Research Project: Genetic Improvement of Lettuce, Spinach, Celery, Melon, and Related Species

Location: Crop Improvement and Protection Research

2025 Annual Report

Objectives
Objective 1: Develop genetic resources and germplasm resistance to key pests and pathogens and abiotic stresses of lettuce, spinach, celery, and melon. Sub-objective 1.A: Breed for resistances to fungal, bacterial, and viral diseases and disorders in lettuce. Sub-objective 1.B: Develop durable resistance to downy mildew in spinach. Sub-objective 1.C: Improve celery for resistance to Fusarium oxysporum f.sp. apii (Foa) races 2 and 4. Sub-objective 1.D: Breed melon for resistance against powdery mildew, virus, and whitefly. Objective 2: Develop new procedures and technologies to evaluate and enhance postharvest quality and shelf life of fresh cut leafy greens, especially lettuce. Objective 3: Improve content and bioavailability of phytonutrients or fiber of leafy vegetables for improved impact on human health nutrition and composition of the gut microbiome. Sub-objective 3.A: Characterize polyphenol and fiber content among lettuce cultivars. Sub-objective 3.B: Breed red spinach for improved nutrient content and disease resistance. Objective 4: Characterize the composition and effects of environment on the microbiota found on leafy vegetables.

Approach
Objective 1: Sub-objective 1A: Map major QTLs for resistance to downy mildew using a genome-wide association mapping (GWAS) approach and develop breeding lines with the improved resistance to lettuce drop and downy mildew. Characterize resistance to Fusarium, Verticillium, and Pythium wilts to develop improved lettuce germplasm. Cross corky root-resistant variety ‘Glacier’ and wild species L. serriola to sources of other disease resistance and select for combined resistances to corky root, leafminer, downy mildew, lettuce mosaic virus, tipburn, and horticultural and nutritional traits in different types of lettuce. Characterize resistance to impatiens necrotic spot virus to develop improved lettuce germplasm. Develop controlled environment and molecular assays to characterize tipburn resistance in lettuce. Sub-objective 1B: Breed open-pollinated (OP) spinach with resistance to all prevalent downy mildew races through crosses in isolators, recurrent selection, and replicated field trials. Sub-objective 1C: Develop celery germplasm with resistance to Foa races 2 and 4 through disease assays, selection, and self-pollination. Sub-objective 1D: Breed western U.S. shipper type melon resistant to powdery mildew races 1, 2, 3.5, 5, and S. Identify and characterize resistance in melon to whitefly-transmitted cucurbit chlorotic yellows virus (CCYV). Characterize antixenosis to sweetpotato B biotype whitefly in melon. Objective 2: Fine-map the locus for slow decay of fresh-cut lettuce, map QTLs for limited oxidative browning, and develop lettuce breeding lines with high postharvest quality. Objective 3: Sub-objective 3A: Characterize polyphenol and fiber content among lettuce cultivars to understand genotype x environment interactions and to identify genetically stable high polyphenol cultivars in replicated greenhouse assays. Sub-objective 3B: Improve the betacyanin content, antioxidant capacity, and downy mildew resistance of different types of spinach through crossing, recurrent selection, and field trials. Objective 4: Determine the relationships in the phyllosphere community among leafy vegetables, nearby weeds, and the soil microbiota by isolating genomic materials from the phyllosphere and rhizosphere for sequencing.

