2006 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? Disease and plant pests limit the production of small fruits and deciduous tree crops in California. For several of the major diseases of almond, grapes, walnuts and strawberry we do not have effective control strategies. To address these phytopathological issues we will identify the etiological agents and develop effective and environmentally friendly control strategies. This research program will include an examination of the fungal, bacterial and viral disease causing agents of small fruit and deciduous tree crops. We will also develop control strategies that eliminate the need for methyl bromide fumigation. Integrated management approaches for key soilborne diseases affecting these crops also will be developed. The results of this work will facilitate the development of both rapid diagnostic and sustainable disease management strategies. The research directly and indirectly benefits growers and consumers by improving economic productivity of the crops and helping to avoid any unfavorable environmental impacts from disease control practices associated with production of the crops. This project is aligned with National Program 303, Plant Diseases and contributes to National Program 308, Methyl Bromide Alternatives. 2.List by year the currently approved milestones (indicators of research progress) FY2004 Milestone 1: Establish and sample fumigated and non-fumigated PRD sites Milestone 1: Culture and identify microbes associated with PRD Milestone 1: Quantify nematodes associated with PRD Milestone 1: Characterize DNA associated with PRD Milestone 1: Soil treatment assays (greenhouse) Milestone 1: Chemical and fractionation assays (greenhouse) Milestone 1: Pathogenicity tests (greenhouse) Milestone 2:Optimize PCR for Phytophthora cactorum using species primers Milestone 2:Test and optimize PCR for P. cactorum in soil Milestone 2:Develop PCR-based detection for P. cactorum with field strawberry and soil samples Milestone 3: Test Methyl Bromide alternatives (almond and walnut nurseries, and almond orchards) Milestone 3: Evaluate cover crop and fallow for PRD control, micro plots Milestone 3: Field test resistance to P. cactorum in strawberry cultivars Milestone 3: Evaluate resistance to P. citricola in Paradox walnut clones Milestone 3: Evaluate resistance to P. citricola in Paradox walnut clones Milestone 3: Field test resistance to P. cinnamomi in Chinese wingnut Milestone 4: Monitor Butte budline performance under irrigation regimes Milestone 6: Inoculate with diseased collections, inspect for wood markings FY2005 Milestone 1: Establish and sample fumigated and non-fumigated PRD sites Milestone 1: Culture and identify microbes associated with PRD Milestone 1: Quantify nematodes associated with PRD Milestone 1: Characterize DNA associated with PRD Milestone 1: Soil treatment assays (greenhouse) Milestone 1: Chemical and fractionation assays (greenhouse) Milestone 1: Pathogenicity tests (greenhouse) Milestone 1: Analyze data, prepare and submit manuscripts Milestone 2:Optimize sampling for PCR, P. cactorum on strawberry plants Milestone 2:Test and optimize PCR for P. cactorum in soil Milestone 2:Develop PCR-based detection for P. cactorum with field strawberry and soil samples Milestone 2:Analyze data, prepare and submit manuscripts Milestone 3: Test Methyl Bromide alternatives (almond and walnut nurseries, and almond orchards) Milestone 3: Evaluate cover crop and fallow for PRD control, micro plots Milestone 3: Field test resistance to P. cactorum in strawberry cultivars Milestone 3: Evaluate resistance to P. citricola in Paradox walnut clones Milestone 3: Evaluate resistance to P. citricola in Paradox walnut clones Milestone 3: Field test resistance to P. cinnamomi in Chinese wingnut Milestone 3:Analyze data, prepare and submit manuscripts Milestone 4: Monitor Butte budline performance under irrigation regimes Milestone 4: Analyze data, prepare and submit manuscripts Milestone 5: Evaluate tree performance (ALS incidence, nut set, nut quality) Milestone 5: Analyze data, prepare and submit manuscripts Milestone 6: Inoculate with diseased collections, inspect for wood markings Milestone 6: Isolate and characterize cDNAs, develop RT-PCR assays Milestone 6: Analyze data, prepare and submit manuscripts FY2006 Milestone 1: Establish and sample fumigated and non-fumigated PRD sites Milestone 1: Culture and identify microbes associated with PRD Milestone 1: Quantify nematodes associated with PRD Milestone 1: Characterize DNA associated with PRD Milestone 1: Soil treatment assays (greenhouse) Milestone 1: Chemical and fractionation assays (greenhouse) Milestone 1: Pathogenicity tests (greenhouse) Milestone 1: Analyze data, prepare and submit manuscripts Milestone 2:Test