Soil samples were collected from four different geographical locations, in Andong, Gyeongsangbuk Province, Korea, i.e., Ongcheon-ri (36°41’35.5”N 128°41’39.8”E), Mulhan-ri (36°40’12.0”N 128°42’34.0”E), Namseon myeon (36°32’06.0”N 128°47’04.6”E), and Seonyeon-ri (36°40’55.6”N 128°44’52.8”E) of apple orchards near mountain regions. The soil sample was collected 50 cm away from the apple tree and 5 cm below the ground. One gram of soil was taken in a Falcon tube (50 mL) containing sterile distilled water (SDW) and shaken (180 rpm) for 1 h. The samples were diluted five times, and 100 µL was spread onto potato dextrose agar (PDA) plates. Fungal colonies were collected 7 d after incubation at 25°C. The internal transcribed spacer (ITS) region served as the basis for the selection and identification of 40 putative colonies.

For molecular identification of the antagonistic Trichoderma spp., PCR for the amplification of the ITS region was performed (El-Sobky et al., 2019). Genomic DNA from Trichoderma isolates was extracted using the Genomic DNA Prep Kit (Biofact Co., Seoul, Korea) following the manufacturer’s instructions. The isolated genomic DNA was stored at –80°C until further use. The ITS region was amplified using the primer pair ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) (White et al., 1990). The PCR was performed in a total reaction volume of 50 μL, containing 50 ng of template DNA (2 μL), 0.5 mM of forward primer (2 μL), 0.5 mM of reverse primer (2 μL), 1.25 U Taq DNA polymerase (0.25 μL) (Solgent, Daejeon, South Korea), 5 μL 10X Taq buffer, and 32.25 μL SDW. It took 35 cycles of extension at 72°C for 1 min, extension at 72°C for 5 min, and cooled at 4°C to complete the PCR amplification of ITS. Initial denaturation at 95°C for 1 min, denaturation at 95°C for 20 s, and annealing at 52°C for 40 s. The PCR products were purified using a PCR purification kit (Biofact Co., Seoul, Korea). The PCR product obtained was sequenced by an automated sequencer (Genetic Analyzer 3130; Applied Biosystems, Carlsbad, CA, USA) at Solgent, Daejeon, South Korea. The same primers as above were used for this step. The analyzed DNA nucleotide sequences were compared with existing DNA nucleotide sequences using the NCBI-BLAST tool. Sequence alignment and phylogenetic tree construction were performed using the DNASTAR software (Version 5.02, DNA Star Inc., Madison, USA) and MEGA 4.0, respectively (Biodesign Institute, Tempe, USA).

Pathogenic fungal cultures including Fusarium oxysporum KACC40043, and Phytophthora capsici KACC40473 were obtained from the Korean Agricultural Culture Collection (KACC), NAAC, RDA, Korea; and the pathogen Colletotrichum acutatum GYUN-10586 used in this study was from our laboratory’s collection. This pathogen was isolated according to the modified method followed by Lin et al. (2021). In brief, the symptomatic red pepper fruit tissue (0.5 cm²) was surface sterilized with 1% sodium hypochlorite (NaClO) for 1 min, washed with SDW twice, plated onto PDA plates, and incubated under 12-h/12-h cycles of light and darkness at 25°C for 4 d. To obtain pure colonies, the fungal colonies were transferred to freshly prepared PDA plates. The cells were cultured on PDA plates and stored at 4°C until further use. Conidial suspensions were prepared by suspending mycelia scraped from 7-day-old cultures of pathogenic fungus culture on a PDA plate. The resulting suspensions were filtered through a double-layered cheesecloth. Before application, the spore suspensions were adjusted to 10⁵ conidia/mL using a hemocytometer. For long-term preservation, spore suspensions of fungal pathogens were maintained at –80°C in potato dextrose broth (PDB) with glycerol (20%).

