Research Project: Mitigating High Consequence Domestic, Exotic, and Emerging Diseases of Fruits, Vegetables, and Ornamentals

Location: Subtropical Plant Pathology Research

2020 Annual Report

Objectives
1. Develop a risk-based index to analyze potential emerging pathogens/pests to identify those with greatest negative impact on agriculture to guide future research. 2. Characterize ecology, biology, epidemiology, genetics, and vector and host (crop and weed) interactions of exotic, newly emerging, re-emerging, and domestic pathogens. 2a. Characterize the basic biology, molecular biology and genetics of ‘Candidatus Liberibacter asiaticus (Las),’ the bacterium associated with citrus huanglongbing (HLB), and molecular mechanisms of host resistance/tolerance to Las infection. 2b. Characterize the basic biology, molecular biology, vector interactions and/or epidemiology of Tomato chlorotic spot virus (TCSV) and other tospoviruses, Squash vein yellowing virus (SqVYV), ilarviruses and other viruses of vegetables, ornamentals, and weeds, and Xanthomonas fragariae [causing angular leaf spot (ALS) on strawberry]. 2c. Characterize meteorological components affecting the epidemiology of Asiatic citrus canker (ACC), and the interactions of the Asian leaf miner with ACC, citrus black spot (CBS), the Asian citrus psyllid with HLB, the interaction of thrips with TSV. 3. Develop/refine and deliver rapid, sensitive reliable detection/sampling methods for high consequence/economic limiting pathogens, including but not limited to citrus canker, huanglongbing, black spot, plum pox virus, seedborne pathogens, and insect vectored viruses of vegetables and ornamentals. 3a. Develop improved detection methods for tospoviruses, Las and Xanthomonads on citrus and strawberry. 3b. Develop new and augment existing surveillance methods and protocols for HLB and the other newly introduced diseases such as CBS and PPV. 4. Develop or improve comprehensive integrated disease management strategies to rapidly find and delimit new disease introductions and develop quarantine and control/mitigation/eradication programs for exotic pathogens. 4a. Develop and use stochastic models to test various disease control strategies for citrus and prunus diseases caused by exotic pathogens. 4b.Develop and implement the most efficacious strategies for disease management of HLB, Xanthomonads of citrus and strawberry, CBS, PPV, and viruses of vegetables and ornamentals.

Approach
The overall approach is to thoroughly characterize plant pathogens causing domestic, exotic and emerging diseases at multiple levels: assessment and prioritization of economic, production and political risk; cellular, molecular and/or biochemical levels of host-pathogen-vector interaction; and traditional and newer stochastic epidemiological analysis at regional and plantation levels. New pathogens will be identified and characterized by biological and traditional cultural methods. Recombinant DNA and genomics technologies will be applied to study host-pathogen interactions. Resulting knowledge will be used to develop new detection and sampling methods, and management strategies, for these pathogens.

