2009 Annual Report 1a.Objectives (from AD-416) 1. Improve management strategies for key soil borne diseases of tree fruit and nut crops. 1A. Develop economical alternatives to preplant use of methyl bromide (MB). 1B. Identify and characterize available walnut and almond rootstock germplasm for resistance to key soil borne pathogens. 2. Characterize the etiology of key soil borne diseases of tree fruit and nut crops, and examine the molecular microbial ecology of both pathogenic and beneficial plant-associated microorganisms. 2A. Determine the etiology of Prunus replant disease (PRD). 2B. Characterize the genetic diversity and ecology of Agrobacterium tumefaciens. 3. Develop enhanced traditional and culture-independent molecular detection methods for key pathogens of fruit and nut crops. 3A. Develop enhanced detection protocols for Agrobacterium tumefaciens. 3B. Develop enhanced detection protocols for Brennaria rubrifaciens. 3C. Develop enhanced detection protocols for Phytophthora cactorum. 4. Examine etiology, epidemiology, and control strategies for systemic/graft transmissible pathogens of fruit and nut trees. 4A. Identify and characterize graft-transmissible pathogens(GTP)in fruit and nut trees with an emphasis on determining the etiology of emerging diseases such as Tieton cherry stunt (TCS) and Plum necrotic union (PNU). 4B. Epidemiology and molecular characterization of PBNSPaV. 4C. Evaluate Marianna 2624 rootstock for tolerance to Xyllela fastidiosa in almond. 5. Examine the structural and functional genomics of Phytophthora ramorum, the causal agent of Sudden Oak Death. 6. Identify and characterize rhizobacterial genes whose expression is mediated by plant root exudates and evaluate involvement of these genes in such ecologically important phenotypes as root colonization, infection, competition, and persistence in the environment. 7. Characterize the biology, genetic diversity, and ecology of key plant pathogenic agents of fruit/nut trees including A. tumefaciens strains, and examine the ecology and significance of molecular microbe-microbe and plant-microbe interactions of key plant associated microorganisms. 1b.Approach (from AD-416) 1. Chemical and crop-rotation-based alternatives to methyl bromide will be tested for control of Prunus replant disease (PRD), and genetic resistance to Phytophthora spp. and Agrobacterium tumefaciens will be explored in promising almond and walnut rootstocks. 2. Examine shifts in soil borne microbial communities associated with PRD incidence using culture-based and culture-independent approaches and complete Koch's postulates for organisms linked to the disease. Examine the molecular microbial ecology of A. tumefaciens under both orchard and nursery conditions in an effort to design effective crown gall control strategies. Examine Ti-plasmid ecology under commercial orchard conditions. Assess the genetic diversity of A. tumefaciens in California. 3. Described and novel PCR primers will be tested for specific amplification of ribosomal and mitochondrial DNA from P. cactorum. Enhance described and develop novel PCR primers for species-specific detection and quantification of A. tumefaciens in soil and inplanta. Develop PCR primers and define the extraction and cycling parameters for the detection and quantification of Brennaria rubrifaciens. 4. Epidemiological studies and molecular characterization of Plum bark necrosis-stem pitting associated virus (PBNSPaV) will be performed. Discarded and healthy almond trees on M2624 plum and peach rootstocks will be compared for tree performance, nut set, nut quality and decline symptoms as a function of X. fastidiosa infection. 5. Structural and functional genomics and/or a proteomics approaches will be used to examine the biochemical and molecular mechanisms of pathogenesis. Analysis of the P. ramorum genome also will focus on gene identification, assignment of putative gene function, and comparative genomics studies using the currently sequenced genomes of Phytophthora spp. FORMERLY CRIS PROJECT #5306-22000-013-00D 3.Progress Report We continued our field, greenhouse and laboratory trials to identify sustainable control strategies for crown gall disease (CG) and phytophthora crown rot of walnut trees in both orchard and nursery conditions. We estimate the economic impact of these diseases to be $20M annual loss in CA alone. This does not include the significant nursery losses that also occur each year. We have focused our efforts in two areas. First,CG- and Phytophthora-resistant breeding material was identified in wild relatives of cultivated walnut, and a Phytophthora-resistant walnut hybrid rootstock was identified. A joint UC/ARS patent has been sought for the rootstock. The disease-resistant walnut relatives will be useful in breeding disease resistant rootstocks. Second, we are examining the ecology of the CG bacterium and determining how it infects the walnuts that are planted and used to generate the trees used in the industry. Our goal is to develop disease resistant rootstocks combined with sustainable disease management strategies for the walnut industry. We continued our characterization of causative agents associated with graft union disorders in almonds, stone fruits, and walnuts and replant disease in almonds and stone fruits. Sustainable management practices to minimize losses caused by these agents were tested. In several counties of California, blackline disease, caused by Cherry leafroll virus, continues to be