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United States Department of Agriculture

Agricultural Research Service


Location: Subtropical Plant Pathology Research

2011 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
Progress was made on all three objectives and their subobjectives. Under objective 1, transmission of Squash vein yellowing virus (SqVYV) and Groundnut ringspot virus (GRSV) is continuing to be elucidated. Host range and genetics of GRSV were investigated. Progress on culture of ‘Candidatus Liberibacter asiaticus’ (Las) in vitro has been made. Genetic diversity of Las has been demonstrated by several molecular markers. New plots to test the interaction of ACC, leafminer, and wind breaks were established in Brazil. Numerous studies were conducted on the dispersal characteristics of ACC via wind and rain to characterize the dispersal plume of inoculum and its disease causing potential downwind. Under objective 2, CTLV strain characterization is complete. Genetic characterization of CTV strains associated with infection of Persian lime is nearing completion. We are evaluating the sensitivity and specificity of a hybridization assay to detect SqVYV and comparing it to RT- polymerase chain reaction (PCR). Under objective 3, we have developed and tested a web-based and mobile technology platform that functions as a disease and insect scouting and management tool. The application will allow growers and scouts to enter global positioning system (GPS)-labeled disease, insect, and production information directly into their mobile device (e.g. smartphone) where it will be processed and used to develop field-specific, pest management recommendations. A citrus canker stochastic model is in the final stages of development and a similar model for huanglongbing (HLB) moved forward, as well as a user-friendly front end to the model which will make it plausible to regulatory agencies and commodity groups. Tests using guava as an intercrop between citrus trees as a mitigation strategy for HLB were somewhat successful in nursery conditions but unsuccessful in citrus groves due to a series of freezes, which severely damaged the guava and demonstrated its infeasibility in a subtropical setting. Work with Brazilian collaborators demonstrated the effectiveness of an area-wide control strategy for HLB. Keying off of this, the surveillance methods discussed above were adapted for statewide sweeps for HLB and its vector, allowing regulatory agencies and commodity groups to target most prevalent hotspots for existing HLB and new disease outbreaks to erupt. Chemical treatments of HLB-affected citrus have proved to effective. Seed transmission of Las has been investigated with several different approaches. One showed Las bacteria are associated with the seed coat and not the seed/embryo tissues so seed transmission of Las likely is not a concern in the overall management strategy of HLB.

4. Accomplishments
1. Groundnut ringspot virus (GRSV) was identified in tomato with severe symptoms in south Florida. This extends the known distribution of this vegetable pathogen beyond South America and South Africa to North America. During the past year, we have documented the northward movement of GRSV up the Florida peninsula and additional hosts including pepper, eggplant and tomatillo. We have also demonstrated that western flower thrips transmit Florida isolates of GRSV. Collectively, this information indicates that a wide range of solanaceous plants is likely to be infected by this emerging viral pathogen in Florida and beyond.

2. Colombian datura virus (CDV) was widespread in Brugmansia and related ornamental solanaceous plants across the United States. These plants are commonly traded worldwide. All CDV isolates sequenced in this study (and those in GenBank) were quite similar. This suggests recent ancestry of the worldwide CDV population, coinciding with anthropogenic collection and dissemination of Brugmansia plants from their center of origin.

3. Colonization of seed coats by huanglongbing (HLB)-associated bacterium Las was detected. Seedlings germinated from these seeds did not become infected. The data showed a much more consistent colonization of seed coats of grapefruit than of sweet orange with no apparent infection of the embryo in the seed or of the emergent seedling during germination. A Fluorescent In Situ Hybridization procedure was developed to fluorescently label Las cells to observe them in plant tissue using the light microscope, allowing visual assessment of the bacterium in citrus tissues.

4. Chemical compounds effective against the citrus HLB-associated bacterium Las has been proved in planta. The combination of penicillin and streptomycin (PS) was effective in eliminating or suppressing Las and provided a therapeutically effective level of control for a much longer period of time than when administering either antibiotic separately. When treated with the PS, Las-infected periwinkle cuttings achieved 70% of regeneration rates versus <50% by other treatments. The Las titers in the infected periwinkle plants decreased significantly following root soaking or foliar spraying with PS. Application of the PS via trunk injection or root soaking also eliminated or suppressed the Las in the HLB-affected citrus plants. This may provide a useful tool for the management of citrus HLB and other Liberibacter-associated diseases.

5. Citrus canker lesion activity was quantified on samples of fruit, stems and leaves. Results show that the proportion of active lesions on fruit of grapefruit decline during the season, although many lesions remained active up to harvest, and the quantity of bacteria produced from individual lesions is highly variable throughout the season. This underscores the need for post-harvest treatment to neutralize the perceived risk of infected material. However, whereas lesions on fruit declined in activity, lesions on leaves were the most active throughout both seasons, suggesting the lesions on mature fruit pose a lower risk. Stem lesions had consistently the lowest activity, and also declined as the season progressed.