Progress Report
Culture of Candidatus Liberibacter asiaticus (Las) in vitro was greatly improved with much more consistent results along with the development of semi-selective medium. Las was maintained and propagated in liquid media for months, and caused Huanglongbing (HLB) via feeding psyllid and inoculating to citrus. Selections of citrus seedlings and bud sports were evaluated for HLB resistance/tolerance via graft-based and psyllid inoculations. The resistant/tolerant selections from greenhouse evaluations were further propagated, and planted in Picos Research Farm and commercial grove for field trials where they have been evaluated for 2-3 years. From Las pathogenesis standpoint, we demonstrated that Las 5315 effector induced extreme starch accumulation and chlorosis, typical symptoms of HLB, which provides new targets for interference. Experiments with a controlled experimental system to study aspects of tissue colonization and population development in different citrus genotypes showed that bacterial populations peaked within 30 days of tissue colonization and did not increase significantly after this time. Calculations indicated that bacterial populations increased slowly in planta, with a doubling time approaching 48 hours, which is very slow. A small scale, short-term protocol for assessing antimicrobial compounds showed that antibiotics touted as effective in publications from various labs did not affect the HLB-associated bacteria. Transmission of tospoviruses, ilarviruses and squash vein yellowing virus (SqVYV) is continuing to be elucidated. Production scale monitoring of tospoviruses and ilarviruses were investigated. Seeds of fruits from watermelon plants infected with SqVYV were collected for virus testing and seed transmission studies. A novel tobamovirus (Hoya chlorotic spot virus) was isolated from the ornamental crop, Hoya spp., and its biology, host range and genome were characterized. A second novel tobamovirus (Brugmansia latent virus) was identified in another ornamental crop, Brugmansia spp., and its genome was sequenced and characterized. The survival time of Xanthomonas fragariae, the causal agent of strawberry angular leaf spot, was examined on 10 common surface materials at both room temperature and -4 °C (the cold storage temperature) to determine the likelihood for those materials to harbor the pathogen and serve as source of inoculum for disease. Survival was determine with a specialized quantitative polymerase chain reaction (qPCR) procedure and the likelihood of disease transmission was evaluated in a bioassay with strawberry plants. The experiment was completed and analysis of the data showed that X. fragariae survived between two to four weeks on most materials tested, with the longest survival and highest disease transmission rate (9 months) being observed on cardboard at -4°C. The information provided by this study can be used to develop improved sanitation protocols for angular leaf spot management in strawberry nurseries. The manuscript is currently in draft form. Plots to test the interaction of Asiatic citrus canker (ACC), leafminer, and wind breaks were established in Brazil and data collection continues with Brazilian collaborators. Results indicated that combinations of windbreaks and leafminer control are highly efficacious for canker mitigation. Fourier transform infrared spectroscopy (FTIR) was fully evaluated as a Huanglongbing (HLB) detection tool and found to be of marginal use, requiring no further study. Fear of spread by hurricanes of both ACC in Texas and Citrus Black Spot (CBS) in Florida led to the development of predictive models to estimate pathogen spread via hurricane events. These are being used by state and federal regulatory agencies to target detection survey post-event. The long distance dispersal model via hurricane (i.e. Irma & Harvey) are incorporated into recent multi-pest survey (MPS) design in Florida and Texas. Model accuracy will be assessed in future years when data of hurricane spread is collected. A stochastic simulation risk-based model for CBS spread by trade pathways has also been developed. It has been tested against trade pathway data sets, reviewed by collaborators, and is now completed. Meteorological, insect count, and virus incidence data (TSV and tospoviruses) are currently and continue to be monitored in trap crop plantings established at the USDA-ARS Picos Farm in Fort Pierce, Florida. New sets of conventional reverse transcription-polymerase chain reaction (RT-PCR) and qRT-PCR primers for all three genomic RNAs of Tomato chlorotic spot virus (TCSV), Groundnut ringspot virus (GRSV) and Tomato spotted wilt virus (TSWV) have been developed and validated. These primers were used for first detection of TCSV in soybean. Full genome sequence of TCSV isolate from peanut was determined. A new detection method based on two different biomarkers from host response to Candidatus Liberibacter asiaticus infection was developed, which may significantly reduce diagnostic time and cost for citrus Huanglongbing, and serves as pre-symptomatic diagnosis. A multi-pest surveillance method for statewide sweeps for Huanglongbing (HLB) and its vector and several other diseases including Citrus black spot (CBS) has been very successful, is continuously adapted to new disease priorities and personnel/resource capacities as requested by USDA APHIS and California Department of Food and Agriculture (CDFA), and is re-deployed yearly with updated survey design. Risk-based residential and commercial survey methods for Asian Citrus psyllid (ACP) and HLB are in the ninth year of deployment in California. Model validation indicated the models and linked surveillance are highly successful as they continually detect new introductions in Southern California which have now exceeded 2100 as of March 31, 2020. At the request of CDFA, a spatiotemporal analysis of HLB detections resulted in a recommendation of delimiting surveys around new detections to be reduced from 800m to 400m with great savings in personnel and fiscal resources. CDFA now routinely uses the reduced delimiting survey methodology to mitigate the disease pressure in new HLB detection locations. A risk-based survey to detect plum pox virus (PPV) outbreaks in New York State is integrated into the State/Federal New York PPV eradication program and completed the eighth year of deployment, successfully eradicating PPV in New York. A risk-based model and survey to detect new PPV introduction was developed and will be deployed in California when personnel/resources allow. Our surveys developed for multiple pathogens are being used by regulatory agencies and commodity groups to target disease/vector hotspots for existing Huanglongbing (HLB) and predict new outbreak locations. These are revised each year to optimize surveys to the changing disease/vector conditions and will be used for multiple years into the future. Models for Asiatic citrus canker (ACC), HLB, PPV and Citrus tristeza continue to be augmented and extended in unique ways to address specific regulatory issues as they arise. These models and their refinement continues to be the foundation for future work. Solar thermotherapy reduces the titer of Candidatus Liberibacter asiaticus (Las) and enhances canopy growth by altering gene expression profiles in Huanglongbing- (HLB-) affected citrus plants. Despite the presence of Las in heat-treated commercial citrus, many trees produced abundant flush and grew vigorously for two to three years after treatment. Transcriptome analysis comparing healthy trees to HLB-affected citrus both before and after heat treatment demonstrated that post-treatment transcriptional expression patterns more closely resembled the expression patterns of healthy controls. In addition, thermotherapy significantly improved the quality of orange juice. Overall, these results indicate that solar thermotherapy can be an effective component of an integrated control strategy for citrus HLB. An agent-based model has also been developed to examine the efficacy of ACP and HLB control strategies in selected locations in California with consideration of social and economic perspectives, area-wide approaches and delimitation protocols. A census-travel model to predict introductions of pathogens from foreign sources has been integrated into HLB, PPV, and other surveys as an added risk factor and has recently been published as a stand-alone method. A Bayesian Belief Network model to assess phytosanitary health of seed commodities has been developed, and has been validated for a tomato seed-borne disease and is currently being extended to other seed bacterial, viral and fungal pathosystems and being incorporated into systems approaches for seed health management. A manuscript documenting the research associated with the “Plant Sauna” and the application of precision thermotherapy to strawberry nursery stock was prepared and submitted. Watermelon and pepper germplasm evaluations for squash vein yellowing virus (SqVYV), powdery mildew and/or tospoviruses are continuing. Virus sanitation trial continued.

