2007 Annual Report 1a.Objectives (from AD-416) The long-term objective of this program is to develop biologically based technology for controlling soilborne pathogens of wheat, barley, and Brassicas, grown as part of cereal-based production systems. Five specific objectives will be addressed over the next five years. Objective 1: Evaluate the genotypic diversity between species and within pathogen populations of root pathogens in direct-seeded cereal-based cropping systems. Objective 2: Identify and characterize microorganisms and mechanisms active in the suppression of soilborne pathogens by rhizobacteria. Objective 3: Identify the determinants responsible for differential rhizosphere competence among genotypes of DAPG-producing Pseudomonas fluorescens strains. Objective 4: Identify and characterize the molecular mechanisms of host-microbe interactions, including the action of host genes governing disease resistance and biological control. Objective 5: Identify and develop biocontrol technology for suppression of root pathogens in cereal-based cropping systems. 1b.Approach (from AD-416) Biological control of root pathogens Gaeumannomyces, Pythium, Rhizoctonia, and Fusarium by naturally occurring and genetically engineered microorganisms will be developed and quantified in different soils. The genetic determinants and molecular mechanisms responsible for root colonization and pathogen suppression will be characterized with emphasis on the genetics and regulation of phenazine and phloroglucinol biosynthesis. Structural loci for these anti-fungal metabolites will be used to engineer improved biocontrol agents. The genetic and physiological diversity of populations of the root pathogens, and influence of cropping system on pathogen populations and diversity will be determined. New sources & mechanisms of resistance will be identified. Practical root diseases control will be accomplished by both maximizing the activity of natural biocontrol agents. Formerly 5348-22000-012-00D (2/07). 3.Progress Report None 4.Accomplishments Precision seeding between relic stubble rows does not reduce Rhizoctonia in direct-seeded wheat: Many soilborne plant pathogens produce inoculum on standing crop residue, and seeding between the rows of the previous year may reduce disease. ARS scientists at Pullman, WA in collaboration with scientists at Washington State University compared disease levels with seeding at different spacing from the old stubble, in natural and fumigated plots. Fumigation but not spacing reduced disease in both field and greenhouse experiments. Rhizoctonia survives on roots between stubble rows, and above-ground crop residue is not a major source of inoculum, hence field burning will not reduce this disease. This accomplishment aligns with Component 4, Problem Statements 4A and 4C of NP 303. Rhizoctonia bare patch pathogen shows a unique distribution in Washington State: Rhizoctonia bare patch and root rot, caused by R. solani AG-8, is a major problem on wheat and barley, especially in direct-seed systems. ARS scientists at Pullman, WA, using species-specific primers they developed for quantitative PCR, surveyed variety testing and grower sites throughout the wheat growing area. Surprisingly, the highest pathogen levels were found in a crescent from Ritzville to Walla Walla, an area with lower precipitation and sandy loam, but the pathogen was present in low but detectable levels in the higher precipitation zones with continuous annual cropping. This information will be useful for growers to determine disease risk, and suggests that natural disease suppression may be functioning in the low risk areas of Washington. This accomplishment aligns with Components 1, Problem Statements 1A and 1B and Component 2, Problem Statement 2C of NP 303. Rhizoctonia is not influenced by burning or residue management in irrigated cereal crops: Growers perceive that they can control diseases by burning the old crop stubble, which creates environmental and health problems. ARS scientists at Pullman, WA in collaboration with researchers at Washington State University studied Rhizoctonia disease on barley and wheat in a 6-year study with no residue removal (standing stubble), burning, or mechanical removal of the straw. Residue management did not affect disease or pathogen levels in the soil, as tested with DNA techniques. Highest Rhizoctonia levels occurred in direct-seeded (no-till) treatments, however with irrigation and crop rotation, the deleterious effects of this pathogen were not manifested. These results show that burning is not needed to control this disease, since the pathogen resides on roots in soil, which are not killed by burning. This accomplishment aligns with Components 4, Problem Statements 4A and 4C of NP 303. Virus-induced gene silencing suppresses COI expression in wheat roots: Transient suppression of two COI genes, which encode regulators of the jasmonate pathway, has been demonstrated in roots of wheat cultivar Scarlet. The jasmonate pathway is important for innate immunity against the root pathogen Pythium in the model plant Arabidopsis. ARS scientists at Pullman, WA in collaboration with researchers at Washington State University developed barley stripe mosaic virus-mediated gene silencing for wheat, and showed that root expression of COI1 and COI2 was reduced about 55% and 25%, respectively, using real-time PCR. This accomplishment is the first step to modulating the expression of specific genes in wheat roots as an alternative to stable transformation, with implications for testing the role of specific genes in pathogen resistance. This accomplishment aligns with Component 3, Problem Statement 3A of NP 303. Exogenous DAPG induces wheat root genes: DAPG, an antifungal metabolite produced by biocontrol bacteria, induced changes in wheat root gene expression at 12 to 24 hours of treatment. Molecular changes that occur in wheat during early stages of root colonization are deemed the most critical for establishing successful interactions, and these findings are a first step in understanding the impact of biocontrol agents on their host. ARS scientists at Pullman used microarray analysis to identify host defense and detoxification genes. The findings provide leads to host pathways that might be modulated to enhance interactions with DAPG-producing biocontrol bacteria. This accomplishment aligns with Component 3, Problem Statement 3A of NP 303. DNA-based identification of Pratylenchus species: Over 50 percent of fields tested in Oregon and Washington are infested by damaging populations of root-lesion