Source: PENNSYLVANIA STATE UNIVERSITY submitted to
INTEGRATED MANAGEMENT OF EMERGING SEEDBORNE BACTERIAL DISEASES OF CUCURBITS AND CHENOPODS (IMDCC)
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
NEW
Funding Source
Reporting Frequency
Annual
Accession No.
1020279
Grant No.
2019-51181-30019
Project No.
PENW-2019-03142
Proposal No.
2019-03142
Multistate No.
(N/A)
Program Code
SCRI
Project Start Date
Sep 1, 2019
Project End Date
Aug 31, 2023
Grant Year
2019
Project Director
Bull, C. T.
Recipient Organization
PENNSYLVANIA STATE UNIVERSITY
408 Old Main
UNIVERSITY PARK,PA 16802-1505
Performing Department
Plant Pathology and Environmen
Non Technical Summary
Bacterial leaf spot (BLS) is caused by a diverse group of Pseudomonas syringae and is responsible for significant economic losses in seed and food crops from the Chenopodiaceae and Cucurbitaceae. For example in 2017, the prevalence of BLS was 75% in New York table beets. Likewise, in 2013-14, BLS affected an estimated 8,000 acres of cucurbits in Florida in major production areas. Because of the seedborne nature of these related pathogens, their genetically monomorphic population structure with wide host range, and similarities in the potential pathogen detection and disease management strategies, it is cost-effective and biologically relevant to research BLS of chenopods and cucurbits simultaneously. This project will develop an economically sound integrated approach to manage BLS across seed and food production systems to ensure high-quality seed and food for public consumption. In this project, we will develop: resistant plant lines for seed and crop production and evaluate whether the traits needed for seed crop resistance are the same as those needed for food crop production (Objective 4); improved IPM strategies for seed and food crops using new technologies and identified inoculum sources in seed and food crops (Objectives 2); biological and genetic data that will enable the development of accurate and sensitive pathogen detection and quantification methods; novel seed treatments to ensure clean seed for both production phases (Objective 1 and 3); cost analysis for implementation of practices developed (Objective 5); and translation of findings for growers, seed companies, scientists, students, and the public (Objective 6).
Animal Health Component
0%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
21214991100100%
Goals / Objectives
We have demonstrated that significant disease losses in cucurbits and chenopods are due to bacterial leaf spot diseases caused by a diverse group of seedborne Pseudomonas syringae strains but our understanding of their diversity and ecology remains poor. The experiments proposed here will provide tools for understanding the breadth of diversity and the ecology and evolution of the pathogens. Our primary goal is to develop economically feasible management strategies for these diseases, we have begun to understand these pathosystems (e.g., they are seedborne) and develop stand-alone management tactics, but our knowledge and tactics require integration. We are taking a three-pronged approach to the development of management strategies through: 1) breeding for resistance (related to legislative focus area on breeding for disease management); 2) development of economically feasible IPM strategies for food and seed production (related to 'efforts to improve production efficiency, handling and processing, productivity, and profitability over the long term'); and 3) the development and use of technologies for efficient seed quality assurance and treatment (related to new innovations and technology and improved efficiency in processing).The major Goals are to:Develop diagnostic methods for detection and quantification of the pathogens in seed and from environmental inoculum sourcesDevelop novel IPM practices for crop production and seed production to reduce seed contamination/infection and diseaseDevelop seed testing protocols and treatments for qualityIdentifynovel sources of disease resistance to CBC pathogensAnalyze the cost-effectiveness for all practices developedProvide international seed health extension, training, and mentorship
Project Methods
Develop diagnostic methods for detection and quantification of the pathogens in seed and from environmental inoculum sources. We will develop sequence-based markers and amplicon-sequencing methods for pathogen detection and quantification. Multiple approaches will be used to identify host- and clade-specific genetic markers useful for identification and quantification of various fractions of CBC P. syringae pathogens. Most will rely on identification of sequences via comparative genomics of whole bacterial genomes. Specificity and sensitivity of methods developed in this study will be evaluated using Constructed Bacterial Communities. Additional methods will be incorporated to differentiate between live and dead cells in samples including viability PCR.Development of novel IPM practices for crop production and seed production to reduce seed contamination/infection and disease. We will identify primary sources of inoculum in food and seed production fields using methods developed in the previous objective. These methods will be will be compared to methods that are currently available for samples from both seed and crop production fields from soil, crop debris, water, and seed among other potential inoculum sources.In addition we will evaluate the population dynamics of CBC pathogens on susceptible and resistant cultivars in growth chamber and field experiments. Pathogen populations on leaves, stems, flower, fruits, and seeds (stored under different conditions) will be monitored over time using methods available. Similar methods will be used to determine the location of the pathogen on the seed coat/pericarp, the embryo/perisperm, or the endosperm. Artificially or naturally infested commercial seed lots will be evaluated using differential seed disinfestation and sterile microdissection to localize the pathogen on seed and fruit (beet and chard) tissues. We will use the information about the taxonomy and population structure of the pathogens to strategically develop novel biological controls including bacteriophage and myxobacteria. Seed contamination will be reduced through integrated treatments in seed production fields. Management strategies and experimental designs may be altered with additional input from industry members of the collaboration team and local growers. Annual field trials will be established using local standard management practices for crop or seed production.Develop seed testing protocols and treatments for quality assurance. The primary goal will be to develop verified pathogen detection and quantification methods for seed testing based on the work described above. These methods will be modified to fit the International Seed Health Initiative for Vegetable Crops (ISHI - Veg) Best Practices for PCR Assays in Seed Health Tests and Guidelines for Validation of Seed Health. This will allow us to identify pathogen population levels that cause economically significant disease. Threshold population is defined