In-vitro study of fungicides, botanicals and bioagents against Sclerotium rolfsii Sacc. causing collar rot disease in tomato (Solanum lycopersicum L.)

Introduction

Tomato (Solanum lycopersicum L.) is a widely cultivated vegetable crop in tropical and subtropical regions, belonging to the family Solanaceae. Tomato cultivation is severely affected by collar rot caused by Sclerotium rolfsii Sacc, a major soil-borne disease responsible for 10-45% yield losses in tropical and subtropical regions. It is one of the most destructive diseases of tomato in Karnataka, causing collar rot, wilting, and plant death, leading to significant yield losses and reduced market value.It is pertinent to generate information on the efficacy of available and new fungicides, botanicals and bioagents for managing the disease. Hence, the present study was undertaken to screen various fungicides, botanicals and bioagents under in vitro conditions against Sclerotium rolfsii.

Materials and Methods

Isolation and purification of S. rolfsii

The fungus was isolated from collar rot infected tomato plants by direct plating method and were aseptically placed on Potato Dextrose Agar (PDA) medium. The plates were incubated at 27 ± 1 °C for 3-5 days. The emerging fungal growth was purified by the hyphal tip technique by transferring a 2-3 mm hyphal tip from the actively growing colony margin onto fresh PDA medium and incubating at 27 ± 1 °C for 5-7 days. The fungal colonies, on development were identified using simple microscope and recorded the mycelial characters. The purified culture was examined for contamination to obtain a pure culture. The pure culture was maintained on PDA slants at 4°C and sub cultured periodically for further studies. The fungalcolonies development was identified and proved pathogenicity.

In vitro evaluation of fungicides

Four systemic fungicides (Difenoconazole 25% EC, Carbendazim50%WP, Propiconazole 25% EC and Tebuconazole 5% EC). Contact fungicides (Captan 50%WP, Chlorothalonil 75%WP, Copper oxychloride 50% WP and Mancozeb 75%WP) combi product fungicides (Tebuconazole 50% + Trifloxystrobin 13.28%) 75% WG, (Carbendazim 12% + Mancozeb 63%) 75% WP, (Carboxin 37.5% + Thiram 37.5%) 75% WP and (Captan 70% + Hexaconazole 5%) 75% WP were tested against S. rolfsii for inhibition of the fungal radial growth on potato dextrose agar by using poisoned food technique against S. rolfsii for inhibition of the fungal radial growth on potato dextrose agar by using poisoned food technique Wedajo (2015) [15] . In vitro condition, Systemic fungicides were tested at 0.025, 0.05 and 0.1 per cent concentrations. For both contact and combi product were tested in 0.1, 0.15, 0.2 per cent concentrations.

In vitro evaluation of botanicals

Nine botanicals viz wild senna (Leaf extract), Datura (Leaf extract), Mint (Leaf extract), Parthenium (Leaf extract), Mulberry (Leaf extract), Garlic (Bulb extract), Tulsi (Leaf extract), Ginger (Rhizome extract) and Kalmegh (Leaf extract) were tested against the growth of S. rolfsii in vitro by poisoned food technique Wedajo (2015) [15] . Fresh healthy plant parts of 50 g (leaves/bulb) collected from field were washed with distilled water and air dried and crushed in 50 ml of sterile water. The crushed product was tied in muslin cloth and the filtrate was extracted. The stock solution was further diluted into desired concentrations of 5.0, 7.5 and 10.0 per cent. The results were expressed in terms of per cent inhibition of mycelial growth over control. Per cent inhibition of growth of the test fungus was calculated by using the formula of Vincent (1947) [13] .

In vitro evaluation of bioagents

The twenty fungal antagonists viz., T. yunnanense (12 isolates), T. atrobrunneum (5 isolates), T. crassum (2 isolates) and T. lixii (1 isolate) were collected from department of plant pathology, Kittur rani chennamma college of horticulture, Arabhavi. Were evaluate their bioefficacy through dual culture technique. (Mc Kinney, 1923) The bioagents and the S. rolfsii. were inoculated side by side on a single Petri dish containing solidified PDA medium. Three replications were maintained for each treatment with one control by maintaining only pathogen and bioagent separately. They were incubated at 27±1 o C. The diameter of the colony of both bioagents and the pathogen was measured in two directions and average was recorded. Per cent inhibition of growth of the test fungus was calculated by using the formula of Vincent (1947) [13] .

