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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

2015 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. New media obtained via a CRADA is being evaluated as substrate to culture Las; experiments are ongoing. 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. In addition, we have identified 4 more effectors of Las bacterium, and they targeted host mitochondria and chloroplast, respectively. Studies on Huanglongbing effects on seedling growth and vitality are ongoing. Work to assess the cell viability of Las in plant tissues is ongoing. Studies of resistance in Murraya paniculata to Las infection are ongoing. Purification of Las from plant and insect tissues is ongoing. Protein-protein interactions between Las and the Asian citrus psyllid vector were characterized; a manuscript was submitted to PLoS One and is under review. 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-and-systemic-infection studies with GFP-transformed X. fragariae complete and manuscript is currently being written. Under sub-objective 1c: Plots to test the interaction of 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 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 Xf was developed and the manuscript was written and has been submitted for publication. The PMA-qPCR protocol is in its final stages of evaluation. Under sub-objective 2b: A multi-pest Surveillance method for statewide sweeps for Huanglongbing and its vector and several other diseases including 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 ACP and Huanglongbing are in the third year of deployment in California, Texas and Arizona, and validation indicates they are highly successful as they continually detect new introductions. Surveys are being used by regulatory agencies and commodity groups to target disease/vector hotspots for existing Huanglongbing 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 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 future work. AgScouter’s management module was improved by purchasing access to Greenbook’s agricultural chemical database for Florida. Access to the database was purchased for one year and we are testing it with collaborators in Florida. Under sub-objective 3b: Tests using guava as an intercrop between citrus trees as a mitigation strategy for Huanglongbing were completed and published. A Citrus Black Spot probabilistic risk model is in the 4th year and fully developed and publication in draft. Citrus Black Spot 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 in greenhouse and field trials. Heat treatment cured Huanglongbing-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. To improve the efficacy for the control of citrus Huanglongbing, field trials are underway with integration of thermotherapy and chemotherapy. For strawberry, a commercial-scale “Plant Sauna” was constructed at a commercial nursery in California, and heat-treatment trials are currently underway.


Accomplishments
1. Continued host range expansion of Tomato chlorotic spot virus (TCSV). An emerging tospovirus TCSV continues to be frequently detected in tomato and pepper tospovirus-like symptoms in south Florida. Five new plant hosts (three ornamental crops, a solanaceous crop and a solanaceous weed) were identified by ARS scientists in Fort Pierce, Florida, in collaboration with state regulatory agency, industry and university scientists. Little genetic diversity was observed in TCSV sequences consistent with its recent introduction to the U.S. Detection of additional host species highlights the continuing spread of TCSV in the U.S.

2. Squash vein yellowing virus (SqVYV). SqVYV is the cause of viral watermelon vine decline, identified for the first time in Central America. Watermelon plants in Guatemala were observed to collapse near harvest leaving non-marketable fruits exhibiting symptoms of internal rind necrosis similar to viral watermelon vine decline originally characterized by ARS scientists in Fort Pierce, Florida and Charleston, South Carolina, in collaboration with state regulatory agency and university scientists. SqVYV was identified in samples from Guatemala using molecular techniques developed by ARS scientists (in Fort Pierce, Florida) in collaboration with state regulatory, industry and university scientists. Expansion of the geographic range of of SqVYV to Guatemala provides further opportunity for spread of this emerging whitefly-transmitted virus.

3. LAMP assay and a new sample preparation procedure developed. ARS Scientists in Ft. Pierce, FL developed a LAMP assay and a new sample preparation procedure to increase the likelihood for detecting low densities of Xanthomonas fragariae in strawberry, the cause of angular leaf spot. X. fragariae is an important disease worldwide and significantly affects the commercial value of the crop. The bacteria survive in or on plants at low densities without producing symptoms, thus reducing the ability to identify infected plants and hampering disease management. LAMP offers the combined benefit of greater sensitivity/specificity, rapid implementation, and/or cost-effectiveness than previous detection tools. It will contribute to improved disease detection and management in the strawberry industry.

4. A commercial-scale “Plant Sauna” was built and is currently being evaluated at commercial strawberry nursery in California. ARS Scientists in Ft. Pierce, FL modified the original design of the Plant Sauna to include a vacuum infiltration system that allows users to heat treat strawberry nursery stock in their final packaging. This modification was of central importance to the industry and now enables heat-treatment to be a viable option for disease and insect management of nursery stock. Field testing of heat-treated plants is being done to evaluate the effect on horticultural and production traits, as well as efficacy on a number of nursery-related diseases, including angular leaf spot, anthracnose, powdery mildew, and gray mold.

