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ARS Home » Southeast Area » Fort Pierce, Florida » U.S. Horticultural Research Laboratory » Subtropical Plant Pathology Research » Research » Research Project #423073

Research Project: EMERGING DISEASES OF CITRUS, VEGETABLES, AND ORNAMENTALS

Location: Subtropical Plant Pathology Research

2014 Annual Report


Objectives
1. Characterize ecology, biology, epidemiology, molecular genetics, and vector and host (crop and weed) interactions of domestic, exotic, newly emerging, and re-emerging pathogens. 1a. Characterize the etiology, molecular biology and genetics of ‘Candidatus Liberibacter asiaticus (Las),’ the bacterium associated with citrus huanglongbing (HLB). 1b. Molecular characterization, vector interactions and/or epidemiology of Groundnut ringspot virus (GRSV), Squash vein yellowing virus (SqVYV) and other viruses of vegetables, ornamentals, and weeds, and Xanthomonas fragariae [causing angular leaf spot (ALS) on strawberry]. 1c. Characterize meteorological components affecting the epidemiology of Asiatic citrus canker (ACC), the interaction of the Asian leaf miner with ACC, the Asian citrus psyllid with HLB, the interaction of whiteflies with SqVYV, and the interaction of thrips with GRSV. 2. Develop/refine rapid, sensitive reliable detection/sampling methods for pathogens. 2a. Develop improved detection methods for GRSV, Las and Xanthomonads on citrus and strawberry. 2b. Develop new and augment existing surveillance methods and protocols for HLB, Xanthomonas citri subsp. citri on citrus, and the other newly introduced citrus diseases such as citrus black spot (CBS) and sweet orange scab (SOS). 3. Develop or improve comprehensive integrated disease management strategies. 3a. Develop and use stochastic models to test various disease control strategies for HLB, ACC, CBS and diseases caused by other exotic pathogens. 3b. Develop and implement the most efficacious strategies for disease management of HLB, Xanthomonads of citrus and strawberry, CBS, Plum pox virus (PPV), and viruses of vegetables and ornamentals.


Approach
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.


Progress Report
Progress made on all three objectives. Under sub-objective 1a: Progress on culture of Candidatus Liberibacter asiaticus (Las) in vitro was made. Genetic diversity of Las was demonstrated with molecular markers to differentiate mild and severe strains/isolates. Two novel effectors and their interactions with host proteins were further characterized. Studies on huanglongbing (HLB) effects on seedling growth and vitality are ongoing. Analysis of Las viability in plant tissues was initiated. Studies of resistance in Murraya paniculata to Las infection are ongoing. Purification of Las from plant and insect tissues is ongoing. Metabolomic analyses to identify compounds associated with early Las infection are ongoing. Under sub-objective 1b: Transmission of tospoviruses and Squash vein yellowing virus (SqVYV) is being elucidated. Host range, geographic range and genetics of tospoviruses were investigated. Crown infections studies with green fluorescent protein (GFP)-transformed Xanthomonas fragariae (Xf) nearly complete and have shown that systemic movement of Xf within the plant is not an active process. Under sub-objective 1c: Plots to test the interaction of Asiatic citrus canker (ACC), leafminer, and wind breaks were established in Brazil in 2010. Data collection continues and is indicating that combinations of windbreaks and leafminer control are highly efficacious for canker mitigation. Packinghouse studies have demonstrated ACC survival is poor and declines in mature fruit. Meteorological, insect count, and virus incidence data continue to be collected in cucurbit/solanaceous crops. Under sub-objective 2a: A new detection method based on a chemical from host response to Candidatus Liberibacter asiaticus infection was developed, which may reduce diagnostic time for citrus HLB, and serves as pre-symptomatic diagnosis. Sensitivity/specificity of multiple virus detection assays are being evaluated. A loop-mediated isothermal amplification (LAMP) detection assay for Xf was developed and its diagnostic performance evaluated. The LAMP assay is nearly as a good of a diagnostic tool as the standard quantitative (qPCR) but can be done without specialized equipment. The propidium monoazide (PMA)-PCR protocol is currently being evaluated. Under sub-objective 2b: A multi-pest Surveillance method for statewide sweeps for HLB and its vector and several other diseases including citrus black spot (CBS) has been very successful, is continuously adapted to new disease priorities as requested by USDA APHIS, and is re-deployed yearly. Risk-based residential and commercial survey methods for Asian Citrus Psyllid (ACP) and HLB are in the second year of deployment in California, Texas and Arizona, and validation indicates they are highly successful. Surveys are being used by regulatory agencies and commodity groups to target disease/vector hotspots for existing HLB and predict new outbreaks locations. These are reviewed, and revised to re-optimize to changing disease/vector conditions. Under sub-objective 3a: Both ACC and HLB stochastic models have been developed, validated and publications in press or in print. A user-friendly front end to both canker and HLB models was completed for use by regulatory agencies and commodity groups and has been submitted for publication. These models and their refinement will be the foundation for future work. AgScouter interface was further improved, along with improvements to smartphone applications for iPhone and Android. Subsequent development continues to focus on field-specific, pest management recommendations in Florida and with collaborators in California. Under sub-objective 3b: Tests using guava as an intercrop between citrus trees as a mitigation strategy for HLB were completed and a publication was submitted and is in press. A CBS probabilistic risk model is in the 3rd year of development and will be important to citrus international trade to determine if fruit are a pathway for disease establishment in new locations. Several compounds were effective for control of HLB in greenhouse and field trials. Heat treatment cured HLB-affected citrus plants in greenhouse settings, providing a simple, effective method for Las control; Field trials of heat treatment demonstrated its effectives on mitigating citrus HLB in groves.