Accomplishments
1. Canine detection of Huanglongbing (HLB) in California potentially mitigates an impending state-wide epidemic. HLB epidemics continue to spread worldwide and devastate all citrus industries. The key to mitigation of HLB is early detection and rapid response. Through the HLB MAC (Multi-Agency Coordination) grants program ARS researchers at Fort Pierce, Florida, have trained 20 dogs for early detection with >99% accuracy within 2 weeks of infection (months to years earlier than other current assays) in part due to the dogs’ ability to sample the entire tree. In 2019-2020 California growers and the California Department Food and Agriculture (CDFA) integrated and deployed the canines into CDFA surveys to detect asymptomatic incipient infections of HLB for early response and tree removal. Simulations demonstrate how the impending epidemic is greatly mitigated by the incorporation of canine detection teams for early detection. Use of Candidatus Liberibacter asiaticus (Las) cultures demonstrated that dogs were detecting the actual pathogen. Canine detection of HLB was featured in FY19 ARS Annual Report on Science and also in FY21 Budget Explanatory Notes.

2. Risk model and resulting risk-based survey direct California program to combat Huanglongbing (HLB). To effectively mitigate the devastating effects of the HLB epidemic on the California citrus industry, ARS researchers at Fort Pierce, Florida, devised and deployed models and surveillance methods to search the large (300,000 acre) commercial citrus industry in addition to the vast residential plantings of citrus (varies by county but about 60 % of residences have at least one citrus tree). At present the state of California has 75-100 surveyors dedicated to state-wide surveys but they cannot reach all commercial and residential properties. ARS researchers have developed a risk-model that uses 10 risk criteria to rank each 1 square mile area in California. Via this model ARS researchers have designed and deployed a risk-based survey in California to target and optimize survey efforts. ARS researchers have also designed a 3-cycle survey based on the risk model to survey the entire state 3 times per year to capture the seasonality of disease development. The risk-based residential and commercial survey methods for Asian Citrus psyllid (ACP) and HLB are in the ninth year of deployment in California and validation indicated they have been highly successful and have become the mainstay of the large-scale statewide survey in California. The survey continually detects new infections in Southern California, which have now exceeded 2100 as of March 31, 2020. These detections are immediately removed and result in a quarantine surrounding the detection source with delimiting response protocols. At present the survey is done visually with PCR confirmation of suspicious trees, however, canine detection will likely be integrated into the risk-based survey.