A total of 11 Trichoderma species (T. atroviride, T. asperellum, T. harzianum, T. hamatum, T. koningiopsis, T. lixii, T. longibrachiatum, T. reesei, T. tomentosum, T. virens, and Trichoderma sp.) were selected based on ITS region and screened for antagonistic activity against mycelial growths of C. acutatum (anthracnose), F. oxysporum (wilt), and P. capsici (blight), using a dual culture plate assay as per the procedure developed by Castillo et al. (2011). An agar plug from a 5-day-old fungal pathogen colony was placed on one side of a Petri dish (9 cm diameter), with an agar plug of each Trichoderma sp. placed on the opposite side, 5 cm distant from the pathogen. PDA plates inoculated with the pathogen alone were used as the controls. The experiment used a complete randomized block design (CRBD) and was performed twice with five replicates. The pathogen’s radial growth was assessed after 7 d of incubation at 25°C, and the percentage inhibition was computed using the method provided by Rahman et al. (2009), using the following equation:

Percentage inhibition (%) = R1−R2/R1 × 100, where R1 and R2 are radial growths of pathogen in the control and treatment, respectively.

Based on conidial characteristics described by Phillips et al. (2013), morphological characterization of the fungal mycelia was examined. After mycelia were fully grown on PDA plates, the formation of microstructures such as conidiophores and conidia was induced by incubation at 28°C, 12/12 photoperiod for 3 weeks. When the colonies had nearly completely covered the plate, the entire plate, the diameters of the colonies were measured along two perpendicular axes, and the data was translated to daily radial growth (mm per day). For microscopy analysis, fungal hyaline hyphae were cut and placed in 100% lactic acid, and morphological characters of the conidia and conidiogenesis were observed under a ProgRes speedXT ^core3 Imager microscope. These images were captured with an AxioCam MRc5 camera (Zeiss, Germany).

To determine the effect of the volatile organic compounds (VOCs) secreted by Trichoderma sp., against the growth of fungal pathogens, exposure of Trichoderma VOCs was performed using the overlapping plates assay. Two Trichoderma isolates, namely T. atroviride ATR696 and T. longibrachiatum LON701, were cultured on PDA plates at 25°C for 7 d. Mycelial discs of Trichoderma were cut using a sterile cork borer (5 mm diameter) and placed at the center of a freshly prepared PDA plate. A mycelial disc (5 mm diameter) of the fungal pathogen C. acutatum GYUN-10586 was placed onto another freshly prepared PDA plate in the same manner. PDA plates inoculated with C. acutatum mycelial plugs were placed on top of the PDA plates inoculated with Trichoderma species and the plates were then sealed with parafilm. The inhibition of mycelial growth of C. acutatum was observed after incubating the plates at 25°C for 14 d. The experiment was performed twice in triplicates (Petri dishes).

To determine the effects of antifungal non-volatile metabolites secreted by the Trichoderma species, a cellophane membrane experiment was performed using the method described by Li et al. (2018). A plug from 7-day-old Trichoderma isolates, ATR696 and LON701 cultured on a PDA plate was inoculated on a cellophane membrane (LABISKOMA Ltd., Seoul, Korea) overlaid onto PDA plates. The cellophane membrane with Trichoderma culture was removed after 3 d of incubation at 25°C, and a plug of C. acutatum culture was inoculated on the same medium to measure the level of fungal mycelia growth inhibition. The experiment was performed twice in triplicates (Petri dishes).

The effect of culture filtrates (CF) of Trichoderma isolates against the growth of the fungal pathogen C. acutatum under in vitro conditions was determined as follows. The two Trichoderma isolates, such as ATR697 and LON701, were cultured in a 500 mL Erlenmeyer flask containing 200 mL of PDB or M9 broth at 25°C under shaking conditions (150 rpm). Six days after incubation, 20 mL was taken and centrifuged at 3000 × g and 4°C for 20 min and filtered using a syringe and 0.22 µm filter using an aseptic technique. Therefore, 200 µL of the CF was spread onto PDA plates, and a 7-day-old cultured C. acutatum mycelium plug was placed onto a PDA plate. Plates treated with PDB alone served as a non-treated control. All the plates were incubated in the dark at 28°C for 7 d. Mycelial growth (mm in diameter) inhibition rate (%) was recorded after incubating the plates at 28°C for 7 d. The experiment was performed twice in triplicates.

