Project Number: 5348-21000-014-04
Project Type: Specific Cooperative Agreement

Start Date: Aug 24, 2004
End Date: Jun 30, 2009

Objective:
Establish an EST library of Sclerotinia sclerotiorum and sequence about 4000 EST clones. Establish an interaction EST library of Pisum sativum tissue infected with S. sclerotiorum and sequence about 2000 EST clones. Develop a procedure using chemical mutagenesis to generate mutant strains of S. sclerotiorum that are non-pathogenic on grain legumes. The mutant strains will be used to generate an EST library.

Approach:
The strain WM1 of S. sclerotiorum collected from pea field will be used to construct a cDNA library. Similarly pea cultivar Franklin will be inoculated with white mold and the infected host tissue will be used to isolate cDNA for library construction. Total RNA from the mycelium and from the infected host tissue will be isolated and purified using the oligotex mRNA isolation kit. A cDNA library will be prepared in plasmid pSPORT1 using the superscript plasmid system for cDNA systesis and cloning. After plasmid ligation, they will be transformed into E. coli. Individual colonies will be selected and transferred to 96-well microtiter plates and used for automatic DNA sequencing. Chemical and physical mutagenic agents, such as 5-bromouravil, N-methyl-N'-nitro-N-nitrosoquanidine, and ultraviolet light, will be used to generate mutants of S. sclerotiorum and the mutants will be screened for pathogenicity. Non-pathogenic mutants will be used in cDNA library construction. Documents SCA with WSU.