Co-transformation of the sclerotial mycoparasite Coniothyrium minitans with hygromycin B resistance and beta-glucuronidase markers

Coniothyrium minitans was successfully co-transformed with the uidA (beta-g lucuronidase) and the hygromycin-resistance (hph) genes. Both were under th e control of the glyceraldehyde-3-phosphate promoter from Aspergillus nidul ans. Hygromycin resistance was used as a selectable marker for transformati on In successive transformation experiments, transformation frequencies of up to 1000 transformants mu g(-1) of plasmid DNA were obtained for isolate A69. Of the ten monospore hygromycin-resistant cultures tested, nine also e xpressed the uidA gene. Expression of hph and uidA was stable in all transf ormants after several months of successive subculturing on non-selective me dium, and after passage through a sclerotium of Sclerotinia sclerotiorum. S outhern hybridization analyses showed all transformants carried multiple co pies of each marker gene. When grown on PDA, the culture morphology of thre e of the transformants of (T x 2, T x 3 and T x 4) was similar to the wild type. Four of the five transformants (T x 3, T x 4, T x 21 and T x 24) grew as well as the wild type on different media, and responded to changes In w ater potential in a similar manner to the wild type. All five transformants were equally parasitic on sclerotia of S. sclerotiorum compared with the w ild type. Transformants T x 3 and T x 4 were the most similar to the wild t ype in biological characteristics and will be used in future studies. The r esults indicate that hph- and uidA-transformed strains of C. minitans will be useful for ecological studies on its survival and dissemination.