Studies on the biology of Sclerotinia sclerotiorum and Cladosporium fulvum

Sclerotinia sclerotiorum is a necrotrophic fungal pathogen causing diseases in over 400 plant species. Although host resistance is the preferred method to control diseases caused by S. sclerotiorum, incorporation of resistance into commercial cultivars has been a difficult objective, due in part to the lack of rapid and effective methods for disease-resistance screening. Isolates of S. sclerotiorum were transformed with the green fluorescent protein (GFP) gene to study whether fluorescence could be quantified and used as a tool to study host resistance. The results demonstrated that fluorescence quantification could differentiate between wild-type and GFP-expressing isolates using mycelium-colonized potato dextrose agar plugs and mycelium protein extracts, but could not be used to measure colonization of tissue or to differentiate partially resistant and susceptible soybean cultivars based on protein extracts of soybean leaves inoculated with GFP-expressing isolates.; Cladosporium fulvum is a biotrophic fungal pathogen causing leaf mold on tomato. The role of nitrogen deprivation and nitrogen response factor 1 (Nrf1) in the induction of Avr and Ecp effector genes in planta and in vitro was investigated. The results demonstrated that expression of most effectors is not affected by varying levels of nitrogen supplementation in vitro. In addition, Nrf1 deletion lines caused reduced virulence compared to wild-type.; Agrobacterium tumefaciens has been adopted for transforming a wide range of cells, including filamentous fungi. Despite this broad appeal, there are no published protocols of A. tumefaciens-mediated transformation of C. fulvum. Conditions for gene transfer from A. tumefaciens to C. fulvum were investigated using beta-glucuronidase (GUS) and a gene encoding resistance to nourseothricin. Co-cultivation with 1x107 condia/ml and A. tumefaciens supplemented with acetosyringone at 22°C was optimal to produce nourseothricin-resistant colonies. In addition, RNA interference (RNAi) silencing vectors were developed and transformed into C. fulvum via the A. tumefaciens-mediated transformation protocol. Of the five putative genes for which RNAi constructs were developed and transformed into C. fulvum, preliminary results show that Bap1 is required for full virulence of the pathogen since fungal growth was severely compromised in in planta virulence assays.