Identification of resistant sources in chilli hybrids and evaluation of copper-chitosan nanocomposites against Colletotrichum truncatum

Introduction

Chilli (Capsicum annuum L.) is one of the most important spice and vegetable crops cultivated all over the world, valued for its culinary, nutritional and economic significance. It is a rich source of vitamins A and C, capsaicin and antioxidants and also plays a vital role in the food processing and pharmaceutical industries (Rajendran and Thomas, 2021) [15] . India is one of the largest producer and exporter of chilli but its productivity is significantly constrained by various biotic stresses, particularly fungal diseases. Among the major diseases affecting chilli, anthracnose caused by several species of the genus Colletotrichum is considered one of the most destructive, causing severe losses in both yield and fruit quality under field and post-harvest conditions (Pakdeevaraporn et al., 2005) [13] . The disease affects leaves, stems and especially fruits, where it produces sunken necrotic lesions that reduce marketability and shelf life. In addition, anthracnose has been found to cause yield losses of more than 50% in Thailand under favorable environmental conditions especially in humid and warm environment (Pakdeevaraporn et al., 2005) [13] and in India, disease incidence levels varied from 66 to 84% and yield losses ranged from 12 to 50% (Thind and Jhooty, 1985, Bagri et al., 2004, Sharma et al., 2005) [23,1,18] . Anthracnose of chilli is caused by a complex of Colletotrichum species, including C. capsici, C. gloeosporioides and C. truncatum. Among these, C. truncatum is highly aggressive and widespread, resulting in severe fruit rot and rapid disease progression (Damm et al., 2012; Cannon et al., 2012) [5,3] . The pathogen colonise host tissues at different growth stages, but fruit infection is of major economic importance. Disease development is influenced by environmental factors such as temperature, humidity and host ontogeny. Ripe fruits tend to be more vulnerable compared to fruits that are not yet matured.

Disease control of chilli anthracnose largely depends on the use of chemical fungicides such as copper oxychloride, carbendazim and mancozeb. Eventhough they are effective in controlling the disease, excessive and indiscriminate use of these chemicals has led to several concerns, including the development of resistance to fungicides in pathogen populations, environmental contamination and accumulation of toxic residues in food products (Lucas et al., 2015) [9] . These constraints underscore the urgent need to develop sustainable and environment-friendly disease management strategies. Nanotechnology has recently proved to be an efficient tool for controlling plant pathogens because it is capable of increasing efficiency in a way that would reduce the negative impact of chemicals on the environment. Chitosan, being a biopolymer formed from chitin, is a substance that has been extensively studied lately owing to its biodegradability, biocompatibility and inherent antifungal properties. It also acts as an elicitor of plant defense mechanisms, inducing the production of phytoalexins and pathogenesis-related proteins (El Hadrami et al., 2010; Pichyangkura and Chadchawan, 2015) [6,14] . The incorporation of metal ions such as copper into chitosan matrices leads to the formation of copper-chitosan nanocomposites, which exhibit enhanced antimicrobial activity. These nanocomposites possess unique properties such as increased surface area, improved adhesion to plant surfaces, controlled release of active ions and better penetration into microbial cells. Copper ions are proven to affect the fungal cellular structure by disrupting their cell walls, denaturing proteins and causing enzymatic activities, thus inhibiting the pathogen growth (Lemire et al., 2013; Salah et al., 2021) [8,16] . The synergistic interaction between copper and chitosan further enhances their antifungal efficacy. Although the nanocomposite material of copper-chitosan shows considerable promise, the number of investigations related to the efficacy of copper-chitosan nanocomposites against Colletotrichum truncatum in chili pepper is limited. In addition, variability in host resistance among different chilli hybrids plays a crucial role in disease development and management. Identification of resistant or moderately resistant hybrids, combined with effective nanocompositebased treatments, can contribute significantly to integrated disease management strategies. Hence, the experiment was planned to investigate the following objectives (i) evaluate the resistance of selected chilli hybrids to anthracnose caused by Colletotrichum truncatum and (ii) assess the antifungal efficacy of copper-chitosan nanocomposites under controlled conditions. The findings of this study are expected to provide insights into sustainable and environmentally safe approaches for managing chilli anthracnose.

