Location: Subtropical Plant Pathology Research
2018 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, Las was maintained and propagated in liquid media for months, and cause Huanglongbing (HLB) via feeding psyllid and inoculating to citrus. An Material Transfer Agreement (MTA) established in the prior year with a new partner at Northeastern University has been inactive pending acquisition of independent funding by the MTA partner. In planta Las growth data collected and growth curves developed. 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 our Picos Research Farm for field trials. 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. A controlled experimental system to study aspects of tissue colonization and population development was developed. A small scale, short term protocol for assessing antimicrobial compounds has been developed.
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.
Results of the X. fragariae survival study showed that survival rates of X. fragariae varied by both the material and storage temperature. X. fragariae was consistently found viable to survive on cardboard stored at -4°C six months after contamination.
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 development of predictive models to estimated hurricane spread of the pathogens. These are being used by state and federal regulatory agencies to target detection survey. 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 been developed. It is being tested against trade pathway data sets and is in review by collaborators.
Meteorological, insect count, and virus incidence data for Tobacco streak virus (TSV) continue to be collected in cucurbit and solanaceous crops.
New sets of conventional 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/or validated. 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. Small tree/single leaf grafting systems applied to study factors involved in controlling bacterial colonization of citrus tissues.
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 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 seventh year of deployment in California and validation indicated highly successful as they continually detect new introductions in Southern California which have now exceeded 675 as of June 1, 2018. At the request of California Dept. of Food and Agriculture (CDFA), a spatiotemporal analysis of HLB detections resulted in a recommendation of delimiting surveys around new detections to be reduced from 800m to 400 m with great savings in manpower and fiscal resources.
A risk-based survey to detect plum pox virus (PPV) outbreaks in New York State is integrated into the State/Federal NY PPV eradication program and is in the sixth year of deployment. A risk-based model and survey to detect new PPV introduction is 50% developed and will be deployed in California and perhaps other states when complete. Our surveys developed for multiple pathogens 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 multiple years into the future. Models for Asiatic citrus canker (ACC), Huanglongbing, PPV and Citrus tristeza continue to be augmented and extended in unique ways to address specific regulatory issues. These models and their refinement continues to be the foundation for future work.
Melded the leaf grafting system with vacuum-assisted infiltration of symptomatic leaves with antibiotics to inhibit transmission of Candidatus Liberibacter asiaticus (Las). Determined that penicillin G and oxytetracycline are effective against Las, but streptomycin is not effective. Solar thermotherapy reduces the titer of Candidatus Liberibacter asiaticus and enhances canopy growth by altering gene expression profiles in 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. 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 Huanglongbing (HLB) control strategies in selected locations in California. A census-travel model to predict introductions of pathogens from foreign sources has been integrated into HLB, Plum Pox Virus (PPV), and other surveys as an added risk factor. A Bayesian Belief Network model to assess phytosanitary health of seed commodities has been developed and is under validation for a tomato seed-borne disease and will be extended to other seed pathosystems.
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. Results reinforced use of the current sauna protocol over traditional hot water treatment.
Watermelon and pepper germplasm evaluations for Squash vein yellowing virus (SqVYV), powdery mildew and/or tospoviruses are continuing.
Virus sanitation trial initiated.
Accomplishments
1. Canine detection of Huanglongbing HLB in California potentially mitigates an impending state-wide epidemic. Citrus Huanglongbing (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 grants program ARS researchers at Ft. Pierce, Florida, have trained 20 dogs for early detection (within 2-4 weeks post infection) of HLB where the prior gold standard, PCR, requires months to detect. The dogs can also detect with 99.16% accuracy whereas PCR detection is ~30-35%, not because the assay is poor, but due to sampling and the problem of selecting the precise infected tissue in a tree with 1-200,000 leaves. California growers and the California Department Food and Ag. (CDFA) have initiated a team of researchers, industry leaders and regulatory personnel to work out final validation and deployment of the dogs to detect asymptomatic incipient infections of HLB for early response (tree removal). Simulations demonstrate how the impending epidemic would be greatly mitigated by the incorporation of canine detection teams for early detection.
