Biocontrol Mechanisms of Trichoderma koningiopsis PSU3-2 against Postharvest Anthracnose of Chili Pepper

Several mechanisms are involved in the biological control of plant pathogens by the soil-borne Trichoderma spp. fungi. The aim of this study was to characterize a new strain of Trichoderma as a potential biological control agent to control the postharvest anthracnose of chili pepper caused by Colletotrichum gloeosporioides. A total of nine strains of Trichoderma spp. were screened for their antifungal activity using a dual culture assay against C. gloeosporioides. Trichoderma koningiopsis PSU3-2 was shown to be the most effective strain, with a percentage inhibition of 79.57%, which was significantly higher than that of other strains (p < 0.05). In the sealed plate method, T. koningiopsis PSU3-2 suppressed the growth of C. gloeosporioides by 38.33%. Solid-phase microextraction (SPME) was applied to trap volatiles emitted by T. koningiopsis PSU3-2, and the GC/MS profiling revealed the presence of antifungal compounds including azetidine, 2-phenylethanol, and ethyl hexadecanoate. The production of cell-wall-degrading enzymes (CWDEs) was assayed through cell-free culture filtrate (CF) of PSU3-2, and the enzyme activity of chitinase and β-1,3-glucanase was 0.06 and 0.23 U/mL, respectively, significantly higher than that in the control (p < 0.05). Scanning electron microscopy of the mycelium incubated in cell-free CF of T. koningiopsis PSU3-2 showed the abnormal shape of C. gloeosporioides hyphae. Application of T. koningiopsis PSU3-2 by the dipping method significantly reduced the lesion size (p < 0.05) after inoculation with C. gloeosporioides compared to the control, and there was no disease symptom development in T. koningiopsis PSU3-2-treated chili pepper. This study demonstrates that T. koningiopsis PSU3-2 is an effective antagonistic microorganism and a promising biocontrol agent against postharvest anthracnose of chili pepper, acting with multiple mechanisms.


Introduction
Rhizosphere soil has long been considered as the main source of isolation of useful beneficial microorganisms [1,2]. At present, numerous soil fungi isolated from soil are employed as biological control agents, especially fungi in the genus Trichoderma. Trichoderma species are widely used to control numerous plant pathogens and reduce disease severity [3,4], due to their capacity for nutrient and space competition [5,6], parasitism [7], secretion of antimicrobial metabolites [7][8][9][10], activation of defense responses [11,12], and promotion of plant growth [8,9,13]. Moreover, metabolites, such as volatile organic compounds (VOCs), secreted from the Trichoderma species have been applied to promote plant growth [8,9,14]. Application of the Trichoderma species has been used to reduce the disease severity of leaf spots on lettuce [12] and sugar beet [15], as well as brown spots on rice [16]. Biological control presents low human health risks, as well as an environmentally friendly method without the excessive use of chemical fungicides in various crops. 2

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Anthracnose is a common plant disease characterized by dark, sunken lesions on fruits, leaves, and stems containing conidia [17]. The causal agents of this disease, identified as Colletotrichum spp., reduce both the quality and the quantity of a harvest yield. Disease severity increases during the rainy season, as conidia of Colletotrichum are splashed and dispersed onto fresh fruit, resulting in secondary infection [18]. Anthracnose disease caused by Colletotrichum spp. has been reported to negatively impact the cultivation and production of mangoes [19,20], bananas [21], tomatoes [22], and chili peppers [23].
Chili anthracnose is a major constraint in chili production leading to huge losses, especially postharvest anthracnose, which causes the decay of chili pepper in tropical and subtropical regions [24,25]. Developing biological management strategies to control chili anthracnose may benefit disease management in chili peppers. This study, therefore, aimed to explore the potential of Trichoderma spp. isolated from soil as a biocontrol agent through dipping application. Multiple mechanisms of Trichoderma strains were tested for antifungal activity against Colletotrichum gloeosporioides.

Dual Culture Assay
Nine strains of Trichoderma spp. were screened for antifungal activities on the mycelial growth of C. gloeosporioides through a dual culture assay on PDA plates [27]. An agar plug of a 5-day-old C. gloeosporioides colony was placed on the side of 9 cm Petri dishes, with an agar plug of each Trichoderma sp. placed on the opposite side 5 cm from the pathogen. PDA plates with pathogen alone served as the control. The experiment was designed according to a complete randomized block (CRD) with five replicates and repeated twice. The tested plates were incubated at ambient temperature (28 ± 2 • C) for 7 days. Colony radii of C. gloeosporioides were measured, and the percentage inhibition was calculated using the method of Rahman et al. [28], as given in Equation (1).
where R1 is the radial growth of C. gloeosporioides in control, and R2 is the radial growth of C. gloeosporioides with treatment.

