The tools use in this study included a Petri dishes, a laminar air flow cabinet, an autoclave, and an incubator. The materials consisted of phyllosphere fungal isolates, Colletotrichum sp., and Potato Dextrose Agar (PDA) medium. Antagonistic activity was evaluated based on the inhibition of pathogen growth by the antagonistic fungi compared with the control treatment.

This study was conducted as a laboratory-based experimental research to evaluate the antagonistic activity of phyllosphere fungi isolated from several medicinal plants against Colletotrichum sp., the causal agent of anthracnose disease in chili plants, using the dual culture method. The experiment was arranged in a completely randomized design (CRD) with three replicates.

a. Media Preparation

Potato Dextrose Agar (PDA) medium was prepared by dissolving 39 g of ready-to-use PDA powder (Merck, Darmstadt, Germany) in 1 L of distilled water, followed by heating and homogenization. The medium was sterilized in an autoclave at 121 °C under a pressure of 2 atm for 20 minutes.

b. Fungi Reculture

Reculture of phyllosphere fungal isolates obtained from the Laboratorium Biota Sumatera culture collection, Universitas Andalas, and Colletotrichum isolates was carried out in test tubes by transferring a portion of fungal hyphae previously grown on Potato Dextrose Agar (PDA) plates into sterile PDA medium in test tubes. The recultured fungi were incubated at room temperature (25–27 °C) for 7 days until abundant mycelial growth was obtained.

c. Morphological Characterization of Colletotrichum sp.

The characterization of Colletotrichum sp. was carried out through macroscopic and microscopic observations. Microscopic characteristics were observed using a photomicrograph (microscope). A small portion of hyphae was carefully taken using a sterile inoculating needle and placed on a glass slide previously dropped with sterile distilled water. The observation was then performed using a photomicrograph (microscope). The microscopic characteristics observed included hyphal structure, conidia, and conidiophores. Meanwhile, macroscopic characteristics observed included colony shape, color, and growth pattern (Halwiyah et al., 2019).

d. Antagonistic Assay

The antagonistic activity of phyllosphere fungal isolates from several medicinal plants against the pathogenic fungus Colletotrichum sp. was evaluated using the dual culture method with three replications. Mycelial plugs (5 mm) of the antagonist and pathogen were taken from 7 day old cultures using a sterile cork borer and placed opposite each other (3 cm apart) on the same Petri dish. Plates were incubated at 25–28°C for 5–7 days, and inhibition was assessed based on radial growth reduction of the pathogen compared to the control (R1 = control; R2 = treatment), following (Dennis & Webster, 1971).

e. Measurement of Percentage Inhibition

The percentage of inhibition (PI) was determined by measuring the radius of the pathogenic fungal colony growing toward and away from the antagonistic fungal colony. According to Ningsih et al. (2016), the percentage of inhibition (PI) was calculated using the following formula:

R = percentage of growth inhibition (%);

R₁ = diameter of pathogen growth in the control treatment (mm);

R₂ = diameter of pathogen growth in each treatment (mm).

f. Data Analysis

The data were analyzed for inhibition and fungi growth diameter using ANOVA. If a significant difference was observed, it was followed by Duncan’s Multiple Range Test (DMRT) at a 5% significance level.

The morphological characteristics of the pathogenic fungus Colletotrichum sp. are shown in Figure 2.

The results of the study showed in Figure 2. the Colletotrichum isolate exhibited distinctive morphological characteristics, with colonies appearing whitish-gray and a darker central area. The colony texture was smooth, with radial growth characterized by even margins and a denser central zone. During the early stage of growth, the conidia appeared orange in color. Microscopically, the isolate showed septate hyphae with smooth and branched structures. The conidiophores were observed in clusters, with terminal structures producing chains of conidia. The conidia were oval to fusiform in shape, small in size, and evenly distributed along the conidiophores.

The colony texture of Colletotrichum varies depending on the species and the growth medium used. Generally, on Potato Dextrose Agar (PDA), Colletotrichum colonies exhibit cottony to velvety textures, with surfaces that are often smooth or slightly fluffy. In some species, such as Colletotrichum gloeosporioides, colonies tend to be grayish to dark in the central area with white margins. In contrast, Colletotrichum acutatum typically forms colonies that are orange to pinkish in color, with a more compact and smoother texture (Wakhidah & Sari, 2021). As the colonies mature, the central region may become darker due to spore formation. Colony growth patterns may also display concentric rings formed as a result of variations in hyphal and conidial production (Marizka et al., 2025-08).

The percentage of inhibition of each phyllosphere fungi isolate against Colletotrichum sp. can be seen in Table 1.