Accomplishments
1. Characterized two newly identified vegetable viruses in the U.S. Multiple biological attributes of recently introduced Groundnut ringspot virus (GRSV) and Tomato chlorotic spot virus (TCSV) including host range, geographic distribution and vectoring capacity of locally important thrips species in the southeastern U.S. were determined. GRSV and TCSV were frequently detected in tomato, pepper and other solanaceous crops and weeds with tospovirus-like symptoms in south Florida. Impatiens and lettuce were the only non-solanaceous GRSV and/or TCSV hosts identified in experimental host range studies. Western flower thrips transmitted GRSV and TCSV. Little genetic diversity was observed in GRSV and TCSV sequences; all GRSV isolates characterized were reassortants and had the same genotype originally detected by ARS scientists (in Fort Pierce, Florida). Further spread of GRSV and TCSV in the U.S. is possible and detection of mixed infections highlights the opportunity for additional reassortment of tospovirus genomic RNAs.

2. Revised and released Maryblyt V.7.1. This is a computer program to forecast fire blight of apple and pear. Version 7.1 will incorporate several cosmetic and functional changes, including the capability to use international numbering and date formats, a spray effectiveness module that allows users to enter a spray efficacy threshold to account for treatments that are less than 100% effective, the ability to turn on and off the effect that spraying antibiotic has on Maryblyt calculations so that users can track symptom development as if they had not sprayed so that disease can be monitored more effectively and spray effectiveness can be evaluated, and, finally, an audible warning to alert users when an infection event has occurred re-established (with the ability to turn it on and off). Maryblyt is free public domain software.

3. Characterized epidemiology of three introduced whitefly transmitted cucurbit viruses. The epidemiology of Squash vein yellowing virus (SqVYV) was studied in a one hectare field of ‘Fiesta’ watermelon over six growing seasons to characterize the spatial patterning of disease, the temporal rate of disease progress, and its association with Cucurbit leaf crumple virus (CuLCrV) and Cucurbit yellow stunting disorder virus (CYSDV), two additional whitefly transmitted viruses that often occur with SqVYV. It was shown that SqVYV progressed in a characteristic logistic fashion and that the rate of disease progress was rapid and very similar during seasons with high disease incidence. A positive correlation between the area under the disease progress curve and whitefly-days was found, where both measures were calculated as a function of thermal time (degree days, base 0°C). SqVYV displayed significant but variable levels of aggregation as indicated by extensive spatial analysis. Association analysis indicated that the viruses were largely transmitted independently. Results of this study provided epidemiological information that will be useful in the development of foundational management strategies for SqVYV-induced watermelon vine decline, and provide new information for CuLCrV and CYSDV.

4. Determined biological characteristics of Candidatus liberibacter asiaticus (Las)-infected plants. Citrus plants were infected with Las by grafting of small pieces of tissue and using intact single citrus leaves; this allowed the calculation of a minimal infectious dose of Las in citrus. Psyllid transmissions and grafting experiments identified Murraya paniculata plants that express resistance to Las infection; resistant plants can be identified by a simple grafting experiment. A protocol to assess viability of Las cells in plant tissue was modified to allow direct evaluation of Las cell viability in intact plant tissues; this modification could allow for the analysis of Las metabolism without having Las in a sustained culture. Metabolomics analysis identified differences in types and levels of plant defense-related metabolites which differentiated Las-infected citrus from healthy citrus and citrus infected with non-Las pathogens.

