Eco-Friendly Management of Chickpea Wilt: In vitro Evaluation of Bioagents and Botanicals against Fusarium oxysporum f. sp. ciceri

Introduction

Chickpea (Cicer arietinum L.) is one of the world's most important pulse crops, serving as a major source of plant protein, dietary fibre, and essential micronutrients for millions of people. In addition to its nutritional significance, chickpea contributes to sustainable agriculture through biological nitrogen fixation, improving soil fertility and reducing dependence on synthetic fertilizers (Zhang et al, 2023) [1] . These characteristics make the crop particularly valuable in lowinput farming systems where maintaining soil health and environmental quality is increasingly recognized as a priority. Despite its agronomic importance, chickpea production is severely constrained by Fusarium wilt, caused by Fusarium oxysporum f. sp. ciceri (FOC) It is an ubiquitous pathogen both soil-and seed-borne, ranks 5th among the 10 most economically important fungal phytopathogens (Sharma et al., 2016) [2] .The pathogen invades the root system and vascular tissues, disrupting water transport and causing progressive wilting, yellowing, and ultimately plant death. Its ability to survive in soil for several years through resilient chlamydospores makes the disease difficult to manage (Chaudhary et al.,2025) [3] . Consequently, the disease cause substantial yield losses in many chickpea-growing regions and poses a persistent challenge to sustainable crop production. Sustainable agriculture emphasizes maintaining crop productivity while preserving ecological integrity. Within this framework, biological control agents have emerged promising because they suppress pathogens through naturally occurring mechanisms such as competition, antibiosis, mycoparasitism, and the induction of plant defense responses. Organisms including Trichoderma spp., Chaetomium globosum, Pseudomonas fluorescens, and Bacillus subtilis have demonstrated considerable potential, many of these microorganisms also improve rhizosphere health and support plant growth (Singh et al., 2016) [4] , making them attractive components of integrated crop management rather than simple replacements for chemical pesticides. Similarly, botanical extracts have gained renewed interest as biodegradable and renewable sources of antimicrobial compounds. Many medicinal and aromatic plants synthesize secondary metabolites capable of inhibiting fungal growth and posses no environmental risks.

(Shukla and Dwivedi, 2012) [5] Extracts derived from garlic, neem, tulsi, ajwain, bael, and other plants have demonstrated antifungal activity against diverse phytopathogens (Cowan, 1999; Khanna et al., 2024) [6,7] . Against this background, the present study evaluated the antagonistic potential of selected microbial bioagents and botanical extracts against FOC under controlled laboratory conditions. The study seeks to contribute to the development of environmentally sustainable and integrated disease management strategies. The approach aligns with the broader goal of reducing dependence on synthetic fungicides while supporting productive, resilient, and ecologically balanced chickpea production systems.

Materials and Methods Isolation and purification of the pathogen

Diseased plants with typical wilt symptoms were taken for isolation. Sample were washed thoroughly in distilled water to remove adhering dirt particles and surface contaminates and thereafter, placed in between blotting paper to remove excess moisture, if any. The infected parts were split open into half and aseptically cut into small pieces. Surface sterilized with Sodium hypochlorite (1%) solution for 30 seconds. Later on, these pieces were taken out from solution and subsequently washed three-time with sterilized distilled water to remove the traces of Sodium hypochlorite solution, if any. To remove the excess moisture the cut tissues were pressed between two folds of sterilized blotting paper under aseptic condition. Prepared sterilized cut pieces were placed at equal distance in petri dishes containing potato dextrose agar medium with the help of sterilized forceps under aseptic conditions in laminar air flow. The inoculated petri plates were incubated at 26±1 °C for five days in the B.O.D. Incubator. The petri dishes were observed regularly for mycelial growth of the fungus. Purification of the fungus was done by taking freshly grown mycelial tips. Sub culture was done on petri plates and subsequently, pathogen was transferred into culture tubes to keep it viable for further use.

