In vitro antagonistic activity of bioagents against Fusarium oxysporum F. sp. capsici in Chilli

Introduction

Chilli (Capsicum annuum L.) is a highly valued vegetable and spice crop, widely cultivated for its pungency, flavor and nutritional benefits. It belongs to the Solanaceae family and has a chromosome number of 2n=24. Commonly referred to as chilli pepper, hot pepper or red pepper, it has been cultivated since ancient times in the Americas and is considered among the earliest domesticated crops (Chiou et al., 2014) [8] . Archaeological studies indicate that chilli peppers existed in Mexico and Peru as early as 7000 BC, with Mesoamerica, particularly Mexico's Tehuacan Valley, identified as the primary center of domestication (Ettenberg, 2019) [13] . The wild ancestor, C. annuum var. glabriusculum, also called the "Mother Chilli," bears small fruits that are dispersed naturally by birds, aiding its widespread distribution (Basu & De, 2003) [2] . Chilli was introduced to Europe in 1493 by Columbus and later reached India through Portuguese traders in the seventeenth century (Pandit et al., 2020) [18] . Chilli fruits are rich in essential nutrients, including vitamins C, A and E, which function as antioxidants, strengthen immunity and support vision and skin health. Capsaicin, the compound responsible for the crop's characteristic pungency, exhibits diverse pharmacological properties such as anti-inflammatory, analgesic, antibacterial and anticancer effects. Additionally, capsaicin contributes to the management of obesity, type-2 diabetes, and cardiovascular disorders (Diaz-Laviada, 2010) [12] . The red coloration of the fruit is primarily attributed to capsanthin, while oleoresins extracted from chilli have extensive applications in food, pharmaceutical, and cosmetic industries (Sambamurty & Subrahmanyam, 1989) [22] . Being a day-neutral crop, chilli can be grown throughout the year in tropical and subtropical climates, with optimal growth occurring between 15 and 35 °C. Among the five cultivated species of Capsicum, C. annuum dominates Indian agriculture, cultivated both for pungent and non-pungent varieties. Other species like C. frutescens, C. chinense and C. baccatum are grown mainly in home gardens or localized areas (Popelka et al., 2017) [20] . India leads global chilli production, consumption, and export, with Andhra Pradesh, Telangana, Madhya Pradesh and Karnataka being the main producing states (National Horticulture Board, 2021-22). Chilli cultivation faces serious challenges from biotic stresses, particularly fungal pathogens. Among these, Fusarium wilt caused by Fusarium oxysporum f. sp. capsici is one of the most destructive diseases affecting yield and quality. This soil-borne fungus can persist in the soil for several years, infects the root system and colonizes the xylem vessels, leading to water transport disruption, leaf curling, wilting, vascular browning and plant death (Booth, 1971; Di Primo et al., 2001; Agrios, 2005) [4,11,1] . In India, Fusarium wilt can cause up to 80% yield losses and in the United States, disease incidence has been reported up to 35% (Najar et al., 2006; Roberts et al., 2004) [16,21] . Management of Fusarium wilt traditionally relies on chemical fungicides, which, although effective, can be costly and environmentally harmful. Biological control using antagonistic microorganisms provides an eco-friendly alternative. Bioagents can suppress the pathogen through mechanisms such as competition, antibiosis and induction of host resistance. In vitro evaluation of these bioagents is essential to identify effective strains that can be incorporated into integrated disease management strategies, offering sustainable solutions for Fusarium wilt control in chilli (Joshi et al., 2013; Thoyajakshi et al., 2018) [14,24] .

Materials and Methods

Isolation and Identification of Fusarium oxysporum F. sp. Capsici

Wilt-affected chilli plants were collected from infected fields located in Malegaon, Baramati. The diseased plants displayed typical wilt symptoms, such as yellowing and desiccation of leaves, along with browning of the vascular tissues. The pathogen was isolated from infected roots and stem portions of chilli using the tissue isolation technique on Potato Dextrose Agar (PDA). After an incubation period of 3-4 days, fungal growth was observed in the Petri plates. A pure culture was obtained through the hyphal-tip technique under aseptic conditions and subsequently preserved on PDA slants in test tubes. Identification of the pathogen was carried out based on its distinct cultural and morphological attributes. After 7 days of incubation at 27±1 °C, the culture attained maximum growth, showing circular, fluffy mycelial growth with whitish to pinkish colonies. The colony pigmentation varied from pink to brown. Microscopic examinations were performed to study mycelial characteristics, conidial morphology (shape and size), and the presence of chlamydospores. The observed features were compared and confirmed using the standard descriptions of Fusarium species given by Booth (1971) [4] .

Pathogenicity of Fusarium oxysporum F. sp. capsici

Pathogenicity of Fusarium oxysporum F. sp. capsici was established in accordance with Koch's postulates using the sick soil method. The mass-cultured pathogen was thoroughly mixed into sterilized soil filled in pots, and chilli seedlings of the cultivar Pusa Jwala were transplanted into the infested soil. Observations were recorded 10-12 days after transplanting. The inoculated seedlings exhibited characteristic wilt symptoms, including yellowing of leaves, browning of vascular tissues, basal stem discoloration, and subsequent wilting. In contrast, the seedlings in uninoculated (control) soil remained healthy and symptomfree. The fungus was reisolated from symptomatic plants and re-cultured, and the morphological characteristics of the reisolated culture were identical to those of the original isolate obtained from naturally infected plants. These results confirmed Fusarium oxysporum F. sp. capsici as the causal organism of chilli wilt, thus fulfilling Koch's postulates.

