Progress report for LNE23-463
Project Information
To be completed for final report.
200 diversified vegetable growers will learn to recognize disorders, pests, and diseases of brassica leafy greens and 100 of these will improve their pest/disease scouting and management skills, resulting in 50 producers adopting brassica pest/disease scouting and effective management techniques, affecting 6 acres. They report 10% improvement in marketable crop yields. 300 mixed vegetable growers in the Northeast will know which experts to contact for information about optimizing arugula and brassica leafy green production. 10 Seed producers, technicians, and distributers will understand the risk of this seedborne pathogen and 5 will select resistant germplasm to market based on this work.
Problem and Justification: Consumers demand fresh high quality local greens. Brassica leafy greens are quick-growing shoulder season crops well suited to the Northeast's cool climate. Arugula (or rocket) is favored partly due to the health benefits associated with consumption [1]. Although, brassica leafy greens and specifically arugula are profitable components of mixed vegetable operations throughout the Northeast and contribute to the overall financial sustainability of these farms, the extent of arugula production and broader leafy greens in mixed vegetable operations is unknown, yet growers are reaching out to members of NEAT to help them identify and manage bacterial blight of arugula due to recent outbreaks.
Solution and Approach:
NEAT was formed to respond to grower requests for identification and management of foliar bacterial diseases of arugula. To meet the larger sustainability needs of mixed vegetable producers we conducted a preliminary grower’s survey (ongoing during the project) of factors limiting brassica leafy greens/arugula production. This project will meet growers’ needs through an iterative process of education, identification of issues, and research to meet those needs. We will use a variety of educational tools to deliver a staged learning Educational Plan.
By the end of the project, we will understand the extent of brassica leafy green production in the Northeast through surveys conducted. The distribution of key pests and issues limiting yield will be mapped through on farm scouting of growers’ fields and submissions of plants to our team for disease and pathogen identification. Growers will be able to identify major pests and diseases of arugula and other brassica leafy greens and will consult experts for management due to our extensive education program and recognition of our team as experts. By screening over 100 lines in greenhouse/growth chamber and field trials, we will identify germplasm with resistance to foliar bacterial diseases, flea beetles, and bolting. Germplasm resistance/tolerance data will be provided to seed professionals through their involvement on the Project Advisory Committee (PAC) and through presentations at seed industry specific meetings to allow for selection of these materials for breeding for elite cultivars. To disseminate project materials, we will use a combination of in-person, written, and virtual outreach approaches. Factsheets in English and Spanish along with short instructional videos, in-person field meetings, webinars will be developed to help growers develop scouting programs and identify problems in their greens, as well as strategies to mitigate disorders and pests. We will quantify our progress toward our learning objectives using audience response technology and surveys that can assist us in measuring grower knowledge gain. We will track the number of growers accessing our virtual materials and survey actively supported growers for changes in practices and financial benefits
Cooperators
- (Researcher)
- (Researcher)
- (Researcher)
- (Researcher)
Research
We have one question and one hypothesis. Question: What is the diversity of pests and pathogens of brassica leafy greens in the Northeast, namely bacterial pathogens, for which we need to screen for resistance in arugula? Hypothesis: Germplasm from Eruca sativa and Diplotaxis tenuifolia will differ for resistance and tolerance to pathogens, flea beetles, and bolting. Pathogens will be identified for growers and a table of resistant or tolerant germplasm will be developed and used by growers to choose cultivars to reduce losses due to these issues in their production systems and by seed companies to develop advanced varieties.
Farmer input: This research was initiated due to the increase in samples sent for diagnosis by growers seeking management strategies. PAC and Grower Network (28 current members) members were identified through a preliminary survey. Grower Network identified limits to sustainability and research needs were reviewed for inclusion in the project during meetings with NEAT and PAC members. Growers and seed professionals are providing replicated field trials, on farm trials, and industry standards for research conducted on university research farms. Our outreach presentations will enlarge our Grower Network. All network and PAC members will receive project updates annually and biannually, respectively, will know how to submit samples for diagnosis/identification, and will receive information about resistant/tolerant germplasm from this research. PAC members will meet with the team annually to update methods, treatments, and educational plans. PAC members will review educational materials prior to release. Other growers will receive this information through our listservs, grower association newsletters, extension newsletters and bulletins, web postings, and our presentations at meetings.
