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 pla smids conferring hygromycin B (HYG) resistance (pAN7-1) and beta-glucu ronidase (GUS) activity (pNOM 102). Of 20 HYG-resistant transformants (pAN7-1), 16 had single integrations of vector, in four of which vecto r DNA had integrated at the same site in each transformant, possibly i n tandem. Two transformants had DNA integrations at two independent lo ci and in the remaining two transformants, vector had either integrate d at two sites, or had integrated at a single site in tandem. Followin g single spore isolation, five transformants were mitotically stable a fter five rounds of sporulation and indistinguishable from the wild ty pe. Growth rates were variable. Co-transformation of T. harzianum with both plasmids resulted in 50% of transformants exhibiting GUS activit y. Of 13 transformants, only two (XTH3.9.3 and XTH3.24.1) retained GUS activity following single spore isolation. These two transformants we re mitotically stable and morphologically indistinguishable from the w ild 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 significantl y lower. Biocontrol ability of XTH1.14 (HYG-resistant) and XTH3.9.3 (G US-positive/HYG resistant) transformants was indistinguishable from th at of the wild type, indicating the feasibility of using transformants to analyse biocontrol agent fitness and mechanisms of biocontrol.