Source: WASHINGTON STATE UNIVERSITY submitted to
STOP THE ROT: COMBATING ONION BACTERIAL DISEASES WITH PATHOGENOMIC TOOLS AND ENHANCED MANAGEMENT STRATEGIES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
NEW
Funding Source
Reporting Frequency
Annual
Accession No.
1020312
Grant No.
2019-51181-30013
Project No.
WNP03104
Proposal No.
2019-03171
Multistate No.
(N/A)
Program Code
SCRI
Project Start Date
Sep 1, 2019
Project End Date
Aug 31, 2023
Grant Year
2019
Project Director
du Toit, L. J.
Recipient Organization
WASHINGTON STATE UNIVERSITY
240 FRENCH ADMINISTRATION BLDG
PULLMAN,WA 99164-0001
Performing Department
Plant Pathology
Non Technical Summary
Onion bulb crops are grown on ~140,000 acres/year in the U.S. at a farm-gate value of $925M. Bacterial pathogens cause >$60M in losses annually to this industry. Losses can be particularly severe for stored bulbs as bacterial bulb rots typically only develop in storage, after all production costs have been incurred. Poor scientific understanding of the diversity and epidemiology of bacterial pathogens, and the lack of systemic bactericides limit industry capacity to mitigate these losses; this is in sharp contrast to the significant work that has been accomplished with fungal pathogens of onion. This 'Stop the Rot' project organizes 24 scientists in diverse disciplines across the U.S. to research the complete system (host, pathogen, and environment) of bacterial diseases of onion. The long-term goal is to support profitability and sustainability of onion production in the U.S. using a coordinated, national survey of bacterial pathogens affecting onion crops combined with a stakeholder-focused, systems approach to investigate how production practices, inoculum sources, and environmental conditions can be managed to develop effective, practical, economically-viable, and environmentally-sound strategies to limit losses to bacterial diseases.The project has two primary objectives linked iteratively in a systems approach. The first objectives utilizes comparative genomics to identify genetic factors that enable some bacteria to cause diseases on onion, and to develop practical diagnostic tools as well as phenotypic resistance screening methods for bacterial pathogens of onion. A survey of onion bacterial diseases over three seasons in each of 12 states representing the seven primary regions of onion production in the U.S. will be used to understand the diversity of onion bacterial pathogens in the U.S., and to develop a National Onion Bacterial Strain Collection. Genomic assessment of this bacterial collection will enable us to understand the genetic basis of bacteria that can cause diseases of onion across the U.S. This, in turn, will be used to design rapid, accurate, and robust methods of detecting and identifying onion bacterial pathogens. The collection also will be used to develop methods of screening onion germplasm for resistance to bacterial pathogens. The screening methods can then be used in breeding programs to develop cultivars with greater resistance than currently available. The second objective focuses on onion bacterial disease management by examining how irrigation practices, fertility practices, pesticide programs, cultural practices, post-harvest practices, and bacterial disease modeling can be managed to develop effective, practical management programs. A 12-person, nation-wide onion Stakeholder Advisory Panel worked with our team from 12 states to prioritize the objectives and develop approaches for this project. Broad, stakeholder-based evaluations of the research results over the duration of the project will ensure results are delivered to constituents and that solutions developed are viable economically and environmentally.
Animal Health Component
0%
Research Effort Categories
Basic
25%
Applied
60%
Developmental
15%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
21214511100100%
Goals / Objectives
The ability of growers to manage bacterial diseases of onion is limited compared to many fungal diseases because of unique epidemiological and management aspects of the bacterial pathogens, which cause >$60 million/year in damages to the U.S. onion industry. Losses can be particularly severe for stored onions as bacterial bulb rots typically develop after harvest, with losses ranging from 5-100% in individual fields. The cost of sorting symptomatic bulbs can result in rejection of entire fields if the incidence of rot exceeds 30%. There are effective tools to manage other factors that can cause storage losses (e.g., sprouting and fungal bulb rots), in contrast to bacterial rots. This project takes a systems approach encompassing the pathogen, host, environment, economics, and stakeholder priorities to address the need to manage onion bacterial diseases far more effectively. The goal of this project is to support long-term profitability and sustainability of onion production in the U.S. using a stakeholder-informed, systems approach by:Undertaking a national survey of onion bacterial diseases;Developing a National Onion Bacterial Strain Collection (NOBSC);Using this resource for genotypic characterization of the pathogens to design rapid, accurate, and robust methods for detecting and identifying onion bacterial pathogens across the U.S.;Developing methods of screening onion germplasm for resistance to these bacteria;Integrating the diagnostic and detection tools into comprehensive integrated disease management research trials;Generating predictive bacterial disease models across diverse regions of onion production in the U.S.; andImplementing a broad, Stakeholder Advisory Panel (SAP)-informed dissemination plan to deliver results to constituents.The goal utilizes a coordinated, national survey of bacterial pathogens affecting onion crops combined with a stakeholder-focused, systems approach to investigating how production practices, inoculum sources, and environmental conditions can be managed to develop effective, practical, economically-viable, and environmentally-sound strategies to limit losses to bacterial diseases. The project has two primary objectives linked iteratively in a systems approach:A: Utilize comparative genomics to identify virulence factors and develop practical diagnostic tools, as well as phenotypic resistance screening methods for bacterial pathogens of onion (60% of the effort for this project). The four activities for this objective entail: A1) national onion bacterial disease surveys and development of a National Onion Bacterial Strain Collection (NOBSC), A2) onion bacterial pathogenomics, A3) development of onion bacterial pathogen detection tools, and A4) development of onion phenotyping (screening) protocols for reactions to bacterial pathogens.B: Examine how key production practices, environmental factors, and inoculum sources that impact bacterial disease outbreaks can be managed to develop practical solutions that are viable environmentally and economically (40% of the project effort). This will entail six areas of investigation: B1) irrigation management, B2) fertility management, B3) pesticide programs, B4) cultural management, B5) post-harvest management, and B6) bacterial disease modeling.Objective A addresses the SCRI focus area of "efforts to identify and address threats from pests and diseases" by clarifying for stakeholders nationwide, using standardized protocols, the diversity of onion bacterial pathogens causing losses in each of three growing seasons. The National Onion Bacterial Strain Collection resulting from this project will provide an invaluable resource for this and future projects on genetic studies of pathogens and associated bacteria in/on onions, e.g., whole genome sequencing to find virulence factors in order to develop robust molecular tools to detect and differentiate pathogenic vs. non-pathogenic bacteria. This will facilitate development of effective phenotypic methods of screening onion germplasm for reactions to diverse bacterial pathogens, alone and in combinations, which will facilitate future efforts to breed for resistance.Objective B addresses the SCRI focus area efforts to identify and address threats from pests and diseases" as well as "efforts to improve production efficiency, handling and processing, productivity, and profitability". This coordinated, multi-state objective will determine how regional onion production practices, environmental conditions, and inoculum sources can be managed using practical, economically-viable, and environmentally-sound strategies to limit losses to bacterial rots in fields, storage, and shipping.