Materials and Methods

Experimental Location

The study was conducted under greenhouse and laboratory conditions in the Department of Plant Pathology during kharif 2025. Laboratory experiments involving pathogen isolation, culturing and inoculum preparation were conducted in a sterile environment under aseptic conditions. Greenhouse experiment was maintained favourable for the development of chilli anthracnose disease.

Plant Material

Six chilli hybrids were selected: Arka Tejasvi, Arka Gagan, Arka Kyathi, Arka Swetha, Arka Meghana, Arka Harita for evaluating their susceptibility to anthracnose disease caused by Colletotrichum truncatum.

Pathogen Isolation and Inoculum Preparation

The pathogen Colletotrichum truncatum was cultured on suitable media. A conidial suspension (1 × 10⁶ cfu mL⁻¹) was prepared from 12 day old cultures. For inoculum preparation, sterile distilled water was added to fully grown fungal cultures and the colony surface was gently scraped using a sterile glass rod to release conidia. The resulting suspension was filtered through double-layered muslin cloth to remove mycelial debris. The concentration of conidia was adjusted to 1 × 10⁶ cfu mL⁻¹ using a haemocytometer. The prepared conidial suspension was used immediately for pathogenicity tests and antifungal evaluation studies.

Resistance Assessment of Chilli Hybrids to Anthracnose Disease

Six different chilli hybrids -Arka Tejasvi, Arka gagan, Arka kyathi, Arka Swetha, Arka Meghana and Arka Harita were used for testing the susceptability of chilli anthracnose pathogen. Plastic bags (15x12 cm) were filled with sterilized soil-sand-FYM (farm yard manure) mix (2:1:1) and autoclaved at 15 psi for two hours for three consecutive days. Simultaneously, the bags were also sterilized with 4 percent formalin solution to eliminate the residual contaminants. Each bag was transplanted with one seedling of six different chilli hybrids. The bags were placed in a greenhouse with five replications. Fertilizers and water applied as per the need to maintain optimum moisture for growth of seedlings in the greenhouse. The conidial suspension (10 6 cfu/ml) was prepared by scraping the 12 days old culture plate of Colletotrichum truncatum. The Red ripe and green fruits of different chilli hybrids was collected. The collected fruits are surface sterilized with 1 per cent sodium hypochlorite and followed by twice washing with distilled water. Then the fruits are placed in a Petri plate containing the moist blotter paper to maintain the humidity and conidial suspension was inoculated by pin pricking method (Manzoor et al., 2023) [11] . After inoculation the Petri plates were incubated at 27±2 °C in BOD incubator and monitored for symptom development; re-isolation was made from the infected fruits and compared with the original to prove Koch's postulates. Observation was taken for no. of lesions, lesion length, lesion width and PDI were calculated to know the susceptible variety of chilli for further evaluation. Further, the scale was converted to per cent disease index (PDI) using the formula by Wheeler (1969) [24] :

Disease Assessment

Sum of individual ratings 100 Percent Disease Index (PDI) = × Total number of plants/Leaves observed Maximum disease grade Percent disease intensity (PD Intensity) was further calculated based on infection levels by using the disease rating scale (Machenahalli et al., 2016) [17] as follows:

Symptomatology of Chilli Anthracnose Caused by

Colletotrichum truncatum

Anthracnose symptoms caused by Colletotrichum truncatum were observed on different plant parts of chilli under both greenhouse and laboratory conditions. The disease progression was found to vary depending on the stage of the host tissue and environmental conditions.

Evaluation of Copper chitosan nanocomposites on detached unripe chilli fruits

The experiment was carried out on detached chilli fruits under controlled laboratory conditions. The best treatments under in-vitro evaluation (poison food technique) were evaluated on detached fruits. Fruits from susceptible chilli cultivar Arka Tejasvi were evaluated under controlled conditions. Fruits were surface sterilized with 1 percent Sodium hypochlorite solution for 30 seconds and washed thoroughly with sterile distilled water. The surface sterilized fruits were dried by placing on sterilized blotter paper. Different treatments were applied on test fruits after 48 hrs of spore (C. truncatum) inoculation. All the fruits were given pin-pricks at three specific points and then spray inoculated with spore suspension (10 6 spores/ml) of seven days old culture of C. truncatum. The treated fruits were kept in moist chamber and incubated at 28±2 0 C for 5 days.

The fungicide served as standard check while as sterilized distilled water (SDW) was applied in case of control. Three replications of each treatment were maintained and observations taken under completely randomized design.