a menace in the production of walnuts. Because of the demands by stake holders, we have initiated a project to examine the potential of genomics approaches to manage this disease. Last year, an outbreak of almond brown line disease occurred in a young California almond orchard on plum rootstock. Preliminary evidence pointed to involvement of a phytoplasma, a species of bacteria without cell wall and difficult to culture on artificial medium. We were able to detect a phytoplasma in one of the diseased trees last fall using molecular bioassays and, based on the sequence identity, identified it as Pear decline phytoplasma. We will soon provide UCD Foundation Plant Services suitable primers and sampling times for the routine detection of this phytoplasma in almond foundation germplasm. Sudden Oak death is caused by a fungus-like organism whose genome has recently been sequenced. Using a combination of comparative genomics, protein motif search and gene expression analysis, we are validating the identification of approx 16,000 genes. In addition we are using a functional genomic approach to predict gene function in about 1800 genes that were not previously identified. This work will facilitate our ability to clearly define the genes that mediate such important parameters as virulence, host range, and survival in both soil and leaf surfaces there by enhancing disease control and pathogen quarantine efforts. 4.Accomplishments 1. Identification of disease resistant walnut germplasm. Crown gall(CG) is an important disease in walnut and resistant germplasm is needed. ARS scientists in Davis, CA found that from continued greenhouse trials, durable crown gall resistance is present in Juglans microcarpa, J. ailantifolia, J. mandischurica and Pterocarya accessions. New bacterial inoculation procedures were developed that will facilitate “high throughput” screening for CG resistance. Approximately 20% of the rooted dormant cuttings from mother trees exhibiting CG resistance continue to exhibit CG resistance. Directed crosses were made generating interspecific hybrids between male and female trees which exhibit various degrees of CG resistance. The potential impact is the development of CG resistant stock that would save $20 million in annual losses. 2. The genetic diversity of A. tumefaciens. Agrobacterium tumefaciens is a soil-borne bacterium that causes crown gall disease on walnut. ARS scientists in Davis, CA continued efforts to define the genetic diversity of A. tumefaciens as a function of geography, host origin and most importantly, nursery origin. This continues to enhance our forensic ability to identify sources of inoculum which aids the industry as it moves towards the cultivation of A. tumefaciens free planting stock and reduction of post plant infections leading to crown gall formation. The potential impact is the development of CG resistant stock that would save $20 million in annual losses. 3. Sudden Oak Death. The majority of the genes sequenced in the fungal pathogen that causes sudden oak death do not have experimental evidence supporting their identification. To validate the gene models, and make the genome data more reliable and useful, ARS scientists at Davis, CA constructed and sequenced cDNA libraries from 3 life stages of the fungus which provided data to predict 75% of sequenced genes. However >20% of the genes contain incorrect sequence data and we detected c.a. 1800 genes which were not identified by gene prediction programs. The majority of gene models do not have functional annotations. We constructed gene expression microarrays for the fungus, and obtained baseline gene expression levels during the 3 life stages which allows us to associate specific genes with specific biological processes. 4. Use of new molecular probes to screen for virus resistance in walnut. In California, black line disease (caused by a virus) adversely impacts nut production and longevity of some orchards. ARS scientists at Davis, CA have incorporated a resistance gene from black walnut into English walnuts and developed virus resistant backcrosses for release in areas with high incidence of black line disease. A marker had been found associated with resistance. We developed and optimized a molecular assay, based on this marker, to identify virus resistant germplasm. In a collaboration with UCD, more than 700 saplings from different back crosses were screened and trees potentially resistant to the virus have been identified for field testing. 5. Identification of the causal agent associated with the almond brownline disease. Almond brown line disease was discovered in California in the Nineties as a graft union disorder in almonds grown on plum rootstock in orchards on marginal land. When the trees are infected by Peach yellow leafroll phytoplasma, a bacterium without cell walls, they develop a brown necrotic line at the graft union resulting in tree death. It has been difficult to prove the association of phytoplasma in infected almond trees because of non-availability of a suitable detection assay. ARS scientists in Davis, CA developed a molecular assay and successfully detected this phytoplasma in almond extracts. This assay can be used to monitor the trees in a commercial orchard impacted by almond brown line disease. 6.Technology Transfer None Review Publications Mcclean, A.E., Kluepfel, D.A. 2009. GENETIC LOCI INVOLVED IN RUBRIFACINE PRODUCTION IN THE WALNUT PATHOGEN BRENNARIA RUBRIFACIENS. Phytopathology. 99:145-151.