Research Project: Genetic Improvement and Cropping Systems of Alfalfa for Livestock Utilization, Environmental Protection and Soil Health

Location: Plant Science Research

2022 Annual Report

Objectives
1. Develop genomic tools to enhance genetic selection of alfalfa for beneficial traits. Subobjective 1.A: Develop a reference genome sequence for cultivated alfalfa to facilitate breeding and genome editing for agronomic traits. Subobjective 1.B: Utilize universal DNA markers for accelerating breeding in alfalfa for improved forage digestibility. Subobjective 1.C: Utilize universal DNA markers for accelerating breeding in alfalfa for root system architecture. 2. Establish innovative, science-based methods and standards for assessing and evaluating alfalfa quality for multiple end uses. Subobjective 2.A: Develop new tools for enhancing nutritive value of alfalfa. Subobjective 2.B: Investigate cell wall lignification and digestion in alfalfa with reduced lignin concentrations. 3. Develop germplasm and crop management strategies to enhance productivity and environmental resiliency of forages. Subobjective 3.A: Evaluate expression of antimicrobial proteins for enhancing disease resistance in alfalfa. Subobjective 3.B: Map genes for resistance to Aphanomyces root rot. Subobjective 3.C: Evaluate seed treatments for enhancing germination and establishment of alfalfa plants. Subobjective 3.D: Evaluate modified alfalfa plants for phosphate uptake to remediate soil and reclaim phosphate. Subobjective 3.E: Evaluate alfalfa plants with an edited disease susceptibility gene for altered responses to pathogens. Subobjective 3F: Develop genomic and management strategies to reduce winterkill and increase persistence of alfalfa. 4. Develop knowledge and tools to increase understanding of the interactions among forage crops, soil nutrients, soil and plant health, and animal productivity. Sub-objective 4.A: Compare conventional and novel crop rotations utilizing forages for their effect on greenhouse gas emissions, C sequestration, nutrient cycling, and beneficial and pathogenic microbial populations. Sub-objective 4B: Measure effects of dairy manure application on productivity of alfalfa and rotational crops, forage quality, and soil health. Subobjective 4.C: Develop and utilize novel methods to identify and characterize pathogens of alfalfa.

Approach
Alfalfa is the most widely grown perennial forage crop in the U.S. and plays key roles in livestock nutrition, protecting water and soil resources, enhancing soil fertility, and sequestering soil carbon. However, there has been slow progress in alfalfa improvement and the contributions of alfalfa to agroecosystem sustainability are undervalued. To meet these needs, we aim to develop tools to accelerate breeding; test strategies to improve stand establishment and persistence; and measure carbon dioxide emissions, carbon sequestration, and nutrient cycling benefits of alfalfa in crop rotations and in the broader dairy production system. We will sequence and assemble the genome of alfalfa and provide web-accessible platforms to retrieve data. New breeding strategies including genome editing will be used for improving forage quality and stress tolerance. New seed treatments will be tested for improving stand establishment and soil indexing methods will be developed to determine risk of soilborne diseases. In-depth analyses of stem cell wall development and ruminal degradation will be done to gain a better understanding of developmental and structural changes that improve forage quality. Conventional and novel crop rotations utilizing forages will be evaluated for their effect on greenhouse gas emissions, carbon sequestration, nutrient cycling, and beneficial and pathogenic microbial populations. The effect of dairy manure application on productivity of alfalfa and rotational crops, forage quality, and soil health will be measured. The research will be of use to public and private plant breeders who will utilize the alfalfa genome sequence, markers, and breeding strategies in developing new cultivars; alfalfa farmers who will use soil indexing methods and seed fungicides to reduce damage from plant diseases; and the dairy food industry which has increased emphasis on reducing the environmental impacts of dairy, especially greenhouse gas emissions.

