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United States Department of Agriculture

Agricultural Research Service


Location: Subtropical Plant Pathology Research

2012 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 sub-objectives. Under sub-objective 1a: Progress on culture of ‘Candidatus Liberibacter asiaticus’ (Las) in vitro has been made. Genetic diversity of Las has been demonstrated by several molecular markers. Two novel autotransporters and a functional flagellin of Las have been identified and/or characterized. Additional studies on seed transmission of Las and psyllid transmission of Las to different citrus varieties are nearly completed. Experiments to purify Las from plant and insect tissues are ongoing and metabolomics analyses to identify compounds associated with early Las infection were initiated. Under sub-objective 1b: Transmission of Groundnut ringspot virus (GRSV) and Squash vein yellowing virus (SqVYV) is continuing to be elucidated. Host range and genetics of GRSV were investigated. Under sub-objective 1c: Plots to test the interaction of ACC, leafminer, and wind breaks were established in Brazil in 2010 and data collection continues. Numerous studies were conducted on dispersal characteristics of Asiatic citrus canker (ACC) via wind and rain to characterize the dispersal plume of inoculum and its disease-causing potential downwind. Under sub-objective 2a: A new detection kit for Las was developed, reducing diagnostic time for Las from several hours to less than an hour, and is going to be commercialized for field detection. Sensitivity and specificity of multiple GRSV and SqVYV detection assays are being evaluated. Under sub-objective 2b: Surveillance methods were adapted for statewide sweeps for Huanglongbing (HLB) and its vector for Florida and risk-based residential survey methods for Asian citrus pysllid (ACP) and HLB are nearing completion, are ready to be deployed and will enter the validation phase. These new survey methods regulatory agencies and commodity groups to target the most prevalent hotspots for existing HLB and new disease outbreaks to erupt. Under sub-objective 3a: A citrus canker stochastic model has been developed and validated. A similar model for HLB is in the final stages of validation, as is a user-friendly front end to both canker and HLB models, which will make them plausible to regulatory agencies and commodity groups. We are currently upgrading a web-based and mobile technology platform (AgScouter) that functions as a disease and insect scouting and management tool using a mobile device (e.g. smartphone) for data entry. Subsequent processing will be used to develop field-specific, pest management recommendations. Under sub-objective 3b: Tests using guava as an intercrop between citrus trees as a mitigation strategy for HLB were marginally 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. Several chemicals (including non-antibiotics) have been demonstrated to be effective for control of HLB in greenhouse trials. Heat treatment cured potted citrus plants under control conditions, providing a simple but effective method for the control of Liberibacter-infected diseases in greenhouse settings.

4. Accomplishments
1. U.S. population of Squash vein yellowing virus (SqVYV; cause of viral watermelon vine decline) is homogenous. SqVYV isolates collected from cultivated and weedy cucurbits representing major hosts and locations in the U.S. were analyzed to better understand pathogen diversity and population structure. No differences in symptoms were observed in field-collected isolate source plants or subsequently inoculated greenhouse plants. Sequence comparison of three genome regions for 41 isolates showed little diversity across seven years of sampling. This homogeneity indicates that the current U.S. population of SqVYV was introduced from elsewhere and validates ongoing resistance breeding efforts by ARS scientists (in Fort Pierce, FL and Charleston, SC) for viral watermelon vine decline management.

2. A web-based and mobile technology platform (known as AgScouter) was developed as a disease and insect scouting and management tool. It is currently being updated (v2.0) to allow growers and scouts to enter 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. Tomato yellow leaf curl virus and whiteflies were initially used to validate this approach. Subsequently the adaptability of this approach has been demonstrated by expanding it to include the emerging Squash vein yellowing virus (cause of viral watermelon vine decline) and Groundnut ringspot virus. Use of this system to promote real-time, regional pest management is now being tested.

