2008 Annual Report 1a.Objectives (from AD-416) To genotypically and phenotypically characterize plant pathogens that cause diseases of crops grown in cereal-based cropping systems; to genotypically and phenotypically characterize microbial biocontrol agents that suppress plant pathogens in these cropping systems; and to determine the molecular genetic and biochemical basis of plant-microbe interactions and plant defense. 1b.Approach (from AD-416) Plant pathogens and their biocontrol agents will be characterized through a combination of molecular fingerprinting techniques and methods of classical plant pathology and microbiology. The population structures of pathogens and biocontrol agents will be determined by statistical analysis of DNA banding patterns generated by techniques such as rep-PCR and RAPDs, RFLPs, and AFLPs. Results of genotypic and phenotypic analyses will be compared. Plant-microbe interactions will be characterized using a combination of laboratory, greenhouse and field studies, and molecular biology and bioanalytical tools. Biologically active metabolites produced by pathogens and antagonists will be characterized using techniques such as high performance liquid chromatography (HPLC) and mass spectrometry. Replaces 5348-22000-012-09S (3/05). Documents SCA with WSU. Formerly 5348-22000-012-15S (03/07). 3.Progress Report Root diseases, including take-all, Pythium, Rhizoctonia and common root rots, and Fusarium crown rot, cause $3 billion in losses annually to U.S. wheat and barley growers. For most of these diseases, there are no resistant varieties and chemical treatments are not available or perform inconsistently. Modern production practices of reduced tillage or direct seeding, used to control soil erosion, exacerbates the severity of root diseases. Researchers at Washington State University, in collaboration with ARS scientists at Pullman, WA, used a combination of molecular fingerprinting techniques, conventional and real-time PCR and methods of classical plant pathology and microbiology to characterize the genetic diversity present in populations of soilborne pathogens including Pythium spp., Gaeumannomyces graminis var. tritici (Ggt) and Rhizoctonia spp. that attack crops grown in cereal-based cropping systems. Methods to rapidly isolate fungal and Pratylenchus DNA from soil were developed, allowing detection and quantification of pathogens in field soil by using real-time PCR and species specific primers. Knowledge of pathogen population structure and virulence insures that research to develop resistant wheat, barley, and canola germplasm utilizes pathogen isolates typical of field populations. This work provides to growers a direct test for determining the risk from pathogens present in their fields. The build-up of naturally-occurring populations of 2,4-diacetylphloroglucinol (DAPG)-producing Pseudomonas fluorescens during wheat monoculture is responsible for the suppression of take-all of wheat known as take-all decline (TAD). The sensitivity of root pathogens to DAPG and other antibiotics produced by rhizosphere bacteria, responsible for soil suppressiveness, was determined. The ED90 of DAPG against most isolates of Ggt from suppressive and conducive fields ranged from 3.14 to 11.1 µg ml-1, and there was no evidence that Ggt becomes insensitive to DAPG after years of exposure to DAPG in TAD fields. A real-time PCR assay to quantify populations and genotypes of DAPG-producers from rhizosphere DNA was refined, thus making it possible to provide growers with an assessment of the suppressiveness of their soil to take-all. Novel antifungal, metabolites such as phloroglucinols and phenazines responsible for suppression of root pathogens were characterized by mass spec. Phenazine-1-carboxylic acid was isolated from roots of wheat collected from multiple fields in the dryland wheat producing region of Washington, suggesting a possible role for the antibiotic in disease suppression. Collectively, these findings will hasten the development of biocontrol technology, a sustainable approach to control root diseases in cereal-based production systems. Accomplishments align with Components 1, Problem Statements 1A and 1B; Component 2, Problem Statements 2C; and Component 4, Problem Statements 4A, 4B and 4C of NP 303. Progress on this project is monitored by weekly conversations with the Washington State University collaborator