here as the lowest population on seed that results in economically significant disease. The relationship between seed or starting leaf population levels and disease (incidence and severity) and final plant population levels will be evaluated in growth chamber and field experiments with infested seed lots and artificially inoculated seed and plants. Treatments will be tested for elimination of the pathogens from seed while maintaining quality. Temperature therapy (dry heat, hot water, and/or steam), commercial biologicals, chemical bactericides (e.g., coppers), Actigard, nanoparticles, and other methods identified by industry partners will be used to treat seed (naturally or artificially infested) using standard or manufacturer protocols. Biologicals developed in this application will be applied by soaking seed in cell, spore, or phage suspensions in buffer or a sticking agent (methylcellulose), and air dried before further treatment or planting. Disease will be evaluated with or without treatment.Identifying novel sources of disease resistance to CBC pathogens. We will describe the relevant host range for the pathogens including relevant cultivars using our previously published methods. Virulence of the strains will be assessed visually after 14 to 21 days. We will develop resistant germplasm using wild and domesticated germplasm for beet, chard, and cucurbits. This will include evaluating differences in resistance needed for seed and food crops. QTLs associated with disease resistance will also be identified. Analyze the cost-effectiveness for all practices developed. Based on the previous research stages, effective interventions will be selected. The costs of these investigated interventions will be calculated and, together with the corresponding efficacy, the cost-effectiveness of each measure will be determined.International seed health extension, training, and mentorship. To facilitate these goals, we will develop a website and listserv to facilitate and foster communication. We will develop a new online course on the historical and international nature of seed production. This newly developed course will leverage scientific inquiry to identify pedagogical models which enable transfer of information from the online course to real-world implications and practices. Training workshops in diagnostic metagenomics for seeds, translational taxonomy, online YouTube videos to teach those techniques, fact sheets. Effectiveness of teaching and dissemination methods will be measured. Common extension channels (e.g., factsheets, newsletter articles, blogs, social media, etc.) will be used to disseminate the results of this project in addition to presentations at local and regional grower meetings. Research results will also be used to augment regional production guides to promote the use of IPM for managing these diseases and extend findings to the seed industry, crop producers, industry supporters, seed saving organizations, and the public. As part of this work we will provide formal mentorship of all undergraduate and graduate students, postdocs and early career faculty.

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

Outputs
Target Audience:We provided information about the project or findings to the following groups through extension materials or in person and virtual presentations provided by individuals, small groups, or the entire team of PIs. Vegetable and fruit growers and professionals across the US Florida Suwannee Valley Watermelon Institute Florida Watermelon Association, field producer members Seed technology companies Seed companies Cucurbit transplant producers, Extension agents and crop consultants, Fungicide industry personnel New York Seneca Foods, Bejo Love Beets USA Cornell extension advisory group New York Vegetable Research Association Council Processing Vegetable Group New York Table Beet Research and Development Committee Pennsylvania PA Vegetable Growers Association Disease identification and management in cucurbit crops. February 28, 2020. Cucurbit School, Cumberland County Penn State Extension Office, Carlisle, PA. 22 participants. Integrated disease management with emphasis on cucurbits, tomatoes, and onions. February 29, 2020. Beginner Vegetable Grower Series, Ag Farm Choice Credit, Lewisburg, PA. 25 participants. Vegetable (bacterial) disease update. April 27, 2020. Pennsylvania Department of Agriculture Inspector Training Webinar. 20 participants. Washington Western Washington Small Seed Advisory Committee and the seed company field representatives or seed growers of this committee. Several Chard and beet seed company field representatives who have visited with team members Chard and beet seed growers, whose crops were visited by team members Four small-scale, diversified farms on San Juan Island in San Juan Co., WA growing table beet and Swiss chard Several fresh market beet and chard crops in Skagit Co., WA whose crops were visited by team members Wisconsin Table beet growers Beet seed industry stakeholders in Wisconsin Internal Advisory CommitteeMembers received information regularly from participating and leading Objective Team Meetings: Philip R. Brown, Pathology Senior Manager, Sakata Seed Co. Burlington, WA, USA; John Henderson, Production Director, Love Beets USA, Rochester, NY; Rick Falconer, Managing Director Research, Rijk Zwaan, Salinas, CA, USA; and Regional Vice President Rick Falconer, American Seed Trade Association Dr. Gerbert Hiddink, Seed Technology Research Manager, Enza Zaden, Enkhuizen, The Netherlands Dr. Christina Dennehy, Monsanto Vegetable Seeds, Woodland, California Dr. Sukhi Pannu, Director, Testing Services, California Seed and Plant Laboratory, Inc., Pleasant Grove, CA, USA. Wilmarie Kriel, Senior Plant Pathologist, Starke Ayres Seed (Pty) Ltd., SA Scientists including those from industry, government, and academiaat all levels (PIs through undergraduate students) through activities at scientific meetings. American Phytopathological Society Australasian Society of Plant Pathology Vegetable pathologists and horticulturists Plant breeders Prospective Postdoctoral and graduate student candidatesfrom the US and abroad for each of the filled positions through interviews. There were approximately 3 trainees per position. Network emails and presentations Additionally, we have developed a network of 155 interested parties. Of these, 53 attended our introductory webinar in May 2020https://psu.mediaspace.kaltura.com/channel/Pseudos%2Bon%2BSeed/169702511. Many responded that they would like to be involved in the research. Describe the stakeholders we are reaching through our emails. Industry- 50 individuals from 25 seed, seed-testing, or allied agricultural companies and two growers.Only four of these companies are also represented in our Internal Advisory Committee.We can share this information but have not hear for purposes of confidentiality. External Advisory Committee Members:Dewey, Mark, President, Dewey Produce, Inc., Dunkle,Ric, Senior Director, Seed Health and Trade, American Seed Trade Association, Jose-PabloDundore Arias, Assistant Professor, California State University Monterey Bay (CSUMB);Hartney, Sierra, Pathology Manager Breeder Support, Sakata Seed America, Inc.;Holaday, Brian, President, Holaday; Seed Company; Koike, Steven, Director, TriCal Diagnostic; Maretzki, Audrey, Co-Director, Penn State's Interinstitutional Center for Indigenous Knowledge; Stearns, Tom, Founder and Owner, High Mowing Organic Seeds; & Harmon, Carrie, National Plant Diagnostic Network. Commodity Groups and other organizations- four commodity and seed saving groups including two that represent indigenous peoples. Government - Twenty-one individuals from eight US Federal and State Government agencies and one international government agency USDA ARS (3) North Central Regional Plant Introduction Station - USDA (1) CDFA (1) Hawaii Department of Agriculture (2) Idaho State Department of Agriculture (2) INRAE (2) Nevada Dept. Agriculture (1) Wisconsin Department of Agriculture Trade and Consumer Protection (1) Washington State Department of Agriculture (8)? Academia- 28 academic contacts from 17 US universities (3 of which are members of the Executive Committee though these members are not counted in total academic contacts) Iowa State University (2) Louisiana State University (3) Michigan State University (2) Mississippi State University (1) Monsanto (1) New Mexico State University (1) Penn State University (2) Southern Illinois University (2) TAMU (3) UC Davis (3) University of Arizona University of Arkansas University of Florida (2) University of Pretoria University of Puerto Rico Washington State University (2) Auburn University (3) Changes/Problems:There were three issues that were not related to the global pandemic. First, we contacted several professional academic evaluation groups, we were encouraged to conduct the evaluation internally because the planned evaluation is just a review of the GANNT chart which we are doing at each objective team meeting. The external evaluators said they had nothing to do. Second, pathogenicity tests and cultivar evaluations were plagued with contamination of the controls. Several team meetings discussed options for preventing the spread of the pathogen. Improved methods are now being used. Lastly, NoPseudomonaswas detected from any of the FL transplant houses repeatedly surveyed. However, strains were recovered from extension team members. The COVID-19 global pandemic has delayed our project by up to 18 months and an extension of at least 18 months will be needed. There were significant restriction on density when labs opened again. The New York State restrictions for the COVID-19 pandemic initiated in March 2020 meant that we received approval to visit commercial farms (in isolation) but not initiate field trials in time for planting, nor gain access to our laboratories for sample processing until August. PPEM at Penn State University was shutdown except for mission critical projects March 2020 through June 2020. Constraints on being able to work in the lab, greenhouse, and fields during the first few months of the COVID-19 pandemic shutdown in Washington State limited the progress for several months. Once allowed back to work, it was under strict safety guidelines and WA surveys were initiated. Boyd was due to have isolations of biological control agents complete in spring and tests completed in summer 2020. However, constraints on the number of people in labs and hours that could be worked after labs were reopened slowed this work considerably. Progress was and strains have been isolated from most soil samples, but testing is just beginning. 1-year additional funding will be needed for this student. At Auburn an undergraduate working on the project only resumed their work in August 2020. Field work was allowed to continue in some cases and in others field work was limited but not restricted by the access to laboratories and preparation of materials. Some level of field experiments delay was experienced by Penn State, Washington State, University of Florida, and Cornell. Recruiting took longer due to the global pandemic ARS Postdoctoral associate was hired in May and has had extremely limited access to our research facility due to the pandemic. PSU postdoc will not start until January 2021 the global pandemic and will have restricted access to the laboratory. International recruits were prevented from entering the country due to the global pandemic and changes in the federal regulations concerning international students and scientists. Both Auburn and Penn State delays of up-to 18 months in hiring international postdocs. GANNT charts can't be finished by teams until all major positions have been recruited. Objective 1 The postdoc to be hired by Penn State who will serve as the manager of the databases has not yet been hired due to COVID and new immigration restrictions. 18-month COVID related delay. Graduate student working on the evaluation of existing markers for specificity acrossP. syringaeCBC pathogens and nonpathogens began with the project in Fall 2020. Currently available methods and pathogens and non-pathogens of interest were identified and will be tested in 2021. 1-year delay. Sequencing for all projects was delayed because genomic facilities were prioritizing COVID sequencing. WGS of first 100 pathogens is delayed because and two postdocs has not yet been hired due to COVID and new immigration restrictions. This person will send identified strains for sequencing. 18-month COVID related delay. Collection of new strains causing these diseases in important to the overall goals of the project. While in WA the disease is the worst that du Toit has seen and isolates were obtained by Pethybridge and Bull labs in New York, in FL it was barely observable. Graduate student to be engaged by NCSU was not recruited due to COVID delay. 18- month COVID related delay. Soils from beet fields in NY will be a good source of phage when the student is engaged. Objective 2 ·Cornell University restrictions for the COVID-19 pandemic were initiated in March 2020. They received approval to visit commercial farms but not initiate field trials in time for planting, nor gain access to laboratories for sample processing until August. Thought they were not able to plant a replicated cultivar trial in the field this year they instead collaborated with Bejo and evaluated BLS incidence and severity in two on-farm demonstration trials in western New York, and a replicated trial at the Bejo facilities at Geneva. Alternate plans will accomplish some of the same goals with minimal impact on timelines. ·Penn State closed labs and limited access delayed Boyd's isolations of biological control agents from soils collected in summer 2019. Predation assays that were to be finished by fall 2020 will be initiated in the second year. Objective 3 Difficulty in recruiting and onboarding Penn State postdoc in a timely manner resulted in 18-month delays in work on this objective. Objective 4 The ARS laboratory is a bit behind in the first year's milestones due to the Covid-19 pandemic.The Postdoctoral associate was hired in May and has had extremely limited access to our research facility due to the pandemic.We should be able to make up the lost experiments if