Progress 09/01/22 to 08/31/23
Outputs
Target Audience:Approximately 100 growers, industry stakeholders, plant breeders, regional extension personnel, students, private industry crop scouts, master gardeners, students, and other scientific peers or collaborators we reached through numerous presentations throughout the US (Florida, New York, North Carolina, Washington, Wisconsin and regionally, at international venues and through the "Seed Pathology Fundamentals" course sponsored financially by this project. Project personnel interacted with industry stakeholders through advisory panel meetings and through surveys conducted in commercial processing fields to quantify the prevalence, incidence and severity of bacterial leaf spot, facilitated by access and information provided by growers and industry stakeholders. Growers, extension personnel and advisory panel members visited trials in some locations. 30 growers were directly reached through visits to individual farms. Specific Grower Organizations served Vegetable Breeding Institute National Association of Plant Breeders Beet Resources Conference Western Washington Seed Workshop Western Washington Small Seed Advisory Committee (WWSSAC) WSU Master Gardener 50th Anniversary Event K-12 and Higher Education Outreach. Additional outreach efforts to students exhbited table beet and Swiss chard plants with bacterial leaf spot and explained the economic importance of Pseudomonas syringae pv. aptata Mount Vernon High School Science Night WSU Graduate student Peer-to-Peer Field Day "Plants get sick too" outreach event for "A scientist in every Florida school" program PSU College of Agricultural Sciences Undergraduate Research Recruiting Night Juntos Academy K- 12 program Cases studies and information taught in graduate level courses PP501 Biology of Plant Pathogens NCSU and PPEM 505 Penn State and mixed graduate/undergraduate course PLPPM 5020 at Cornell University. Seminars in several academic departments throughout the US including: University of Maryland, Professional meetings - posters were presented at the following scientific meetings: APS Southern Division Meeting, Durham, North Carolina International Conference of Plant Pathology 2023 in Lyon France Plant Health 2023, Denver Colorado National Association of Plant Breeders Changes/Problems:Over the course of the project graduate students and postdocs important to the success of the project left the project prematurely due to issues related to COVID and stress of international research. This has delayed several aspects of the project. We are requesting for an additional year extension for the grant that should be sufficient for completion of the project. We have had significant difficulty isolating bacteriophage that lyse the pathogen targets in this project. Preliminary data in the Huerta Lab indicate that P. syringae pathogens are resistant to phage infection. To better understand the molecular mechanisms that contribute to P. syringae pathogen resistance to bacteriophage he will conduct a comparative genomic analysis of hundreds of P. syringae strains sequenced through this grant by collaborators to conduct a genomic analysis of molecular bacterial mechanisms of resistance to phage infection. Furthermore, he will be responsible for performing a comparative genomics analysis on these same genome sequences to predict prophage activity in P. syringae strains. For field trials of IPM practices in Florida, later than usual frosts and severe and uncontrollable outbreaks of Alternaria fungus in test plots, resulted in only two weeks of usable disease severity data being collected. From the limited data collected, no trends in disease control efficacy were able to be observed. Our ability to analyze the cost-effectiveness of existing interventions is limited due to the confidential nature of the data collected by the industry. The researchers on this team will participate in discussions to facilitate the analysis this year. Administrative staff and faculty member managing the website are no longer working on the project. We have been delayed in updating the website. There is a plan for all PSU participants to be involved in the update. What opportunities for training and professional development has the project provided?Please see objective 6 accomplishments as that objective is a professional development objective. Information about this is split between this section and that. Many student and postdoc members of the team and some PIs attended the course sponsored financially by our project titled "Seed Pathology Fundamentals". Sponsorship by this project has enabled a reduced registration fee to be offered to attendees from developing nations. Of the 421 registrants, 128 reside (30%) in developing nations in addition to our team members. This course is the first step in meeting the seed health training needs of the industry. Postdoctoral Mentorship: Postdocs Gabriel Rennberger and Raymond Garcia Rodriquez prepared and published manuscripts (Rennberger et al., 2022) with mentorship from their PIs, Wechter and Bull, respectively. Both were offered prestigious jobs with Beyer Crop Science. Drs. Prasanna Joglekar, Mozhde Hamidizade, and Manish Ranjan were recruited, onboarded, and mentored to prioritize and accomplish objectives for this grant. Graduate Student Mentorship: At the request of graduate students from this project and beyond, Bull and Rioux developed the webinar 'Mentoring Relationships' https://www.apsnet.org/edcenter/resources/Webinars/Pages/Mentoring-Relationships-Video.aspx Audrey (Kruse) Morrison was chosen to attended the American Seed Trade Association Leadership Summit- chosen to attend ASTA's Leadership summit in Sacremento, CA from June 10-15, 2023. Audrey was assigned a mentor for the program. The Leadership summit focused on resilience in agriculture, a business, and in personal life. Speakers were brought in, and there were of attendee facilitated discussions throughout the week. There was a graduate student track as well, where graduate students could hear from young professionals about the transition from academia to industry. NAPB Borlaug Scholar- Audrey (Kruse) Morrison was Chosen as a Borlaug Scholar Class of 2023. Was assigned a year-long mentor. Attended the annual meeting for NAPB (National Association of Plant Breeders) in Greenville, SC, from July 16-21, 2023. Attended farm operation tours, Clemson facility and breeding site tours, and listened to several days of speakers and panelists. Participated in the poster presentations. Mohammad Afiq Hafiy Mohammad Taufiq, Plant Pathology, North Carolina State University, Raleigh, NC, 2022-2023- Graduated and is now a technician at the Food Venture Center in Geneva NY. Students, PIs, and industry partners met in Denver, CO for dinner and discussion about the projects during the APS meeting. Rachel Herschlag was mentored to write a proposal for her dissertation based on research partially funded by this grant. Additionally, she received grant writing training and submitted a competitive grant program Penn State College of Ag. Sci Competitive Grant. She is also receiving Microbiome Science training through the Penn State One Health Microbiome Center. Samuel Osabutey joined PSU as a PhD student in July. He went through onboarding training and mentorship for priortizing research from this grant. A Cornell University graduate student was trained in bacterial isolations and conducting a small plot replicated trial, disease evaluation, statistical analysis, and yield assessments. Graduate and undergraduate students of the course, PLPPM 5020 (Systems Epidemiology for Plant Pathology) also participated in a table beet foliar disease case study, featuring bacterial leaf spot. In this course, students were exposed to concepts surrounding risk management, decisions facing growers on whether to intervene with disease control tactics, inoculum sources, and epidemiology. How have the results been disseminated to communities of interest?Results have been disseminated to stakeholders, colleagues, and K-12-postdoc groups through scientific and grower presentations or posters described in the Target Audience, Products, Other Products, and Accomplishments sections of the report. Here we provide more information about some of these events or products because of their added value and space limitations. Importantly, results were disseminated to communities of interest through one-on-one visits to farmers, field days, and interactions with extension agents and crop scouts primarily in Florida, New York, Wisconsin Washington. In addition to the project website, factsheets are available for these diseases and their management. Several major accomplishments need to be described in detail due to limited space in the accomplishments section of the report. Most of these directly contribute to objective 6. First, the CC-SCRI team sponsored financially an online course titled "Seed Pathology Fundamentals", developed by a member of the CC_SCRI team (Lindsey du Toit) and a member of the advisory team (Gerbert Hiddink from Enza Zaden), in collaboration with the American Phytopathological Society and the American Seed Trade Association. The course is being offered online in fall 2023 (Sep. 19 to Dec. 5, every