Volatile Antifungal Bioassay and Solid-Phase Microextraction GC/MS Analysis
The effect of volatiles emitted by Trichoderma spp. was examined using the sealed plate method [10,29]. The most effective Trichoderma isolate was cultured in a 20 mL chromatography vial, 20 mm in diameter (PerkinElmer, Waltham, MA, USA), and incubated at 28 ± 2 • C for 10 days. Volatiles emitted by Trichoderma were trapped by solid-phase microextraction (SPME) fibers and inserted into the injection port of an SQ8 gas chromatograph (PerkinElmer, Waltham, MA, USA). GC/MS conditions adhered to the method previously described by Phoka et al. [9] and Intana et al. [10]. Total volatiles released from Trichoderma were tentatively identified by a computer search of the National Institute of Standards and Technology (NIST) Mass Spectral Library Search Chromatogram.

Liquid-Phase Cultivation and Enzyme Assay
The effective Trichoderma spp. were cultivated in potato dextrose broth (PDB) and incubated at 28 ± 2 • C for 5 days according to the method of Wonglom et al. [6]. The PDB-cultured Trichoderma spp. were filtrated with a 0.45 µm Minisart ® Syringe Filter (Sigma-Aldrich, St. Louis, MO, USA) and used as cell-free culture filtrate (CF). An enzyme assay was conducted to confirm that the cell-free CF of Trichoderma spp. contained cellwall-degrading enzymes (CWDEs) responsible for the fungal cell-wall degradation, while chitinase and β-1,3-glucanase activities were assayed with 3,5-dinitrosalicylic acid (DNS), as suggested by Miller [30]. Reaction mixtures containing colloidal chitin were used as the substrate in the chitinase assay, whereas mixtures containing laminarin (Sigma-Aldrich, St. Louis, MO, USA) were used as the substrate in the β-1,3-glucanase assay. An assay with PDB alone served as the control. Reducing sugar released in the test reaction mixtures was measured using an ultraviolet/visible light (UV/Vis) spectrophotometer UV5300 (METASH, Shanghai, China) at 550 and 575 nm for β-1,3-glucanase and chitinase, respectively. Enzymes were assayed in three replicates, and the experiments were repeated twice.

Scanning Electron Microscopy
To test the effect of cell-free CF on fungal mycelia morphology, a scanning electron microscope (SEM) was utilized according to the method of Baiyee et al. [12]. A mycelial plug (0.5 cm) of a 7-day-old colony of C. gloeosporioides was incubated in the cell-free CF of effective Trichoderma strains at 37 • C for 1 h, whereas the control was incubated with PDB only. The mycelial plugs were fixed in 3% glutaraldehyde at 4 • C for 24 h and then dehydrated in a 30%, 50%, 60%, 70%, 80%, 90%, and 100% alcohol series, three times each. The samples were coated with gold and observed using a JSM-580 LV SEM (JEOL, Peabody, MA, USA) at the Science Equipment Center, Prince of Songkla University, Songkhla, Thailand.

In Vivo Test
A spore suspension of effective Trichoderma was prepared, and the concentration was adjusted with sterile distilled water (DW) to 1 × 10 6 conidia/mL. A spore suspension of the Colletotrichum sp. was prepared in the same manner. Chili peppers were surface-disinfected with 70% ethanol, dipped in the spore suspension of Trichoderma spp., and air-dried in a laminar airflow cabinet. Chili peppers dipped in DW alone and the spore suspension of the Colletotrichum sp. served as the negative and positive controls, respectively. Then, 20 mL spore suspensions of C. gloeosporioides were sprayed onto the chili peppers after being dipped in the spore suspension of Trichoderma for 24 h and incubated in a moist box for 5 days, at which time the lesion development of all treated chili peppers was measured. Each treatment included five chili peppers (five replicates), and each experiment was repeated three times.

Statistical Analysis
The results regarding fungal inhibition, the enzyme assay, and lesion development were subjected to one-way analysis of variance (ANOVA). Statistically significant differences among treated samples were determined by Tukey's test.

