2007 Annual Report 1a.Objectives (from AD-416) To develop genomic resources for S. sclerotiorum and P. sativum through the random sequencing of expressed genes. 1b.Approach (from AD-416) Pure cultures of S. sclerotiorum and pea infected with S. sclerotiorum will be used to created libraries of expressed sequence tags. We will use a pilot-scale cDNA sequencing project to provide the first foray into gene diversity of the white mold pathogen and the host-pathogen interaction with P. sativum. 3000 ESTs will be generated for S. sclerotiorum grown in culture. 3.Progress Report This report documents research conducted under a specific cooperative agreement between ARS and Washington State University. Additional details of research can be found in the report for the in-house associated project 5442-21220-023-00D, “Sclerotinia Diseases.” This project was initiated on June 1, 2006, and research is ongoing. The overall objective is to sequence expressed sequence tags of Sclerotinia sclerotiorum and Pisum sativum. Sclerotinia sclerotiorum WMA-1 isolate was compared genetically to eight other S. sclerotiorum isolates collected from various geographic locations by random amplified polymorphic DNA (RAPD) analysis. RAPD analysis demonstrated that WMA-1 was representative of S. sclerotiorum genotypes infecting pea, and thus suitable for expressed sequence tag (EST) library construction. Three cDNA libraries have been constructed from culture-grown mycelium on Potato Dextrose Broth (PDB) and Yeast Peptone Galactose medium (YPG) and have been transformed into E. coli. We have determined the sequences of approximately 250 clones and most clones had significant matches to bacterial sequences. Interestingly, the ratio of bacterial to fungal sequences in our EST libraries was reduced when the fungus was grown in the nutrient-poorer YPG medium. This suggested the possibility of bacteria associated with the fungus or contamination of our fungal isolates prior to EST library construction. The hypothesis of culture contamination was ruled out when we were unable to. 1)detect the bacteria microscopically in the culture filtrates,. 2)culture any bacterium from these filtrates, and. 3)PCR amplify bacterial DNA from the culture filtrates. We were able to amplify bacterial DNA from strain WMA-1, but not from strain A1-5 which was obtained as a subculture of WMA-1 on Gentamicin or strain 1980 which was the strain used for genomic sequencing and kindly provided by Jeffrey Rollins, University of Florida. These preliminary results may indicate that field strain WMA-1 has associated symbiotic bacteria while lab strains 1980 and sub-cultured strain A1-5 do not. Lack of bacteria may result from extended sub-culturing and laboratory maintenance. In addition, we have attempted bacterial specific staining of the bacteria (BacLight Bacterial Viability Kit, Molecular Probes) inside the fungal hyphae. We plan to confirm the presence of the bacteria with specific probes using fluorescence in situ hybridization (FISH). Preliminary results have indicated that it is possible to cure WMA-1 of the bacteria by Gentamicin antibiotic treatment and subsequent sub-culturing. We have developed an inoculation method of pea with the S. sclerotiorum and have begun experimenting with various extraction protocols from infected tissue. ADODR monitoring activities to evaluate research progress included phone calls, meetings with the cooperator, and an annual meeting held each year in January. Publications: Kawabe, M., Peever, T. L., Chen, W. and McPhee, K. 2007. Symbiotic Bacteria Associated with Sclerotinia sclerotiorum from Pea. 2007 Sclerotinia Initiative Annual Meeting (January, Bloomington, USA).