Project : USDA ARS

ARS Home » Southeast Area » Fort Pierce, Florida » U.S. Horticultural Research Laboratory » Subtropical Plant Pathology Research » Research » Research Project #442004

Research Project: Mitigation of Domestic, Exotic, and Emerging Diseases of Subtropical and Temperate Horticultural Crops

Location: Subtropical Plant Pathology Research

2023 Annual Report

Objectives
Objective 1: Characterize ecology, biology, and epidemiology of domestic, exotic, newly emerging, and re-emerging pathogens of horticultural crops. (NP303, C1, PS1A; C2, PS2A, PS2B, PS2C, PS2D) 1.A: Characterize the basic biology of ‘Candidatus Liberibacter asiaticus (Las),’ the bacterium associated with citrus huanglongbing (HLB) by in vitro culture, and characterize new seed-transmissible diseases of citrus. 1.B: Characterize the basic biology, molecular biology, vector interactions and/or epidemiology of orthotospoviruses, Xanthomonas fragariae [cause of angular leaf spot (ALS) on strawberry] and other pathogens of vegetables, citrus, ornamentals, and weeds. Objective 2: Develop and improve reliable detection and sampling methods for pathogens of subtropical and temperate horticultural crops. (NP303, C1, PS1A, PS1B) 2.A: Develop and/or improve detection and sampling methods for orthotospoviruses, Las and Xanthomonas fragariae on strawberry. 2.B: Train canines to detect thrips-transmitted TCSV and whitefly-transmitted SqVYV. Objective 3: Develop or improve comprehensive integrated disease management strategies to mitigate existing or emerging diseases of horticultural crops. (NP303, C3, PS3A, PS3B) 3.A: Develop and implement the most efficacious strategies for disease management of HLB, Xanthomonas fragariae on strawberry, fungal foliar diseases on cucurbits, and viruses of vegetables and ornamentals. 3.B: Develop new and/or augment existing surveillance methods and protocols for HLB and other new citrus diseases, and areawide management of insect-vectored viral diseases of vegetables.