An ex vivo experiment was conducted to examine the preventive and curative effects of Trichoderma isolates ATR697 and LON701 on anthracnose suppression in wounded red pepper fruits (cv. green light). Fully ripened red peppers were selected for the experiments. The fruits were surface-sterilized in 70% ethanol for 3 min, submerged in 1% NaOCl for 1 min, and then rinsed 2–3 times in SDW. The red pepper fruits were then air-dried at room temperature. Wounds were created on the surface of the fruits using a sterilized needle. To test for preventive effects, the wounded fruits were treated with antagonistic Trichoderma suspensions of ATR697 and LON701 at different concentrations (10⁷, 10⁶, C10⁵, and 10⁴ spores/mL) by spot inoculating the wounded site with a micropipette. To prepare the suspensions, the two Trichoderma isolates were individually cultured on PDA plates. Seven days after incubating the plates at 25°C, the spore suspensions were washed with SDW before filtering with double-layered gauze. The concentration was estimated using a hemocytometer and adjusted appropriately. After drying at room temperature, 10 μL of C. acutatum spore suspension (10⁵ conidia/mL) was inoculated on the wounded sites. They were then placed on square plates (40 × 40 cm) containing moist filter paper to maintain humidity. Fruits treated with pyraclostrobin and SDW served as positive and negative controls, respectively. The disease index was measured 10 d after incubation at 25°C and compared to a non-treated control; the control value (%) was computed from this. Wounded red pepper fruits were inoculated with 10 μL of C. acutatum spore suspension (10⁵ conidia/mL), dried at room temperature, and placed on square plates (40 × 40 cm) with moist paper to maintain humidity. After incubating the plates at 25°C for 1 d, the wounded sites of the fruits were treated with 10 μL of spore suspensions of two antagonistic Trichoderma isolates ATR697 and LON701 at various concentrations (10⁷, 10⁶, C10⁵, and 10⁴ spores/mL), and incubated at 25°C for another 7 d. Fruits treated with pyraclostrobin and SDW served as the positive and negative controls, respectively. The disease severity (%) was calculated as follows: A disease index scale was established based on the score from 0 to 5 (0 = no symptoms, 1 = lesions with<10% disease incidence, 2 = symptoms with 11–20%, 3 = 21–40%, 4 = 41–70%, and 5 = symptoms with >71-100%. Disease severity (%) = [sum (class frequency × score of rating class)]/[(total number of fruits) × (maximal disease index)] × 100.

To investigate the fungicidal sensitivity of the two Trichoderma isolates, ATR697 and LON701, three chemical fungicides (pyraclostrobin, tebuconazol, and kresoximmethyl) that are currently used to control red pepper anthracnose in Korea were used. These chemicals (0.2%) were dissolved in SDW and mixed with autoclaved PDA (cooled to approximately 50°C). Before the medium solidified, the medium containing the fungicide was added to Petri dishes. The control plates contained PDA without any fungicides. A mycelium plug (5 mm diameter) of two Trichoderma isolates cultured for 7 d was placed on the center of the PDA plates with or without fungicides, and the plates were incubated at 25°C for 7 d. Mycelia growth was observed, and results were compared to mycelia grown on PDA plates without fungicides. The diameter of each colony of the microorganism was measured once the fungicide-free control plates’ colonies had covered 75% of the agar surface. Trichoderma resistance and sensitivity to fungicides were determined on the basis of the inhibition zone. The experiment was carried out twice with three replicates for each treatment. The mycelial growth rate inhibition was determined as a percentage (%).

Wounded red pepper fruits were treated with a combined application of a chemical (20% WG pyraclostrobin) and Trichoderma isolates ATR697 and LON701 at two different concentrations (10⁵ and 10⁴ spores/mL) using the preventive method described above. The chemical (pyraclostrobin) was dissolved in SDW (0.335mg/mL) and from this; 10 µL was placed on the wounded site of the red pepper fruit, an hour after air-dried, 10 µL suspensions (10⁵ and 10⁴ spores/mL) of Trichoderma isolates were placed on the same site. After 24 h of incubation at 25°C, the treated red pepper fruits were inoculated with a 10 μL of fungal pathogen suspensions (10⁵ spores/mL) of C. acutatum, and fruits were kept in square plates (40 × 40 cm) containing moist paper to maintain humidity, were incubated at 25°C for 7 d. The disease index representing the disease severity (%) was then recorded. Fruits treated with pyraclostrobin and SDW served as the positive and negative controls, respectively. Each experiment was performed twice, with ten replicates (fruits) per treatment.