Antifungal Activity of Trichoderma spp.
After incubation for 7 days, a smaller growth of C. gloeosporioides was observed in the dual culture plate than in the control plate. Nine strains of Trichoderma spp. inhibited the fungal growth of C. gloeosporioides in dual culture plates with inhibition percentages ranging from 60.84 to 79.57% ( Figure 1). T. koningiopsis PSU3-2 was shown to be the most effective strain, with a percentage inhibition of 79.57%, statistically higher than that of other strains (p < 0.05) in this assay ( Figure 1); therefore, the T. koningiopsis PSU3-2 strain was selected for further bioassays.
After incubation for 7 days, a smaller growth of C. gloeosporioides was observed in the dual culture plate than in the control plate. Nine strains of Trichoderma spp. inhibited the fungal growth of C. gloeosporioides in dual culture plates with inhibition percentages ranging from 60.84 to 79.57% ( Figure 1). T. koningiopsis PSU3-2 was shown to be the most effective strain, with a percentage inhibition of 79.57%, statistically higher than that of other strains (p < 0.05) in this assay ( Figure 1); therefore, the T. koningiopsis PSU3-2 strain was selected for further bioassays.

Production of Volatile Antifungal Compounds
The sealed plate method showed that T. koningiopsis PSU3-2 inhibited the fungal growth of C. gloeosporioides, with a percentage inhibition of 38.33%. This result reveals that T. koningiopsis PSU3-2 produced volatile organic compounds which were responsible for suppressing the mycelial growth of C. gloeosporioides in vitro. A total of 16 volatile compounds were detected in T. koningiopsis PSU3-2 through GC/MS analysis. The volatile compounds contained carbon numbers ranging from C1 (fluoro(trinitro)methane) to C20 (ethyl (E)-octadec-9-enoate). The major compounds found in this study were 2-phenylethanol followed by fluoroethane and 1-oxacyclotetradeca-4,11-diyne, with percentage peak areas of 14.94, 12.85, and 11.588%, respectively (Table 1). According to previous literature reviews, only three compounds were reported as volatile antifungal compounds (VOCs), namely, azetidine (1.507% peak area), 2-phenylethanol (14.941%), and ethyl hexadecanoate (9.036%). Figure 2 shows the mass spectrum of volatile antifungal compounds and their structures. No major peaks were observed in PDA alone, which served as the control group.

Production of Volatile Antifungal Compounds
The sealed plate method showed that T. koningiopsis PSU3-2 inhibited the fungal growth of C. gloeosporioides, with a percentage inhibition of 38.33%. This result reveals that T. koningiopsis PSU3-2 produced volatile organic compounds which were responsible for suppressing the mycelial growth of C. gloeosporioides in vitro. A total of 16 volatile compounds were detected in T. koningiopsis PSU3-2 through GC/MS analysis. The volatile compounds contained carbon numbers ranging from C1 (fluoro(trinitro)methane) to C20 (ethyl (E)-octadec-9-enoate). The major compounds found in this study were 2-phenylethanol followed by fluoroethane and 1-oxacyclotetradeca-4,11-diyne, with percentage peak areas of 14.94, 12.85, and 11.588%, respectively (Table 1). According to previous literature reviews, only three compounds were reported as volatile antifungal compounds (VOCs), namely, azetidine (1.507% peak area), 2-phenylethanol (14.941%), and ethyl hexadecanoate (9.036%). Figure 2 shows the mass spectrum of volatile antifungal compounds and their structures. No major peaks were observed in PDA alone, which served as the control group.

Effect of Cell-Free CF on Fungal Mycelia
SEM analysis was conducted to confirm the nature of the cell-free CF of T. koningiopsis PSU3-2 containing CWDEs or antifungal compounds responsible for inhibiting the fungal growth of C. gloeosporioides. The SEM micrograph of the control (PDB alone) exhibited no morphological change in the fungal mycelia of the Colletotrichum sp. (Figure 4), whereas the fungal mycelia incubated in the cell-free CF of T. koningiopsis PSU3-2 displayed abnormal shapes and mycelial distortions (Figure 4).

Effect of Trichoderma on Lesion Development
Treatment of T. koningiopsis PSU3-2 using the dipping method prior to inoculation with Colletotrichum sp. significantly reduced the size of anthracnose lesions (p < 0.05) analyzed for all chili peppers in all treatments. The lesion sizes developed on the chili pepper of the untreated control group, the Trichoderma PSU3-2-treated chili pepper, and C. gloeosporioides inoculation alone (control) were 0, 0, and 1.28 cm in diameter, respectively ( Figure 5). There was no disease development in the T. koningiopsis PSU3-2-treated chili pepper fruit after incubation for 5 days.

