Source: OREGON STATE UNIVERSITY submitted to
A SYSTEMS APPROACH FOR MANAGING BACTERIAL BLIGHT OF CARROT
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
NEW
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1023567
Grant No.
2020-51181-32154
Project No.
OREXHC2020
Proposal No.
2020-02656
Multistate No.
(N/A)
Program Code
SCRI
Project Start Date
Sep 1, 2020
Project End Date
Aug 31, 2024
Grant Year
2020
Project Director
Dung, J. K.
Recipient Organization
OREGON STATE UNIVERSITY
(N/A)
CORVALLIS,OR 97331
Performing Department
Central Oregon Ext
Non Technical Summary
Carrots (Daucus carota subsp. sativus) are one of the United States' leading root crops. The 2018 farm-gate value of carrots in the U.S. was $731,504,000 and average per capita consumption of fresh carrots in the U.S. is 12.4 lb. per person. Carrot seed production is limited to only a few regions of the world where suitable climatic conditions exist for hybrid seed production of this biennial species. In fact, over 50% of the world's hybrid carrot seed is produced in central Oregon, central Washington, southern Idaho, and northern California of the U.S. Pacific Northwest region. Stakeholders have consistently identified bacterial blight caused by Xanthomonas hortorum pv. carotae (Xhc) as the most significant threat to the sustainability and economic viability of US carrot seed production. However, because Xhc is seed-borne, it is not only a major concern for the hybrid carrot seed industry in the U.S. but also to regions that import carrot seed for fresh or processed carrot production. Xhc can survive and reproduce epiphytically on the leaves, flowers, and stems of host and non-host plants without causing disease symptoms. When weather conditions are sufficiently warm and humid, Xhc can incite disease, which can lead to defoliation and significant yield losses. In the case of bacterial blight, symptoms are often not observed on carrots until a relatively high population is attained (> 104 Xhc/g leaf tissue). One reason why Xhc is so problematic in carrot and carrot seed production is that the process by which the pathogen infects plants and infests seeds is largely unknown. Critical needs associated with bacterial blight in carrot and carrot seed include: i) improving disease control in the field and post-harvest; ii) learning how Xhc spreads in seed production systems; iii) determining if carrot seed becomes infested externally, infected internally, or both; iv) identifying sources of Xhc in production systems and quantifying the extent of inoculum dissemination via propagative materials; and v) quantifying the impact of bacterial blight on yield and/or quality of carrot seed, as well as the costs associated with mitigation of these effects.We propose to use a transdisciplinary research approach, deeply integrated with Extension and outreach, to address these critical needs. For Objective 1, we will evaluate and implement sustainable Integrated Pest Management (IPM) approaches for bacterial blight in carrot and carrot seed crops. To improve Xhc IPM in the field we will identify cultural practices that reduce Xhc survival and sources of inoculum, screen natural compounds and biorational products for the biocontrol of Xhc, and assess a multi-pronged IPM approach. We will also test complementary approaches that can be used with existing post-harvest seed disinfestation practices. In Objective 2, we will identify and model environmental factors and physical processes related to Xhc aerobiology and epidemiology. To understand Xhc and general bacterial aerobiology at multiple scales, we will determine the particle sizes, agriculture events, and environmental conditions associated with Xhc aerial dispersion and develop or adapt transport models that accurately describe pathogen dispersion between and within fields. The goal of Objective 3 is to characterize the nature and extent of Xhc colonization in carrots to inform and enable more precise and effective control measures. We will use various microscopy methods (light, laser confocal fluorescence, and scanning electron microscopy) to trace the Xhc infection process and gain insights into where the pathogen colonizes plants, when and where Xhc initiates infection, and how the pathogen infests seeds. We will use population genomics approaches in Objective 4 to identify sources of bacterial blight inoculum and dispersal potential of Xhc in carrot and carrot seed production systems. The genomic diversity of Xhc from U.S. and international carrot and carrot seed production areas, wild carrot populations, propagative materials, environmental samples, and historical collections will be determined and this information will be used to model transmission chains and inoculum sources. Finally, the activities in Objective 5 will calculate the economic impact of bacterial blight and associated management practices on carrot seed stakeholders. Outreach and Extension are integrated throughout the project.This project will address multiple goals that will lead to actionable items that can be implemented in the short-, medium-, and long-term in both conventional and organic systems. The proposed activities will foster changes in practices, increase the number and efficacy of bacterial blight control methods, and introduce potentially transformative technologies that can be used at different stages in the process of carrot seed and root production. The expected deliverables resulting from this project include: i) enhanced IPM approaches for bacterial blight control in the field and post-harvest; ii) particle transport models that explain factors associated with Xhc dispersal; iii) an understanding of plant and seed infection processes to inform future management strategies; iv) identification of sources and transmission pathways of Xhc in carrot production systems for targeted mitigation efforts; and v) an economic analyses of the costs and benefits associated with bacterial blight and related IPM efforts to guide future stakeholder decisions. Our long-term goal is to develop a suite of IPM approaches for the carrot industry to reduce risks associated with bacterial blight caused by Xhc. An integrated approach towards mitigating Xhc will reduce industry reliance on hot water treatment of seed lots, which is expensive and difficult to perform at large scales, reduce economic losses by limiting the number of seed lots rejected at ports of entry, and increase agricultural income and associated activities in rural areas in which carrot and carrot seeds are produced. In carrot and carrot seed production, it is anticipated that an IPM approach will decrease copper use thus reducing the indirect impacts to soil and the water table and direct impacts to bees, native pollinators, and other beneficial microorganisms that promote the health of crop plants. A sustainable carrot seed supply is required to continue production of this nutritious and popular vegetable, and the grower community will benefit through an enhanced understanding and applications of IPM approaches.
Animal Health Component
0%
Research Effort Categories
Basic
45%
Applied
45%
Developmental
10%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2121452110040%
2121452117040%
2121452301010%
2121452201010%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
1452 - Carrot;