Project Methods
Objective A: A1. Survey onion bulb crops for bacterial pathogens. Onion surveys will be completed in 12 states over 3 seasons with samples sent to 7 Regional Bacterial Diagnostic Labs. A team workshop in Year 1 will standardize protocols. Bacterial strains will be shipped to UGA for the NOBSC. Directed surveys in Seasons 2-3 will use molecular diagnostic tools developed. ~1,000 strains from Regional Labs will be tested for pathogenicity to onion using a red onion scale assay for high throughput screening of bulb colonization phenotype, and a foliar pathogenicity test. Species will be identified by MLSA.A2. Pathogenomic analyses, virulence factors, and bacterial community analyses. Pathogenomic analyses and detection of virulence factors will focus on the Pantoea species complex as a model system. Genomic DNA will be sequenced for 100 strains of Pantoea (pathogenic and non-pathogenic) from the NOBSC by Illumina shotgun, assemblies annotated, and the Pantoea pangenome analyzed. Candidate gene loci associated with pathogenic strains of Pantoea will be identified by correlating virulence phenotypes with gene presence/absence and nucleotide variations of virulence-associated genes recently identified in P. ananatis. Virulence-associated loci of each species will be characterized using mutation analysis and genetic complementation, and loci tested to design robust, molecular diagnostic assays for pathogenic strains. To understand interactions of bacterial pathogens in complex microbial communities associated with onion bulbs, DNA extracts of bulb samples from 3 Regional Labs will be examined using Illumina MiSeq.A3. Molecular diagnostic tools for pathogenic strains of P. agglomerans and P. ananatis. Onion virulence loci identified in P. ananatis will be targets for developing rapid diagnostic markers in P. ananatis and P. agglomerans strains for robust and sensitive Taqman PCR and LAMP assays.The primer/probe sets will be evaluated with strains from the NOBSC. Optimized assays will be validated at Regional Labs. Diagnostic tools will be tested in directed surveys for assessing inoculum sources and development of bacteria on onion and other plant species.A4. Development of phenotypic resistance screening methods for onion bacterial diseases. Lab and greenhouse phenotypic screening at UGA will screen short-, medium-, and long-day cultivars and diverse Allium germplasm from the USDA NPGS with virulent strains of P. ananatis, P. agglomerans, and B. gladioli pv. alliicola. A detached fleshy bulb scale assay will be compared with foliar cut-tip and atomizing methods. In Season 1, ~50 cultivars will be screened with P. agglomerans and B. gladioli pv. alliicola in a WA field trial. Field screening also will be completed in GA, TX, and WA in Season 2, and in 7 states in Season 3 to compare among regions, seasons, pathogens, and assays.Objective B: B1. Irrigation practices. Trials in 6 states will evaluate frequency and duration of irrigation, and timing of final irrigation relative to crop maturity on onion bacterial diseases, with input from SAP and regional stakeholders. Results will be used to inform bacterial disease modeling (B6) and identify practices associated with increased risk of bulb rots.Weeds and irrigation water sources will be tested using diagnostic tools developed. Greenhouse studies will evaluate impacts of elevated soil temperature and frequency of irrigation on bacterial diseases. Season 3 on-farm trials will compare growers' practices with recommendations we develop.B2. Fertility practices. Trials in 5 states will evaluate effects of N application rates and N application after bulb initiation on bacterial diseases. These treatments also will be evaluated in irrigation trials (B1) to assess interactions of these practices. Trials in 3 states will assess how onion sulfur concentration affects resistance to bacteria. Results will be used in risk modeling (B6).B3. Pesticide programs. In Season 1, trials in 9 states will evaluate bactericide efficacy compared to regional growers' pesticide programs. Promising treatments will be refined in Season 2. Herbicides used in onion crops can alter the leaf cuticle. Trials in 7 states in Seasons 2-3 will compare efficacy of bactericides on plants treated or not treated with herbicides. Trials in 5 states will evaluate the degree to which thrips management reduces losses to bacterial pathogens.B4. Cultural practices. Trials in 4 statesin Seasons 1-3 will evaluate how undercutting, rolling onion tops, and topping necks at different stages of field curing affect bacterial bulb rots. Greenhouse trials will evaluate elevated soil temperature, light intensity, and mulch treatment effects.B5. Postharvest practices. Onion packers commonly inject desiccants or sanitizers into storage facilities to limit bulb rots. Based on conflicting grower practice vs. researcher recommendations, trials in 4 states will assess the degree to which this practice with conventional and organic disinfectants is efficacious.B6. Bacterial disease prediction modeling. Data on production practices, crop microclimate, and bulb rot records from growers/packers will be mined for environment- and management-related drivers of bacterial diseases, from which hypotheses will be developed to test in trials. Predictive models for onion bacterial diseases will be developed and evaluated by growers for quantifying risks of bacterial diseases. This will inform stakeholders whether to sell bulbs immediately after harvest or store bulbs. Data from onion farms in WA, ID, OR (~45,000 acres) and other regions will be analyzed using algebraic topology and interactive visual analytics to identify key drivers and interactions of bacterial diseases, develop testable hypotheses, and develop empirical predictive models.B7. Economic assessments. A stakeholder survey in Year 1 will assess perceived economic impacts of bacterial diseases, management practices, and research and extension needs. SAP members will be surveyed annually. In Year 4, packers/shippers will be surveyed again to gauge adoption of recommendations and changes in impacts of bacterial diseases resulting from knowledge generated by this project. Economic analyses will be integrated into trials. Input costs and outputs synthesized into a cost-benefit framework will estimate efficacy of management strategies developed, and be incorporated into outeach activities.Evaluation: Results will be shared with SAP members twice annually and via Alliumnet to refine the project. At field days and regional meetings, assessments will be distributed for feedback. Grower/packer survey results annually will assess knowledge of and practices used for these diseases, and research priorities. A U.S. stakeholder survey in Year 4 will assess adoption of recommendations from the project. Short-term metrics: Progress in monthly, quarterly, and annual reports; SAP feedback; number of strains in the NOBSC; effectiveness of standardized protocols; development of robust phenotypic screening methods; identification of virulence-associated genetic loci; development of molecular diagnostic tools; effectiveness of risk models for predicting bacterial diseases; changes in stakeholder understanding of bacterial diseases; numbers of reports, Extension materials, and journal articles published. Medium-term metrics: Degree of change in understanding onion bacterial pathogens across the U.S.; extent of adoption of integrated management practices; number and robustness of molecular diagnostic tools developed; adoption of phenotypic screening methods by breeders; number of graduate students/postdoctorates trained in onion production, pathology, genomics, soil science, and Extension. Long-term metrics: Number of onion cultivars developed with resistance to bacterial diseases; extent of reduction in losses to bacterial diseases across the U.S.; greater profitability and sustainability of onion production in the U.S.