5. Whole Leaf Grafting to Transmit Las. ARS Scientists in Ft. Pierce, FL developed a method of transmitting Las to citrus plants by grafting single, intact leaves to each plant. This method reduces the size of plants which can be infected and in which full symptoms of Huanglongbing develop. The rate of infection is comparable to that of the standard grafting procedure. This method uses smaller plants so more can be included in experiments in a reduced space. In addition, changes in gene expression and other physiological changes can be analyzed in both roots and shoots of an entire intact plant, and not just samples of these tissues from a plant as is currently done.

6. Asian Citrus Psyllid Transmission of Las to Small Citrus Trees as an Early and Rapid Screen for Susceptibility to Infection. ARS Scientists in Ft. Pierce, FL exposed small grafted trees of 16 citrus cultivars to Asian citrus psyllids for up to 45 weeks. Statistically significant differences were observed between cultivars in the number of infected trees, the time needed for infection to occur, and the titer of bacteria that developed. The use of small plants in this experimental system can facilitate screening for citrus cultivars/species which have a decreased susceptibility to infection by insects or an increased resistance to the growth of Las bacteria.

7. Genes related to huanglongbing (HLB) tolerance were identified by transcriptome profiling HLB tolerance and HLB susceptible citrus plants. ARS Scientists in Ft Pierce, FL examined the expression differences between HLB-tolerant ‘Jackson’ grapefruit hybrid trees and HLB susceptible ‘Marsh’ grapefruit trees after HLB infection using RNA-seq. These two cultivars are very closely related, and are very similar in phenotype, but differ markedly in susceptibility to HLB. A total of 686 differentially expressed (DE) genes between two groups were identified. Among them, 247 genes were up-regulated and 439 were down-regulated in tolerant citrus trees. We also identified a total of 619 genes with significantly differential expression of alternative splicing isoforms between HLB tolerant and HLB susceptible citrus trees. We analyzed the functional categories of DE genes using two methods. We found that several DE genes are related to the activation of basal resistance in HLB tolerant citrus trees.

8. Thermotherapy using a portable greenhouse. ARS Scientists in Ft Pierce, FL implemented field trials using integrated strategy of thermotherapy and chemotherapy with several selective chemicals using a newly developed delivery system. Thermotherapy using a portable greenhouse was one of the heat treatment approaches that are currently used by the Florida growers.

9. Canine disease detection research. 15 years of canine disease detection research has culminated in training 10 dogs to detect Huanglongbing and three dogs to detect citrus canker all at > 99.97% reliability. Dogs will be deployed over the next two years to various affected states and commercialization plans are in progress with dog training companies. USDA APHIS PPQ Farm Bill project.

10. Census travel basead predictive model. ARS Scientists in Ft Pierce, FL developed a census travel based predictive model to predict the introduction of plant pathogens and pests that also works well on human and animal pathogens. This has resulted in strong interest of DHS, U.S. military, and medical researchers for multiple human diseases including Ebola virus and Chagas disease plus tracking introductions and spread of feral swine.

11. Multi-pest surveillance method developed for Huanglongbing (HLB). ARS Scientists in Ft Pierce, FL developed a multi-pest surveillance method for statewide sweeps for HLB, its vector and several other diseases including citrus black spot (CBS) has been very successful, is continuously adapted yearly 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 4th 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. 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 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.