Field Of Science
2010 - Physics; 1100 - Bacteriology; 3010 - Economics; 1170 - Epidemiology;
Goals / Objectives
Bacterial blight, caused by the plant pathogenic bacterium Xanthomonas hortorum pv. carotae (Xhc), is an important seedborne disease of carrot. Our long-term goals are to improve our understanding of Xhc biology and bacterial blight epidemiology, develop and implement a suite of integrative pest management approaches for the pathogen, and inform the carrot industry on these methods to reduce risks and losses associated with the disease. Towards this end, the objectives of this project are to:Evaluate and implement sustainable IPM approaches for bacterial blight in carrot and carrot seed cropsHypothesis: An IPM approach that integrates chemical, biological, and cultural practices with model-based decision support systems will improve control of Xhc. Model Xhc aerobiology and epidemiologyHypothesis: Characterizing environmental conditions and cultural practices that contribute to Xhc dispersal will allow us to identify pathogen sources, model dispersion and dissemination of the pathogen, and forecast and mitigate risk factors that promote the disease.Visualize plant infection processes to determine the nature and extent of Xhc colonization in carrotsHypothesis: Understanding the processes of plant and seed infection by Xhc will inform chemical, biological, and post-harvest management practices.Assess the population genomic diversity of Xhc in the U.S. to infer sources of inoculum and dispersal potential of bacterial blight in carrot and carrot seed production systemsHypothesis: Characterizing the genomic diversity and structure of Xhc across diverse geographic areas and sources will improve our understanding of inoculum sources and the extent of seedborne pathogen dispersal in carrot production systems.Calculate the economic impact of bacterial blight and associated management practices on carrot seed stakeholdersHypothesis: Increasing grower and stakeholder knowledge of the economic impacts of bacterial blight in seed production systems will foster the evaluation, refinement, and adoption of coordinated IPM practices to reduce the impact of Xhc on carrot and carrot seed production.
Project Methods
For Objective 1, we will: a) identify cultural practices that reduce Xhc survival and sources of inoculum; b) use biochemical-, culture- and plant-based assays to evaluate natural products for biocontrol of Xhc; c) assess a multi-pronged IPM approach for Xhc control; and d) test complementary approaches that can be used with existing post-harvest seed disinfestation practices.1a) The effects of crop debris management practices on Xhc survival after harvest will be evaluated in plots planted to a hybrid carrot seed line. Three post-harvest carrot seed crop debris removal practices (debris left on the soil surface and plots not tilled, debris removed from plots and plots not tilled, and debris removed from plots and plots tilled) will be compared and a viability qPCR assay will be performed to enumerate Xhc levels in soil and debris collected from each plot. The effect of fungicides used to control powdery mildew and other fungal diseases on the epiphytic carrot microbiome, including Xhc and potential antagonists of Xhc, will be investigated by performing fungicide applications and timings that are typical for seed-to-seed carrot production in the U.S. and represent fungicides with different modes of action. Changes in microbiome diversity and Xhc populations after each treatment will be measured using MiSeq and viability qPCR, respectively. 1b) we will cultivate bacteria antagonistic to Xhc in different production media and under various growth conditions to assess further their growth and inhibitory activity against Xhc. Antagonistic bacterial cultures will be harvested, centrifuged, and the supernatants fractionated. The extracts will be screened for antibacterial activity to Xhc using an agar diffusion assay and on carrot plants in the greenhouse. Active compounds from effective extracts will be isolated using bioassay-guided fractionation procedures and chromatography methods, and characterized structurally by employing NMR and mass spectrometry. In addition to the natural extracts discovered during the course of the studies described above, commercially-available biocontrol products will be evaluated alone or in combination during the course of the project. 1c) IPM approaches for control of Xhc will be evaluated in carrot seed field trials in Oregon and Washington and carrot root field trials in California. Treatments will consist of the most effective biocontrol extracts and products from the trials described above used in alternation or tank-mixed with copper bactericides and/or MgO nanoparticles. 1d) Samples of Xhc-infested carrot seed lots will be obtained from commercial sources and assayed for Xhc using a qPCR-based bulk seed wash assay to determine pathogen levels. Infested seed will be subjected to one of the following treatments: a soak in hot water (52°C) for 25 minutes; trisodium phosphate (10% vol/vol) for 15 minutes; or cold plasma-activated hydrogen peroxide (7.8% vol/vol) aerosol for 15, 30, or 60 seconds. Treated seeds will be subjected to a viability qPCR to enumerate live Xhc from each seed sample and the effect of each treatment on seed germination will be tested using the method of the Association of Official Seed Analysts. For Objective 2, we will: a) determine the particle sizes, agriculture events, and environmental conditions associated with Xhc aerial dispersion; and b) develop or adapt transport models that accurately describe pathogen dispersion between and within fields. 2a) We will examine the Xhc distribution and factors associated with dispersal using both long-term (months) and short-term (daily) studies in two commercial carrot seed fields. At each cardinal direction a sampling station will be created which will consist of a LEMS micrometeorological station, an Alphasense OPC-N23 particle counter, a Burkard volumetric sampler, a modified Anderson Cascade Sampler, and a 5m vertical impact trap array. DNA will be extracted from each sample and Xhc will be quantified using viability qPCR. To estimate field-to-field or regional spread and/or deposition of Xhc, an understanding of the 3D particle plumes over carrot canopies will be determined. Particle release experiments will be conducted over various atmospheric conditions by introducing fluorescent microspheres into the flow at three different heights and captured using a vertical impaction sampler array, and then enumerated microscopically. An eddy covariance tower with sonic anemometers will provide high-resolution turbulent fluxes of momentum and heat required to deduce the turbulent dispersion parameters used for adapting the QUIC row-crop model. 2b) Dispersion will be modeled in a Lagrangian reference frame using the open-source QUIC dispersion modeling system and a recent modification for row-oriented crops. Metrics of plume spreading and decay derived from microsphere release experiments will be used to tune QUIC for prediction field to field and region dispersion.For Objective 3, we will use microscopy methods to trace the Xhc infection process and determine where the pathogen colonizes plants, when and where Xhc initiates infection, and how the pathogen infests seeds. The model Xhc strain, M081, will be sprayed onto plant leaves and leaf colonization will be examined by light microscopy and the localization of β-glucoronidase-expressing Xhc. Xhc M081 expressing GFP or mCherry will be visualized using laser confocal microscopy to further characterize host colonization. High-resolution imaging of leaf and stem colonization will be obtained using established fixation and SEM protocols. We will use standard dilution plating and viability qPCR to determine bacterial levels in and on leaves, stems, umbels, and seeds to corroborate the colonization patterns visualized by microscopy. For Objective 4, we will: a) characterize Xhc diversity from U.S. and international carrot and carrot seed production areas, wild carrot populations, propagative materials, environmental samples, and historical collections; and b) use this information to model transmission chains and inoculum sources. 4a) Whole genomes of Xhc isolates from historical and contemporary collections will be sequenced and classified to characterize diversity in the carrot production system. 4b) Minimum spanning networks will be constructed to identify patterns and determine epidemiological links within the carrot production system. For Objective 5, we will: a) quantify the economic impacts of bacterial blight on carrot seed yield and quality; and b) compare the costs, efficacy, and profitability associated with current management practices to the IPM approaches evaluated in this project. Project evaluation will occur through regular meetings with an established Stakeholder Advisory Group (SAG) and via industry and grower feedback at Extension and outreach events. Metrics include the number of IPM practices and strategies that are evaluated, the number of dispersion models developed and their accuracies, characterization of temporal and spatial Xhc colonization in carrot seeds and plants, measures of gene flow among Xhc populations within carrot and carrot seed production systems, econometrics of improved IPM strategies, and the number of Extension publications produced; these metrics will be reported on annual reports submitted to USDA, SAG, and other interested parties. The long term metrics include the number of US growers adopting the IPM practices and strategies developed in this project; the number of undergraduate students, graduate students, and post-doctoral scientists conducting research associated with this project, the number of women and/or underrepresented groups involved in this project; the number of papers presented at stakeholder, Extension, and professional meetings, the number of peer-reviewed scientific research papers published; the number of carrot seed lots that show reduced Xhc; the incidence and severity of bacterial blight in carrot and carrot seed production systems.

