Antagonistic Potential of Trichoderma Isolates from Bamboo and Cardamom Rhizospheres Against Chili Anthracnose Pathogen

Chili is a vital agricultural commodity in Indonesia, essential for domestic consumption and the national economy. The demand for chili continues to grow with the increasing population and expanding industries that use chili as a raw material. Economic analysis indicates that chili farmers can achieve substantial profits, averaging around IDR 73.39 million per growing season after costs. However, chili production has decreased in recent years, from 1.5 million tons in 2020 to 1.39 million tons in 2021, reflecting a concerning decline [1-6].

Chili production in Indonesia faces significant challenges from various diseases, with anthracnose, caused by Colletotrichum spp., being one of the most destructive [7-10]. Recent studies have identified new pathogens causing chili anthracnose, such as Colletotrichum sojae in China [11] and Colletotrichum queenslandicum in Mauritus [12]. These findings highlight the need for continuous monitoring and research to understand the evolving epidemiology of chili anthracnose and to develop innovative control methods to mitigate its impact on chili yield and quality.

Anthracnose leads to substantial yield losses and affects the quality of chili fruits, posing significant challenges for farmers and the agricultural industry. The disease impact varies with environmental conditions, especially during the rainy season when losses can range from 10% to 80% due to favorable humidity and temperature. In contrast, during the dry season, yield losses are typically lower, ranging from 2% to 35%. In extreme cases, particularly in swampy areas during the rainy season and in highly susceptible varieties, yield losses can reach as high as 50% to 100% [3, 13-15].

Chili anthracnose manifests with various symptoms affecting leaves, stems, and fruits. The disease typically begins on fruits with small, slightly sunken, dark yellow spots that darken and enlarge, often merging in high humidity or wet weather. These spots may develop concentric rings resembling a target due to spore mass arrangements. Severe infections lead to extensive necrotic tissue, causing significant marketability issues and post-harvest rots, further diminishing quality. Irregularly shaped brown spots with dark borders can appear on leaves, though less common than fruit symptoms, still indicating severe infection. Infected plants may also experience dieback, where parts of the plant, including stems and branches, start to wilt and die [16-19]. The epidemiology of chili anthracnose is influenced by environmental factors such as rain, which can favor disease development [20].

Adopting sustainable and eco-friendly strategies is crucial for mitigating anthracnose in chili. Utilizing biological control techniques, particularly involving native antagonists like Trichoderma species, offers a viable alternative to chemical fungicides. Trichoderma species are recognized for their ability to control pathogens through methods like mycoparasitism, competition, and production of antimicrobial substances. Additionally, they enhance plant growth and boost disease resistance [18, 21-23].

Research indicates that Trichoderma harzianum and Trichoderma asperellum are particularly effective against Colletotrichum species. For example, T. harzianum has been shown to significantly reduce disease incidence and enhance plant growth in chili peppers infected with Colletotrichum [24, 25]. Similarly, Trichoderma viride has been reported to significantly reduce the severity of anthracnose on chili fruits, with culture filtrates demonstrating effective inhibition of mycelial growth and spore germination [26], suggesting that the metabolites produced by Trichoderma are directly antagonistic to the pathogen [27, 28]. Field trials have corroborated these findings, revealing that pre-treatment of chili seeds with Trichoderma not only reduces the incidence of anthracnose but also improves overall plant health and yield [25, 29].

The integration of Trichoderma spp. into agricultural practices can thus be seen as a sustainable approach to managing anthracnose in chili peppers, aligning with the growing emphasis on environmentally friendly disease control methods [30, 31]. Previous research has shown promising results when using Trichoderma spp. From agricultural soils in managing other Colletotrichum-related diseases, but the unique microbiomes of bamboo and cardamom rhizospheres may provide novel strains with improved efficacy against Colletotrichum spp. According to studies [32, 33], bamboo and cardamom plants are known to support diverse microbial communities within their rhizospheres, which may contain powerful biocontrol agents, harbor unique Trichoderma isolates with enhanced biocontrol potential. Furthermore, this study aims to address a critical gap in current knowledge by investigating whether isolates from these unique rhizospheres exhibit distinct or enhanced biocontrol properties compared to existing agents.