3. Host response to ‘Candidatus Liberibacter asiaticus’ (Las) bacterium associated with Huanglongbing (HLB) was characterized. Citrus rootstock varieties, but not scion varieties, are propagated from seed. Seed transmission studies of the HLB-associated Las in sixteen popular citrus rootstock varieties found no trace of the pathogen in dissected seed embryos or cotyledons, or in germinated seedlings even when pathogen DNA was found in seed coats and in foliar samples. Analysis of the susceptibility of genetically diverse varieties of citrus to infection with Las via psyllid transmission found specific varieties which had a common genetic background that consistently were first to have detectable levels of Las. These data suggest either a psyllid feeding preference or that susceptibility to infection has a host genetic component. Initial protocols were developed to isolate Las cells from plant tissue by enzyme treatment, homogenization and fractionation on Percoll gradients.

4. Genetic variation among global isolates of ‘Candidatus Liberibacter asiaticus’ (Las) bacterium associated with Huanglongbing (HLB) indicates that multiple Las populations exist. Eight different types of sequences (A to H) were identified in two prophages (FP1and FP2) in Florida isolates. Sequence analysis revealed variations that were the likely result of frequent recombination and reassortment. Results using primers specific to the different types of sequences indicated that types A and B were the major groups located in FP1 and FP2, respectively. Las-infected periwinkle and dodder contained all types of Las populations, whereas psyllids only contained selected types A. Las-infected citrus plants contained various types of Las populations, but only one type was associated with typical HLB symptoms. Similar results were obtained from global samples, indicating the dramatic variations of these prophage regions among the isolates. These intraspecies variations derived from the prophage activities may be important for the bacterial adaptation to their host plants and insects.

5. The effectiveness of several antibiotics and combinations thereof has been demonstrated for eliminating or suppressing ‘Candidatus Liberibacter asiaticus’ (Las) bacterium associated with Huanglongbing (HLB). Field trials indicated each of the antibiotics and their combinations were effective at Las suppression for four months following treatment when applied with a syringe injector as measured by decrease in Las titer. Effects on Las diversity caused by the antibiotic treatment were also investigated.

6. ‘Candidatus Liberibacter asiaticus’ (Las) encodes two hypervariable genes (hyvI and hyvII). Bioinformatic analyses revealed that HyvI and HyvII share the characteristics of an autotransporter family, which contains large tandem repeats of a passenger domain and a C-terminal translocator domain. When expressed in E. coli, both proteins were localized to the bacterial poles, similar to members of autotransporters from other bacteria. Despite the absence of signal peptides, HyvI was found to localize at the cell surface. When transiently expressed in tobacco leaf cells, the HyvI protein did not target the cell nucleus, but did target organelles. This is the first evidence that Las may employ the type V pathway.

7. Thermotherapy was demonstrated to suppress ‘Candidatus Liberibacter asiaticus’ (Las) populations in citrus. Results of quantitative polymerase chain reaction (qPCR) after treatment in a growth chamber showed significant decreases in the Las titer, reaching an undetectable level, combined with healthy vigorous tree growth. Repeated surveys confirmed previously infected plants showed no detectable Las, whereas untreated plants remained infected. Continuous thermal exposure of 40-42°C for at least 48h was sufficient to significantly reduce Las in citrus. Heat treatment of dooryard citrus and commercial groves using portable greenhouses indicated suppression rather than elimination of Las in the field plants.

8. Due to the fastidious nature of ‘Candidatus Liberibacter asiaticus’ (Las), little is known about prophage response to stress conditions. Real-time polymerase chain reaction (PCR) was used to investigate the potential conversions of FP1 and FP2 prophages under stress conditions by comparing to the copy number of 16S rDNA in the HLB-affected periwinkle and citrus. Relative copy number of FP1 and FP2 following both heat and antibiotic stresses. Results suggest Las prophages may be converted from a lysogenic cycle to a lytic cycle by stress induction, providing a potential mechanism to explain inability to detect Las in periwinkle following heat treatment.

