)
R Isolate-007	68.73 (56.30)	59.15 (49.78)	55.80 (47.21)	61.22 (51.10)
Mean	63.04 (52.16)	57.66 (48.58)	49.78 (43.77)	56.83 (48.17)

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Effect of antagonists on the growth of Rhizoctonia solani on Czapek's sucrose nitrate agar medium in dual culture

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Conclusion

This study demonstrates that these biocontrol agents have potential for managing root rot diseases. Future research should focus on more comprehensive investigations into the relationships within various pathosystems and the interactions between microorganisms and host plants. Such studies are essential for developing effective biocontrol strategies for specific plant diseases.

Acknowledgement

Acknow2edgementThe author expresses gratitude to our esteemed research supervisors for their invaluable guidance and the provision of essential laboratory facilities throughout this project.

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