Progress Report
This project made significant progress on all objectives. In support of Objective 1, genome assemblies from ten different alfalfa accessions were generated that capture >99% of expected single-copy plant genes. The RegenSY27x reference genome assembly was further improved using Hi-C high-throughput sequencing and optical mapping data to scaffold assembly pieces into larger fragments. Initial results of scaffolding show 89% of the genome assembly has been scaffolded, improving its continuity by more than 8-fold. The RegenSY27x assembly was uploaded to the Legume Information Systems website (LIS) and integrated into the blast alignment tool, the Genome Context Viewer tool and other LIS tools. Genotyping with 3,000 SNP markers from Breeding Insight was carried out on over 1,300 plants that varied in forage digestibility and initial analysis showed clustering based on expected phenotypes. Field grown materials for phenotyping are being collected and association of markers and traits will be carried out. Field plots were established for phenotyping alfalfa populations differing in root architecture and will be genotyped and evaluated in fall 2022. An additional 3,000 SNP markers will be delivered by the end of the year by Breeding Insight from plant materials submitted by ARS scientists. To develop genomic and management strategies to reduce winterkill and increase the persistence of alfalfa, 1,280 landraces/cultivars/PI accessions in the greenhouse were transplanted to two field locations and tissues were sampled for genotyping with Breeding Insight SNP markers. Four global fall dormancy germplasm pools with 1,680 plants selected worldwide were screened for winter survival, biomass, and persistence; with tissues sampled for genotyping. Image and computer vision analysis will be used on photos from each plant, taken in early and late spring to develop new phenotyping methods. In support of Objective 2, transgenic plants previously selected for expression of genes to increase lipid accumulation in alfalfa leaves, fatty acyl-coenzyme A (CoA) diphosphatase (FIT2) and acyl-CoA:diacylglycerol acyltransferase (DGAT2), were further tested for lipid concentration and lipid characterization. Microscopic images of leaf tissue at various ages were stained with a neutral lipid identification stain to determine the location within the plants where lipid droplets form. Additionally, transgenic plants containing high expression of FIT2 and DGAT2 were crossed and viable seed for an F1 generation were collected. For evaluating stem composition and digestibility, two plants were selected from diverse genetic backgrounds planted in the previous year. These two plants and three other cultivars with known digestibility (high or low) were cloned in the greenhouse during the winter. From each of the five selected cultivars/lines, 800 clones were space planted in two different geographical locations. These plants will be used for the evaluation of stem traits after establishment in the field. In support of Objective 3, to identify markers associated with resistance to Aphanomyces root rot, a backcross population was phenotyped for disease reaction and genotyped using diversity arrays technology (DArT) markers. Separate loci and candidate genes for resistance to race 2 isolates were identified. DNA sequence from the resistant parent was assembled for comparison with susceptible alfalfa lines. Analysis of data from field experiments and growth chamber experiments testing seed fungicides found that most treatments did not improve stand establishment or establishment year forage yields over the conventional treatment. Two treatments had activity against seed rot and seedling damping-off pathogens like the conventional treatment, which would give growers additional tools for disease control. Plants with mutated PHOSPHATE2 (PHO2) genes, which play pivotal roles in phosphate signaling in plants, were mated with wildtype plants to transfer the phosphate hyperaccumulation trait into different genetic backgrounds. Analysis of progeny plants is underway to identify the number of mutated genes and to select plants for analysis under field conditions. In support of Objective 4, winter camelina was harvested from field plots in July 2021 and short-season soybean was immediately planted, then harvested in the fall. In spring 2022, a spring wheat crop was planted in this field, to be followed by fall seeding winter camelina in September 2022. In field 10, alfalfa termination was delayed from fall 2021 to fall 2022 to collect an additional year of carbon and water flux data on this crop before transitioning to Kernza intermediate wheatgrass in August 2022. Data collection continued in 2022 with spring soil sampling to 90-cm depth in the two research fields and ongoing eddy covariance, crop leaf area index, height, and yield data collection. In support of measuring the effects of dairy manure application on alfalfa productivity and soil health, the fourth year of spring soil sampling was conducted at the three research locations across Minnesota. Soils have been dried, ground, and analyzed for soil fertility, soil organic carbon, and mineralizable carbon. DNA samples were extracted and have been sent for metabarcode sequencing. Analyses to date suggest that single applications of relatively large amounts of manure had minor impacts on soil communities by the end of the growing season in the year after application. Location, field-scale variability, and cropping phase were strong drivers of soil microbial community composition in both years. The role of manure amendments on the composition, diversity, and assembly of soil microbial communities was studied using lab incubations. Chemical inputs from manure promoted the growth of potentially beneficial indigenous microbes. Soil abiotic factors, rather than indigenous microbial communities, played a primary role in preventing the establishment of manure-borne microbes. In support of process-based modelling to examine tradeoffs in carbon, water, and nutrient flows associated with dairy forage production, baseline alfalfa management and crop rotation scenarios have been modeled and alternative scenario modeling is ongoing, in partnership with collaborators. Simulation of baseline scenarios is complete and long-term (100 year) impacts on soil carbon are being integrated into a life-cycle assessment (LCA) of alfalfa in dairy supply chains being conducted by a collaborator at the University of Minnesota. Establishment problems are limiting for alfalfa production in some fields and are thought to be a result of oomycete and fungal pathogen complexes. Amplicon sequencing was used to characterize oomycete communities in fields with histories of establishment problems and a novel alfalfa oomycete pathogen, Phytophthora sansomeana was identified. Quantitative PCR assays and bioassays confirmed the population density of major alfalfa pathogens from amplicon sequencing. Characterization of alfalfa fungal and bacterial communities also include several potential alfalfa pathogens as targets for isolation/pathogenicity assays.