Question. What is the diversity of pests and pathogens, namely bacterial pathogens, for which we need to screen for resistance in arugula for the Northeast?
Treatments: For all tests, treatments include isolates collected or sent in from growers’ fields, reference strains of Pseudomonas, Xanthomonas, and any other bacterial pathogens related to the isolates according to 16S rDNA sequencing.
Methods: Each year, ten farm surveys of brassica leafy greens including arugula and brassica salad mixes containing tatsoi, frilly mustards, Tokyo bekana will be conducted by Cedeno, Hodgdon, Sideman, for bacterial and fungal diseases and insect pests. The percent disease incidence or insect damage, host/cultivar, location of the farm, and management conditions will be recorded. The hosts/cultivars of heathy brassica leafy green plantings will also be recorded from nearby plantings. For each field or high tunnel with bacterial diseases, five plants for each diseased brassica cultivar or isolates from five plants of each cultivar will be sent to PSU for characterization and identification of the pathogen. Additional symptomatic plants from at least five additional locations will be provided in response to a request from our grower network and the NE-NPDN. All methods will follow our standard protocols [5]. Bacteria will be isolated on both semi selective media to specifically isolate unknown bacteria and Xanthomonas campestris, or Pseudomonas cannabina pv. alisalensis, respectively [3,6-11]. All methods will follow our standard protocols [5]. Bacteria will be isolated on both semi selective media to specifically isolate unknown bacteria and Xanthomonas campestris, or Pseudomonas cannabina pv. alisalensis, respectively [3,6-11]. Fluorescent isolates will be characterized phenotypically using LOPAT and all isolates will be genotyped for identification by 16S rDNA sequencing, rep-PCR, and MLSA. Pathogenicity of the isolates will be confirmed and differences in virulence will be investigated for diverse genotype using a RCB design with at least three replicates per treatment [2,3,12].
Data Collection:
For each location, the identity and genotype of pathogens will be reported for each brassica leafy green host and arugula cultivar. These data will be reported back to the cooperating growers and be used to identify regional or host/cultivar trends. These data will be used to select germplasm and strains for testing the hypothesis. A complete description of diseases and pests of brassica leafy greens and their impact is expected. Photos taken during surveys will be used in educational materials.
Hypothesis: Germplasm of Eruca sativa and Diplotaxis tenifolia will differ for resistance and tolerance to pathogens, flea beetles, and bolting.
Treatments: In growth chamber/greenhouse trials, 100 cultivars and germplasm lines will be screened for resistance to Xanthomonas campestris, Pseudomonas cannabina pv. alisalensis, and other bacterial pathogens identified in surveys. In field trials (year 2 and 3), 20 lines including those showing resistance to bacterial pathogens in growth chamber/greenhouse trials and those resistant or tolerant to bolting or flea beetles, will be inoculated with the dominant pathogen(s). Uninoculated cultivars will be treatments to evaluate bolting and flea beetle damage each year.
Methods: The following experiments will be conducted at PSU and UNH. Growth chamber/greenhouse trials (controlled conditions) – Seeds planted in propagation trays will be grown for 3 weeks in a standard soil mix before bacterial inoculation using standard methods [6-8,11-13]. In addition to disease incidence and severity, the date of flowering for each plant will be recorded [14]. Each cultivar or germplasm line will have 3 replicates and each replicate will consist of 6 plants and be arranged in a RCB design. Field trials – For year one, 20 commercially available Eruca sativa and Diplotaxis tenifolia cultivars will be evaluated for flea beetle and bolting resistance or tolerance. At least four replicates of each cultivar will be planted in two passes with a Jang five row planter on 40-inch beds and each plot will be 10 feet long. All field trials will be established as RCB designs. In year two and three, we will repeat the trial from the first year and add germplasm lines that were found to be resistant or tolerant to bacterial disease under controlled conditions. Additionally, we will plant two independent plots (inoculated with two different pathogens) to evaluate disease resistant cultivars identified under controlled conditions for resistance to Xanthomonas campestris and Pseudomonas cannabina pv. alisalensis. Plants at the four-leaf stage will be inoculated in the late afternoon. Plants will be kept moist with irrigation for 48 hrs following inoculation and will be reinoculated if needed.