2. Risk model and resulting risk-based survey direct California program to combat HLB. To effectively mitigate the devastating effects of the Huanglongbing epidemic on the California citrus industry, ARS researchers at Ft. Pierce, Florida, must search the large (300,000ac) commercial citrus industry in addition to the vast residential plantings of citrus (60 % of residences have at least one citrus tree). At present the state of California has 75-100 surveyors dedicated to this task but they cannot reach all commercial and residential properties. We have developed a risk-model which uses 10-15 risk criteria to rank each 1 mi sq area in California. Via this model we have designed and deployed a risk-based survey in California to target and optimize survey efforts. We have also designed a 3-cycle survey based on the risk model to speed the entire state 3 times per year. The risk-based residential and commercial survey methods for Asian Citrus psyllid (ACP) and HLB are in the seventh year of deployment in California and validation indicated they have highly successful and have become the mainstay of the large state-wide survey in California. The survey continually detects new introductions in Southern California which have now exceeded 675 as of June 1, 2018. These new introductions are immediately removed and result in a quarantine surrounding the detection. At present the survey is done visually with PCR confirmation of suspicious trees, however, canine detection (see above accomplishment) will likely be integrated into the risk-based survey.
3. Epidemiological models predict the spread of 2 severe citrus diseases by hurricanes. Asiatic citrus canker (ACC) and citrus black spot (CBS) are 2 serious diseases that only causing economic damage but impediments to international trade of citrus is a commodity. Both ACC, caused by a bacterium, and CBS, caused by a fungus, are 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 Ft. 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. We also developed risk-based surveys to target areas rejected by the hurricane models. As result Texas and Florida state regulatory agencies in collaboration with USDA, APHIS have deployed the surveys for early detection of potential spread of these diseases in both states.
4. Genotyping methods developed and validated for emerging tospoviruses. Vegetable and ornamental crops in the southeastern U.S. have recently been impacted by the emergence of Tomato chlorotic spot virus (TCSV) and Groundnut ringspot virus (GRSV), in addition to the long-present Tomato spotted wilt virus (TSWV). Currently available commercial diagnostic reagents for any one of these viruses were observed to crossreact with the others. In addition, commercial diagnostic reagents were not able to identify genetic reassortments of these viruses that have been identified by ARS researchers at Ft. Pierce, Florida. Newly designed primers for conventional reverse transcription-polymerase chain reaction developed by ARS scientists in Fort Pierce, Florida, in collaboration with university scientists address both issues. Each of the three genome segments of each of these viruses are able to be unequivocally identified to genotype the tospovirus(es) present. This level of virus identification is necessary to provide growers with effective recommendations for deployment of resistant crop cultivars (because resistance genes react differently with each virus species) and management of thrips vector species (because different thrips species are the most effective vector for each virus species).
5. Xanthomonas fragariae can survive up to nine months in cold storage on cardboard. X. fragariae causes strawberry angular leaf spot, an important disease in strawberry nursery production. To identify potential inoculum sources, the ability of X. fragariae to survive was examined by ARS researchers at Ft. Pierce, Florida, on ten common materials typically associated with strawberry nurseries (cardboard, glass, latex gloves, strawberry leaves, medal, plastic bags, rubber, T-shirt, Tyvek, and wood). Each surface was artificially contaminated with the bacteria and stored at room temperature and -4°C, respectively. The bacteria were collected from each surface with PBS-soaked cotton balls at ten time points from the day of contamination until 1 year after inoculation. During each sampling, the survival rates were examined by testing the bacteria isolated from the PBS-soaked cotton balls by PMA-qPCR, and by rub-inoculating strawberry leaves with the cotton balls. Results showed that X. fragariae survival rates varied by both the material and storage temperature, and the longest survival was consistently observed on cardboard stored at -4°C, the conditions which dormant plants are stored in strawberry nurseries. The information provided by this study may contribute to the development of improved sanitation and disease management strategies for strawberry nurseries.
