2008 Annual Report 1a.Objectives (from AD-416) Develop pathogen detection arrays in support of certification programs for Prunus and Citrus crops and provide innovative diagnostic systems for new and emerging plant pathogens; investigate molecular and biological factors in pathogen/host/vector systems that affect host adaptation, vector adaptation, and evolution of new pathogenic forms; and, investigate critical factors that influence developmental and circulative processes of vector transmission in new or emerging plant pathogenic diseases (pathogen ingestion and fate in persistent and non-persistent transmission systems) i.e., Huanglongbing and citrus psyllids, Soybean dwarf virus and soybean aphid, Plum pox virus and aphid vectors, Citrus tristeza virus and brown citrus aphid, and other foreign and emerging plant pathogens. Contribute to curation of microbial collections for all CRIS projects at the unit, perform physical audits of pathogens, monitor APHIS permit status, oversee and track regulations regarding transport and storage of APHIS Select Agents, and liaison with APHIS for containment inspections and certifications. PER PDRAM NAA2 FY07 Program Redirection for Plum Pox Research Adding this objective: Investigate virus adaptation to changing hosts using plum pox as a working model and develop a fluorescent viral labeling system for tracking viruses in aphids. 1b.Approach (from AD-416) Establish and maintain foreign (exotic) and emerging insect-transmitted plant pathogens under quarantine containment and determine factors involved in pathogen change and adaptation, mechanisms of transmission, and novel detection strategies. Specific approaches will include using microarray format to select optimal probes for multiple Prunus pathogen detection macroarrays and adaptation of TIGER diagnostics for the detection of potyviruses. Viral adaptation to host and vector will be studied experimentally using repeated passages of Plum pox virus and Soybean dwarf virus as model systems. Virus/vector interactions will be studied using fluorescently tagged virions of PPV and SbDV to study viral movement in aphids. The presence or absence of transovarial transmission of HLB by the citrus psyllid will be determined by following the developmental stages of hundreds of progeny of infective psyllids from egg to adult on non-HLB hosts using Real-time PCR and specific primers for HLB. The presence, pathway, and location of HLB in citrus psyllids will be monitored by real-time PCR on whole psyllids, dissected psyllid organs, and use of fluorescently tagged HLB bacteria. 3.Progress Report The Prunus diagnostic array has been developed and tested against 19 diseases of Prunus. The array contains probes that successfully detect all pathogens tested. The number of optimal probes has been reduced significantly, and continues to be refined. The pathogens for the citrus array have been selected, and sequences collected where available. Probe selection for citrus pathogens has begun. The effects of changing hosts on the Plum pox virus genome were monitored through six (6X) passages in black cherry and the Soybean dwarf virus genome through six passages in clover and soybean. Genomic sequences were compared at the initial passage and after six passages. A number of mutations have been identified. A real-time PCR assay was developed for both Soybean dwarf virus D and Y strains. Two endemic field isolates of Soybean dwarf virus collected in Maryland and Virginia were transmitted by the soybean aphid through 4 serial passages. Both isolates were cloned and sequenced when first transmitted and after 4 passages. Both isolates were determined to be initial mixtures of dwarfing and yellowing strains. Transovarial transmission of the causal agent of Huanglongbing (HLB) disease in citrus through the eggs of infectious citrus psyllids was investigated using two different approaches. Within each approach initial adult psyllids were tested for the presence of the HLB bacteria at the start. Egg masses, first and second instar nymphs, and subsequent adult psyllids were analyzed by PCR for the bacterium. Experiments were replicated at least two times. Location of the bacterium in the psyllid vector was studied by fixing individual healthy and infected psyllids in glutaraldehyde, staining with osmium tetroxide, and examining by transmission and scanning electron microscopy. Research accomplished under National Program 303, (Plant Diseases), Action Plan Program Component I, “Disease Diagnosis: Detection, Identification, and Characterization of Plant Pathogens”. 4.Accomplishments 1. Alternative hosts of Huanglongbing: Citrus psyllids (insects), recognized as the only vectors of Huanglongbing (HLB), the most economically devastating disease of citrus, use ornamental Murraya species as preferred food hosts. In controlled transmission experiments, the susceptibility of Murraya paniculata (Jasmine orange), M. exotica, and M. (Bergera) koenigii (curry leaf) to HLB infection was determined using infectious psyllids. All plants and psyllids were assayed by PCR before and after each transmission experiment to confirm the presence or absence of the HLB bacteria. Although Murraya plants did not show evidence of infection, inoculations from Murraya plants to sweet orange resulted in infected sweet orange, indicating that the Murraya plants were carrying the disease bacteria. Murraya plants in citrus production areas can thus serve as a host for the insect vector and a source of the HLB pathogen for infecting citrus trees. This research accomplished under National Program 303, Action Plan Program Component IV, Milestone Objective 3-B. 5.Significant Activities that Support Special Target Populations NONE 6.Technology Transfer Number of New Commercial Licenses Executed 3 Review Publications Wallis, C.M., Schneider, W.L., Stone, A.L., Sherman, D.J., Damsteegt, V.D., Gildow, F.E. 2007. Identification of a mutation in the plum pox potyvirus nib gene associated with adaptation to pea (pisum sativum). Journal of General Virology. 88:2839-2845. Pita, J., De Miranda, J., Schneider, W.L., Roossinck, M.J. 2007. Fidelity of a viral RNA polymerase in an intact host: template structure and host factors affect rates of deletion mutations. Journal of Virology. 81:9072-9077.