Location: Subtropical Plant Pathology Research
2021 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 by ARS researchers in Fort Pierce, Florida, for HLB resistance/tolerance via graft-based and psyllid inoculations. 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 3-4 years. A number of new citrus lines with improved HLB-resistance/tolerance have been obtained from the budsport selection and evaluation. From Las pathogenesis standpoint, ARS researchers in Fort Pierce, Florida, demonstrated that Las 5315 effector induced extreme starch accumulation and chlorosis, typical symptoms of HLB, and revealed that host DEAD-box RNA helicase (DDX3) was targeted by SDE1 (Las5315), and that downregulation of host DDX3 gene was associated with leaf chlorosis. These findings facilitate our understanding of Las pathogenesis and HLB symptom development. A controlled experimental system was used 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 by ARS researchers in Fort Pierce, Florida, 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 with host range ongoing.
Survival time of Xanthomonas fragariae, the causal agent of strawberry angular leaf spot, was examined by ARS researchers in Fort Pierce, Florida, 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 determined with a specialized quantitative polymerase chain reaction (qPCR) procedure and the likelihood of disease transmission was evaluated in a bioassay with strawberry plants. 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. Information from this study can be used to develop improved sanitation protocols for angular leaf spot management in strawberry nurseries. The manuscript is in final draft form.
Plots to test the interaction of Asiatic citrus canker (ACC), leafminer, and windbreaks were established in Brazil. Results indicated that combinations of windbreaks and leafminer control are highly efficacious for canker mitigation. Fourier transform infrared spectroscopy (FTIR) was fully evaluated by ARS researchers in Fort Pierce, Florida, 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 hurricanes are incorporated into recent multi-pest survey (MPS) design in Florida and Texas. Model accuracy has been assessed for Hurricanes Irma and Harvey, and will continue to be analyzed for recent hurricane activity when survey data 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 by ARS researchers in Fort Pierce, Florida. 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 by ARS researchers in Fort Pierce, Florida, 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 2,673 as of mid-2021. At the request of CDFA, a spatiotemporal analysis of HLB detections resulted in a recommendation of delimiting surveys around new detections to be reduced to 250m with significant 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 was integrated into the State/Federal New York PPV eradication program for 8 years, 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. Regulatory agencies and commodity groups are implementing our risk-based survey models for multiple pathogens to predict new outbreak locations and target disease/vector hotspots. These are refined each year to optimize surveys to the changing disease/vector conditions, available manpower and resources, and will continue to be used for many years into the future. Models for Asiatic citrus canker (ACC), HLB, PPV and Citrus tristeza virus (CTV) 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 by ARS researchers in Fort Pierce, Florida, to examine the efficacy of ACP and HLB control strategies in select 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 by ARS researchers in Fort Pierce, Florida, 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 submitted and published by ARS researchers in Fort Pierce, Florida.
Watermelon and pepper germplasm evaluations for squash vein yellowing virus (SqVYV), powdery mildew and/or tospoviruses are continuing. Virus sanitation trial continued.
Accomplishments
1. Detector dog development and deployment for exotic plant diseases offers potential to mitigate epidemics. Early detection and rapid response is the key to mitigation of all plant diseases. Representative samples for testing can be difficult to collect, especially early in the growing season or epidemic when the pathogen exists at a low incidence. This same caveat applies at the individual plant scale when a pathogen is incompletely distributed in its plant host, particularly with large and/or perennial host plants. Both of these sampling scenarios can lead to false negative test results. Canine detection of plant pathogens is a novel approach demonstrated by ARS researchers in Fort Pierce, Florida, and industry cooperators to be rapid, sensitive and reliable for bacterial and viral plant pathogen detection. Since canines holistically and non-destructively assay the entire plant, sampling issues can be avoided at both agro-ecosystem and individual plant scales while maintaining testing accuracy. Canine detection occurs in real-time, with immediate results that can be translated into immediate corrective actions by growers. Detector dogs for Huanglongbing (HLB) and vegetable viruses were featured in the USDA “Scientific Discoveries” video series in spring 2021.
