Trichoderma Species Identification Protocol

According to Abuhena et al. (2022b) (link), the PrepMan^® Ultra Sample Preparation Reagent Kit (Applied Biosystems, United States) was used to isolate genomic DNA from a single colony of a Trichoderma isolate. PCR was performed in a MyGeneTM Series Peltier Thermal Cycler using genomic DNA isolated from a Trichoderma isolate and a positive control of Saccharomyces cerevisiae with standard primers provided in the Fast MicroSEQ^® D2 LSU rDNA Fungal PCR Kit (Applied Biosystems, United States). The MicroSeq^® D2 LSU rDNA Fungal Sequencing Kit’s Forward Primer Reaction Mix and purified PCR product were used in cycle sequencing (Applied Biosystems, United States). Using the NCBI BLAST system,¹ DNA similarity was examined. To identify species, a criterion of 98% sequence similarity was applied. The isolated strains’ D2 LSU rDNA sequences were added to the NCBI GenBank database and given an accession number. The phylogenetic analyses were conducted in MEGA11.0 (Maitra et al., 2021 (link)).