Observations on number of lesions and size (length) of lesions were recorded on 5 th day after inoculation.

Results and Discussion

Pathogenicity and Resistance screening of Chilli Hybrids against Colletotrichum truncatum

The pathogenicity of C. truncatum was successfully confirmed through detached fruit inoculation assays and greenhouse pathogenicity tests. Typical anthracnose symptoms developed consistently across all inoculated chilli hybrids under both experimental conditions. The first primary symptoms observed as small, water-soaked lesions at the site of inoculation, which gradually enlarged into sunken necrotic spots with dark brown discoloration. Under humid conditions, the presence of characteristic acervuli with salmon-pink spore masses were observed on infected fruits, confirming the typical symptomatology of chilli anthracnose caused by C. truncatum. The development of disease was showed greater effects in case of matured red ripe fruits compared to green fruits, thus indicating the influence of fruit maturity on host susceptibility. The re-isolations of fungus from artificially infected fruits resulted in colonies of fungi that showed morphological resemblance with the initial isolation, thereby fulfilling Koch's postulates for the causal organism and confirming C. truncatum as the causal agent of anthracnose in chilli.

Assessment of Disease Reaction in Chilli Hybrids against Anthracnose

Considerable variation in resistance to chilli anthracnose was observed among the evaluated chilli hybrids under detached fruit and greenhouse conditions. The differential disease response indicated the presence of genetic variability among hybrids with respect to resistance against C. truncatum infection. Based on Percent Disease Index (PDI), the hybrids were categorized as follows: Highly susceptible were Arka Tejasvi, Arka Swetha, Arka Harita; Susceptible were Arka Gagan; moderately susceptible were Arka Kyathi; moderately resistant were Arka Meghana. From the hybrids tested, Arka Tejasvi was found to have the highest disease severity with values of PDI going up to 100 percent in both greenhouse condition and in detached red fruit conditions, implying high susceptibility to the anthracnose pathogen infection (Plate 3 and Plate 2). This hybrid exhibited rapid lesion expansion, extensive tissue necrosis and profuse sporulation of the pathogen. Similarly, Arka Swetha and Arka Harita also showed severe disease symptoms with high lesion development and elevated PDI values. On the other hand, Arka Meghana exhibited the lowest disease severity with comparatively smaller lesions and reduced pathogen colonization, suggesting the presence of partial resistance mechanisms against C. truncatum as mentioned in Table 3, Plate 2. The lower susceptibility observed in Arka Meghana may be attributed due to inherent structural and biochemical defense responses that restrict fungal establishment and disease progression. Therefore, this hybrid may serve as a potential genetic source in future resistance breeding programs aimed at developing anthracnose-resistant chilli cultivars. Furthermore, disease severity was consistently higher in detached red fruits compared to green fruits across all hybrids, indicating that fruit maturity plays a significant role in the development and progression of anthracnose disease in chilli.

Symptomology difference between different hybrids

Some of the easily distinguishable and economically significant symptoms of anthracnose on chilli fruit caused by C. truncatum were observed on fruits, in case of susceptible hybrids initial infection appeared as small, circular, water-soaked lesions, particularly at the site of inoculation. These lesions progressively enlarged, becoming sunken, necrotic spots with dark brown to black discoloration and eventually coalesced to form large depressed areas that markedly reduced fruit quality. Under high humid conditions, characteristic concentric rings of acervuli developed within the lesions, appearing as black pinhead-like structures that produced salmon-pink spore masses, a diagnostic feature of Colletotrichum infection. Similar observations were reported by Damm et al. (2012) [5] , who described C. truncatum as a highly aggressive species associated with severe anthracnose symptoms in chilli and other solanaceous hosts. In advanced stages, infected fruits exhibited severe shrivelling, rotting and premature abscission, resulting in substantial yield losses (Plate 3). The moderately resistant chilli hybrids exhibited small, white necrotic restricted lesions at the infection site, suggesting localized cell death and no further colonization was evident for C. truncatum. However, the manifestation of symptoms depended on the maturity status of the fruits, such that green fruits showed slower disease development with smaller lesions and delayed sporulation, whereas ripe (red) fruits were highly susceptible to the pathogen, expressing rapid lesion expansion, profuse sporulation and increased disease severity, indicating the critical role of physiological maturity in host susceptibility. Although infection was predominantly fruit-based, mild symptoms were also observed on vegetative parts, where leaves developed small, irregular necrotic spots surrounded by chlorotic halos that could enlarge under severe infection, leading to blighting and premature senescence. Similar observations were reported by Than et al. (2008) [22] and Temiyakul et al. (2012) [21] , who observed enhanced lesion development and faster disease progression in mature chilli fruits infected by Colletotrichum spp. In detached fruit assays, symptom development was evident within 3-5 days after inoculation, particularly at pin-pricked sites, confirming wounds as major entry points for the pathogen; lesion number and size increased progressively in positive control, whereas fruits treated with copper-chitosan nanocomposites exhibited delayed symptom onset and reduced lesion development, demonstrating effective disease suppression.