2. Risk model and resulting risk-based surveys 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 about 50 surveyors dedicated to state-wide surveys, but they cannot reach all commercial and residential properties. ARS researchers in Fort Pierce, Florida, have continued developing a risk-model that uses 10 risk criteria to rank every 1 square mile area in California and validation has indicated the high success rate of the predictive model. Via this model ARS researchers in Fort Pierce, Florida, have designed and deployed a risk-based survey in California to target and optimize survey efforts. ARS researchers in Fort Pierce, Florida, have also designed a 3-cycle survey based on the risk model to survey across 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 has become the mainstay of the large-scale statewide survey in California. The survey continually detects new infections in Southern California, which have now exceeded 2,673 as of mid-2021. These detections are immediately removed and result in a quarantine surrounding the detection source with delimiting response protocols initiated. 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 integrated to MPS survey design for improved decision-making by regulatory agencies and science advisory committees. 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. There are ongoing analyses of the recent hurricane events to refine survey design for disease management.
4. Areawide pest management (AWPM) is an alternative for whitefly-transmitted virus management in vegetable crops. A number of whitefly-transmitted viruses, particularly tomato yellow leaf curl virus (TYLCV) in tomato, have severely impacted vegetable production in Florida and the southeastern U.S. over the last 20 years. 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. ARS researchers at Fort 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. In addition, ARS researchers in Fort Pierce, Florida, are working with a high-resolution satellite image provider to develop an algorithm that can classify the landscape by crop type and planting stage. Having the ability to fully characterize the landscape in real time (new satellite images are available every 3-5 days) will allow us to identify areas of the landscape that may serve as sources or reservoirs for pests and pathogens, and subsequently use this information for areawide management. Canine detection of whitefly-transmitted vegetable viruses is currently being developed.
5. Xanthomonas fragariae, the causal agent of angular leaf spot (ALS) of strawberry, can be found routinely on strawberry nursery stock. ARS researchers at Fort Pierce, Florida, designed, built and tested a commercial-scale precision thermotherapy unit (PTU) for applying a new thermotherapeutic protocol to 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 reduced in thermotherapy-treated plots. Several commercial nurseries have adopted and are scaling-up the thermal treatment for use on their nursery stock. The procedure has not only proven effective against ALS, but also against other pests and pathogens. The net effect permits simultaneous management of several pests and pathogens, reducing or eliminating pesticide use against a broad range of threats common to strawberry production.
6. Early detection of citrus HLB is critical for disease control. Pennsylvania State University and ARS researchers in Fort Pierce, Florida, have used cutting-edge CRISPR/Cas technology to develop a diagnostic test that could enable early diagnosis of citrus greening, or HLB. The new assay can detect the presence of the disease's causal agent - the bacterium Candidatus Liberibacter asiaticus (Las) - at a sensitivity level 100 to 1,000 times greater than the commonly used qPCR diagnostic test. The DNA endonuclease-targeted CRISPR trans reporter (DETECTR) assay's compatibility with lateral flow technology holds promise for providing rapid and economical testing for HLB in the field.
7. Field evaluation of chemotherapy for mitigating citrus Huanglongbing (HLB) in Florida. ARS and University of Florida researchers in Fort Pierce, Florida, investigated the effects of different antimicrobial chemicals on mature HLB-affected citrus trees with emphasis on fruit yield and quality. By extensive examination of parameters such as disease index, tree canopy, fruit yield and quality, it was found that penicillin (PEN) with surfactant was most effective in killing the HLB bacteria in infected citrus trees, followed by Fosetyl-Al (ALI), Oxytetracycline (OXY), Carvacrol (CARV), Validamycin (VA). ARS researchers in Fort Pierce, Florida, further analyzed fruit yield and quality, and determined that PEN treatment increased the soluble solids content (SSC), whereas OXY treatment significantly reduced titratable acidity (TA) level and increased the SSC/TA ratio compared to the control. In addition, nutrient analysis showed increased N and Zn levels in ALI and PEN treatments, and OXY treatment increased leaf P, K, S, and Mg levels compared to untreated control. These findings revealed that some of the chemical treatments were able to reduce the levels of HLB pathogen, to enhance nutritional status in leaves, and to improve tree growth and fruit quality of HLB-affected trees. Altogether, chemotherapy is effective to a certain degree and may serve as a component of integrated control for HLB.
