Indicator microorganisms. Indicator microorganisms for antagonistic activity testing, i.e., Bipolaris sp. strain RCT, Colletotrichum spp. strain TCT, Corynespora cassiicola strain CCT, Fusarium sp. strain SCT, and Ralstonia solanacearum strain CBL, were provided by the Plant Gene Technology Laboratory, Biotechnology Research and Development Institute, Can Tho University, Can Tho City, Vietnam.
Isolation of Trichoderma spp. from lichen samples. Samples of Cryptothecia spp. and Dirinaria spp. were collected in the Mekong Delta, Vietnam, including 12 provinces: An Giang (VAG), Bac Lieu (VBL), Ben Tre (VBT), Ca Mau (VCM), Dong Thap (VDT), Hau Giang (VHG), Kien Giang (VKG), Long An (VLA), Soc Trang (VST), Tien Giang (VTG), Tra Vinh (VTV), and Vinh Long (VVL) and the city of Can Tho (VCT). Bark samples with lichens growing on the surface were collected into paper bags and rapidly transported to the laboratory. Identification at the genus level was performed using the taxonomic key described by Elix (2009a, 2009b). The lichen samples were washed under running tap water for 5 min. Then, the lichen thalli were separated from tree bark with a sterile surgical blade under a stereo microscope (ZEISS, Germany). The surface of the thalli was sterilized as follows: 95% ethanol for 30 s, 0.5% NaClO for 2 min, 70% ethanol for 30 s, and rinsed three times with sterile distilled water (Oh et al., 2020). The thalli were dried on sterilized paper and cut into 1 cm2 pieces using sterile surgical blades (Oh et al., 2020). The lichen fragments were placed on potato dextrose agar (PDA) plates (five fragments per plate), and incubated at room temperature for 2 weeks. The names of the endolichenic fungal isolates were coded corresponding to the location where the lichen samples were collected. The isolates were identified as Trichoderma spp., based on the description by Chaverri and Samuels (2003).
Antifungal activities. The Trichoderma isolates were screened for antifungal activities using the dual culture methoddescribed by Hamzah et al. (2018) with minor changes. Agar discs (6 mm diameter) of the fungal pathogens and the test Trichoderma isolates were obtained from the growing margin of the colonies. Prepared potato dextrose agar (PDA) plates were divided into two halves. On each plate, a pathogenic fungal disc was placed 1.5 cm from the margin on one half. On the other half, a disc of the tested Trichoderma spp. was placed similarly but directly opposite the pathogen. The plates inoculated with pathogens without antagonistic fungi were used as controls. After seven days, the radial growth of the test pathogen mycelia was measured on the control plate (r1) and the plate with the antagonistic Trichoderma isolate (r2). The experiment was repeated three times. The growth inhibition percentage (GI%) was calculated by the equation: GI% = [(r1– r2)/r1] × 100.
The zone of inhibition (if any) was recorded and the interactions between the indicator strains and the isolates were evaluated according to the following criteria (Mejía et al., 2008; Hamzah et al., 2018): (1) mutual intermingling growth: both fungi grow in separate sections without any signs of macroscopic interaction (Fig. 1a); (2) antibiosis: growth inhibition is determined by the presence of an inhibitory zone (Fig. 1b); (3) competition for substrate: overgrowth of one organism over another (Fig. 1c); and (4) mycoparasitism: direct parasites on the mycelia of pathogens (Fig. 1d).
Antibacterial activities assay. A suspension of R. solanacearum was prepared in a sterile physiological saline solution with turbidity equal to 0.5 McFarland. Fifty µL of the bacterial suspension was spread evenly onto the Mueller-Hinton agar plate. Fungal discs (6 mm in diameter) from one-week-old Trichoderma plates were placed in the center of the R. solanacearum lawn. Plates inoculated with R. solanacearum alone were used as controls. The test plates were incubated at 2--8°C for 14--18 h for the diffusion of the active substances from the fungal disc into the medium plate. Then, the test plates were incubated at 30°C, and antagonism was observed after 48-h incubation (Nedialkova and Naidenova, 2005). A zone of inhibition (if any) was noted, and the interactions between Trichoderma isolates and R. solanacearum were evaluated according to the criteria described above (Hamzah et al., 2018; Mejía et al., 2008). The experiment was repeated in triplicate.