Two Trichoderma isolates, ATR697 and LON701, were used in a field experiment to investigate the biological control of red pepper anthracnose at Andong National University, Andong, Korea. Three-week-old red pepper seedlings were transplanted in a field with 100 cm between rows and 40 cm between the individual plants. Non-woven fabric was used to cover planting rows in order to prevent weed growth. The selected field was a naturally disease-infested field with red pepper anthracnose, caused by C. acutatum. The treatments with antagonistic Trichoderma isolates were categorized into two groups, namely, foliar spray and foliar spray + soil drenching. The experiment was conducted in two consecutive years, 2021 and 2022. Trichoderma suspensions prepared at a concentration of 10⁷ spores/mL were used for all the treatments. The first dose was administered on July 2^nd and June 25^th, in 2021 and 2022, respectively. Six treatments were administered at 10 d intervals in both years until August 31. Pyraclostrobin emulsion and tap water were used as positive and negative controls, respectively. Disease-infected fruits were collected 10 d after the last treatment. Disease severity (%) was calculated using a disease index. The disease severity (%) of the treatments was compared with that of non-treated and chemical controls. Each treatment consisted of 20 replicates (red pepper plants). The disease rate (%) was calculated using the following formula:

Data were analyzed using the R software [(RStudio “Prairie Trillium” Release (2022-05-20)] for Windows. The independence test was used based on Pearson’s chi-squared test (Agresti, 2007). One-way analysis of variance (ANOVA) was used (Chambers et al., 1992). Means were separated using the least significant difference test (LSD) at P< 0.05.

From a total of 40 fungal isolates obtained from the soil sample, only 11 isolates have been identified as Trichoderma species. Of which, only two Trichoderma isolates, such as T. atroviride ATR697 (ATR697) and T. longibrachiatum LON701 (LON701) were selected based on in vitro antagonistic activity against three fungal pathogens, C. acutatum, F. oxysporum, and P. capscici ( Figure 1 ). The two Trichoderma isolates, ATR697 and LON701 were found to inhibit the mycelial growths of the three above said fungal pathogens. These two Trichoderma isolates have the highest in vitro antagonistic activity against C. acutatum, with a 100% inhibition value. Whereas, the growth inhibitions of F. oxysporum and P. capsici were displayed as 56.7% and 63.3%, respectively, by treatment with ATR697. Meanwhile, in treatment with LON701, the mycelial growth inhibition rates of F. oxysporum and P. capsici were 57.4% and 66.1%, respectively ( Figure 2 ).

The two Trichoderma isolates were identified by PCR using an internal transcribed spacer (ITS)-specific primer sequence. A total of 11 Trichoderma isolates were identified, including T. asperellum, T. atroviride, T. hamatum, T. harzianum, T. koningiopsis, T. lixii, T. longibrachiatum, T. reesei, T. tomentosum, T. virens, and T. sp. ( Table 1 ), of which two effective antagonist strains (ATR697 and LON701) were subjected to phylogenetic analysis. Based on this, a phylogenetic tree was constructed using ITS sequences from Trichoderma isolates with sequences retrieved from GenBank. Gene sequences of both isolates ATR697 and LON701 were deposited in NCBI with GenBank accession numbers OQ772187 and OQ772191, respectively. BLAST analysis yielded 99.8%, 99.8%, and 99.8% identity with T. atroviride strains (accession no. ON203997.1, KT852808.1, and OP162786.1); while 100%, 100%, and 100% identity with T. longibrachiatum strains (accession no. MN416777.1, OP938775.1, and DQ200259.1). Phylogenetic analysis of these isolates clustered with other Trichoderma spp. The isolates were confirmed to be Trichoderma atroviride ATR697 and Trichoderma longibrachiatum LON701 ( Figure 3 ).

