2007 Annual Report 1a.Objectives (from AD-416) The overall objective of this project is to identify mechanisms by which the biological control agent Pseudomonas fluorescens Pf-5 suppresses plant disease. Strain Pf-5 is a rhizosphere bacterium that suppresses numerous soilborne plant pathogens and produces an array of secondary metabolites. Recently, the complete genome of Pf-5 was sequenced. The proposed research will employ microarrays representing each gene in the Pf-5 genome to study genome expression profiles of Pf-5 on seed surfaces, and to identify novel genes involved in biological control of Pythium damping-off, an important disease effecting seedlings of many crops. Genes potentially involved in biological control of Pythium damping-off of cucumber will be identified by two criteria: a) their expression in the spermosphere, and b) their control by two global regulatory genes known to affect the biological control. Specific objectives of the research are to: . 1)Identify genes expressed by Pf-5 inhabiting seed surfaces under the control of two global regulatory genes. . 2) Construct mutants of Pf-5 with genetic lesions in genes identified in objective 1. Resultant mutants will be compared to the wildtype strain for biological control of plant disease. 1b.Approach (from AD-416) The planned approach is to identify molecular determinants of biological control based upon their transcriptional regulation in the spermosphere and knowledge of the key regulators of biological control. The experiments will monitor gene expression in Pf-5 and derivatives on seed surfaces over time using oligonucleotide arrays. The goals of these experiments are (1) to identify genes expressed under the control of two regulatory genes on seeds, and. 2)gain a view of the changes in patterns of gene expression that occur during the process of seed and root colonization. RNA will be isolated from Pf-5 growing on seed surfaces and analyzed in microarray experiments, followed by validation experiments using a combination of quantitative, reverse-transcriptase PCR and transcriptional fusions. The nutritional composition of seed exudates (i.e., carbohydrates, amino acids, and organic acids) will be assessed so that gene expression data can be related directly to the nutritional environment of the spermosphere. Candidate genes with a potential role in biological control will be identified from the microarray experiments as those expressed by Pf-5 on seeds surfaces under the control of the global regulatory genes. Candidate genes will be mutagenized through recombinant DNA techniques (PCR amplification, cloning, and allelic exchange mutagenesis) and resultant mutants of Pf-5 will be compared to the wildtype strain for biological control of plant disease.Documents Reimbursable with CSREES. Log 30407 3.Progress Report This report serves to document research conducted under a reimbursable agreement between ARS and CSREES. Additional details of research can be found in the report for the parent project 5358-12220-003-00D, Biology and Management of Soilborne Diseases of Horticultural Crops Dr. Loper and collaborators conducted the following research towards the agreements objectives: The development of reliable and effective biologically-based strategies for American agriculture depends on an understanding of the mechanisms by which biological control operates. The objective of this project is to identify mechanisms by which the biological control agent Pseudomonas fluorescens Pf-5 suppresses plant disease. Strain Pf-5 is a rhizosphere bacterium that suppresses numerous soilborne plant pathogens and produces an array of secondary metabolites. Recently, the complete genome of Pf-5 was sequenced. The proposed research will employ microarrays representing each gene in the Pf-5 genome to study genome expression profiles of Pf- 5 on seed surfaces, and to identify novel genes involved in biological control of Pythium damping-off, an important disease effecting seedlings of many crops. Genes potentially involved in biological control of Pythium damping-off of cucumber will be identified by two criteria: a) their expression in the spermosphere, and b) their control by two global regulatory genes known to affect the biological control. An approach for making unmarked mutations was adapted so that derivatives of Pf-5 having multiple mutations in specific genes could be generated efficiently. Using the new approach, unmarked mutations in the pyrrolnitrin and 2,4- diacetylphloroglucinol gene clusters were generated. Using the new approach, specific mutants can be constructed very efficiently, and this method will allow us to test the role of many secondary metabolism and other genes in biological control of plant disease by Pf-5. We expect to complete a sub-objective of the project to generate a strain of Pf-5 with mutations in all known secondary metabolism genes. We also expect to complete Subobjective 1a, using microarrays to identify genes expressed by Pf-5 on seed surfaces under the control of a global regulatory gene. ADODR Statement: The ADODR met with the cooperating PI and project personnel at meetings during the year and discussed results through phone calls, e-mail, and in person.