Accomplishments
1. Demonstrating the ecohydrological benefits of alfalfa in Midwest tile-drained landscapes. Increased reliance on corn silage as a primary dairy forage in place of perennial crops like alfalfa may result in tradeoffs with on-farm water balances and water quality. ARS scientists at Saint Paul, Minnesota, demonstrated that, while crop evapotranspiration and tile drainage flows were similar between silage corn and alfalfa, the latter reduced drainage loads of nitrate-nitrogen, phosphorus, and sediment by 72%, 33%, and 37%, respectively. However, overall productivity and water-use efficiency were higher for silage corn than alfalfa. These results highlight the value of alfalfa for managing on-farm water flows and water quality during critical periods of the year, though these environmental benefits must be balanced by farmers and regulators against the production gains that have driven the increased use of corn silage in the dairy industry.

2. Discovery of alfalfa pathogens using high throughput DNA sequencing. Stand failures from soil-borne diseases are major problems for alfalfa farmers. Identification of pathogens by isolation in pure culture is difficult, time consuming, and may overlook pathogens that are slow growing or that have specific growth requirements. ARS scientists at Saint Paul, Minnesota, and University of Minnesota collaborators identified entire microbial communities in soil and plant samples from eight sites with poor alfalfa seedling establishment by sequencing specific genes used for microbe identification. The relative population densities of specific pathogens from DNA sequencing were confirmed using quantitative PCR assays. Several novel alfalfa pathogens were identified and a widespread soybean pathogen, Phytophthora sansomeana, present in most samples was shown for the first time to cause root rot of alfalfa. Thus, microbial community analysis was found to be a rapid and robust way to identify pathogens that had previously not been known to cause disease in alfalfa seedlings. The identification of widespread seedling pathogens is useful for plant breeders to develop disease resistant cultivars and for seed marketers to use appropriate fungicidal seed treatments so that farmers can achieve vigorous, dense alfalfa stands.

3. Manure microbes have limited survival in field soils. Livestock manure is a common soil amendment in forage and row cropping systems used to provide plant-available nutrients, build soil organic matter, and enhance soil health. Many of the benefits of manure applications are thought to be driven by their impacts on soil microbial communities; however, manure amendments may also introduce potential undesirable microbes to soils. ARS scientists at Saint Paul, Minnesota, constructed soil microcosms with five soils from distinct locations using combinations of sterilized and unsterilized manure and soils to determine the importance of abiotic and biotic factors in the response of indigenous soil communities to manure and the fate of manure-borne microbes. Initial shifts in soil communities were driven by the introduction of manure-borne bacteria but the introduced microbes died off rapidly, within 30 days of application, while some indigenous populations increased in relative abundance over time. Introduced microbes died off even in the absence of indigenous microbiota, suggesting that the soil abiotic environment is a strong barrier to colonization by manure-borne bacteria. The response of indigenous soil populations to sterilized manure was like that of unsterilized manure, indicating that organic matter, nutrients, or other physiochemical factors associated with manure amendments are primary drivers of shifts in indigenous soil populations. These studies increase knowledge of the dynamics of soil microbial populations after manure amendments and the role of manure in improving soil health.