The morphological characteristics of two Trichoderma species, such as T. atroviride ATR697 and T. longibrachiatum LON701, were observed on the PDA medium. Conidia of each strain were observed under a microscope ( Figure 4 ). The mycelia of T. atroviride ATR697 appeared as greenish at the center and edges of the plate as well; it appears in light grey on the reverse side of the plate ( Figure 4A ). The conidia were round and measured 3.0-4.0 × 2.0-3.0 (length × width) µm. Whereas the growth of mycelia of T. longibrachiatum LON701 appears light yellow on the front side of the PDA plate, but it appears yellow color on its reverse side ( Figure 4B ). The conidia were round shaped and measured 4.0-5.0 × 2.0-3.0 (length × width) µm.

We tested the effect of VOCs emitted by Trichoderma isolates ATR697 and LON701 against the growth of C. acutatum using the overlapping pates assay in vitro ( Figure 5A ). The pathogen C. acutatum was inhibited by the VOCs produced by ATR697 significantly (P< 0.05) to a greater level than the other isolate LON701; while there was no suppression of mycelial growth of C. acutatum in a non-treated control. Overall, mycelial growth of the fungal pathogen C. acutatum was inhibited to a greater level when a 7-day-old Trichoderma isolates culture plate overlapped with C. acutatum culture plate than the mycelial growth when Trichoderma isolates were inoculated and overlapped on the same day. The mycelial growths were 1.07 and 1.43 cm in the ATR697 and LON701 treatments, respectively, while the mycelial growths were observed as 1.93 and 2.67 cm in ATR697 and LON701 treatments, respectively, when both the pathogen and antagonistic Trichoderma isolates were inoculated on the same day.

We evaluated the ability of Trichoderma isolates to secrete metabolites through the cellophane membrane that can suppress the growth of the fungal pathogen C. acutatum. When we cultured two Trichoderma isolates ATR697 and LON701 on a cellophane membrane overlaid on PDA plates, the mycelial growth of the pathogen was inhibited when cultured onto the site of the cellophane membrane after taking out the cellophane membrane ( Figure 5B ). The growth inhibition rate (%) was found to be significantly (P< 0.05) to a greater level in the ATR697 treatment than in the LON701 treatment and the non-treated control. The mycelial growth inhibitions were 5.5 and 6.3 cm in diameter on the ATR697- and LON701-treated plates, respectively. Meanwhile, the inhibition was observed as 8.6 cm in a control, suggesting that the increased mycelial growth inhibition was predominantly from metabolites secreted into the medium.

As assessed by in vitro antagonistic activities of Trichoderma isolates against the fungal pathogen, the ability of the cell-free culture filtrates (CFs) of Trichoderma isolates obtained from the two different culture media (PDB and M9) to inhibit the growth of the fungal pathogen, C. acutatum, was tested on PDA plates. After 7 d of incubation at 28°C, the colonies were measured for diameter. The colony diameter of the ATR697-CF-treated fungal growth was reduced significantly (P< 0.05) to a greater extent than that of the other treatments and the untreated control ( Figure 5C ). The resulting colony diameters were 61.6 and 7.26 cm, in ATR697 and LON701 treatments, respectively, of PDB. Meanwhile, the colony diameters in the ATR697 and LON701 treatments on M9 media were 7.1 and 6.93 cm, respectively. However, there was no significant inhibitory effect of CFs of the two Trichoderma isolates from either of the media.