6. The citrus industry maintains an interest in identifying antimicrobial compounds effective against Las. A vacuum-assisted infiltration assay with individual HLB-symptomatic leaves followed by grafting demonstrated by an ARS researchers at Ft. Pierce, Florida, to be an assay system that identified two compounds with antimicrobial activity effective against Las and one compound that was ineffective. Based on this initial success, screening of additional compounds is underway.
7. Solar thermotherapy reduces the titer of Candidatus Liberibacter asiaticus (Las) and enhances canopy growth by altering gene expression profiles in HLB-affected citrus plants. Using portable clear plastic greenhouses and homemade tents made from opaque plastic sheeting, ARS researchers at Ft. Pierce, Florida, tented infected citrus plants. Within the tent, temperatures increased above 40°C for 3 to 8.5 hours per day through solarization. As a result of the thermotherapy treatment, bacteria titers were decreased in trees and the citrus trees produced new leaves which resulted in a denser canopy for up to two years. Although solar thermotherapy did not cure residential and commercial HLB-affected citrus trees, the treatment decreased the amount of bacteria in the trees and caused the citrus trees to grow better for a period of time.
8. Emerging tobamovirus identified in ornamental crop hoya in Florida. Ornamental crops are the highest value sector of Florida agriculture. Chlorotic spots were observed on leaves of vegetatively propagated hoya leading to loss of marketability. ARS researchers at Ft. Piece, Florida, identified Hoya chlorotic spot virus in symptomatic hoya plants from several south Florida ornamental production areas. Host range and genome were characterized in collaboration with state regulatory agency and local college scientists. Little genetic diversity was observed in virus sequences consistent with recent introduction. Molecular diagnostic test was developed, validated and published to improve detection by regulatory agencies and producers.
9. LasDelta5315 effector induces extreme starch accumulation and chlorosis in Ca. Liberibacter asiaticus infection in Nicotiana benthamiana. ARS researchers at Ft. Pierce, Florida, demonstrated that Las can experimentally infect N. benthamiana via dodder transmission. Furthermore, they revealed another key function of the LasDelta5315 effector by demonstrating that transient expression of the truncated form of the effector, LasDelta5315, induced excessive starch accumulation by 6 fold after 8 dpi in N. benthamiana. The induction mechanisms of LasDelta5315 in N. benthamiana were attributed to the up-regulation of ADP-glucose pyrophosphorylase, granule-bound starch synthase, soluble starch synthase, and starch branching enzyme for increasing starch production, and to the significant down-regulation of the starch degradation enzymes: alpha-glucosidase, alpha-amylase, and glycosyl hydrolase for decreasing starch degradation. This is the first report that Las can infect the model plant N. benthamiana. Using this model plant, we demonstrated that the LasDelta5315 effector caused the most prominent HLB symptoms, starch accumulation and chlorosis as Las infection in N. benthamiana.
Review Publications
Adkins, S.T., D'Elia, T., Fillmer, K., Pongam, P., Baker, C. 2018. Biological and genomic characterization of a novel Tobamovirus infecting Hoya spp.. Plant Disease. 102(12):2571-2577. https://doi.org/10.1094/PDIS-04-18-0667-RE.
Fillmer, K., Adkins, S.T., Pongam, P., D'Elia, T. 2018. Using tobamoviruses for phylogenetic instruction in undergraduate biology courses. Journal of Microbiology and Biology Education. https://doi.org/10.1128/jmbe.v19i2.1519.
Tantiwanich, Y., Chiemsombat, P., Naidu, R., Adkins, S.T. 2018. Integrating local lesion assays with conventional RT-PCR for detection of interspecies tospovirus reassortants and mixed tospovirus infections. Plant Disease. 102:715-719.
Gazis, R., Poudel, B., Dey, K.K., Zhang, S., Palmateer, A., Campoverde, V.E., Baker, C.A., Adkins, S.T. 2018. First report of Cactus virus X in Hylocereus undatus (dragon fruit) in Florida. Plant Disease. 102:2666. https://doi.org/10.1094/PDIS-05-18-0725-PDN.
