2009 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 Prunus diagnostic array has been developed and tested against 19 diseases of Prunus. The array contains probes that successfully detect all pathogens tested. The array has been tested with several artificial mixtures of Prunus pathogens. 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. Broad range potyvirus primers were assembled from the literature and from sequence alignments. Only one of these primer sets is fully efficient at amplifying all known potyviruses, and few other primer sets are capable of amplifying a significant number of potyviruses. This will make the TIGER analysis infeasible. 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. Work was initiated to establish a technique for labeling virus particles with fluorescent tags (Q-dot fluorescent particle from Invitrogen) that can be monitored in real time as aphids feed. Labeling purified soybean dwarf virus particles with Q-dots was not successful because the labeling procedure degraded the virus particles. Transovarial transmission of the causal agent of Huanglongbing (HLB) disease in citrus through the eggs of infectious psyllids was investigated using different approaches. Following a 3-week acquisition feeding on infected sweet orange, more than 100 female psyllids were transferred to M. paniculata seedlings and allowed to lay eggs. Females were removed when oviposition was evident. Egg masses per female and individual females were analyzed by PCR for the presence of HLB. In a second set of experiments, infected and healthy psyllids were transferred to M. koenigii plants. Adult psyllids were removed after oviposition and analyzed for the presence of the HLB organism. Second generation adult psyllids were moved to new M. koenigii plants, observed for oviposition and then removed. This was repeated through 4 generations after which psyllids were transferred to healthy Madam Vinous plants. 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. Several non-citrus Rutaceous species were tested as potential hosts of the HLB organism. Research initiated on proof of pathogenicity of a suspected causal bacterium of HLB using psyllids as vectors.
Huanglongbing (HLB), the most economically devastating disease of citrus, is transmitted by citrus psyllids: In response to stakeholder requests, a study was conducted to determine the susceptibility of Murraya paniculata (Jasmine orange), M. exotica (Jessamine orange), and M. (Bergera) koenigii (Curry leaf) to the Asian species of the HLB bacterium when transmitted by the Asian citrus psyllid. None of the test plants showed diagnostic symptoms of disease, while 34/36 Jasmine orange, 21/23 Jessamine orange and 0/16 Curry leaf plants tested positive for the HLB bacterium. The results indicate that Murraya species could serve as potential sources of the HLB bacteria in addition to hosts for rearing psyllid populations.