EMERGING DISEASES OF CITRUS, VEGETABLES, AND ORNAMENTALS

We have exceeded the milestones originally set out in the project plan for all objectives by augmenting models beyond projected applications, developing additional spinoff models, developing extended or new survey methods, characterizing pathogens' biology and/or epidemiology more fully, exceeding the depth of molecular analyses of pathosystems, developing more extensive detection methods, additional thermotherapy methods, extending canine detection to other diseases such as Plum Pox Virus (PPV), and extending disease control approaches for multiple diseases. Where we have exceeded the milestones has also lead to augmentation of or additional technology transfer not outlined in the initial project goals or objectives. Progress on culture of Candidatus Liberibacter asiaticus (Las) in vitro was slow. New media obtained via a CRADA did not support growth of Las in experiments at ARS or in labs of the CRADA partner; the CRADA partner did not renew the CRADA upon expiration. An MTA with a new partner was established to culture Las and experiments are in progress. Genetic diversity of Las was demonstrated with molecular markers to differentiate mild and severe strains/isolates. Three novel effectors and their interactions with host proteins were further characterized. In addition, we have identified two more effectors of Las bacterium, and they targeted host mitochondria and chloroplasts, respectively. Studies on Huanglongbing effects on seedling growth and vitality are ongoing. A controlled experimental system to study Las population characteristics and growth was developed. A small scale, short term protocol for assessing antimicrobial compounds is being developed. Metabolomic analyses to identify compounds associated with early Las infection are ongoing. Transmission of Squash vein yellowing virus (SqVYV), tospoviruses and ilarviruses is continuing to be elucidated. Host range, geographic range and genetics of tospoviruses and ilarviruses were investigated. Crown-and- systemic infection studies with green fluorescent protein (GFP)- transformed Xanthomonas fragariae (Xf) were completed and manuscript is currently being written. Plots to test the interaction of Asiatic citrus canker (ACC), leafminer, and wind breaks were established in Brazil in 2010. Data collection complete and Brazilian collaborators have decided to extend project to collect even more data. Results indicated 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. This has resulted in international phytosanitary regulations lessening for ACC and has kept domestic citrus markets open worth $40-100 million per year. Meteorological, insect count, and virus incidence data continue to be collected in cucurbit and solanaceous crops. 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 LAMP detection assay for Xanthomonas fragariae (Xf) was developed and the manuscript was published. The PMA-qPCR protocol was completed and the manuscript is in preparation. A multi-pest surveillance method for statewide sweeps for Huanglongbing 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 Huanglongbing are in the fifth year of deployment in California, Texas and Arizona, and validation indicated highly successful as they continually detect new introductions especially in Southern California. Deployed surveys are being used by regulatory agencies and commodity groups to target disease/vector hotspots for existing Huanglongbing and predict new outbreak locations. These are revised each year to optimize surveys to changing disease/vector conditions and will be used for several years into the future. Both ACC and Huanglongbing stochastic models have been developed, validated and publications are in print. A user-friendly front end to both canker and Huanglongbing models is completed for use by regulatory agencies and commodity groups and has been published. These models and their refinement will be the foundation for continued future work. AgScouter development is complete and is commercially available online. Tests using guava as an intercrop between citrus trees as a mitigation strategy for Huanglongbing were completed and published. A Citrus black spot (CBS) probabilistic risk model is in the 5th year and fully developed and publication in draft. CBS model 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 Huanglongbing (HLB) in greenhouse and field trials. Heat treatment improved HLB-affected citrus plants in greenhouse settings, providing a simple, effective method for control. Field trials of heat treatment demonstrated its effectiveness on mitigating HLB in citrus groves. To improve the efficacy for the control of HLB, field trials are underway with integration of thermotherapy and chemotherapy. Second generation hydro-solar thermotherapy equipment has been developed and is under validation that can treat large acreages simultaneously. Analysis of virus sanitation trial data is continuing. For strawberry, a commercial-scale �Plant Sauna� was constructed at a commercial nursery in California. Heat-treatment trials were repeated in California with three nursery collaborators. Accomplishments 01 Emerging tospovirus Tomato chlorotic spot virus (TCSV) identified in new geographic areas and hosts in Florida and Caribbean. TCSV was first found in in $437 million annual fresh-market tomato crop in Florida in 2012 by ARS scientists in Fort Pierce, Florida. The TCSV host range has expanded in Florida and Puerto Rico to include all major solanaceous vegetable crops, and in the last year non-solanaceous ornamentals, herbs and weeds. Portulaca, Marsdenia, sweet basil, erect spiderling, Asian spiderflower and sweet chili pepper have been identified as TCSV hosts in collaboration with state regulatory agency, industry and university scientists highlighting potential for further spread in the U.S. Thrips vector species have been collected and identified in locations of TCSV outbreaks. Little genetic diversity was observed in TCSV sequences consistent with its recent introduction. 