5. Characterized Candidatus liberibacter asiaticus (Las) populations in infected plants. Multiple populations of Las were found to co-exist in citrus Huanglongbing (HLB)-affected citrus plants. The dynamics of these populations were primarily prophage-mediated. Molecular markers were developed to differentiate these populations, and their associations with HLB development. Using these markers, we have isolated severe and mild strains/isolates of Las, and developed a method to generate a mild strain. Demonstrated that mealybug (Ferrisia virgata) carries Las populations that do not cause disease in host plants. The potential application of a mild strain for cross protection is under evaluation.

6. Tested novel therapeutics for Candidatus liberibacter asiaticus (Las)-infected citrus. Field trials of selective chemicals indicated some new antibiotics/chemicals and their combinations were effective on elimilating or suppressing Las bacteria in Huanglongbing (HLB)-affected citrus plants when applied with a new delivery system and nanoemusion technologies. Heat treatments of dooryard citrus and commercial groves using portable greenhouses indicated thermotherapy mitigated HLB in citrus plantings. Florida citrus growers are widely applying this simple technology.

7. Characterized molecular biology of Candidatus liberibacter asiaticus (Las) effectors. A number of important effectors of Las were further characterized. Molecular interactions between these effectors and host proteins were elucidated. Specific antibodies against these effectors proteins were generated, which will be used for further characterization of their role in the pathogenesis of Las bacterium.

8. Deployed statewide risk models and surveys for Huanglongbing (HLB) in the Southwest U.S. New statewide residential and commercial risk-based models and related surveys were deployed for HLB and its Asian citrus psyllid (ACP) vector to 1) Southern California, 2) Central and Coastal California, 3) Arizona, and 4) Texas. These models were re-optimized for a 2nd year and re-deployed. Survey data and model risk predictions provide the empirical evidence on which management and regulatory decisions are being made. 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, swap meets, border crossings with Mexico, and transportation corridors. In addition, California, Arizona, and Texas citrus industries have been parsed into strata and sampled based on a multi-pest risk-bias algorithm previously designed and deployed in Florida.

9. Optimized area-wide citrus health management. The citrus industry in Florida has been divided into Citrus Health Management Areas (CHMA), i.e., groups of growers who coordinate in control of Huanglongbing and ACP. The program has been somewhat successful. We have developed a CHMA optimization algorithm that can increase CHMA efficacy many fold. It is being considered to help California, Texas and Arizona develop optimal CHMAs and for Florida to redeploy CHMAs with greater efficacy.

10. Developed model for Asian citrus psyllid (ACP) spread. As part of a NIFA/SCRI grant aimed at developing and releasing genetically modified ACP (NuPsyllid) to outcompete and/or decimate wild-type ACP populations, we have developed a geographic information system-based, ACP spread simulation model. The new model can be used to test and optimize points of release of NuPsyllid and to predict the efficacy of ACP chemical, biological, and horticultural control strategies.

11. Continued development of canine detection for major citrus diseases. Over the prior 12 years, we have demonstrated the efficacy of canine detection of citrus canker, both in field and packinghouse environments. This year, canine detectors successfully distinguished multiple odor signatures (i.e., infected foliage vs. fruit) in replicated citrus field, nursery and packinghouse trials with a demonstrated detection accuracy of >99.6%, with nearly equal numbers of false-positive and false-negative non-detections. The method has been commercialized by one firm for citrus nursery canker detection. We have now moved into Huanglongbing (HLB) detection via canine detectors with a new firm that has procured dogs and began training. We have just been awarded USDA, Farm Bill multi agency coordinating funding to train 20 dogs for HLB detection/validation.

12. Continued development of holistic models to optimize canker and Huanglongbing (HLB) Control. Susceptible, exposed, infected, diseased, recovered models have been finalized, validated, and published to predict the spatial and temporal dynamics of citrus HLB and canker using Markov-chain Monte Carlo methods, and extensive data from infected areas in Florida. A front-end (web-based version of the model) has also been completed and submitted for publication that can be used to test various control strategies. These models are the foundation for continued work on holistic models to test and optimize canker and HLB control strategies by regulatory agencies and stakeholders.