Effect of Cell-Free CF on Fungal Mycelia
SEM analysis was conducted to confirm the nature of the cell-free CF of T. koningiopsis PSU3-2 containing CWDEs or antifungal compounds responsible for inhibiting the fungal growth of C. gloeosporioides. The SEM micrograph of the control (PDB alone) exhibited no morphological change in the fungal mycelia of the Colletotrichum sp. (Figure 4), whereas the fungal mycelia incubated in the cell-free CF of T. koningiopsis PSU3-2 displayed abnormal shapes and mycelial distortions (Figure 4).

Effect of Trichoderma on Lesion Development
Treatment of T. koningiopsis PSU3-2 using the dipping method prior to inoculation with Colletotrichum sp. significantly reduced the size of anthracnose lesions (p < 0.05) analyzed for all chili peppers in all treatments. The lesion sizes developed on the chili pepper of the untreated control group, the Trichoderma PSU3-2-treated chili pepper, and C. gloeosporioides inoculation alone (control) were 0, 0, and 1.28 cm in diameter, respectively (Figure 5). There was no disease development in the T. koningiopsis PSU3-2-treated chili pepper fruit after incubation for 5 days.

Effect of Cell-Free CF on Fungal Mycelia
SEM analysis was conducted to confirm the nature of the cell-free CF of T. koningiopsis PSU3-2 containing CWDEs or antifungal compounds responsible for inhibiting the fungal growth of C. gloeosporioides. The SEM micrograph of the control (PDB alone) exhibited no morphological change in the fungal mycelia of the Colletotrichum sp. (Figure 4), whereas the fungal mycelia incubated in the cell-free CF of T. koningiopsis PSU3-2 displayed abnormal shapes and mycelial distortions (Figure 4).

Effect of Trichoderma on Lesion Development
Treatment of T. koningiopsis PSU3-2 using the dipping method prior to inoculation with Colletotrichum sp. significantly reduced the size of anthracnose lesions (p < 0.05) analyzed for all chili peppers in all treatments. The lesion sizes developed on the chili pepper of the untreated control group, the Trichoderma PSU3-2-treated chili pepper, and C. gloeosporioides inoculation alone (control) were 0, 0, and 1.28 cm in diameter, respectively (Figure 5). There was no disease development in the T. koningiopsis PSU3-2-treated chili pepper fruit after incubation for 5 days.

Discussion
Postharvest anthracnose of chili pepper is reportedly caused by Colletotrichum spp., leading to a reduction in both the quality and the quantity of chili pepper production [24,25]. This study investigated the antifungal activity of Trichoderma spp. against postharvest anthracnose of chili pepper fruit. T. koningiopsis PSU3-2 effectively suppressed the fungal growth of the C. gloeosporioides, revealing a competition mechanism (Figure 1). This isolate was documented as being capable of emitting VOCs to restrict the mycelial growth of the C. gloeosporioides (Figure 3), along with overproduction of CWDEs leading to a morphological change in the C. gloeosporioides (Figure 4). Furthermore, treatment with T. koningiopsis PSU3-2 protected chili peppers from postharvest anthracnose decay ( Figure 5).
The ability to compete for nutrients and space is commonly found in several Trichoderma spp. to overcome the growth of fungal pathogens through a dual culture assay [3,4,6,31]. In vitro studies revealed the competition mechanism of Trichoderma spp. against Sclerotium sclerotiorum [32], Rhizoctonia solani, Macrophomina phaseolina [33], and Curvularia oryzae [3]. Our findings in this study are in agreement with previous publications that found that T. koningiopsis PSU3-2 grew faster than the C. gloeosporioides, effectively inhibiting the growth of the C. gloeosporioides in PDA-assayed plates, thereby suggesting a competition mechanism involved in biocontrol activity (Figure 1).
VOCs have been reported as being produced and released by several Trichoderma species with a diversity of volatile compounds [31]. The VOCs emitted by Trichoderma species display multiple functions; they have antifungal properties, induce a defense response, and promote plant growth [8,9]. Among the 16 VOCs produced by T. koningiopsis PSU3-2, three compounds, namely, azetidine, 2-phenylethanol, and ethyl hexadecanoate, have been reported to have antimicrobial activity [34][35][36]. For instance, 2-phenylethanol emitted from T. asperellum T76-14 was reported to control the postharvest fruit rot of

