Development of Species/Genus specific Primers for Identification of Three Trichoderma Species and for Detection of Trichoderma Genus

Abstract

Trichoderma is a widely used bio-control agent, and has excellent ability to antagonize plant pathogens and promoting plant growth. It has been successfully used to control various plant diseases. The premise of using Trichoderma is accurate identification of it. In this study, four sets of species/genus-specific primers were designed based on the translation elongation factor 1-alpha (tef1) gene and internal transcribed spacer (ITS) region, in order to identify Trichoderma harzianum, T. koningiopsis, and T.virens, and detect the genus of Trichoderma. Here, the rapid, simple, and reliable PCR methods were development using the species-specific primers EHarF2/EHarR2, EKoisF/EKoisR, and EVireF/EVireR to produce 253 bp (tef1 gene), 255 bp (tef1 gene), and 263 bp (tef1 gene) DNA bands to identify T. harzianum, T. koningiopsis, and T. virens, respectively. The genus-specific primers ITricF/ITricR produces a single DNA band in the range of 103–113 bp (ITS region) to distinguish Trichoderma from other genera fungi, and the size of the band is related to the species of Trichoderma. In addition, ITricF/ITricR could use for real-time PCR amplification for detection the quantity of Trichoderma spp..