New Zealand Journal of Crop and Horticultural Science abstracts
Molecular markers for a Trichoderma harzianum biological control agent:
introduction of the hygromycin B resistance gene
and the [[beta]]-glucuronidase gene by transformation
J. K. BOWEN1*
R. N. CROWHURST2
M. D. TEMPLETON2
A. STEWART1+
1School of Biological Sciences
University of Auckland
Auckland, New Zealand
2The Horticulture and Food Research
Institute of New Zealand
Mt Albert Research Centre
Private Bag 92 169
Auckland, New Zealand
*Present address: The Horticulture and Food Research Institute of
New Zealand, Mt Albert Research Centre, Private Bag 92 169, Auckland, New
Zealand.
+Present address: Department of Plant Science, Lincoln University,
Canterbury, New Zealand.
Abstract A biocontrol isolate of Trichoderma harzianum
was transformed with plasmids conferring hygromycin B (HYG) resistance (pAN7-1)
and [[beta]]-glucuronidase (GUS) activity (pNOM102). 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.
Keywords Trichoderma harzianum; Armillaria
novae-zealandiae; Armillaria limonea; Sclerotium rolfsii;
Pinus radiata; Actinidia deliciosa; biological control agent;
transformation; molecular marker
New Zealand Journal of Crop and Horticultural Science, 1996, Vol. 24:
219-228
0114-0671/96/2403-0219 $2.50/0 (c) The Royal Society of New Zealand
1996
PDF file of entire paper: medium quality (2243K); (scanned from paper original: notes about this process)
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