Progress 09/01/19 to 08/31/20

Outputs
Target Audience:Stakeholders involved in the U.S. onion industry are the primary audience for this project. This includes onion producers, packers, shippers, and associated stakeholders engaged in various capacities in onion production, distribution, and marketing, e.g., agronomists, crop consultants, farm managers, field workers; personnel associated with agricultural supply companies (fertilizer and pesticide dealers, irrigation supply companies, etc.), seed companies, and dealers; onion breeders (public and private); and onion storage and shipping/transport personnel and companies. Public and private research and extension specialists, undergraduate students, graduate students, and postdoctorates working with diverse aspects of onion production are another key target audience for this project. The target audiences reached during this reporting period included: Onion growers, packers, processors and associated stakeholders in all 7 onion-growing regions of the US; Extension professionals in the 12 onion-growing states that are represented in this project; The project's Stakeholder Advisory Panel members represent onion farms, regional onion associations, and major vegetable seed companies (including onion breeders and plant pathologists) from across the U.S. Panel members conducted further outreach to their own networks on behalf of the project. Panel members who are industry representatives include Greg Bird (President of the Michigan Onion Committee), Bob Ehn (California Garlic and Onion Research Advisory Board), Charles Hall (Executive Director of the Georgia Fruit and Vegetable Growers Association), Robert Sakata (President of the Colorado Fruit and Vegetable Growers Association). Four Masters students, one PhD student and three postdoctorates began working on research directly related to the Stop the Rot project objectives in this reporting period. They are conducting their research in Washington State, Georgia, Colorado and South Africa. We have used several channels to reach our target audience during this reporting period, including grower meetings, conferences and workshops, the Alliumnet.com website, industry newsletters and trade publications. Project team members also reached out directly to growers in each of the onion-growing regions to recruit them for participation in the bacterial surveys during the first season. Approximately 900 growers, agronomists and industry professionals were reached through in-person presentations at conferences, workshops and grower meetings in this reporting period. From early spring, methods of communication and presentation were necessarily shifted away from in-person to virtual format due to COVID-19 constraints across the USA and in South Africa, which made it difficult to accurately count the number of people who were reached. Changes/Problems:1. LATE RELEASE OF FUNDS FOR THE PROJECT The final release of funds in April 2020 by NIFA was later than expected as a result of the USDA NIFA headquarters being relocated from Washington DC to Kansas City, MO in 2019-20. This led to delays in issuing the subcontracts for the collaborating institutions, compounded further by delays associated with COVID-19 constraints implemented at the various institutions involved in this project. Consequently, invoicing and expenditure on the project for Year 1 has been delayed, and a significant portion of the Year 1 funds will be rolled over into Year 2 to cover work that was done in the last four months of Year 1 (May - August 2020). 2. PROBLEMS DUE TO COVID-19 IMPACTS, AND RESULTING CHANGES COVID-19 impacts on project work in this reporting period were principally due to temporary closures of labs and restrictions on travel for some team members. Most field work and field trials have proceeded as planned, with additional safety precautions and social distancing requirements in place for researchers, technical staff and students that added limitations and/or additional expenses to some of the work completed in Year 1. Overall, COVID-19 impacts have led to some of the work originally planned for Year 1 being rescheduled to Year 2 or later as some labs had to shut down completely during the first ~3 months of the pandemic. Several programs were unable to hire technical support for the project in a timely manner, which affected the capacity to get as much work done as had been planned. While this will require shifting of funds from Year 1 to Year 2 budgets, it is not expected to result in substantial changes in the scope of work, nor in any changes in the total budget for the work. We expect to be able to meet all the project's planned deliverables within the original approved budget. However, we will likely need to make use of the permitted one-year no-cost extension of the project because of delays in some aspects of the project. Specific changes in this reporting period as a result of COVID-19 impacts include the following: As a result of the labs closing at the University of Georgia, early work on pathogenomic tools was unavoidably delayed and technician salary will be shifted from Year 1 to Year 2 to complete this work. In addition, while field work proceeded as planned, the team was unable to access the labs to work on identification of the bacterial genera and species collected in the field. However, this will not lead to overall delays or changes in the work program. Collaborator Colson at the University of Georgia was unable to recruit a graduate student to work on economic assessments in Year 1 and Year 2 as planned. The recruitment has been rescheduled to Years 3 and 4 with no change in the planned scope of work on this objective. Field trials at The Pennsylvania State University have been rescheduled from Year 1 to Year 2 due to the university's COVID-19 restrictions on research and hiring of new employees. The salary allocated for the field technician will be used to cover the salary of a lab technician to process onion samples from PA and NY. Summer wages for the graduate student will be reallocated to project Year 4 and summer wage payroll wages will be reallocated to Year 2. Materials and supplies allocated for field trials in Year 1 will be reallocated to Year 2. Collaborator Malla completed one round of field surveys of 4 onion crops in the Rio Grande Valley before his lab at Texas A&M Agrilife Research was shut down and his team's travel was restricted. However, their field trials were not affected and proceeded as planned. As a result of the TX lab closure, bacterial isolates collected by the CA team in their field surveys were rerouted from the TX lab to PD du Toit's regional lab in WA for processing. Due to this change, WA requested $5,000 moved from TX to WA in the second year's budget. In New Mexico, co-PI Cramer was able to identify and visit only 4 of the 5 planned fields in the bacterial survey, and the fields were not visited at the exact times proposed due to a shortage of labor. Due to the shutting down of co-PI Kvitko's lab in Georgia and collaborator Coutinho's lab in Pretoria, the WA regional lab extracted DNA from four sets of nine onion bulbs, and sent the DNA to Genohub in TX for sequencing, so that collaborator Coutinho's students could continue their research into onion bulb microbiome characteristics. Funds will be shifted from the University of Pretoria budget allocation to UGA to cover this. Co-PI Hoepting requested a shift of funds saved on her nitrogen rate trial to travel expenses, since the mileage costs for her to travel to her nitrogen timing trial increased significantly as a result of COVID-19 restrictions. Co-PI Aegerter requested to shift funds from travel to lab supplies as she did isolations for all CA fields surveyed in 2020. 