we can get mor time in the Unit. No overall delay Goldman - We will be revising the way we perform inoculations in the greenhouse. Data from the pilot study showed very little difference for the overall disease rating or rate of senescence between control and inoculated plants. This suggests contamination from inoculated plants and several sources were identified and mediated. Methods are clear and no overall delay us expected. Objective 6 Call - Planned to attend several different seed testing meetings, trainings and committee meetings to get feedback about the materials and kind of course need in summer 2020. ISTA was cancelled due to COVID-19 pandemic and other meetings were virtual and provided less opportunity for side discussions and meetings. However, during the course of other objective meetings, ideas for presentations were developed that are going to be produced and provided on our website. These can be used for the course. All members - SACNAS 2020 was held virtually this year and would have been more difficult to attend. Instead, we invited CSUMB to work with us as an opportunity to recruit their students as part of our projects. We will plan to attend the following year and present a poster about our project. We would like to direct funds from the project to this effort. What opportunities for training and professional development has the project provided?Objective 6.4 of the project is explicitly professional development and training opportunities created by the grant. They are reported here. Training Caroline Lacault from INRAE, the University of Angers, France and HM Clouse presented her previous and current research. She provided a one-hour webinar on2/26/20,which was recorded for future viewing by the Collaboration Team. She presented her work on Zucchini vein clearing disease caused by several lineages withinPseudomonas syringaesensu strictoand which are related to the pathogens with which we are working. This provided training for the entire team. New graduate students and postdocs in all programs received mentorship into labs via in-person and Zoom modalities starting in Spring 2020. All graduate students, postodocs, and facultyare involved in all Objective Team meetings. We held two all hands meeting since the beginning of the project and one or two objective team meetings in 2020. These meetings provide project management training for all. Students and postdocs and early career faculty presented a group poster about this project and at least one additional poster related to the project at Plant Health 2020. Emilee Gaulke visitedthe Bull Lab at Penn State on 02/24-27/2020to learn techniques for preparation of pathogen inoculum, storage, and plant inoculation. This was invaluable for her investigations in 2020. She received additional training via email and zoom from du Toit and Bull labs. Marilen Nampijja, PhD student, started on this project in January 2020 when she began a PhD program at WSU. Marilen has been learning how to work withPsand with table beet and Swiss chard. Despite major constraints on research progress this year as a result of the COVID-19 pandemic, Marilen was able to complete a fairly extensive survey of beet and chard seed crops in 2020, and has been learning how to complete pathogenicity tests on beet and chard, how to assay seed lots forPs, and the process of MLSA for pathovar determination. Stephanie Crane, MS in Ag student, started on this project in spring semester of 2020. Stephanie is doing an online degree as she works full-time for Sakata Seed America, and is using her work experience withPsover the past five years to do a thesis project onPsfocused on management of the pathogen in beet and chard seed crops. Madeleine Nielsen, undergraduate research intern, started work on this project in July 2020 and has been working full-time in the WSU-Vegetable Pathology lab. During her time working in the lab, she has learned how to do bacterial isolations from symptomatic plant tissues, prepare media for fungi and bacteria, extract DNA, set up PCR reactions, prepare and run gels, and has assisted in planting and harvesting of field trials. Madeleine is an incoming freshman in the Medical Anthropology and Global Health program at the University of Washington. She will continue working in the Vegetable Pathology lab on a part-time basis after her classes start. Susannah Da Silva, a new Ph.D student starting Fall 2020, and another Ph.D student has been assisting and training in the program. A biological scientist in the project in plant pathology had opportunity to work with cucurbit transplant producers for the first time which provided experience in handling and collecting samples for diagnostic purposes. Bull Lab SCRI sub-team has been meeting monthly to ensure students and postdocs understand the background for all parts of the project and especially bacterial taxonomy components. Meena Gurung, a new PhD student joined the lab in Fall 2020 and has been trained by Lindsay Boyd and others to cultivate and store plant pathogenic bacteria and to select and evaluate rifampicin resistant variants for growth equivalent to the wild type strains. Rachel Hershlag moved from another project in the Bull lab in Fall 2020 as a PhD student. Hershlagand Bull are working to ensure that Hershlag understands all of the currently available detection and identification methods for the pathogens. Postdocs and students had opportunities to learn to mentor students and others. Eric Newberry started working on this project in December 2019. Eric has also mentored a graduate student, Shreya Sadhukhan, on protocols for mutant construction, and pathogenicity assays. Lindsay Boyd started working on this project prior to its initiation the project in summer 2020. Her project was supposed to end by the end of the summer but due to the COVID-19 pandemic her progress has been delayed and now anticipates graduating in summer 2021. However, this allowed her to mentor Meena Gurung through her first experiments. Undergraduate students being trained by Boyd and others were not allowed back in the lab after March 2020 due the COVID-19 pandemic. Penn State students are being trained to conduct microbiome analysis. Hershlag and Gurung attended the Penn State Microbiome Center Kickstart program that allowed them to sign up for computer cluster accounts, analyze simplified data sets and become familiar with all the training programs available August 4-7, 2020. They are also attending weekly Penn State Microbiome Center meetings. Professional Development Bull reviewed Collaboration and Team Science: A Field Guide from NIH, NCIhttps://www.cancer.gov/about-nci/organization/crs/research-initiatives/team-science-field-guideat the October 2020 Collaboration Team meeting. This document outlines the best practices for team research and the guiding principles for how our team functions. Gurung participated in a professional development workshop taught by Bull called, How to Be Your Own Best Mentor on August 21, 2020 (Boyd and Hershlag have taken this previously). Attendees developed personal mission statements and developed action plans to make progress toward