Molecular markers for a Trichoderma harzianum biological control agent: Introduction of the hygromycin B resistance gene and the beta-glucuronidase gene by transformation

A biocontrol isolate of Trichoderma harzianum was transformed with plasmids conferring hygromycin B (HYG) resistance (pAN7-1) and beta-glucuronidase (GUS) activity (pNOM 102). Of 20 HYG-resistant transformants (pAN7-1), 16 had single integrations of vector, in four of which vector DNA had integrated at the same site in each transformant, possibly in tandem. Two transformants had DNA integrations at two independent loci and in the remaining two transformants, vector had either integrated at two sites, or had integrated at a single site in tandem. Following single spore isolation, five transformants were mitotically stable after five rounds of sporulation and indistinguishable from the wild type. Growth rates were variable. Co-transformation of T. harzianum with both plasmids resulted in 50% of transformants exhibiting GUS activity. Of 13 transformants, only two (XTH3.9.3 and XTH3.24.1) retained GUS activity following single spore isolation. These two transformants were mitotically stable and morphologically indistinguishable from the wild type. The growth rate of XTH3.9.3 was not significantly different from that of the wild type, whereas that of XTH3.24.1 was significantly lower. Biocontrol ability of XTH1.14 (HYG-resistant) and XTH3.9.3 (GUS-positive/HYG resistant) transformants was indistinguishable from that of the wild type, indicating the feasibility of using transformants to analyse biocontrol agent fitness and mechanisms of biocontrol.