Review Publications
Gottwald, T.R., Luo, W., Mcroberts, N. 2013. Risk-based residential HLB/ACP survey for California, Texas and Arizona. Plant Management Network. Webcast.
Matos, L.A., Hilf, M.E., Chen, J., Folimonova, S.Y. 2013. Validation of ‘Variable Number of Tandem Repeat’ analysis and its applications for examination of ‘Candidatus Liberibacter asiaticus’ populations in areas of recent introduction. PLoS One. 8-11:e78994.
Neri, F.M., Cook, A.R., Gottwald, T.R., Gilligan, C.A. 2014. Bayesian analysis for inference of an emerging epidemic: citrus canker in urban landscapes. PLoS Computational Biology. 10(4):e1003587.
Anco, D.J., Poole, G.H., Gottwald, T.R. 2014. Effects of postharvest treatments on recovery of Xanthomonas citrisubsp. citri from infected grapefruit leaves. Crop Protection Journal. 62:115-123.
Bock, C.H., Graham, J.H., Gottwald, T.R., Cook, A.Z., Parker, P.P. 2014. Effect of the duration of inoculum exposure on development of citrus canker symptoms on seedlings of Swingle citrumelo. European Journal of Plant Pathology. 138:237-245.
Estevez De Jensen, C., Adkins, S.T. 2014. First report of Tomato chlorotic spot virus in lettuce in Puerto Rico. Plant Disease. 98:1015.
Goncalves, F.P., Stuchi, E.S., Lourenco, S.A., Kriss, A.B., Gottwald, T.R., Amorim, L. 2014. The Effect of Irrigation on the Temporal Increase of Citrus Variegated Chlorosis. International Research Conference on Huanglongbing. 57:8-14.
Graham, J.H., Gottwald, T.R., Timmer, L.W., Filho, A.B., Van Den Bosch, F., Irey, M.S., Taylor, E.L., Magarey, R.D., Takeuchi, Y. 2014. Response to the recently published article “Potential distribution of citrus black spot in the United States based on climatic conditions". European Journal of Plant Pathology. 139(2).
Kousik, C.S., Ling, K., Adkins, S.T., Webster, C.G., Turechek, W. 2014. Phytophthora fruit rot-resistant watermelon germplasm lines: USVL489-PFR, USVL782-PFR, USVL203-PFR, and USVL020-PFR. HortScience. 49:101-104.
Niedz, R.P., Hyndman, S.E., Chellemi, D.O., Adkins, S.T. 2013. Production of Brugmansia plants free of Colombian datura virus by in vitro ribavirin chemotherapy. ARPN Journal of Agricultural and Biological Science. 8(11):751-755.
Pitino, M., Hoffman, M.T., Zhou, L., Stocks, I.C., Duan, Y. 2014. The phloem-sap feeding mealybug (Ferrisia virgata) carries 'Candidatus Liberibacter asiaticus' populations without transmitting disease. PLoS One. 9(3):e92757. https://doi.org/10.1371/journal.pone.0092757.
Stanghellini, M.E., Mohammadi, M., Mathews, D.M., Adkins, S.T. 2014. First report of vine decline of mature watermelon plants caused by Olpidium bornovanus. Plant Disease. 98:852. https://doi.org/10.1094/PDIS-11-13-1123-PDN.
Stover, E.W., Richardson, M.L., Driggers, R., Hall, D.G., Duan, Y.P., Lee, R.F. 2014. Incidence and severity of Asiatic citrus canker on citrus and citrus–related germplasm in a Florida field planting. HortScience. 49:4-9.
Tantiwanich, Y., Baker, C.A., Turechek, W., Adkins, S.T. 2014. Detection of Papaya ringspot virus type W infecting the cucurbit weed Cucumis melo var. dudaim in Florida. Plant Health Progress. 15(1):29-30. https://doi.org/10.1094/PHP-BR-13-0126.
Turechek, W., Mcroberts, N. 2013. Considerations of scale in the analysis of spatial pattern of plant disease epidemics. Annual Review of Phytopathology. 51: 453-472.
Turechek, W., Roberts, P.D., Stansly, P.A., Webster, C.G., Kousik, C.S., Adkins, S.T. 2014. Spatial and temporal analysis of squash vein yellowing virus infections In watermelon. Plant Disease. 98(12):1671-1680. https://doi.org/10.1094/PDIS-10-13-1094-RE.