Progress Report
In support of Sub-objective 1.A, Goals 1.A.1 and 1.A.2, the GWAS (genome-wide association study) population and F1 lines were tested in replicated field trials in the Salinas Valley, California. Plants of 500 accessions plus 57 F1 lines were evaluated for resistance to downy mildew caused by Bremia lactucae using a 0-5 rating scale that combines disease severity and incidence. Lettuce drop caused by Sclerotinia minor was evaluated as the percentage of plants showing disease symptoms at whole-head maturity. F2 seeds were produced in a hoophouse and a greenhouse. In support of Sub-objective 1.A, Goal 1.A.3, researchers at Salinas, California, in collaboration with university researchers, characterized multiple Fusarium wilt isolates collected from commercial lettuce fields that represent the new variant race. Variation was identified in multiple greenhouse tests, and the most aggressive isolate was targeted for resistance breeding efforts. Discussions with ARS researchers and industry partners resulted in the formation of a global lettuce Fusarium wilt organization: the International Lettuce Fusarium Evaluation Board. Researchers at Salinas, California, planted a field trial in June to evaluate USDA breeding lines and check varieties under Verticillium race 1 and Fusarium race 1. Selections for disease resistance and horticultural traits will be conducted in September and promising lines will be moved forward for germplasm development and release. Previously selected lines were crossed in the greenhouse and seed generations were advanced for future evaluation. Researchers at Salinas, California, in collaboration with university researchers, developed a Pythium wilt inoculation protocol to evaluate lettuce germplasm for resistance in the greenhouse. For Sub-objective 1.A, Goal 1.A.4, lettuce breeding lines in advanced generations were tested in replicated field trials with control varieties and commercial cultivars. The corky root and leafminer resistances of the breeding lines were similar to or better than resistant controls, while their plant weight, height, core length, tipburn, and downy mildew resistance traits were comparable or better than control cultivars. In support of Sub-objective 1.A, Goal 1.A.5, researchers at Salinas, California, conducted greenhouse tests in April, June, and August to evaluate selected USDA breeding lines, commercial varieties, wild accessions, and check varieties for resistance to impatiens necrotic spot virus (INSV). Researchers at Salinas, California, collaborating with industry researchers under an agreement, initiated a fine mapping project for the INSV resistance from ‘Eruption’, which will advance breeding and development efforts towards germplasm release. Previously selected lines were crossed in the greenhouse and seed generations were advanced for future evaluation. Researchers at Salinas, California, will plant fields trials in August to evaluate commercial varieties, USDA breeding lines, and check varieties for resistance to INSV. We will harvest in early FY26. Tipburn is a physiological disorder of lettuce that is characterized by necrotic tissue at the tips of growing lettuce leaves that can lead to significant crop loss when the environmental conditions are conducive. Field trials for resistance to this trait are difficult due to the prevalence of other diseases and variable environmental conditions, therefore tests within growth chambers or other controlled environments are necessary to develop germplasm with tolerance to this physiological disorder. In support of Sub-objective 1.A, Goal 1.A.6, develop controlled environment and molecular assays to characterize tipburn resistance, researchers in Salinas, California grew susceptible cultivars in growth chambers with warm days/warm nights, hot days/warm nights, warm days/cool nights, and cool days/cool nights and under two different watering regimes. Warm days/cool nights with light watering regimes induced the most tipburn. This information will be used to induce tipburn under controlled conditions to better understand environmental conditions that cause this detrimental condition in the field. For Sub-objective 1.B, thirty-four spinach populations of western and oriental types were planted in the field to confirm previous results demonstrating downy mildew resistance, and 10-50 plants were selected from each population and transplanted into isolators to produce seeds for selection next year. Some plants were self-pollinated to produce inbred lines. Fusarium wilt of celery is the major pathogen affecting celery production in California, and it is caused by the soil-borne pathogen Fusarium oxysporum f.sp. apii (FOA) races 2 and 4. There is some resistance to FOA race 2 in existing celery cultivars, however there is no resistance to FOA race 4 and the pathogen causes complete crop loss in infested fields. In support of Objective 1.C, and to improve celery for resistance to Fusarium oxysporum f.sp. apii races 2 and 4 in celery, researchers in Salinas, California, planted approximately 200 plants from previously self-pollinated celery germplasm in infested fields, and made selections in late 2023. Approximately 40 plants were selected for vernalization, and were maintained in isolators for self-pollination. Seed was harvested in the fall of 2024, and future development of these lines will develop germplasm with resistance to FOA race 2 and race 4. In support of Objective 2, the GWAS population and F1 lettuce breeding lines of eight horticultural types were tested in replicated field trials in the Salinas Valley, California. Plants of 500 accessions plus 21 F1 lines were evaluated for postharvest quality, including oxidative discoloration (pinking and browning) and tissue deterioration of fresh-cut lettuce in modified atmosphere packaging. F2 seeds were produced in a hoophouse and a greenhouse. Polyphenols are important antioxidants in the human diet that are derived entirely from plants. Polysaccharides in the plant cells that provide important dietary fiber in the human diet bind polyphenols, and prevent their absorption into the human gut. In support of Objective 3.A, characterize polyphenol and fiber content among lettuce cultivars, researchers in Salinas, California assayed total polyphenol content in 52 lettuce cultivars over two years, and total dietary fiber content of 6 cultivars. There was significant variation among years; however, some varieties were found to have higher total polyphenol content than others, and these may be suitable as parental lines for germplasm with improved nutrition for human health. In support of Sub-objective 3.B, ARS researchers continued to improve the color, betacyanin content, and disease resistance of red spinach by conducting recurrent selection in 69 populations (20-40 plants selected from each population of 400-800 plants) and self-pollinating individual plants. The community of microorganisms, including bacteria and fungi that live on the leaves of plants is termed the phyllosphere community, and the community that live near the roots are the rhizosphere community. In support of Objective 4, characterize the composition and effects of environment on the microbiota found on leafy vegetables, researchers in Salinas, California, grew cultivated lettuce and weedy wild lettuce side-by-side in growers fields, and sampled leaf tissue and root tissue for microbiome sequencing. Growers requested a pause of 6 months before submitting the samples for high-throughput sequencing to ensure the crops in the fields had moved through the supply chain. However, by the time this period elapsed, there were significant purchasing restrictions preventing submission of the samples for sequencing. The samples are currently stored at ultra-low temperatures and awaiting sequencing.