8. To further develop thermotherapy into a field-applicable control strategy, practical methods were devised for treating mature trees in a field setting. ARS researchers in Fort Pierce, Florida, initial study utilized a solar thermal technology, whereby a portable greenhouse structure was placed over the trees to harness the sun’s heat energy and expose residential and commercial citrus to temperatures >40°C. This method of solar thermotherapy not only reduced the titer of Las for 18 to 36 months, but also increased the vigor of HLB-affected trees. Furthermore, the effects of thermotherapy treatments on the plants beyond the scope of the associated pathogen titer were also investigated in HLB-affected commercially grown Valencia trees. ARS researchers in Fort Pierce, Florida, not only monitored tree canopy density and Las titer in leaves but also determined fruit yield and juice quality after treatment. The results showed that solar thermotherapy resulted in improved juice quality and flavor in well-managed groves. The outcome of this study emphasizes the utility of solar thermotherapy as a part of an integrated management strategy for HLB-affected citrus.
Review Publications
Li, Y., Zhou, Y., Liu, Z., Zhuo, T., Fan, X., Duan, Y., Zou, H. 2020. 'Candidatus Liberibacter asiaticus' SED1 effector induces Huanglongbing chlorosis by downregulating host DDX3 gene. International Journal of Molecular Sciences. 21(7996):1-13. https://doi.org/10.3390/ijms21217996.
Mastin, A.J., Gottwald, T.R., van den Bosch, F., Cunniffe, N.J., Parnell, S.R. 2020. Optimising risk-based surveillance for early detection of invasive plant pathogens. PLoS Biology. 18(10): e3000863. https://doi.org/10.1371/journal.pbio.3000863.
Batuman, O., Yilmaz, Y., Roberts, P., McAvoy, M., Hutton, H., Dey, K., Adkins, S.T. 2020. Tomato brown rugose fruit virus (ToBRFV): Potential threat for tomato production in Florida. Extension Digital Information Source (EDIS). https://edis.ifas.ufl.edu/pp360.
Gottwald, T., Poole, G., Taylor, E.L., Luo, W., Posny, D., Adkins, S.T., Schneider, W., McRoberts, N. 2020. Canine olfactory detection of a non-systemic phytobacterial citrus pathogen of international quarantine significance. Entropy. 22:1269. https://doi.org/10.3390/e22111269.
Zhang, M., Karuppaiya, P., Zheng, D., Sun, X.N., Bai, J., Ferrarezi, R., Powell, C., Duan, Y. 2021. Field evaluation of chemotherapy on hlb-affected citrus trees with emphasis on fruit yield and quality. Frontiers in Plant Science. 12:611287. https://doi.org/10.3389/fpls.2021.611287.
De Marchi, B., Smith, H., Turechek, W., Riley, D. 2021. A maximum dose bioassay to assess susceptibility of bemisia tabaci meam1 to key insecticides. Journal of Economic Entomology. https://doi.org/10.1093/jee/toab016.
Clohessy, J.W., Sanjel, S., O'Brian, G., Barocco, R., Kumar, S., Adkins, S.T., Tillman, B., Wright, D.L., Small, I.M. 2021. Development of a high-throughput plant disease symptom severity assessment tool using machine learning image analysis and integrated geolocation. Computers and Electronics in Agriculture. 134:106089. https://doi.org/10.1016/j.compag.2021.106089.
Yang, C., Hu, H., Wu, Y., Lin, X., Fan, G., Chen, J., Duan, Y., Powell, C., Ancona, V., Zhang, M. 2020. Metagenomic analysis reveals the mechanism for the observed increase in antibacterial activity of penicillin against uncultured bacteria candidatus liberibacter asiaticus relative to oxytetracycline in planta. Antibiotics. 9:874. https://doi.org/10.3390/antibiotics9120874.
Wheatley, M.S., Duan, Y., Yang, Y. 2021. Highly sensitive and rapid detection of citrus huanglongbing pathogen (Candidatas Lliberibacter asiacticus) using Cas12a-based methods. Phytopathology. https://doi.org/10.1094/PHYTO-09-20-0443-R.
