2007 Annual Report 1a.Objectives (from AD-416) Combining strategically selected genes conferring diverse modes of action against plant pathogens into one organism can improve the efficacy of biological control agents. This strategy eliminates the costly process of screening new strains of uncertain value, simplifies formulation, avoids competition among introduced strains, and enables agents already adapted to a particular agroecosystem to reliably deliver metabolites with known activity against the target pathogen. In this project, we will introduce known biocontrol genes into the well-characterized rhizobacteria Pseudomonas fluorescens Q8r1-96 and Serratia plymuthica IC1270 to broaden their range of target pathogens and enhance their antifungal and plant growth-promoting activities. 1b.Approach (from AD-416) Construct site-specific integrative expression cassettes carrying: (i) the chiA gene for a 58-kDa endochitinase, (ii) the pyrrolnitrin biosynthetic operon, and (iii) the acdS gene encoding 1-aminocyclopropane-1-carboxylate (ACC)-deaminase (which confers plant growth-promoting activity); (2) employ these constructs and an existing cassette carrying the 2,4-diacetylphloroglucinol biosynthetic operon to engineer stable recombinant strains with an expanded repertoire of beneficial activities; (3) evaluate the rhizosphere competence and antifungal activity of the wild-type and modified strains against Rhizoctonia solani, Pythium aphanidermatum, Pythium irregulare, and Gaeumannomyces graminis var. tritici on cucumber, wheat, and pea under laboratory and greenhouse conditions; and (4) use molecular and traditional approaches to monitor the persistence and impact of the introduced strains on culturable and nonculturable rhizosphere microbial populations in the greenhouse and the field. Documents BARD Grant. Log 27564. Formerly 5348-22000-012-16R (6/07). 3.Progress Report This report serves to document research conducted under a Reimbursable between ARS and United States-Israel Binational Agricultural Research and Development Fund (BARD). Additional details of the research can be found in the report for the parent project 5348-22000-013-00D, NP 303, Plant Diseases. This project is assigned to National Program Plant Diseases, NP 303. Most food, fiber and ornamental crops grown in the U.S. are infected by soilborne pathogens that cause wilts, root and crown rots, and damping-off diseases. These diseases result in billions of dollars in losses to American farmers. There is increasing interest in the use of introduced microorganisms to control soilborne plant pathogens. However, inconsistent performance due to inefficient root colonization or inadequate antagonistic activity continues to hinder commercial implementation of the technology. Combining strategically selected genes conferring diverse modes of action against plant pathogens into one organism can improve the efficacy of biocontrol agents. This strategy eliminates the costly process of screening new strains of uncertain value, simplifies formulation, avoids competition among introduced strains, and enables agents already adapted to a particular agroecosystem to reliably deliver metabolites with known activity against target pathogens. The goal of this project is to introduce and express known biocontrol genes into Pseudomonas fluorescens Q8r1-96 and Serratia plymuthica IC1270 to broaden their range of target pathogens and enhance their antifungal and plant growth-promoting activities. Plasmids and site-specific integrative expression cassettes carrying the chiA gene for a 58-kDa endochitinase, the pyrrolnitrin biosynthetic operon, and the acdS gene encoding 1-aminocyclopropane-1-carboxylate (ACC) deaminase have been constructed and engineered into strain Q8r1-96. In greenhouse studies, Q8r1-96 expressing pyrrolnitrin from a plasmid was more effective against Rhizoctonia solani on bean and no less rhizosphere competent than the parental strain. Results of these studies complement ongoing efforts to identify factors governing the efficacy, competitiveness and persistence of natural and engineered biocontrol agents on different plant hosts and facilitate the development of novel and environmentally friendly pest management technologies that address an increasingly urgent problem facing agriculture in the US and Israel. Accomplishments align with Components 4, Problem Statement 4A of NP 303.