Delineation of species within the complex by UP-PCR cross-blot hybridization

Abstract

Sequences from the ribosomal DNA ITS regions have shown that there is limited variation among the species in the Trichoderma viride/atroviride/koningii species complex also known as Hypocrea rufa complex, and that infraspecies variation sometimes is larger than interspecies variation. Strains belonging to T. viride, T. atroviride, T. koningii, T. asperellum, and respective teleomorphs were analyzed using cross-blot hybridization of PCR products generated by the universally primed PCR (UP-PCR) technique. The hybridization results showed that the morphologically defined species could be delineated molecularly, suggesting that the species are more separated than indicated by ITS sequence phylogeny. However, cross-hybridization signals at infra- and interspecies level within this species complex was overlapping, again raising the question of how many species there are. Due to the heterogeneity of T. viride revealed, a further revision of this species is needed. In addition, this study shows that a macroarray (DNA chip) containing membrane-bound UP-PCR products for reference strains can be developed for routine identification of Trichoderma strains.