This research aims to identify and evaluate Trichoderma isolates from the rhizospheres of bamboo and cardamom plants. These isolates are being studied for their ability to counteract Colletotrichum species, which are the pathogens responsible for chili anthracnose. This study seeks to isolate and characterize these Trichoderma isolates to find effective biocontrol agents that can be utilized in sustainable management practices for chili anthracnose.

The objectives of this research are twofold: (1) to isolate and identify Trichoderma spp. from the rhizospheres of bamboo and cardamom, and (2) to evaluate the antagonistic potential of these isolates against Colletotrichum sp. under in vitro conditions. Through this approach, we aim to contribute to developing integrated disease management strategies that are environmentally friendly and effective in reducing the impact of chili anthracnose.

3.1 Morphological characterization of Trichoderma spp.

The results of the isolation obtained two Trichoderma isolates, BmMG and KpGR. All two isolates demonstrate fast growth rates and exhibit green pigmentation with various textures and margins, the differences in their macroscopic characteristics and growth speeds highlight their unique adaptations to their respective rhizosphere environments. Understanding these traits is essential for distinguishing between the isolates and for exploring their ecological roles. Different Trichoderma isolates exhibit variations in growth rates, pigmentation, textures, and margins, reflecting their adaptations to specific rhizosphere environments [43]. For instance, the morpho-cultural and molecular characterization of Trichoderma species from various regions has allowed for the identification of distinct isolates like T. koningiopsis, T. viride, T. asperellum, T. asperelloides, T. hamatum, and T. harzianum [44].

As seen in Table 2 and Figure 1(a, b, e, f), BmGr, isolated from the bamboo rhizosphere, exhibited colonies that were olive green to dark green in color with a cottony or powdery texture and an irregular or fringed margin. On the reverse side, the colonies showed a pale yellow to dark green concentric ring. The growth rate of BmGr was approximately 8.98 mm per day, indicating a fast growth speed. In contrast, KpGr, originating from the cardamom rhizosphere, showed colonies that were bright green to dark green, with a fluffy or woolly texture and an irregular or fringed margin. The reverse side displayed a pale yellow to light green concentric ring with dark green in the middle. KpGr grew at a rate of 8.35 mm per day, which was slightly slower compared to BmGr and CbGr but still within the fast growth category. The growth rate of Trichoderma is a key factor influenced by environmental conditions such as pH, nutrient availability, and interactions with other microorganisms in the rhizosphere. Trichoderma isolates are characterized by rapid growth rates and the production of secondary metabolites, which play a crucial role in their antagonistic activity against pathogens. These secondary metabolites not only act as antagonistic agents against pathogens but also serve as sporogenic factors and growth promoters affecting morphological differentiation in Trichoderma [45, 46].

The growth patterns and colony appearance of Trichoderma are influenced by various factors, including the nutrient media used for cultivation. Studies have shown that Trichoderma isolates display early sporulation and maximum mycelial development on specific solid media such as corn meal agar and oat meal agar [47]. Additionally, the differentiation of Trichoderma isolates based on mycelia growth rate, colony appearance, and microscopic morphological features like phialides and phialospores contribute to understanding the adaptability and diversity within the genus [48]. Furthermore, the morphological identification of Trichoderma species relies on observations of colony appearance, growth rate, and microscopic characteristics such as conidiophore branching patterns, phialide organization, and conidia shapes and sizes [49-52].

1.png

Figure 1. Macroscopic and microscopic characteristics of Trichoderma spp. isolates

Note: BmGr isolate (a-d); KpGr isolate (e-h); front colony (a,e); reverse colony (b, f); conidiophore and phialide (c, g); conidia (d, h, i). The black scale bar is 10 µm.

Table 3. Microscopic characteristics of Trichoderma spp. isolates from bamboo and cardamom rhizosphere

