Molecular Identification of Trichoderma Isolated from the Rhizosphere of Cacao (Theobroma cacao L.) in Central Sulawesi, Indonesia

Morphology-based identification methods, especially in cocoa, often have limitations, especially in similar overlapping symptoms. DNA-based molecular identification is required to ascertain effective fungal biocontrol species. This study aimed to identify and characterize Trichoderma isolates from the rhizosphere of cacao from three different altitudes in Central Sulawesi using a molecular DNA barcoding approach.

The method used in this study consists of various stages, namely sampling, isolation, purification of Trichoderma, DNA extraction, DNA amplification, electrophoresis, sequencing and phylogenetic analysis. Quick-DNA Magbead Plus Kit (Zymo Research, D4082) was used for genomic DNA extraction, primers ITS-1 (5'-TCCGTAGGTGAACCTGCGG-3’) and ITS-4 (5'-TCCTCCGCT TATTGATATGC-3’) were used for PCR amplification and DNA sequence analysis using Basic Local Alignment and Search Tool (BLAST) on NCBI database.

The results obtained showed that the amplified DNA electrophoresis had high quality with thick and clear bands with a size range of 575–578 bp. The five Trichoderma isolates based on sequencing analysis were identified as Trichoderma asperellum with different strains, namely T. asperellum strain NG125, T. asperellum strain NECC30406, T. asperellum strain IIPR-81, T. asperellum strain BHU203 and T. asperellum strain WZ-184, with a similarity rate (query cover and identity percentage) of 100%. The results of this study enrich the sequence data of Trichoderma asperellum in NCBI and can be a reference for the development of Trichoderma- based biological agents.