Progress 09/01/23 to 08/31/24

Outputs
Target Audience:Organic vegetable growers, vegetable seed companies, vegetable plant breeders, plant science researchers in horticulture, plant breeding, plant pathology, soil science, and botany. Changes/Problems:The Arkansas location had weather issues during our winter 2023 trial in Fayetteville, AR. We expect to have fewer issues with the weather during our Fall trial. What opportunities for training and professional development has the project provided?The project provides training and professional development opportunities for graduate students, postdoctoral positions at Texas A&M, and research internship positions at OSA. As part of education activities, interns, graduate students, and post-doctorates are being trained in genomics, crop and seed production, disease protection and diagnosis, and soil science with a focus on organic systems as they participate in research projects critical to the project activities, and present in professional conferences. This project also provides external training (Extension) and professional development for farmers, seed producers, and seed companies through educational resources, webinars, field days, and conference presentations. Educational outreach is delivered through the project website on eOrganic (www.eorganic.info/spinach ) and in year 1 spinach seed production resources were posted and promoted through the website and partner-organization newsletters. Collaborating farmers gained first-hand experience evaluating crop diversity through on-farm trials and gained training and professional development through coordination of field research with the project team. Trials were conducted with organic spinach farms in WA, . Each trial provided training on carrot trialing and seed production for employees and interns. These trials allowed expansion of project impacts, testing materials in new regions, and soliciting input on evaluations from two organic seed companies. Project collaborators also hosted public farmer field days with trials at the project research sites. Dr. Gehendra Bhattarai, a postdoctoral researcher at the University of Arkansas, led this project. Under the supervision of Dr. Ainong Shi (Co-PD) and Dr. Vijay Joshi (PD), Dr. Bhattarai played a crucial role in executing project tasks. Dr. Haizheng Xiong, a research associate in Dr. Shi's lab, was responsible for field management and contributed to the project. As a senior researcher in Dr. Shi's lab, Dr. Bhattarai provided guidance and mentorship to a Ph.D. student (Ibtisam Alatawi) and two MS students (Teresha Phiri and Kenani Chiniwa) in completing their research projects and gained valuable experiences through their involvement in the lab's ongoing activities. The graduate students gained valuable experiences by participating in the lab's ongoing activities. All three graduate students were involved in field trial activities and data collection for this project to enhance the nitrogen use efficiency (NUE) and nutrient components How have the results been disseminated to communities of interest?Educational events and conference presentations delivered include: Article: Formiga, A., V. Joshi, M. Colley. 2023. Challenges in the market for organic spinach seed. eOrganic article. Available at https://eorganic.org/node/35758 ?ASHS conference: Oral presentation and a poster Exploiting the Natural Variation in Spinach Germplasm to Enhance Organic Productivity D Thompson, M Moreno, M Ramezani, A Shi, V Joshi - 2023 ASHS Annual Conference, 2023 Outreach: eOrganic resources: Guide to organic spinach seed production, video of field evaluations at Texas A&M, photo gallery of spinach seed production morphological stages. Field Days and Training: OSA Washington Farmer Field Day, Sep 11, 2023Blogposts/ Media: Organic Seed Alliance, June 19, 2023: Spinach trial underway to inform organic spinach seed production. https://seedalliance.org/2023/spinach-trial-launch/ What do you plan to do during the next reporting period to accomplish the goals?In year 2, the OSA research team will repeat the seed production trials in Chimacum, WA. Trial results from year 1 will be integrated into a multi-year data set. A webinar on organic seed production delivered through eOrganic will train farmers and seed companies and share preliminary project results. A project poster will be developed to present at agricultural conferences with the target of presenting at X events. Field evaluations of the USDA spinach accessions are underway at the Uvalde and Fayetteville farms. By conducting a second-year trial in both locations, we aim to address potential limitations and improve the value of the phenotypic dataset generated by this project. The preliminary dataset generated from these evaluations will be combined with the dataset from the Uvalde trial and used for further analysis and reporting. The phenotype data collected from the multi-location and multi-year trials will be utilized in subsequent genomic analyses and identification of trait-associated markers, as well as improving genomic selection models and breeding and selection of high-yielding nutrient-denser improved cultivars fitting the organic production system. These genetic analyses will help us identify markers linked to specific traits and enhance our genomic selection models.