Progress 09/01/22 to 08/31/23

Outputs
Target Audience:Target audiences of this project included carrot and carrot seed growers, carrot seed contractors, professional crop consultants, agri-chemical company representatives, vegetable seed company representatives, University Extension educators and faculty, and the public. Changes/Problems:Covid travel restrictions and inflation have increased costs and difficulty in conduction field campaigns. What opportunities for training and professional development has the project provided?One M.S. student (Thesis Title: Epidemiology of Bacterial Blight in Carrot Seed Production Systems,), one undergraduate, and one Branch Experiment Station undergraduate intern were trained in plant pathology and seed pathology .The project has also provided training opportunities to two postdoctoral scholars and one summer intern from Bakersfield College. One PhD student, four undergraduates, and one high school student worked on various aspects of data collection/analyses or equipment design. How have the results been disseminated to communities of interest?A total of 3 conference presentations and one symposium presentation were made to scientists and stakeholders. Two additional presentations were made for industry stakeholders (January and July 2023). The cyclonic sampler is being utilized to examine airborne pathogen movement in tomato production and occurrence of DMI tolerant Aspergillus spp. in compost production and other crops. The "Cost of Carrot Seed Production Worksheets/Analysis" were made available to stakeholders via the Pacific Northwest Vegetable Association Website. What do you plan to do during the next reporting period to accomplish the goals?1. We will complete the chemical synthesis of the most active antibiotic compound and its diastereomers and test their anti-Xhc activity, identify active compounds from isolates 16-135, 16-304, and 16-357, and test active compounds against Xhc infections in carrot plants. 2. We will conduct phenotypic screening of ~50 strains of Xhc for tolerance to a range of copper concentrations (0 to ~400 ppm, in 50 ppm increments) to compare with genotypic data on the presence/absence of various copper tolerance genes detected in a diversity of Xhc strains from three clades in a phylogenetic tree built using whole genome sequences derived from Objective 4. Strains of Xanthomonas perforans with known sensitivity and tolerance to copper were obtained from Jeff Jones, phytobacteriologist at the University of Florida, to serve as control strains in the phenotypic screening. 3. We will conduct a field trial to further evaluate novel and existing chemical and biological control options for Xhc. 4. We will conduct a particle release experiment to further test and characterize all sampling devices and understand plume dynamics by deploying a large scale sampling array to tract fluorescent particles placed in the debris stream of the harvester. 5. Activities conducted in the next reporting period will lead to improved understanding of the potential dollar value impact of alternative management strategies on the carrot seed industry.