fulfilling their personal missions. How have the results been disseminated to communities of interest? Six students, two postdoc, and three earlier career PIs attended Plant Health 2020 and presented a poster on our project (Nampijja et al., 2020). Boyd et al., 2020 was likewise presented at the meeting. A public webinar was presented on 5/20/20 to familiarize interested parties with the project. The webinar was advertised through our listserv,twitter, participating commodity groups, and major seed organizations. Additionally, PD Bull sent emails to seed saving organizations and seed companies run by Indigenous organizations. Presenters provided an overview of the project and what is currently known about pathogen biology and life cycles, and disease management. Seventy-four attendees included government, industry, and academic professionals including 21 from our research team participated. The recording of the event is availablehttps://www.pseudosonseed.org/ Pethybridge -The reached target audiences through this reporting period are table beet growers and industry stakeholders, regional extension personnel, and other scientific peers. All grower participants and industry stakeholders (Seneca Foods, Bejo and Love Beets USA) provided access to their fields for quantifying the prevalence and incidence of bacterial leafspot in spring and evaluating cultivar susceptibility. These personal visits to growers during the season afforded multiple opportunities to talk to growers and crop scouts informally regarding the status of BLS and whether control was warranted, and the objectives of this project. Pethybridge -In addition, the objectives of this project were discussed at extension advisory group meetings for the New York Vegetable Research Association and Council Processing Vegetable Group Meeting (December 2019), and the New York Table Beet Research and Development Meeting (March 2020). Pethybridge - Results were disseminated to communities of interest through the publication of an extension article in the Cornell VegEdge newsletter. This newsletter is distributed to all registered vegetable growers in New York on a weekly basis and is available online for growers and extension agents from other states to access. Pethybridge - This project was featured at regional agricultural events such as the Empire Expo (Syracuse, New York) and the Mid-Atlantic Fruit and Vegetable Grower Convention (Hershey, PA) in January 2020. These talks and conference publications enhanced our outreach of the concepts and ideals of the project to vegetable growers in New York and surrounding states. du Toit - On March 2ndof 2020, information on the CC_SCRI project was shared with the Western Washington Small Seed Advisory Committee, along with a request for approval to survey table beet and Swiss chard seed crops in the 2020 field season for BLS. Seed company field representatives and seed growers were encouraged to engage with the project and team members. du Toit - Seed company field representatives who accompanied Marilen Nampijja and Lindsey du Toit to survey their table beet and Swiss chard seed crops in June, July, and August 2020 were engaged in discussions about the CC_SCRI project, in particular BLS in beet and chard seed crops. Information was exchanged on seed production practices, experience of the field reps with this disease, and management recommendations. Some seed growers whose crops were visited also engaged in discussion with Nampijja and du Toit. du Toit - Eight members of Dr. du Toit's Vegetable Seed Pathology program at the WSU Mount Vernon NWREC, including four graduate students, a technician, a postdoctorate, and a project manager, visited four small-scale, diversified farms on San Juan Island in San Juan Co., WA on July 14, 2020, where table beet and Swiss chard was grown as seed crops (one farm) or fresh market crops (three farms). The team assisted each farm with diverse disease diagnoses and management recommendations on different crops. The graduate students and postdoctorates participated in the exchange. On two of the farms, symptoms of BLS were observed in table beet and/or chard crops, with very severe symptoms in fresh market beet and chard crops on one farm, to the extent the farmer had abandoned at least one planting. The group discussed the disease cycle and recommended management practices with the farmers. Samples of seed used to plant the beet and chard fresh market crops were provided to PhD student, Marilen Nampijja, to assay forPsand assess if infected seed might have been the primary source of inoculum. Additional samples of BLS were received from fresh market beet and chard crops in Skagit Co., WA during the 2020 season, with details of the disease and management recommendations provided to each farm. du Toit -After processing of suspect leaf samples collected from symptomatic table beet and Swiss chard seed crops and fresh market crops in western WA, growers and field representatives received feedback on the diagnoses and were provided with relevant recommended management strategies for those fields from which suspectPscolonies have been isolated. Goldman - Shared the results of pilot studies with local growers and grower groups, such as the Midwest Food Products Association members, who meet regularly throughout the growing season, and with the members of the Vegetable Breeding Institute, a public-private consortium that includes scientists and representatives from many vegetable seed companies. These virtual meetings, held during the spring, summer, and early fall of 2020, allowed us to share some of our approaches in pathogen screening and some of the preliminary results. Paret- Presented with colleagues at adisease management workshop for Florida watermelon production. Suwannee Valley Watermelon Institute, Dec 3, 2019, Gainesville, FL. Paret - Provided information about the project during visits with members of the Florida Watermelon Association, field producer members; Seed technology companies; Seed companies; Cucurbit transplant producers; Extension agents and crop consultants; Fungicide industry personnel. Gugino - Presented, Disease identification and management in cucurbit crops on February 28, 2020 at theCucurbit School, Cumberland County Penn State Extension Office, Carlisle, PA. 22 participants. Gugino - Presented, Integrated disease management with emphasis on cucurbits, tomatoes, and onions onFebruary 29, 2020 at the Beginner Vegetable Grower Series, Ag Farm Choice Credit, Lewisburg, PA. 25 participants. Gugino - Presented, Vegetable (bacterial) disease update on April 27, 2020 to thePennsylvania Department of Agriculture Inspector Training Webinar. 