Review Publications
Tomassoli, L., Manglli, A., Tiberini, A., Adkins, S.T. 2015. Investigation on the phytosanitary status of major ornamental hibiscus species in Italy to assess virus infection. Acta Horticulturae. 1072:29-35.
Adkins, S.T., Webster, C.G., Mellinger, H.C., Frantz, G., Turechek, W., Mcavoy, E., Reitz, S.R., Funderburk, J.E. 2015. Detection and characterization of tomato viruses: A case study of emerging tospoviruses in Florida. Acta Horticulturae. 1069: 83-85.
Tyler-Julian, K.A., Funderburk, J.E., Olson, S.M., Paret, M.L., Webster, C.G., Adkins, S.T. 2015. A stimulo-deterrent method of thrips and Tomato spotted wilt virus management in tomatoes. Acta Horticulturae. 1069:251-258.
Zhang, M., Guo, Y., Powell, C., Doud, M.S., Yang, C., Duan, Y. 2014. Effective antibiotics against 'Candidatus Liberibacter asiaticus' in HLB-affected citrus plants identified via the graft-based evaluation. PLoS One. 9(11):e111032. doi: 10.1371/journal.pone.0111032.
Webster, C.G., Frantz, G., Reitz, S.R., Funderburk, J.E., Mellinger, H.C., Mcavoy, E., Turechek, W., Marshall, S.H., Tantiwanich, Y., Mcgrath, M.T., Daughtrey, M.L., Adkins, S.T. 2015. Emergence of Groundnut ringspot virus and Tomato chlorotic spot virus in Vegetables in Florida and the Southeastern United States. Phytopathology. 105:388-398. https://doi.org/10.1094/PHYTO-06-14-0172-R.
Rosskopf, E.N., Lucas, L., Mellinger, H., Adkins, S.T. 2014. First report of tomato mottle mosaic virus infecting tomato in the United States. Plant Health Progress. 15:151-152.
Puttamuk, T., Zhou, L., Thaveechai, N., Zhang, S., Armstrong, C.M., Duan, Y. 2014. Genetic diversity of Candidatus Liberibacter asiaticus based on two hypervariable effector genes in Thailand. PLoS One. 9(12):e112968. doi: 10.1371/journal.pone.0112968.
Baker, C.A., Adkins, S.T. 2015. First report of tomato chlorotic spot virus in Hoya wayetii and Schlumbergera truncata. Plant Health Progress. 16(1):29-30. https://doi.org/10.1094/PHP-BR-14-0043.
Fillmer, K., Adkins, S.T., Pongam, P., D'Elia, T. 2015. Complete genome sequence of a Tomato mottle mosaic virus isolate from the United States. Genome Announcements. 3(2):e00167-15. doi: 10.1128/genomeA.00167-15.
Adkins, S.T., Baker, C.A., Badillo-Vargas, I.E., Frantz, G., Mellinger, H.C., Roe, N., Funderburk, J.E. 2015. Necrotic streak disease of tomato in Florida caused by a new ilarvirus species related to Tulare apple mosaic virus. New Disease Reports. 31:16.
Warfield, C.Y., Clemens, K., Adkins, S.T. 2015. First report of Tomato chlorotic spot virus on annual vinca (Catharanthus roseus) in the United States. Plant Disease. 99(6):895. https://doi.org/10.1094/PDIS-12-14-1269-PDN.
Fillmer, K., Adkins, S.T., Pongam, P., D'Elia, T. 2015. Complete genome sequence of Tomato mosaic virus isolated from jasmine in the United States. Genome Announcements. 3(4):e00706-15. doi: 10.1128/genomeA.00706-15.
Badillo-Vargas, I.E., Roe, N., Funderburk, J.E., Adkins, S.T. 2015. First report of Tomato chlorotic spot virus in scarlet eggplant (Solanum aethiopicum) and American black nightshade (Solanum americanum) in the United States. Plant Disease. Available: http://dx.doi.org10/1094/PDIS-04-15-0385-PDN.
Funderburk, C., Funderburk, J.E., Tyler-Julian, K., Srivistava, M., Knox, G., Andersen, P., Adkins, S.T. 2015. Population dyanamics of Frankliniella bispinosa (Thysanopatera: Thripidae) and the predator Orius insidiosus (Hemiptera: Anthocoridae) as influenced by flower color of Lagerstroemia (Lythraceae). Environmental Entomology. 44(3):668-679.
Jeyaprakash, A., Baker, C.E., Schubert, T.S., Badillo-Vargas, I.E., Roberts, P.D., Funderburk, J.E., Adkins, S.T. 2015. First report of Squash vein yellowing virus in watermelon in Guatemala. Plant Health Progress. 16(3):113-114.. https://doi.org/10.1094/PHP-BR-15-0019.
Anco, D.J., Poole, G.H., Gottwald, T.R. 2015. Postharvest quarantine treatments for Diaphorina citri on infested curry leaves. Plant Disease. 99(7):926-932. doi: 10.1094/PDIS-12-14-1271-RE.
Cunniffe, N.J., Koskella, B., Metcalf, J.E., Parnell, S., Gottwald, T.R., Gilligan, C.A. 2014. Thirteen challenges in modelling plant diseases. Elsevier. 10:6-10. doi: 10.1016/j.epidem.2014.06.002.