Development of Species Specific Markers For Detection of Trichoderma Species

A set of 72 Trichoderma isolates from India were morphologically and molecularly characterized and confirmed as Trichoderma virens, T. harzianum, T. longibrachiatum and T. asperellum. ITS1 and ITS2 regions were amplified using universal ITS primers. The amplicon size varied from 550 to 600 bp in the isolates studied. Cluster analysis based on amplicon sequence of ITS region could not clearly differentiate the Trichoderma species into distinct group. Thus tef1 was used for differentiating the Trichoderma sp. The sequence analysis based on tef1 gene primers could differentiate T. longibrachiatum, T. virens, T. harzianum and T. asperellum species and the phylogenetic analyses were in conformity with the morphological observations. Species specific tef1primer sets T1F/T1R and T2F/T2R were designed for T. virens and T. longibrachiatum isolates respectively and validated against other species of Trichoderma. T1F/T1R specifically amplified in T. virens giving fragment size of 330 bp and T2F/T2R gave amplification of 452 bp only in T. longibrachiatum.