Impacts
What was accomplished under these goals? Objective 1 - Enhancing the nitrogen use efficiency (NUE) of organic spinach Objective 1.1 Exploring the natural variation for NUE and associated traits. Around 300 USDA spinach germplasm panels and four check varieties were planted at the Organic Farm in Uvalde, TX (Dec 2022) and Fayetteville, AK farm (Feb, 2023) in an augmented designs. Uvalde Farm: 277 Spinach accessions from the USDA National Germplasm Repository and 4 commercial checks. 30 plants of each accession were grown in 2 rows in a 4 ft plot with a plant-to-plant distance of 2.5 inches following augmented design with repeated checks, namely, Acadia F1, Corvair F1, Space F1, Tundra F1 at locations (Uvalde, TX) in 2022-23. Chlorophyll content (MC-100 chlorophyll concentration meter; Apogee), stomatal conductance (gsw), photosynthetic electron transport rate (ETR), and PSII actual photochemical quantum yield (PhiPS2) using LI-600 Porometer/Fluorometer. Individual per plant basis fresh and dry-biomass were recorded at physiological maturity by pooling 3 plants per accession. The data analysis confirmed a normal distribution for all the traits relevant to organic productivity in the selected germplasm. Several significantly associated markers and candidate genes were identified for the selected traits. SNP markers will facilitate the breeding of organic spinach varieties with enhanced productivity. Fayetteville farm: The four check varieties were randomly replicated twice within each block. Each block consisted of 22 plots, and approximately 20 seeds of each accession were planted in each plot to ensure around 10 plants per accession for phenotyping. The trial was planted later than usual, in late February of this year, due to poor weather conditions, and the project will start later in 2022. Typically, our trials are conducted in late September or early October, but we had to adjust our schedule this year due to heavy snowfall in January and February. The campus facilities were closed during this time, and the fields were covered in snow. To obtain soil nutrient parameters, we collected samples from the field before planting at 30-60 cm depth. The samples will be examined for factors such as total nitrogen (N), free nitrates, and other macro- and micronutrients. We used MultispeQ (https://www.photosynq.com/) to measure the relative chlorophyll content of all plants by estimating chlorophyll fluorescence parameters. All plants were harvested at physiological maturity to collect fresh and dry biomass to understand spinach's nitrogen use efficiency under an organic production system. However, we did not evaluate the nutrient content composition, which we plan to perform during our second trial performed in Fall 2023, detailed below. Pre-sowing soil analysis was performed to evaluate the nutrient content of the soil. Around 300 USDA spinach germplasm panels were planted at the Fayetteville farm in October 2023, along with four check varieties replicated twice in each block in an augmented design. The plants are growing well so far, and we plan to measure the chlorophyll content of all plants and the fresh and dry weight biomass. We will homogenize the dried samples of each accession to estimate the total Kjeldahl N (TKN) concentrations using the Kjeldahl method and the levels of free nitrates (NO3, NH4, total N)) at PD-Joshi's lab. The mineral contents of 14 elements will be determined, including Ca, Fe, Zn, Mg, Mn, N, P, K, Cu, I, Se, Cr, Cl, and Mo. Further, targeted metabolome profiling will be performed to estimate the nutraceutical traits for free amino acids, oxalic acids, Vitamin C/ Ascorbic acid, Vitamin A (Retinol), and Vitamin E (alpha-tocopherol). Objective 1.2 Genome-wide association study (GWAS) and genomic prediction (GP) of NUE and associated traits in organic systems Using the traits collected from the accessions screen at Uvalde, TX, principal component analysis (PCA) and genetic diversity were analyzed with GAPIT 3 (Wang and Zhang, 2021) by setting PCA = 2 to 10 and NJ tree = 2 to 10. Phylogenetic trees were drawn by using the neighbor-joining (NJ) method. Variation in the spinach population was apparent for all the measured traits, which showed normal distributions. 88,288 SNPs distributed on 6 chromosomes were used to perform GWAS for organic productivity and photosynthetic traits after filtering and keeping MAF>2%, Missing < 9%, and Het < 15%. Significant SNPs and candidate genes identified under the organic production system for the selected traits would help unlock the resource use efficiency and develop spinach varieties adapted to organic production. Objective 3 - Enhancing the seed production and availability of organic spinach: At the Organic Seed Alliance Research farm in Chimacum, WA, researchers evaluated a single replicate trial with 3 repeated check cultivars of more than 200 accessions from the Germplasm Resources Information Network (GRIN) collection for phenotypic traits related to seed production. Evaluation of each plot for included assessment of the timing of bolting, the ratio of pollen and seed-producing plant types, and per plot seed yield. All plots were evaluated for field traits in Yr 1, are currently dried, and will be processed for seed yield and quality in the winter of 2023. Seed types varied across accessions with pointy and round seed shapes. Photo documentation captured overall plant stature per plot and seed samples from each accession collected.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Exploiting the Natural Variation in Spinach Germplasm to Enhance Organic Productivity. D Thompson, M Moreno, M Ramezani, A Shi, V Joshi - 2023 ASHS Annual Conference, 2023
• Type: Websites Status: Published Year Published: 2023 Citation: Formiga, A., V. Joshi, M. Colley. 2023. Challenges in the market for organic spinach seed. eOrganic article. Available at https://eorganic.org/node/35758