Accomplishments
1. Unlocking lettuce's natural disease resistance. Lettuce drop, caused by the fungus Sclerotinia minor, is a major disease inflicting significant financial losses to the lettuce industry, valued at over $4.6 billion annually. Current methods for controlling lettuce drop are limited because the fungus can survive in soil for long periods and spread easily by wind. Developing lettuce varieties that are naturally resistant is the most effective way to manage this disease, but such options are currently scarce. ARS researchers in Salinas, California, have discovered that the makeup of lettuce stem cell walls plays a key role in the plant's ability to resist lettuce drop. Through extensive laboratory analysis, they found a strong connection between certain components within the cell walls, like specific fibers and sugars, and the plant's resistance to the disease, as well as the stem's physical strength. This finding means that measuring these cell wall components can help identify more resistant lettuce plants, providing a valuable tool for breeders to develop new, robust lettuce varieties that better withstand this devastating disease.

Review Publications
Sandoya, G.V., Trent, M., Hayes, R.J., Lebeda, A., Rosenthal, E., Simko, I., Bull, C.T. 2025. Differential sources of resistance from Lactuca serriola against three races of Xanthomonas hortorum pv. vitians causing bacterial leaf spot of lettuce. Plant Disease. 109(3):615-622. https://doi.org/10.1094/PDIS-06-24-1239-RE.
Das, M.K., Park, S., Adhikari, N.D., Mou, B. 2024. Genome-wide association study of salt tolerance at the seed germination stage in lettuce. PLoS ONE. 19(10). Article e0308818. https://doi.org/10.1371/journal.pone.0308818.
Eriksen, R.L., Kumar, P., Sandoya, G., Adhikari, N.D., Mou, B. 2025. Effects of mesophyll conductance and nitrogen content on carbon assimilation during low-water stress in leaf lettuce cultivars. Horticulturae. 11(4). Article 414. https://doi.org/10.3390/horticulturae11040414.
Simko, I. 2024. Spatio-temporal dynamics of lettuce metabolome: A framework for targeted nutritional quality improvement. Plants. 13(23). Article 3316. https://doi.org/10.3390/plants13233316.
Peng, H., Lavelle, D.O., Truco, M., Michelmore, R.W., Simko, I. 2025. Differential low oxygen response and transcriptomic shifts drive fresh-cut lettuce deterioration in modified atmosphere packaging. Postharvest Biology and Technology. 227. Article 113571. https://doi.org/10.1016/j.postharvbio.2025.113571.
Hwang, S., Simko, I., Mou, B. 2024. QTL mapping and transcriptome analysis of seed germination under PEG-induced water stress in Lactuca spp. Scientific Reports. 14. Article 27157. https://doi.org/10.1038/s41598-024-77972-9.
Ravelombola, W., Xiong, H., Bhattarai, G., Manley, A., Cason, J., Pham, H., Zia, B., Mou, B., Shi, A. 2025. Genome-wide association study for drought tolerance in cowpea (Vigna unguiculata (L.) Walp.) at seedling stage using a whole genome resequencing approach. International Journal of Molecular Sciences. 26(12). Article 5478. https://doi.org/10.3390/ijms26125478.