3. OTHER CHANGES IN THIS REPORTING PERIOD Bacterial strain database Instead of purchasing Filemaker Cloud to serve as the data platform for the proposed National Onion Bacterial Strain Collection (NOBSC), the team decided to purchase a license for LabGuru for each regional lab. The LabGuru software is already in use in some of the project's regional labs, provides an online system that can streamline management of onion bacterial strain data within the project. The total cost for LabGuru licenses for each regional lab is slightly less than the amount budgeted for Filemaker Cloud. Irrigation trials in NY Co-PI Hoepting proposed that since NY does not have many onion growers who irrigate their crops, she would not undertake irrigation trials in NY as a priority in Year 1. The team agreed. The resources will be directed to other aspects of the field research in NY. Directed surveys in Year 2 Soil, water, weeds and contaminated onion seed can all act as sources of inoculum for bacteria that may be pathogenic on onion. At our first annual meeting and in subsequent team videoconferences, the team discussed modifying the planned directed bacterial surveys in Year 2 to also include concurrent sampling of soil, water, weeds and seeds. Collaborator Woodhall's lab has the capacity for high-throughput analyses of bacterial DNA found in soil and water and Dr Woodhall is developing sampling protocols to share with the team, who will send their soil and water samples to him for analysis in Year 2. This work will complement the surveys of bacterial pathogens in onion foliage and bulbs. The proposed change to incorporate sampling of other sources of inoculum will not require any changes to the project budget. New project team member Prof. Steven Beer of Cornell University joined the project team this year as a collaborator. His lab is producing inoculum for field trials in NY, in support of co-PI Hoepting's field work. Prof. Beer will also be sending selected pathogenic onion bacterial strains from his own extensive collection to co-PI Kvitko's lab for the National Onion Bacterial Strain Collection (NOBSC). During the past 10 years, his program received hundreds of symptomatic onion samples from NY, PA, MI, GA, ME and OR. To cover the costs of producing inoculum and sending strains to the NOBSC, $5,000 will be shifted from the WA regional budget allocation to NY. The value of Beer's bacterial strains added to the NOBSC will be added as part of the cost-share budget once the final number of strains sent to UGA has been determined. Prof. Beer's program has NY onion stakeholder funding for continuing his assessment of onion genotypes for relative susceptibility and resistance to bacterial pathogens. This work is strongly complementary to the Stop the Rot project, and he will be collaborating with the project team in the development and testing of methods for phenotypic resistance screening in onions. There will be no cost to the Stop the Rot project budget for this. What opportunities for training and professional development has the project provided?STUDENTS The grant has provided the opportunity for several graduate students and post-doctorates to work on different aspects of the project, as described below. Verushka Ibanez, 1st year MSc student at the University of Pretoria, South Africa. Dissertation title: 16S rRNA profiling and functional genomics of onion bulbs displaying symptoms of center rot from Georgia. Advisors: Teresa Coutinho, Pedro Lebre. Expected completion date: December 2021 Christopher Liakos, 1st year MSc student at the University of Pretoria, South Africa. Dissertation title: 16S rRNA profiling and functional genomics of onion bulbs displaying symptoms of center rot from Washington State. Advisors: Teresa Coutinho, Jacquie van der Waals, Pedro Lebre. Expected completion date: December 2021 Tessa Belo joined the Soils & Water program at the Northwestern Washington Research and Extension Center of Washington State University as a MS student in the summer of 2020. She is advised by collaborator LaHue and co-advised by PD du Toit. Her project focus is on combatting bacterial rot in onions through fertilizer and irrigation management. Antoinette Machado started as a MS student at Colorado State University in January 2020, supervised by co-PI Uchanski. Shaun Stice, supervised by co-PI Kvitko, was a PhD student at the University of Georgia at the start of the project period. He has been conducting research on the genetic basis of P. ananatis virulence of onion as well as distribution of identified virulence loci among Pantoea isolates. Dr. Mei Zhao was recruited to co-PD Butta's program at the University of Georgia as a post-doctorate in January 2020, following completion of her PhD at that institution. She is focusing on the bacterial survey, identification and characterization of isolates in GA, and will also assist in greenhouse and field evaluations. Dr. Gaurav Agarwal is a current post-doctoral research associate under PI Dutta who will assist in pathogenomics of Pantoea spp. Dr. Hossein Noorazar is a postdoctorate at Washington State University who is focusing on data analysis and disease risk modeling efforts, supervised by co-collaborators Rajagopalan and Kalyanamaran. His appointment on the project (part time) is expected to start in fall 2020. Hossein will initially focus on getting trained on the hypothesis extraction tools and exploring relevant satellite imagery. PROFESSIONAL DEVELOPMENT In February 2020 a workshop was held at UGA to provide hands-on training for 10 project team members. The training covered basic methods for bacterial isolations from onion tissue, pathogenicity testing and sample processing. How have the results been disseminated to communities of interest?In this reporting period, our focus has been on sharing information and engaging with as many onion growers and industry stakeholders as possible. We have been actively reaching out to growers and stakeholders through different channels (described below) in order to share current scientific understanding of onion bacterial diseases and their management, to understand growers' current state of knowledge about causes and management of bacterial diseases of onion, and to identify priority concerns for the onion industry as regards management of bacterial diseases. By December 2020, we expect to have compiled the results and findings from the first season's bacterial surveys (except potentially for some bulbs in storage in several northern states) and the first season's field trials. We plan to share that information with growers and stakeholders through many of the same channels we have used for communication in this first year, i.e., Alliumnet website content, research summaries, extension bulletins, articles in trade publications and presentations at growers' meetings and field days. REACHING GROWERS AND ONION INDUSTRY STAKEHOLDERS: Outreach to growers and onion industry stakeholders and extension staff at informal meetings, grower meetings, field days and technical workshops has included the following activities: du Toit, Lindsey (2020). Shared information on the Stop the Rot SCRI project with two onion farms in Uvalde, TX on 19 Feb. 2020. Discussed opportunities to engage in the project. 