Tuesday for 75 minutes). After 421 people from ~40 countries in 6 continents had registered for the online course (Fig. 9), registration was closed. There is a waiting list for access to the recordings of the 12-session course that will be made available in spring 2024. Sponsorship by the CC-SCRI project has enabled a reduced registration fee to be offered to attendees from developing nations. Of the 421 registrants, 128 reside (30%) in developing nations. This course is the first step in meeting the seed health training needs of the industry. Additionally, Drs. Huerta provide hands on research opportunities to students that do not traditionally have access to internships, workshops, or learning modules that focus on the science behind agriculture. In the lab we endeavor to expose youth from the LatinX community in North Carolina and beyond to expose them to the fact that plant get sick and that we need talent to help find solutions to the complex problems that arise from bacterial plant pathogens. They accomplish this through hand-on workshops, learning modules, and seminars. They reach our target populations through collaboration and support of Juntos https://juntos.dasa.ncsu.edu/ director Diana Urieta. Dr. du Toit co-organized and moderated a special session on The Potential of Seed Microbiomes at the 2023 International Congress of Plant Pathology in Lyon, France on 20-25 August 2023. The session was attended by >150 people (standing room only, so a second room was opened up with online access to the session). What do you plan to do during the next reporting period to accomplish the goals?Develop diagnostic methods for detection and quantification of the pathogens in seed and from environmental inoculum sources. Will publish proof of concept paper with phage-based semi selective medium. Will evaluate phage-based media for increased selection of P. syringae pv. aptata from beet and chard seed. We are currently testing additional methods for increasing selectivity of available semi-selective media including the use of the prospector with semi-selective media and will add phage-based methods. Genomic analysis of P. syringae strains sequenced through this grant will be used to describe the presence and absence of prophage in genomes and evaluate resistance to phage infection. We will target phage isolation based on the findings of these results. Additional phage may be used for management or detection depending on the specificity and spectrum of kill of the phage. Will modify and test molecular detection methods described in previous reports or previously published work (phylogroup 2b qPCR) and develop detection methods based on comparative genomic analyses (hrp and other identified pathogen markers). This is an alternative to the phage-based methods described above. These methods should allow for faster identification of pathogen infested seed lots. Describing the complete diversity of pathogens that can cause bacterial diseases on chenopods and cucurbits is important for detection and management. We will complete identification of novel pathogens and describe changes in the organisms causing these diseases by completing the following tasks. We will submit a paper describing the diversity of pathogenic and non-pathogenic strains from beet and chard seeds. This is a priority. Additionally, a publication describing P. syringae diversity in beet and seed production fields in the Northwest is expected. Further characterization of new species with MALDI-TOF MS will be completed. Two publications describing new species will be completed and the paper currently under review by Plant Disease will be published. Completion of publication describing similarities and differences of new species P. alabamensis isolated from cantaloupe and an isolate of the same species from a cow. We will finish the characterization of strains collected from 2023 watermelon BLS outbreak. These and other isolates will be analyzed in context of previous outbreaks by submitting a publication describing genetic differences between P. syringae isolates collected from 2020-2022 and those collected from previous BLS outbreaks in Florida (2013-2014). Bacteriocin interactions will be explored and investigated for impacts on seed microbiomes and diversity. Develop novel IPM practices for crop production and seed production to reduce seed contamination/infection and disease Will submit Non-thermal Atmospheric Plasma for the Inactivation of Pseudomonas syringae pv. aptata on Beet (Beta vulgaris) Infected Seeds and Its Impact on the Seed Bacterial Microbiome for publication and work with collaborators on new prototypes. Although the machines producing Non-thermal Atmospheric Plasma are currently prototypes, this work shows promise for reducing seed pathogen populations. Nanomaterials will continue to be tested. Specifically, we will continue of in vitro testing of efficacy of metal-containing nanomaterials against P. syringae. We will repeat field experiments comparing efficacy of metal-containing nanomaterials against grower standard controls. Greenhouse testing of efficacy of metal-containing nanomaterials against P. syringae will progress. An extension article currently under review about nanomaterials will be published. A replicated field trial is planned to evaluate the yield effects of bacterial leaf spot on table beet using selected cultivars (from 2021 and 2022 results) and pesticide efficacy trial findings (2023) to develop an integrated pest management program and answer questions from growers surrounding at what stage of plant growth is BLS control worthwhile. Biological fractions in soil decrease foliar P. syringae plant pathogens. These fractions will be identified and if possible isolated using microbiome and empirical approaches. Develop seed testing protocols and treatments for quality Table beet seed crop trial in western Washington will be threshed and the seed combined, dried, cleaned, and sized. The seed will be tested to assess the amount of rif-Psa infection, and the location of rif-Psa infection on/in seed determined using component seed assays (pericarps vs. embryos) as previously described. These and previously produced seed will be used as test material for various treatments. Bleach and hot water seed treatments will be repeated with the 2023 table beet seed lot. The effect of decortication on recovery of rif-Psa will be evaluated for this seed lot, as was done for the 2021 seed lot. Myxobacteria and other biologicals isolated from soil will be evaluated as seed treatments. Improved semi-selective media will be used to evaluate isolation of pathogens from these beet seed and naturally infested seed. This will include the incorporation of phage-based media using additional phage. Additional seed treatments additional phage that are isolated after evaluation of barriers to isolation will be evaluated as seed treatments. Additionally, we will continue the development and testing of nanomaterial-containing seed coatings for control of seedborne P. syringae in cucurbits. Identify novel sources of disease resistance to CBC pathogens Analysis will be performed on the Cucumis pepo data using Genome-Wide Association Study to identify QTN associated with resistance. Resistant germplasm will be crossed with a susceptible line to generate an F1. F2 individuals from the Citrullus amarus resistant-by- susceptible population will be tested for segregation of resistance. This will provide needed information for understanding resistance to these pathogens in cucurbits. GWAS results for beet and chard resistance will be written in a manuscript. Genotype file will be refiltered (merge resequenced material into master file). Other software for GWAS results will be compared (i.e. GAPIT, GWASpoly, rMVP). Candidate gene searches will be conducted. For linkage mapping of chenopod resistance: Self F1 roots in the greenhouse this winter, harvest seed in the spring, plant out F2 seed next May in order to harvest F2 roots next summer. These data will help lead to marker assisted selection. Beet and chard interactions with strains from seed diversity studies will be evaluated for host by pathogen interactions. This will provide further information needed for resistance breeding to a broader set of pathogens described by this project. Analyze the cost-effectiveness for all practices developed. All researchers involved in disease management research will participate this year in helping to estimate costs. Provide international seed health extension, training, and mentorship Several extension materials and presentations at winter conferences are planned for 2023/24 in Washington, New York, and Florida. The international seed pathology course will be completed and established as an online at will course. We have designed a Pseudomonas taxonomy webinar which will be produced in 2024. The project website will be updated by August 2024. Two students will be mentored for their qualifying exam and one student for their comprehensive exam at PSU. One student will be mentored to write a federal grant for graduate students at PSU. One student is scheduled to graduate in Fall 2023 and two others are scheduled to graduate from WSU and UF in 2024.
Impacts
What was accomplished under these goals?
