Optimization of antagonistic fungal epiphyte for management of walnut anthracnose

Introduction

Trichoderma spp. have emerged as successful and extensively studied fungal antagonists owing to their broad-spectrum activity against numerous plant pathogens. Trichoderma spp. naturally colonize the phyllosphere and other ecological niches, where they effectively compete with phytopathogens [1] . Their success as biological control agents is attributed to multiple mechanisms, including mycoparasitism, competition for nutrients and space, production of antifungal metabolites, secretion of cell wall-degrading enzymes and induction of systemic resistance in host plants [2] . Initial inoculum dose plays a critical role in determining successful colonization, persistence and subsequent suppression of the pathogen. An inadequate inoculum dose may fail to establish sufficient propagule density, whereas excessively high concentrations may not provide proportional improvement and may increase production costs. Therefore, optimization of inoculum dose is a prerequisite for the development of an effective and economically viable biocontrol strategy. In order to develop such a strategy against walnut anthracnose (Marssonina juglandis), this investigation was undertaken with the objective to optimize the inoculum dose of fungal epiphyte namely Trichoderma pleuroticola, a predetermined highly antagonistic walnut epiphyte against Marssonina Juglandis.

Material and methods

The study was conducted during 2025 cropping season in the Division of Plant Pathology, Faculty of Agriculture, Wadura, (SKUAST-K). The promising fungal epiphyte NK-8(Trichoderma pleuroticola) was grown on Potato Dextrose Agar (PDA) medium at 25±1°C. Spore mass was harvested in sterile distilled water to prepare a stock of spore suspension. Spore load of the stock suspension was determined by using haemocytometer and working suspension were prepared in sterile distilled water after applying formula n 1 v 1 =n 2 v 2 .where n1= initial stock concentration, v1= volume of stock solution, n2=final concentration after dilution, v2= final volume of diluted solution. As such eight levels of the inoculum dose viz., 5x10 2 , 1x10 3 , 5x10 3 , 1x10 4 , 5x10 4 , 1x10 5 , 5x10 5 , and 1x10 6 were prepared for the evaluation besides one untreated control. Each treatment consisted of spraying the designated inoculum load of NK-8 spore suspension uniformly on the foliage of tagged branches. Following a 12 hour interval to facilitate establishment of the antagonist, the treated branches were challenge inoculated with a conidial suspension of Marssonina juglandis adjusted to 1 × 10⁶ conidia ml⁻¹ using a haemocytometer [3] . Disease severity was assessed 14 days after pathogen inoculation using 0-9 disease rating scale and the disease intensity (%) was calculated by formula [∑(n× v)/N×G] ×100, where n = number of diseased leaves in each category, v = numerical value of the category N = total number of leaves examined, and G = highest grade value. The experiment was conducted under open field and greenhouse conditions. The treatment allocation was done on tagged branches in RBD and CRD in field and greenhouse environments respectively. The data were statistically analyzed using analysis of variance (ANOVA), and treatments means were separated by least significant difference (LSD) using R software R 4.6.1. Trichoderma pleuroticola (NK-8) produced a rapidly growing colony on Potato Dextrose Agar (PDA), covering the Petri plate within 3-4 days of incubation at 25±1°C. The colony was initially light green and powdery with circular margins, gradually turning olive green to dark green due to profuse conidial production.

Distinct concentric zonation was also evident on the colony surface. Microscopic examination revealed that the isolate NK-8 produced hyaline spores that were globose, sub-globose to ellipsoidal in shape with 2.8-4.0 × 2.2-3.0 µm in size (Fig 1). These morphological characteristics were consistent with those described for Trichoderma pleuroticola. The Trichoderma pleuroticola, irrespective of inoculum dose, suppressed walnut anthracnose under both field and controlled conditions (Table 1, Fig: 2). The data revealed a positive dose response with increased disease suppression at higher test doses. Lower inoculum densities (5 × 10² and 1 × 10³ spores ml⁻¹) resulted in comparatively high periodic disease increase of 17.22 per cent and 16.48 per cent and provided only 18.89 per cent and 19.15 per cent disease suppression over the control respectively. The application of 5x10 3 and 1x10 4 spores ml⁻¹ reduced periodic disease increase to 15.11 and 12.25 per cent with disease suppression of 22.31 and 34.35 per cent respectively. Further increase in inoculum density of 5x10 4 and 1x10 5 spores ml⁻¹ further reduced periodic disease increase to 11.34 and 9.96 with disease suppression of 40.70 and 46.84 per cent respectively. The highest inoculum density (1x 10 6 ) was more effective in reducing periodic disease increase to 5.61 per cent with disease suppression of 68.49 per cent over untreated control. However, two densities 5x 10 5 and 1x 10 6 showed non-significant (p ≤ 0.05) effects. In greenhouse condition, the similar trend was also observed. So, the dose density of 5x 10 5 was considered as optimum dose for providing comparable efficacy with higher dose, while requiring lower inoculum density.

Discussion

This study was conducted to assess, for the first time, the efficacy of Trichoderma pleuroticola at different inoculum density against Marssonina juglandis on walnut. Our results clearly demonstrated that, upon increasing the inoculum density, the disease suppression was significantly enhanced, indicating that the efficacy of the antagonist was closely associated with its population density on the walnut phylloplane. Higher inoculum levels likely facilitated rapid establishment and colonization of the leaf surface, thereby limiting disease development through competition for nutrients and space, mycoparasitism, and the production of antifungal metabolites [4] . Although the highest disease suppression was recorded at 1 × 10⁶ spores ml⁻¹, this treatment was statistically at par with 5 × 10⁵ spores ml⁻¹, indicating that further increasing the inoculum concentration beyond 5 × 10⁵ spores ml⁻¹ did not provide a significant additional reduction in disease intensity. Therefore, 5 × 10⁵ spores ml⁻¹ may be considered the optimum inoculum density for practical application, as it offers comparable disease control while requiring a lower quantity of inoculum, making it more economical and suitable for large-scale use. The improved disease suppression observed at higher inoculum densities may also be attributed to the greater production of antimicrobial compounds and lytic enzymes by Trichoderma, including volatile and nonvolatile metabolites, hydrolytic enzymes, ammonia, hydrogen cyanide, and other secondary metabolites that inhibit pathogen growth and infection [5] . These mechanisms collectively contribute to effective biological control once the antagonist has established a sufficient population on the host surface. The present findings are consistent with earlier reports demonstrating the positive relationship between antagonist population density and disease suppression. Padder et al. reported that increasing the concentration of endophytic microorganisms significantly reduced the incidence and severity of apple scab [6] . Similarly, Bonaterra et al. and Francés et al. observed that higher populations of Pantoea agglomerans EPS125 effectively suppressed powdery mildew and reduced disease incidence in stone fruits [7,8] . Comparable observations were also reported by Navaneetha et al. found that increasing the conidial concentration of Trichoderma spp. enhanced the management of Alternaria leaf blight of sunflower and gray mold of castor, with higher inoculum levels providing superior disease control [9] .

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