Trichoderma isolates, ATR697 and LON701 were tested for their ability to suppress anthracnose disease caused by C. acutatum using preventive and curative methods. Red pepper fruits treated with ATR697 or LON701 were inoculated with conidial suspensions of C. acutatum. In the curative method, the disease severity (%) was suppressed moderately in both concentrations of Trichoderma spore suspensions without any significant differences between Trichoderma treatments and the non-treated control ( Figure 6A ). The disease severity in ATR697-treated red pepper fruits was 55.8% and 53.3% at 10⁶ and 10⁷spores/mL, respectively; while the disease severity (%) was 44.1 and 51.6% in the LON701-treated red pepper fruits at 10⁶ and 10⁷spores/mL, respectively. Whereas, the disease severity was 94% in the non-treated control. This result indicates that preventive treatment had the highest disease suppression effect when the Trichoderma was inoculated on the wounded red peppers before pathogen inoculation. The percentage of disease severity was suppressed significantly with all treatments and at all concentrations (10⁶ and 10⁷spores/mL) of Trichoderma isolates ( Figure 6B ). The disease severity in the preventive method was 11.66% in the ATR697-treated red pepper fruits at both concentrations (10⁶ and 10⁷ spores/mL), while the disease severity was 5% and 3.3% in the LON701-treated red pepper fruits at 10⁶ and 10⁷ spores/mL, respectively. Whereas, non-treated red pepper fruits presented 60% disease severity (%). Based on the effective results of the preventive method, the concentrations of the Trichoderma isolates were further reduced to 10⁴ and 10⁵ spores/mL to check their efficacy against pathogen growth. The two Trichoderma isolates, ATR697 and LON701, at both concentrations, have displayed the suppression of red pepper anthracnose drastically ex vivo, while non-treated control fruits did not exhibit disease suppression ( Figure 6C ). Among all the treatments, there were no diseased lesions in the LON701 treatment at 10⁵ spores/mL. Based on these results, two Trichoderma isolates display effective biocontrol activity against red pepper anthracnose.

The two Trichoderma isolates, ATR697 and LON701, were tested for their resistance to various commercial fungicides under in vitro conditions. The isolates, ATR697 and LON701 were fully grown on control and kresoxim-methyl-treated PDA plates, while both Trichoderma strains were found to be sensitive to tebuconazole ( Figure 7 ). However, the LON701 strain was found to be more sensitive than ATR697. The mycelial growth of the ATR697 strain was spread to 50% on a pyraclostrobin-treated plate, while the mycelial growth of the LON701 strain was spread more than 50% of the plate, suggesting the higher resistance to pyraclostrobin than the ATR697 stain.

When Trichoderma isolates along with a chemical (pyraclostrobin), are used as treatments to manage red pepper anthracnose ex vivo, the four treatments have demonstrated their ability to manage anthracnose disease caused by C. acutatum in red pepper fruits using a preventive method ( Figure 8 ). The disease severity of anthracnose was reduced to a significant level (P< 0.05) when two Trichoderma isolates at lower concentrations (10⁴ and 10⁵spores/mL) were used in combination with the chemical fungicide (pyraclostrobin). This finding suggested that the two Trichoderma isolates could be used in conjunction with chemical fungicides to boost the effectiveness of biocontrol.

Based on in vitro and ex vivo results of disease suppression of anthracnose by two Trichoderma isolates, ATR697 and LON701, these two isolates were investigated for their ability to suppress C. acutatum-caused red pepper anthracnose in the field in 2021 and 2022. In 2021, a reduced disease rate (%) was observed in all treatments. However, the treatment with LON701 significantly reduced the red pepper anthracnose infection (P< 0.05) by the foliar spray + soil drench method with a disease rate of 14%, when compared to chemical control (pyraclostrobin) and the non-treated control, presented 46.1% and 55.3% of disease rate, respectively ( Figure 9A ). The disease rate was 51.5% and 37.4% by foliar spray and foliar spray + soil drench, respectively, in the ATR697 treatment 10 d after the last treatment. Overall, a significant (P< 0.05) reduction of the disease rate (%) was indicated by the LON701 treatment under field conditions, compared to the ATR696 treatment in 2021. However, in 2022, there was no statistically significant difference (P< 0.05) between the treatments for two isolates ATR696 and LON701 using foliar spray and foliar spray + soil drench methods for reducing the disease rate of anthracnose. Whereas, the two Trichoderma isolates have been found to be more effective in disease suppression, compared to the chemical and the non-treated control with disease rates of 64% and 74.6%, respectively ( Figure 9B ). Between the two years, the disease rate (%) had reduced to some extent in the year 2021 than in 2022, because of changes in weather parameters.