9. A new statewide residential risk-based model and related survey was developed and is ready to be deployed for citrus huanglongbing (HLB) and its Asian citrus psyllid (ACP) vector. Risk factors include ACP and human population density, Asian populations, and proximity to commercial plantings, military installations, retail sales stores, citrus nurseries, green waste areas and transportation corridors. For residential survey, overall risk estimates are made and survey protocols developed to deploy survey teams to areas with the highest risk. For commercial citrus, similar risk factors are calculated and the risk reversed to reflect the risk of urban populations and activities on commercial plantings. The California citrus industry will be parsed into strata and sampled based on a risk-bias algorithm previously designed and deployed in Florida. Survey data will be used for validation and subsequent refinement of the existing predictive model in California. Survey data and model risk predictions provide the empirical evidence on which management decisions can be made.

10. Studies were initiated in 1999 on canine detection of citrus canker, and recently, we have demonstrated canine detection of citrus canker, both in field and packinghouse environments. Canines were cross trained by positive reinforcement to discriminate between target (canker-infected) and non-target (non-infected) odor signatures of citrus foliage and fruits. Canines have no trouble distinguishing multiple odor signatures (i.e., foliage vs. fruit) and will alert within a few seconds and confirm detection by sitting next to the target. Replicated field trials with spatially randomized plots with various disease incidences demonstrated detection accuracy is ~98%, with nearly equal numbers of false-positive and false-negative non-detections. Commercial field plots had similar results. The method can detect much lower levels of canker infections than human visual detection, availing opportunities for commercial producers to detect canker, clean groves and certify more groves free of canker for international shipment, providing tremendous marketing opportunities for fresh fruit growers. The methodology has gone to commercialization, with commercial canine handlers developing dog team surveys to detect citrus canker in citrus plantations.

11. An SEIDR model was finalized and validated to predict the spatial and 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. Model 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 (web-based version of the model) has also been developed for non-researcher use. Model can be used to test various control strategies. For instance, 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.

Review Publications
Hilf, M.E. 2011. Colonization of Citrus Seed Coats by 'Candidatus Liberibacter asiaticus' the bacterium associated with citrus Huanglongbing; Implications for seed transmission of the bacterium. Phytopathology. 101:1242-1250.

Bar-Joseph, M., Robertson, C., Hilf, M.E., Dawson, W.O. 2011. A novel method for citrus propagation: Seed Grafting. Journal of Horticultural Science and Biotechnology. 86:616-618.

Webster, C.G., Adkins, S.T. 2011. Low genetic diversity of Squash vein yellowing virus in wild and cultivated cucurbits in the U.S. suggests a recent introduction. Virus Research. doi:10.1016/j.virusres.2011.11.017.

Gottwald, T.R., Graham, J.H., Mccollum, T.G., Wood, B.W., Irey, M.S. 2012. Inconsequential effect of nutritional treatments on Huanglongbing control, fruit quality, bacterial titer and disease progress. Crop Protection Journal. 36:73-82.

Scholthof, K.G., Adkins, S.T., Czosnek, H., Palukaitis, P., Jacquot, E., Hohn, T., Hohn, B., Saunders, K., Candresse, T., Ahlquist, P., Hemenway, C., Foster, G.D. 2011. Top 10 Plant Viruses in Molecular Plant Pathology. Molecular Plant Pathology. 12:938-954.

Bock, C.H., Parker, P.E., Gottwald, T.R. 2011. Distribution of canker lesions on the surface of diseased grapefruit. Plant Pathology. 60:986-991.

Parnell, S., Gottwald, T.R., Irey, M., Van Den Bosch, F. 2011. A stochastic optimisation method to estimate the spatial distribution of an invasive plant pathogen. Journal of Phytopathology. 101:1184-1190.