Objective 1 BLS symptoms were not observed and Psa was not recovered from any of nine table beet and three Swiss chard seed crops surveyed in 2023 in Skagit and Snohomish Counties of WA. - In NY, 2023 was hot and dry in early spring which negatively impacted plant populations and reduced foliar disease incidence in early spring. BLS was detected in only four of 30 table beet fields sampled. -We identified nine species, P. syringae,P. alliivorans, P. capsici, P. viridiflava, P. lijiangensis, and the four new species, P. boreofloridensis, P, alabamensis, P. citrullus, and P. serbiens among isolates collected isolates from cucurbits symptomatic for bacterial leaf spot. These include with eight species were never found in association with BLS of cucurbits before and four entirely new species. -We described the diversity of pathogens from beet and chard crops and described two MLST groups that are broadly present on infested seed grown on three continents (publication in preparation). However, the diversity of P. syringae pathogens and non-pathogens is much broader than expected. -MLSA and ortholog-based core genome phylogenies have been constructed. Nonpathogenic chenopod or cucurbit strains are not restricted to specific phylogroup strains as earlier assumed. -PopCOGenT was used to create networks of horizontal gene transfer between P. syringae strains isolated from seed and other sources. - For chenopod strains, hrp genes are candidate markers to distinguish pathogenic and nonpathogenic strains from PG2. We have identified some unique effectors present in pathogenic strains of chenopods. -We identified a phage that aggressively kills, mild pathogens or non-pathogens of beet and chard and are incorporating this into semi-selective media. -We established protocol for isolating phage from soil, plant tissue, and seed. However, the data from this research indicates that there may be major barriers to phage infection. Objective 2. The BLS pathogen was not detected in irrigation water or rainwater in beet/chard seed production region of western WA in 2023. However, BLS was not detected in field plantings. After a cold, wet April, conditions turned unusually dry for western Washington the rest of the 2023 season, and were highly unfavorable for BLS. -Of ten bactericides evaluated in five chard seed crops in WA, none reduced severity of BLS symptoms or Psa infection levels of the harvested seed, except ManKocide in only one trial with moderate BLS severity. -Theia, Theia + Kocide, Curezin, and SeCurezin spray programs were identified that provided moderate control of bacterial leaf spot in table beet in NY. -Efficacy of conventional copper-based and plant-defense-activator grower standard controls (Mankocide, Kocide, Actigard) for bacterial leaf spot of cucurbits (BLS) were compared to novel metal-containing nanomaterials (Mg-Cu, Fixed quat. Cu, Mg-Cu DB) in a field setting. Objective 3. The location of rif-Psa infection on/in seed determined using component seed assays (pericarps vs. embryos) was assayed. -We finished evaluation of: i) thresholds for seedborne Psa that result in development of BLS in baby leaf beet and chard, ii) duration of survival of Psa in beet and chard seed, and iii) efficacy of foliar applications of bactericides for control of BLS in seed crops. The threshold for seedborne Psa, resulting in ≥5% severity of BLS, ranged from 0 to ~6 x 104 CFU/g seed, depending on environmental conditions. -Recovery of seedborne Psa in naturally infected beet and chard seed lots revealed the rate of decline in seedborne Psa ranged from -0.07 to -0.19 (log10CFU)/g seed/month in storage. Therefore, storing seed for 12 to 24 months may be a viable option to reduce Psa to negligible levels prior to selling the seed. -Population levels of Psa rapidly declined in natural but not in autoclaved soils. The biological fractions responsible for this decline will be identified and if possible isolated using microbiome and empirical approaches with the hopes of using these as seed treatments. -A paper titled 'Non-thermal Atmospheric Plasma for the Inactivation of Pseudomonas syringae pv. aptata on Beet (Beta vulgaris) Infected Seeds and Its Impact on the Seed Bacterial Microbiome' was prepared for publication. Objective 4. A second year BLS disease screens on Beta vulgaris diversity panel were completed. We have identified beet and chard accessions that exhibit ranges of susceptibility or tolerance to BLS. This data is being analyzed for a manuscript. A significant QTL was found on chromosome 2 associated with resistance to BLS. This QTL explains 10.1% of the variation of this trait. The most significant SNP has recessive property. When the allele is present in two copies (homozygous recessive), it confers a lesser degree of amount of diseased leaf area. Candidate gene search is underway and crosses of accessions used in this diversity panel to create mapping populations is underway. -The host range of pathogen causing BLS on cucurbits and chenopods are being assessed. Whereas breeding or screening for resistance to BLS in beet and chard have been conducted with a single genotype, we are evaluating multiple bacterial haplotypes on the most resistant and susceptible beet and chard germplasm. We also summarized host range data available for pathotypes of Pseudomonas syringae pathovars in Pseudomonas syringae sensu stricto (genomospecies 1 phylogroup 2) for a manuscript. Green fluorescent protein-tagged strains of Pseudomonas syringae (GFP-Psa) were used to examine micro-scale colonization of table beet and Swiss chard foliage to determine whether there are differences that could be attributed to the differences in disease severity seen with two strains of the pathogen. The results indicate that the differences in severity or host range is not a result of restriction of Pap014 colonization to the leaf surface, but may be associated with ability of Pap009 to increase to greater populations on beet and chard leaves compared to Pap014. -Three hundred plus accessions of Cucumis pepo from the USDA germplasm collection were screened for reaction to inoculation with a pathogenic isolate of Pseudomonas syringae. Data has been collected and are awaiting GWAS analysis. Resistant Citrullus amarus lines (determined in last years studies) have been crossed to susceptible C. amarus lines to generate an F1. The F1s are being selfed to generate F2 individuals. Objective 5 Research on IPM in objective 2 is coming to fruition and will be used for completing this objective in the coming year. Objective 6 One major objective was to develop a course on seed pathology for which 461 people registered. This objective was achieved through collaboration with a number of different organizations and is described in detail in 'How have results been disseminated section of the report. -Collaboration team meetings (a minimum of 5 a year) continue to be an excellent source of tacit mentorship across labs. Industry members actively participate and PIs and other members of the SCRI team meet with seed companies to share our latest work. -Lindsey du Toit attended the 1-day Seed Health Workshop preceding the 2023 International Congress of Plant Pathology in Lyon, France on 19 August 2023. -Sarah Pethybridge visited 10 growers (of the 35 fields) and discussed individual disease diagnosis to differentiate BLS symptoms from other late-season foliar diseases. -Lindsey du Toit co-organized and moderated a special session on The Potential of Seed Microbiomes at the 2023 International Congress of Plant Pathology in Lyon, France on 20-25 August 2023. -WSU collaborators are using the chenopod BLS pathosystem to mentor a high school student, Ryan McCloud, at Franklin Pierce HighSchool in Tacoma, WA in 2023 with a science project.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2023
Citation:
Nampijja, M., Jacobs, J., and du Toit, L. 2023. Host colonization by strains of Pseudomonas syringae pv. aptata with differential pathogenicity on table beet and Swiss chard. Poster presented at Plant Health 2023, Annual Meeting of the American Phytopathological Society, 12-16 August 2023, Denver, CO. Phytopathology 113: accepted.
- Type:
Other
Status:
Published
Year Published:
2023
Citation:
News article about research: https://www.apsnet.org/members/community/phytopathology-news/2023/July/Pages/default.aspx
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Fullem et al., 2023 Unexpected diversity of pseudomonads associated with bacterial leaf spot of cucurbits in the southeastern United States https://doi.org/10.1094/PDIS-06-23-1081-SR
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Bushong et al., 2023. Characterization of nine Pseudomonas species associated with foliar disease of cucurbits. https://www.apsnet.org/members/community/divisions/south/Pages/MeetingHistory.aspx
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Bushong et al., 2023. Identification Of Four Novel Species Of Pseudomonas
Associated With Bacterial Leaf Spot Of Cucurbits https://www.icpp2023.org/programme/abstracts-book
- Type:
Websites
Status:
Published
Year Published:
2023
Citation:
Pethybridge and Kikkert 2023. Bacterial leaf spot of table beet. Cornell Vegetables Factsheet. Updated 2023.
factsheet template (cpb-us-e1.wpmucdn.com)
- Type:
Book Chapters
Status:
Awaiting Publication
Year Published:
2023
Citation:
Pethybridge, S. J., and Harveson, R. M. 2023. Diseases of table beet. Handbook of Vegetables and Herb Diseases. Elsevier Publishing.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2023
Citation:
Pethybridge, S. J., Murphy, S. P., and Kikkert, J. R. 2023. Efficacy of pesticides for bacterial leaf spot control in table beet, 2023. Plant Dis. Manage. Rep. Submitted.