20 participants. What do you plan to do during the next reporting period to accomplish the goals?Objective 1 Finalize strain collection organization and determine management of sequence databases. Evaluate existing markers for specificity acrossP. syringaeCBC pathogens and nonpathogens. WGS for first 250 pathogens and 50 non-pathogens, initial comparative genomics to identify novel markers. Identify additional candidate genes fromcomparative genomic analyses andevaluate by mutant construction followed by pathogenicity on the host plants. Graduate student, Shreya Sadhukhan, is constructing in-frame deletion mutant of avrRpt2and complement construct to confirm this phenotype and the host specificity. Bacteriaphage will be isolated and host ranges of isolated phages will be determined Drafted manuscripts will be published on the diversity of strains isolated from various regions from beet and chard crops. Objective 2 Surveys of table beet and Swiss chard seed crops in western WA forPswill continue 2021 and fields surveyed in 2020 will be revisited to collect seed crops residues to assess survival ofPson residues. Weeds will be sampled for their ability to serve as alternate hosts. Sources of surface-water used to irrigate seed crops will also be tested forPs. Research on chemical and physical methods of treating table beet and Swiss chard seed crops forPswill be initiated in 2020-21. Bactericide trials and seedborne threshold trials will continue in 2020-21 in WA. In 2017 and 2018, rainwater was collected from the shoulders from a mulched cucurbit field study at WSU-NWREC after each rain event. Genetic diversity of isolates will be analyzed to determine if rainwater is a potential source of inoculum. Experiments testing water and alternative hosts will be repeated. NY Beet surveys will be repeated in 2021 with the additional of collecting soil, water, and plant samples from selected positive and negative fields to facilitate a multivariate analysis approach to identifying inoculum sources. Cornell will work with Penn State to look at the genetic variation of the pathogen across NY. Cucurbit transplant facilities and field production sites will continue to be surveyed and sampled in Florida. Isolates from 2020 and 2021 cucurbit sampleswill be characterized. In Florida, in vitro studies on nanomaterials will be conducted in Fall 2020 and the data generated will be used to establish treatments for a field trial which will be conducting during spring 2021. Seed coating experiments will be standardized in lab and in collaboration with seed technology companies. Findings will be disseminated through grower association meetings In vitro predation assays with myxobacteria will be complete. Those with promising attributes will be used as seed treatments in growth chamber studies. A replicated glasshouse trial is planned for fall/winter to evaluate product efficacy for BLS control.One field trial will be conducted in 2021 to evaluate the efficacy of selected products for BLS control in table beet. Products will be selected based on coordination with replicate trials conducted in other states. Annual field trials will incorporate new management tools. Objective 3 A review article is being written on threshold populations and seed testing methods. After written, the team will select standard seed testing methods to use and these will be modified as new methods are developed. Initial threshold population tests for seed inoculation will be repeated. Activity within this objective will be initiated as planned in 2021 with a replicated field trial evaluating control of BLS in table beet root crops at the Cornell AgriTech research facilities. Ps rifampicin resistant variants will be used in plant population dynamics, andsoil and water survival assays. Amplicon sequencing and metagenomics will be used to determine changes related to decline of soil populations ofPs. Efficacy ofstandard seed treatments for beet and chard disases will be evaluated. Bull is working with collaborators on a unique engineering process for seed treatments. We will start the work with artificially inoculated seeds including varieties used in microgreens. If the treatment is promising other crops and varieties will be tested. Objective 4 ·Pethybridge -A replicated glasshouse trial is planned for fall/winter to evaluate table beet cultivar efficacy for BLS control.One field trial in each year will be conducted to evaluate the susceptibility of selected table beet cultivars to BLS. Tymon and Bull are finishing a manuscript which evaluates the host range of the wart strains and pathotypes from this species. The methods used are available for consideration across labs Bull, du Toit, Goldman, Tymon, and Pehtybridge have conducted preliminary experiments using spray inoculation and seed inoculation methods. We will harmonize these methods at our annual meeting and select standard protocols for disease rating. Bull - The PSU team will evaluate population dynamics of bacteria on resistant and susceptible cultivars and colonization of seedling surfaces and pumpkin tissue using rifampicin resistant isolates. Bull -The PSU team will evaluate a variety of methods for confirming visual HR responses in plants for the host range studies. Pethybridge -A replicated glasshouse trial is planned for fall/winter to evaluate table beet cultivar efficacy for BLS control.One field trial in each year will be conducted to evaluate the susceptibility of selected table beet cultivars to BLS. Goldman - The University of Wisconsin team will be conducting a screen comprised of plants in the reproductive phase of their life cycle. The reason for this is that a great deal of the table beet and Swiss chard seed produced in the US is subject to infection from this pathogen, and has the potential to spread the disease through transmission of the seed. Because infection by this pathogen can be a problem in both the vegetative and reproductive phases of the crop, we will compare reaction to inoculation with the pathogen in both phases of the life cycle in a large collection of cultivars, breeding lines, and germplasm accessions. Wechter - plans to begin the initial screening of theCitrullus amaruspopulation this next year, as well as begin acquiring squash lines for testing. Bull lab will test cultivars used in microgreens for susceptibility. Objective 5 ·Analyze the cost-effectiveness for all practices developed. ·Meet and discuss opportunities for economic analysis with the beet and chard seed industry.The potential issue here might be confidentiality problems with industry participants. Objective 6 The website pseudosonseed.org will be completed, maintained, and continually expanded. Social media and listservs will continually be used to market the website. The targeted official launch is January 2021. Asyllabus and marketing report for a seed pathology short course will be developed. This will includeexploration of a full semester seed pathology courseusing gap data collected from other similar courses. Students and postdocs will develop videos describing different aspects important to seed testing. Team will submit a proposal for a special session on seedborne bacterial diseases and seed treatment in 2021 for Plant Health 2022. APseudomonastaxonomy video will be finished and presented on our website. Annual mentorship and professional development webinars will include How to Write a Cover letter and Research Statement. Recruitment will be expanded to include research at CSUMB. Attendance at SACNAS will resume as the pandemic allows. Review progress and GANNT chart at Annual Meeting December 2020.

