Bacteria belonging to the order Actinomycetales produce most microbial metabolites thus far described, several of which have found applications in medicine and agriculture. However, most strains were discovered by their ability to produce a given molecule and are, therefore, poorly characterized physiologically and genetically. Thus, methodologies for genetic manipulation of actinomycetes are not available and efficient tools have been developed for just a few strains. This constitutes a serious limitation to applying molecular genetics approaches to strain development and structural manipulation of microbial metabolites. To overcome this hurdle, we have developed bacterial artificial chromosomes (BAC) that can be shuttled among Escherichia coli, where they replicate autonomously, and a suitable Streptomyces host, where they integrate site-specifically into the chromosome. The existence of gene clusters and of genetically amenable host strains, such as Streptomyces coelicolor or Streptomyces lividans, makes this a sensible approach. We report here that 100 kb segments of actinomycete DNA can be cloned into these vectors and introduced into genetically accessible S. lividans, where they are stably maintained in integrated form in its chromosome.

The potential use of the commercial biocontrol products Serenade (Bacillus subtilis QST-713) and Trichodex (Trichoderma harzianum Rifai strain T39) to inhibit the postharvest pathogenic molds Penicillium crustosum and Mucor circinelloides was investigated. Both products exhibited antagonistic activity in vitro against the pathogens, reducing their growth at different levels. In addition, epiphytic yeasts isolated from peaches were identified as Candida maltosa, Pichia fermentans, and Pichia kluyveri by PCR-restriction fragment length polymorphism of internal transcribed spacer regions and screened for antagonistic activity against the same molds. The efficacy of biocontrol in vitro was dependent on the concentration of the yeast cells. Optimal yeast concentrations were above 10(7) CFU ml(-1). However, C. maltosa and P. fermentans were more effective than P. kluyveri in inhibiting molds. The exclusion of antifungal metabolite production and direct competition for nutrients or space with the pathogens was proposed as the mechanism of biocontrol. Application of biocontrol agents directly on artificially wounded peach fruits significantly reduced the incidence of mold rot during storage at 20 degrees C.