Efficacy of Copper Chitosan Nanocomposites against

Colletotrichum truncatum

CuChNCs exhibited significant antifungal activity against C. truncatum under detached fruit conditions and effectively suppressed anthracnose symptom development in green chilli. The nanocomposites demonstrated a clear dosedependent response, wherein increasing concentrations resulted in progressive reduction of lesion development and disease severity. Among the different concentrations tested, CuChNCs at 500 ppm completely inhibited disease development, recording zero Percent Disease Index (PDI), indicating absolute suppression of pathogen infection and lesion formation. Similarly, treatments with 300 ppm and 200 ppm CuChNCs significantly reduced disease severity, recording only 3.70% and 7.41% PDI, respectively. The treated fruits exhibited reduced lesion number, smaller lesion size and delayed symptom expression compared to untreated control fruits (Table 4 andPlate 4). Similar synergistic effects of copperchitosan nanocomposites against phytopathogens have been reported by recent studies on Fusarium species (Mosa and El-Abeid, 2023; Tarakanov et al., 2023) [12,20] . Conventional treatments such as copper sulphate (CuSO₄), COC and chitosan applied individually were comparatively less effective in suppressing anthracnose disease. Copper sulphate treatments recorded high disease severity with PDI values reaching up to 92.59%, while chitosan alone showed moderate disease reduction with maximum PDI of 77.78%. The untreated positive control fruits exhibited severe symptom development with 100% PDI, characterized by extensive tissue necrosis, sunken lesions and profuse sporulation of the pathogen (Plate 4). In contrast, nanocomposite-based formulations provide improved efficacy at lower dosages while minimizing environmental contamination and excessive copper usage. Recent studies have emphasized the potential of chitosan-based nanocomposites as sustainable alternatives to conventional fungicides in integrated disease management programs (Chiquito-Contreras et al., 2026; Hashim et al., 2024) [4,7] . The enhanced antifungal efficacy of CuChNCs against C. truncatum may be attributed due to the synergistic interaction between copper ions and chitosan polymer matrix. The nanoscale formulation likely improved the surface area and interaction of the active compounds with fungal cells, resulting in disruption of membrane integrity, leakage of intracellular contents and inhibition of fungal growth. In addition, the sustained release of copper ions from the chitosan matrix may have contributed to prolonged antifungal activity and effective suppression of pathogen colonization. These effects have also been observed in other recent nanofungicide studies by Shoala et al. (2025) [19] and Malook et al. (2026) [10] , who reported enhanced pathogen suppression through controlled ion release and reactive oxygen species (ROS)-mediated cellular damage. The findings clearly demonstrate the superior antifungal potential of CuChNCs over conventional treatments and highlight their promise as eco-friendly nanofungicides for sustainable management of chilli anthracnose caused by C. truncatum.

Conclusion

The study demonstrates that C. truncatum is highly pathogenic to chilli, with significant variability among hybrids.

Copper-chitosan nanocomposites showed exceptional antifungal efficacy, achieving complete disease suppression at higher concentrations. These findings suggest that CuChNCs are a promising eco-friendly alternative to conventional fungicides and hold significant potential for integrated disease management strategies in chilli cultivation.

Acknowledgement

AcknowledgementThe authors are grateful to Department of Plant Pathology, Department of Forestry and Environmental sciences, Soil Science and Agricultural Chemistry, College of Agriculture, V.C. Farm, Mandya, Organic Farming Research Station, Naganahalli, Department of Horticulture, ICAR, Krishi Vigyan Kendra (KVK), V. C. Farm, Mandya for the financial support and for providing laboratory facilities to conduct the research.

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