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 09/01/20 to 08/31/21

Outputs
Target Audience:Stakeholders involved in the U.S. onion industry are the primary audience for this project. This includes onion producers (farmers), 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 also a 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., with one international member. 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, two PhD students and four postdoctorates have worked on research directly related to the Stop the Rot project objectives in this reporting period. They are conducting their research in Washington State, Georgia, Texas, Pennsylvania, Colorado, and South Africa. We used several channels to reach our target audience during this reporting period, including grower meetings and field days, conferences and workshops, the Alliumnet.com website, industry newsletters, trade publications and extension videos. Project team members also reached out directly to growers in each of the onion-growing regions to recruit them for participation in the bacterial field surveys. Approximately 1,008 growers, agronomists and industry professionals were reached directly through presentations at conferences, workshops, field days and grower meetings in this reporting period. Many of the events 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 through presentations. Approximately 2,796 people have viewed our series of project videos. Communication and outreach materials and specific activities are reported in detail in the list of products. Changes/Problems:Bacterial surveys: managing workloads in the regional labs Two meetings were held in December 2020 (virtual) where the regional labs reviewed Season 1 work on bacterial surveys and planned for Season 2 surveys. The workload in Season 1 associated with processing onion survey samples proved to be very challenging for the labs, but the Season 2 survey program was greatly streamlined by reducing the number of visits to each survey location as well as the number of strains to be isolated from each sample collected in the field or in storage. Postdoctorate Shin (UGA) prepared a review session for the team on 30 June 2021 on using the SILVA database (in comparison with RDP and GenBank) for identifying bacterial isolates to genus-level based on 16S rDNA sequences. Following the session, the co-PIs agreed to switch from using RDP to SILVA as the preferred database for identifying bacterial isolates to genus-level that are collected in each regional lab for the state surveys each season. Risk-based modeling: access to commercial production data Access to production data from growers and packers has been very limited as stakeholders are reluctant to share proprietary crop information in a highly competitive market. In addition, growers do not always have detailed environmental data records, particularly for soil moisture status which we hypothesize is one of the most important risk factors in bacterial disease development. This has affected the ability of the team to develop and evaluate bacterial disease risk models. To resolve this, we plan to focus on collecting more data from our project field trials in Year 3, including deploying additional sensors to monitor environmental conditions in the trial plots. Weather-related problems Winter storms in Texas in January 2021 led to severe commercial onion crop losses and curtailed the bacterial survey in commercial fields for Year 2. Excessive heat and drought in Utah in summer of 2021 also caused significant commercial crop losses there, but bacterial surveys were still conducted. In UT research trials, onion plants stopped growing just as the crops were at bulb initiation due to extreme heat, and by harvest the plants were still very stunted. Final results are not available yet. Cancellation of LabGuru licenses During Year 2, we decided to centralize and simplify the compilation of regional bacterial survey data, using an Excel spreadsheet instead of proprietary software from LabGuru. We did not renew the LabGuru licenses for Year 2 so the funds ($3,800) were instead allocated to support additional staff time for redesign of the Alliumnet website. COVID19 impacts The lack of in-person SAP meetings has led to a shortfall in reported cost-share funding, since almost all SAP members had committed their travel and time to attend team meetings as part of their match. We plan to replace this with the value of strains donated to the project from pre-existing collections held by several team members and collaborators - these strains allowed us to move ahead with preliminary pathogenomics work while the Season 1 bacterial surveys were completed. In WA, restrictions regarding travel and hotel stays increased in-state travel expenses considerably (no sharing of hotel rooms, more cars needed to travel between Mount Vernon and Pasco each time. In addition, a positive COVID-19 incident in the WA program in Nov. 2020 resulted in that individual being on medical leave for 3 weeks, which added to the workload of others in the program working on this project, with delayed ability to make as much progress on the survey work (bacterial strain identifications) as planned. In PA, the University mandated COVID-19 restrictions delayed processing of the Year 1 bacterial survey samples from PA and NY. In addition, the inability to extract DNA for sequencing and conduct pathogenicity testing prior to putting the isolates into long-term storage significantly increased the labor and material costs required for this portion of the survey. However, as restrictions are beginning to lift and more people can access the lab, PA team members are finalizing the genera identifications of select isolates as well as running the onion pathogenicity tests. PA and other states anticipate there will be more opportunities for Extension and outreach in Season 3. At the University of Pretoria, the research undertaken in Year 2 was largely computer-based so both MSc students and their advisors either worked from home or from the lab (there was a rotational scheme in place because of COVID restrictions so the University of Pretoria lab is limited