The microscopic examination of the isolates BmGr and KpGr, as is shown in Table 3 and Figure 1 (c, d, g, h) revealed distinct morphological characteristics that were important for their identification and differentiation. Both isolates produced smooth-walled, green conidia and exhibited variability in conidial shape and size, phialid dimensions, and conidiophore characteristics. BmGr conidia were ellipsoidal to cylindrical, smooth-walled, and varied from hyaline to bright green in color. They measured between 3.49-5.17 µm in length and 2.33-3.94 µm in width, with average dimensions of 4.23 µm in length and 2.98 µm in width. The phialides of BmGr were flask-shaped, hyaline, and ranged in length from 14.33-20.76 µm and in width from 4.02-7.88 µm, with mean values of 18.26 µm and 5.60 µm, respectively. The conidiophores were branched and colorless to pale yellow, producing conidia in whorls or clusters on the flask-shaped phialides. Compared to BmGr, KpGr conidia were ellipsoidal to cylindrical, smooth-walled, and green, with sizes ranging from 3.27-6.14 µm in length and 2.61-4.52 µm in width, and average dimensions of 5.16 µm in length and 3.29 µm in width. The phialides were slender, flask-shaped, and hyaline, ranging from 16.91-20.75 µm in length and 2.2-3.63 µm in width, with mean dimensions of 18.33 µm in length and 3.06 µm in width. The conidiophores of KpGr were erect, branched, and hyaline to pale green, producing conidia in dense clusters on slender, flask-shaped phialides. Although KpGr's conidial shape and color were similar to those of BmGr, the differences in size and density of the clusters suggested unique adaptations among these isolates. These differences are crucial for accurate identification and understanding the biological behavior of the fungi, which have implications for disease management and ecological studies.

The dimensions of conidia, including their length and diameter, vary significantly among different Trichoderma species, and these morphological traits are often used to classify and identify isolates. A study conducted by López-Quintero et al. [53] has demonstrated that conidial dimensions can be measured accurately, allowing for differentiation among closely related species based on these metrics. Additionally, the density of conidial clusters plays a significant role in species identification and ecological adaptation. Higher conidial densities can indicate particular species' adaptations to environmental conditions, influencing their growth and reproductive strategies. For instance, some Trichoderma isolates exhibit a strong ability to respond to diverse environmental signals, which correlates with their growth rates and conidial production [54].

Environmental factors such as temperature, chemical pressure, ultraviolet radiation, and cold stress influence conidial density and germination [55]. The regulation of gene expression networks also affects conidial production and density, impacting both asexual lifecycles [56]. Furthermore, factors like incubation temperature can alter conidial density, with higher temperatures leading to a decrease in conidial length [57]. This intricate interplay of morphological traits and environmental conditions underscores the complexity of Trichoderma species' adaptations and their classification.

3.2 Molecular identification of Trichoderma spp.

The provided phylogenetic tree (Figure 2), supported with BLAST results (Table 4 and Table 5), illustrates the evolutionary relationships among different isolates of Trichoderma, including Trichoderma virens and Trichoderma hamatum, with Colletotrichum asianum GY20 as an outgroup. The tree shows a well-supported clade (bootstrap value of 100) that includes all the T. virens strains: TR039, QT22122, OTU51, KY22016, F1d5c1, and BmGr. Another distinct clade, with strong bootstrap support (99), includes T. hamatum strains: CEN1334, CC13A, CEN1350, EEK D8, KpGr, and TR090. These results indicated that the BmGr isolate was closely related to Trichoderma virens, while the KpGr isolates was T. hamatum. The T. virens BmGr and T. hamatum KpGr sequences were then submitted to GenBank with accession numbers PQ066098 and PQ066099, respectively. The tree also reveals that T. virens clade was more distantly related to T. hamatum and T. asperellum compared to the relationship between T. hamatum and T. asperellum clade. The tight clustering and high bootstrap values within the T. virens and T. hamatum clades indicate strong intraspecies conservation. This suggests that these isolates are either under similar selective pressures or have not had enough time to accumulate significant genetic differences. The larger genetic distances and distinct clades between T. virens and the other two species imply a greater evolutionary divergence. These findings are crucial for understanding the evolutionary biology of these fungi and their potential applications in biological control and agriculture.

2.png

Figure 2. The maximum likelihood phylogenetic tree of Trichoderma spp. bootstrap values (1000 replications) above 50% shown at the nodes

Colletotrichum asianum serves as the outgroup. Isolates from this study are marked in red. The scale bar represents the number of substitutions per nucleotide position

Table 4. BLAST result of Trichoderma isolate KpGr ITS sequence

Table 5. BLAST result of Trichoderma isolate BmGr ITS sequence

As can be seen in Table 6, among the Trichoderma virens isolates, which included TR039, QT22122, OTU51, KY22016, F1d5c1, and BmGr, the pairwise distance was consistently 100%. This indicated that these strains were genetically identical or extremely similar, suggesting a high level of genetic conservation within this group. Such genetic similarity might have reflected their adaptation to similar ecological niches or their recent divergence from a common ancestor. In contrast, the Trichoderma hamatum isolates, which consisted of TR090, KpGr, EEK_D8, CEN1350, CEN1334, and CC13A, also showed high similarity among themselves, with pairwise distances around 100%. When compared to T. virens isolates, the T. hamatum isolates exhibited a pairwise distance of approximately 83%, indicating a moderate genetic difference between these two species. This moderate difference suggested that T. hamatum and T. virens might have diverged from a common ancestor at a more distant point in time than the divergence within each species. 