Results and Discussion

In vitro evaluation Fungicides Among the systemic fungicides, Tebuconazole 5% EC and Propiconazole 25% EC exhibited 100% inhibition at all concentrations (0.025, 0.05 and 0.1%) respectively and were significantly superior to others. (Table 1) Difenoconazole 25% EC showed that next best effective fungicide (up to 92.42%), while Carbendazim 50% WP was least effective (13.17%). Interaction effects revealed complete inhibition with Tebuconazole 5% EC and Propiconazole 25% EC at all concentrations and with Difenoconazole 25% EC at 0.1% were shown cent percent inhibhition. The Carbendazim 50% WP remained significantly inferior to inhibiting fungus. Where as all the tested combi product fungicides showed complete inhibition (100%) of mycelial growth of S.rolfsii at all concentrations. The fungicides Captan 70% + hexaconazole 5% WP, Carboxin 37.5% + Thiram 37.5% WP, Carbendazim 12% + Mancozeb 63% WP, and Tebuconazole 50% + Trifloxystrobin 13.28% WG recorded cent per cent mycelial inhibition, indicating that all fungicides were equally effective against the pathogen under vitro conditions (Table 1). This may be because by these fungicidal combinations inhibit mycelial growth disrupting essential fungal processes such as ergosterol biosynthesis, cell division, enzyme activity, and mitochondrial respiration, providing both protective and systemic action. These findings agree with earlier reports by Chowdhury et al. (1998) [4] , Virupaksha Prabhu and Hiremath (2003) [14] , Kulkarni (2007) [9] and Arunasri et al. (2011) [2] . Among contact fungicides evaluated, captan 50% WP, chlorothalonil 75% WP and mancozeb 75% WP completely inhibited the mycelial growth of Sclerotium rolfsii (100%) at all tested concentrations (0.1, 0.15, and 0.2%) and were significantly superior to copper oxychloride. Copper oxychloride 50% WP was the least effective, recording mycelial inhibition of 2.96%, 3.95%, and 8.64% at 0.1, 0.15, and 0.2% concentrations, respectively, with a mean inhibition of 5.18%. The overall mean mycelial inhibition increased slightly with increasing concentration, from 75.74% at 0.1% to 75.99% at 0.15% and 77.16% at 0.2%. This fungicides shows strong inhibition may be attributed to their multi-site mode of action. Karthikeyan et al. (2006) and Reddy et al. (2010) [11] .

In vitro evaluation of botanicals

The nine botanicals tested against Sclerotium rolfsii. Tulsi (Leaf extract) recorded the highest mean mycelial inhibition of 65.81%, followed by Garlic (Bulb extract) with 54.77% and Ginger (Rhizome extract) with 40.75%. Moderate inhibition was observed in Mulberry (Leaf extract) (25.11%), Kalmegh (Leaf extract) (20.69%), Parthenium (Leaf extract) (19.14%) and Datura (Leaf extract) (18.95%). The least inhibition was recorded in Mint (Leaf extract) (15.83%) and Wild senna (12.12%). The high antifungal activity of Tulsi may be attributed to bioactive compounds such as eugenol and phenolics, which inhibit fungal growth (Kaur et al., 2024) [8] . In which all tested botanicals are stastically significant with each other. Similar observations were reported by Sahana et al. (2017) [12] , who found that aqueous extracts of medicinal plants effectively suppressed the growth of Sclerotium rolfsii under in-vitro conditions (Table 4).

In vitro evaluation of bioagents

The antagonistic activity of Trichoderma isolates against Sclerotium rolfsii varied significantly. Among the twenty isolates tested, T. atrobrunneum (Ta-5) recorded the highest mycelial inhibition (72.12%), followed by T. yunnanense (Ty-4) (68.88%). These found more effective than remaining isolates (Table 3) In contrast, T. yunnanense Ty-3 and Ty-9 showed the lowest inhibition (7.47% and 5.74%, respectively). The superior performance of Ta-5 and Ty-4 may be attributed to their rapid growth, may be attributed to their rapid mycelial growth, production of antifungal metabolites, and competitive ability to colonize the substrate (Harman et al 2004) [6] antagonistic ability. These results agree with earlier reports on the effectiveness of Trichoderma spp. against S. rolfsii (Dubey and Patel, 2001) [5] .

Conclusion

The present study evaluated the efficacy of fungicides, Trichoderma isolates, and botanicals against Sclerotium rolfsii Sacc causing collar rot of tomato. Among the fungicides, tebuconazole 5% EC, propiconazole 25% EC, and all combiproduct fungicides completely inhibited mycelial growth (100%), while mancozeb 75% WP, chlorothalonil 75% WP, and captan 50%WP were the most effective contact fungicides. Tulsi leaf extract exhibited the highest antifungal activity (91.98%) among the botanicals, and Trichoderma atrobrunneum Ta-5 proved to be the most effective bioagent against S. rolfsii. These findings indicate that effective fungicides, along with promising bioagents and botanicals can be integrated for the eco-friendly and sustainable management of tomato collar rot. However, further evaluation under greenhouse and field conditions is required to validate their efficacy before recommending them for large-scale cultivation.

Acknowledgement

AcknowledgementI owe my heartfelt gratitude to Dr. Prashantha A, Chairman, members of my advisory committee, all the teaching and nonteaching staff, my friends, batch mates, seniors and juniors of KRCCH, Arabhavi for their constant and consistent support during my research work.

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