2007 Annual Report 1a.Objectives (from AD-416) 1) Identify and sequence key genes involved in ochratoxin biosynthesis from Aspergillus alliaceus and A. ochraceus. 2) Evaluate environmental regulation of expression of ochratoxin biosynthesis genes. 3) Develop molecular markers for detection of potentially ochratoxin-contaminated food products and environmental samples. 1b.Approach (from AD-416) 1) We will identify ochratoxin biosynthetic genes by PCR and/or DNA hybridization using DNA sequences of homologous genes from Aspergillus ochraceus, provided by our collaborators. We will identify gene clusters that may be involved in toxin synthesis by sequencing DNA flanking the initial gene sequences. We will investigate the involvement of these genes in ochratoxin biosynthesis by making site-directed mutations in each gene, and analyzing mutants for loss of ochratoxin production. 2) We will use genes involved in ochratoxin synthesis from Aspergillus alliaceus and other fungi to create specific DNA microarrays for gene expression studies. Expression of these genes will be examined under varying conditions (temperature, pH, nutrient sources) and substrates (grains, tree nuts, soil, etc.) and compared to levels of ochratoxin produced under these conditions. 3) Using genes identified in objective 1 we will design PCR primers to detect ochratoxin-producing fungi, quantify gene expression in environmental and food samples, and address time intervals between detection of gene expression and toxin production. Documents reimbursable with FAS. Log 26151. Formerly 5325-42000-032-05R (3/06). 3.Progress Report This report serves to document research conducted under a reimbursable agreement between ARS and the USDA Foreign Agricultural Service. Additional details of research can be found in the report for the parent project 5325-42000-035-00D, Molecular and Genetic Approaches to Suppressing Fungal Pathogens and Mycotoxin Contamination. The effects of several phenolic antioxidant compounds on ochratoxin production and fungal growth rate were tested using 12 different isolates of Aspergillus, consisting of 9 ochratoxigenic species. Results showed that several of the antioxidant compounds resulted in reduced ochratoxin production by these fungi, and that the relative activity of each antioxidant was strain-dependent. Several polyketide synthase (PKS) gene fragments were identified from Aspergillus alliaceus during the previous reporting period. To further investigate the involvement of one or more of these PKS genes in ochratoxin production, RNA was isolated from A. alliaceus grown in the presence or absence of ochratoxin-suppressing antioxidant compounds. Quantitative reverse transcription-PCR (qRT-PCR) was used to determine the relative expression of each PKS gene. Results indicate that the qRT-PCR primers used were not specific enough to each of the identified PKS genes, and so the suppression of PKS expression by antioxidants was not clearly determined. Efforts to construct PKS-specific insertional mutants in A. alliaceus, to determine which PKS is specific to ochratoxin biosynthesis, are ongoing.