nematodes. ARS collaborators at Oregon State University, Pendelton, OR found that published Pratylenchus species-specific primers were of limited application for clearly differentiating P. neglectus and P. thornei especially in mixed cultures derived from extractions of soil naturally infested with many plant-parasitic and saprophytic species. They optimized new species-specific primers for detecting and providing ratios of prevalence for the two Pratylenchus species of importance to dryland agriculture in the Pacific Northwest. A multiplexing assay was also developed to enable each species to be quantified from soil extracts using real-time PCR. Detection sensitivity was refined to enable detections at 50% of the economic threshold for damage by each species. This technology will be adopted by commercial nematode diagnostic laboratories, most of which currently avoid microscopically-based identifications of Pratylenchus species due to the great difficulty and time requirement for this differentiation, and the economic liability associated with incorrect species assignments that are easily made using microscopy alone. This accomplishment aligns with Component 1, Problem Statement 1B of NP 303. Real-time PCR detection and quantification of DAPG-producing Pseudomonas fluorescens in the plant rhizosphere and soil: The build-up of 2,4-diacetylphloroglucinol (DAPG)-producing Pseudomonas fluorescens during wheat or barley monoculture is responsible for take-all decline, which is a natural and sustainable biocontrol of take-all utilized by many growers worldwide to manage take-all disease of wheat. ARS scientists at Pullman, WA in collaboration with researchers at Washington State University developed a quantitative real-time PCR assay that quantifies populations of DAPG-producing P. fluorescens in the soil and rhizosphere. This culture-independent technique is as effective as culture-based methods currently used to detect and quantify these important biocontrol agents. The results of this study provide a method for determining the suppressiveness of a soil to take-all simply by assaying total DNA isolated from the wheat or barley rhizosphere. Knowledge about the suppressiveness of a field will allow wheat growers to make greater use of TAD to control take-all. This accomplishment aligns with Component 4, Problem Statement 4A of NP 303. Biodiesel production by The Confederated Tribes of the Colville Reservation: The Colville Reservation, largest Native American Reservation in Washington State, traditionally has relied on logging as the primary source of employment even though the reservation has nearly 200,000 acres of farmable land with irrigation. At the invitation of the Colville Tribal Business Council, ARS scientists at Pullman, WA initiated studies of canola production for biodiesel on the reservation in 2006 and greatly expanded those studies in 2007. Results demonstrated the ability to grow canola using minimal inputs and without pesticides, a request by the Business Council to preserve tribal traditions about the land. This research has resulted in allocation of funds by the Business Council to purchase a canola crusher and the development of an infrastructure to produce biodiesel for the reservation’s 150+ logging trucks. This partnership with the Colville Reservation also resulted in ARS scientists teaching science modules in reservation schools once a month, hosting high school science interns in ARS laboratories, and sponsoring science camps on the reservation. This project has attracted statewide attentions as a model for science outreach to groups of Americans that currently are underrepresented in the scientific workforce. This accomplishment aligns with Component 4, Problem Statement 4A of NP 303. 5.Significant Activities that Support Special Target Populations ARS scientists at Pullman, WA lead an outreach program, with participation by USDA, NRCS, Washington State University Cooperative Extension, WSU Plant Pathology Department, and Bellevue Community College, to The Confederated Tribes of the Colville Reservation (largest Native American reservation in Washington State). The program entitled “Pumping-Up the Science Pipeline: Grade School to College” has four objectives: i) development of energy independence on the reservation through the production of biofuels; ii) hands-on science education in reservation schools by research scientists and technicians; iii) development of and participation in on-reservation summer science camps; and iv) employing Native American high school summer science interns in ARS laboratories. Examples of these activities include field tests of spring and winter canola varieties and biodiesel production on reservation land; monthly visits to Nespelem Elementary School and Pascal Sherman Indian School on the Colville Reservation to present science modules, December 2006 to May, 2007; sponsorship of and participation in the Skwant Life Science Camp at the Pascal Sherman Indian School, June 2007 (first science camp ever conducted on a reservation); and molecular biology training for 3 summer high school interns (July 2007) in ARS labs at Pullman. This award-winning program is promoting economic development on the Colville Reservation, enhancing science awareness among Native American youth, and training the next generation of Native American scientists. Native American reservations throughout the Pacific Northwest are requesting expansion of this ARS program to their communities. 6.Technology Transfer Number of new CRADAs and MTAs 4 Number of active CRADAs and MTAs 13 Number of patent granted 1 Number of new commercial licenses granted 1 Number of non-peer reviewed presentations and proceedings 26 Number of newspaper articles and other presentations for non-science audiences 24 Review Publications Gohain, N., Thomashow, L.S., Mavrodi, D.V., Blankenfeldt, W. 2006. The purification, crystallization and preliminary structural characterization of FAD-dependent monooxygenase PhzS, a phenazine-modifying enzyme from Pseudomonas aeruginosa. Acta Crystallographica F 62:989-992 Mavrodi, O.V., Mavrodi, D.V., Weller, D.M., Thomashow, L.S. 2006. The role of ptsP, orfT, and sss recombinase in root colonization by Pseudomonas flurescens Q8r1-96. Applied and Environmental Microbiology. 72:7111-7122 Weller, D.M., Landa, B.B., Mavrodi, O.V., Schroeder, K.L., De La Fuente, L., Bankhead, S.B., Molar, R.A., Bonsall, R.F., Mavrodi, D.M., Thomashow, L.S. Role of 2,4-diacetylphloroglucinol-producing fluorescent pseudomonas spp. in plant defense. Plant Biology 9 (1): 4-20 Jan, 2007.