Progress 09/01/22 to 08/31/23

Outputs
Target Audience:Field day conducted at AgriLife Research and Extension Center in Uvalde May 11, 2023 attended by over 60 participants representing growers, processors, shippers, packers and general public interested in organic production. Changes/Problems:Finding a suitable post-doctoral candidate has been difficult. Further, the candidate hiring was substantially delayed due to visa processing delays. The new hire is likely to join Joshi Lab in mid-July 2023. We anticipate the delay won't slow down the project plan but if needed, we may request a no-cost extension at the end of 3rd year. Weather problems delayed planting at the Arkansas location. We are re-sowing the experiments in order to generate quality data. The sowing will be performed again in the fall of 2023. What opportunities for training and professional development has the project provided?University of Arkansas: Dr. Gehendra Bhattarai, a postdoctoral researcher at the University of Arkansas, led this project. Under the supervision of Dr. Ainong Shi (Co-PD) and Dr. Vijay Joshi (PD), Dr. Bhattarai played a crucial role in executing project tasks. Dr. Haizheng Xiong, a research associate in Dr. Shi's lab, was responsible for field management and contributed to the project. As a senior researcher in Dr. Shi's lab, Dr. Bhattarai provided guidance and mentorship to a Ph.D. student (Ibtisam Alatawi) and two MS students (Teresha Phiri and Kenani Chiniwa) in completing their research projects and gained valuable experiences through their involvement in the lab's ongoing activities. The graduate students gained practical experience by participating in the lab's ongoing activities. They helped in field trial activities and data collection for this project to enhance spinach's nitrogen use efficiency (NUE) and nutrient components. Texas A&M AgriLife Research: A Technician, Dalton Thompson, post-doc Mazi Ramazani, and an undergraduate student, Matte Moreno, were trained on several project-related activities research activities. Two new students (MS and Ph.D.) will join Joshi's lab later in the year, participate in the ongoing research activities, and be trained on organic production practices at the AgriLife Research Center. A field day organized at the AgriLife Research Center (May 2023) allowed producers and the general public to learn about the project. As a part of the 4-H STEM club organized by PD Joshi, students will be allowed to get familiar with project activities and learn about organic production and practices. Organic Seed Alliance (OSA): OSA staff holds skills and experience in organic spinach seed production, including authorship of the Guide - Principles and Practices of Organic Spinach Seed Production by Program Director Micaela Colley. OSA's research farm manager, Kayla Ierlan, and field research assistant, Johanna Willingham, bring spinach seed production experience but are gaining professional development in the project through knowledge exchange with project partners and respective research on spinach nutrition and pathology as well as first-hand knowledge of the diversity of spinach phenotypes from GRIN. OSA's annual interns will benefit from learning seed production practices and the research methodology. How have the results been disseminated to communities of interest?Project outreach will continue in year two with annual field days showcasing the project and online communications through eOrganic, the OSA website, and social media. In year 2, a webinar on organic. Spinach seed production will advance seed growers' knowledge of production practices, including horticultural management, isolation, disease and pest management, and harvest and cleaning practices. This project will expand the number of organic producers with skills and knowledge in spinach seed production and seed companies' knowledge of relative seed yields of spinach varieties and breeding lines. The long-term impact will be to expand organic spinach seed availability. eOrganic will provide peer-and National Organic Program compliance review and editorial support for at least three publicly available articles by project members, which will be published on the eOrganic website and announced in our newsletter and social media. We will also conduct at least three webinars and evaluate them for quality and utility. In addition, eOrganic has been preparing a report on the organic spinach seed market for the project website. What do you plan to do during the next reporting period to accomplish the goals?Texas A&M AgriLife Research: Joshi lab will undertake heat stress evaluations and speed breeding research under controlled environments in 2023-24, as described in the work plan in the proposal. The hiring of a postdoctoral research associate has been significantly delayed due to visa approval and processing times. The position has been offered to a candidate expected to arrive at Joshi's lab beginning July 2023. The new hire will focus on objective 2, to characterize the role of the microbiome in nutrient uptake for sustainable spinach production. Samples will be collected to analyze selective rhizosphere (soil) and endospheric (root) microflora acclimatized to commercial organic spinach at multiple locations. The field evaluation of organic spinach will be performed in 2023-24 using the previously established processes (described before). University of Arkansas: Dr. Bhattarai (post-doc in Shi's lab) planted over 300 accessions from the USDA germplasm panel at the Fayetteville farm. Our objective is to evaluate these accessions for various important physiological traits. We will conduct the trial again next year to obtain additional data. The preliminary dataset generated from this evaluation will be combined with the dataset from the Uvalde trial and used for further analysis and reporting. Our plan for the next year is to plant our trial during the fall season. By conducting a second-year trial in both locations, we aim to address potential limitations and improve the value of the phenotypic dataset generated by this project. As a part of our project, we will use the phenotype data collected from the multi-location and multi-year trials to perform genomic analysis. This analysis will help us identify markers linked to specific traits and enhance our genomic selection models. This USDA NIFA Organic Research and Extension Initiative-funded research project will lay a foundation to develop spinach varieties for organic producers and consumers with an improved nutrient acquisition, improve our understanding of how spinach leaf and root-associated microbiomes interact with organic fields dedicated to spinach production, and create a breeding model to develop varieties with higher organic seed productivity. Organic Seed Alliance (OSA). OSA will continue to replicate the seed yield and quality trial annually. We will refine horticultural and evaluation protocols based on year one outcomes, though no major modifications are anticipated. Entries were all sourced from GRIN with enough seed for multi-year trials. Entries not germinating in the first year will be dropped in year 2.