Antibacterial and Antifungal Activities of Non-Volatile Compounds from the Trichoderma Isolates
Preparation of the cell-free supernatant. Three fungal discs (6 mm) were inoculated into a 250-mL conical flask containing 100 mL of potato dextrose broth (PDB) and incubated at room temperature for 15 days on a shaker at 120 rpm. The culture aliquot was filtered through no. 1 Whatman filter paper under sterile conditions.
Investigation of the ability to inhibit indicator fungi. The cell-free supernatant (CFS) obtained from the Trichoderma isolates was mixed with molten PDA to a final concentration of 20% (vol/vol) and distributed into petri dishes (Hamzah et al., 2018). After solidification, a fungal disc (6 mm in diameter) of the tested pathogen was placed in the center of the freshly prepared medium plates. The pathogenic fungal strains were cultured on a PDA medium as controls. After 7 days, the radial growth of the tested pathogens on a PDA supplemented with CFS (d2) and on the control plates (d1) was measured. The percentage inhibition of mycelia growth was calculated using the formula: GI% = (1 - d2/d1) × 100.
Investigation of the ability to inhibit Ralstonia solanacearum. The cell-free supernatant obtained from the Trichoderma isolates was mixed thoroughly with Mueller-Hinton broth (MHB) to achieve the final concentration of 20% (vol/vol). R. solanacearum suspension prepared as described above (50 µL) was added to 5 mL of the MHB with the CFS (treatment). For control, 5 mL of MHB was used, followed by inoculation of 50 µL of R. solanacearum suspension. After 18 h of incubation, 1 mL of bacterial culture was centrifuged at 13,000 g for 5 min. The pellet was resuspended in 1 mL of sterile physiological saline. The optical density (OD600) of the bacterial suspension was measured using a GENESYS 10 UV Scanning UV/Visible spectrophotometer (Thermo Fisher Scientific, United States). The experiment was repeated three times. Growth inhibition of indicator bacteria of the CFS was determined by the formula: GI% = (1 - OD600 treatment/OD600 control) × 100.
Cultivation on Polymeric Substances
A fungal disc with a diameter of 6 mm was placed in the center of a petri dish containing the medium used for investigation and incubated at room temperature. The experiment was repeated in triplicate.
Starch. The nutrient agar with 5% starch was used to investigate the amylolytic activity of the Trichoderma spp. After 48 h of incubation, the test plates were flooded with 1% Lugol solution, and the clear zone size was recorded (Kannangara et al., 2009).
Cellulose. Cellulose degradation activity of the Trichoderma isolates was investigated on carboxymethyl cellulose (CMC) medium (Eggins and Pugh, 1962) after 3-day incubation at room temperature. Cellulose utilization was assessed by clear zones around the fungal colonies after staining with 1% Lugol solution.
Pectin. The basal medium described by Eggins and Pugh (1962) and supplemented with 0.5% pectin from citrus peel was used. After 2 days of incubation, the plates were coated with 1% cetyltrimethylammonium bromide solution, and clear zones around the fungal colonies were measured (Kannangara et al., 2009).
Lignin. The cultures were inoculated on a PDA medium and incubated for 48 h. The presence of the enzyme was quantified with the corresponding reagent as described by Stalpers (1978) with a minor change. The reaction with α-naphthol (on laccase) revealed a purplish discoloration. A yellowish-brown color occurred in the case of the reactions with pyrocatechol and H2O2 (on peroxidase). The color turns orange-brown when tyrosinase reacts with p-crezol.
Identification of the selected strain VDT6 using ITS sequence analysis. Based on the assessment results, the DNA of strain VDT6 was isolated by the method described by Al-Samarrai and Schmid (2000). Amplification of the internal transcribed spacer (ITS) region was done by the set of primers ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) (Fujita et al., 2001). PCR was carried out in a reaction mixture containing 15.75 µL double distilled water, 5 µL 5× MyTaq DNA polymerase buffer (Bioline, United States), 1 µL 10 μM forward and reverse primers, 1.25 U MyTaq DNA polymerase (Bioline, United States), and 1 µL genomic DNA. PCR was carried out in ABI 9800 Fast Thermal Cycler, with the following conditions: 94°C for 3 min, 35 cycles of 94°C for 30 s, 55°C for 30 s, elongation at 72°C for 1 min, and the final elongation step at 72 °C for 7 min. The amplified product was sent for sequencing (Apical Scientific, Malaysia). The obtained sequences (GenBank deposition number: OP108343) were used for homology search against the NCBI database by BLASTn.
Statistical analysis. One-way ANOVA was used to analyze the antagonistic activities of the Trichoderma strains,using MINITAB 16 statistical packages (Minitab, Inc.).