Approach
The overall approach is to thoroughly characterize plant pathogens causing domestic, exotic and emerging diseases at multiple levels: cellular, molecular and/or biochemical levels of host-pathogen-vector interaction and traditional and newer stochastic epidemiological analysis at field and regional 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
Work on all objectives commenced with the new project plan in late March 2022 and we made progress this year that allowed us to fully meet all of our milestones during the first complete year of the project. Candidatus Liberibacter asiaticus (Las) basic and molecular biology, and genetic characterization, has continued from the previous project. In vitro Las culture was greatly improved with simpler results and much more consistent results and revealed Las-titer independent Huanglongbing (HLB) progression, and long-lasting low Las titer infection in planta when cultured Las was inoculated back to citrus via psyllid feeding. Citrus bud sport and seedling selections continue to be evaluated, and undergo genomic and transcriptomic analyses, for HLB resistance/tolerance via graft-based and psyllid inoculations. More than 10 new citrus lines with improved HLB-resistance/tolerance have been obtained from the bud-sport selection and evaluation after seven years of greenhouse and field trials. These newly selected lines have gone through a pathogen clean-up by the Florida Department of Agriculture and Consumer Services-Division of Plant Industry and are ready for future release. Some lines are now in a second round of field trials on different rootstocks. Citrus, vegetable and strawberry pathogen surveys, and meteorological data compilation continued. A new citrus-infecting virus was identified via high throughput sequencing. This virus was determined to systemically replicate in citrus plants with relatively high titers, while not inducing apparent symptoms in most species of citrus tested. Importantly, the virus was found to suppress citrus HLB symptoms in infected plants. Tospovirus and tobamovirus characterization continued at virus and field levels. A tobamovirus (chili pepper mild mottle virus) was detected in the U.S. for the first time in calibrachoa, a popular ornamental crop. Two quarantine tobamoviruses (cucumber green mottle mosaic virus and watermelon green mottle mosaic virus) were detected in Florida for the first time in specialty cucurbit crops. Sequencing and reports were made in cooperation with state and Federal regulatory agencies. A screening of a Xanthomonas fragariae isolate collection to identify phenotypical differences in disease development on several different cultivars was completed. X. fragariae causes angular leaf spot on strawberry, and anecdotal evidence suggests that differences in the pathogen population exist that allow some strains to go systemic in the plant vascular tissue – allowing the pathogen to remain cryptic – whereas other strains simply cause the classic leaf spotting systems and do not go systemic. Should differences be identified, additional studies will look to determine genetic and morphological differences among the two populations. Bacterium and virus detection assay development has continued. Tissue elasticity screening of different citrus genotypes for HLB diagnostics was completed. Although the degree of elasticity varied among citrus species, the parameters for differentiation of infected from healthy citrus were found to be measurable and reproducible. A rapid field detection assay for the tospovirus, tomato chlorotic spot virus (TCSV), was developed and published. New sets of conventional reverse transcription-polymerase chain reaction (RT-PCR) and qRT-PCR primers for TCSV were developed and evaluated. High throughput sequencing of many Florida TCSV isolates, including those collected from tospovirus-resistant tomato varieties, is facilitating sequence analysis for marker development for potential resistance-breaking TCSV isolates. Canine virus detection trials have continued for vegetable and grape viruses. Detector dog demonstrations with virus-infected vegetable transplants were made at the Florida Citrus and Vegetable Show in front of hundreds of growers, industry and regulatory members. Management trials and collection of spatial distribution data and development of epidemic models have continued. Development and validation of a model/algorithm to identify and map the regional distribution of crops on satellite imagery and produce landscape-level risk maps of whitefly and whitefly-transmitted viruses continued. Initial testing in southwest Florida showed the algorithm is a good predictor of crop type once the crop has grown sufficiently to be “seen” by the satellite. Early/young plantings are often incorrectly predicted as fallow ground. Thus, we have expanded the algorithm to identify fallow fields that are transitioning into a planting and newly planted fields by focusing on the raised, plastic-mulched beds that are visible in satellite imagery. Once refined, the landscape-level risk maps can be incorporated into an areawide pest management (AWPM) program for management of whitefly-transmitted viral epidemics. Working with local crop consultants and agricultural technology companies, we have continued to develop the tools for real-time mapping of pest and diseases, 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 southeastern U.S. Recent efforts have focused on off-season scouting to identify areas that may serve as sources of whitefly or virus for the following production season, including the use of canine detection. Having the ability to fully characterize the landscape in real time is a necessary component for AWPM. A thermotherapeutic protocol that combined a conditioning thermal treatment with an eradicative thermal treatment for managing several pathogens and some pests on strawberry nursery stock was developed and refined. Commercial-scale precision thermotherapy units designed to apply the specific thermal treatment are being built by a European company, shipped to U.S. strawberry nurseries and put into service. In cooperation with local breweries, we have been studying the production of hops in Florida. Several varieties and production systems have been tested for production under Florida conditions, but an essential component common among the varieties is the need for supplemental lighting to extend the daylength to suppress flowering until bines have elongated to maximize yield. Commercial analysis of the hops chemical content show that the hops produced under Florida conditions have sufficient alpha and beta acids for beer production. The hops are used to produce “fresh-hopped” beer, and private taste testing ensued to evaluate consumer attitudes. A risk-based survey (RBS) method for statewide sweeps in California to detect HLB and its vector, the Asian citrus psyllid (ACP), has successfully been implemented in cooperation with USDA Animal and Plant Health Inspection Service (APHIS) and the California Department of Food and Agriculture (CDFA). The RBS is continuously adapted to survey priorities and personnel/resource capacities, and it is redeployed twice yearly with updated survey designs. The RBS residential and commercial methods for HLB and ACP have been in use for 11 years in California. Model validation indicates that the models and linked surveillance have been highly successful, consistently detecting new introductions in Southern California, which have now exceeded 5,500 as of May 2023. At the request of CDFA, a spatiotemporal analysis of HLB detections resulted in a recommendation to reduce the delimiting surveys around new detections to 250 meters, leading to significant savings in personnel and fiscal resources. CDFA now routinely utilizes the reduced delimiting survey methodology to mitigate disease pressure in new HLB detection locations. An agent-based model to assess the effectiveness of ACP and HLB control strategies in selected locations in California has also been developed. The model takes into account social and economic perspectives, area-wide approaches, and delimitation protocols. Through simulation modeling and comprehensive data analysis, this tool informs management decisions and provides valuable insights. Moreover, the project includes an online platform that facilitates prompt collaboration among researchers within University of California-Davis, the California-based Citrus Research Board, and the Fort Pierce development team. This platform serves as a scenario-based guide to address priority questions and concerns related to citrus management efficacy. Mitigation strategies for citrus pests and diseases can vary in effectiveness based on several factors, including location (e.g., Southern vs. Central CA, urban vs. semi-urban vs. rural areas, and coastal vs. desert regions), the phase of the epidemic (ACP and/or HLB), climatic conditions, compliance or risk perception, and social and economic aspects. Collectively, this increases our understanding of the pathways and landscape drivers that influence ACP dynamics and HLB progression. A predictive model that builds upon previously established methodologies to estimate the dispersal of plant pests and diseases driven by major weather events is under construction. To predict areas potentially affected by severe storm events, we considered the "dispersal cone." Instead of assuming a uniform dispersal risk within the cone, we are employing a dispersal gradient function that incorporates penalty terms for distance and angle to better represent the probability of infection.