Impacts
What was accomplished under these goals? The production of active compounds against Xanthomonas hortorum pv. carotae (Xhc) in bacterial isolates 17-044 and 17-049 has been inconsistent. However, after an extensive medium optimization study, we found that NB and KB media supplemented with 0.5% L-proline and L-leucine gave the optimal production of the active compounds, particularly in isolate 17-049. We have scaled up the culture and produced active extracts ready for in planta testing, which will be done by the Dung lab at Oregon State University and the du Toit lab at Washington State University. We have also started working on the chemical synthesis of the most active compound and its diastereomers and will evaluate their activity against Xhc. We have screened 14 new isolates received from the Dung lab for their activity against Xhc. Each isolate was cultured in three different media. The best results were observed in isolate 16-135 (cultivated in modified 2XYT medium), 16-304 (cultivated in KB medium), and isolate 16-357 (cultivated in KYPG medium). Currently, we are in the process of isolating and identifying the active compounds from these isolates. Given the history of very poor efficacy of copper-based spray programs for control of Xhc in carrot seed crops in the Pacific Northwest, combined with evidence from an onion SCRI project of the presence of copper tolerance genes in the strains of multiple bacterial pathogens of onion in the Columbia Basin of Washington State, the genomes of >300 Xanthomonas strains associated with carrot were screened by the Chang lab at Oregon State University for the presence, absence, and allelic diversity of the following copper tolerance genes: copR, copS, smmD, cusB, copB, copC, copD, copG, copK, copM, copL, cusA, copA, copF. Only 4 of these genes were detected in the >350 genomes. A subset of 49 of these strains from each of 3 phylogenetic clades (determined using whole genome sequences produced in Objective 4) with various permutations of the presence or absence of these four copper tolerance genes was selected to test phenotypically for tolerance to copper. The strains will be tested in fall 2023 on agar media amended with a range of concentration of copper sulfate, from 0 to 400 ppm, in 50 ppm increments. A field trial was conducted at the Kern County Extension Research Station in Shafter, CA to evaluate the efficacy of various bactericide products. In the trial, nine bactericide and biological plant activators were tested for efficacy against Xhc. Pathogen population levels in the foliage at different sampling points following bactericide applications was assessed. Our team implemented a refined design of a 3D printable cascade particle settling trap (CST) that separates airborne particulates into size classes. Using these tools, we designed and executed another field experiment using CSTs to test the hypothesis that airborne Xhc concentration is a function of both distance from particle generating source and particle size. This experiment was carried out independently during three combining events. We also deployed a 10-meter and a 5M micro-meteorological tower and 4 smaller micro-meteorological stations to evaluate the surface fluxes and atmospheric transport characteristics during harvesting events. A system of laser-based optical particle counters was used to evaluate how particle concentration changes as a function of height and time during tractor-based field events. We performed preliminary analysis on these data to plot the spatiotemporal evolution of dust concentration during the combining events and refine experimental design for the 2023 field campaign. Finally, we developed a mobile 3D printed cyclonic sampling sampling device for capture particles over a carrot crop and tested its suitability for use as a scouting method to monitor disease presence and development during the production season To improve understanding of the cost of Xhc to seed companies, buyers, and growers and to evaluate the impact of sanitation practices on seed quality we collected and analyzed germination data before and after hot water treatment from 65 seed lots spanning a 5-year timeframe. A forecasting model to predict germination rates after hot water treatment was developed. Results indicated that variety had a significant impact on germination rates after hot water treatment. One variety stood out as having a very favorable response to hot water treatment while on average, all other varieties exhibited a 3.5 % lower germination rate after treatment as compared to the highest performing variety. Stressful growing conditions have been found to impact germination rates after hot water treatment, but analysis from our sample suggested that the unique and unusual heat and haze experienced by carrot seed producers in the western US in 2021 and 2022 did not have a significant impact on germination rates after hot water treatment. Treatment batch size was also not found to have a significant impact on seed germination rates. Ultimately, seed quality before treatment had a positive significant impact on germination rates after treatment. The economic impact of reductions in germination rate are dependent on buyer. In some cases, the decrease in germination rates after treatment can lead to rejection of the entire seed lot, in other cases the price may be discounted. An important implicit cost of hot water treatments was identified within the seed germination analysis. The amount of time required to treat and test seed lots can create significant time lags between delivery of the crop and receipt of payment; a financial stress that could be partially alleviated by improved in field treatments to reduced dependency on hot water treatments.

Publications

  • Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2023 Citation: Baldino. K., Huckins, M., Chang, E., Pardyjak, E., Stoll, R., Mahaffee, W., and Dung, J.K.S. 2023. Still gathering dust: Monitoring airborne Xanthomonas hortorum pv. carotae during carrot seed harvesting events in Central Oregon. American Phytopathological Society Plant Health 2023. Meeting. August 12-16, 2023. Denver, CO.
  • Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2023 Citation: Hobson, E., Bernal, E., Dung, J.K.S., du Toit, L.J., and Jacobs, J.M. 2023. Examining the influence of carrot cultivar resistance on host colonization by Xanthomonas hortorum pv. carotae. American Phytopathological Society Plant Health 2023. Meeting. August 12-16, 2023. Denver, CO.
  • Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2023 Citation: Scott, J.C., Sullens, K.L., Pardo, S.M., and Dung, J.K.S.. 2023. Leaf spot disease of Nepeta cataria caused by a distinct pathovar of Xanthomonas hortorum. American Phytopathological Society Pacific Division Meeting. March 14-16, 2023. Tucson, AZ.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: An Update on SCRI-Funded Bacterial Blight Research. Invited speaker. 2023 Carrot Research Symposium. February 14, 2023. Remote/virtual (~50 attendees)