The pairwise distances indicated minimal variation within the T. virens and T. hamatum groups, suggesting that these groups were highly conserved. The interspecies variation indicated moderate genetic differences between T. virens and T. hamatum. The pairwise distance matrix provided valuable insights into the genetic relationships and evolutionary distances among different isolates of Trichoderma. This information was crucial for understanding their taxonomy, evolutionary biology, and potential applications in biological control and agriculture [58]. The observed genetic distances could help guide future research on the evolutionary history of these fungi and their potential use in biocontrol strategies.

Table 6. Pairwise distance between Trichoderma spp. isolates from bamboo and cardamom rhizosphere

Note: 1= Trichoderma hamatum CEN1334; 2 = Trichoderma hamatum CC13A; 3 = Trichoderma hamatum CEN1350; 4 = Trichoderma hamatum EEK_D8; 5 = Trichoderma hamatum KpGr; 6 = Trichoderma hamatum TR090; 7 = Trichoderma virens TR039; 8 = Trichoderma virens QT22122; 9 = Trichoderma virens OTU51; 10 = Trichoderma virens KY22016; 11 = Trichoderma virens F1d5c1; 12 = Trichoderma virens BmGr.

Table 7. Antagonism of Trichoderma spp. against Colletotrichum sp. in the dual culture test

Note: R = Replication.

3.3 Antagonistic potential of Trichoderma spp.

3.png

Figure 3. Dual culture test of Trichoderma spp. against Colletotrichum sp.

Description: a = control; 1 = Colletotrichum sp. isolate BCG1; 2 = Trichoderma spp. isolates; b = Trichoderma virens isolate KpGr; c =Trichoderma hamatum isolate BmGr. Red arrow is competition zone between Trichoderma spp. and Colletotrichum sp.

The results presented in Table 7 and Figure 3 illustrated the antagonistic effects of two Trichoderma virens isolate BmGr and Trichoderma hamatum isolate KpGr against Colletotrichum sp. in a dual culture test. Isolate BmGr showed inhibition percentages of 71.42%, 33.33%, and 85.71% for the three replicates (R1, R2, R3), respectively, with a mean inhibition percentage of 63.49% and a strong degree of inhibition. Isolate KpGr exhibited inhibition percentages of 54.28%, 66.66%, and 58.57%, with a mean inhibition percentage of 59.84%, also indicating a strong degree of inhibition. Both isolates employed competition and mycoparasitism as antagonistic mechanisms, with no evidence of antibiosis. BmGr demonstrated higher variability in inhibition percentages across replicates, while KpGr displayed more consistent performance.

Mycoparasitism was confirmed by direct microscopic examination, which revealed physical interactions between Trichoderma hyphae and Colletotrichum sp. during these observations, Trichoderma hyphae were seen coiling around the pathogen's hyphae, consistent with known mechanisms of mycoparasitism. Additionally, the absence of antibiosis was supported by dual culture assays, where no clear inhibition zones were observed between Trichoderma spp. and Colletotrichum sp. on the agar medium. This suggested that the isolates did not produce diffusible antimicrobial compounds under the experimental conditions. Instead, the reduced pathogen growth in dual cultures was attributed to nutrient competition and direct mycoparasitic interaction, as evidenced by the reduction in pathogen biomass near Trichoderma. These findings highlighted the multifaceted mechanisms of Trichoderma spp. in pathogen control, emphasizing the role of physical interaction and resource competition rather than antibiosis.

