2009 Annual Report 1a.Objectives (from AD-416) Three broad objectives guide this project: Objective 1: Characterize ecology, biology, epidemiology, genetics and host interactions of domestic, exotic, newly emergent and re-emerging pathogens. Objective 2: Develop/refine rapid, sensitive reliable detection/sampling methods for pathogens. Objective 3: Develop or improve comprehensive integrated disease management strategies. 1b.Approach (from AD-416) The overall approach is to thoroughly characterize new exotic and emerging plant pathogens at multiple levels: epidemiologically epidemics will be followed and modeled by traditional and newer stochastic methods at the regional, and plantation levels, biologically the pathosystems will be characterized at the level of host-pathogen-vector interaction, as well as at the cellular, molecular and/or biochemical levels. New pathogens will be identified and characterized by molecular biological and traditional cultural methods. Recombinant DNA and genomics technologies will be applied to study host/pathogen interactions and to investigate virulence differences between strains of a pathogen. New CTV genotypes will be identified by cloning products obtained by PCR and degenerate primers and also by hybridization to a sequencing microarray. Primers for PCR diagnostics will be devised from novel CTV genotypes. An immunocapture-based PCR protocol will be developed for CTLV for assessment of genetic variability of CTLV populations from the US and from international locations. 3.Progress Report Progress was made on several diseases that plague citrus, vegetables and ornamentals. A second set of cDNA libraries was created to screen for molecular sequences of the uncharacterized virus-like pathogen causing the citrus disease citrus concave gum. Sequences are being screened for molecular signatures indicative of pathogens to develop probes for improved disease detection. Watermelon vine decline caused by Squash vein yellowing virus (SqVYV) has been responsible for losses in Florida over the past several growing seasons, and Cucurbit leaf crumple virus (CuLCrV) is now established in Florida. The progress of SqVYV, CuLCrV and whitefly density was monitored in field trials over the last 4 growing seasons. Analyses indicated that SqVYV was distributed randomly at low incidences, but became more aggregated as incidence increased. The degree of association between SqVYV and CuLCrV was typical of a random arrangement of the 2 viruses. Results indicate that the viruses are being introduced independently by whiteflies, although the whiteflies may be emigrating from the same source, with secondary spread being dominated by within-field populations of whiteflies. HLB, the most devastating citrus disease, is now widespread in Florida, and is an impending threat to the citrus industries in California and Texas. Due to the fastidious nature of the HLB bacterium, a high throughput detection technology is critically important for HLB research and disease management. Monoclonal antibodies against HLB pathogen and a series of primers and probes for qPCR and nested PCR were developed and evaluated for better detection of HLB. These technologies have been transferred to university and state regulatory laboratories for diagnostic and basic research purposes. The full genome sequence of Candidatus Liberibacter asiaticus has been completed. All sequences have been submitted to GenBank and shared with ARS, university, and other researchers to facilitate HLB research. Quarantines due to citrus canker have long restricted national and international markets. In 2007 through 2009 studies on the survival of citrus canker bacteria on fruit as a pathway for bacterial spread and establishment in new areas were conducted. The bacteria were found to decrease rapidly on fruit surfaces and bacterial lesions on fruit post harvest that are passed through the packinghouse were not found to be epidemiologically significant as a pathway for establishment of the disease in new areas. These findings were reviewed by USDA, APHIS and new fruit shipping rules are presently out for comment, which if adopted would open up both national and international markets for fruit originating from citrus canker infected areas. State and federal regulatory agencies needed methods to quickly survey the entire state for newly introduced exotic pathogens. In 2009 we expanded a previous stochastic statewide stratified sampling/survey program for HLB/canker/citrus variegated chlorosis/citrus leprosis virus, to include Black Spot; the survey is further adaptable if needed for future pest and pathogen. 