Accomplishments
1. ARS researchers in Fort Pierce, Florida, developed a model for predicting whitefly mortality in response to freeze events in southwest Florida vegetable production fields. This is a critical new tool for growers, crop consultants and Extension personnel to use for prediction of whitefly numbers and whitefly-transmitted virus incidence for vegetable crops. The model will be incorporated into a risk prediction model for management of whitefly and whitefly-transmitted viruses in vegetable production as part of an areawide pest management research effort. Other components include proximity to whitefly or whitefly-transmitted viruses and satellite identification of hosts for the insect and/or the virus. Together this novel management approach should be useful for whitefly-transmitted viruses like tomato yellow leaf curl virus and squash vein yellowing virus.

2. Detector dog development and deployment provides early pathogen detection and rapid response to offer the possibility to mitigate plant diseases before they become epidemic. ARS researchers in Fort Pierce, Florida, and industry cooperators, initiated a program to cross-train detector dogs for squash vein yellowing virus (cause of viral watermelon vine decline) in watermelon, and tomato chlorotic spot virus in tomato and pepper, rather than relying on traditional field scouting. Measurement and analysis of performance metrics including accuracy, sensitivity and specificity demonstrated no loss in effectiveness for detection of either virus following cross-training. Public demonstration of canine detection at a well-attended public stakeholder event provided an ideal demonstration of this concept. A proof-of-concept has also been demonstrated for canine detection of viruses in wine grapes, and a major strawberry producer in California has initiated a project for use of canines for pest detection in strawberry nursery production.

3. ARS researchers in Fort Pierce, Florida, identified an ~8.3 kb DNA region in the Las genome as a mosaic island associated with genome plasticity (instability). To study the population dynamics of this mosaic island, specific primers were developed to target both the presence (Las wild-type) and absence (Las mutant) of this region within its hosts. From this, Las populations with and/or without the wild-type strain were detected and differentiated in >2,300 samples that encompassed infected psyllids, periwinkle, and several different species of citrus plants. In psyllids, although a mixed population of both the wild-type and mutant was observed in most of the samples (88%), the wild-type Las was detected alone at a rate of 11%. In contrast, the wild-type alone was not detected in infected citrus plants, which tended to harbor either the mutant strain alone (8%) or a mixed population where the mutant and wild-type coexist (92%). The dynamics of these two major Las populations varied by host and geographical location, with the highest ratio of mutant:wild-type in grapefruit, the most susceptible citrus host. Overall, these results indicate that the deletion or excision of this mosaic island is critical for Las fitness in planta.

4. ARS researchers in Fort Pierce, Florida, in cooperation with University of Florida, Clemson University assembled a chromosome-level phased Valencia sweet orange (SWO) genome. With ~98.5% completeness and high accuracy, it has the highest annotation completeness (99.2%) thus far in citrus. This research found that citrus HLB shifted the allelic expression patterns in HLB-affected leaves and calyx abscission zones from the SWO healthy status. With long-read sequencing and RNAseq, the allelic structural mutations and corresponding allelic expression alterations were detected in HLB-tolerant mutant T19, a more susceptible mutant T78, and a normal Valencia SWO. Most structural mutations induced by irradiation displayed double-strand breaks, while the majority of natural structural mutations were associated with transposon insertions. 84.0% of genes had significant allelic expression ratio alterations (= 1.5 fold), and all of them had one allele directly affected by the structural mutation. In T19, alleles encoding three heat shock proteins (HSPs) were identified at one terminal of a translocated segment. The expression of these proteins along with other 65 HSPs was all upregulated. Therefore, increased levels of small HSPs may directly affect T19’s HLB tolerance by reducing phloem necrosis/plugging and promoting phloem homeostasis. More importantly, the Valencia SWO will advance further allelic level studies in citrus.

5. Agent-based model enhanced for timely analysis of scenarios in real-world California landscapes. Resulting analyses by ARS researchers in Fort Pierce, Florida, in collaboration with University of California-Davis, have provided location-based guidance on effective management programs for citrus landscapes at various scales, optimizing resources under different climatological conditions, ACP/HLB pressures, and drivers of ACP/HLB propagation. These analyses are regularly revisited and updated with new detection data and changes in management protocols/capabilities. This ensures that growers and stakeholders receive timely information about short- and long-term performance and sustainability. Concurrently, an online interface has been developed to propose rapid response guidelines and evaluate various scenarios, such as management strategies (including multiscale, delimitation, and detection technologies), epidemiological and environmental factors, social behaviors, and economic considerations, in real-world California citrus landscapes.

U.S. DEPARTMENT OF AGRICULTURE

Subtropical Plant Pathology Research: Fort Pierce, FL