Each year an on-farm field trials on one commercial farm in New York will be designed in accordance with the host-farmer's field layout, with side-by-side plantings of each 4-5 common cultivars selected by the PAC using grower production practices. Grower practices will be followed for repeating seedings of these trial varieties over the course of two seasons. Farms will apply nutrients based on UVM recommendations after soil testing. Flea beetle damage and bolting will be evaluated, and samples will be sent to the lab for pathogen analysis. Farm harvest records from the trial plots will be kept comparing yield, quality, and customer preference between varieties. In year two, the varieties used in the on-farm trials may vary based on farmer preference and input from the Grower Network.
Data collection and analysis: For growth chamber/greenhouse studies, diseases incidence will be assessed as the percent of the total number of plants that show typical symptoms and disease severity will be assessed via a modified Horsfall-Barratt scale at 6-, 10-, and 12-days post inoculation and with the Plantdoctor phone app at 12 days post inoculation. Statistical analysis of disease severity and incidence calculated as area under the disease progress curve will follow previously published procedures [15,16]. Average days to bolting will be analyzed by ANOVA and Tukey’s test. Additionally, the hypersensitive reaction will be assessed visually at 30 and 48 hours. For field experiments, Disease incidence, severity, flowering, and flea beetle damage will be assessed from two 4x4 subplots internal to each plot weekly for at least 6 weeks or until all germplasm lines have initiated flowering. Data analysis for disease and bolting will be similar to analyses conducted for experiments under controlled conditions. Additionally, non-parametric analyses similar to disease progress will be used to analyze progress of flea beetle damage.
Three different research goals were initiated this year: 1) Identification of arugula and brassica leafy green bacterial pathogens; 2) Field germplasm evaluation; 3) Greenhouse germplasm evaluations.
1) Identification of arugula and brassica leafy green bacterial pathogens. Bacterial disease outbreaks were recognized through NEAT scouting & collection efforts, collections made by an extension personnel, and growers contacting us to scout their farms. Plants were transported back to our lab in a cooler. In the 2023 growing season, we collected isolates from 62 samples from 5 Pennsylvania arugula growing locations and none from other states. From each plant sample, bacteria were isolated using published methods using KBBC, a semi-selective medium for Pseudomonas and general nutrient agar media. Pure cultures were stored and they were used in hypersensitivity test on tobacco to determine if isolates are likely to be plant pathogens. Organisms inducing an HR in 24-48 hours were stored. Once the bacteria are identified, we will conduct pathogenicity tests on the plants from which they were isolated (arugula or other brassica leafy green). Of these 62 samples, 13 plant pathogenic bacteria were isolated, 6 of which have been successfully genotyped using multi locus sequence analysis. Six Pseudomonas allivorans isolates or a closely related member of the P. syringae species complex were identified. We are in the process of analyzing the data for the other seven isolates.
To improve our detection methods, we have been working with a new collaborator (Dr. Hehe Wang) to verify a rapid detection method for Pseudomonas cannabina pv. alisalensis, the causal agent of bacterial blight in brassica leafy greens. Specifically, we identified this pathogen and related pathogens in collections by MLSA and rep-PCR after a real-time PCR assay determined whether they were P. cannabina pv. alisalensis or not in a rapid test.