6. Control of citrus canker studied. A new series of plots to examine the effect of windbreaks, the finder control and copper on citrus canker outbreaks were established via a USDA/ARS specific cooperative agreement with the University of Sao Paulo at a farm, in Xambrê, Parana state. The cultivar used is Pêra on Rangpur lime, two years of age at the beginning of the experiment. Windbreaks were established in Mid April 2010, but severe winds damaged the initial windbreaks which have now been rebuilt. We anticipate running these plots for 2-3 more years to collect all necessary data.

7. Spread of citrus huanglongbing (HLB) explored. A model was developed to predict the spatial an temporal dynamics of citrus Huanglongbing (HLB) using Markov-chain Monte Carlo methods, and extensive data from infected areas of South Florida. Transmission rates and dispersal kernel were estimated for HLB. A working model was developed that focuses on the differential effects of host age on epidemiological parameters as well as variability across the plantation and that allows for uncertainty in the parameters as well as variability over time and through space. A front-end (a web based version of the model) was developed for non-researcher users has been nearly finalized and is in validation testing. Model output suggests that controlling secondary infections by diseased tree removal and insecticide applications plus controlling primary infection from new insect immigrations by areawide control strategies, can reduce disease increase to a manageable 2 to 5% increase per year, which appears to be economically sustainable.

8. To examine the potential disease control of citrus huanglongbing (HLB) by interplanting citrus with guava. Data analysis to date indicates no differences were observed among treatments, i.e., guava interplanted vs. non-interplanted plots prior to the final demise of the plots. Our interpretation is that Florida is actually a subtropical environment, prone to intermittent freezes and cool or cold temperatures. Cool or freezing temperatures inhibits volatile production and thus the citrus crop is left unprotected from psyllids. While guava not be a viable deterrent as an intercrop, it still may be possible to identify individual volatiles from guava that might be useful under field applications as chemical applications.

9. Accurate detection of citrus huanglongbing (HLB)-associated bacteria can be challenging due to low and uneven titer distribution within the infected plants. An improved real-time polymerase chain reaction (PCR) method for detection of Las was developed from citrus and psyllid hosts. By targeting unique characteristics of specific Las genes, methods were devised that dramatically enhance the detection of the bacterium. Via this method =100 fold increase in the detection sensitivity can be achieved compared with the present standard 16S rDNA-based method. The increased sensitivity provided by this method allows detection of Las from some HLB samples that are negative by alternate detection methods.

10. The huanglongbing (HLB)-associated bacterium Las is both diverse and variable as revealed by DNA sequence analyses of its prophage genes. The variations among global isolates indicate multiple Las populations exist. This is perhaps best explained by multiple source introductions of the Las bacterium into Florida. More importantly, the finding documents that different Las populations can co-exist in a single HLB-infected plant/sample.

Review Publications
Bock, C., Gottwald, T.R., Parker, P., Ferrandino, F., Welham, S. 2010. Some consequences of using the Horsfall-Barratt scale for hypothesis testing. Phytopathology. 100:1030-1041.

Webster, C.G., Kousik, C.S., Roberts, P., Rosskopf, E.N., Turechek, W., Adkins, S.T. 2011. Cucurbit yellow stunting disorder virus detected in pigweed in Florida. Plant Disease. 95(3):360.

Webster, C.G., Reitz, S.R., Perry, K.L., Adkins, S.T. 2011. A natural M RNA reassortant arising from two species of plant-and-insect-infecting bunyaviruses and comparison of its sequences and biological properties to parental species. Virology. 413:216-225.

Zhang, S., Flores-Cruz, Z., Kang, B., Fleites, L., Wulff, N., Davis, M.J., Zhou, L., Duan, Y., Gabriel, D. 2011. Ca. Liberibacter asiaticus carries two prophage lysogens - one replicating as an excision plasmid and another that becomes lytic in plant infections. Molecular Plant-Microbe Interactions. 11(4):458-468.

Koh, E., Zhou, L., Kang, B., Williams, D.S., Duan, Y. 2011. Callose deposition and inhibited symplastic transport in the phloem of citrus leaves infected with Candidatus Liberibacter asiaticus. Plant Physiology. 249:687-697.

Adkins, S.T., Webster, C.G., Kousik, C.S., Webb, S.E., Roberts, P.D., Stansly, P.A., Turechek, W. 2011. Ecology and management of whitefly-transmitted vegetable viruses in Florida. Virus Research. 159:110-114.

Sundaraj, S., Srinivasan, R., Webster, C.G., Adkins, S.T., Perry, K.L., Riley, D. 2011. First Report of Tomato chlorosis virus in Tomato in Georgia. Plant Disease. 95(7):881.

Webster, C.G., Turechek, W., Mellinger, C.H., Frantz, G., Roe, N., Yonce, H., Vallad, G.E., Adkins, S.T. 2011. Expansion of Groundnut ringspot virus host and geographic ranges in solanaceous vegetables in peninsular Florida. Plant Health Progress. DOI: 10.1094/PHP-2011-0725-01-BR.

Last Modified: 2/23/2016
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