Coniothyrium minitans, a mycoparasite of sclerotia of Sclerotinia sclerotiorum and Sclerotium cepivorum, produced four closely related metabolites inhibitory to fungal growth. The major metabolite, identified as macrosphelide A, had IG(50) values (the concentration of metabolite to inhibit growth by 50%) of 46.6 and 2.9 microgram ml(-1) against S. sclerotiorum and S. cepivorum, respectively. This is the first report of both antifungal activity due to macrosphelide A as well as isolation of macrosphelide A from C. minitans.

An efficient Biolistic® transformation technology was developed to stably transform Picea abies (L.) Karst. Several embryogenic tissue lines were tested for proliferation on standard embryogenesis media. Transient transformation studies with these lines were performed to optimize the parameters for genetic transformation. Selection conditions for transgenic tissue based on the nptII resistance gene in combination with the antibiotic geneticin were defined such that only transgenic P. abies lines were able to develop. Nontransgenic tissue was completely inhibited under these conditions. Stable integration of a uidA reporter gene and a nptII resistance gene into the genome of P. abies was achieved and more than 200 mature embryos were regenerated for every transformation event. Histochemical and fluorometric analysis indicated strong expression of the uidA gene in transgenic material. ELISA studies to detect and quantify the nptII gene product as well as polymerase chain reaction and Southern blotting confirmed the presence and integration of uidA and nptII genes into the P. abies genome. Transgenic P. abies plants from nine independent transformation events were recovered and are currently growing in a greenhouse for genetically modified organisms, awaiting field release.

The ability of the mycoparasit e Coniothyriu m minitans to utilize a range of C and N sources and vitamins for growth, pycnidia l formation and antifunga l metabolite production was examined using a deWned liquid medium. Coniothyriu m minitans was able to use all the C sources tested, with the exception of d-xylose, and all the N sources tested, although growth was generally better on organic N sources rather than NO 3 ± N. Increasing C:N ratios from 9:1± 202:1 with N constant (2.0 g L 2 l l-alanine) resulted in steadily increasing yields, whereas increasing C:N ratios with C constant (40.0 g L 2 l d-glucose) gradually decreased yield. Addition of thiamine to the glucose± alanine basal medium resulted in the greatest increase in growth but biomass was still less than that achieved using an undeWned molasses± yeast medium. Pycnidial production was generally low or failed to occur in the basal medium + C + N sources in the absence of vitamins, but addition of thiamine consistently led to abundan t pycnidial formation. Molasses± yeast static culture provided greater biomass and conidial yields than molasses± yeast shaken culture. Incorporation of C. minitans culture medium into potato dextrose broth (10% v/v) resulted in consistent reduction in growth of Sclerotinia sclerotiorum irrespective of C, N or vitamin content of the basal medium or whether molasses± yeast medium was used. This is the Wrst report of consistent production of antifunga l metabolites by C. minitans.

Verticillium biguttatum, a mycoparasite of the ubiquitous soil-borne plant pathogen Rhizoctonia solani, excreted chitinase and beta-1,3-glucanase into liquid medium when grown on laminarin and chitin, respectively. Neither chitinase nor beta-1,3-glucanase was produced by the mycoparasite when grown on cell walls of two isolates of R. solani representing anastomosis groups (AG)-3 and AG-8. Extracellular protease was induced by growth on cell walls of the pathogen, whereas beta-1,3-glucanase and chitinase were produced bound to the cell wall of V. biguttatum. This is the first report of chitinase, beta-1,3-glucanase and protease production by V. biguttatum. These enzymes may play a previously unforeseen role in dissolving and penetrating the cell walls of R. solani.