2) Field germplasm evaluation. The primary goal of the fall 2023 field season was to determine nutrient management, irrigation techniques, and pest management strategies for our farming situation where we had not previously grown brassica leafy greens. We were able to successfully grow commercial arugula cultivars under the parameter recommended to NEAT by our PAC & cooperator Bill Boone. We successfully grew 10 cultivars in randomized plots which we did preliminary evaluations on for bolting and flea beetle damage, and scouted regularly for bacterial diseases. The cultivars used included: five Eruca sativa cultivars (Astro, Runaway, Esmee, Standard, Balboa), five Diplotaxis tenuifolia cultivars (Bellezia, Sylvetta, Wild, Wasabi, Dragon’s Fire). Arugula was planted in plots at high density (3-5 seeds per inch) and in 36 inch raised beds as recommended to us by farmers from NEAT’s PAC as industry standards. Plots are randomized throughout raised beds and each raised bed within the research field is considered a replicate block. Overhead irrigation was utilized, a 20-10-10 NPK nutrient treatment was applied, and no forms of pest control have been applied. Observations were made twice a week to record bolting and pest damage. Dates were recorded for the first signs of bolting within a plot and once >50% of the plants within a plot have bolted. However, late season plantings, cold weather & shorter days delayed bolting in most cultivars. Consistent bolting throughout the field was only observed in four Diplotaxis tenuifolia cultivars (Bellezia, Sylvetta, Wild, Wasabi) out of 10 cultivars by the time frost terminated the plantings in December 2023. Additionally, no flea beetle damage was seen in any cultivar in this field. This maybe due to the colder weather and late season start or low pressure from the lack of history of leafy green plantings. Likewise, no natural disease outbreaks occurred in the arugula plantings. However, we planted radish & rapini cultivars with known susceptibility to Pseudomonas cannabina pv. alisalensis as trap crops at the edge of the field plot. In these plantings, we observed disease outbreaks and processed the symptomatic tissue. From these isolations, we detected three plant pathogenic bacteria isolates that we suspect to be P. allivorans or a related pathogen.
3) Greenhouse germplasm evaluations. During fall 2023, we established a greenhouse misting system for ensuring conducive conditions for bacterial diseases of arugula and initiated our first greenhouse experiment at PSU. Five Eruca sativa and five Diplotaxis tenuifolia arugula varieties were grown in six packs until early maturity at 21 and 35 days for E. sativa and D. tenuifolia, respecitively. Pseudomonas cannabina pv. alisalensis was grown on KMB agar and suspended in buffer to an OD of 600nm which is approximately 108 CFU/ml. The bacteria were sprayed onto the tops & undersides of arugula foliage, and the arugula is placed in high humidity for 48 hours to create optimal conditions for disease infection. Disease severity is currently being recorded at 7, 14, & 21 days after inoculation. Disease severity data will be analyzed to using an AUDPC and appropriate statistical analysis. Additionally, we began to analyze data from previous greenhouse experiments that were conducted in California. We are in the process of determining which varieties were the best and worst performers to use in comparisons in the spring greenhouse trials.
Education
Engagement
Our goal (300 grower contacts) during the project is largely met through the thousands of subscribers to the NH Vegetable & Fruit Newsletter, PVGA newsletter (with 300 leafy green farms), Vermont Veg and Berry Listserv, and the Eastern NY Commercial Horticulture Program listserv. Advertisement through these sources resulted in over 180 growers attending our first NEAT presentation at the New England Vegetable and Fruit Conference in 2021. We will use these venues to recruit growers to our grower network and actively supported grower Groups.
We currently have a 37 member grower network which will expand to at least 200 from those attending presentations, via our extension networks, grower meetings, webinars, and submitting samples for diagnosis. By filling out our previously developed interest survey, they will enroll/commit. At a minimum these growers will regularly receive educational materials and annual reports.
100 actively supported growers will be further supported by NEAT members as part of their extension or research roles and through disease diagnosis. As requested, NEAT members or our trainees will visit farms to provide on-farm technical assistance to at least 50 growers for implementation of management strategies and pest/disease identification. Annual virtual or in person surveys will evaluate progress toward performance targets.
Learning
We will use a multipronged staged learning approach where growers will sequentially achieve the following goals:
1) Learn to identify arugula/brassica leafy greens production problems;
2) Recognize available management options;
3) Become adept at tapping additional expert support; and
4) Determine how to apply appropriate management options.
Learning will be achieved through:
- Videos: Hodgdon will create a short instructional video on how to identify arugula bacterial diseases that will demonstrate a scouting program for growers. Longer format virtual field day webinar with videos that will cover background information and research trial results on arugula cultivar pest susceptibility and talks by project grower collaborators on their leafy green production recommendations will cover all learning stages.
