Location: Subtropical Plant Pathology Research2010 Annual Report
1a. Objectives (from AD-416)
1. Characterize ecology, biology, epidemiology, genetics and host interactions of domestic, exotic, newly emergent and re-emerging pathogens. 2. Develop/refine rapid, sensitive reliable detection/sampling methods for pathogens. 3. Develop or improve comprehensive integrated disease management strategies. a. Assess the impact of a wide array of control/mitigation/therapeutic strategies on Huanglongbing (HLB) and the Asian citrus psyllid epidemiology to evalute optiumu means of disease control.
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
Watermelon vine decline, caused by Squash vein yellowing virus (SqVYV), has been responsible for losses in Florida over the past several growing seasons. Cucurbit leaf crumple virus (CuLCrV) and Cucurbit yellow stunting disorder virus (CYSDV) are recent introductions. Improved detection methods for SqVYV, CuLCrV and CYSDV have supported within-plant distribution analyses, facilitated identification of a new weed host, and a field study to characterize spatial and temporal patterns of disease, quantify viral associations, and determine how environmental factors affect SqVYV epidemic development. Not all viruses appeared each growing season, and certain environmental parameters may be useful in predicting the occurrence of SqVYV. The level of association between SqVYV and CuLCrV was not greater than what would be expected from random associations, indicating that disease may be introduced by separate whiteflies, although the whiteflies may be emigrating from the same source. CYSDV was discovered only in the last planting, but like CuLCrV the severity of disease appeared to be limited by rapidly advancing SqVYV. The data gathered from this study along with transmission parameters are being used to predict epidemic development and test management strategies. Continuing disinfectant trials have identified several effective sanitation protocols for eliminating tobamoviruses from cutting tools during propagation and production of ornamentals. Huanglongbing (HLB), the most devastating citrus disease, is widespread in Florida, and is an impending threat to the citrus in California and Texas. Monoclonal antibodies against the HLB pathogen and reagents for qPCR and nested PCR were developed and have been transferred to university and state regulatory laboratories for diagnostic and basic research purposes. Genome sequencing of Ca. L. solanacearum (Las), the causal agent of potato zebra chip, has been initiated and a complete genome of 1.25 Mb has just been obtained. Functional genomics of Las, such as characterization of ATP translocase, putative Zinc uptake operons has been carried out. The genetic diversity of the Las bacterium has been revealed, which is associated with phenotype variations of the HLB diseases. Seed transmission of Las is being investigated using several different approaches. A stochastic statewide stratified survey program for HLB/canker/citrus variegated chlorosis/citrus leprosis virus/black spot continued to be used in 2010 and was further adapted to determine the distribution of HLB and its vector the Asian citrus psyllid to target areawide control efforts. The method will be used for a delimiting survey for black spot in Florida to determine how far the disease has spread. A new predictive model has been developed based on travel and US census information to predict introductions of plant pathogens and pests and has possible adaptation to animal and human disease prediction as well. Rapid detection methods for HLB were developed based on a spectral analyses of chemical changes in plant tissues that could aid in both early detection and the processing of large numbers of samples.
1. 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) and Cucurbit yellow stunting disorder virus (CYSDV) are now established in Florida. The progress of SqVYV, CuLCrV, CYSDV and whitefly density was monitored using newly developed detection methods in designed field trials over the last six 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 two 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.
2. Full genome sequence of Candidatus Liberibacter asiaticus completed. Only limited (less than 50 kb) of three genetic loci were available in Genebank previously. 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.
3. New exotic bacterial disease of citrus identified and characterized. The causal agent of bacterial brown leaf spot (BBLS) of citrus in Florida was identified and characterized. The publication from the research on BBLS addressed the confusion between citrus canker and the BBLS disease, and provided tools to differentiate these two pathogens for growers and regulatory agencies
4. A novel chemical screen system for control of citrus Huanglongbing (HLB) developed. Because of the fastidious nature of the HLB bacterium and the difficulty of propagating and maintaining a large number of HLB-infected plants, it is extremely difficult to screen chemicals for control of HLB using naturally infected citrus plants. A novel system to screen potential chemicals for control of citrus HLB has been developed using Las-infected periwinkle cuttings and by application of uniform design that significantly reduces the number of trials. In this system, the optimal regeneration conditions for Las-infected periwinkle cuttings were elucidated. By using this system, a cocktail of two chemicals has proven to be effective for elimination of HLB pathogens.
5. High throughput detection technology for citrus huanglongbing (HLB) developed. 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.
6. A stochastic statewide stratified sampling/survey program known as the Multi Pest Survey (MPS) for HLB/canker/citrus variegated chlorosis/Citrus leprosis virus/Black Spot continued to be used in 2010 and was further adapted to determine the distribution of HLB and its vector the Asian citrus psyllid to target areawide control efforts. The method continues to be adapted for new purposes and will be used for a delimiting survey for black spot in Florida to determine how far the disease has spread. A new predictive model has been developed based on travel and US census information to predict introductions of plant pathogens and pests and has possible adaptation to animal and human disease prediction as well. New very rapid detection methods for HLB have been developed and published based on a spectral analyses of chemical changes in plant tissue that could potential aid in both early detection and the processing of large number of suspect samples.
Hall, D.G., Gottwald, T.R., Nguyen, N.C., Ichinose, K., Le, Q.D., Beattie, G.A.C., Stover, E. 2008. Greenhouse investigations on the effect of guava on infestations of Asian citrus psyllid in grapefruit. Proceedings Florida State Horticultural Society. 121:104-109