2008 Annual Report 1a.Objectives (from AD-416) The overall goals of this project are to reduce losses due to Xf-caused diseases during crop production and to develop effective, sustainable disease and insect vector management strategies by characterizing the host-pathogen-vector-environment interactions of these complex pathosystems. Specific objectives for the project are outlined below: Objective 1: Determine the epidemiology of exotic, emerging, re-emerging, and invasive diseases in California, including (but not necessarily limited to) Xf-caused diseases of horticultural, agronomic, and ornamental crops. Objective 2: Determine the nature and mechanism(s) of susceptibility/resistance to Xf infection and subsequent disease development in horticultural and agronomic crops, including (but not necessarily limited to) Vitis species and Prunus species. Objective 3: Develop effective, economical, environmentally sound strategies to manage exotic, emerging, re-emerging, and invasive diseases, including (but not necessarily limited to) xylella diseases. 1b.Approach (from AD-416) Determine the epidemiology of Xylellae diseases in California. Characterize the complex host-pathogen-insect vector-environment interactions of these pathosystems. Determine the biochemical, physiological, genetic and mechanistic bases of resistance to Xf infection and subsequent disease development in Vitis and selected Prunus species. Formerly 5302-22000-007-00D. 3.Progress Report Primary focus of the research is on diseases of horticultural and landscape crops caused by Xylella fastidiosa with emphasis on Pierce’s disease (PD) of grapes, almond leaf scorch disease (ALSD), and sharpshooter insects that vector the pathogen, including the glassy-winged sharpshooter (GWSS), green sharpshooter, and smoketree sharpshooter. This project contributes to National Program 303 (Plant Diseases); Component 2 (Biology, Ecology, Epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors); Problem Statements 2A (Pathogen Biology, Virulence Determinants, and Genetics of the Pathogen, 2B (Plant-Microbe Interactions), and 2C (Population Dynamics, Spread, and Epidemiology of Pathogens). Progress has been made in the areas of X. fastidiosa-plant interactions, development of PD resistance in grape, pathogen genetic diversity/genomics, mechanisms of vector transmission, and modeling of disease and vector populations. X. fastidiosa multiplication, aggregation and biofilm formation were inhibited in xylem sap from PD-resistant grapevines, indicating that xylem sap composition affects disease progression. Furthermore, specific components of plant cell walls had positive or negative effects on X. fastidiosa growth. Mobile genetic elements of X. fastidiosa, important for gene transfer among bacteria, were characterized. Mulberry-infecting strains of X. fastidiosa were shown to harbor a 25 kilobase pair plasmid, encoding DNA transfer genes, that may be modified as a gene vector system for X. fastidiosa. Viruses of X. fastidiosa, potentially useful as control agents or as DNA transfer agents, were identified by particle form. Gene families derived from wild-species crosses with table grapes generated PD-resistant hybrids that are being selected for resistance and agronomic characteristics. Geographic Information Systems (GIS) maps plotting the occurrence of citrus (a GWSS reservoir host) and grape (susceptible to PD) were analyzed to identify grape growing regions at risk for new epidemics of PD should GWSS abatement efforts be abandoned. Deficit irrigation practices on citrus have been evaluated, indicating that moderate levels of water stress reduce GWSS reproduction and feeding without significantly affecting citrus production. Critical components of GWSS saliva that may facilitate X. fastidiosa transmission to plants were identified. Limited research also has been conducted on Huanglongbing (HLB) disease of citrus. As HLB associated bacteria, classified as “Candidatus Liberibacter” spp. are non-culturable, listed as select agents, and do not occur in California, research has been limited to DNA based genomic studies and development of PCR detection assays. Genome-walking approaches have resulted in cloning and sequencing of “Ca. Liberibacter” spp. genome fragments. PCR assays for the 16S rDNA region have been developed that amplify DNA of all three “Ca. Liberibacter” spp. associated with HLB disease. 