3. Epidemiological models predict the spread of two severe citrus diseases by hurricanes. Asiatic citrus canker (ACC) and citrus black spot (CBS) are two serious diseases that are not only causing economic damage but are severe impediments to international trade of citrus as a commodity. ACC, caused by a bacterium, and CBS, caused by a fungus, are both dispersed by rain splash. In the presence of hurricanes such rain splash can be spread over many miles. Two such hurricanes, Harvey in southeast Texas, and Irma in southwest Florida potentially spread ACC and CBS, respectively in 2017. ARS researchers at Fort Pierce, Florida, adapted and extended a previously developed hurricane dispersal model to address both diseases and make predictions for where these infections may have spread due to the hurricanes. The results of these model predictions were presented to the appropriate regulatory agencies and science advisory committees. ARS researchers also developed risk-based surveys to target areas rejected by the hurricane models. As a result Texas and Florida state regulatory agencies in collaboration with USDA, Animal and Plant Health Inspection Service have deployed the surveys for early detection of potential spread of these diseases in both states.

4. Areawide pest management (AWPM) is an alternative for whitefly-transmitted virus management in vegetable crops. Vegetable production in Florida and the southeastern U.S. has been severely impacted by a number of whitefly-transmitted viruses over the last 20 years, particularly tomato yellow leaf curl virus (TYLCV) in tomato. Current strategies for management focus on farm-centric tactics that rely mainly on strategic insecticide application for whitefly vector control, use of virus-resistant cultivars, rogueing infected plants and various cultural controls. With the exception of resistant cultivars, these approaches have had limited success for controlling both virus and whitefly. Recent research characterizing the spatial and temporal dynamics of TYLCV and its whitefly vector revealed that AWPM may be a viable alternative for managing TYLCV and likely other whitefly-transmitted viruses of vegetables. A strong correlation between TYLCV and whitefly pressure was found in neighboring fields, and could extend to approximately 3 kilometers, particularly in the latter part of the season when whitefly pressure increased. ARS researchers at Ft. Pierce, Florida, are currently working with local crop consultants and an agricultural technology company to develop the tools for real-time mapping and a system for information delivery to foster development of AWPM as a new strategy for managing whitefly-transmitted virus epidemics in Florida and the southeast U.S. We are also working to establish canine detection of whitefly-transmitted vegetable viruses.

5. Xanthomonas fragariae, the causal agent of angular leaf spot (ALS) of strawberry, can be found routinely on strawberry nursery stock. Heat treatment has been shown previously to be an effective treatment for managing ALS on nursery stock in small-scale experimental trials. ARS researchers at Ft. Pierce, Florida designed, built and tested a commercial-scale precision thermotherapy unit (PTU) for applying a new thermotherapeutic protocol on strawberry nursery stock that combined a conditioning thermal treatment with an eradicative thermal treatment. Several trials were conducted in cooperation with commercial nurseries to determine the impact of thermotherapy on plant health and on the natural development of ALS. Overall, precision thermotherapy had a negligible effect on plant growth and yield. Angular leaf spot, when it occurred, was always lower in thermotherapy-treated plots. Successfully scaling-up thermal treatment of nursery stock to commercial levels could have a unique impact on controlling pests and diseases while simultaneously reducing or eliminating pesticide use against a broad range of threats to sustainable strawberry production.

6. The citrus industry maintains an interest in identifying antimicrobial compounds effective against Candidatus Liberibacter asiaticus (Las) the cause of Huanglongbing (HLB). A vacuum-assisted infiltration assay developed by ARS scientists in Fort Pierce, Florida, with individual HLB-symptomatic leaves followed by grafting was used to assay a range of antimicrobial compounds and found no additional effective compounds.