1) Brian King, Dixondale Farms, which produces ~1 billion onion transplants/year for distribution to all 50 states in the USA; 2) Jay Carnes, Wintergarden Produce, which produces short- and medium-daylength, non-storage type onion cultivars. Reitz, Stuart (2020). Provided information on the project at the Idaho-Eastern Oregon Onion Growers' Meeting, Ontario OR, February 5, 2020. Nischwitz, Claudia (2020). Presented information about the project at the Utah Onion Association meeting, February 11, 2020 (Nischwitz, n=40 attendees) Lahue, Gabe (2019). Shared irrigation expertise with onion growers at the WSU Onion Cultivar Field Day, August 29, 2019 (LaHue). Uchanski, Mark (2020). The project was summarized at the Colorado Fruit and Vegetable Growers Association (CFVGA) Produce Day in Greeley, Colorado on Jan. 28, 2020. Grower introductions and recruitment also occurred at CFVGA annual conference Feb. 24-25, 2020 in Denver, CO. Dutta, Bhabesh (2020). Provided updates on onion bacterial disease management options in 5 different county meetings in Georgia, January-March 2020 (n=314 attendees in total) Dutta, Bhabesh (2020). Provided information at county meeting, Orange County NY, on management of bacterial bulb rot with growth-stage targeted copper management in onion (n=40 attendees). Meeting arranged by Cornell University Cooperative Extension. Uchanski, Mark (2020). 06/08/2020, Field visit, Eaton Colorado, with Fagerberg Produce. du Toit, Lindsey (2020). Visited in person or by phone with 12 individual onion growers, agronomists, and field reps in central WA regarding onion bacterial diseases between March and July 2020. Outreach to onion stakeholders and the industry though trade publications and newsletters has included the following informational articles: National Onion Association. 2019. $4M given for onion bacterial disease research. Vegetable Growers News, 11 Sep. 2019. https://vegetablegrowersnews.com/news/onion-researchers-earn-4-million-grant-to-combat-bacterial-disease/ USDA Announces Specialty Crop Award to Combat Onion Diseases. California Garlic & Onion Research Advisory Board News, Fall 2019, p. 4. https://www.cagarlicandonion.com/resources/newsletters/detail/id/17 Keller, Denise. 2019. Stop the rot. Combating onion bacterial diseases. Onion World, November 2019, pp. 24-26. https://issuu.com/columbiamediagroup/docs/onion_world_november_2019 Aegerter, Brenna (2019). Bacterial Bulb Rots of Onion. In: Field Notes newsletter, UCCE San Joaquin County, CA, November 2019 issue (mailed or emailed to 1,029 stakeholders, primarily growers and allied industry members). http://cesanjoaquin.ucanr.edu/newsletters/Field_Notes_Newsletter82270.pdf Thompson, C. 2019. Center rot disease in organic onion growing studied. Vegetable Growers News, November 2019. Arboleda, K. 2019. UGA Researchers working on disease control in Vidalia onions. Vegetable and Specialty Crop News, December 2019. AgNetWest February 2020 issue: Stop the Rot Project to Combat Onion Bacterial Diseases AgNetWest March 2020 issue: Samples Needed to Help 'Stop the Rot' Project In California, a newsletter article was written and one presentation given at a regional onion extension meeting in spring 2020, and a 1-page flyer produced to announce the project and ask that our CA team be alerted to possible bacterial problems in commercial onion fields. Onion World magazine May/June 2020 issue has a short overview of the Stop the Rot project in the report on the Utah growers meeting https://onionworld.net/magazine/ du Toit, Lindsey & Hoepting, Christy (2020). "Stop the Rot: National U.S. Team to Combat Bacterial Diseases of Onion with Fierce Collaboration". Comprehensive article about the project, published in the July/August 2020 issue of Onion World magazine. https://onionworld.net/magazine/ Onion World July/August 2020 issue has a short 'Disease Quiz' submitted by Bhabesh Dutta and Ron Gitaitis to highlight onion bacterial diseases. https://onionworld.net/magazine/ du Toit, L.J., Waters, T., Derie, M., and Darner, J. 2020. Battling onion bacterial diseases with bactericides. Onion World, in press (to be published in the September/October 2020 issue). Outreach to growers and industry stakeholders through the Stop the Rot Stakeholder Advisory Panel: Our 14-member Stakeholder Advisory Panel (SAP) brings a diverse range of expertise and experience to the project. The Panel includes growers, pathologists and onion breeders from onion growing regions across the US. Panel members are active in sharing information about the project to growers and onion industry stakeholders through their own regional and national networks, and in bringing insights and information into the project from their networks. Our first annual meeting/videoconference with the SAP was held on February 3, 2020 in Athens GA. Since then, SAP members have actively participated in our monthly project team meetings by videoconference and they continue to receive project updates, notes from the monthly meetings and our internal project team newsletter. REACHING ACADEMIC RESEARCHERS, EXTENSION PROFESSIONALS AND GRADUATE STUDENTS The project team now includes 25 research collaborators from 12 states, representing all seven major U.S. onion growing regions, as well as onion bacteriologist Prof. Teresa Coutinho from South Africa. Since our first annual in-person meeting in February 2020, we have held monthly team videoconferences to share preliminary results and experiences and discuss recent findings in our Year 1 work. These monthly meetings of 1 to 2 hours have facilitated research and Extension collaboration within the project. Almost all the project collaborators are also involved in extension services and extension education in their regions, which makes for efficient transfer of new information and research results from the team to extension professionals and thence to growers. Technicians and graduate students from each of the regional teams also join our monthly meetings and contribute actively to the discussions, providing feedback on methods and protocols and sharing their latest results with the full team in a collegial setting. What do you plan to do during the next reporting period to accomplish the goals?OBJECTIVE A: PATHOGENOMICS TOOLS A1. Bacterial surveys: The bacterial survey will be repeated in all 12 states. In addition, a directed survey will be completed two crops in each of ID, southern CA, and GA to monitor development of bacteria from asymptomatic plants to development of symptoms, using molecular diagnostic tools developed for Objective A3 during the first year. Selected strains generated from the survey in each state will be shipped to the UGA Athens campus on nutrient agar (NA) stab cultures, re-streaked, characteristics checked, and stored in 15% glycerol at -80oC. Nutrient broth cultures will be used to isolate