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

Abstract

Coniothyrium minitans was successfully co-transformed with the uidA (b-glucuronidase) and the hygromycin-resistance (hph) genes. Both were under the control of the glyceraldehyde-3-phosphate promoter from Aspergillus nidulans. Hygromycin resistance was used as a selectable marker for transformation. In successive transformation experiments, transformation frequencies of up to 1000 transformants lg’ of plasmid DNA were obtained for isolate A69. Of the ten monospore hygromycin-resistant cultures tested, nine also expressed the uidA gene. Expression of hph and uidA was stable in all transformants after several months of successive subculturing on non-selective medium, and after passage through a sclerotium of Sclerotinia sclerotiorum. Southern hybridization analyses showed all transformants carried multiple copies of each marker gene. When grown on PDA, the culture morphology of three of the transformants of (T‹2, T‹3 and T‹4) was similar to the wild type. Four of the five transformants (T‹3, T‹4, T‹21 and T‹24) grew as well as the wild type on dierent media, and responded to changes in water potential in a similar manner to the wild type. All five transformants were equally parasitic on sclerotia of S. sclerotiorum compared with the wild type. Transformants T‹3 and T‹4 were the most similar to the wild type in biological characteristics and will be used in future studies. The results indicate that hph- and uidA-transformed strains of C. minitans will be useful for ecological studies on its survival and dissemination. Sclerotinia sclerotiorum (Lib.) de Bary is a soil-borne fungal pathogen which attacks a wide range of fruit and vegetable crops (Purdy, 1979). The sclerotia of S. sclerotiorum are crucial for the pathogen’s survival, serving as a primary inoculum source and remain viable in soils for many years (Merriman, 1976). Mature sclerotia germinate either myceliogenically to produce mycelium which infects host plants directly (Huang & Dueck, 1980), or carpogenically to produce apothecia which discharge wind-borne ascospores which subsequently infect the host (Huang & Kokko, 1992). Control of Sclerotinia spp. has been obtained by fungicide application, in particular the use of members of the dicarboximide group, but enhanced degradation of the fungicides in soils by other microorganisms has led to reduced ecacy of control in Europe (Martin et al., 1990). Enhanced degradation of dicarboximides has been reported in onion paddocks in New Zealand (Slade et al., 1992) and is likely to occur in other vegetable paddocks where these chemicals are being used to control Sclerotinia diseases. Soil sterilisation by stem or methyl bromide is eective but costly. Further, environmental concerns over the use of methyl bromide mean