4.Accomplishments 1. Identification of California grape growing regions at risk for Pierce’s disease epidemics. Introduction of the glassy-winged sharpshooter (GWSS) vector of Xylella fastidiosa resulted in destructive epidemics of Pierce’s disease (PD) at a magnitude not previously experienced in California. In an effort to describe why and where previous epidemics of PD occurred and to predict where future epidemics are likely to occur, Geographic Information Systems maps on the distribution of grape (PD-susceptible) and citrus (GWSS reservoir host) in California and historical insecticide application databases were analyzed. These analyses identified which counties in California have the greatest overlap of citrus and grape and which counties applied the least amount of insecticides to citrus. Based on these results, Riverside, Kern, and Tulare counties (where previous outbreaks occurred) have the highest level of grape-citrus interface and appear to be at greatest risk for future epidemics of PD. These findings will facilitate efforts to control Pierce’s disease by identification of areas where GWSS vector populations would reach high levels in close proximity to vineyards should the current Area-Wide program for GWSS control be abandoned. This project contributes to National Program 303 (Plant Diseases); Component 2 (Biology, Ecology, Epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors); Problem Statement 2C (Population Dynamics, Spread, and Epidemiology of Pathogens). 2. Xylem sap source and cell wall constituents effect Xylella fastidiosa growth. Differential response of grapevines to infection with Xylella fastidiosa is poorly understood, but key to understanding processes that lead to either disease susceptibility or resistance. Xylella fastidiosa growth, aggregation, and biofilm formation was measured in media amended with different cell wall constituents or with xylem sap from Pierce’s Disease resistant or susceptible grapevines. Xylem sap from resistant plants resulted in reduced growth, cell aggregation and biofilm formation compared to xylem sap from susceptible plants. Responses of X. fastidiosa to media amended with cell wall compounds was variable. The results indicate that xylem sap composition may play a role in plant defense to X. fastidiosa and that chemical composition of xylem cell walls may be critical with respect to nutrients available to X. fastidiosa during infection and disease progression in plants. These results will facilitate efforts to control Pierce’s disease through plant breeding by providing an assay to score plants for xylem sap antagonistic to Xylella fastidosa growth. This project contributes to National Program 303 (Plant Diseases); Component 2 (Biology, Ecology, Epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors); Problem Statement 2A (Pathogen Biology, Virulence Determinants, and Genetics of the pathogen). 3. Continuous deficit irrigation of citrus reduces glassy-winged sharpshooter (GWSS) feeding and reproduction. Unabated, GWSS populations reach high levels in citrus that subsequently move into vineyards where they may transmit Xylella fastidiosa to grape. Research was conducted, by ARS scientists in the Crop Diseases, Pests and Genetics Research Unit in Parlier, CA, to assess effects of irrigation schedules on feeding preference and reproduction of GWSS on citrus. Moderate levels of continuous deficit irrigation resulted in reduced feeding, lower population levels, and lowered reproduction. Thus, irrigation management may be useful in reducing GWSS populations in citrus when used in conjunction with other cultural/biological control strategies in an integrated pest management program targeting GWSS. This project contributes to National Program 303 (Plant Diseases); Component 2 (Biology, Ecology, Epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors); Problem Statement 2C (Population Dynamics, Spread, and Epidemiology of Pathogens). 4. A DNA-based assay to discriminate among G-genotype strains of Xylella fastidiosa. G-genotype strains of Xylella fastidiosa are able to cause both Pierce’s disease (PD) of grapevines and almond leaf scorch disease (ALSD). However, the ability to distinguish separate lineages of G-genotype strains is limited. Hyper-variation, due to differences in simple sequence repeats, within the psbB gene encoding a protease was identified, by ARS scientists in the Crop Diseases, Pests and Genetics Research Unit in Parlier, CA, and used to distinguish among G-genotype isolates. Pending verification of stability in simple sequence repeat differences, the assay may be useful for tracking individual X. fastidiosa lineages during epidemics of PD or ALSD. This project contributes to National Program 303 (Plant Diseases); Component 2 (Biology, Ecology, Epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors); Problem Statement 2A (Pathogen Biology, Virulence Determinants, and Genetics of the Pathogen). 