Discussion
Postharvest anthracnose of chili pepper is reportedly caused by Colletotrichum spp., leading to a reduction in both the quality and the quantity of chili pepper production [24,25]. This study investigated the antifungal activity of Trichoderma spp. against postharvest anthracnose of chili pepper fruit. T. koningiopsis PSU3-2 effectively suppressed the fungal growth of the C. gloeosporioides, revealing a competition mechanism (Figure 1). This isolate was documented as being capable of emitting VOCs to restrict the mycelial growth of the C. gloeosporioides (Figure 3), along with overproduction of CWDEs leading to a morphological change in the C. gloeosporioides (Figure 4). Furthermore, treatment with T. koningiopsis PSU3-2 protected chili peppers from postharvest anthracnose decay ( Figure 5).
The ability to compete for nutrients and space is commonly found in several Trichoderma spp. to overcome the growth of fungal pathogens through a dual culture assay [3,4,6,31]. In vitro studies revealed the competition mechanism of Trichoderma spp. against Sclerotium sclerotiorum [32], Rhizoctonia solani, Macrophomina phaseolina [33], and Curvularia oryzae [3]. Our findings in this study are in agreement with previous publications that found that T. koningiopsis PSU3-2 grew faster than the C. gloeosporioides, effectively inhibiting the growth of the C. gloeosporioides in PDA-assayed plates, thereby suggesting a competition mechanism involved in biocontrol activity (Figure 1).
VOCs have been reported as being produced and released by several Trichoderma species with a diversity of volatile compounds [31]. The VOCs emitted by Trichoderma species display multiple functions; they have antifungal properties, induce a defense response, and promote plant growth [8,9]. Among the 16 VOCs produced by T. koningiopsis PSU3-2, three compounds, namely, azetidine, 2-phenylethanol, and ethyl hexadecanoate, have been reported to have antimicrobial activity [34][35][36]. For instance, 2-phenylethanol emitted from T. asperellum T76-14 was reported to control the postharvest fruit rot of muskmelon [10]. Therefore, the VOCs of T. koningiopsis PSU3-2 containing azetidine, 2-phenylethanol, and ethyl hexadecanoate may be associated with the suppression of the mycelial growth of the C. gloeosporioides, suggesting the antibiosis mechanism of T. koningiopsis PSU3-2. Several Trichoderma species produce and secrete hydrolytic enzymes responsible for degrading the fungal cell wall. The main CWDEs produced by Trichoderma species are chitinase and β-1,3-glucanase [37]. Chitinase restricts fungal growth by degrading chitin, the major component within the fungal cell wall [38], whereas β-1,3-glucanase hydrolyzes β-glucan to oligosaccharide and glucose [39]. A combination of both enzyme activities strongly suppresses the growth of several plant fungal pathogens [4]. Our results demonstrate a high activity of CWDEs in the cell-free CF of T. koningiopsis PSU3-2 ( Figure 3), possibly related to the inhibition of fungal growth. We confirmed through SEM analysis that the cell-free CF of T. koningiopsis PSU3 contained CWDEs, which caused lysis and distortion of the C. gloeosporioides hyphae (Figure 4). The ability to produce CWDEs capable of creating mycelial lysis (holes), further resulting in fungal penetration in the host fungi, suggests mycoparasitism [40]. Baiyee et al. [4] similarly observed high activities of chitinase and β-1,3-glucanase, which caused abnormal changes in the fungal mycelia. These findings may be the result of CWDEs or some type of antifungal compound released by T. koningiopsis PSU3-2. However, we only studied the effects of cell-free CF, and we did not observe other metabolites in this study.
The application of a Trichoderma spore suspension has been shown to successfully control several plant diseases [3,16,41]. Treatment with a spore suspension of Trichoderma spirale T76-1 reduced the disease severity of lettuce leaf spots caused by Corynespora cassiicola and Curvularia aeria [4]. Root dipping with a T. asperellum T1 spore suspension was reported to activate defense responses in lettuce against leaf spot disease [12]. Treatment with Trichoderma protected tomato plants from infection by Phytophthora nicotianae [42]. Jogaiah et al. [43] demonstrated that the application of a Trichoderma virens spore suspension mediated resistance in tomatoes against Fusarium wilt by activating the jasmonic and salicylic pathways. Our study showed that chili peppers dipped in a spore suspension of T. koningiopsis PSU3-2 displayed no anthracnose lesions ( Figure 5). Therefore, the biological activity of T. koningiopsis PSU3-2 is able to limit fungal infections, thereby controlling postharvest anthracnose of chili pepper fruit.

Conclusions
This study revealed the potential of a new strain of T. koningiopsis PSU3-2 isolated from soil as a biocontrol agent against anthracnose of chili pepper fruit caused by a C. gloeosporioides. The ability to compete for nutrients and space (competition), the production of VOCs (antibiosis), and the production of CWDEs (mycoparasitism) were the main factors contributing to its success in controlling the postharvest anthracnose of chili pepper fruit. The potential to develop a biopesticide to control chili anthracnose using T. koningiopsis PSU3-2 needs to be verified in the near future.