4.Accomplishments 1. Citrus concave gum is a continuing production problem causing decline of sweet orange, mandarin, and tangelo trees. A second set of cDNA libraries were created to screen for molecular sequences of the uncharacterized virus-like pathogen causing the disease. Sequences are being screened for molecular signatures indicative of pathogens. New identified sequences will be used to develop molecular probes for improved disease detection. 2. Watermelon vine decline caused by Squash vein yellowing virus (SqVYV) has been responsible for losses in Florida over the past several growing seasons, and Cucurbit leaf crumple virus (CuLCrV) is now established in Florida. The progress of SqVYV, CuLCrV and whitefly density was monitored in designed field trials over the last 4 growing seasons. Analyses indicated that SqVYV was distributed randomly at low incidences, but became more aggregated as incidence increased. The degree of association between SqVYV and CuLCrV was typical of a random arrangement of the 2 viruses. Results indicate that the viruses are being introduced independently by whiteflies, although the whiteflies may be emigrating from the same source, with secondary spread being dominated by within-field populations of whiteflies. The research will provide critical information necessary to develop decision management strategies. 3. Developing high throughput detection technology for citrus huanglongbing (HLB). HLB, the most devastating citrus disease, is now widespread in Florida, and is an impending threat to the citrus industries in California and Texas. Due to the fastidious nature of the HLB bacterium, a high throughput detection technology is critically important for HLB research and disease management. Monoclonal antibodies against HLB pathogen and a series of primers and probes for qPCR and nested PCR were developed and evaluated for better detection of HLB. These technologies have been transferred to university and state regulatory laboratories for diagnostic and basic research purposes. 4. Full genome sequence of Candidatus Liberibacter asiaticus is completed. Only limited (less than 50 kb) of three genetic loci were available in Genebank in the past. Annotation of the 1.23Mb genome revealed 1186 open reading frames, of which 81.0% had functional assignment. All sequences have been submitted to GenBank and shared with ARS, university, and other researchers. Knowledge of the full genome will greatly facilitate HLB research and the development of new strategies for disease control of this devastating pathogen. 5. Packing house and field experiments on citrus canker leads to proposed new national regulations and expansion of markets for citrus fruit. Quarantines due to citrus canker have long restricted national and international markets. In 2007 through 2009 studies on the survival of citrus canker bacteria and on fruit as a pathway for bacterial spread and establishment in new areas were conducted. The bacteria were found to decrease rapidly on fruit surfaces and in bacterial lesions on the fruit post harvest that are passed through the packinghouse were not found to be epidemiological significant as a pathway for establishment of the disease in new areas. These findings were reviewed by USDA, APHIS and new fruit shipping rules are presently out for comment, which if adopted would open up both national and international markets for fruit originating from citrus canker infected areas. 6. Multi-pest statewide (MPS), citrus exotic pest sampling method. State and federal regulatory agencies needed methods to quickly survey the entire states for newly introduced exotic pathogens. In 2009 we expanded a previous stochastic statewide stratified sampling/survey program for HLB/canker/citrus variegated chlorosis/Citrus leprosis virus, to include Black Spot; the survey is further adaptable if needed for future pest and pathogens. The revised multi-pest sampling/survey program is being utilized immediately to determine the distribution of these diseases in commercial citrus throughout Florida and the MPS is adaptable to California and Texas statewide citrus industries. 