5. Characterization of a novel plasmid in Xylella fastidiosa. Xylella fastidiosa (the causal agent of Pierce’s disease and other vascular occlusion diseases of perennial plants) exists in nature as distinct strains able to exchange genetic material resident on extrachromosomal plasmids. Mulberry-infecting strains of X. fastidiosa were found, by ARS scientists in the Crop Diseases, Pests and Genetics Research Unit in Parlier, CA, to harbor a 25 kilobase pair (kpb) plasmid encoding Type IV secretion system genes known to facilitate DNA transfer among bacteria. Sequence analysis indicated that the 25 kbp plasmid shared significant sequence identity to the corresponding Type IV secretion system encoded by a 31 kbp plasmid resident in an unrelated bacterial species (Verminephrobacter eiseniae) that is a symbiont of earthworms. These results suggest that X. fastidosa may acquire DNA from sources much more divergent than previously recognized. In addition, portions of the 25 kbp plasmid may be useful as components of a shuttle vector for delivery of foreign DNA to X. fastidiosa. This project contributes to National Program 303 (Plant Diseases); Component 2 (Biology, Ecology, Epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors); Problem Statement 2A (Pathogen Biology, Virulence Determinants, and Genetics of the Pathogen). 6. High-throughput detection of X. fastidiosa from plant tissues. Xylella fastidiosa is responsible for Pierce’s disease of grapevines and almond leaf scorch disease. Although the pathogen may be cultured from diseased plants, isolation of the pathogen from field samples is difficult and not suitable for evaluation of large sample sizes needed for epidemiological studies. A rapid and simple procedure for X. fastidiosa DNA extraction from diseased almond tissue was developed by ARS scientists in the Crop Diseases, Pests and Genetics Research Unit in Parlier, CA, and found to be both reliable and suitable for high throughput PCR analysis. The technique will facilitate epidemiological studies in which X. fastidiosa infection status for a large number of samples are required to track pathogen spread and disease progression in the field. This project contributes to National Program 303 (Plant Diseases); Component 2 (Biology, Ecology, Epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors); Problem Statement 2C (Population Dynamics, Spread, and Epidemiology of Pathogens). 7. Characterization of ribosomal RNA genes from “Candidatus Liberibacter” associated with citrus Huanglongbing (HLB) disease. HLB disease of citrus represents a serious threat to the US citrus industry. The disease is associated with an unculturable bacterium, “Candidatus Liberibacter” spp. Sequencing of ribosomal RNA genes of the bacterium facilitated development by ARS scientists in the Crop Diseases, Pests and Genetics Research Unit in Parlier, CA, of an improved PCR assay for detection of the bacterium. The assay will be useful in studies designed to evaluate the association of the bacterium with HLB disease. This project contributes to National Program 303 (Plant Diseases); Component 2 (Biology, Ecology, Epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors); Problem Statement 2A (Pathogen Biology, Virulence Determinants, and Genetics of the Pathogen). 8. Immunocytochemical protocols developed for detection of Xylella fastidiosa and glassy-winged sharpshooter (GWSS) watery saliva in grape tissue. The mechanism by which the Pierce’s disease bacterium, Xylella fastidiosa, is inoculated (injected into plants) by sharpshooters is presently unknown. Evidence to date supports that bacteria are carried into the plant by injected watery saliva, which is invisible in classically stained plant tissues. Immunocytochemical protocols were developed by ARS scientists in the Crop Diseases, Pests and Genetics Research Unit in Parlier, CA, for confocal microscopy to separately localize in grape tissue either X. fastidiosa cells or the primary enzymatic constituent of GWSS watery saliva, a type of cellulase called beta 1,4-glucanase. These new methods will enable testing of the role of salivation in the inoculation mechanism, and may result in potential targets for disruption of pathogen transmission to grapes. This project contributes to National Program 303 (Plant Diseases); Component 2 (Biology, Ecology, Epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors); Problem Statement 2B (Plant-Microbe-Vector Interactions). 