7. Field evaluation of integrated management for mitigating citrus Huanglongbing (HLB) in Florida. ARS and University of Florida researchers in Fort Pierce, Florida, implemented an integrated strategy that includes chemotherapy, thermotherapy, and additional nutrition treatment in field trials over three years. To assess the complex interactions, they used several methods for evaluating the effectiveness of integrated management, including the slopes of the cycle threshold (Ct) increase, the pathogenic index and the decline index from Ct value and tree scores, and the antimicrobial efficacies from pathogenic and decline indices. This comprehensive analysis showed that most of the tested chemicals were effective to some degree in killing or suppressing the Candidatus Liberibacter asiaticus (Las) bacterium. Trunk-injected penicillin was the most effective chemical treatment in all groves, followed by oxytetracycline, and silver nitrate delivered as foliar sprays. Although the steam heat treatment and additional nutrition did not eliminate or suppress Las bacteria over the long term, these treatments did positively affect tree growth and recovery in the short term. Overall, our results provide new insights into HLB control method and strategy for integrated management for HLB epidemic plantations.

8. Symptom inducer of Candidatus Liberibacter asiaticus (Las) infection determined. LasDelta5315 effector induces extreme starch accumulation and chlorosis in Las infection in Nicotiana benthamiana, and the effector SDE1 (5315) targets DEAD-box RNA 2 helicase DDX3 to modulate chlorosis. ARS researchers at Fort Pierce, Florida, demonstrated that the key function of the LasDelta5315 effector induced excessive starch accumulation. Furthermore, this study demonstrated that SDE1 is controlled by a complex promoter, and targets DEAD-box RNA helicase DDX3 to induce chlorosis by downregulating the DDX3 gene expression.

