2012 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.
The Probe selection for a citrus pathogen array was completed, and probe validation was done on a limited basis; however, actual array construction is delayed by financial considerations. Broad range potyvirus primers were assembled from the literature and sequence alignments. Only one of these primer sets is fully efficient at amplifying all known potyviruses. This will make the TIGER analysis infeasible, so other routes of broad range potyvirus detection are being explored (arrays and sequencing). In order to salvage the objective, the available broad range primers were used to characterize a relatively unknown potyvirus from tobacco. Soybean dwarf virus (SbDV), a significant pathogen of soybeans in Japan, has been identified in multiple locations in the Eastern and Midwestern U.S. It was determined that several isolates of U.S. SbDV were transmissible by Aphis glycines, the soybean aphid. The effects of changing hosts on the Plum pox virus genome were monitored through six passages in black cherry and the Soybean dwarf virus genome through six passages in clover, soybean and peas. Genomic sequences were compared at the initial passage and after six passages. SbDV populations in peas incur low levels of mutations compared to SbDV populations in soybeans. The mutations that occur in SbDV populations in soybean render the virus population non-transmissible, suggesting a trade-off between replication and transmission selection pressures. Five (5) mutations have been identified in the readthrough protein that is associated with A. glycines transmission. The reversion rate of these mutations was determined for populations that were back inoculated onto the original host, clover. SbDV populations were assessed for diversity and penetrance of host and vector adaptive mutations using cloning and next generation sequencing. Several non-citrus Rutaceous species were tested as potential hosts of the HLB organism. Dodder, (Cuscuta sp.), was allowed to attach to infected citrus and then become attached to potatoes, tomatoes, Vinca, Murraya sp., sweet potato, Nicotiana tabacum, N. benthamiana, N. clevelandii, and radish. Subsequent RT-PCR assays indicated the bacterium was transferred to each new host, some with symptoms. Utilizing specific and generic primers considerable biodiversity was found in Liberibacter isolates from Florida and foreign countries with evidence of mixed Liberibacter populations in many isolates.
Soybean dwarf virus (SbDV), which significantly affects soybean crops in Japan and other Asian countries, has been identified in multiple locations within the U.S. The outbreaks of SbDV in the U.S. have always been limited, perhaps because the primary aphid colonizing soybean (Aphis glycines) is reported to be a poor vector of SbDV. An experimental analysis conducted by ARS researchers at Frederick, Maryland demonstrated that specific mutations must occur in U.S. isolates to enable soybean outbreaks, and these mutations tend to prevent effective transmission. This work demonstrates that SbDV outbreaks in the U.S. would require adaptation of the virus to the vector, and that risks associated with SbDV for U.S. soybean production are limited.