Impacts
What was accomplished under these goals? A culture collection of 2332 pathogenic and nonpathogenicPseudomonas syringae(Ps) strains from chard, beet, and cucurbit field and seed crops,transplants and seed were collected from over 70 surveys conducted in FL, NY, WA, from PA clinic samples, and from industry collections. Thisrepresents an invaluable resource of diverse strains ofPs strains fromcrops grown in the USA and several other countries.Bacterial leaf spot severity and incidence was at a 20-year highin WA. Preliminary studies established cultivar screening assays for use by the breeding and pathology programs for identifying resistant germplasm, host range, and treatment screening.Potential chemical and biological treatments for management of diseases have been tested and effective treatments identified for cucurbits that may be transferable tochenopods.Experimental data indicate new avenues fordetection and treatment methods.For example, a candidate gene has been identified that may serve as a specific pathogenicity marker for specific detection of pathogens which infect squash and predators were isolated that kill the pathogens. These will be useful to seed, seed treatment, and seed testing industries. 1.1a:A strain collection of >2332Psstrains from cucurbits and chenopods was established. New strains came from over 70 surveys conducted in FL, WA, NY, and PA as well as from industry collections. This represents an invaluable resource ofPsstrains from seeds and seed and field crops grown in the USA and abroad. 1.1b: AvrRpt2 was identified as a potential candidate for host specific marker ofpathogens of squash. strains.This is an important first candidate for detection of host specific pathogens and may be useful to all aspects of the seed industry. 2.1a. Survivorship ofPswas evaluated on plastic mulch films used in cucurbit cropping systems. Data are available for analysis. 2.1b.Seed transmission of from seed artificially inoculated withPscausing wart on cucurbit fruit andPspv.lachyrmansto seedlings was evaluated. Data are available for analysis. 2.1c.Weeds growing along the shoulders of artificially inoculated cucurbit production fields were collected and sampled forPs. Weeds were identified to species and isolated bacterial colonies that were cream-white were purified are available for further analysis. 2.2a. Variants resistant to the antibiotic rifampicin variants of five pathogens were generated. Strains are available for to monitor population dynamics of pathogens on susceptible and resistant hosts, survival in soil, and other experiments. 2.4a.> 180 predatory myxobacteria were isolated from soils from beet fields heavily infested with BLS.The collection is available to identify the best predators ofPsandfor potential use as a seed treatment. 2.5aFive to seven bactericide treatments were investigated in field trials for beet and chard seed crops are being conducted, in three locations in western Washington in 2020. Plots were rated for evidence of phytotoxicity and BLS severity. Seed harvested from the trials is available for detection and quantificationofPsinfection levels. 2.5b:Thirteen chemical treatments including a novel nano-copper compounds and a non-formulated MgO compound were evaluated for management ofPson watermelons.Pson watermelon was best suppressed with ManKocide and Kocide than with any of the copper and magnesium compounds tested in this trial, although the data may suggest that FQ-Cu in TEOS and MgO nano powder may provide good suppression with some adjustment of the chemical concentration. Potential chemical treatments effective for management of diseases have been identified and may be useful for management ofPson other crops. 3.1a: Several seed sampling protocols are being evaluated at WSU, UFL, and PSU for beet, chard, and cucurbits using naturallyinfested seed lots collected from fields sampled and industry partners. A list of seed sampling protocols is in development and infested seed lots are available for testing new seed treatment methods. 3.2a. The impact of beet seed inoculum concentration on disease incidence was evaluated in growth chamber studies.There were no statistically significant differences in disease incidence between seeds treated withPsconcentrations ranging from 103to 107CFU, with 107having a disease incidence of 50% on seedling cotyledons. Seed inoculated with as low as 101resulted in 10% disease under controlled conditions. Even very low seed populations will lead to significant disease under controlled conditions. 3.2b.Seedborne thresholds ofPswere evaluated in baby leaf field trials in Mount Vernon, WA in spring 2020. Samples of a chard seed lot infected withPswere mixed with a non-infected seed lot of the sample cultivar in ratios ranging from no infected seed to 105CFU ofPs/g seed. Under the cool, wet spring conditions in western WA this year, fairly severe symptoms of BLS were observed in some of the plots in the spring baby leaf trial. Data are available to be analyzed. 4.1a:Host range testing demonstrated difference between the cucurbit wart pathogen,PsPsa_2015 and the pathotype strain ofPspv.aptata.Although theywere both pathogenic on leaves, although the lesions were distinct and Psa_2015 did not cause symptoms on beet seedlings whereasPspv.aptatadid. We may be able to find host specific markers for detection of these pathogens. 