- Factsheets: Hazelrigg,
BibiCedeno, and Bull will create at least 2 factsheets (one also in Spanish) that: 1) outlines key disease and pest management information for brassica leafy greens, and 2) describes germplasm resistance/tolerance. - Grower conference presentations: Bull,
BibiCedeno, Sideman, Hazelrigg, and Hodgdon will attend and present on brassica leafy greens. Depending on the stage of learning of growers at the events, they will present best management practices, disease and pest identification, and research results at the: Mid-Atlantic Fruit and Vegetable Conference, New England Vegetable and Fruit Conference, and the Eastern NY Commercial Horticulture Program Winter Meeting. At least two growers or PAC members will participate as speakers or through slideshow creation, sharing their experiences. - Farmer-to-farmer trainings: Trainings including twilight or other farmer-to-farmer training meetings at on-farm research and survey locations will be organized by NEAT members each year in their home state or a neighboring state. Meetings will reach beginner to advanced growers, cover a broad range of topics on disease diagnosis and management using a staged learning approach, and will feature growers presenting their solutions to brassica leafy green production problems. These meetings will be held on farm sites identified through surveys.
- Train-the-trainers: To further our impact we will present a workshop in collaboration with NE NPDN and extension educations to address the recognition, diagnosis and management of pest and pathogens.
Evaluation:
At individual events, we will quantify progress toward our learning objectives using surveys and audience response technology, e.g. Poll Everywhere, that can assist us in measuring attendee knowledge gain. Specifically, we will ask attendees to identify key arugula pests and diseases, and effective management strategies at the start of the conference, and again at the end of the conference. 100 growers will correctly identify symptoms of arugula bacterial blight and recommended management practices after our programs.
Annual surveys of actively supported or network members will determine that extent of grower learning and implementation of knowledge on their individual farms. They will indicate whether: 1) They were able to identify any arugula pests and disease on their farm as a result of our outreach; 2) They adopted any of our recommended arugula pest and disease management practices; and 3) Their arugula yield changed as a result and by how much. Documentation of achievements and progress toward performance targets will be tracked through our validation tool.
Annually the PAC will review assessments and request changes in approaches to educational and technical outreach programs. Additional data will be collected or verified as requested by the PAC each year.
Milestones
Outreach and Education
- By July 1, 2023, over 300 agriculturalists will receive a short presentation about the project, including a description of how to submit samples, and a QR code to join our Grower Network. All NEAT and PAC members will share this through their networks including: NH Vegetable & Fruit Newsletter; Pennsylvania Vegetable Growers Association Newsletter, Vermont Veg and Berry Listserv; CCE Eastern NY Commercial Horticulture Program and others. This will meet the first grower performance target. Status: Delayed until March 2024 due to hiring delays.
- The NEAT Grower Network will grow from 28 to 50 growers by September 1, 2023 and will expand to 200 members by August 2024. Grower Network members are defined as brassica leafy green producers or seed professionals who give us their contact information for additional information from the project. Members will be solicited by all NEAT members during on-farm visits and during all outreach events. Status: In Progress due to hiring delays we have only added 9 farmers to the network as of January 2024. We still hope to meet our goal for August 2024.
- Each winter >60 growers attend NEAT presentations at grower meetings such as the New England Vegetable & Fruit Conference and Mid-Atlantic Fruit and Vegetable Conference. By the end of the project 200 growers will have achieved our second performance target. The will have learned increased scouting skills and how to diagnose diseases, pests, and production problems in brassica leafy greens. Status: In progress - three meetings were attended and/or presented at in winter 2024 and results are just being analyzed.
- At least 10 growers/farms each year have bacterial diseases of brassica leafy greens identified through surveys and submissions. By the end of the project at least 30 growers will have bacterial diseases of brassica leafy greens diagnosed and pathogens identified. This will be the direct result of achieving the first and second performance targets. Status: In progress - samples from 5 locations in Pennsylvania were collected in 2023.
- By July 1, 2024, requests for information will be sent to produce auctions, major retailers, and other outlets to try to determine the volume and acreage of arugula production in participating states. Additional information will be gathered through our grower’s survey. By November 2024, we will have estimates of arugula or brassica leafy green production by state. Status: In progress cold calls to several produce actions were made to start this goal.
- Through the life of the project, we will reach 30 growers via downloads of our factsheets, and 100 viewers for our videos. We anticipate that 1/3 of these will occur each year. Status: In progress - website was developed and information sheets were provided to growers at meetings this year. We are still analyzing the number distributed.
- By December 2024, NEAT will produce a Plant Disease Feature Article which will describe diseases, pests and production issues of brassica Leafy Greens and their management practices. By the end of the project, 100 reprints of this article will be distributed to growers. Status: In progress The scope of this article will be for bacterial diseases of brassica leafy greens. The timeline is delayed due to hiring delays.