- Type:
Book Chapters
Status:
Submitted
Year Published:
2024
Citation:
Pethybridge, S. J. 2024. Cornell Integrated Crop and Pest Management Guidelines for Commercial Vegetable Production.
Chapter 14 Beets. Updates
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Kruse Morrison, A., Goldman, I. L, 2023.QTL on Chromosome 2 Associated with Bacterial Leaf Spot Resistance in Beta Vulgaris. National Association of Plant Breeders Annual Meeting, Greenville, SC
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Rennberger, G., Branham, S.E., Wechter, W.P. 2023. Genome-wide association study of resistance to Pseudomonas syringae in the USDA collection of Citrullus amarus. Plant Disease https://doi.org/10.1094/PDIS-04-23-0795-RE
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2023
Citation:
Crane, 2023. Seed transmission of Pseudomonas syringae pv. aptata, and efficacy of bactericides for control of the pathogen in beet and Swiss chard seed production. Webinar of thesis defense. https://wsu.zoom.us/rec/share/mBcOUem4alh_RpXVf3l47kd4tfctCYfsEGzx6nbU_YvlpGXuHa2894oPTkv0Igdo.64TAp-iaRRxTI0Fh
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Progress 09/01/21 to 08/31/22
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. Over 1600 public and private vegetable, fruit, and seed growers, allied ag and seed industry scientists, and public scientists in the US and abroad received information from this project in this year. Additionally, our team from North Carolina provided significant outreach to approximately 180 domestic, URM, and international students. Vegetable and fruit growers and professionals across the US Florida, approximate numbers reached 595: 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 Vegetable growers New York, Approximate number of people reached 200: table beet root growers and industry stakeholders, regional extension personnel, and other scientific peers and collaborators Vegetable growers seed companies Private industry crop scouts. New York Table Beet Research and Development Committee, Cornell extension advisory group Cornell Extension Educators North Carolina, Approximate number of people reached 180: Juntos Acadamy, University of Sao Paulo Brazil, NCSU, Dominican University IL, and Doane University NE. Washington, Approximate number of people reached: 169 Table beet and Swiss chard seed crop and vegetative growers, seed industry members, consultants, extension educators, State legislators and aides, and regulatory agency staff (EPA, WSDA, L&I, DOE). Western Washington Small Seed Advisory Committee Western Washington Seed Workshop Puget Sound Seed Growers' Association (PSSGA) Western Washington Small Seed Advisory Committee WSU Filed Plant Pathology course participants (Graduate Students) Wisconsin - Approximate number of people reached 350: Private and public vegetable growers and breeders. Vegetable growers Plant breeders Vegetable pathologists and horticulturists Molecular biologists and geneticists Vegetable industry personnel Seed producers, farmers Seed industry personnel Midwest Food Products Association Vegetable Breeding Institute National Association of Plant Breeders World Beta Network Various scientists at national and international Conferences and Symposia American Phytopathological Society American Society for Horticultural Science International Congress of Plant Pathogenic Bacteria, Assisi, Italy International conference on smart plant protection, organized by Muhammad Nawaz Shareef University of Agriculture, Pakistan University of Guelph, Canada University of Tuscia, Italy Grant Team Taking the Seed Health Dogma for a Walk - Webinar 10/14/2021. developed by postdocs and graduate students in collaboration with seed industry scientists. 75 growers, seed industry, allied ag industry, students, government, and academics attended. APS Special Session - Unraveling and Tackling Bacterial Seedborne Pathogens. Organizer and Moderator - Raymond O. García-Rodríguez and Rachel Herschlag 40+ Plant pathologists attending. Plant Health 2022 meeting of the American Phytopathological Society. Industry representative from our team presented with other experts. Changes/Problems:Objectives 2.1: "Identification of primary sources of inoculum" and 4.3: "Evaluating potential differences in resistance needed for food and seed crops" were not addressed during this review period. Members of our team will meet to discuss how to ensure these objectives are addressed in the future review periods. We are finding some very interesting reactions to infection when studied over a period of 6-8 weeks. Not much disease spread is noted after inoculation, suggesting that this bacteria does not spread readily in our assay, although symptoms are quite strong during the week following inoculation. We are assessing whether AUDPC is the best way to characterize disease reaction. There was a slight delay in progress with the WSU Chenopodiaceae component of the project when PhD student, took maternity leave in spring 2022. However, she has been making very good progress since returning. Portions of the project delayed significantly due to the impact of the global pandemic. This has significantly impacted accomplishments in objective 1, 2, 3, and 4. These included delays in the hiring and recruiting of postdocs, graduate students, and undergradaute students. This also included significant delays due to supply chain issues. We will need a two year no cost extension in order to complete all aspects of the project. What opportunities for training and professional development has the project provided? Students, postdocs, and PIs attended the ISTA Seed Health Testing Training (Online Workshop, February 2022) Trainees received significant mentorship in developing two programs that extended the results of this work. They worked with faculty from other institutions and with industry scientists to produce: 1) the APS Special Session - Unraveling and Tackling Bacterial Seedborne Pathogens (Moderators - Raymond O. García-Rodríguez and Rachel Herschlag); and 2) Taking the Seed Health Dogma for a Walk - Webinar 10/14/2021. Two Postdocs and an MS student took positions as scientists in industry. 10 undergraduates, including URM students from McNair and other programs, have been trained in our laboratories. They received training in lab safety, aseptic techniques, plant husbandry, and basic plant pathology skills. Besides the general training, each one received specific training based on their research interests or undergraduate majors and one is now independently working on a research project that will helps understand better the pathogenic diversity of P. syringae sensu lato. Two students trained were part of the Agroecology Scholars Program in Research and Extension (ASPIRE). Two postdocs participated as trainers in the Penn State Data Analysis Working Group (DAWG), for the Penn State Microbiome Center. One postdoc was a leader in the Penn State Interdisciplinary Initiative on Plasma Agriculture. One postdoc attended the InternationalPhytobiomes Conference 2022 and associated metagenomics workshop. 7 trainees (postdocs, graduate and undergraduate students) attended the Plant Health 2022 American Phytopathological Society annual meeting. Contributed to the knowledge, networking, and skills. They attended a networking dinner with industry. Marilen Nampijja traveled to Jonathan Jacobs' lab at the Ohio State University in Dec. 2021 to learn how to work with a confocal microscope to monitor colonization of beet and chard plants inoculated with GFP-tagged isolates of Ps. One MS student graduated, and one finished their research and is working to finish their thesis. Two students passed their qualifying exams. Michelle Pena received specific training from Potnis on developing computational pipelines for genome analyses. Potnis has been mentoring her designing approaches for computational analyses and manuscript writing. Sarah Pethybridge visited 10 growers (of the 35 fields) and discussed individual disease diagnosis to differentiate BLS symptoms from other late-season foliar diseases. How have the results been disseminated to communities of interest?Drs. Huerta and D'Amico-Willman have worked together to provide hand on research opportunities to students that do not traditionally have access to internships, workshops, or learning modules that focus on the science behind agriculture. In the lab we endeavor to expose youth from the LatinX community in North Carolina and beyond to expose them to the fact that plant get sick and that we need talent to help find solutions to the complex problems that arise from bacterial plant pathogens. We do this through hand-on workshops, learning modules, and seminars. We would not be able to reach our target populations without the support of Juntos https://juntos.dasa.ncsu.edu/ director Diana Urieta. Results were disseminated to communities of interest through the publication of a factsheet to facilitate BLS identification, modification of the Cornell Vegetable Production Guidelines, and one-on-one visits to farmers and interactions with extension agents and crop scouts. The factsheet is freely available for download from the Cornell Vegetables website. Results of the BLS trials at WSU