Windham, W.R., Poole, G.H., Park, B., Heitschmidt, G.W., Albano, J.P., Gottwald, T.R., Lawrence, K.C., Hawkins, S.A. 2011. Rapid screening of huanglongbing-infected citrus leaves by near infrared reflectance spectroscopy. Transactions of the ASABE. 54(6):2253-2258.

Hall, D.G., Gottwald, T.R. 2011. Pest management practices aimed at curtailing citrus huanglongbing disease. Outlooks on Pest Management. 22(4):189-192.

Kousik, C.S., Donahoo, R.S., Webster, C.G., Turechek, W., Adkins, S.T., Roberts, P.D. 2011. Outbreak of powdery mildew on watermelon fruit caused by podosphera xanthii in southwest Florida. Plant Disease. 95:1586.

Islam, M., Glynn, J.M., Bai, Y., Duan, Y., Coletta-Filho, H., Kuruba, G., Civerolo, E.L., Lin, H. 2012. Multilocus microsatellite analysis of ‘Candidatus Liberibacter asiaticus’ associated with citrus Huanglongbing worldwide. BMC Microbiology. Available:

Walter, A.J., Hall, D.G., Duan, Y. 2012. Low incidence of Candidatus Liberibacter asiaticus in Diaphorina citri and its host plant Murraya paniculata. Plant Disease. 96:827-832.

Niedz, R.P., Evens, T.J., Hyndman, S., Adkins, S., Chellemi, D.O. 2011. In vitro shoot growth of Brugmansia x candida Pers. Physiology and Molecular Biology of Plants. 18:69-78.

Hall, D.G., Hentz, M.G., Meyer, J.M., Boucias, D.G. 2012. Observations on the entomopathogenic fungus Hirsutella citriformis attacking adult Diaphorina citri (Hemiptera: Psyllid) in a managed citrus grove. Biocontrol. 57:663-675.

Bock, C.H., Parker, P. E., Cook, A. Z., Graham, J. H. and Gottwald, T. R. Infection and decontamination of citrus canker-inoculated leaf surfaces. Crop Protection 30: 259-264. 2011.

Cheng Kao, C., Adkins, S.T. 2012. Bromovirus. The Springer Index of Viruses. 12:173-177.

Turechek, W., Madden, L.V., Gent, D.H., Xu, X.M. 2011. Comments Regarding the Binary Power Law for Heterogeneity of Disease Incidence. Phytopathology. 101:1396-1407.

Webb, S.E., Adkins, S.T., Reitz, S.R. 2012. Semipersistent whitefly transmission of Squash vein yellowing virus, causal agent of viral watermelon vine decline. Plant Disease. 96:839-844.

Nunes, M.A., De Oliveira, C.L., De Oliveria, M.L., Kitajima, E., Hilf, M.E., Gottwald, T.R., Freitas-Astua, J. 2012. Transmission of Citrus leprosis virus C by Brevipalpus phoenicis (Geijskes) to Alternative Host Plants Found in Citrus Orchards. Plant Disease. 96:968-972.

Srinivasan, R., Riley, D., Diffie, S., Sparks, A., Adkins, S.T. 2012. Whitefly population dynamics and evaluation of whitefly-transmitted Tomato yellow leaf curl virus (TYLCV)-resistant tomato genotypes as whitefly and TYLCV reservoirs. Journal of Economic Entomology. 105(4):1447-1456.

Luo, W., Pietravalle, S., Parnell, S., Van Den Bosch, F., Gottwald, T.R., Irey, M.S., Parker, S.R. 2012. An improved regulatory sampling method for mapping and representing plant disease from a limited number of samples. Elsevier. 4:68-77.

Parnell, S., Gottwald, T.R., Gilks, W.R., Van Den Bosch, F. 2012. Estimating the spatial distribution of a plant disease epidemic when it is first discovered and the design of early detection monitoring. Journal of Theoretical Biology. 305: 30-36.

Last Modified: 04/25/2017
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