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United States Department of Agriculture

Agricultural Research Service

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Location: Foreign Disease-weed Science Research

2010 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 successfully tested against 21 diseases of Prunus, as well as several artificial mixtures of Prunus pathogens. The number of optimal probes has been reduced significantly, and continues to be refined. A successful macroarray format has been developed for plum pox virus. Probe selection for citrus pathogens is continuing, actual array construction is delayed by financial considerations. Broad range potyvirus primers were assembled from the literature and from sequence alignments. However, only one of these primer sets is fully efficient at amplifying all known potyviruses. This will make PCR-mass spectrometry infeasible, so other routes of broad range potyvirus detection are being explored (arrays and sequencing). 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 tradeoff between replication and transmission selection pressures. Five mutations have been identified in the readthrough protein associated with A. glycines transmission. Labeling purified soybean dwarf virus particles with Q-dots was not successful because the labeling procedure degraded the virus particles. Alternative approaches are being investigated in collaboration with Stuart Grey. Analyses of hundred of clones from mixed infections of endemic SbDV isolates indicated a predominance of the dwarfing genotype. 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. Proof of pathogenicity of ‘cultivated’ bacteria from HLB infected plants was investigated using psyllids as vectors. Bacteria were extracted from infected citrus, dodder, and citrus psyllids, grown on selected media to obtain ‘clean’ cultures, assayed by RT-PCR with generic and specific primers. Culture extracts were inoculated into test plants by psyllids which had obtained the bacteria by probing stretched parafilm membrane feeding sachets or by micro-injection of the extracts into psyllid abdomens. Psyllids were allowed to colonize orange seedlings for 14 days, removed and assayed for the presence of Las by RT-PCR, and plants were allowed to grow for at least 6 months. Positive infections were recorded from both membrane fed and micro-injected psyllid inoculations. 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.

4. Accomplishments
1. The use of insect vectors to provide indirect proof that the Candidatus Liberibacter asiaticus bacterium causes the disease Huanglongbing of citrus. The efforts to combat citrus greening (HLB) have long been slowed by the lack of confirmation of the causal agent. Liberibacters have been identified as the likely cause, but cultured liberibacters rarely generated normal infections when re-injected into healthy trees, on the rare occasions when infections occurred. Experiments were conducted by ARS scientists at the Foreign Disease-Weed Science Research laboratory at Ft. Detrick, Maryland to bridge this knowledge gap, using the psyllid vector as an intermediate step. Healthy Asian citrus psyllids were either membrane fed on ‘Candidatus Liberibacter asiaticus’ (Las) cultures or directly injected with the cultures using a microinjector. In both cases the psyllids successfully transmitted the pathogen, reinforcing the HLB causal agent status of Las. This demonstration represents a critical step in confirming the causal agent of HLB to focus research and control efforts.

Review Publications
Damsteegt, V.D., Postnikova, E.N., Stone, A.L., Kuhlmann, M., Wilson, C., Sechler, A.J., Schaad, N.W., Brlansky, R.H., Schneider, W.L. 2010. The relevance of Murraya paniculata and related species as potential hosts and inoculum reservoirs of Candidatus Liberibacter asiaticus, causal agent of Huanglongbing (HLB). Plant Disease. 94:528-533.

Last Modified: 2/23/2016
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