were shared at the Western Washington Seed Workshop on 14 January 2022; with the Western Washington Small Seed Association (seed company field reps) on 1 June 2022; and with state legislators and aides, federal agency staff (EPA, DOE, L&I, USDA APHIS, etc.), and ag industry people in the Washington Pest Control Tour on 26 July 2022. Refer to the list of events under the 'target audience' section above. What do you plan to do during the next reporting period to accomplish the goals? Isolate and characteirze phage, conduct host range experiments with Penn State bacterial isolates (visit Penn State), present our results in upcoming meeting, identify a phage and develop a protocol to engineer phage with a florescence reporter to detect and potentially quantify bacterial populations at low number in seed. Two manuscripts are currently being written: one describing diversity of pseudomonads associated with beet and chard seeds and fresh market crop and computational analyses to inform putative markers for diagnostics. And another one with similar focus on cucurbit Pseudomonas spp. Genome analyses will help identify candidate primers indicative for host specificity, which could be further evaluated by constructing mutants in multiple strain backgrounds to confirm contribution of the candidates in host specificity. We will write a publication describing the diversity of pathogens found on beet and chard seed. Use alternatives to phylogenetic analyses to determine bacterial populations. Write publication describing impact of plasma on seed pathogens. Continue collecting and characterizing strains from bacterial leaf spot outbreaks on cucurbits in the southeastern US Sequence and perform genetic analysis on recently acquired strains Write a publication describing strain characterization work and new species discovered Write a publication exploring genetic differences between recently collected strains with P. syringae strains recovered from Florida and Georgia in 2013-14. Continue evaluating the in vitro effects of metal-containing nanomaterials on P. syringae Test efficacy of nanomaterial seed coatings to reduce or eliminate P. syringae populations in cucurbit seed In 2022, when diseased fields are identified we will do comprehensive sampling within the field of foliage, soil, seed and water. This was hindered by low BLS prevalence and incidence this year. This will be completed in 2023. Objective 2.5/3.1. A replicated field trial is planned to evaluate the efficacy of foliar sprays for BLS control in table beet root crops. Test recovery of P. syringae strains from inoculated seeds. A replicated field trial is planned to evaluate the efficacy of foliar sprays for BLS control in table beet root crops. We will be obtaining GBS data on 230 accessions that have been tested once in our screen, and these data will be used to map QTL for resistance to BLS. The diversity panel of 230 accessions will be tested again in the summer of 2023, and these two phenotypic datasets will be used to analyze the SNP data for QTL. During the next reporting period we will use the analyses of the watermelon data as guidance and perform GWAS analyses of the data collected from the screening tests with the collection of C. pepo. We will write a manuscript about that part of the project and submit the manuscript for publication. In addition, we will develop KASP markers for the SNPs found to be significantly associated with resistance in both cucurbit species and validate the markers on a validation population. Participate in discussions to facilitate the analyses of this objective for table beet. Present research at Cucurbitaceae 2022 conference Finish and publish UF EDIS extension publication on bacterial leaf spot of cucurbits Continuation of the online seed pathology student course utilizing multi-faculty support and APS support. Continuation of seed pathology webinar series, post doc and graduate student mentorship, and continual website upgrades to support current and newly published data.
Impacts
What was accomplished under these goals?
We described the diversity of BLS pathogens from beet and chard seed lots world-wide. >1600 were considered. Preliminary ortholog-based phylogenies and MLSA of 336 sequenced strains demonstrated that the strains comprised 78 MLSTs. Seed lots contained strains from multiple MLSTs. MLSTs 1 and 3 had the highest number of strains. Strains within MLST1 were non-fluorescent, MLST3 strains were fluorescent, and both were highly virulent on either, beet or chard. The remaining 76 MLSTs were more pathogenically diverse with strain ranging from non-pathogenic to highly virulent. All P. syringae sensu stricto pathogens were consistant for LOPAT (Levan: Positive, Oxidase: Negative, Potato Soft Rot: Negative, Arginine Dihydrolase: Negative, Tobacco HR: Positive). Non-pathogenic members of P. syringae sensu stricto were also isolated from beet and chard seed. Whole-genome analyzes revealed diversity of Pseudomonas spp. isolated from beet and chard includes typically opportunistic species beyond P. syringae sensu stricto co-colonizing with pathogenic strains. Whole genome sequences of 50 strains from bacterial leaf spot of cucurbits were characterized and whole genomes were sequenced. Analyses of cucurbit pathogens revealed previously unknown diversity in Pseudomonas strains associated with bacterial leaf spot of cucurbits. Three new species of Pseudomonas may have been discovered. Field outbreaks observed on cucurbits in the past two years are caused by P. syringae sensu stricto and also by P. cichorii and P. viridiflava like strains. Three manuscripts describing pathogen diversity are in progress. Targets have been identified for pathogen specific primers using an empirical approach. We established three protocols for isolating phage from soil, plant tissue, and seed. Processed a total of 66 samples (seed, plant tissue, and seed) - 43 samples (seed and plant tissue) led to zones of lysis zones on two target strains. A green fluorescent protein (GFP)-tagged strain of Pap014, that is pathogenic on table beet but not Swiss chard, was generated through a collaboration at Ohio State University. Colonization of table beet cotyledons by GFP-Pap014 was assessed in a preliminary experiment using a confocal microscopy. ?Based on these preliminary results, P. syringae colonizes the intercellular spaces of beet cotyledons in close proximity to stomata. Two table beet seed crops were generated from vernalized stecklings inoculated with a rifampicin resistant variant of Pap010. Weather patterns were very different in 2021 and 2022. Pathogen colonization crops and development of BLS were assessed during two seasons. Warm and dry conditions (e.g., most of the 2021 season) do not favor the pathogen, and very few BLS symptoms were observed in the 2021 field trial. Rif-Ps was recovered from 54% of seeds evaluated. Bacteria were recovered from the pericarps but none of the embryos of the 2021 beet seed lot. Alternative weed hosts were evaluated for colonization and disease in inoculated fields. Several weeds were shown to potentially add to inoculum reservoirs and disease pressure in beet and chard crops. Extended moist and cool weather in the first half of the 2022 table beet field season enabled proliferation of rif-Ps in the seed crop, which was reflected in abundant symptoms of BLS through the season (peaking at 80% incidence), even after conditions turned dry and warm the second half of that season. A second comprehensive survey of table beet root crops in NY was conducted. Thirty-five fields were visited on one occasion (early spring). Data will be used as a basis for the economic analysis (objective 5). Disease prevalence, incidence and severity data was obtained in leaf spring in 5 organic and 30 conventional fields. The incidence of bacterial leaf spot in 2022 was very low. The table beet cropping season in 2022 was cold and wet in early spring which delayed crop planting. BLS was detected in one organic and three conventional table beet fields. Average BLS incidence in organic and conventional table beet fields was 4% and 5%, respectively. Average BLS severity in organic and conventional table beet fields was 3% and 5%, respectively. Seed coatings including metal-containing nanomaterials were synthesized and applied to two varieties of watermelon seed. Germination assays were performed to evaluate the effect of the seed coatings on germination. Nanomaterial-containing (copper-zinc, magnesium-copper, and magnesium-copper double) seed coatings were created and a protocol was developed for their optimal application to watermelon seeds. Nanomaterial-containing seed coatings were found to not affect seed germination. Protocols and antibiotic-resistant P. syringae strains and inoculation and bacterial recovery protocols were developed for future seed-coating testing with infested seed. Seedcoat topography seems to impact seed coat coverage. Seed coating has no obvious effect on seed germination; this is promising for use of nanomaterial-containing seed treatments for control of seedborne P. syringae. Three strains of myxobacteria were identified which had greater killing of pathogen than the most well studied myxobacteria. Dielectric