Review Publications
Gottwald, T.R., Gabriel, D. 2020. Bacterial pathogens of Citrus: Citrus canker, Citrus Variegated Chlorosis and Huanglongbing. In: Lopes, S.A., Wulff, N.A., editors. The Genus Citrus. 1st Edition. Sawston, Cambridge, UK. Woodhead Publishing. Chapter 18:371-389. https://doi.org/10.1016/b978-0-12-812163-4.00018-8.
Milne, A., van den Bosch, F., Gottwald, T.R., Parnell, S.R., Chavez, V.A. 2020. What makes or breaks a campaign to stop an invading plant pathogen? PLoS Computational Biology. 16(2):e1007570. https://doi.org/10.1371/journal.pcbi.1007570.
Hughes, H.R., Adkins, S.T., Alkhovsky, S.V., Beer, M., Blair, C.D., Calisher, C.H., Drebot, M., Lambert, A.J., Marciel De Souza, W., Marklewitz, M., Nunes, M.T., Shí, X., ICTV Consortium. 2020. ICTV Virus Taxonomy Profile: Peribunyaviridae. Journal of General Virology. 101:1-2. https://doi.org/10.1099/jgv.0.001365.
Hughes, H.R., Adkins, S.T., Alkhovsky, S.V., Beer, M., Blair, C.D., Calisher, C.H., Drebot, M., Lambert, A.J., de Souza, W. M., Marklewitz, M., Nunes, M.T., Shí, X. Peribunyaviridae. In: Virus Taxonomy: The Classification and Nomenclature of Viruses. 10th Report of the Internaional Committee on Taxonomy of Viruses. Available: https://talk.ictvonline.org/ictv-reports/ictv_online_report/negative-sense-rna-viruses/bunyavirales/w/peribunyaviridae 2019. (Online Book Chapter).
Anco, D., Rouse, L., Lucas, L., Parks, F., Mellinger, C., Adkins, S.T., Kousik, C.S., Roberts, P., Stansly, P., Ha, M., Turechek, W. 2019. Spatial and temporal physiognomies of whitefly and tomato yellow leaf curl virus epidemics in southwestern Florida tomato fields. Phytopathology. 110(1):130-145. https://doi.org/10.1094/PHYTO-05-19-0183-FI.
Gottwald, T.R., Luo, W., Posny, D., Poole, G.H., Louws, F., Mccollum, T.G., Hartung, J.S., Bai, J., Duan, Y., Taylor, E.L., Da Graça, J., Schneider, W., Polek, M., Hall, D. 2020. Canine olfactory detection of a vectored phytobacterial pathogen, Liberibacter asiaticus, and intergration with disease control. Proceedings of the National Academy of Sciences. 117(7)3492-3501. https://doi.org/10.1073/pnas.1914296117.
Wang, H., Turechek, W. 2020. Detection of viable Xanthomonas fragariae cells in strawberry ssing propidium monoazide and long-amplicon quantitative PCR. Plant Disease. 104(4):1105-1112. https://doi.org/10.1094/PDIS-10-19-2248-RE.
Yang, C., Huang, J.Y., Powell, C., Duan, Y., Ancona, V., Zhang, M. 2020. Transcriptomic analysis reveals root metabolic alteration and induction of Huanglongbing resistance by Sulfonamide antibiotics in Huanglongbing-affected citrus plants. Plant Pathology. https://doi.org/10.1111/ppa.13154.
Sheng, L., Feng, W., Duan, Y., Zhengfei, G., Singerman, A. 2020. Citrus greening: Management strategies and their economic impact. HortScience. p. 1-9. https://doi.org/10.21273/HORTSCI14696-19.
Miller, M., Deiulio, A., Holland, C., Douthitt, C., Mcmahon, J., Wiersma-Koch, H., Turechek, W., D'Elia, T. 2020. Complete genome sequence of Xanthomonas phage RiverRider, a novel N4-like bacteriophage that infects the strawberry pathogen Xanthomonas fragariae. Archives of Virology. 165:1481-1484. https://doi.org/10.1007/s00705-020-04614-6.
Graham, J., Gottwald, T.R., Setamou, M. 2020. Status of Huanglongbing (HLB) outbreaks in Florida, California and Texas. Tropical Plant Pathology. 2020:e-ISSN 1983-2050. https://doi.org/10.1007/s40858-020-00335-y.
Gadhave, K.R., Gautam, S., Dutta, B., Coolong, T., Adkins, S.T., Srinivasan, R. 2020. Low frequency of horizontal and vertical transmission of cucurbit leaf crumple virus in whitefly Bemisia tabaci Gennadius. Phytopathology. 10. https://doi.org/10.1094/PHYTO-09-19-0337-R.
Ippolito, S., Laborde, J., Gottwald, T.R., Irey, M.S. 2020. Studying the spatial temporal spread of the citrus tristeza virus through ODEs and Bernoulli trials. Journal of Theoretical Biology. 497:1-13. https://doi.org/10.1016/j.jtbi.2020.110279.
Gautam, S., Gadhave, K.R., Buck, J.W., Dutta, B., Coolong, T., Adkins, S.T., Srinivasan, R. 2020. Virus-virus interactions in a plant host and in a hemipteran vector: Implications for vector fitness and virus epidemics. Virus Research. 286 (2020) 198069. https://doi.org/10.1016/j.virusres.2020.198069.
Adegbola, R.O., Marshall, S.H., Batuman, O., Ullman, D.E., Gilbertson, R.L., Adkins, S.T., Naidu, R.A. 2019. Sequence analysis of the medium and small RNAs of impatiens necrotic spot virus reveals segment reassortment but not recombination. Archives of Virology. https://doi.org/10.1007/s00705-019-04389-5.
Hilf, M.E., Luo, W. 2020. Inoculation period and citrus host effect establishment of new infections of ‘Candidatus Liberibacter asiaticus’ transmitted via vegetative grafting. Plant Disease. 104:1894-1899. https://doi.org/10.1094/PDIS-09-19-2022-RE.
Scott-Brown, A.S., D'Elia, T., Devey, D.S., Funderburk, J.E., Adkins, S.T. 2020. Genome characterization of Brugmansia latent virus, a novel tobamovirus. Archives of Virology. https://doi.org/10.1007/s00705-020-04718-z.