in the number of staff/students that can be present in the lab each day). Due to COVID-based restrictions, Postdoctorate Shin was not able to secure a visa to allow her to start her position with the Kvitko group at UGA in September 2021. A temporary position was created for her in the lab of collaborator Coutinho at the University of Pretoria until early 2021, when she was granted a visa to travel to Georgia. Dr. Shin contributed to project objectives from South Africa by genotyping and phenotyping South African Pantoea strains, which will ultimately add diversity and robustness to our analysis, and she conducted bioinformatics analysis. Shin also conducted a meta-analysis of published and unpublished Pantoea species-specific PCR primer sets, which will simplify Pantoea species identifications. Many of the states involved in this project could not hold regular in-person meetings and other events such as field days for sharing results or demonstrating field trials from this project. If COVID constraints continue to ease up over the next year, we anticipate more in-person events will take place for all team members. Other changes New SAP members joining in this period included: Larry Duell (CO), Margreet Asma (Netherlands), Kalie Christensen (WI), David Burrell (GA). Russell Hamlin and Kerrick Bauman stepped down from the SAP due to time constraints. New team members joining in Year 2 included: Paul Stodghill and Jo Ann Asselin of the USDA-ARS in Ithaca, NY. Their work is not funded through Stop the Rot but their current research is highly complementary to this project, so they are collaborating with the Stop the Rot team on the pathogenomics objective. 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, MSc student at the University of Pretoria, South Africa. Dissertation title: Metagenome analysis of healthy and diseased onion bulbs from Georgia. Anticipated completion date: October 2021. Advisors: Teresa Coutinho, Pedro Lebre. Christopher Liakos, MSc student at the University of Pretoria, South Africa. Dissertation title: Metagenome analysis of healthy and diseased onion bulbs from Washington State. Anticipated completion date: October 2021. Advisors: Teresa Coutinho, Jacquie van der Waals, Pedro Lebre. Tessa Belo, MS student in the Soils & Water program at the Northwestern Washington Research and Extension Center of Washington State University. Advised by collaborator LaHue and co-advised by PD du Toit. Thesis topic: Impacts of irrigation and fertilization practices on bacterial diseases in onion bulb crops in the Columbia Basin. Expected completion date: Spring 2022. Antoinette Machado, MS student at Colorado State University in January 2020, supervised by co-PI Uchanski. Thesis topic: Evaluation of bactericides and plant defense inducers to manage center rot of onion in Colorado. Expected completion date: Spring 2022. Bed Prakash Bhatta, a PhD student at Texas A&M AgriLife Research - Uvalde is being trained on isolation, culture, and inoculation of bacteria under this project. Thesis topic: Breeding for anthracnose and Fusarium wilt resistance in watermelon. Ram Neupane, Plant Pathology PhD student at Pennsylvania State University (started Spring 2021). Thesis topic will be related to bacterial diseases of onion. Dr. Mei Zhao, postdoctorate in co-PD Dutta's program at the University of Georgia, is focusing on the bacterial survey, identification and characterization of bacterial isolates in GA, and assisting with greenhouse and field evaluations. Dr. Hossein Noorazar, postdoctorate at Washington State University, is focusing on data analysis and disease risk modeling efforts, supervised by co-collaborators Rajagopalan and Kalyanamaran. His part-time appointment on the project started in fall 2020. He initially focused on hypothesis extraction tools and exploring satellite imagery, and is now working with data provided by members of the team to identify factors that influence development of bacterial diseases. Dr. Navdeep Singh, postdoctoral research associate at Washington State University (started 16 Sep. 2020), primarily is working on other projects but is contributing to this work, particularly Objective B1, and will be engaged in this research until Fall 2022. Dr. Gi-Yoon (Gina) Shin, postdoctorate in co-PI Kvitko's lab at the University of Georgia, is assisting with the bacterial survey, investigating the pathogenomics of Pantoea spp. that are pathogenic and non-pathogenic on onion, and identifying target regions of the genomes for developing molecular diagnostic tools for onion bacterial pathogens. In Utah, five undergraduate students have been working on this project with collaborator Nischwitz (making media, culturing bacteria, storage evaluations, molecular identification of bacteria, conducting red scale necrosis tests, and helping with foliar and bulb assays). One student is writing an undergraduate research grant proposal for a side project identifying the gut bacteria of thrips in Utah and their potential as bacterial bulb rot pathogens. In New Mexico, two graduate students assisted with bacterial surveys in four autumn-sown onion fields. Shaun Stice, supervised by co-PI Kvitko, was a PhD student at the University of Georgia at the start of the project. He completed research on the genetic basis of P. ananatis virulence of onion as well as distribution of identified virulence loci among Pantoea isolates. He graduated in 2021. Dr. Gaurav Agarwal was a post-doctoral research associate under PI Dutta who assisted with the pathogenomics investigation of Pantoea spp. PROFESSIONAL DEVELOPMENT WSU team members (du Toit, Derie, and Waters) were hosts for training on 21-22 July 2021 for two team members from Texas A&M University (technician Khamal and PhD student Bhatta) to review methods for lab, greenhouse, and field trials on onion bacterial diseases. Postdoctorate Shin (UGA) prepared a review session for the team on 30 June 2021 on using the SILVA database (in comparison with RDP and GenBank) for identifying bacterial isolates to genus-level based on 16S rDNA sequences. Following the session, the co-PIs agreed to switch from using RDP to SILVA as the primary database for identifying