In vitro assessment of bio-agents and fungicides for the management of cowpea wilt caused by Fusarium oxysporum f. sp. tracheiphilum

Introduction

Cowpea (Vigna unguiculata (L.) Walp.) is an annual dicotyledonous legume belonging to the family Fabaceae and order Fabales. It is widely cultivated in tropical and subtropical regions and is valued for its edible dry seeds and green pods. The crop is also recognized for its potential use as a green manure, contributing to the improvement of soil fertility and properties (Langyintuo et al., 2003) [7] . In India, cowpea is predominantly cultivated in the central and southern regions and is well adapted to warm climatic conditions. Its ability to withstand periods of drought and relatively heavy rainfall makes it an important pulse crop under variable environmental conditions. The crop performs well under temperatures ranging from approximately 27 to 35 °C and grows satisfactorily on a range of soil types, particularly well-drained loamy and moderately heavy soils (Kumar and Narain, 2005) [6] . From a nutritional perspective, cowpea is an important source of dietary protein, carbohydrates, minerals and vitamins. The crop provides substantial nutritional value while having relatively low energy and fat content (Goncalves et al., 2016) [3] . Cowpea leaves and seeds contain several essential nutrients, with seeds generally containing about 23-25% protein, 50-67% carbohydrates and approximately 8-9% moisture, while the fat content remains comparatively low (Rangel et al., 2003) [11] . These nutritional attributes contribute considerably to the importance of cowpea as a food and multipurpose crop. Despite its adaptability and nutritional significance, cowpea is affected by several fungal, bacterial and viral diseases. Among the major biotic constraints, wilt caused by the soil-borne pathogen Fusarium oxysporum f. sp. tracheiphilum is of considerable economic importance.

The pathogen invades the vascular tissues of infected plants, interfering with the movement of water and nutrients and resulting in symptoms such as yellowing, drooping and premature plant death. Disease development is particularly favoured by warm and humid environmental conditions, high soil moisture and the acidic lateritic soils prevalent in the Konkan region. During the last two to three years, wilt incidence in cowpea Cv. Konkan Safed has resulted in considerable crop losses, estimated at approximately 30-40%, at the Research Farm, Botany field, College of Agriculture, Dapoli. The disease has also received limited attention under the specific conditions of this location. Therefore, identification of effective and environmentally compatible management measures is essential for reducing the impact of the disease. Management of Fusarium wilt generally involves the use of chemical fungicides and biological control agents. Bioagents such as Trichoderma spp., Pseudomonas fluorescens and Bacillus subtilis can restrict pathogen development through mechanisms including competition, antibiosis and mycoparasitism, thereby providing an alternative or complementary approach to chemical disease management. However, information regarding the comparative efficacy of different fungicides and bioagents against F. oxysporum f. sp. tracheiphilum under the agroclimatic conditions of the Konkan region is limited. Therefore, the present investigation was undertaken to evaluate selected fungicides and bioagents under in vitro conditions against F. oxysporum f. sp. tracheiphilum, with the objective of identifying effective treatments for further evaluation under greenhouse and field conditions and for developing an integrated approach to the management of cowpea wilt.

Materials and Methods

In vitro exploration of bio-agents

Six bioagents, namely Trichoderma viride, T. harzianum, T. longibrachiatum, T. koningii, Pseudomonas fluorescens and Bacillus subtilis, were assessed for their antagonistic activity against Fusarium oxysporum f. sp. tracheiphilum (Table 1). The experiment was conducted using the dual culture technique described by Dennis and Webster (1971) under a Completely Randomized Design (CRD) with three replications. For the assay, 5 mm diameter mycelial discs of the test pathogen and individual bioagents were obtained from seven-day-old cultures using a sterilized cork borer. The discs were placed at equal distances and opposite to each other on Petri plates containing Potato Dextrose Agar (PDA) medium. Plates inoculated only with the pathogen were maintained as the untreated control. All inoculated plates were incubated at 28±2 °C. The radial growth of F. oxysporum f. sp. tracheiphilum was recorded after seven days of incubation, and the percentage inhibition of mycelial growth over the control was calculated according to the formula proposed by Vincent (1947) [14] .

Per cent Growth Inhibation

Where, C = Growth (mm) of the test fungus in control plate T = Growth (mm) of the test fungus in treatment plates.

In vitro exploration of fungicides

Ten fungicides belonging to systemic, contact and combination groups were tested against F. oxysporum f. sp. tracheiphilum using the poisoned food technique described by Nene and Thapliyal (1993) [8] . The experiment was conducted in a Completely Randomized Design (CRD) with three replications. The required quantity of each fungicide was incorporated into molten PDA medium to obtain the respective test concentration. Approximately 20 ml of the fungicide-amended medium was dispensed into each sterilized Petri plate. A 5 mm diameter mycelial disc taken from a seven-day-old actively growing culture of the pathogen was placed at the centre of each plate. PDA medium without fungicide was maintained as the untreated control. The inoculated plates were incubated at 28±2 °C for seven days. Following incubation, radial colony growth was measured and the percentage inhibition of mycelial growth over the control was calculated using the formula of Vincent (1947) [14] given above.

