**2.2.2 Molecular analysis**

Initially, it was presumed that sequences of the ITS regions of rDNA were sufficient for identification of most fungal species (e.g. Lieckfeldt & Seifert, 2000). However, the popular application of BLAST (in Genbank for example) to identify species based on ITS sequence homologies can provide misleading identifications. Kopchinskiy et al. (2005) found numerous identification errors among sequences deposited in Genbank, which from time to time also does not include all species of the genus. It is now apparent that ITS alone is not sufficiently informative to resolve closely related species in *Trichoderma*, illustrated by the more recent studies employing GCPSR based on multiple gene phylogenies to resolve cryptic species. In addition, paralogous copies of RNA coding genes have been found in some genera of Hypocreales which can result in misleading identifications based on ITS alone (O'Donnell, 2000; Lieckfeldt & Seifert, 2000, Chaverry et al., 2003b, Hoyos et al., 2009a). Numerous genes have now been investigated for the application of GCPSR to resolve species limits in *Trichoderma,* with genes such as translation elongation factor 1-α (TEF), RNA polymerase II subunit B2 (RPB2), chitinase 18-5 (ECH42), calmodulin 1 (CALM1), actin, β-tubulin2 (TUBB2), LAS1 nuclear protein and ATP citrate lyase subunit A (ACLA) providing informative loci for multi-gene studies.

Other molecular tools have been developed for identification of *Trichoderma*. Druzhinina et al. (2005) presented a unique 'bar-code' system for *Trichoderma,* based on 'hallmark' regions in sequences of ITS 1 and 2, and using several of these oligonucleotide regions as genetic fingerprints. These are stored in a MySQL database and integrated with their TrichOKey program for identification (www.isth.info). This program can be used to supplement traditional identification methods. For other gene loci they have developed the program TrichoBLAST (Kopchinskiy et al., 2005), which allows alignment and comparison of sequences of ITS 1 and 2 and fragments of *tef*1α and RPB2. Complementing these methods they developed TrichoMARK to detect one or more sequence fragments of these genes as phylogenetic markers. The latter program is capable of distinguishing the five groups of species haplotypes that have identical ITS 1 and 2 sequences, *viz*.: *T. tomentosum / T. cerinum, T. longipile / T. crassum, T. koningii / T. ovalisporum / T. muroiana, H. lutea / H. melanomagna,*  and *T. longibrachiatum / H. orientalis / H. cerebriformis.* In the case of *H. lixii / T. harzianum*, the program detects intraspecific differences accurately in this cluster, which contains several putative phylogenetic species. The ISTH website (www.isth.info) also provides the primer sequences and protocols necessary for sequencing the genes used for identification.