Based on the data in Table 1,all isolates of phyllosphere fungi from several medicinal plants showed antagonistic activity against the pathogen Colletotrichum sp. The highest inhibition percentage was produced by the Trichoderma sp. (2) isolate (92.78%), and it was not significantly different from Trichoderma sp. (1) (89.67%), Trichoderma sp. (5) (89.00%), Trichoderma sp. (4) (86.89%), Trichoderma sp. (3) (85.77%) and Trichoderma sp. (6) (85.55%) isolates. However, it was significantly different from the Trichoderma sp. (7) (65.44%) isolates. All isolates of phyllosphere fungi have the potential to inhibit the growth of the pathogen Colletotrichum sp.

Competition occurs when two microorganisms require nutrients and living space in limited quantities. In this mechanism, antagonistic fungi are able to gain more control over resources than pathogenic fungi, thereby inhibiting their growth. The suppression of pathogens by antagonistic fungi can occur through competition for nutrients and space, the production of antibiotic compounds (antibiosis), and parasitic mechanisms (Ainy et al., 2015). According to a study by (Kamel et al., 2020), microscopic observations of mycoparasitic interactions show various forms of different interactions. Examples include the abundant growth of antagonistic hyphae around the pathogenic hyphae, hyphae coiling around pathogenic hyphae, and lysis of the hyphae.

The process of antagonistic mechanisms through competition occurs because two or more microorganisms require nutrients and space with limited availability (Karim et al., 2020). The antibiosis process involves the production of chemical compounds by antagonistic fungi that are toxic to the pathogen. These compounds can be antibiotics that directly inhibit or kill the pathogen. In parasitism, antagonistic fungi directly attack the pathogen by attaching to, coiling around, or penetrating the pathogen's hyphae, which ultimately causes damage or death to the pathogen (Muhibuddin et al., 2021).

The rapid growth of antagonistic fungi plays a crucial role in suppressing target pathogens through competition for space and nutrients. Factors that influence the growth rate of a pathogen are its viability and conidia density. Antagonistic fungi with slow growth generally have a lower inhibition potential against faster-growing pathogens (Yulianto, 2014).

Figure 3 shows the results of the antagonistic test between the phyllosphere fungi (yellow arrow, A) and the pathogen Colletotrichum sp. (red arrow, B). All isolates of the phyllosphere fungi showed inhibitory activity against the growth of Colletotrichum sp., which was indicated by the presence of an inhibition zone or suppressed growth around the pathogen. In the isolates of Trichoderma sp. (1), Trichoderma sp. (2), Trichoderma sp. (5), and Trichoderma sp. (7). The phyllosphere fungi were observed to occupy the entire Petri dish, indicating that the fungi can inhibit the growth of pathogenic fungi through competition for space and nutrients.

According to (Cendrawati et al., 2020), the grouping of antagonistic mechanisms can be based on the growth rate of the fungi in occupying the media surface in a Petri dish. If the antagonistic fungi grow faster until they occupy the entire Petri dish, this indicates a competition mechanism. This is a factor that triggers antagonism through competition. This is due to the presence of several microorganisms that compete with each other to obtain nutrients and space with limited availability.

In Trichoderma sp. (3), Trichoderma sp. (4), and Trichoderma sp. (7), the inhibition zone (empty space) separating the mycelium of the antagonistic and pathogenic fungi was not visible, but a change in the color of the media indicated antibiosis activity in the dual culture test.According to Jayalakshmiet al. (2021), Previous studies reported that Trichoderma spp. produce antifungal secondary metabolites such as gliotoxin that contribute to the inhibition of plant pathogenic fungi. The production of these metabolites during fungal interaction may also influence changes in the culture medium. The antibiosis mechanism of antagonism is suspected to occur as a result of compounds produced by a microorganism, such as antibiotics, alkaloids, metabolites, or toxins, which can inhibit the growth of other microorganisms(Berlian et al., 2013).

An organism can be called a biological control agent if it is able to multiply in conditions that are not favorable for other organisms and can inhibit their growth and development (Muslim, 2019). Antagonistic fungi groups are known for their ability to control or suppress the growth of plant pathogens (Widiyanti et al., 2022). Antagonistic isolates with high inhibition are those whose colonies grow faster than the pathogen's colonies, thus inhibiting pathogen growth. This rapid growth allows antagonistic fungi to dominate in utilizing nutrients and space, ultimately suppressing the pathogen's development (Sari & Setiawanto, 2015).

Overall, these results show that the 7 isolates of phyllosphere fungi have the potential to be used as biological control agents against Colletotrichum sp., although their effectiveness can vary depending on the type of isolate used. These findings affirm the importance of selecting isolates with the highest antagonistic activity, as each isolate may have a different mechanism of action, such as nutrient competition, production of antifungi compounds, or parasitism, which need to be studied further before being applied in the field. Thus, the utilization of phyllosphere fungi can be a promising and environmentally friendly alternative for biological control.