Fungal isolation
The fungus was isolated from soil samples collected from a dragon fruit (Hylocereus cactus) farm in Villanueva, Misamis Oriental, Philippines (8.59654° N, 124.79971° E), in November 2019. To this end, the insect-baiting technique was used according to the procedure developed by Bedding and Akhurst (1975). Briefly, moistened soil samples were placed in plastic containers. Then, 5 last instar larvae, Z. morio, were added into each container (Navarez et al. 2021). Containers were sealed and stored in the dark at 25 ± 2 °C. Containers were inspected every other day, for 15 days, until visible symptoms of fungal infection were observed. Insect cadavers showing fungal infection were transferred to Sabouraud dextrose agar (SDA-Scharlau) media for purification (Herlinda et al. 2020). To inhibit the growth of bacteria, the media was supplemented with streptomycin, a broad-spectrum antibiotic, at a concentration of 20 µg/l (Goettel and Inglis 1997).
Morphological identification
Isolated fungi were identified based on phenotypic characteristics in SDA and PDA (Potato dextrose agar) media. Characteristics such as surface texture, surface margin, surface topography, surface pigmentation, reverse pigmentation, and growth rate were taken into account. In addition, microscopic characteristics such as the length and shape of conidia were taken into account for initial identification using the key described by Humber (1997).
Molecular identification
Genomic DNA (gDNA) of the fungal isolate was extracted using PureLink® Genomic Plant DNA Purification Kit (Thermo Fisher Scientific). Gene amplification by PCR was carried out with the following components: 3 µl of gDNA, 1 µl of ITS1 (5′tccgtaggtgaacctgcgg-3′) and 1 µl of ITS4 (5′-tcctccgcttattgatatgc-3′) primers, 2.5 µl of Taq buffer, 0.5 µl of DNA polymerase, 1 µl of dNTP mix, and 1 µl nuclease-free dH2O. Cycling parameters on the thermal cycler were set at 95 °C for 5 min, followed by 30 cycles at 95 °C for 60 s, 58 °C for 45 s, 70 °C for 60 s, and a final extension at 72 °C for 10 min. Amplicons generated had sizes ranging from 400 to 600 bp, which was expected for ITS1 and ITS4 primers (Additional file 1: Fig. S1). Capillary sequencing was performed in a bidirectional manner at the Philippine Genome Center-Core Sequencing Facility (Manila, Philippines). Fluorescent-labeled chain terminator dNTPs with the reaction components, viz. amplicons, primers, and ABI BigDye® Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific), were used. Cycling parameters on Bio-Rad T100 Thermal Cycler include: pre-hold at 4 °C; 96 °C for 60 s; 25 cycles of 96 °C for 10 s, 50 °C for 5 s, 62 °C for 4 min and hold at 4 °C. Ethanol precipitation was carried out to remove unincorporated dNTPs, excess primers, and primer dimers. Capillary electrophoresis was carried out on the ABI 3730×l DNA Analyzer using a 50 cm 96-capillary array, POP7TM Polymer, and 3730×l Data Collection Software v3.1. Base-calling was carried out on the Sequencing Analysis Software v5.4.
Sequences alignment and phylogenetic analysis
The generated sequences were manually curated and trimmed in BioEdit (Hall, 1999). The obtained sequences were searched in the National Centre for Biotechnology (NCBI) data bank using the nucleotide basic local alignment search tool (BLASTn) (Altschul et al. 1990). Sequences of Trichoderma asperellum RMCK01 were aligned with sequences of other related fungi species using default ClustalW parameters in MEGA 7.0 (Kumar et al. 2016) and optimized manually in BioEdit (Hall 1999), and sequences were only trimmed at both extremes after alignment to maintain fragments with similar length. Pairwise distances were computed using MEGA 7.0 (Kumar et al. 2016). Codon positions included were 1st + 2nd + 3rd + Non-coding. The base substitution model was evaluated using jModelTest 0.1.1 (Posada 2008).
The phylogenetic tree was inferred from the datasets using the Bayesian inference method (BI). All characters were treated as equally weighted and gaps as missing data. Trichoderma longibrachiatum (EU401556.1) and T. brunneoviride (EU518659.1) were used as out-groups. Bayesian phylogenetic reconstructions were performed using MrBayes 3.2.7 (Ronquist et al. 2012). The best fit model was identified as the GTR + G model test using the MrModeltest 2.0 program (Nylander 2004). Metropolis-coupled Markov chains Monte Carlo (MCMCMC) generations were run for 1 × 106 cycles, and one tree was retained every 2000 generations. The Bayesian tree was visualized using the FigTree program 1.4.4 (Rambaut 2018). In addition, genetic pairwise distances were estimated using MEGA-7 software (Kumar et al. 2016).
Mass production of spores
Mass production of spores was carried out following the method of Podder and Gosh (2019). For this, 50 g of rice was washed thoroughly under running tap water and soaked in water for 3 h. The soaked rice was transferred to a 15 × 30 cm polypropylene plastic bags and then autoclaved, maintaining a temperature of 121 °C for 15 min under 15 psi. The sterilized rice was allowed to cool down at room temperature. Then, the rice was inoculated by T. asperellum spores under a biological safety cabinet. Trichoderma asperellum spores were produced by growing the fungus on the rice. The inoculated rice was incubated at 28 ± 1 °C for 15 days (Fig. 1A).
Preparation of inoculum and quantification
Conidial concentrations were estimated based on the protocol of Goettel and Inglis (1997), with slight modifications. Briefly, mycelium was harvested, washed with distilled water, and filtered through a cheesecloth. Then, Tween 80 (0.05%) was added and 100 µl of conidial suspension was pipetted and mixed with 100 µl of sterile lactophenol blue stain solution (Barta 2010). This suspension was vortexed for 30 s and serially diluted with sterile deionized water. The number of conidia in each sample was determined using a hemocytometer (Neubauer, Marienfeld, Germany). Then, 3 conidial suspensions were prepared. Spores were counted under microscopic power of 40× and calculated using the formula (Kamaruzzaman et al. 2016):
$${\text{Total spores/ml}} = \frac{{{\text{Number of spores counted}} \times {\text{dilution factor}}}}{{\text{Number of smallest square counted}}} \times 4000$$
Finally, the suspension was standardized to 3 final concentrations using C1V1 = C2V2 formula.
Pathogenicity tests
To test the pathogenicity of T. asperellum isolate RMCK01, the last instar Z. morio larvae were treated with 3 conidial suspensions: 2.68 × 107, 6 × 108, or 3 × 109 conidia/ml. These 3 concentrations were selected based on previous studies (Mohamed 2019). Zophobas morio larvae were immersed into one conidial suspension for 60 s and then let to air dry. All insects were then transferred to 9 cm diameter clean glass Petri dishes. A piece of filter paper (9 cm in diameter) was placed at the bottom of the dish with a few drops of water. Controls were dipped into a 0.02% solution of Tween-80 prepared with distilled water. Experiments were repeated 4 times with a new batch of insects and new conidial suspensions, and for each repetition, there were 5 Z. morio larvae. Bioassays were incubated at 26 °C and > 95% relative humidity (RH). Insect mortality and fungal infection symptoms were recorded at 12 h intervals for 21 days (Fig. 1B).
Statistical analysis
Results of statistical analysis are expressed as mean ± standard deviation (n = 3) with 5 replications. To determine whether there were significant differences (p < 0.05), one-way ANOVA was used, followed by Tukey’s HSD test (XLSTAT version 2020.1.3) for post hoc analysis.