Progress 09/01/21 to 08/31/22

Outputs
Target Audience:Target audiences of this project included carrot and carrot seed growers, carrot seed contractors, professional crop consultants, agri-chemical company representatives, vegetable seed company representatives, University Extension educators and faculty, and the public. Changes/Problems:COVID-19 travel restrictions have increased costs and difficulty in conducting field campaigns. Costs of laboratory supplies and consumables have also increased significantly. What opportunities for training and professional development has the project provided?1. One summer intern from Fresno State University trained on bactericide applications, sampling, and data collection in the trial. One M.S. student and one undergraduate student were trained in plant pathology, bacteriology, and epidemiology. The project has provided training opportunities to two postdoctoral scholars in biochemistry and pharmacology. 2. One PhD student, four undergraduates, and one high school student worked on various aspects of data collection/analyses or equipment design. 3. A graduate student and postdoctoral researcher were trained as part of this year's project work. 4. One postdoctoral researcher and one undergraduate worked on genomic epidemiology. 5. None to report. How have the results been disseminated to communities of interest?A total of nine conference presentations, one technical report, two popular press articles, and one peer-reviewed manuscript were published. One workshop was also held. Outreach was conducted at the International Carrot Conference, the Columbia Basin Vegetable Seed Association annual meeting. Audiences for these presentations and publications include(d) seed company representatives, scientists, students, agri-chemical company representatives, and farmers/producers. What do you plan to do during the next reporting period to accomplish the goals?1. We will investigate the impacts of infected/infested carrot seed, stecklings, and carrot seed crop debris on the development of bacterial blight epidemics and evaluate cultural, chemical, and IPM practices to mitigate these sources of inoculum. We will continue to screen bactericide products for bacterial blight control. We will also determine the MIC of the active compounds against Xhc, conduct in planta assay of the active compounds against Xhc, explore the possibility of improving the production of active compounds through genetic engineering, and investigate anti-Xhc compound(s) from another isolate, 16-317. 2. The designed sampler is being utilized to examine airborne pathogen movement in tomato production. We will conduct another field campaign during harvest attempting to capture up to 8 harvest plumes from each of 3 sperate fields. 3. We will make steps towards defining seed colonization by Xhc with electron microscopy. 4. We are approximately 75% done writing this work up. Our goal is to have a manuscript submitted before the end of 2022. 5. Within the next reporting period preliminary field data will be evaluated and the estimation of economic impacts to seed companies and buyers will be completed.