- November 2025, chart indicating resistance and tolerance of various lines of germplasm will be published for dissemination to growers and seed companies. Status: Not Begun
- Bull and Cedeno will present results of germplasm evaluation to 54 members of the Seed Pathology Committee of the American Phytopathological Society using PSU travel funds in August 2024. Additional remote presentations to members of the International Seed Federation Vegetable working group and the American Seed Trade Association will occur between August 2024 and February 2025. Status: Not Begun - Note that this milestone previously was for Bibi and Bull but Cedeno replaced Bibi on this project.
- By February 2026, seed companies will begin to provide seeds with resistance/tolerance to bacterial diseases, flea beetles, or bolting, in their germplasm to consumers. Status: Not Begun
- Starting in December 2024 growers will begin to report increased profits with the greatest profits due to pest and disease management occurring in 2025. Status: Not Begun
EVALUATION
- Each Month Cedeno will review our Verification Tool to make sure that data are entered appropriately for each interval for Stakeholder Knowledge Tracking (milestone number tracking), GANTT Chart (measuring milestone completion), Bull Strain Intake Form (documenting diagnostic responses to growers), and Grower Network Members (to continue to increase our network). Status: In progress
- At the time growers enroll and in December of each year NEAT will survey the Grower Network for changes in knowledge, practices, changes in crop choices, profitability, and losses. Status: Not Begun -will begin in December 2024.
- Within one year of our presentations, we will contact >150 growers for follow up surveys following our presentations. Status: Not Begun
- Consult PAC members NEAT members will review changes in knowledge, practices, changes in crop choices, profitability, and losses. NEAT will use PAC recommendations to change outreach and education to achieve Performance targets for the following year. Status: In progress
Research
- By July 2024 domesticated cultivars and accessions of Eruca and Diplotaxis and at least 100 accessions of Eruca from the US National Plant Germplasm System will be evaluated for resistance in greenhouse/growth chamber trials at PSU and UNH. Replications of these trials will be conducted before November 2024. Status: In progress UNH faculty member who was going to replicate these has taken another job. We are proposing to replicate trials at 4 locations in Pennsylvania instead.
- November 30, 2023, 2024, and 2025 – Data will be analyzed and summarized for each year’s field trial accessing flea beetle and bolting resistance or tolerance on at least 20 Eruca sativa and Diplotaxis tenifolia cultivars/germplasm lines in replicated trials conducted at PSU or in parallel on-farm field trials conducted by UVM with 10 or more cultivars. Bacterial and other diseases will be assessed if they are present in these plots without inoculation. Status: In progress - Note that we are proposing to replicate trials at 4 locations in Pennsylvania instead of replicated trials at UVM.
- November 30, 2024, and 2025 - Data will be analyzed and summarized for each year’s field trial accessing 20 lines resistant to bacterial diseases in inoculated field trials at PSU and for on-farm trials conducted by Hodgdon. The findings will be incorporated into presentations to improve all performance targets. Status: Not begun
See GANTT chart for details of scheduled milestones.
Milestone Activities and Participation Summary
Educational activities:
Participation Summary:
Learning Outcomes
Performance Target Outcomes
Target #1
Additional Project Outcomes
We were approached by two different research teams working on brassica leafy greens to collaborate. A manuscript describing a new detection method for Pseudomonas cannabina pv. alisalensis is in progress because of this.
Since the project start date of March 1, 2023, NEAT members have Anissa Poleatewich (Co-PI, Assistant Professor), & Shaheen Bibi (Co-PI, Post-Doc) have left the project due to career changes. Anissa Poleatewich (University of New Hampshire) was responsible for germplasm evaluations, grower outreach, scouting, & grower surveying. Shaheen Bibi (Penn State University) was responsible for project management, pathogen identification and diversity, germplasm screening, webinar development, & presentations for grower meetings. UNH will no longer be responsible for conducting arugula germplasm evaluations. In place of those evaluations, PSU will conduct additional on-farm germplasm evaluations on 4 additional farms from 4 different climate zones across Pennsylvania.
Personnel changes have disrupted the initial phases of NEAT’s project in Q1-Q3 of 2023, but we are now making progress on these goals and are looking forward to meeting our 2024 performance targets.