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


Location: Subtropical Plant Pathology Research

2016 Annual Report

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.

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
For all objectives we have exceeded the milestones initially set out in the project plan by augmenting models beyond their projected applications, developing new 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 project goals or objectives. 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. Three novel effectors and their interactions with host proteins were further characterized. In addition, we have identified 2 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. Metabolomic analyses to identify compounds associated with early Las infection are ongoing. Transmission of tospoviruses and Squash vein yellowing virus (SqVYV) is continuing to be elucidated. Host range, geographic range and genetics of tospoviruses were investigated. Similar studies on ilarviruses were initiated. Crown-and-systemic-infection studies with green fluorescent protein (GFP)- transformed Xanthomonas fragariae (Xf) complete 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. Meteorological, insect count, and virus incidence data continue to be collected in cucurbit/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 Xf was developed and the manuscript was published. The PMA-qPCR protocol is in its final stages of evaluation. 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 fourth year of deployment in California, Texas and Arizona, and validation indicated highly successful as they continually detect new introductions. Deployed 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 revised each year to re-optimize 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’s management module was improved by purchasing access to Greenbook’s agricultural chemical database for Florida. AgScouter is now 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 probabilistic risk model is in the 5th 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 improved 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. Analysis of virus sanitation trial data is continuing. For strawberry, a commercial-scale “Plant Sauna” was constructed at a commercial nursery in California, and heat-treatment trials are currently underway.

1. Emerging ilarvirus identified in fresh-market tomato in Florida. $437 million of fresh-market tomatoes are produced annually in Florida, ranking the state first in the U.S. ARS scientists in Fort Pierce, Florida, identified Tomato necrotic streak virus in two south Florida production areas. Host range and genome were characterized, and seed transmission in tomato was demonstrated, in collaboration with state regulatory agency, industry and university scientists. Little genetic diversity was observed in virus sequences consistent with its recent introduction to the U.S. Seed transmission highlights potential for spread in the U.S.

2. Genomics of emerging tobamoviruses of vegetables and ornamentals. Although tobamoviruses were the first virus discovered over 100 years ago, species in this virus group remain widespread economic problems because of the ease of seed transmission and stability of virus particles. The genome sequences for the first U.S. isolate of Tomato mottle mosaic virus, an ornamental plant isolate of Tomato mosaic virus, and Tropical soda apple mosaic virus (originally characterized by ARS scientists in Fort Pierce, Florida) were completed in collaboration with university scientists. Phylogenetic analysis shows all three viruses are most closely related to other solanaceous plant-infecting tobamoviruses. Availability of these sequences allows development of improved detection reagents for deployment by regulatory agencies.

3. Detection tools for Squash vein yellowing virus (SqVYV). ARS scientists in Fort Pierce, Florida, developed an enzyme linked-immunosorbent assay (ELISA) and a quantitative reverse transcription-PCR (qRT-PCR) for the identification of SqVYV, the cause of viral watermelon vine decline. SqVYV was first found in Florida, but the known geographic range of SqVYV has since increased with findings in Indiana, South Carolina, Georgia, California, Puerto Rico, Guatemala and Israel. The disease is difficult to identify based on symptoms alone, thus rapid and sensitive diagnostic tools are needed to facilitate detection. The two new procedures were capable of detecting SqVYV in a wide range of cucurbit hosts. qRT-PCR was able to detect the virus in over 99% of the samples where the virus was known to be present, whereas ELISA was able to detect the virus in 58-76% of known infections, depending on the host plant. Both tests had very similar false positive rates of <6% with ELISA sometimes outperforming qRT-PCR. The newly developed diagnostic tools provide more options for the detection of SqVYV and will assist in improved management of SqVYV-induced watermelon vine decline.

4. Heat treatment of strawberry nursery stock. A large steam chamber was built at a commercial strawberry nursery in California for 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 of the protocol showed minimal impact on horticultural and production traits, but a reduction in disease. This modification enables heat-treatment to be a viable option for disease management of nursery stock and could lead to a significant reduction in the use of pesticides for disease management if adopted by the industry.

5. Whole leaf grafting to transmit Las, the causative agent of citrus Huanglongbing (HLB). ARS scientists in Fort Pierce, Florida, 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 HLB develop. The rate of infection is comparable to that of the standard grafting procedure. We are adapting this method to evaluate anti-microbial compounds. Experiments have been established to assess impact on transmission of bacteria from treated compared to non-treated leaves.

6. 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.