genomic DNA and for differential virulence phenotyping. Strains will be tested at each of the six regional labs in the team using a red onion scale assay as a high throughput screening for bulb colonization phenotype, with results photographed. Isolates will be compared with a standardized panel of onion bacterial pathogens. Strains will be identified to species using MLSA schemes, and tested for ability to cause symptoms on onion leaves using a 5-day leaf blade inoculation protocol. Metadata and results will be added to the NOBSC database and made available publicly on Alliumnet. A2. Pathogenomic analyses, virulence factors & bacterial community analyses The strains of Pantoea agglomerans added to the expanding NOBSC will be sequenced and examined to identify putative virulence factors associated with the ability to cause diseases on onion (confirmed by pathogenicity tests described for Objective A1. The presence and identification of putative virulence factors will be completed using similar methods to those published by members of this team for identifying virulence factors of the related pathogen, P. ananatis. As described in the work accomplished/initiated in the first year of this project, the asymptomatic and symptomatic bulbs collected from the same field in each of GA and WA are being used to assess the microbial communities associated with symptomatic and healthy bulbs. This will help assess potential interactions among the complex of microflora in onion bulbs that can lead to development of bulb rots. Results of the first year's microbial community analyses will be used to design additional microbial community analysis work to increase our understanding of how non-pathogenic, pathogenic, and opportunistic bacteria and other microflora interact in onion bulbs. A3. Molecular diagnostic tools Recently identified loci of P. ananatis associated with onion virulence were named the HiVir loci and Onion Virulence Regions (OVR) A-D. These will be targets for developing rapid diagnostic markers. The same approach will be used by Dutta, Kvitko, Coutinho, and Woodhall to identify DNA targets in P. ananatis and P. agglomerans strains for developing robust and sensitive Taqman PCR and LAMP assays, i.e., virulence-associated DNA regions of each speciesfor designing primers and probes.The primer/probe sets will be evaluated with additional strains from the NOBSC. The most sensitive and specific (optimized) assays will be subjected to validation according to internationally recognized parameters (EPPO 2014; Roenhorst et al. 2018) using additional strains at UGA and UI-Parma to ensure assays are robust enough for use at all six Regional Labs for this team. Diagnostic tools developed in Year 1 will be tested in directed surveys in Seasons 2 and 3 to monitor P. agglomerans and P. ananatis in crops and bulbs. The tools will enable detailed epidemiological assessment of inoculum sources and development of bacteria on onion plants and other species, including weeds. This will be the foundation to extend the approach to other genera and species of onion bacterial pathogens, which is beyond the timeframe and resources of this project. A4. Phenotypic resistance screening methods Onion breeders from the project team and the Stakeholder Advisory Panel met by videoconference in July 2020 to review preliminary results and plan Year 2 work on developing resistance screening methods. Lab and greenhouse onion phenotyping protocols will be evaluated by Dutta at UGA to screen commercial short-, medium-, and long-day cultivars as well as diverse Allium germplasm from the USDA NPGS. Field phenotypic screening trials will be completed in GA, TX, and WA in Season 2 with pathogens predominant in each state, based on the surveys. Cultivars planted will reflect those suitable to each region as well as germplasm with diverse phenotypic reactions from Season 1 trials. Dr. Beer at Cornell University will do additional phenotypic screening studies in growth chambers to complement the work by Dutta et al. in Georgia as well as field trials under growers' production environments to validate correlation of lab, growth chamber, and greenhouse trials with field trials. OBJECTIVE B: ONION BACTERIAL DISEASE MANAGEMENT Lab, greenhouse, and field trials will be continued in Year 2 on irrigation, fertility, pesticide programs, cultural practices, and postharvest practices. Studies have demonstrated these practices to have some influence on bacterial diseases, but they remain to be translated into integrated, practical management tools for regions across the U.S. Specific aspects investigated in each region will be prioritized based on regional practices and constraints, and on stakeholder priorities as well as results from Year 1 of the project. Asymptomatic colonization and symptomatic development of pathogens on plants and stored bulbs will be monitored with isolations onto agar media, use of molecular diagnostic tools, and visual ratings. Standardized protocols will be used to facilitate comparison of results among states, regions, and seasons. Results from Season 1 trials will besharedat the team and SAP meetings to refine research plansfor Season 2. This will be repeated after Season 2 to plan for Season 3 in an iterative approach guided by stakeholders to ensure translation of results into practical tools. Data from NY field trials in Year 1 will be used to inform and validate disease risk models in Year 2. Detailed records on onion production practices, crop microclimate, and bacterial diseases will be sought from growers to assess whether the risks of diseases can be modeled statistically, to predict if a grower should sell bulbs at harvest, or can store bulbs with minimal risk of bacterial rots. ECONOMIC ASSESSMENTS The potential for adoption of management recommendations based on results of trials will be assessed annually by the SAP and stakeholders with economist Colson. Economic analyses will be integrated into various trials based on inputs (expenses) and outputs (marketable yield). OUTREACH AND EXTENSION The redesigned Alliumnet website will be ready by December 2020. Results from Year 1 work will be shared in research summaries; on Alliumnet.com; in project newsletters, trade publications and presentations at growers' meetings and field days. The results of pesticide field trials in this project will be added to information in the current literature survey. The final product is expected to be a quick guide where a grower, Extension Educator or researcher could look up an active ingredient or product and quickly see how it performed in replicated field trials. A 5-minute extension video will be released in November 2020, covering: symptoms caused by different bacterial pathogens in inoculated bulbs and in naturally infected and inoculated onion crops; causes and disease pathways as well as current management options; Stop the Rot project objectives. The video will be shot in the Columbia Basin but will have broad applicability in other onion growing regions. PROJECT MANAGEMENT AND COORDINATION The second annual project team meeting and the Stakeholder Advisory Panel videoconference are scheduled for January 2021.Regular monthly videoconferences for the full team and monthly meetings for the co-PIs will continue in Year 2.