and optimize PCR for P. cactorum in soil Milestone 2:Develop PCR-based detection for P. cactorum with field strawberry and soil samples Milestone 2:Analyze data, prepare and submit manuscripts Milestone 3: Test Methyl Bromide alternatives (almond and walnut nurseries, and almond orchards) Milestone 3: Evaluate cover crop and fallow for PRD control, micro plots Milestone 3: Field test resistance to P. cactorum in strawberry cultivars Milestone 3: Evaluate resistance to P. citricola in Paradox walnut clones Milestone 3: Evaluate resistance to P. citricola in Paradox walnut clones Milestone 3: Field test resistance to P. cinnamomi in Chinese wingnut Milestone 3:Analyze data, prepare and submit manuscripts Milestone 4: Analyze data, prepare and submit manuscripts Milestone 5: Evaluate tree performance (ALS incidence, nut set, nut quality) Milestone 5: Analyze data, prepare and submit manuscripts Milestone 6: Inoculate with diseased collections, inspect for wood markings Milestone 6: Isolate and characterize cDNAs, develop RT-PCR assays Milestone 6: Analyze data, prepare and submit manuscripts FY2007 Milestone 1: Establish and sample fumigated and non-fumigated PRD sites Milestone 1: Culture and identify microbes associated with PRD Milestone 1: Characterize DNA associated with PRD Milestone 1: Pathogenicity tests (greenhouse) Milestone 1: Analyze data, prepare and submit manuscripts Milestone 2:Analyze data, prepare and submit manuscripts Milestone 3: Field test resistance to P. cactorum in strawberry cultivars Milestone 3: Evaluate resistance to P. citricola in Paradox walnut clones Milestone 3: Field test resistance to P. cinnamomi in Chinese wingnut Milestone 3:Analyze data, prepare and submit manuscripts Milestone 4: Analyze data, prepare and submit manuscripts Milestone 5: Evaluate tree performance (ALS incidence, nut set, nut quality) Milestone 5: Analyze data, prepare and submit manuscripts Milestone 6: Inoculate with diseased collections, inspect for wood markings Milestone 6: Isolate and characterize cDNAs, develop RT-PCR assays Milestone 6: Analyze data, prepare and submit manuscripts Milestone 7: Develop species-specific DNA primers for culture independent detection/quantification of Agrobacterium and Brennaria species FY2008 Milestone 1: Establish and sample fumigated and non-fumigated PRD sites Milestone 1: Culture and identify microbes associated with PRD Milestone 1: Characterize DNA associated with PRD Milestone 1: Pathogenicity tests (greenhouse) Milestone 1: Analyze data, prepare and submit manuscripts Milestone 2:Develop PCR-based detection for P. cactorum with field strawberry and soil samples Milestone 2:Analyze data, prepare and submit manuscripts Milestone 3: Field test resistance to P. cactorum in strawberry cultivars Milestone 3: Evaluate resistance to P. citricola in Paradox walnut clones Milestone 3: Field test resistance to P. cinnamomi in Chinese wingnut Milestone 3:Analyze data, prepare and submit manuscripts Milestone 5: Evaluate tree performance (ALS incidence, nut set, nut quality) Milestone 5: Analyze data, prepare and submit manuscripts Milestone 6: Inoculate with diseased collections, inspect for wood markings Milestone 6: Isolate and characterize cDNAs, develop RT-PCR assays Milestone 6: Analyze data, prepare and submit manuscripts Milestone 8: Identify Crown Gall resistant walnut germplasm Milestone 9: Characterize root erudate modulated bacterial gene expression 4a.List the single most significant research accomplishment during FY 2006. A discovery of, determining disease incidences of, and evidence for rapid spread of a new disease (necrotic union disorder) on grape (Pinot noir/110R grapevines) in California. This discovery addressed NP303 Component 1. Disease Diagnosis Detection, Identification and Characterization of Plant Pathogens, problem b) Detection, Identification, Characterization, and Classification of pathogens. In a research collaboration with Dr. Adib Rowhani of UC Davis, ARS Scientist Dr. Uyemoto surveyed several vineyard blocks planted to Pinot noir clones 667, 777, pommard, and 2A and disease incidence ranged from, respectively 5, 12, 25, and 45%. A research project has been established to study the etiology, epidemiology, and control of this new viral disease and to identity and characterize the etiological agent. 