MOLECULAR MARKERS FOR A TRICHODERMA-HARZIANUM BIOLOGICAL-CONTROL AGENT - INTRODUCTION OF THE HYGROMYCIN-B RESISTANCE GENE AND THE BETA-GLUCURONIDASE GENE BY TRANSFORMATION
Jk. Bowen et al., MOLECULAR MARKERS FOR A TRICHODERMA-HARZIANUM BIOLOGICAL-CONTROL AGENT - INTRODUCTION OF THE HYGROMYCIN-B RESISTANCE GENE AND THE BETA-GLUCURONIDASE GENE BY TRANSFORMATION, New Zealand journal of crop and horticultural science, 24(3), 1996, pp. 219-228
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.