Impacts
What was accomplished under these goals? OBJECTIVE A: PATHOGENOMICS TOOLS A1. Bacterial disease surveys Onion crop surveys were initiated in all 12 states during this reporting period. In southern states, crops were sampled from December 2019 to July 2020; in northern states, field sampling will be completed by October 2020 and post-harvest surveys by January 2021. In each state, we collect 10 samples from symptomatic onion plants in up to 5 fields, at least twice during the growing season and then again at harvest or in storage. Preliminary survey results in GA indicate seasonal changes in the predominant bacterial genera isolated from onion foliage (January to May 2020). In January and February, the predominant bacterial pathogens isolated were Pseudomonas spp. (P. viridiflava and P. coronafaciens). By March and April, Pantoea was the predominant genus isolated from 64% and 68% of the symptomatic foliage, respectively. When symptomatic onion bulbs in storage were sampled in May and June, Burkholderia spp. (65%), Rahnella spp. (18%), Pantoea spp. (14%) and Enterobacter spp. (3%) were isolated. A2. Onion bacterial pathogenomics We assembled a panel of previously collected Pantoea agglomerans strains associated with onion crops from PA (3 strains); WA (17 strains); GA (11 strains) and additional strains from CO, MI and NY. This strain panel, along with strains collected in Year 1 of the project, will be used for preliminary phenotyping and genotyping for identification of onion-virulence associated traits for P. agglomerans, to compare with onion-virulence genetic traits of P. ananatis identified recently by Kvitko and Dutta. Experiments to investigate the role played by bacterial communities in center rot involve meta-profiling (16S rRNA profiling) of onion bulbs.In GA and WA, nine healthy and nine diseased bulbs were collected from the same field in each state 16s rDNA sequencing was conducted on DNA extracted from these bulbs. Sequencing data are being analyzed by two graduate students in Coutinho's lab. A3. Diagnostic tools First iteration PCR primers were designed to identify the presence of two known P. ananatis virulence factor loci, HiVir and alt. PCR assays using these primers, combined with the red scale necrosis assay, were used to test a collection of Georgia isolates (n=90) and an international collection of Pantoea isolates (n=140) from Co-PI Coutinho. We observed a strong statistical correlation for the presence of alt, which confers tolerance to thiosulfinates, among Pantoea isolates collected from onion vs. non-onion isolation sources. Among P. ananatis isolates there was nearly perfect statistical correlation between positive HiVir PCR assay results and a positive red scale necrosis pathogenicity phenotype. Collaborator Woodhall assembled 20 DNA samples from onion bacterial isolates collected in the Treasure Valley area, to be used for assay validation purposes in Year 2. Methods for sampling and DNA extraction from soil, plant material, seed and water are being evaluated for use in Year 2 and a filter funnel trap was developed for sampling rain and irrigation water. A4. Phenotypic screening methods In GA greenhouse studies, team members evaluated methods of leaf inoculation (cut-tip and foliar spray) on cultivars Vidalia and Sapelo using a highly pathogenic GA strain of P. ananatis, for potential use in phenotypic screening trials. Twelve onion cultivars were selected for a phenotypic resistance screening field trial planted in April 2020 near Pasco, WA. Cultivars are grouped by daylength sensitivity for bulb initiation, so that inoculation can be timed for each of 4 maturity groups. This minimizes the risk of confounding cultivar maturity with susceptibility to bacterial infection at the time of inoculation, which will be important in developing phenotypic resistance screening methods that are valid under field conditions. Plants in the trial plots were inoculated on July 30 and August 13 with a CO2-backpack spray application of Burkholderia gladioli pv. alliicola and P. agglomerans. OBJECTIVE B: ONION BACTERIAL DISEASE MANAGEMENT B1-B5: Influence of onion production practices on bacterial diseases Field trials were established to investigate the influence on onion bacterial diseases of: irrigation practices (WA, CO, OR in progress; GA completed); fertility (WA, NY in progress; GA completed); cultural practices (WA, CO in progress); pesticide programs (CO, UT, WA, NY in progress; GA, TX completed); and postharvest practices (trials in CO, NY, OR, WA will begin in late summer-fall 2020). Field trials have prioritized practices and products currently used by growers, that reflect stakeholder priorities and concerns. When appropriate, inoculated and non-inoculated plots have been included in trials using local bacterial strains to ensure adequate disease pressure for differentiating treatment effects. The WA team is developing bins in which onion bulbs can be enclosed and treated with post-harvest products, to mimic treatments applied in commercial storage units. Preliminary results from GA bactericide trials indicated that the incidence of bulbs with center rot after one month of storage at 4oC was significantly less from plots treated with a copper product (Mankocide, Kocide 3000, Champ, Nordox, Mastercop, or NuCop), compared with plots treated with Oxidate 5.0, Leap, or Actigard (which had similar incidences of bacterial bulb rot as the non-treated check plots). Phytotoxicity to onion foliage was observed in plots treated with Oxidate 5.0 but not in plots with other treatments. B6. Bacterial disease prediction modeling The data modeling team received detailed crop production and disease records from a Columbia Basin farm for the 2017 season. The data will be used to develop and test preliminary bacterial disease risk models. ECONOMIC ASSESSMENTS A baseline grower survey was developed with the project team and the Stakeholder Advisory Panel, to assess onion growers': (a) experience with losses due to bacterial diseases and rots, (b) strategies used to reduce losses, and (c) perceptions of the efficacy of management practices that potentially could reduce losses.The survey is being distributed by project team and SAP members and online through the National Onion Association and other industry communication channels, and will establish a baseline against which to measure the impact of this project on growers' knowledge about bacterial diseases. OUTREACH AND EXTENSION Outreach focused on increasing general awareness of regional bacterial pathogens of onion, through: Newsletters, articles and presentations at grower meetings and conferences; Recruitment of growers in all onion-growing regions of the US to report symptoms of bacterial disease in their crops; A short video "How to Diagnose Onion Bacterial Bulb Rots", at the request of stakeholders (SAP members); A review of published pesticide efficacy trials for control of bacterial diseases of onion across the U.S. (in progress). PROJECT MANAGEMENT AND COORDINATION A fulltime Project Manager (PM) was hired in November 2019 for 4 years, to assist with communication, coordination and budget management. The following meetings were held: The annual team meeting, a training workshop and the annual SAP videoconference in Athens, GA in February 2020; Monthly videoconferences for the project team to share results, discuss progress and plan research. SAP members regularly join our monthly team videoconferences and receive the meeting notes to stay abreast of the team's work. Monthly videoconferences of the PD and co-PIs to discuss research direction and collaboration; Six work sessions on specific research topics, with SAP members participating as subject matter experts.