bacterial isolates to genus-level that are collected in each regional lab for the state surveys each season. How have the results been disseminated to communities of interest?In this reporting period, we have been actively reaching out to growers and stakeholders to: share new information and learning from the project as well as current scientific understanding of onion bacterial diseases and their management; understand growers' current state of knowledge about causes and management of bacterial diseases of onion, and identify priority concerns for growers and the onion industry as regards management of bacterial diseases. Information has been shared with growers and stakeholders through a range of communication channels, including the newly revamped Alliumnet website, research summaries, extension bulletins, articles in trade publications and presentations at growers' meetings and field days. Results were also shared at professional scientific meetings (American Phytopathological Society's Plant Health 2021 Online. REACHING GROWERS AND ONION INDUSTRY STAKEHOLDERS: Outreach to growers and onion industry stakeholders, field representatives and extension staff has occurred through informal visits with growers during field surveys, grower meetings, field days and technical workshops. Our Stop the Rot video playlist https://www.youtube.com/playlist?list=PLajA3BBVyv1zf2obB16bNEdQPQeLW_XB_ now contains several videos aimed at growers and the onion industry (introduction to rot identification, in both English and Spanish; a longer video providing an overview of bacterial diseases in onion and current management options; a short video of the experimental equipment for our field trials of post-harvest disinfection treatments.) A short time-lapse video of the Red Scale Necrosis (RSN) assay has been added. We plan to include more short videos from field surveys and laboratory work during the next year. Refer to details above for statistics on number of views for each video and two awards given for the 14-minute video on the whole project produced by WSU. Outreach and dissemination of preliminary results to onion stakeholders and the industry has been conducted a number of informational articles in trade publications and extension newsletters (see list of publications) and websites/online alerts. 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. Four new members joined the Panel in Year 2: Larry Duell of Gowan Seeds in Colorado; Margreet Asma, seed pathologists with Bejo Zaden in the Netherlands; David Burrell of National Onion Labs in Georgia; and Kalie Christensen of Gumz Farms in Wisconsin. Panel members are active in sharing information about the project to growers and onion industry stakeholders through their own regional and national networks in 12 states, and in bringing insights and information into the project from their networks. Our second annual meeting with the SAP was held on February 5th, 2021 by videoconference. Several panel members have participated in team work sessions on phenotypic screening methods as subject matter experts. Panel 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 newsletters. REACHING ACADEMIC RESEARCHERS, EXTENSION PROFESSIONALS AND GRADUATE STUDENTS The project team now includes 24 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. During this year, Prof Steven Beer of Cornell and Drs. Paul Stodghill and Jo Ann Asselin of the USDA-ARS also joined the team as collaborators. We hold monthly team videoconferences to share preliminary results and experiences and discuss recent findings. These monthly meetings of 1.5 to 2 hours have facilitated research and Extension collaborations within the project. Almost all the project collaborators are also involved in extension services and 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. Once final results of 2020 field trials became available in spring 2021 after evaluation of bulbs in storage, we compiled results from the first season's bacterial surveys and field trials into a summary of Year 1 research findings. The summary is available on Alliumnet.com https://alliumnet.com/wp-content/uploads/2021/09/StoptheRot_Year1_summaries.pdf. Preliminary results of the national bacterial survey were shared at the American Phytopathological Society annual meeting, 2021 Plant Health Online, and will be shared with relevant agencies in each participating state. The results of Year 1 field trials in participating states have been published in 12 peer-reviewed Plant Disease Management Reports (see list of publications). Five articles have been published in scientific journals (see list of publications). Four Research on Demand presentations were shared at Plant Health 2021 Online, the annual conference of the American Phytopathological Society. What do you plan to do during the next reporting period to accomplish the goals?OBJECTIVE A: PATHOGENOMICS TOOLS A1. Bacterial surveys: In the next reporting period, we plan to repeat the bacterial surveys in all the onion regions. In addition, directed surveys will be carried out using a real-time PCR assay being developed for Pantoea agglomerans strains virulent on onion, based on whole genome sequencing of bacterial strains collected in this survey and strains received from other collections. Regional labs will again send both pathogenic and non-pathogenic isolates of Pantoea to UGA, endeavoring to represent the diversity found in their regions. As strains of the other genera are characterized, each lab will send pathogenic and non-pathogenic strains that represent the diversity detected in the samples. Strains sent to UGA will be identified to species using MLSA schemes and tested for pathogenicity on onion. Metadata and results will be added to the NOBSC database and made available publicly on Alliumnet. During the next two years of the project, regional labs will also plan for long-term storage and back-up of their regional collections. The NOBSC will only include strains that are in the national collection. A2. Pathogenomic analyses, virulence factors & bacterial community analyses Additional strains of P. agglomerans will continue to be added to the NOBSC as Year 2 survey