barrier non-thermal pressure plasma (DBDP) significantly reduced the number of bacteria per gram of seeds. DBDP significantly reduced seed population levels of the pathogen on naturally infested or artificially inoculated seed as well as the levels of indigenous, culturable bacteria. We evaluated the efficacy of 1.2% NaOCl as a seed treatment for beet seed infected with the BLS pathogen. Germination was not impacted by NaOCl treatment. Bleach treatment for as little as 5 minutes eradicated the pathogen from the 2021 seed lot tested that had a low level of infection by a rif-Ps strain after the record hot, dry 2021 season in western WA. Significant differences in BLS severity between cultivars table beet cultivars were observed in both the inoculated and non-inoculated field trials conducted in New York. Of the selected cultivars, Pablo was the most susceptible. Identified resistant and susceptible beet and chard germplasm, developed a large diversity panel to assess QTL for resistance via GWAS, and started breeding for resistance in beet and chard at the University of Wisconsin. Although cultivars varied less than PIs, good BLS resistance (low disease scores) was noted for 'Touchstone Gold', 'Kestrel', 'Bull's Blood', 'Rainbow' chard, as well as PIs 222234, and NSL 28026. Accessions W451C, Red Cloud, Detroit Dark Red, and NSL 28020 were highly susceptible. Demonstrated that both the vegetative and reproductive phases of beets and chard (biennial crops) are susceptible to the pathogen. Cucurbita pepo (n = 206) was screened for leaf spot resistance. Disease phenotyping was conducted and DNA from all C. pepo accessions were submitted GBS genotyping. GWAS found a total of eight SNPs that are significantly associated with resistance to Pseudomonas leaf spot. A total of 43 candidate genes were identified near 7 of the 8 significant SNPs, of which 11 have been previously reported to play a role in defense against P. syringae in other plant species. Two resistant female parents (PI 482301and PI 482307) and two susceptible male parents (PI 296342 and PI 596668) were identified and the F2 seed of these crosses was harvested in April 2022. Factsheets are currently being developing for major cucurbit crops based on available NASS agricultural Census and survey data. Team members are meeting on a regular basis to incorporate findings into the economic analyses. Members of the research team are working with the American Phytopathological Society to develop an online seed pathology course. Webinars developed provided educational opportunities and trained postdocs and students developing the trainings with industry collaborators.
Publications
- Type:
Conference Papers and Presentations
Status:
Awaiting Publication
Year Published:
2022
Citation:
Bushong et al., 2022 Characterizing genetic diversity of Pseudomonas strains causing foliar diseases of cucurbits in Florida and Alabama. Phytopathology 112: In Press
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Nampijja, M., Derie, M.L., and du Toit, L.J. 2021. First report of bacterial leaf spot caused by Pseudomonas syringae pv. aptata on Swiss chard, Beta vulgaris subsp. vulgaris, in Arizona. Plant Dis.105:3738.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Gaulke, E., and Goldman, I.L. 2022. Screening Table Beet and Swiss Chard for Resistance to Pseudomonas syringae pathovar aptata. HortScience. https://doi.org/10.21273/HORTSCI16777-22
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2022
Citation:
Garc�a-Rodr�guez et. al. (2022). Dielectric barrier discharge plasma (DBDP) as a seed treatment against seedborne Pseudomonas syringae and modulation approach for seed bacterial communities. Changing Microbiomes Symposium. Boalsburg, PA.
- Type:
Other
Status:
Published
Year Published:
2021
Citation:
Pethybridge, S. J., Sharma, P., & Kikkert., J. R. (2021). Spot the differences on table beet leaves! Cornell VegEdge 17(12):6-7 (Factsheet). Updated in 2022.
- Type:
Other
Status:
Published
Year Published:
2022
Citation:
ethybridge, S. J., Sharma, P., and Kikkert. J. R. 2022. Bacterial leaf spot of table beet. Cornell Vegetables Factsheet. Updated 2022.factsheet template (cpb-us-e1.wpmucdn.com)
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Progress 09/01/20 to 08/31/21
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. Approximately 9,507 public and private vegetable, fruit, and seed growers, as well as professionals across the US have received information from this project in this year. Florida - Approximate number of people reached: 111 Florida Watermelon Association, Suwannee Valley Watermelon Institute, industry personnel (i.e. - seed technology and fungicide companies), cucurbit transplant producers, extension agents, and crop consultants. Bushong, K., Paret, M., Da Silva, S., Freeman, J., Jones, J. and Hochmuth, B. (2021, January 27). Bacterial and angular leaf spot diseases of watermelon and other cucurbits caused by Pseudomonas syringae: Disease cycle and comparative assessment of control options. https://www.flfwa.com/post/industry-research-article. New York - Approximate number of people reached: 240 Scientific peers, vegetable growers, industry stakeholders (Seneca Foods, Bejo, Love Beets USA), New York Vegetable Research Association, Council Processing Vegetable Group, New York Table Beet Research and Development Committee, and Cornell extension advisory group. Pennsylvania - Approximate number of people reached: 9,041 PA and mid-Atlantic Vegetable Growers García-Rodríguez, R. and Gugino, B. (2021, August 25). Bacterial Leaf Spot of Cucurbits, Beets, and Swiss Chard. https://extension.psu.edu/pennsylvania-vegetable-disease-update-august-25-2021. The extension newsletter cited above was distributed electronically through the PA Vegetable Growers Association (550 registered members), the PA Vegetable Marketing and Research Program (500 registered members), and the Penn State Extension Vegetable Gazette (2,500 registered members) listserv. It was also distributed as a hardcopy through mail to a distribution list of 3,400 stakeholders. Washington - Approximate number of people reached: 115 Western Washington Small Seed Advisory Committee and the seed company field representatives or seed growers of this committee. Western Washington Seed Workshop and Puget Sound Seed Growers' Association (PSSGA). Nampijja, M. (2021, January 8). Epidemiology and management of bacterial leaf spot caused by Pseudomonas syringae pv. aptata in table beet and Swiss chard seed production. Approximately 50 participants attended this 20-minute presentation offered in a meeting held online due to COVID-19 restrictions. Table beet and Swiss chard seed crop and vegetative growers, seed industry members, consultants, etc. in western Washington. Nampijja, M. and Crane, S. (2020, October 28). Epidemiology and integrated management of bacterial leaf spot caused by Pseudomonas syringae pv. aptata in table beet and Swiss chard seed production in the Pacific Northwest. https://mtvernon.wsu.edu/event/lunch-seminar-epidemiology-and-integrated-management-of-bacterial-leaf-spot-caused-by-pseudomonas-syringae-pv-aptata-in-table-beet-and-swiss-chard-seed-production-in-the-pacific-northwest. Approximately 40 participants, including farmers, seed companies, consultants, and extension specialists, attended the webinar cited above that was offered as part of the WSU Lunch and Learn webinar series. Wisconsin - Approximate number of people reached: Number reached not provided Private and public vegetable growers through virtual meetings held by the Midwest Food Products Association members and Vegetable Breeding Institute. Changes/Problems:COVID remained a major issue limiting progress on this entire project through June 2021. We will require two full years of No Cost Extensions to make up for the losses. One issue we experienced in 2020-2021 was that light colored leaves, such as those on yellow-rooted table beet accessions and light green leaved chard accessions, show lesions that are a bit different in color than those on red or darker backgrounds. Chards like Silverado seemed to show very little in the way of disease in our studies, but after consultation with others on the team, we realized that these leaf types were in fact susceptible but the lesions simply looked different. We have gone back to re-screen all beet and chard accessions that have light colored leaves (such as yellow petioles / green leaf types). Those studies are finishing now (September, 2021) and will be reportable soon. It was very helpful to have a consultation and collaboration with Lindsey's lab to pinpoint this issue and come to a solid conclusion for our studies. 