Publications

  • Type: Other Status: Published Year Published: 2019 Citation: Arboleda, K. 2019. UGA Researchers working on disease control in Vidalia onions. Vegetable and Specialty Crop News, December 2019.
  • Type: Other Status: Published Year Published: 2020 Citation: AgNetWest February 2020 issue: Stop the Rot Project to Combat Onion Bacterial Diseases
  • Type: Other Status: Published Year Published: 2020 Citation: AgNetWest March 2020 issue: Samples Needed to Help Stop the Rot Project
  • Type: Other Status: Published Year Published: 2019 Citation: USDA Announces Specialty Crop Award to Combat Onion Diseases. California Garlic & Onion Research Advisory Board News, Fall 2019, p. 4. https://www.cagarlicandonion.com/resources/newsletters/detail/id/17
  • Type: Other Status: Published Year Published: 2019 Citation: Keller, Denise. 2019. Stop the rot. Combating onion bacterial diseases. Onion World, November 2019, pp. 24-26. https://issuu.com/columbiamediagroup/docs/onion_world_november_2019
  • Type: Other Status: Published Year Published: 2020 Citation: du Toit, Lindsey & Hoepting, Christy. 2020. Stop the Rot: National U.S. Team to Combat Bacterial Diseases of Onion with Fierce Collaboration. Comprehensive article about the project, published in the July/August 2020 issue of Onion World magazine. https://onionworld.net/magazine/
  • Type: Other Status: Published Year Published: 2020 Citation: Dutta B. and Gitaitis R. 2020. Onion World July/August 2020 Disease Quiz to highlight onion bacterial diseases. https://onionworld.net/magazine/
  • Type: Other Status: Awaiting Publication Year Published: 2020 Citation: du Toit, L.J., Waters, T., Derie, M., and Darner, J. 2020. Battling onion bacterial diseases with bactericides. Onion World, in press (to be published in the September/October 2020 issue).
  • Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: du Toit, Lindsey (2020). Onion Bacterial Diseases: Knowledge Gaps and a Federal Grant to Stop the Rot. Presentation on February 5, 2020 at University of Wisconsin & WI Potato and Vegetable Growers Association Grower Conference, Stevens Point WI.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Aegerter, Brenna (2020). New Research Project on Bacterial Diseases of Onion. Presentation on February 10, 2020 at California Garlic & Onion Symposium, Tulare CA.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Uchanski, Mark (2020). Whats New in CSU Specialty Crops Research? Presentation on January 28, 2020 at Colorado Fruit & Vegetable Growers Association Produce Day, Greeley CO.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: du Toit, Lindsey (2019). Stop the rot: Combating onion bacterial diseases with pathogenomic tools and enhanced management strategies. WSU Onion Cultivar Field Day, 29 Aug. 2019, Connell, WA
  • Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: du Toit, Lindsey (2019). Bacterial rots of onion: USDA SCRI project update and knowledge gaps. Pacific Northwest Vegetable Assoc. Annual Convention & Trade Show, 20-21 Nov. 2019, Kennewick, WA.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Waters, Tim, and du Toit, Lindsey (2019). Onion bulb crop production and IPM practices. 3-hour workshop on 13 Dec. 2019 to review onion IPM program at CSS Farms, Kahlotus, WA.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: du Toit, Lindsey (2020). Onion fungal and bacterial bulb rots, and efficacious use of fungicides for disease control in onion production. Invited presentation, 5 Feb. 2020, Kennewick, WA
  • Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Dutta, Bhabesh (2020). Stop the Rot Research Project on Bacterial Diseases of Onion. Invited presentation at the Annual Meeting of the National Onion Association, August 11, 2020 (virtual meeting).
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Stice SP, Thao KK, Khang CH, Baltrus DA, Dutta B, Kvitko BH. Thiosulfinate Tolerance Is a Virulence Strategy of an Atypical Bacterial Pathogen of Onion. Current Biology 2020;S0960-9822(20)30779-X. doi:10.1016/j.cub.2020.05.092
  • Type: Other Status: Published Year Published: 2019 Citation: National Onion Association. 2019. $4M given for onion bacterial disease research. Vegetable Growers News, 11 Sep. 2019. https://vegetablegrowersnews.com/news/onion-researchers-earn-4-million-grant-to-combat-bacterial-disease/
  • Type: Other Status: Published Year Published: 2019 Citation: Aegerter, Brenna (2019). Bacterial Bulb Rots of Onion. In: Field Notes newsletter, UCCE San Joaquin County, CA, November 2019 issue (mailed or emailed to 1,029 stakeholders, primarily growers and allied industry members). http://cesanjoaquin.ucanr.edu/newsletters/Field_Notes_Newsletter82270.pdf
  • Type: Other Status: Published Year Published: 2019 Citation: Thompson, C. 2019. Center rot disease in organic onion growing studied. Vegetable Growers News, November 2019.