4.2a:BLS incidence and severity were evaluated in three cultivar trials (two on-farm and one replicated) in western New York. The disease was found in table beet cultivars Manolo and Pablo in plots established at all three plantings at low incidence (<3%).Low disease incidence might be correlated to environmental conditions and indicates the need for greenhouse trials to complement field research. 4.2b:Preliminary cultivar screening experiments were conducted to evaluate plant reaction differences in beet and Swiss chard germplasm toPs. Two varieties each of table beet and Swiss chard were used in the experiment.Cultivar screening assays are in place and can be used in selecting resistant beet and chard germplasm. 4.2c:Pathogenicity experiments were conducted using fivePsisolates of pathogenic to watermelon, squash and cantaloupe.Psstrains cause different levels of disease onC.amarusvariety Sugar Baby.Isolate 13-139B caused the most severe symptoms and was selected for future workto screen the PI collection of 126 accessions ofC. amarus. 5:At an objective team meeting in fall 2020 the team discussed data collection options for economic analysis. Field treatment data collected for objectives 2, 3, and 4 will be used to facilitate the economic analyses planned for this objective. The main output of the analysis will be assessing the cost-effectiveness of the interventions in the last year of the project, pending the other objectives' outcomes. 6.1a:A project websitehttps://www.pseudosonseed.org/is established and is ready for a January 2021 public launch. Group webinars and presentations by team members are posted there along with publication and information about the pathogens and diseases. 6.2. APublic Webinar was held discussing the epidemiology, etiology and disease management options for BLS on cucurbits and chenopods. Currently, 155 members of government, industry, academia and NGOs are receiving information about our project through our listserv and the webinar. Many more are receiving information through direct contact and presentations at local meetings. 6.3:Industry partners conducted a survey to determine global occurrence ofPs. Depending on the country of origin and year of production between 0 to 59% of seed lots of pumpkin and squash were infested withPs.Seed grown in all parts of the world may be infested withPs. 6.4See Professional Development and Training.

Publications

  • Type: Other Status: Published Year Published: 2019 Citation: Kikkert, J. R., and Pethybridge, S. J. 2019. Leaf diseases identification and management on table beets in NYS. Cornell VegEdge 15(10):1-3. https://rvpadmin.cce.cornell.edu/pdf/veg_edge/pdf164_pdf.pdf
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Pethybridge, S.J., and Kikkert, J. R. 2020. Identification of foliar pathogens and best management practices. Proc. of the Empire Expo, Syracuse, New York. 16 January 2020. Pp. 6. http://www.hort.cornell.edu/expo/pdf/Kikkert_Pethybridge_Table_Beet_Foliar_Disease.pdf
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Boyd, L., Kikkert, J., Bull, C.T. 2020. Strategies for isolation of Myxobacteria and specificity of biological control of Pseudomonas syringae seedborne pathogens of chenopods and cucurbits. Plant Health 2020.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Nampijja, M., Boyd, L. N., Crane S., Dundore-Arias, J. P., Gaulke, E., Herschlag, R., Huerta, A. I., Kulesza, E., Kan, K., Newberry, E. A., and Potnis, N. 2020. Integrated management of emerging seedborne Pseudomonas syringae pathogens of Cucurbits and Chenopods. Plant Health 2020
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Vegetable Breeding Institute annual meeting reports, 2020 https://plbrgen.cals.cornell.edu/research-extension/vegetable-breeding-institute/
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Bull, C.T. 2019. Translational Taxonomy: Balancing Utilitarian and Theoretical Taxonomies of Plant Pathogenic Bacteria; Australasian Plant Pathology Society Conference, Melbourne Australia
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Bull, C. T. 2019 Translation of contemporary species and pathovar concepts for disease management. Plant Health 2020
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Pethybridge, S. J., and Kikkert, J. R. 2020. Identification and management of foliar diseases of table beet. Proc. of the Mid-Atlantic Fruit and Vegetable Growers Convention, Hershey, Pennsylvania. 28 January 2020. Pp. 35-37.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Goldman, I. Vegetable Breeding Institute annual meeting reports, 2020 https://plbrgen.cals.cornell.edu/research-extension/vegetable-breeding-institute/
  • Type: Journal Articles Status: Under Review Year Published: 2021 Citation: Tymon, L.S., Morgan, P., Gundersen, B., Inglis, D.A., and Bull, C.T. Potential of Pseudomonas syringae genomospecies 1 phylogroup 2b to infect pumpkin (Cucurbita pepo) fruit and seeds as a result of seed, flower, or fruit infection.
  • Type: Journal Articles Status: Under Review Year Published: 2021 Citation: Tymon, L.S., Bophela, K., Martins, S.J., Ramos-Sep�lveda, L., Inglis, D.A., Coutinho, T.A., and Bull, C.T. Fruit warts and leafspots of cucurbits caused by diverse strains within Pseudomonas syringae pv. aptata.