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. Carolee Bull offered the workshop "How to write a Cover Letter and Research Statement" to all postdocs in the grant and those associated with the labs that are members of the grant. This workshop intended to help postdocs form their vision and share their strategies to achieve that vision, so that future employers can see their potential in their organization. Pratibha Sharma, a graduate student, have been working one-on-one with her mentor Sarah Pethybridge, and have been trained on bacterial leaf spot identification, pathogen isolation, field trial establishment, disease ratings, and data analysis. Sarah Pethybridge, responded to 8 requests for technical advice from crop scouts and extension agents to distinguish BLS from CLS and other foliar diseases affecting table beet in NY. Michelle Pena started working in Potnis' lab as a postdoctoral researcher on this project in February 2021 and has been working on developing computational pipelines for genome analyses. Also, she has enrolled in the Omics in Agriculture class where she is learning genome and metagenome analyses. Auston Holland, a new research associate in Potnis' lab, has been working on this project since Nov 2020. Auston has been responsible for the strain collection and conducting pathogenicity assays. He has been getting trained in molecular cloning to construct deletion mutants. Emilee Gaulke, a graduate student, has traveled to Michigan State University to talk with sugar beet researchers there and learn about pathology approaches, and has incorporated some of these ideas into her work at the University of Wisconsin. Kiersten Bushong, a graduate student, have been trained on lab, greenhouse, and field experimental set up and data analysis. Marilen Nampijja, a graduate student, attended the 2021 annual meeting of the American Phytopathological Society, in addition to completing Ph.D. coursework and presenting a webinar in October 2021 on the CC-SCRI project. Stephanie Crane, MS in Ag student, who works full-time for Sakata Seed America in Skagit Co., WA, is working on Pseudomonas syringae associated with table beet and Swiss chard seed production. Her project is funded by Sakata, but she has engaged in some outreach activities with Marilen Nampijja, a Ph.D. student supported on this grant. Meena Gurung, a graduate student, have participated on the "Kickstart workshop by Penn State Microbiome Center, 2020", an activity designed to help students build and enhance their knowledge regarding microbiome analysis from planning to sequencing, amplicon sequencing, whole genome shotgun analysis. She also participated on the "How to be your own best mentor" a workshop on professional development offered by Dr. Carolee Bull. Raymond O. García-Rodríguez, a postdoc, have trained two undergraduates and one graduate student as part of this grant. Both undergraduates were trained on lab safety, aseptic techniques, and basic plant pathology skills. Besides the general training, each one received specific training based on their research interests or undergraduate majors. For example, Cole E. Benovy, a plant science major, was trained on greenhouse vegetable production, whereas Emma Stockham, a biochemistry major, was trained on DNA extractions, conventional and rep-PCR, and bioinformatics. Additionally to the training activities, Raymond have participated on professional development activities, including the Penn State Data Analysis Working Group (DAWG), Microbiome Center, and the Penn State Interdisciplinary Initiative on Plasma Agriculture. All these have contributed to further improving his knowledge, networking, and skills in his area of expertise. How have the results been disseminated to communities of interest?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 2021, allowed us to share some of our approaches in pathogen screening research plans. Pethybridge - Results were disseminated to communities of interest through the publication of an extension article in the Cornell VegEdge newsletter, a set of factsheets to support foliar disease identification, and one-on-one visits to farmers and interactions with extension agents and crop scouts. 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. This project was also featured at the NY Vegetable Research Association and Council Meeting, and NY Table Beet Advisory Group Meetings. du Toit - Marilen Nampijja and Stephanie Crane presented a 'lunch and learn' webinar on 28 October 2020 regarding the epidemiology and integrated management of bacterial leaf spot caused by P. syringae pv. aptata in table beet and Swiss chard seed crops in the Pacific Northwest USA. Marilen also presented results of this project at the Western Washington Seed Workshop on 8 January 2021, attended by vegetable seed growers and seed company representatives. Results have also been diseminated through the project webpage. What do you plan to do during the next reporting period to accomplish the goals?Objective 1 Further testing of primers evaluated for detection and quantification will be conducted using all pathogens and nonpathogens from seed. Live and dead differentiation methods will be evaluated with chosen primers. WGS of next 250 pathogens and 50 non-pathogens, comparative genomic analysis to identify diagnostic markers. Identify additional candidates for host specificity, evaluate them by constructing mutants in multiple strain backgrounds to confirm contribution of the candidates in host specificity. Objective 2 For NY beet surveys, when diseased fields are identified we will do comprehensive sampling within the field of foliage, soil, seed and water. Continued collection of P. syringae strains from Southern cucurbit outbreaks will continue Bacterial isolates obtained from cucurbit seeds will be identified using 16S and will be evaluated for their interaction with Pseudomonas syringae pathogenic isolates in vitro and in planta. Pathogenicity and Phenotype testing of strains from beet and chard seeds will be completed. WGS of these strains will help develop strain specific markers to be employed on molecular diagnostic methods, including the use of droplet digital PCR for the quantification of the pathogen in seed and environmental samples. We will evaluate population dynamics of rifampicin resistant variants in soil and cultivars of varying resistance and determine threshold populations. The table beet seed lot harvested at the WSU Mount Vernon NWREC trial inoculated with a rif-variants will be used to examine the location of the pathogen in the table beet seed Rifampicin resistant variants will be used to monitor population dynamics on table beet and chard. We will repeat the table beet seed crop trial in 2022 with inoculation with a rif-Psa strain, and survey beet and chard seed crops for Psa in 2022. Objective 3 A replicated field trial is planned to evaluate the efficacy of foliar sprays for BLS control in table beet root crops in NY and will help meet goals of obj 2, also. Additional nanomaterials will be evaluated in vitro, in greenhouse settings, and in field trials in Florida for cucurbits and WA for beet and chard. Seed coatings containing nano materials will be tested in germination and seedling health experiments. Semispecific media will be evaluated for use in massively parallel isolation procedures. We will continue working on evaluating plasma as an alternative seed treatment for the management of seedborne P. syringae strains. For future experiments, we will consider naturally-infested seeds and determine the effect of different treatment lengths on pathogen's cell density, seed quality, and the seed bacterial community. Objective 4 Replicated glasshouse trials evaluating beet BLS cultivar resistance in 2021 will be repeated in 2022 in NY to examine the reproducibility of results. The variation in reaction to the pathogen in beet or chard cultivars suggests we will have ample opportunity to create mapping populations that are segregating for response to the pathogen, that can be used for genetic mapping studies in the coming year. We have harvested contrasting resistant and susceptible roots from field trials in 2021 and are vernalizing them now. We will be crossing resistant and susceptible parents during the January - April, 2022 greenhouse season and producing F1 seed for planting in the field in the summer. We plan to self pollinate these F1 plants to make segregating populations to grow in late 2022 and in 2023 for genetic mapping studies. The goal of these studies will be to identify genes / QTL associated with BLS reaction in beet and chard. The phenotype data from the two screening tests was used to select two resistant female parents (PI 482301and PI 482307) and two susceptible male parents (PI 296342 and PI 596668) to The C. amarus F1 seed of crosses created in 2021 will be harvested and screened. Objective 5 Data collected in objectives 2.1 and 4.1 will be used to facilitate the economic analyses planned for this objective. All members of objective teams 2 and 4 will participate in discussions to facilitate the analyses of this objective for table beet and cucurbit. Objective 6 APS special session on developing tests for bacterial diseases and trainees will present at both national and regional meetings. Taking the seed testing dogma for a walk webinar series will be initiated. Updating website will occur. Mentoring of graduate students, postdocs, junior and senior faculty will continue through objective team meetings and through laboratory efforts. Emilee Gaulke will defend her M.S. thesis by January of 2022 at University of Wisconsin. Lindsay Boyd is scheduled to graduate Spring 2022. Audrey Kruse from Purdue University will be joining the Goldman lab to take over the project as a new M.S.student.