collections are finalized at each regional lab. A subset of those strains will be subjected to whole genome sequencing and examined to identify putative virulence factors associated with the ability to cause diseases on onion, as detailed above for strains evaluated in Year 2 to validate the virulence factors identified in the first 2 years of this project and for testing the molecular diagnostic tools being developed for onion-pathogenic strains of Pantoea spp. .. Results of the bacterial community analyses from asymptomatic and symptomatic bulbs collected from a field in each of GA and WA will be published in Year 3, when the two MS students working on this aspect of the project will graduate. The results will help assess potential interactions among the complex microflora associated with asymptomatic and symptomatic onion bulbs. Results from both students' work will be used to design additional microbial community analyses to increase our understanding of how non-pathogenic, pathogenic, and opportunistic bacteria and other microflora interact in onion bulbs. A3. Molecular diagnostic tools We will use the results of initial pilot studies in Year 2 to evaluate how to undertake directed surveys for onion bacterial pathogens using molecular diagnostic tools. This will entail collecting onion seed lots as well soil, debris, and water trap samples from fields at critical stages of onion production. Real-time PCR assays of DNA extracted from these materials, using the molecular diagnostic tools in development, should inform us of the relative importance of potential inoculum sources of onion bacterial pathogens in different regions of the US. Results of the pathogenomics work in Years 1 and 2 revealed that it will be challenging to design a sensitive real-time PCR assay for Pantoea spp. pathogenic on onion that works across all onion production regions. We have detected substantial regional variation in the pathogenicity of P. agglomerans strains collected across 12 states during the Season 1 and 2 surveys. For example, all the Idaho and Oregon strains of P. agglomerans tested to date were RSN-, and very few P. agglomerans strains collected in Washington have been RSN+, even when isolated from symptomatic plants or bulbs. This is in contrast to GA, where a majority of P. agglomerans strains collected were RSN+. Therefore, our approach in Year 3 will be to design an assay which detects the presence of the HiVir cluster and, potentially, other genes associated with virulence to onion, and then use a decision support system to identify the associated risk of that bacterial species being pathogenic on onion. The regional survey results will be important for developing a decision support system relevant to different onion production regions of the US. A4. Phenotypic resistance screening methods Based on results of the lab and greenhouse onion phenotyping protocols evaluated in Years 1 and 2, the focus of this project will shift to field evaluations. Dutta at UGA will screen commercial short-, medium-, and long-day cultivars as well as diverse Allium germplasm from the USDA NPGS for resistance to P. ananatis by treating the seed of these cultivars and inoculating plants in the field at various growth stages. Field phenotypic screening trials also will be completed in TX and WA in Season 3 with pathogens predominant in each state. Cultivars planted will reflect those suitable to each region as well as germplasm with diverse phenotypic reactions from Season 1 trials. Based on results of the WA Season 2 cultivar trial, modifications will be incorporated into the Year 3 cultivar field trial in WA. OBJECTIVE B: ONION BACTERIAL DISEASE MANAGEMENT Lab, greenhouse, and field trials will be continued in Year 3 to evaluate irrigation practices, fertility practices, pesticide programs, cultural practices, and postharvest bulb treatments for control of bacterial diseases of onion. Specific aspects investigated in each region will be prioritized based on regional practices and constraints, and on stakeholder priorities as well as results from Years 1 and 2 of the project. Results from Season 1 and 2 trials will besharedat the team and SAP meetings to refine research plansfor Season 3, in an iterative approach guided by stakeholders to ensure translation of results into practical tools. Data from Stop the Rot field trials in Years 1 and 2 will be used to inform and validate disease risk models. Data on onion production practices, crop microclimate, and bacterial diseases will be sought 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 these 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). Work on this objective will include: Drafting an academic article reporting survey findings. Assisting the Extension team and grant leadership to incorporate findings from field trials into website and outreach materials. Working with team members as field trial data becomes available to translate findings into cost-benefit analyses. OUTREACH AND EXTENSION Results from Year 2 work will be shared in research summaries; on Alliumnet.com; in project newsletters, trade publications, regional websites, and presentations at growers' meetings and field days. Plans are underway to develop more videos, "quick guides", highlights of annual project results, training and extension materials, and an interactive map of the USA with results of the bacterial disease surveys. The newly redesigned Alliumnet.com website will be maintained to provide a home for national onion research collaborations, including USDA projects, National Allium Research Conference information and proceedings, links to National Onion Association meetings and events, and the W-1008, W-2008, and W-3008 (and the future W-4008) onion multi-state project reports, activities, and meeting details. PROJECT MANAGEMENT AND COORDINATION The third annual project team meeting is scheduled for March 1, 2022 in Denver CO, and the Stakeholder Advisory Panel videoconference is scheduled for March 25, 2022.Regular monthly videoconferences for the full team, regular meetings for the co-PIs every two months, and weekly meetings of the PD and Project Manager will continue in Year 3.