4b.List other significant research accomplishment(s), if any. Prototype Global Positioning System-assisted Site Fumigation Rig Developed. A prototype Global Positioning System-assisted site fumigation rig was developed for field testing. This development addressed NP308 Methyl Bromide-Component 1. Preplant Soil fumigation alternatives, problem a) chemical controls. This project was accomplished in collaboration with Shrini Upadhyaya at the University of California, Davis, private industry, and ARS Scientist, Dr. Greg Browne. In conjuction with prescriptive risk management approaches to remediation of replant problems, the GPS technology is anticipated to lead to reduced and optimized fumigant use for commercial orchard crops. New Stem Markings on Rootstock Recognized. Four new stem markings on rootstocks have been recognized; stem lesion, stem necrosis, necrotic union, and stem pitting. Rootstock stem necrosis is expressed on several rootstocks and involves different graft-transmissible pathogens (GTPs. This discovery addressed NP303 Component 1. Disease Diagnosis Detection, Identification and Characterization of Plant Pathogens, problem b) Detection, Identification, Characterization, and Classification of pathogens. Work is being done at UC Davis and in commercial vineyards located in Napa and Sonoma counties. Uyemoto’s collaborators includes: Adib Rowhani and Maher Al Rwahnih (UC Davis) and Rhonda Smith and Ed Weber (UCCE Farm Advisors). Outcome of accomplishments are development of a rapid assay for GLRaV-2RG and an indication of a vector for the 110R-necrotic union agent. Genetic and Phenotypic Diversity of Two Phytophthora Species Characterized. We have completed the characterization of the genetic and phenotypic diversity of two Phytophthora Species. This addresses NP303 Component 1. Disease Diagnosis Detection, Identification and Characterization of Plant Pathogens, problem b) Detection, Identification, Characterization, and Classification of pathogens. At the USDA, ARS, Crops Pathology/Genetics Research Unit, several gram negative bacteria have been shown to be associated with the incidence of Almond replant disease. We identified several juglans species that are resistant to Crown Gall Disease. Species-specific primers were developed for Agrobacterium tumefaciens and Brennaria tumefaciens, a potential benefit to the Almond industry. DNA Extraction and Purification Protocol Developed. A DNA extraction and purification protocol was developed to overcome inhibitors of PCR in diseased strawberry plants. This addresses NP303 Component 1. Disease Diagnosis Detection, Identification and Characterization of Plant Pathogens, problem b) Detection, Identification, Characterization, and Classification of pathogens. At the USDA, ARS, Crops Pathology/Genetics Research Unit DNA extraction and a purification protocol was developed to overcome inhibitors of PCR in diseased strawberry plants. This is enabling improved detection of soilborne pathogens in strawberry. Improved diagnostic capability will lead to enhanced disease management. Foliar Spray/Chemigation Phosphonate Treatment Efficacy. Field research demonstrated efficacy of phosphonate treatments applied by foliar spray and/or chemigation for management of Phytophthora crown rot of walnut. This addresses the NP308 Methyl Bromide-Component 1. Preplant Soil fumigation alternatives, problem a) chemical controls. At the USDA, ARS, Crops Pathology/Genetics Research Unit field plots were tested using a variety of phosophonate treatments. This is leading to improved management of Phytophthora crown rot in commercial orchards. 4c.List significant activities that support special target populations. None. 4d.Progress report. None. 5.Describe the major accomplishments to date and their predicted or actual impact. Determined the efficacy of phosphonates for management of Phytophthora diseases on almond and strawberry; growers of these crops often use these treatments due to demonstration of the efficacy in field trials-NP 303 Plant Disease, Componet I. Disease Diagnosis Detection, Identification and Characterization of Plant Pathogens, a) New Diagnostic Methods and Tools Determined efficacy of spot treatments with chloropicrin for prevention of replant disease of almond and peach; growers of these crops have been increasing their use of this fumigant, largely due to the field-demostrated efficacy- NP 303 Plant Disease, Component 1. Disease Diagnosis Detection, Identification and Characterization of Plant Pathogens, a) New Diagnostic Methods and Tools Determined clonal selections of Paradox walnut rootstocks with superior resistance to Phytophthora crown rot; nurseries are beginning to propagate and growers are beginning to plant some of the superior rootstocks for commercial evaluation and use-NP303 Plant Disease, Component III. Plant Disease Resistance, b) Disease resistance in new germplasm and varieties. With our publication reporting partial characterization of a closterovirus, Plum bark necrosis-stem pitting associated virus (PBNSPaV), PBNSPaV has been found in Italy and several Mediterranean countries associated with stem pitting disease. In Morocco, the virus was detected in a wild population of almond trees, suggesting presences of a