Impacts
What was accomplished under these goals? 1. Potential interactions between foliar applications of ManKocide (mancozeb + copper hydroxide) with foliar applications of each of three fungicides commonly used in carrot and carrot seed production were evaluated in replicated greenhouse trials conducted in Mount Vernon, WA. The hypothesis was that foliar-applied fungicides reduce the epiphytic population of fungi on carrot leaves, which might increase the risk of colonization of carrot foliage by X. hortorum pv. carotae (Xhc), thereby minimizing the efficacy of ManKocide for control of bacterial blight. Plants treated with fungicides and then inoculated with Xhc had numerically greater, but not significantly greater, Xhc levels than inoculated plants that had not been treated with fungicides. Tank-mix application of ManKocide with the fungicides Quilt, Rally, or Rovral did not alter the efficacy of ManKocide against Xhc. The trial was repeated in Madras, OR. Results from both greenhouse trials suggest that foliar applications of fungicides to control diseases like powdery mildew and Alternaria leaf blight in carrot seed crops do not affect the efficacy of ManKocide against Xhc. However, ManKocide has limited efficacy and only when used preventatively, as there is no curative efficacy. A field trial was conducted in Shafter, CA to evaluate the efficacy of nine bactericide and biological plant activators for efficacy against the pathogen, Xhc. Pathogen population level in the foliage at different sampling points following bactericide applications was assessed. Compared to the non-treated control, Xhc populations were slightly lower on carrot foliage in all other treatments. Although some treatments such as Kocide and Nordox performed slightly better than others, Xhc populations increased across all treatments (>105 CFU/g dry leaf tissue) in the second sampling. The pathogen levels decreased relatively in the third and final sampling. In the final sampling, treatment Cueva had the lowest pathogen population (1.47×102) followed by Nordox (9.52×103), and Kocide (2.46×104). These levels are lower than the levels of colonization (>106CFU/g of leaf tissue) necessary for the development of symptoms in the field. Significant differences in the Area Under Colonization Progress Curve (AUCPC) were observed with treatments Kocide, LifeGard, Cueva, and Nordox compared to the non-treated control. We have isolated a pure active compound (AP1) from bacterial isolate 17-044 and characterized its chemical structure. We have also isolated two active compounds (AP2 and AP3) from isolate 17-049 and characterized their chemical structures. All of these compounds were active against Xhc, with AP1 and AP2 were significantly more active than AP3 in an agar diffusion assay. We attempted to improve the production of the active compounds by optimizing the culture condition, however, this approach was not successful. In collaboration with Objective 4, we obtained the genome sequences of isolates 17-044 and 17-049. We are exploring the possibility of using a genetic approach to improve the production of the active compounds. These compounds have strong growth inhibitory activity against Xhc in vitro and have great potential to be developed as crop protectants against Xhc infections in carrots. 2. We prototyped, tested, and implemented a 3D printable cascade particle settling trap (CST) that separates airborne particulates generated during tractor-based field events into size classes using stainless-steel woven fabric that is suitable for downstream processing for molecular and culturing assays. Field experiments using the CSTs were executed to test the hypothesis that airborne Xhc concentration is a function of both distance from particle generating source and particle size. This experiment was carried out independently during threshing, swathing, and combining events. We deployed a 10-meter micro-meteorological tower and 8 smaller micro-meteorological stations to evaluate the surface fluxes and atmospheric transport characteristics during harvesting events. Our team designed and configured a system of laser-based optical particle counters to evaluate how particle concentration changes as a function of height and time during tractor-based field events. We also performed preliminary analysis on these data to plot the spatiotemporal evolution of dust concentration during the combining events and refine experimental design for the 2022 field campaign. Our group developed a sampling method that allowed for capturing large volumes of particles discharged from harvest equipment that is suitable for downstream pathogenesis and viability assessments. Findings from these accomplishments demonstrate that the CSTs are suitable for monitoring spread on airborne particulates in carrot and other production systems. These traps are already being deployed to monitor movement of other airborne pathogens. The use of relatively inexpensive laser-based optical particle counters will enable more refined characterization of particle plumes of the finer particles that can move longer distances than larger particles. Results demonstrate that viable and pathogenic inoculum is present in all particles sizes and that long distance dispersion will need to be accounted for to prevent "green bridging" from one carrot-seed field to another due to the 18-month production cycle. 3. We collected confocal imaging data to describe the inner leaf colonization by Xhc. We determined that quantitative resistance significantly slowed down the infection progress compared to susceptible carrot varieties. This imaging also provided stronger evidence that stomata guard cells are the primary entry point for Xhc leaf colonization. 4. In the last report, we had completed the sequencing and analyses of 184 strains. As of this year, we have now sequenced and analyzed a total of 323 strains. Most are Xhc, though some are different species of Xanthomonas, and some were biocontrol strains. Newly sequenced strains were collected from more diverse locations, various tissues, various sources, including airborne particles, international seed production companies, and culture collections. For the latter, we sequenced time stamped historical strains. All genome sequences have been analyzed to infer genus-, species-, and genotype-level relationships, predict effector encoding genes, and presence of plasmids. Moreover, we have constructed time trees and mapped on important historical events in carrot/carrot seed agriculture. The key findings from our analyses are that: 1) there is very limited genetic diversity in Xhc associated with carrot seed production, 2) Xhc strains continue to be disseminated across the world, 3) Xhc has likely been associated with carrots throughout the history of carrot domestication and globalization, 4) the three main lineages of Xhc present in the Pacific Northwest were likely bottlenecked during establishment of the industry in this region, and 5) Xhc is endemic to carrot seed production in the Pacific Northwest and potentially beyond. Findings from this aim are important. They suggest that Xhc is continually dispersed with carrot seeds as regions are being established for carrot seed production. Secondly, use of control methods or development of new disinfection methods are necessary to reduce the continual movement of Xhc on carrot seeds. 5. Cost of production estimates for all major carrot growing regions were completed and updated to reflect current economic conditions allowing growers the ability to accurately assess the impacts of various management strategies in an environment characterized by high input price volatility. We began evaluation of the economic impacts of Xhc to seed companies and buyers by collecting data to estimate the costs of Xhc disinfection practices. The impact of Xhc on seed quality and marketability will also be estimated.

Publications

  • Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: du Toit, L.J., and Dung, J. 2022. Bacterial blight in carrot seed crops. Columbia Basin Vegetable Seed Association Annual Meeting, 13 Jan. 2022, Moses Lake, WA. (~25 people)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Sidhu, J., Dung, J., and Scott, J. 2022. Evaluation of bactericides for bacterial blight control in carrots. Poster presentation at the 40th International Carrot Conference, August 29-30, 2022 Mount Vernon, Washington, USA.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Dung, J., and Scott, J. 2022. The effect of seedborne Xanthomonas hortorum pv. carotae on seed germination and seedborne transmission of bacterial blight in carrot. Oral presentation at the 40th International Carrot Conference, August 29-30, 2022 Mount Vernon, Washington, USA.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Hobson, E., Bernal, E., du Toit, L.J., and Dung, J.K.S. 2022. Characterizing internal carrot leaf colonization patterns by Xanthomonas hortorum pv. carotae. Oral presentation at the 40th International Carrot Conference, August 29-30, 2022, Mount Vernon, Washington, USA.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Baldino, K., Huckins, M., Scott, J., Stoll, R., Pardyjak, E., Mahaffee, W., and Dung, J. 2022. The places Xanthomonas will go: Examining Xanthomonas hortorum pv. carotae in airborne debris and on non-carrot crops in central Oregon. Oral presentation at the 40th International Carrot Conference, August 29-30, 2022 Mount Vernon, Washington, USA.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Mahaffee WF, Margairaz F, Ulmer LD, Bailey BN, Stoll R. Catching spores: Linking Epidemiology, Pathogen Biology, and Physics to Ground-Based Airborne Inoculum Monitoring. Plant Dis. 2022 Jun 9. doi: 10.1094/PDIS-11-21-2570-FE. Epub ahead of print. PMID: 35679849
  • Type: Conference Papers and Presentations Status: Other Year Published: 2021 Citation: Huckins, M., Baldino, K., Dung, J., Mahaffee, W., Morrison, T., Pardyjak, E., and Stoll, R. 2021. New Approach for Capturing and Characterizing Large Aerosolized Particles in Agricultural Settings. GU Fall Meeting 2021, held in New Orleans, LA, 13-17. 2021AGUFM.A45C1866H. https://ui.adsabs.harvard.edu/abs/2021AGUFM.A45C1866H/abstract
  • Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Baldino. K., Huckins, M., Pardyjak, E., Stoll, R., Mahaffee, W., and Dung, J.K.S. 2022. Gathering dust: Exploring the aerobiology of Xanthomonas hortorum pv. carotae in carrot seed crops of central Oregon. 2022 APS Plant Health Meeting, August 6-10, 2022, Pittsburgh, Pennsylvania, USA
  • Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Baldino. K., Scott, J.C., and Dung, J.K.S. 2022. Exploring the epiphytic colonization capability of Xanthomonas hortorum pv. carotae on non-carrot crops. 2022 APS Pacific Division Meeting (virtual).
  • Type: Other Status: Published Year Published: 2021 Citation: Dung, J., Scott, J., Williams, H., and Sagili, R. 2021. Can honeybees become contaminated by Xanthomonas hortorum pv. carotae, causal agent of bacterial blight of carrot? Central Oregon Agricultural Research and Extension Center 2021 Annual Report:24-28.
  • Type: Other Status: Published Year Published: 2022 Citation: Dung, J., and Scott, J. 2022. Blowin' in the wind: Insights into bacterial blight epidemiology. Carrot Country Magazine (Fall):4-5.
  • Type: Other Status: Published Year Published: 2022 Citation: Greenway, G. 2022. Estimating the economic impact of bacterial blight. Carrot Country Magazine (Fall):8-9.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Hobson, E. and Jacobs, J.M. 2022. Plants as Living Landscapes for Microbes: Examining the Role of Plant Immunity on the Bacterial Colonization of the Inner Leaf. Plant Health 2022. Portland OR USA (oral presentation)