Impacts
What was accomplished under these goals? OBJECTIVE A: PATHOGENOMICS TOOLS A1. Bacterial disease surveys, NOBSC Onion foliage and bulbs were surveyed in southern states in 12/2020 to 06/2021, and in northern states from spring to fall 2021. To date, 84 bacterial genera have been identified from 2,594 isolates. Distribution and pathogenicity vary among regions. Few genera produce symptoms on onion. Prevalent genera include: Pantoea, Pseudomonas, Burkholderia, Enterobacter and Erwinia. The GA survey showed temporal progression in genera on foliage. A subset of 612 strains were sent to UGA for the NOBSC. A2. Onion bacterial pathogenomics To design molecular diagnostic tools for onion-pathogenic Pantoea strains, we targeted P. agglomerans. We showed that P. agglomerans strains use similar onion-virulence genes as P. ananatis, but strains with these genes are uncommon. Whole genome sequencing for 32Pantoeastrains showed a perfect correlation between red scale necrosis phenotype and presence of the HiVir operon. PCR primers were designed to detect the HiVir cluster in 3 Pantoea species, which appears necessary for infection. Strains without the alt gene, which imparts tolerance to sulfur compounds, do not colonize onion well. Bacterial DNA extracted from symptomatic and asymptomatic onion bulbs from GA and WA revealed differences in bacterial communities between the types of bulbs, and between GA and WA bulbs. Genes involved in pathogenicity, detoxification and secretions systems were identified in the metagenome-assembled genomes of the known pathogens of center rot. A3. Diagnostic tools In Year 2 we: Verified the specificity and sensitivity of a real-time PCR assay for P. agglomerans; Developed a low-cost water trap to sample irrigation and rain water for onion pathogens; Demonstrated that a bulk soil DNA extraction method will need optimization for muck soils. Public Pantoea species-specific PCR primers were evaluated and new PCR primers designed for P. agglomerans using the strains described above. HiVir operon sequences generated for 58 Pantoea strains are being used to design primers for HiVir+ onion-pathogenicstrains of Pantoea. Genomic DNA was purified from 20 genomes sequenced for P. agglomerans, P. ananatis, and P. allii strains (15 HiVir+/RSN+ and 5 HiVir+/RSN-) to design diagnostic tools for onion-pathogenic strains. A4. Phenotypic screening methods Four cultivars (short-day [SD] dehydration, SD sweet, long-day [LD] dehydration, and LD sweet) were selected to represent what might be susceptible (sweet) vs. partially resistant (dehydration) to bacterial pathogens. Plants were inoculated with P. ananatis in a greenhouse with spray inoculation and cut leaf-tip methods at two inoculum concentrations. No consistent pattern emerged in disease severity among cultivars with either method or inoculum concentration. A cultivar field trial in WA was repeated in Season 2 using 3 cultivars from each of 4 maturity groups. The trial was irrigated with sprinklers instead of center-pivot to cease irrigation at the recommended time for each maturity group. Bulbs are in storage to be rated for bacterial rot in Feb. 2022. OBJECTIVE B: ONION BACTERIAL DISEASE MANAGEMENT Results of Year 1 trials were finalized once onion bulbs in storage were rated for bacterial storage rots. Field trials in Year 2 investigated the impacts of irrigation practices (WA, OR, GA trials), fertility practices (WA, NY, GA, OR, PA), cultural practices (WA, GA, NY), pesticide programs (CO, UT, WA, NY, GA, TX), and postharvest disinfection practices (WA) on development of onion bacterial diseases. In both years, field trials focused on practices and products currently used by onion growers to reflect stakeholder priorities and concerns. When feasible, trials involved inoculated and non-inoculated plots, using local bacterial strains to ensure adequate disease pressure for differentiating treatment effects. Some valuable results with potential application have been shared with stakeholders: Using drip vs. overhead irrigation reduced internal bacterial rot incidence without significant effects on marketable yield or size class distribution. Earlier termination of final irrigation reduced bacterial rot without affecting marketable bulb yield. Earlier termination of the final N application resulted in less bacterial rot in GA trials, but not in WA trials due to high residual N at the WA site. Copper-based bactericides significantly reduced internal bulb rot incidence in the 2020 GA trial. In the WA and NY 2020 trials, copper and other bactericide treatments had no effect on incidence or severity of bacterial bulb rot at harvest or after 5 months of storage. WA trials on undercutting, rolling tops, and the timing of topping showed no significant effects on marketable yield or bacterial bulb rots. GA trials evaluating harvesting methods showed significantly less bacterial bulb rot when using: a chain digger vs. a straight-blade undercutter; mechanical harvest vs. manual harvest; topping onion necks to a length of 3 or 5 inches vs. 1 inch. NY trials showed rolling tops had no effect on bacterial bulb rots, while quick artificial curing with forced heated air reduced bacterial bulb rots by 