Trichoderma are saprophytic and thrive in nutrient-rich environments, allowing them to outcompete pathogenic fungi for essential resources such as nutrients and space. By occupying the ecological niche that pathogens would otherwise exploit, Trichoderma spp. effectively inhibit pathogen growth [59, 60]. One of the primary mechanisms by which Trichoderma spp. exert their biocontrol effects is through competition for nutrients and space. This competitive advantage is crucial, as it allows Trichoderma to establish itself in the rhizosphere, thereby limiting the resources available to pathogenic fungi. For instance, Manandhar et al. [61] highlighted that Trichoderma's ability to bind to pathogens plays a significant role in this competitive dynamic. Additionally, Bhandari [62] noted that Trichoderma species demonstrate strong biocontrol activities under both in vitro and field conditions, emphasizing their capacity to outcompete pathogens for essential nutrients.

Despite this variability, T. virense isolate BmGr and T. hamatum isolate KpGr consistently exhibited strong inhibition, primarily through competition and mycoparasitism. These findings suggest that both isolates could serve as potent biocontrol agents against Colletotrichum sp., providing an eco-friendly alternative to chemical fungicides. The dual culture test results affirmed the antagonistic potential of Trichoderma spp. against Colletotrichum sp., supporting their use as biological control agents. Further research and field trials could help optimize their application and effectiveness in various agricultural contexts.

The Trichoderma isolates in this study exert their antagonistic effects primarily through competition and mycoparasitism, rather than antibiosis. The absence of antibiosis suggests that the inhibition of Colletotrichum sp. is largely due to competitive exclusion of the pathogen and direct parasitism, rather than the production of antimicrobial compounds. This finding aligns with Ahmad et al. [63], who demonstrated that an endophyte reduced corm-rot through similar mechanisms, highlighting the importance of competitive exclusion and direct parasitism in pathogen management. Neupane et al. [64] supported this view by noting that antibiosis involves antimicrobial compound production, which was not observed in our study. Additionally, Adhikari [65] emphasized the need to understand various biological control mechanisms, including antibiosis, for effective pathogen management.

The variability in the performance of T. virens isolate BmGr may be due to environmental conditions or genetic differences between replicates. In contrast, the consistent inhibitory performance of T. hamatum KpGr suggests a more stable interaction with the pathogen, potentially making it more suitable for practical applications. Despite this variability, both isolates offer promise as eco-friendly alternatives to chemical fungicides, supporting the shift towards sustainable agricultural practices.

Genetic diversity within Trichoderma isolates might contribute to the variability observed in their antagonistic performance against phytopathogenic fungi. This diversity is primarily attributed to the genetic variability among different Trichoderma species and strains, which can arise from mutations, chromosomal rearrangements, and recombination processes within their genomes [66]. For instance, studies have shown that multiple introductions of Trichoderma isolates into various environments can enhance genetic diversity, leading to the formation of distinct genotypes that exhibit varying levels of antagonistic activity [67]. The presence of diverse genetic backgrounds allows for a broader range of biochemical and physiological traits, which are crucial for effective biocontrol [68].

Moreover, the heterogeneity among fungal propagules, such as spores and mycelial fragments, plays a vital role in determining the efficacy of Trichoderma as biocontrol agents. Different isolates exhibit variations in morphological and physiological characteristics, which can influence their ability to compete for resources and establish dominance in the rhizosphere [69]. For example, certain isolates may produce higher levels of lytic enzymes, such as chitinases and glucanases, which are essential for degrading the cell walls of pathogenic fungi [70, 71]. This enzymatic activity is often linked to the genetic makeup of the isolates, suggesting that those with a more diverse genetic background may possess enhanced antagonistic capabilities [72].

Research has shown that T. virens is an effective biocontrol agent that inhibits plant pathogens through multiple mechanisms. This includes inducing localized or systemic resistance responses in plants, explaining its non-pathogenic nature. Studies [73, 74] have demonstrated its ability to control plant pathogens, particularly fungi, through mycoparasitism and bioactive compound production. Furthermore, T. virens enhances plant growth and induces plant defense-related enzymes, contributing to its pathogen resistance capabilities [75].

Similarly, T. hamatum is known for its biocontrol efficacy against various plant pathogens. It has been shown to inhibit pathogens such as Sclerotinia sclerotiorum [76], Fusarium oxysporum [77], Meloidogyne incognita [78], Lasiodiplodia theobromae [79], and Rhizoctonia solani [80]. Studies have also highlighted its effectiveness in reducing disease in crops like strawberries [81] and its ability to induce systemic resistance in plants [82]. Additionally, T. hamatum promotes plant growth and produces hydrolytic enzymes that contribute to its antagonistic potential [83].