virus vector, presumably a mealybug species. Also, discovery and characterization of the rootstock stem lesion associated virus, GLRaV-2RG, has lead to its detection in vineyards in South America, New Zealand, and Italy-NP303 Plant Disease, Component 1. Disease Diagnosis Detection, Identification and Characterization of Plant Pathogens, b) Detection, Identification, Characterization, and Classification of Plant Pathogens. Using our newly developed PCR based detection system we are now able to provide science based guidance to both orchard and nursery operation on the incidence of several important pathogens. Identification of crown gall resistance walnut germplasm for use as a rootstock will have tremendous impact on the walnut industry-NP303 Plant Disease, Component III. Plant Disease Resistance, a) Mechanisms of Plant Disease Resistance. 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Research-tested phosphonate treatment methods were transferred to the California almond, strawberry, and walnut industries. (The walnut technology transfer was new for 2006; that for the other crops occurred previously). Research-tested soil fumigation with effective alternatives to methyl bromide were transferred to the California almond and strawberry industries. (This has occurred in part before 2006 but was intensified in 2006). Grapevine viruses/virus diseases. In 2006, several talks (see below) were given at an UCCE grape farm advisors workshop; two society meetings in Boise ID and Sacramento CA; and two seminars in Valenzano, Italy and Aguascalientes, Mexico. The technology is available to end-users. Constraints involved adoption in the State’s or countries official certification of rules and regulations. These detection based technologie will likey be available to the industry within 12-18 months. Crown Gall resistance rootstocks may be available to the industry in 5-7 years in a commercially acceptable form. Developing new cultivars/rootstock of perennial deciduous trees typically requires 7-10 years. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Uyemoto, J.K. 2006. Necrotic union and stem necrosis associated with grafted Pinot noir grapevines. Invited talk to the UC Davis-Integrated Grape Production Workgroup, moderator Mark Battany on April 18th. Uyemoto, J.K. 2006. Newly discovered stem disorders in grapevines. Invited seminar at the CIHEAM Instituto, Valenzano (Bari) Italy on April 24th. Uyemoto, J.K. 2006. Grapevine lethal GTAs – work for the future. Invited talk at the Grapevine Clean Stock Workshop, UC Davis’s DaVinci Building on June 20th. Uyemoto, J.K. 2006. Grapevine rootstock stem disorders in California vineyards. Invited seminar at the INIFAP Campo Experimental Pabellon, Aguscalientes-Zacatezas, Pabellon de Arteaga, Ags on July 14th. Uyemoto, J.K. and C.F. Luhn. 2006. In-season variation in transmission of cherry x-phytoplasma and implication in certification programs. Journal of Plant Pathology (galley proofs in review). Uyemoto, J.K. and A. Rowhani. 2006. New and emerging grapevine viruses in California vineyards. Invited paper presented in the Mini-Conference New, Emerging Pathogens in the Pacific West area at APS Pacific Division Meetings, Boise ID on June 14th. Uyemoto, J.K., R. Smith, and A. Rowhani. 2006. Grapevine necrotic union disease. Paper at the American Society of Enology and Viticulture, 57th Meeting, Sacramento Convention Center on June 29th. Browne, G., Duniway, J., Westerdahl, B., Martin, F., Ajwa, H., and Fennimore, S. 2005. Effects of VIF and chloropicrin and Inline rates on survival of nematodes and pathogens in soil. Paper no. 50, International Conference for Alternatives to Methyl Bromide, San Diego, CA; presented Nov. 2005. Lampinen, B., Browne, G., Schneider, S., Shrestha, S., Hotz, B., and Simon, L. 2005. Alternative pre-plant soil fumigation treatments for deciduous tree crops. International Conference for Alternatives to Methyl Bromide, San Diego, CA; Presented Nov. 2005. Bhat, R. G., Colowit, P. M., Tai, T. H., Aradhya, M. K., and Browne, G. T. 2006. Genetic and pathogenic variability in Phytophthora cactorum affecting fruit and nut crops in California. Plant Disease 90:161-169. Browne, G.T., Connell, J.H., and Schneider, S.M. 2006. Almond replant disease and its management with alternative pre-plant soil fumigation treatments and rootstocks. Plant Disease Plant Disease 90: 869-876. Browne, G.T., Schmidt, L.S., Prichard, T.L., and Krueger, W.H. 2006. Effects of phosphonate treatments on disease caused by Phytophthora citricola on English walnut. Phytopathology 96:S17 (Abstract). Review Publications Wechter, W.P., Kluepfel, D.A. 2005. Global gene expression of Pseudomonas Putida KT2440 during growth in the rhizosphere of Arabidopsis Thaliana [abstract]. Phytopathology 95 (6):S110.