50%. WA trials indicated no benefit to applying ozone or hydrogen peroxide + peroxyacetic acid products to onion bulbs in storage for managing bacterial rots, probably because the products do not penetrate the dry wrapper scales. RISK MODELING The risk modeling team received crop and weather data for the 2017 production season from 5 onion fields planted to the same cultivar in the Columbia Basin of the Pacific Northwest. Excess late-season irrigation was hypothesized as a contributor to bacterial rots in these fields. Large amounts of field data are needed on plant water uptake and transpiration needs based on soil moisture status, weather, and plant growth stage, in order to extract signals from data and help identify hypotheses as the basis for risk-based decision-making. To this end, data was collected in NY trials by team members in Year 2. ECONOMIC ASSESSMENTS Surveys were delivered to onion growers, the project team and the SAP. Growers estimate that, on average over the past 5 years, >10% of the onion crop in their area was lost to bacterial diseases. Approx. 20% of growers reported losses of >50% of the onion crop in their area in the past five years due to bacterial diseases. Respondents consider the most effective management strategies are irrigation and water management, and post-harvest curing (forced air and heating). The average grower is very concerned about bacterial diseases, limited in their ability to identify the diseases or bulb rot pathogens, and feels only moderately equipped to manage them. Growers rely on information from University-based extension services to manage onion bacterial diseases. OUTREACH AND EXTENSION Year 1 outreach focused on increasing awareness of bacterial pathogens of onion, and sharing results of Season 1 field trials. Outreach has included (for a total of 3,897 direct contacts): Articles and quizzes in Onion World and Veg News, with wide circulation in the onion industry; Articles in newsletters of the National Onion Association, regional grower associations and extension services; Presentations at grower meetings and conferences; Release of 2 videos on diagnosing, understanding and managing bacterial bulb rots; Field days and tours in 7 states; A session on onion bulb rot at the NY Empire Producers Expo in Jan. 2021; Overhaul of the Alliumnet.com website serving members of the Allium research and extension community. PROJECT MANAGEMENT Coordination, communication and integration across the team and with SAP members have been supported by monthly videoconferences, bi-monthly conference calls, annual team and SAP meetings, and the adoption of the Project Charter. Work sessions included: streamlining work in regional laboratories; developing phenotypic screening methods; developing diagnostic tools.

Publications

  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Agarwal Gaurav, Choudhary Divya, Stice Shaun P., Myers Brendon K., Gitaitis Ronald D., Venter Stephanus N., Kvitko Brian H., Dutta Bhabesh (2021). Pan-genome-wide analysis of Pantoea ananatis identified genes linked to pathogenicity in onion. Frontiers in Microbiology vol 12:2381 https://www.frontiersin.org/article/10.3389/fmicb.2021.684756
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Koirala, S., Zhao, M., Agarwal, G., Stice, S., Gitaitis, R., Kvitko, B., & Dutta, B. 2021. Identification of two novel pathovars of Pantoea stewartii subsp. indologenes affecting Allium sp. and millets. Phytopathology. 10.1094/PHYTO-11-20-0508-R. https://www.researchgate.net/publication/349422413_Identification_of_two_novel_pathovars_of_Pantoea_stewartii_subsp_indologenes_affecting_Allium_sp_and_millets
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Stumpf, S., Leach, L., Srinivasan, R., Coolong, T., Gitaitis, R., Dutta, B. Foliar chemical protection against Pantoea ananatis in onion is negated by thrips feeding. Phytopathology. 2021 Feb. 111(2):258-267. doi: 10.1094/PHYTO-05-20-0163-R. Epub 2021 Jan 13. PMID: 32748732. https://pubmed.ncbi.nlm.nih.gov/32748732/
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Stice, S., Shin, G.Y., Armas, S.D., Koirala, S., Galvan, G.A., Siri, M.I., Severns, P.M., Coutinho, T.A., Dutta, B., and Kvitko, B. 2021. The distribution of onion virulence gene clusters among Pantoea spp. Frontiers in Plant Science https://doi.org/10.3389/fmicb.2021.00184
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Zhao, M., Kvitko, B.H., Gitaitis, R.D., and Dutta, B. 2021. Bacterial streak and bulb rot of onion. Plant Health Instructor. DOI: 10.1094/PHI-E-2021-0421-01
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: da Silva, A., H.I. de Jesus, B. Dutta. 2021. Effects of irrigation method, nitrogen rate, and fertilizer application timing on bacterial diseases in Vidalia onion, Georgia 2020. Plant Disease Management Reports 15:V012.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Dutta, B., Tyson, C., Edenfield, J., Williams, Z., Tanner, S., Shirley, A., Reeves, B., and Powell, S. 2020. Evaluation of onion growth stage directed chemical applications and thrips management program on center rot incidence in onion bulbs in Georgia, 2019. Plant Disease Management Reports 14:V091