Mandal, M.K., Koike, S.T., Tsuchida, C., Stanghellini, H., Guerrero, J., Sandoya, G., Klosterman, S.J., Simko, I., Subbarao, K.V. 2024. Distribution of three Verticillium dahliae races in coastal California and evaluation of resistance in lettuce. Plant Disease. 108(7):2170-2180. https://doi.org/10.1094/PDIS-01-24-0193-RE.
Simko, I., Mamo, B.E., Foster, C.E., Adhikari, N.D., Subbarao, K.V. 2024. Host cell wall composition and localized microenvironment implicated in resistance to basal stem degradation by lettuce drop (Sclerotinia minor). BMC Plant Biology. 24. Article 717. https://doi.org/10.1186/s12870-024-05399-5.
Nayak, S., Richardson, K.L., Putman, A., LeBlanc, N.R., Martin, F.N., Li, N., McCreight, J.D. 2024. Detection of novel pathogenic variants of Fusarium oxysporum f. sp. lactucae in California. Plant Pathology. 74(2):295-307. https://doi.org/10.1111/ppa.14019.
Bornhorst, E.R., Luo, Y., Park, E., Zhou, B., Turner, E.R., Teng, Z., Simko, I., Fonseca, J.M., Trouth, F.J. 2024. In search of optimum fresh-cut raw material: Using computer vision systems as a sensory screening tool for browning resistant romaine lettuce accessions. Horticulturae. 10(7). Article e10070731. https://doi.org/10.3390/horticulturae10070731.
Oh, S., Ahn, E.J., Shi, A., Mou, B., Park, S. 2025. Genome-wide association studies for seed thermoinhibition in lettuce reveals the interplay of seed age, color, and germination under high temperatures. Scientific Reports. https://doi.org/10.1038/s41598-024-84197-3.
George, A.S., Simko, I., Brandl, M. 2025. Escherichia coli O157:H7 multiplication in the latex of diverse lettuce genotypes is negatively correlated with plant peroxidase activity. International Journal of Food Microbiology. 431. Article 111095. https://doi.org/10.1016/j.ijfoodmicro.2025.111095.
Park, E., Luo, Y., Bornhorst, E., Simko, I., Trouth, F.J., Fonseca, J.M. 2024. Leveraging observations of untrained panelists to screen for quality of fresh-cut romaine lettuce. Horticulturae. 10(8):830. https://doi.org/10.3390/horticulturae10080830.
Simko, I., Mamo, B., Cantu, S.L., Peng, H., Grube Sideman, R., Hayes, R.J., Subbarao, K.V. 2025. Integrative path modeling and QTL mapping identify maturity, stem strength, and cell wall composition driving lettuce resistance to Sclerotinia minor. Scientific Reports. 15. Article 19824. https://doi.org/10.1038/s41598-025-03775-1.
Simko, I., Zhao, R.B., Peng, H. 2025. Differential impact of SiO2 foliar application on lettuce response to temperature, salinity, and drought stress. Plants. 14(12). Article 1845. https://doi.org/10.3390/plants14121845.
Richardson, K.L., McCreight, J.D., Nayak, S. 2025. Registration of six lettuce breeding lines with resistance to Fusarium wilt race 1. Journal of Plant Registrations. 19(1). Article e220423. https://doi.org/10.1002/plr2.20423.