In vitro evaluation of bio control agent against FOC

The effect of five bio-agents were tested for their efficacy against FOC in three replications in completely randomized design under in vitro condition in Post graduate laboratory, Department of Plant Pathology, SVPUAT, Meerut. The bioagents were isolated from soil using serial dilution method. Chemicals were procured from store room, in the Department of Plant Pathology of S.V.P University of Agriculture & Technology Meerut (U.P.). For culture multiplication PDA was used. Dual culture techniques as given by Morton and Stroube (1955) was used for testing antagonistic activities of bio-agents against FOC on PDA.

Dual culture technique

A mycelial disc of 5 mm diameter, obtained from the peripheral region of 5 to 7-day old culture of FOC on PDA, was placed on fresh PDA plate (3 cm from the center) and incubated at 28 ⁰C for 48 hr to initiate growth. Then a mycelial disc (5 mm), obtained from the periphery of a 5 to 7-day old culture of Trichoderma viride, Trichoderma harzianum, Chaetomium globosum, Bacillus subtilis and Pseudomonas fluorescence, was placed 3 cm away from the inoculum of the pathogen, the plates were incubated at 26±1 °C, the measurements were taken after 5 days. In control experiment a sterile agar disc (5 mm) was placed in the dish instead of bio-agents. At the end of incubation period, radial growth was measured. Radial growth reduction was calculated in relation to growth of the control as follows: Percent inhibition over control was worked out using the formula given by Vincent (1947).

Where, C = Growth of pathogen mycelium in control T = Growth of pathogen mycelium in treatment

Observation recorded

• Radial growth of the pathogen.

• Percentage (%) growth inhibition.

In vitro screening of different botanicals against FOC 6 different plants were selected for the study. The study was conducted in three replications in completey randomized design in vitro in the Department of Plant Pathology, SVPUAT, Meerut. Mature leaves were collected from at least five different individual plants from campus of SVPUAT, Meerut. The leaves (200g) were properly washed with distilled water to remove any dirt adhering to it followed by surface sterilization of leaves in 0.1% sodium hypochlorite solution and washing thrice with distilled water. Then the plant part along with equal amount of water were macerated in sterilized mortar and pestel. The macerated extract was filtered through three-folded muslin cloth to remove fibrous and suspended material, this extract was subjected to centrifugation at 12,000 rpm for 10 minutes. The pellets were discarded and the required amount of aqueous extract were mixed with sterilized PDA. 20 ml of poisoned media was poured into 90 mm sterilized petri plates and allowed to solidify in aseptic condition. All plates were inoculated with actively growing 5 mm mycelial disc of FOC. Three replications were maintained for each treatment. These plates were incubated at 26±1 °C for five days in a B.O.D. Incubator and colony diameter was recorded. Percent inhibition of mycelial growth over control was calculated by using the formula given by Vincent (1947).

% Inhibition of mycelial growth = C -T C × 100

Where, C = Growth of mycelium in control T = Growth of mycelium in treatment

Conclusion

The result revealed that there was significant mycelia growth inhibition of FOC by all the tested bio-agents. The maximum mycelial growth inhibition was recorded by Chaetomium globosum (83.33%) which was superior to all the tested bioagents, followed by and Pseudomonas fluorescens (78.89%) and Trichoderma harzianum (77.78%). The minimum mycelial growth inhibition was recorded by Bacillus subtilis (60.00%) followed by Trichoderma viride (76.67%) at 144 hours. Garlic clove extract found to be most efficient against FOC 82.22%, 75.56% growth inhibition was recorded by at 10% and 5% w/w concentration respectively followed by 10% Ajwain seed extract (67.78%) and 10% neem leaf extract (62.22%). The results revealed that there is significant difference in per cent inhibition of mycelial growth of FOC with tested plant extracts. Minimum inhibition was found in bael leaf extract at 46.67% and 51.11% at both 5% and 10% concentrations respectively. Although all treatments significantly reduced fungal growth compared to control, differences in their effectiveness highlighted the importance of selecting suitable biological inputs for disease management. These findings suggest that selected microbial antagonists and botanical extracts possess considerable potential as environmentally compatible tools for suppressing Fusarium wilt. While laboratory results cannot be directly extrapolated to field conditions, they provide a strong foundation for developing sustainable, integrated disease management strategies that reduce reliance on fungicides and promote resilient chickpea production systems.

References

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