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Dutta, B., Tyson, C., Edenfield, J., Williams, Z., Tanner, S., Shirley, A., Reeves, B., and Powell, S. 2021. Evaluation of onion growth stage directed chemical applications and thrips management program on center rot incidence in onion bulbs in Georgia, 2020. Plant Disease Management Reports 15:V023. https://doi.org/10.1094/PDMR15
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Dutta, B. and Tyson, C. 2021. Evaluation of harvesting methods on post-harvest incidence of center rot and sour skin in onion, Georgia 2020. Plant Disease Management Reports 15:V025. https://doi.org/10.1094/PDMR15
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Dutta, B. and Tyson, C. 2021. Evaluation of digging methods on post-harvest incidence of center rot and sour skin in onion, Georgia 2020. Plant Disease Management Reports 15:V026. https://doi.org/10.1094/PDMR15
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Dutta B. and Foster M.J. 2021. Evaluation of bactericides and plant defense inducers to manage center rot of onion in Georgia, 2020. Plant Disease Management Reports 15:V027. https://doi.org/10.1094/PDMR15
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: du Toit, L.J., Derie, M.L., Gundersen, B., Waters, T.D., and Darner, J. 2021. Efficacy of bactericides for management of bacterial leaf blight and bulb rots in an onion crop in Pasco, WA, 2020. Plant Disease Management Reports 15:V107. https://doi.org/10.1094/PDMR15
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: du Toit, L.J., Derie, M. L., and Gundersen, B. 2021. Efficacy of disinfectants applied to onion bulbs in storage for control of bacterial bulb rots, Pasco, WA, 2020-2021. Plant Disease Management Reports 15:V102. https://doi.org/10.1094/PDMR15
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: du Toit, L.J., Derie, M. L., and Gundersen, B. 2021. Efficacy of late-season cultural practices on bacterial leaf blight and bulb rots in an onion bulb crop in Pasco, WA, 2020. Plant Disease Management Reports 15:V100. https://doi.org/10.1094/PDMR15
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Belo, T., du Toit, L., Waters, T., Derie, M., and LaHue, G. 2021. Effects of irrigation frequency and final irrigation timing on onion bacterial diseases in the Columbia Basin of Washington, 2020. Plant Disease Management Reports 15:V109 https://doi.org/10.1094/PDMR15
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Hoepting, C.A., Caldwell, S. K., and van der Heide, E. R. 2021. Evaluation of selected pesticides for control of bacterial bulb rot in onion, 2020. Plant Disease Management Reports 15:V108 https://doi.org/10.1094/PDMR15
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Hoepting, C.A., Caldwell, S. K., and van der Heide, E. R. 2021. Variety evaluation and effect of copper bactericide on bacterial bulb rot in onion, 2020. Plant Disease Management Reports 15:V107. https://doi.org/10.1094/PDMR15
  • Type: Other Status: Published Year Published: 2020 Citation: Dutta, B., and Gitaitis, R. 2020. Bacterial diseases of onion in Georgia. University of Georgia Cooperative Extension Bulletin B1534.
  • Type: Other Status: Published Year Published: 2021 Citation: Dutta, B., and Gitaitis, R.D. 2021. Disease quiz II. Onion World, February 2021:13, 20.
  • Type: Other Status: Published Year Published: 2021 Citation: MacKay, H.M. 2021. Stop the Rot! Researchers finish first year of field, lab work in 4-year project. Update in January 2021 newsletter of the National Onion Association.
  • Type: Other Status: Published Year Published: 2021 Citation: Dutta, B. 2021. Onion disease management in Georgia. Vegetable and Specialty Crop News, February 2021.
  • Type: Other Status: Published Year Published: 2021 Citation: Nischwitz, C. 2021. Bacterial Bulb Rot Diseases in Utah, Utah Pests Quarterly Newsletter, Vol. 15, Spring 2021. https://extension.usu.edu/pests/files/up-newsletter/2021/UtahPestsNews-spring21.pdf
  • Type: Other Status: Published Year Published: 2021 Citation: MacKay, H., and du Toit, L. 2021. Progress report. Stop the Rot: A national collaboration to research, combat bacterial diseases of onion. Onion World, May/June 2021:12-14. https://issuu.com/columbiamediagroup/docs/onion_world_may_june_2021/12
  • Type: Other Status: Published Year Published: 2020 Citation: Hoepting, C.A. 2020. Stop the Rot: New York part of ambitious multi-state, multi-disciplinary, multi-year, multi-million-dollar project to combat bacterial diseases of onion. Cornell Cooperative Extension Cornell Vegetable Program extension newsletter, Veg Edge August 5, 2020: 16(18), pp 6-7. https://rvpadmin.cce.cornell.edu/pdf/veg_edge/pdf198_pdf.pdf
  • Type: Other Status: Published Year Published: 2020 Citation: LaHue, G.T., Waters, T., and du Toit, L., 2020. Revisiting nitrogen management recommendations in the context of onion bacterial diseases. Progress report for Columbia Basin Onion Research Committee. Submitted 31 October 2020.
  • Type: Other Status: Published Year Published: 2021 Citation: Hoepting, C.A. 2021. Pesticides for onion bulb rot  Do they Work? Featuring 2020 field trial results. Cornell Cooperative Extension Cornell Vegetable Program extension newsletter, Veg Edge July 21, 2021: 17(15), pp 6-7.