Progress 09/01/20 to 08/31/21

Outputs
Target Audience:Target audiences of this project included carrot and carrot seed growers, carrot seed contractors, professional crop consultants, agri-chemical company representatives, vegetable seed company representatives, University Extension educators and faculty, and the public. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?1. Objective 1 of this project has provided training opportunities to a graduate (M.S.) student, a postdoctoral scholar, and a Fulbright visiting scholar. 2. One graduate (Ph.D.) student is currently funded by Objective 2 of this project and is being trained in particle modeling and aerobiology of plant pathogens. 3. One postdoc is currently funded by Objective 3 of this project and is being trained in Xhc inoculation and imaging of carrot. 4. Objective 4 has funded one postdoc trained in genomic epidemiology and one undergraduate trained in computational biology. 5. None to report. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?1. The next steps are to complete the structure elucidation of the active compound; obtain the genome sequences of the five variants of strain 17-044; improve the production of the active compound by further optimizing the culture condition; and conduct in planta assays of the active compound against Xhc. 2. The next steps are to conduct particle sampling and analysis when growers are threshing males (late July) and harvesting seed (late August). A series of sampling experiments will be run to collect particles during farm operations to determine particle size associated Xhc movement in the field and the distance particles move in the air mass. Further, refinements to current custom samplers and development of other novel particle sampling approaches will be explored. 3. For the next period, we will continue defining how these bacteria colonize leaf surfaces using microscopy techniques. We will perform a quantitative analysis of bacterial numbers and where they are located in and on leaf surfaces. Seed transmission is an important mechanism in Xhc dissemination. We will begin defining carrot seed colonization by Xhc. We will perform analysis using microscopy of contaminated seed and also develop methods for seed infection analysis from in house inoculation. 4. For the next period, we will continue to analyze whole genome sequences of Xhc to examine alternative explanations for transmission patterns. We will also mine the data for candidate virulence loci and plasmid sequences and determine variations in plasmids to generate hypotheses on the role that these mobile genetic elements have on disease of carrots. We also anticipate receiving more strains from other parts of the world and will analyze their genome sequences along with those reported herein. Last, we will mine genome sequences of putative biocontrol strains and work with members of the research team to identify loci predicted to be involved in the biosynthesis of natural products that could be used to control against Xhc on carrot plants and seeds. 5. Our current focus has been to estimate the grower level impacts of Xhc. Within the next reporting period we expect to evaluate impacts at the next link in the supply chain by developing a hedonic pricing model to estimate the value of quality attributes impacted by Xhc to seed buyers/exporters.

