**2.2.3 Metabolic tests**

306 The Dynamical Processes of Biodiversity – Case Studies of Evolution and Spatial Distribution

phylogenetic structure within these complex species groupings must be taken into account in selecting potential isolates to use in industrial applications. For example, although the name "*T. harzianum*" has been uniformly applied to the biological control agent in the past, there is now increasing evidence that several, genetically diverse species are used in

Different laboratories have used a variety of media culture for morphological observations in *Trichoderma*. In general, a relatively simple media, such as malt extract agar 2% (MEA), is useful for the production of conidia and the observation of complex branching conidiophores (macronematous). A rich culture medium such potato dextrose agar (PDA) is useful for observing pigment production and harvesting mycelium to isolate DNA. Conidiophore structure and morphology is observed from conidiophores taken from the edge of conidiogenous pustules or fascicles when conidia were maturing (usually after 4-7 days of incubation). The morphology and size of conidia should be observed at maturity after approximately 14 days of growth (Bissett 1984, 1991a, b, c, 1992; Gams and Bissett, 1998). The preliminary identification of species or species aggregates based on characteristic morphologies can be attempted using keys and descriptions in the available taxonomic

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

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

biocontrol (Druzhinina and Kubicek, 2005).

**2.2.1 Morphological analysis** 

literature (e.g. Gams & Bissett, 1998).

(ACLA) providing informative loci for multi-gene studies.

**2.2.2 Molecular analysis** 

**2.2 Methods to identify species in** *Trichoderma*

These may be based on the profiles of particular enzyme classes such as chitinases or cellulases, although other metabolic profiling techniques have been developed to validate new species which can also potentially provide data on the ecological roles of species (e.g. Kubicek et al. 2003, Hoyos-Carvajal et al., 2009a). The latter studies employed Biolog FF ® microplates (Biolog Inc., Hayward CA) comprising 96 cells containing different carbon sources and redox reagents sensitive to the activity of the enzyme succinate dehydrogenase in the citric acid cycle. Photometry at 590 nm and 750 nm provide quantitative measurements of assimilation and growth (measuring mycelial density) and respiratory activity on the different substrates. The metabolic profiles obtained may be characteristic of species, and the assimilation of specific substrates may allow hypotheses on the ecological role of species. For example, the assimilation of polyols such as maltitol and adonitol could indicate activity of dehydrogenases relevant to survive droughty conditions.