Evaluation of biocontrol agents against test pathogen 2.3.1 Antagonistic activity of bioagents against chilli wilt pathogen

The antagonistic potential of different biocontrol agents-Trichoderma viride, T. harzianum, T. hamatum, T. virens, Bacillus subtilis and Pseudomonas fluorescens against Fusarium oxysporum f. sp. capsici was assessed using the dual culture assay on PDA, following the method described by Dennis and Webster (1971) [10] .

Dual Culture Technique

The antagonistic interaction between bioagents and Fusarium oxysporum F. sp. capsici was evaluated using the dual culture assay. Sterilized molten Potato Dextrose Agar (20 mL) was dispensed into 90 mm Petri plates and allowed to solidify. After solidification, 5 mm mycelial discs of the pathogen and the fungal antagonists were excised from the margins of 7-day-old cultures using a sterile cork borer and placed on opposite sides of the plate simultaneously at equal distance from the center. In the case of bacterial antagonists, a 5 mm disc of the pathogen was positioned on one side of the PDA plate, while the bacterial bioagent was streaked on the opposite side at the same time. Plates containing only the pathogen, without any antagonist, served as the untreated controls. Each treatment consisted of four replications, and all plates were incubated at 27±2 °C under a completely randomized design. The percentage inhibition of mycelial growth of the pathogen, relative to the control, was computed using Vincent's formula (1927) [25] .

I= C-T C x 100

Where, I= Per cent Inhibition of fungal growth C= Colony growth diameter (mm) in control plate T= Colony growth diameter (mm) in treatment plate

Experimental Design and Statistical Analyses

The experiment was laid out in a Completely Randomized Design (CRD) comprising seven treatments with four replications each. The dual culture technique described by Dennis and Webster (1971) [10] was employed for evaluating the antagonists. The recorded data were subjected to analysis following the procedures outlined by Panse and Sukhatme (1967) [19] . Standard error (SE) and Critical Difference (CD) at the 1% probability level (P=0.01) were calculated to determine the significance of differences among treatment means.

Results and Discussion

Antifungal Efficacy of bioagents against Fusarium oxysporum F. sp. Capsici

The antagonistic potential of seven bioagents comprising fungal species (Trichoderma spp.) and bacterial species (Bacillus subtilis and Pseudomonas fluorescens) was assessed against Fusarium oxysporum F. sp. capsici. The evaluation was performed using the dual culture and streak plate techniques on PDA as the basal medium. The experimental outcomes are presented in Table 1, Figure 1, and Plate 1. A marked variation in the antagonistic response of the bioagents was recorded. Among all treatments, Trichoderma harzianum proved to be the most effective and produced the smallest pathogen colony diameter (23.25 mm), corresponding to the highest mycelial growth inhibition of 74.17%. The next best performer was Trichoderma viride, which exhibited a colony diameter of 27.63 mm and 69.30% growth inhibition. Trichoderma hamatum and T. virens showed moderate suppression of the pathogen, with colony diameters of 31.38 mm and 33.68 mm and inhibition rates of 65.13% and 62.63%, respectively. In contrast, the bacterial antagonists were comparatively less effective; B. subtilis and P. fluorescens recorded colony diameters of 53.50 mm and 57.13 mm and inhibition percentages of 40.55% and 36.52%, respectively. These results are consistent with earlier findings. Singh et al. (2018) reported that T. harzianum showed maximum inhibition of F. oxysporum (75.7%), followed by T. viride under in vitro conditions. Dar et al. (2013) also observed similar trends, with T. harzianum and T. viride recording growth inhibition of 92.5% and 86.2%, respectively. Comparable reports by Chakraborty and Chatterjee (2008) [5] , Bhat et al. (2016) [3] , Mishra et al. (2017) [15] , Cherkupally et al. (2017) [7] , and Coral et al. (2017) [6] further confirmed that T. harzianum consistently displayed the greatest antagonistic effect against the wilt pathogen through its mycoparasitic ability.

Conclusion

Under in vitro conditions, all the bioagents evaluated exhibited notable antagonistic effects against Fusarium oxysporum F. sp. capsici. Among them, Trichoderma harzianum proved to be the most effective, recording the highest mycelial growth inhibition (74.17%) with the minimum colony diameter (23.25 mm). This was closely followed by T. viride, T. hamatum and T. virens, which also displayed substantial suppressive activity against the pathogen. In contrast, the bacterial bioagents, Bacillus subtilis and Pseudomonas fluorescens, were comparatively less effective, inhibiting only 40.55% and 36.52% of pathogen growth, respectively. Overall, the results that T. harzianum holds the greatest potential for the biological management of chilli wilt under laboratory conditions. The results emphasize the potential use of compatible biocontrol agents as an eco-friendly and sustainable alternative to chemical control strategies for managing wilt disease in chilli.

Acknowledgments

I express my sincere gratitude to Dr. Sharadchandra Pawar College of Agriculture, Baramati, for providing the necessary facilities and support that facilitated the successful completion of this research.

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