Impacts
What was accomplished under these goals? Objective 1: Enhancing the nitrogen use efficiency of organic spinach: Recognizing suitable genetics with higher nitrogen use efficiencies for low-input organic spinach production. Seeds of 278 diverse spinach genotypes consisting of obtained from the USDA Germplasm repository (GRIN) were used for field evaluation at the certified organic field at the Texas A&M AgriLife Research and Extension Center in Uvalde on Nov, 2022. Pre-sowing soil analysis was performed to evaluate the organic matter, total nitrogen, and other micro-elemental analyses. Each genotype/accession was grown in a field with 18 rows (each 230 ft), each with 42 sub-plots. Each sub-plot (4 ft) had two rows accommodating 30 plants. Each check within a row was randomized in the augmented design using four checks (Acadia F1 OG, Corviar F1 OG, Space F1 OG, and Tundra F1 OG). Additionally, six commercial varieties (Regiment F1, Renegade F1, Minkar, Nembus, Kiowa, Frontier) were also included in the design. The pests and diseases were controlled manually and OMRI-approved products. At physiological maturity, plants were harvested to collect biomass, chlorophyll content, stomatal conductance (gsw), photosynthetic electron transport rate (ETR), and PSII actual photochemical quantum yield (PhiPS2). Variation in the spinach population was apparent for all the measured traits, which showed nearly perfect normal distributions.Around 300 USDA spinach germplasm panels and four check varieties were planted at the Fayetteville, AK farm in an augmented design across 14 blocks. Within each block, the four check varieties were randomly replicated twice. Each block consisted of 22 plots, and approximately 20 seeds of each accession were planted in each plot to ensure around ten plants per genotype for subsequent data measurement. Our trial was planned later than usual, in late February of this year, due to poor weather conditions and the project starting later in 2022.We used MultispeQ (https://www.photosynq.com/) to measure the relative chlorophyll content of all plants by estimating chlorophyll fluorescence parameters. The measurements for fresh weight and dry weight are ongoing. Objective 3. Enhancing seed production and availability of organic spinach: Assessing selected spinach germplasm for enhanced seed production potential using seed traits through genome wide-association analysis and genomic prediction. Organic Seed Alliance (OSA) (Micaela Colley's Lab): The first project year focused on establishing project protocols, communications, and partnerships and launching the first year of field research. Project outreach included establishing a project website with eOrganic, distributing a project press release, and a blog post in Organic Seed Alliance's (OSA) summer newsletter. At OSA, the field trials of 270 varieties were planted in the greenhouse in mid-March and transplanted into the field in late April. Accessions varied in bolting response and seed morphology. Of all seed, accessions documented the varied seed phenotypes. Trial protocols are in place to evaluate the timing of flowering, the ratio of pollen and seed-producing plants in the populations, the timing of seed maturation/ harvest, and seed yield (total weight and 1000 seed weights). Plans are underway to feature the project in a fall field day at the OSA research farm in Chimacum, WA. Objective 4: Developing an outreach plan to provide relevant information to stakeholders in the organic spinach industry: Establishing a platform in partnership with eOrganic and Organic Seed Alliance to share project progress and outputs with stakeholders. eOrganic (Alice Formiga Lab, Oregon State University) created a website for this project which describes the objectives and collaborators, photos of trials and the different types of spinach, and project publications. Additional resources and webinar recordings will be added to this website as the project progresses. The website is available at https://eorganic.org/spinach.

Publications

• Type: Conference Papers and Presentations Status: Submitted Year Published: 2023 Citation: "Exploiting the Natural Variation in Spinach Germplasm to Enhance Organic Productivity," for the 2023 Annual Conference of the American Society for Horticultural Science (ASHS), has been accepted for a POSTER presentation. https://ashs.confex.com/ashs/2023/meetingapp.cgi/Paper/40044