7. Bactericide demonstrated to have dual functions for control of citrus canker. ARS scientists in Fort Pierce, Florida, demonstrated that bismerthiazol can effectively control citrus canker, a disease responsible for economic damages to citrus crops in subtropical production areas of the world. This compound inhibits growth of the causal bacterium Xanthomonas citri ssp. citri and triggers plant host defense response through expression of several pathogenesis-related genes (PR1, PR2, CHI and RpRd1) and NPR genes (NPR1, NPR2 and NPR3) in ‘Duncan’ grapefruit plants. This provides another tool for management of citrus canker.

8. Canine disease detection research is highly successful. Fifteen years of canine disease detection research has culminated in training 10 dogs to detect Huanglongbing and three dogs to detect citrus canker all at >99.17% accuracy. Ten more dogs will be fully trained to detect HLB for a total of 20 dogs by end of 2016. Dogs will be commercially deployed over the next two years to various affected states especially Texas and California. Commercialization plans by detector dog training companies are nearly complete. This is a USDA APHIS PPQ HLB MAC project.

9. 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 as the basis of statewide surveys for citrus Huanglongbing, citrus canker, citrus scab and other diseases. The model is being tested and also validated to predict introductions of 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, Zika, Malaria, Dengue and Chagas diseases. The model has also been adapted for plus tracking introductions and spread of feral swine.