9. Morphological evidence for viruses (phages) in Xylella fastidiosa. As with many bacteria, the genome of Xylella fastidiosa has integrated prophage DNA sequences but it is unknown whether these sequences are nonfunctional remnants or competent to produce active phage infections able to destroy bacterial cells. X. fastidiosa was cultured under stress, by ARS scientists in the Crop Diseases, Pests and Genetics Research Unit in Parlier, CA, in an attempt to induce active phage reproduction. Senescent cultures of X. fastidiosa produced low levels of virus-like particles which, based on transmission electron microscopy, resemble particles of phages known to occur in other bacteria. X. fastidiosa phages hold promise as both a means of disease control and as tools for introduction of foreign DNA to X. fastidosa. This project contributes to National Program 303 (Plant Diseases); Component 2 (Biology, Ecology, Epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors); Problem Statement 2A (Pathogen Biology, Virulence Determinants, and Genetics of the Pathogen). 5.Significant Activities that Support Special Target Populations 2008 PWA Summer Intern Program: Mentored one female Hispanic student on project concerning molecular characterization of phages from Xylella fastidiosa. 6.Technology Transfer Number of New Commercial Licenses Executed 12 Number of Web Sites Managed 2 Review Publications Chen, J., Civerolo, E.L., Tubajika, K.M., Livingston, S., Higbee, B. 2008. Hyper-variation of Tandem Repeats at the PD0218 (pspB) locus of Xylella fastidiosa Almond Leaf Scorch and Grape Pierce¿s Disease Strains. Applied and Environmental Microbiology. 74:3652-3657. Deng, X., Chen, J., Luo, Z., Feng, Z., Li, H., Civerolo, E.L. 2008. First report of graft-transmission and PCR detection of Candidatus Liberibacter asiaticus from Atalantia buxifolia in Guangdong, China. Plant Disease. 92: 314. Doddapaneni,, H., Lin, H., Walker, M., Yao, J., Civerolo, E.L. 2008. vitisExpDB: A database resource for grape functional genomics. Biomed Central (BMC) Plant Biology.8:23doi:10.1186/1471-2229-8-23 Nadel, H., Seligmann, R., Johnson, M.W., Hagler, J.R., Stenger, D.C., Groves, R.L. 2008. Effects of citrus and avocado irrigation and nitrogen-form soil amendment on host selection by adult Homalodisca vitripennis. Environmental Entomology. 37(3):787-795. Gassmann, A.J., Stock, P.S., Sisterson, M.S., Carriere, Y., Tabashnik, B.E. 2008. Synergistic interactions between entomopathogenic nematodes and Bt crops: integrating biological control and resistance management. Journal of Applied Ecology 45:957-966. Chen, J., Civerolo, E.L. 2008. Morphological evidence for phages of Xylella fastidiosa. Virology Journal. 5:75. Habibi, J., Coudron, T.A., Backus, E.A., Brandt, S.L., Wagner, R.M., Wright, M.K., Huesing, J.E. 2008. Morphology and histology of the alimentary canal of Lygus hesperus Knight (Heteroptera: Cimicomoropha: Miridae). Annals of the Entomological Society of America. 101(1):159-171. Sisterson, M.S., Chen, J., Civerolo, E.L., Ledbetter, C.A., Groves, R.L. 2008. Effects of Almond Leaf Scorch Disease on Almond Yield and Implications for Management. Plant Disease 92:409-414. Sisterson, M.S. 2008. Effects of Insect Preference for Healthy or Infected Plants on Spread of an Inspect Vectored Plant Pathogen: Insights from a Model. Journal of Economic Entomology 101: 1-8. Chen, J., Groves, R.L., Zheng, Y., Civerolo, E.L., Viveros, M., Freeman, M. 2007. Colony morphology of almond leaf scorch strains of xylella fastidiosa and its epidemiological application. Canadian Journal of Plant Pathology. 29:225-231. Chen, J., Livingston, S., Groves, R.L., Civerolo, E.L. 2008. High throughput PCR detection of Xylella fastidiosa directly from almond tissues. Journal of Microbiological Methods. 73:57-61. Deng, X., Chen, J., Feng, Z., Shan, Z., Guo, H., Li, H., Civerolo, E.L. 2007. Identifiction and Characterization of Huanglongbing Bacterium in Pummelo [citrus grandis (L.) Osbeck] from Multiple Locations in Guangdong, P.R. China. Plant Disease. 92:513-518. Deng, X., Chen, J., Shan, Z., Zhou, G., Li, H., Civerolo, E.L. Identification of Candidatus Liberibacter asiaticus from Foshou (Citrus medica) in China. Plant Pathology. New Disease Report, 15:Article July 2007. Available: http://www.bspp.org.uk/ndr/july2007/2007-66.asp Lin, H., Doddapaneni, H., Takahashi, Y., Walker, A. 2007. Comparative analysis of est's involved in grape responses to Xylella fastidiosa infection. Biomed Central (BMC) Plant Biology 2007, 7:8, doi:10.1186/1471-2229-7-8 Fritschi, F., Cabrera-La Rosa, J.C., Groves, R.L., Lin, H., Johnson, M.W. 2007. Behavioral Responses of Homalodisca vitripennis (Hemiptera: Auchenorrhyncha: Cicadellidae) on Four Vitis Genotypes. Environmental Entomology. Vol. 36, Issue 4, pp. 926-936. Sisterson, M.S., Carriere, Y., Dennehy, T.J., Tabashnik, B.E. 2007. Non-target effects of bt crops: implications of source-sink population dynamics. Environmental Entomology. 36:121-127. Sisterson, M.S., Biggs, R.W., Manhardt, N.M., Carriere, Y., Dennehy, T.J., Tabashnik, B.E. 2007. Effects of Transgenic Bt Cotton on Insecticide Use and Predatory Insect Abundance. Entomologia Experimentalis et Applicata. 124:305-311.