6.Technology Transfer Number of Invention Disclosures Submitted 1 Review Publications Young, J., Allen, C., Alvarez, A., Coutinho, T., Denny, T., Fegan, M., Gillings, M., Gottwald, T.R., Graham, J., Janse, J., Leach, J., Lopez, M., Morris, C., Parkinson, N., Rodrigues Neto, J., Scortichini, M., Takikawa, Y., Upper, C. 2008. Plant Pathogenic Bacteria as Bioterror Weapons – a Real Threat? Phytopathology. 98:1060-1065. Novelli, V., Freitas-Astua, J., Segatti, N., Mineiro, J., Arthur, V., Bastaniel, M., Hilf, M.E., Gottwald, T.R., Machado, M. 2008. Effects of radiation (Cobalt-60) on the elimination of Brevipalpus phoenicis (Acari: Tenuipalpidae) Cardinum endosymbiont. Experimental and Applied Acarology. DOI 10.1007/s10493-008-9176-4. Gottwald, T.R., Graham, J., Bock, C., Bonn, G., Civerolo, E.L., Irey, M., Leite, R., Lopez, M., Mccollum, T.G., Parker, P., Ramallo, J., Riley, T., Schubert, T., Stein, B., Taylor, E.L. 2009. The epidemiological significance of post-packinghouse survival of Xanthomonas citri ssp. citri for dissemination of Asiatic citrus canker via infected fruit. Crop Protection. 29:508-524. Duan, Y., Sun, X., Zhou, L., Gabriel, D., Benyon, L.S., Gottwald, T.R. 2008. Bacterial brown leaf spot of citrus, a new disease caused by Burkholderia andropogonis. Plant Disease. 93:607-614. Adkins, S.T., Polston, J.E., Turechek, W. 2008. Cucurbit leaf crumple virus Identified in Common Bean in Florida. Plant Disease. 93:320. Li, W., Lewandowski, D.J., Hilf, M.E., Adkins, S.T. 2009. Identification of domains of the Tomato spotted wilt virus NSm protein involved in tubule formation, movement and symptomatology. Virology. 390:110-121. Gowda, S., Tatineni, S., Folimonova, S.V., Hilf, M.E., Dwason, W.O. 2009. Accumulation of a 5’ proximal subgenomic RNA of Citrus tristeza virus is correlated with encapsidation by the minor coat protein. Virology 389, 122-131. Tatineni, S., Afunian, M.R., Gowda, S., Hilf, M.E., Bar-Joseph, M., Dawson, W.O. 2009. Characterization of the 5’- and 3’-terminal subgenomic RNAs produced by a capillovirus: evidence for a CP subgenomic RNA. Virology 385 (2009) 521–528. Tatineni, S., Afunian, M.R., Hilf, M.E., Gowda, S., Dawson, W.O., Garnsey, S.M. 2009. Molecular Characterization of Citrus tatter leaf virus Historically Associated with Meyer Lemon Trees: Complete Genome Sequence and Development of Biologically Active In Vitro Transcripts. Phytopathology Volume 99, Pages 423-431. Turechek, W., Peres, N. 2009. Hot Water Treatment to Reduce Angular Leaf Spot of Strawberry, Caused by Xanthomonas fragariae, in Nursery Production. Plant Disease. 93:299-308. Pethybridge, S., Gent, D.H., Esker, P., Turechek, W., Hay, F., Nutter, F. 2009. Site-specific risk factors for ray blight in Tasmanian pyrethrum fields. Plant Disease. 93:299-308. Bock, C., Parker, P., Cook, A., Riley, T., Gottwald, T.R. 2009. Comparison of Assessment of Citrus Canker Foliar Symptoms by Experienced and Inexperienced Raters. Plant Disease. 93:412-424. Duan, Y., Zhou, L., Hall, D.G., Li, W., Doddapaneni, H., Lin, H., Liu, L., Gabriel, D., Vahling, C.M., Williams, K., Dickerman, A., Sun, Y., Gottwald, T.R. 2009. Complete Genome sequence of citrus huanglongbing bacterium, ‘Candidatus Liberibacter asiaticus’ obtained through metagenomics. Molecular Plant-Microbe Interactions. 22(8):1011-1020. Cook, A., Gibson, G., Gottwald, T.R., Gilligan, C. 2008. Constructing the effect of alternative intervention strategies on historic epidemics. Journal of the Royal Society Interface, 5:1203-1213. doi:10.1098/rsif.2008.0030 Kousik, C.S., Adkins, S.T., Turechek, W., Roberts, P.D. 2009. Sources of Resistance in U.S. Plant Introductions to Watermelon Vine Decline Caused of Squash Vein Yellowing Virus. HortScience. 44:256-262. Overholt, W., Markle, L., Rosskopf, E.N., Manrique, V., Albano, J.P., Cave, E., Adkins, S.T. 2009. The interactions of Tropical soda apple mosaic tobamovirus and Gratiana boliviana (Coleoptera: Chrysomelidae), an introduced biological control agent of tropical soda apple (Solanum viarum). Biological Control. 48:294-300.