10. Multi-pest surveillance (MPS) method developed for Huanglongbing (HLB). ARS scientists in Fort Pierce, Florida, developed a multi-pest surveillance method for statewide sweeps for HLB, its vector and several other diseases including citrus black spot (CBS) and it 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 4th year of deployment in California, Texas and Arizona, and validation indicates they are successful in detecting new HLB outbreaks. Surveys are being used by regulatory agencies (CDFA and APHIS) 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
Yang, C., Powell, C., Duan, Y., Shatters, R.G., Lin, Y., Zhang, M. 2016. Mitigating citrus huanglongbing via effective application of antimicrobial compounds and thermotherapy. Crop Protection Journal. 84:150-158.
Badillo-Vargas, I.E., Baker, C.A., Turechek, W., Frantz, G., Mellinger, H., Funderburk, J.E., Adkins, S.T. 2016. Genomic and biological characterization of Tomato necrotic streak virus, a novel subgroup 2 ilarvirus infecting tomato in Florida. Plant Disease. 100(6):1046-1053.
Adegbola, R., Fulmer, A., Williams, B., Brenneman, T., Kemerait, R., Sheard, W., Woodward, J., Adkins, S.T., Naidu, R. 2016. First report of the natural occurrence of tomato chlorotic spot virus in peanuts in Haiti. Plant Disease. 100(8):1797.
Fillmer, K., Adkins, S.T., Pongam, P., D'Elia, T. 2016. The complete nucleotide sequence and genomic characterization of tropical soda apple mosaic virus. Archives of Virology. 161:2317. doi: 10.1007/s00705-016-2888-6.
Armstrong, C.M., Dung, J.K., Humann, J.L., Schroeder, B.K. 2016. Effects of postharvest onion curing parameters on bulb rot caused by Pantoea agglomerans, Pantoea ananatis, and Pantoea allii in storage. Plant Pathology. 65:536-544. doi: 10.1111/ppa.12438.
Pitino, M., Armstrong, C.M., Duan, Y. 2015. Rapid Screening for Citrus Canker Resistance Employing Pathogen-Associated Molecular Pattern-Triggered Immunity Responses. Horticulture Research. 2:15042.
Wang, Y., Zhou, L., Li, D., Dai, L., Lawton-Rauh, A., Srimani, P.K., Duan, Y., Lou, F. 2015. Genome-wide comparative analysis reveals similar types of NBS genes in hybrid Citrus sinensis genome and original Citrus clementine genome and provides new insights into non-TIR NBS genes. PLoS One. 10(3):e0121893. doi: 10.1371.
Magarey, R.D., Hong, S., Fourie, P.H., Holtz, T., Fowler, G., Takehuchi, Y., Christie, D., Miles, A., Schutte, T., Gottwald, T.R. 2015. Prediction of Phyllosticta citricarpa using an hourly infection model and validation with prevalence data from South Africa and Australia. Crop Protection. 75(2015):104-114.
Taylor, E.L., Gottwald, T.R. 2015. Using Monte Carlo simulation to examine the economic cost and impact of HLB. Plant Disease. 99(7):926-932.
Rimbaud, L., Dallot, S., Gottwald, T.R., Decroocq, V., Jacquot, E., Soubeyrand, S., Thebaud, G. 2015. Sharka epidemiology and worldwide management strategies: learning lessons to optimize disease control in perennial plants. Annual Review of Phytopathology. 53:357-378. doi: 10.1146/annurev-phyto-080614-120140.
Turechek, W., Biggs, A. 2015. Maryblyt v. 7.1 for Windows: an improved fire blight forecasting program for apples and pears. Plant Health Progress. 16(1):16-22.
Yang, C., Power, C.A., Duan, Y., Shatters, R.G., Zhang, M. 2015. Antimicrobial nanoemulsion formulation with improved penetration of foliar spray through citrus leaf cuticles to control citrus Huanglongbing. PLoS One. 10(7):e0133826. doi: 10.1371/journal.pone.0133826.
Jeyaprakash, A.A., Baker, C.A., 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. doi: 10.1094/PHP-BR-15-0019.
Wang, Y., Zhou, L., Li, D., Lawton-Lauh, A., Srimani, P.K., Dai, L., Duan, Y., Luo, F. 2015. Genome-Wide Comparative Analysis Reveals Similar Types of NBS Genes in Hybrid Citrus sinensis Genome and Original Citrus clementine Genome and Provides New Insights into Non-TIR NBS Genes. PLoS One. 10(3):e0121893.
Parnell, S., Gottwald, T.R., Riley, T.D., Van Den Bosch, F. 2014. A generic risk-based surveying method for invading plant pathogens. Ecological Applications. 24(4):779-790.
Parnell, S., Gottwald, T.R., Cunniffe, N.J., Chaves, V.A., Van Den Bosch, F. 2015. Early-detection surveillance for an emerging plant pathogen: a rule of thumb to predict prevalence at first discovery. Proceedings of the Royal Society B. 282:20151478.
Hilf, M.E., Lewis, R.S. 2016. Transmission and propagation of ‘Candidatus Liberibacter asiaticus’ by grafting with individual citrus leaves. Phytopathology. 106:452-458.
Wang, H., Turechek, W. 2016. A loop-mediated isothermal amplification assay and sample preparation procedure for sensitive detection of Xanthomonas fragariae in strawberry. PLoS ONE. 11(1):e0147122.
Zhang, M., Guo, Y., Powell, C.A., Doud, M.S., Yang, C., Zhou, H., Duan, Y. 2016. Zinc treatment increases the titre of ‘Candidatus Liberibacter asiaticus’ in Huanglongbing-affected citrus plants while affecting the bacterial microbiomes. Journal of Applied Microbiology. 120:1616-1628. doi: 10.1111/jam.13102.
Anco, D.J., Gottwald, T.R. 2015. Within-orchard edge effects of the azimuth of the sun on Diaphorina citri adults in mature orchards. Journal of Citrus Pathology. 2(1):1-9.
Mccollum, T.G., Hilf, M.E., Irey, M., Weiqu, L., Gottwald, T.R. 2016. Susceptibility of sixteen citrus genotypes to Candidatus Liberibacter asiaticus. Plant Disease. 100(6):1080-1086.
Yu, X., Armstrong, C.M., Zhou, M., Duan, Y. 2016. Bismerthiazol inhibits Xanthomonas citri subsp. citri growth and induces differential expression of citrus defense-related genes. Phytopathology. 106(7):693-701. doi: 10.1094/PHYTO-12-15-0328-R.
Kousik, C.S., Adkins, S.T., Turechek, W., Webster, C.G., Stansly, P.A., Roberts, P.D. 2015. Influence of insecticides and reflective mulch on watermelon vine decline caused by squash vein yellowing virus (SqVYV). Plant Health Progress. 16:43-48.
Silva, J.A., Hall, D.G., Gottwald, T.R., Andrade, M.S., Maldonado, Jr, W., Alessandro, R.T., Lapointe, S.L., Andrade, E.C., Machado, M.A. 2016. Repellency of Psidium guajava cultivars to the Asian citrus psyllid, Diaphorina citri. Crop Protection. 84:14-20.
Ding, F., Duan, Y., Paul, C., Brlansky, R., Hartung, J.S. 2015. Localization and distribution of ‘Candidatus Liberibacter asiaticus’ in citrus and periwinkle by direct tissue blot immuno assay with an anti-OmpA polyclonal antibody. PLoS One. 10(5):e0123939. doi:10.1371/journal.pone.0123939.
Hao, G., Pitino, M., Duan, Y., Stover, E. 2016. Reduced susceptibility to Xanthomonas citri in transgenic citrus expressing the FLS2 receptor from Nicotiana benthamiana. Molecular Plant-Microbe Interactions. 29:132-142.