02 An efficient and high fidelity method for amplification, cloning and sequencing of complete tospovirus genomic RNA segments was developed by ARS scientists in Fort Pierce, Florida, in cooperation with university scientists. This experimental tool was subsequently employed for phylogenetic analysis of worldwide tospovirus isolate collections. Demonstration of the first transmission of Tomato spotted wilt virus (TSWV) from infected tomato fruits by western flower thrips, and the first resistance-breaking strain of TSWV in California, also benefited from this technique. Continuing widespread economic losses from TSWV reflect its nationwide importance. 03 Heat treatment of strawberry nursery stock. A large steam chamber (�Plant Sauna�) was built at a commercial strawberry nursery in California for use in heat treating strawberry nursery stock to manage nursery-borne diseases, including angular leaf spot, anthracnose, powdery mildew, and gray mold. ARS scientists in Fort Pierce, Florida, developed a heat-treatment protocol that combines a conditioning, sub- lethal heat treatment of plants followed by a higher temperature heat treatment targeting the pathogens. The conditioning treatment is used to induce a chemical response in the plant that serves to protect plants from damage when treated at higher temperatures. Initial field testing with a single nursery collaborator showed minimal impact on horticultural and production traits, but a reduction in disease. Further testing with three additional nurseries confirmed initial results. Overall, it appears that steam heat-treatment is a viable option for disease management of nursery stock that could lead to a significant reduction in the use of pesticides for disease management if adopted by the industry. 04 Whole leaf grafting was used to transmit the Huanglongbing (HLB) bacterium, Candidatus Liberibacter asiaticus (Las). This transmission system was used to develop a small scale assay to find effective antimicrobial compounds. This method reduces the size of plants which can be infected and in which full symptoms of HLB develop. Single, infected symptomatic leaves were exposed to antimicrobial compounds under various conditions and grafted to receptor trees in which infections and disease development were monitored. Experiments assessing antimicrobial compounds are ongoing. 05 Selected and evaluated natural variants of commercial varieties of citrus seedlings and bud sports with greater Huanglongbing (HLB) tolerance/resistance. ARS scientists in Fort Pierce, Florida, have initiated the screening of natural mutant citrus plants or bud sports of commercially grown citrus cultivars with greater HLB tolerance/ resistance by taking advantage of existing genetic variations, and selected and evaluated a number of natural variants of commercial citrus seedlings and bud spots with greater HLB tolerance/resistance. This provides another tool for management of HLB. 06 Resistance gene identified for Huanglongbing bacterium, Candidatus Liberibacter asiaticus (Las). ARS scientists in Fort Pierce, Florida, identified 16 putative Las effectors (proteins secreted by Las into plant host cells) and revealed diverse subcellular localization with different shapes and aggregation patterns of these effector candidates in plants. One of these efforts was shown to function as an avirulence protein that induced plant host cell death. This facilitated identification of a resistance gene (member of the NB-LRR family) in tobacco plants. 07 Canine disease detection research is highly successful. Sixteen years of canine disease detection research has culminated in training 20 dogs to detect Huanglongbing (HLB) at >99.17% accuracy. Three HLB detector dogs have been commercially deployed to California and are gaining interest in the citrus industries of California, Texas, Arizona, and Mexico. Commercialization plans by detector dog training companies are complete. This is a USDA APHIS PPQ HLB MAC project. 08 Census travel based model developed to predict new disease introductions. The census travel based model combines U.S. census data and international travel data from multiple �big data� sources to predict the introduction of plant pathogens and pests. The model has been deployed to predict points of introduction and forms the basis of statewide surveys for citrus Huanglongbing, Plum Pox, citrus canker, citrus scab and other diseases. The model has been tested and also validated to predict introductions of human and animal pathogens as well. This has resulted in strong interest of DHS, U.S. military, and medical researchers for multiple human diseases including Ebola virus, Zika, malaria, Dengue and Chagas diseases. The model is also being adapted for tracking introductions and spread of feral swine. 09 Multi-pest surveillance (MPS) method developed for Huanglongbing (HLB). ARS scientists in Fort Pierce, Florida, in collaboration with UK researchers developed a multi-pest surveillance method for statewide sweeps for HLB, its vector and several other diseases including citrus black spot (CBS). The methodology has been very successful, is continuously adapted yearly to new disease priorities as requested by USDA APHIS, and is re-deployed yearly. California regulatory agencies have asked that we develop a MPS specifically for California. Risk- based residential and commercial survey methods for Asian Citrus Psyllid (ACP) and HLB are in the 5th year of deployment in California, Texas and Arizona, and validation indicates they are successful in detecting new HLB outbreaks (over 60 detections to date). Surveys are being used by regulatory agencies (CDFA and APHIS) and commodity groups to target disease/vector hotspots for existing HLB and predict new outbreak locations. Survey data and model risk predictions provide the empirical evidence on which management and regulatory decisions are being made. A CBS probabilistic risk model is in the fifth year of development and will be important to citrus international trade to determine if fruit are a pathway for disease establishment in new locations.