Impacts
What was accomplished under these goals?
Two primer pairs of eight tested for amplification of BLS pathogens and pathotypes show promise for detection of pathogens. Sequenced a collection of 130 pathogenic and nonpathogenic Pseudomonas syringae strains from cucurbits, chenopods and other plant hosts. Two effector homologues were identified as potential host-specific markers for pathogens of squash or watermelon. In 2021 bacterial leaf spot was not found in any of 11 table beet seed crops surveyed in western WA during this dry season. In contrast to the survey in 2020, which was cool and wet from planting until early July, and the disease was prevalent in seed crops. Described phenotypes for 601 isolates table beet and Swiss chard seed crops, fresh market crops, and seed lots surveyed or sampled. 288 were tested for pathogenicity on beet and chard and MLSA and a subset were submitted for WGS. Between ~50 to 100% and 18 to 70% of leaf samples from 2020 and 2021 beet and Swiss chard crops, respectively, tested positive for the target pathogen. 53 and 250 isolates from 15 and 27 commercial seed lots, respectively, of beet and chard grown in France, Denmark, New Zealand, & the US, were selected for analysis. We determined fluorescens and LOPAT phentoypes and pathogenicity on beet and chard for the first 53 and these were sent for WGS. 20 strains of Pseudomonas of which 10 strains were tentatively identified as P. syringae from BLS cucurbit outbreaks 2020 and 2021. Organic fields had a higher observed BLS severity and incdence in organic vs conventional beet production fields in a comprehensive survey in 2021 in NY of 15 organic and 15 conventional fields. Identification of BLS reduced fungicide applications and improved profitability, productivity, and sustainability. No evidence of overwintering of pathogen in table beet seed crop residues in fields from infected seed crop the fall prior was observed in WA. The pathogen was recovered from symptomatic volunteer table beet seedlings. Chard seed transmission trials demonstrated that most severe disease was observed in plots with the highest level of seed infection. Data indicate that the seedborne pathogen is very readily seed transmitted. Diversity of pathogens causing warts on Baby Boo pumpkins was described. Seed tests were conducted to determine whether the symptoms observed in the fields resulted from planting infected seed lots. We determined that the symptoms observed in several vegetative table beet crops in western WA in 2020 may not have been the result of planting infected seed lots, based on the limited amount of seed available for testing. The results suggest other sources of inoculum may be important in outbreaks of bacterial leaf spot in some crops, particularly in locations with a history of beet and chard production. Rifampicin resistant variants of seven beet and chard pathogens were generated and compared to the wildtype for growth rates. A beet seed crop was inoculated with a rifampicin-resistant variant and seed harvested from the inoculated beet seed crop trial will be tested for Psa to quantify the level of rif-Psa and the location of the pathogen on seed. 180 Myxobacteria isolated from 6 agricultural fields were used in predation assays against 3 Pseudomonas syringae strains. The best predators have been identified for use in biocontrol and seed treatment assays. Enrichment techniques used did not appear to result in better predators of the pathogens. 92 bacterial isolates from watermelon seeds were obtained for biocontrol studies. None of the bactericides tested in 6 experiments has proven effective at limiting development of pathogen in beet or chard seed crops. Laboratory tests of 3 Nanomaterials against P. syringae in vitro generally gave complete control of the copper-sensitive P. syringae strain after 1 hour of incubation. All 3 materials showed promise as potential control. Some difference in efficacy between suppliers was noted with Cu-Zn, this will also be explored. 3 Nanomaterials at 1000 ppm performed as well as conventional treatments against P. syringae in watermelon in field trials. No phytotoxicity by the 3 nanomaterials were observed at the tested concentrations. A table beet seed crop planted from vernalized seedlings was inoculated with a rifampicin variant of the pathogen. Due to the dry 2021 season the populations were low and few symptoms were observed. Infested seed were planted mixed with various ratios of uninfested seed. For severity (AUDPC) and the final disease rating, symptoms were more severe the higher the ratio of infested seed planted. Negative control plots demonstrated how readily the pathogen can spread under rainy conditions and confounded the ability to determine a threshold for seedborne infection. Evaluated the use of non-thermal dielectric barrier discharge plasma for the elimination of the pathogen from artificially and naturally infested beet seed. We also evaluated the effect of different treatment lengths on seed quality and bacterial microbiome. Plasma-treated seeds had a significantly lower pathogen's cell density (CFU/gram) when compared to the non-treated control. In New York, two field trials evaluated found significant differences in BLS severity of eight table beet cultivars in both inoculated and noninoculated trials. Also, these trials served as materials for research at PSU and NCSU. Thirteen table beet cultivars, five table beet inbred lines from the University of Wisconsin-Madison table beet breeding program, seven Swiss chard cultivars, and twenty-seven table beet Plant Introductions (PIs) were evaluated in three experiments inoculated with Pseudomonas syringae BP1542. Table beet varieties including Early Wonder and Blushing Not Bashful along with two inbred lines were among the most resistant beet cultivars. For Swiss Chard accessions, Magenta Sunset showed more susceptibility than other cultivars in multiple trials. The variation in reaction to the pathogen suggests we will have ample opportunity to create mapping populations that are segregating for response to the pathogen, that can be used for genetic mapping studies. The most susceptible accessions during reproductive growth experiments were similar to those in the vegetative screen. Two tests screened the USDA PI collection for Citrullus amarus (n = 114) for resistance to BLS. Candidate genes associated with resistance to BLS are present in the C. amarus USDA germplasm collection. The phenotype data from the two screening tests was used to select two resistant female parents and two susceptible male parents to cross and develop biparental mapping populations. C. amarus phenotype data was used to perform GWAS analyses and identified that the most likely locations for the QTLs associated with disease resistance are on chromosomes 1 and 5 but further analyses are necessary to confirm and narrow down these regions. Data collected in objectives 2.1 and 4.1 are used for economic analyses for objective 5. Materials developed for the successful 'Taking the Seed Health Dogma for a Walk' seminar series will be modified for the course. Planning of Webinar series 'Taking the Seed Health Dogma for a Walk" occurred throughout the summer of 2021. This was a cocreated event by objective 3 team members that was led by a postdoc and industry member. An industry collaborator surveyed of members of this group. P. syringae was frequently seen on cucurbit and chenopod seedlots around the world. Several additional lines of research were suggested. The trainees had the opportunity to present to and interact with all PIs and industry partners during these 19 meetings.
Publications
- Type:
Book Chapters
Status:
Submitted
Year Published:
2021
Citation:
Pethybridge, S. J. 2021. Bacterial leaf spot. Chenopodiaceae Chapter in World Handbook of Vegetables. Submitted to March 2021.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Nampijja, M., Derie, M. L., and du Toit, L. J. 2021. First report of bacterial leaf spot caused by Pseudomonas syringae pv. aptata on Swiss chard, Beta vulgaris subsp. vulgaris, in Arizona. https://doi.org/10.1094/PDIS-12-20-2554-PDN
- Type:
Websites
Status:
Published
Year Published:
2021
Citation:
https://www.pseudosonseed.org
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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.
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