  • Type: Other Status: Published Year Published: 2021 Citation: Hoepting, C.A. 2021. The rot race: Harvest practices to avoid bacterial bulb rot in onion. Cornell Cooperative Extension Cornell Vegetable Program extension newsletter, Veg Edge July 28, 2021: 17(16), pp 8-9.
  • Type: Other Status: Published Year Published: 2020 Citation: Stop the Rot newsletter #1 in September 2020, which was also posted to the project page on Alliumnet.com https://bugwoodcloud.org/mura/alliumnet/assets/File/Newsletters/StopRotteamNewsletterSept2020(final).pdf
  • Type: Other Status: Published Year Published: 2020 Citation: du Toit, L.J., Waters, T., Derie, M., and Darner, J. 2020. Battling onion bacterial diseases with bactericides. Onion World, December 2020:6-11. https://issuu.com/columbiamediagroup/docs/onion_world_december_2020
  • Type: Other Status: Published Year Published: 2020 Citation: Thompson, C. 2020. Organic Vidalia Onion Industry: Challenges in sour skin management. Vegetable and Specialty Crop News, 16 December 2020 (edits provided by B. Dutta)
  • Type: Other Status: Published Year Published: 2021 Citation: du Toit, L., and Waters, T. 2021. To disinfect or not? Can postharvest applications of disinfectants reduce bacterial bulb rots in storage? Onion World, July/August 2021:6-9.
  • Type: Other Status: Published Year Published: 2021 Citation: Stop the Rot Collated Summaries of Year 1 Technical Reports. Compilation of the final Year 1 topic summaries, August 2021. https://alliumnet.com/wp-content/uploads/2021/09/StoptheRot_Year1_summaries.pdf
  • Type: Other Status: Published Year Published: 2021 Citation: Wohleb, C.H., Waters, T.W., du Toit, L.J., and LaHue, G. 2021. Washington State University Extension Onion Alert, 1 September 2021. https://mailchi.mp/wsu/wsu-onion-alert-sept1-2021-1305808?e=72ba613792
  • Type: Conference Papers and Presentations Status: Published 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). (28 participants)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: November 19, 2020. WA, OR, ID Pacific Northwest Vegetable Growers (virtual) Annual Convention. Stop the Rot SCRI project update (Lindsey du Toit). 185 participants
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: December 4, 2020. OR, WA Hermiston Farm Fair. Understanding and managing Iris yellow spot virus and onion bacterial rots in Columbia Basin onion production (Lindsey du Toit). 213 participants
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Gaurav Agarwal, Santosh Koirala, Brian H. Kvitko and Bhabesh Dutta. 2021. Pan-genome of novel Pantoea stewartii subsp. indologenes identifies genes involved in onion pathogenicity and lateral gene transfer. Research on Demand presentation for American Phytopathological Societys annual meeting, Plant Health 2021 Online, 2-6 August 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: December 9, 2020. CO. Eastern Colorado Crop Production (virtual) Conference. Stop the rot project update (Antoinette Machado). 59 participants
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: January 6, 2021. GA Southeast Fruit and Vegetable (virtual) Conference. Onion disease update (Bhabesh Dutta). 45 participants
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Collaborator (Subas Malla) participated in South Texas Onion Committee meetings on June 12, 2020 and October 13, 2020. He highlighted and updated TX findings of the project to the Committee members. There were around 20 attendees at each meeting.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: MacKay et al. 2021. "Combating onion bacterial diseases with pathogenomics tools and enhanced management strategies: Research objectives and preliminary results." Research on Demand presentation for American Phytopathological Societys annual meeting, Plant Health 2021 Online, 2-6 August 2021. (All topic leads are co-authors).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: January 14, 2021. NY. Empire State Producers Expo  Onion Bulb Rot Session. (Lindsey du Toit, Bhabesh Dutta, Brian Kvitko, Christy Hoepting). 86 participants. -Rotten Onions 101 Part I  What kind of rot you got -Rotten Onions 101 Part II  The many ways onions rot -Stop the Rot: Rot-free onions in the making  Overcoming challenges to identify varietal tolerance to bulb rot.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Brendon Myers, Gaurav Agarwal, Ronald D. Gitaitis, Brian H. Kvitko and Bhabesh Dutta. 2021. Pan-genomic approaches to predicting virulence factors in Pantoea ananatis affecting Allium porrum and Allium fistulosum. Research on Demand presentation for American Phytopathological Societys annual meeting, Plant Health 2021 Online, 2-6 August 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Santosh Koirala, Mei Zhao, Gaurav Agarwal, Ron Gitaitis, Shaun Stice, Brian Kvitko, and Bhabesh Dutta. 2021. Identification of two novel pathovars of Pantoea stewartii subsp. indologenes affecting Allium sp. and millets. Research on Demand presentation for American Phytopathological Societys annual meeting, Plant Health 2021 Online, 2-6 August 2021.


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: Other Status: Published Year Published: 2019 Citation: Thompson, C. 2019. Center rot disease in organic onion growing studied. Vegetable Growers News, November 2019.
  • 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