Statistical analysis

The data obtained from both in vitro experiments were statistically analyzed according to the Completely Randomized Design. Standard error of the mean (S.Em.±) and critical difference (C.D.) were calculated at the 1% level of significance. The analysis was performed using OPSTAT software, following the statistical procedures described by Panse and Sukhatme (1978) [9] .

Results and Discussion

In vitro antagonistic activity of bio-agents

The six bioagents tested in the present study exhibited significant inhibitory effects on the mycelial development of Fusarium oxysporum f. sp. tracheiphilum when compared with the untreated control, which recorded a colony diameter of 90.00 mm (Table 3, Plate I and Fig. 1). Among the treatments, Trichoderma harzianum demonstrated the strongest antagonistic activity, restricting the pathogen colony to 37.33 mm and resulting in 58.52 percent inhibition of mycelial growth. The next highest inhibitory effect was observed with T. viride, which recorded a colony diameter of 41.67 mm and 53.70 percent inhibition.

Trichoderma longibrachiatum and T. koningii produced 47.67 and 49.67 mm colony diameters, corresponding to 47.03 and 44.81 percent inhibition, respectively. Among the bacterial bioagents, Bacillus subtilis exhibited moderate antagonistic activity, with 54.17 mm colony growth and 39.81 percent inhibition. In contrast, Pseudomonas fluorescens was comparatively less effective, recording the highest pathogen growth (59.83 mm) and the lowest inhibition (33.52%) among the bioagents evaluated. The superiority of T. harzianum over other Trichoderma species and bacterial antagonists corroborates several previous studies. Gadhave (2020) [2] reported T. harzianum as the most effective bio-agent against F. oxysporum f. sp. lycopersici (52.33% inhibition), followed by T. koningii, T. viride and P. fluorescens. Khillare et al. (2020) [5] similarly found T. harzianum most inhibitory to F. oxysporum f. sp. udum (85.62%), followed by T. hamatum, T. asperellum, T. koningii, T. longibrachiatum and P. fluorescens in decreasing order of efficacy. Khatun (2018) reported complete inhibition of F. oxysporum f. sp. sesami by T. harzianum, followed by T. viride, and Patra and Biswas (2017) [10] similarly recorded the highest inhibition of F. oxysporum f. sp. ciceri by T. harzianum, followed by T. koningii, T. viride and P. fluorescens. Although absolute inhibition percentages vary with the host plant and forma specialis of the pathogen across these studies, the consistent ranking of T. harzianum as the most antagonistic species, and of P. fluorescens as comparatively weaker, suggests a genuinely superior and broadly applicable antagonistic capability of T. harzianum against Fusarium oxysporum, likely mediated through a combination of mycoparasitism, antibiosis and competition for space and nutrients.

In vitro evaluation of fungicides against the pathogen

All ten fungicides included in the experiment substantially restricted the mycelial growth of F. oxysporum f. sp. tracheiphilum compared with the untreated control, which showed 90.00 mm colony growth ( These results are in close agreement with several earlier reports. Shinde (2025) [13] similarly found carbendazim 50% WP, tebuconazole 25.9% EC, hexaconazole 4% + zineb 68% WP, carbendazim 12% + mancozeb 63% WP and carboxin 37.5% + thiram 37.5% WS gave complete inhibition of Fusarium oxysporum f. sp. sesami, the sesamum wilt pathogen. Khillare et al. (2020) [5] reported that tebuconazole 25% WP, carboxin + thiram and carbendazim + mancozeb combinations gave 100 percent inhibition of F. oxysporum f. sp. udum, the pigeon pea wilt pathogen, followed by carbendazim 50% WP and thiophanate methyl. Ravichandran and Hegde (2015) [12] reported complete inhibition of F. oxysporum f. sp. ciceri by tebuconazole 25.9% EC and carbendazim + mancozeb. The consistently superior performance of systemic and combination fungicides over contact fungicides such as mancozeb and Bordeaux mixture across these studies, and in the present investigation, likely reflects the deeper penetration and broader mode of action of systemic and combi-product active ingredients against Fusarium oxysporum relative to the surface protective action of straight contact fungicides.

Conclusion

From the results of present in vitro study, it is concluded that Fusarium oxysporum f. sp. tracheiphilum causal agent of wilt disease of cowpea can be effectively controlled by fungicides screened in vitro, tebuconazole 25.9% EC, carbendazim 50% WP, captan 50% WP, carbendazim 12% + mancozeb 63% WP and carboxin 37.5% + thiram 37.5% WS gave complete inhibition of the pathogen and are recommended as priority candidates for field evaluation. Among the bio-agents, Trichoderma harzianum was clearly the most promising, offering a viable biological alternative or complement to chemical management. These findings provide a sound basis for subsequent field-level testing of the most effective fungicides and bio-agents identified here, towards developing an integrated management strategy for cowpea wilt in the Konkan region.

Acknowledgement

AcknowledgementThe authors sincerely acknowledge the Head, Department of Plant Pathology, Dr. Balasaheb Sawant Konkan Krishi Vidyapeeth, Dapoli, Maharashtra, for providing the necessary facilities and support to carry out this research work.

References

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