Impacts
What was accomplished under these goals? 1. Growth chamber and greenhouse trials were conducted to evaluate foliar applications of phages effective against other Xanthomonas species for activity againstX. hortorum pv. carotae (Xhc). Foliar applications of phages did not reduce epiphytic populations of Xhc on carrot, but several adjuvants were identified that increased phage persistence on leaves 100-fold. Plaque assays showed that the phage mixture is not effective against all strains of Xhc. Field studies have identified several sources of potential reserviors in central Oregon seed production, including carrot crop debris, umbelliferous seed crops such as parsley and parsley root, and non-umbelliferous crops such as Kentucky bluegrass and roughstalk bluegrass. Studies are underway to further understand the survival of Xhc in carrot crop debris and potential epiphytic hosts other than carrot. Extracts from the culture broths of eight environmental isolates (bacterial strains) were screened for their antibacterial activity. The extracts were tested against five pathogenic bacteria including Xhc. Seven out of the eight bacterial isolates were able to produce antibacterial compounds under laboratory conditions. In particular, extracts from strain 17-044 showed high activity against the Gram-negative bacteria Pseudomonas aeruginosa, Escherichia coli, and Xhc. The active compound was isolated using bioassay-guided fractionation, followed by purification with HPLC. Elucidation of the chemical structure of the compound is currently on-going. Interestingly, while investigating the bacterium for the production of bioactive compounds, we found that cultures of strain 17-044 showed some phenotypic differences, two of which showed strong antibacterial activity towards Xhc. Antibacterial assay and HPLC analysis of the extracts showed that culture J2A is the most productive variant. Further studies of these variants including detailed analysis of their genetic makeup is being pursued. 2. An eddy covariance tower was setup in a carrot field near Madras, Oregon that consisted of four 3D sonic anemometers with a fine wire thermal couple and a HMP60 temperature and humidity sensor located at 10, 3, 2, and 1m above ground. An Irgason was located at 5m to measure carbon and vapor flux in addition to 3D momentum and temperature fluxes. The tower also had sensors for longwave, shortwave, and photosynthetically active radiation. All data was logged on a Campbell Scientific CR3000 datalogger. Airborne particle concentrations data (particles with diameters um was collected using five Alphasense optical particle counters each in custom housings collocated with the 3D sonics at 10, 5, 3, 2, and 1m. In addition, 6 LEMS (lowcost energy measurement stations) were deployed around the field perimeter to measure the spatial distribution of 2D wind speed and direction, temperature, and humidity. To collect data on the movement of larger particles (<1000 µm), a custom particle sampler was designed and built that can collect and bin particles into size classes of >800µm, 800-400, 400-250, 250-100, 100-50 and <50 µm. The samplers are readily constructed using 3D printing and off-the-shelf electronic components. Collected particles are captured on a matrix (nylon mesh) suitable to process for enumeration of Xhc through either culturing or DNA extraction. Xhc was detected in all particle sizes tested. 3. We gathered resistant and susceptible seed (germplasm) from industry partners and collaborators from this project and tested virulence of Xhc strain 14.007 from Oregon on multiple carrot varieties. We are comparing various genotypes to help inform our understanding of bacterial infection of leaves. There is variation of leaf colonization across genotypes, which could affect transmission and spread. An important aspect to the objectives is understanding the infection cycle by Xhc. We developed both artificial and naturalistic inoculation methods for carrot leaf infection by Xhc. We used blunt infiltration by a needleless syringe for artificial inoculation and rapid phenotyping. We used a spray inoculation method to allow bacteria to enter naturally through openings (e.g. hydathodes, stomata or wounds). We transformed Xhc 14.007 and 19.053 with pNEO-GFP and are beginning to define the specific tissues colonized by Xhc cells with confocal and scanning electron microscopy. 4. We gathered 184 strains of Xhc collected from diverse geographic locations and sources, including airborne debris, soils, seeds, and plants without disease symptoms. Their genomes were sequenced in two channels of an Illumina HiSeq3000 and analyzed along with 96 that we had sequenced previously. Those of the latter were collected from carrots showing disease symptoms and seeds primarily from one geographic location. Short reads were processed for quality and assembled into draft genome sequences, which have been annotated. We used average nucleotide identity, multi-locus sequence analysis maximum likelihood trees and single nucleotide polymorphism (SNP) differences to cluster strains into species-level groups and genotypes. Last, we constructed a minimum spanning network and projected geographic information to identify potential epidemiological links. There is a remarkably limited amount of diversity among Xhc analyzed. A total of 267 of the 280 strains grouped into a single species-level group. The remaining 13 strains are distributed throughout the Xanthomonas genus tree. Within the well-represented species-level group, there are a total of 158 genotypes, defined based on having ≤15 SNP differences relative to a common reference sequence, though most of these genotypes differ by no more than 400 SNP differences. Additional investigation of genome composition confirmed that strains within and between genotypes are very closely related and are essentially clonal or nearly clonal. Preliminary analysis of epidemiological patterns suggests that the Xhc population experienced a bottleneck in the Pacific Northwest (PNW) where much of the carrot seeds are produced. Moreover, we identified links between the PNW, other states, and foreign countries, indicative of possible transmission routes for Xhc. We also sequenced the genomes for eight strains identified as potential biocontrol bacteria. Preliminary analyses of their genome sequences confirmed their original taxonomic classifications based on analyses of 16S rRNA-encoding regions. 5. We surveyed growers and crop consultants to document current Xhc management practices in major growing regions and to evaluate perceptions of the impact of Xhc on carrot seed yield in recent production cycles. Preliminary results suggest that on average, the value of lost yield attributable to Xhc ranges between $8 and $11 million annually. Expenditure on the most widely used product for Xhc management is estimated to range between $500,000 and $600,000 annually; application costs are estimated to exceed $250,000 annually. This data highlights the significant role improved Xhc management practices could play in stabilizing some of the volatility and risk associated with carrot seed production. Improvements in the overall financial wellbeing of the carrot seed industry can create positive spillover effects in the rural communities that depend on the industry as an economic driver. Baseline cost, return, and breakeven estimates associated with current production practices under various types of irrigation in major carrot seed growing regions were developed. The downloadable cost of production workbooks allow for evaluation of the short- and long-term economic impacts associated with failure of seed to meet germination standards. Users can also evaluate the impacts of carrot seed management decisions on profitability. This tool will assist in improving stakeholder understanding of the costs and benefits of current and future treatment programs developed as this project progresses.

Publications

  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Weisberg, A.J., Gr�nwald, N.J., Savory, E.A., Putnam, M.L. and Chang, J.H., 2021. Genomic Approaches to Plant-Pathogen Epidemiology and Diagnostics. Annual Review of Phytopathology, 59.