4. High potassium fertilization in alfalfa decreases boron concentrations but does not increase yield or reduce disease. Alfalfa has a high requirement for potassium with each ton of alfalfa herbage removing 50 pounds of potassium from the soil. Potassium fertilization on low potassium soils increases alfalfa yields, but the effects of additional fertilization to potassium sufficient soils are unknown. With the current increases in fertilizer costs, an understanding of when potassium fertilization is economical is needed. ARS scientists at Saint Paul, Minnesota, in collaboration with University of Minnesota scientists conducted a multi-year rotational study on the effects of potassium fertilization on soils containing sufficient potassium for alfalfa. The alfalfa plants continued to take up the available potassium, but without additional yield increases. Concentrations of boron, needed for plant growth and nitrogen fixation, decreased dramatically, as did calcium and sodium concentrations, suggesting that high levels of potassium fertilization might also require farmers to add boron to maintain alfalfa yields. There was no benefit to crown rot disease resistance with increased K fertilization. These results will help guide farmers, crop consultants, and extension personnel in recommendations to balance fertilizer requirements, expenses, and crop yields in alfalfa production systems.

Review Publications
Bucciarelli, B., Xu, Z., Ao, S., Cao, Y., Monteros, M., Topp, C., Samac, D.A. 2021. Phenotyping seedlings for selection of root system architecture in alfalfa (Medicago sativa L.). Plant Methods. 7. Article 125. https://doi.org/10.1186/s13007-021-00825-3.
Wang, Y., Yu, L., Zhang, J., Xu, Z., Samac, D.A. 2022. Overwintering and yield responses of two late-summer seeded alfalfa cultivars to phosphate supply. Agronomy. 12(2). Article 327. https://doi.org/10.3390/agronomy12020327.
Gamble, J.D., Baker, J.M., Dalzell, B.J., Wente, C.D., Feyereisen, G.W. 2022. Ecohydrology of irrigated silage maize and alfalfa production systems in the Upper Midwest US. Agricultural Water Management. 267. Article 107612. https://doi.org/10.1016/j.agwat.2022.107612.
Miller, S.M., Dornbusch, M.R., Farmer, A., Huertas, R., Gutierrez-Gonzalez, J.J., Young, N.D., Samac, D.A., Curtin, S.J. 2022. Alfalfa (Medicago sativa L.) pho2 mutant plants hyperaccumulate phosphate. G3, Genes/Genomes/Genetics. 12(6). Article jkac096. https://doi.org/10.1093/g3journal/jkac096.
Xu, Z., York, L.M., Seethepalli, A., Bucciarelli, B., Cheng, H., Samac, D.A. 2022. Objective phenotyping root system architecture using image augmentation and machine learning in alfalfa (Medicago sativa L.). Plant Phenomics. 2022. Article 9879610. https://doi.org/10.34133/2022/9879610.
Lipps, S.M., Samac, D.A. 2021. Pseudomonas viridiflava: An internal outsider of the Pseudomonas syringae species complex. Molecular Plant Pathology. 23(1):3-15. https://doi.org/10.1111/mpp.13133.
Medina, C., Samac, D.A., Yu, L. 2021. Pan-transcriptome identifying master genes and regulation network in response to drought and salt stresses in alfalfa (Medicago sativa L.). Scientific Reports. 11. Article 17203. https://doi.org/10.1038/s41598-021-96712-x.
Williams, M.R., Welikhe, P., Bos, J.H., King, K.W., Akland, M., Augustine, D.J., Baffaut, C., Beck, G., Bierer, A.M., Bosch, D.D., Boughton, E., Brandani, C., Brooks, E., Buda, A.R., Cavigelli, M.A., Faulkner, J., Feyereisen, G.W., Fortuna, A., Gamble, J.D., Hanrahan, B.R., Hussain, M., Kohmann, M., Kovar, J.L., Lee, B., Leytem, A.B., Liebig, M.A., Line, D., Macrae, M., Moorman, T.B., Moriasi, D.N., Nelson, N., Ortega-Pieck, A., Osmond, D., Pisani, O., Ragosta, J., Reba, M.L., Saha, A., Sanchez, J., Silveira, M., Smith, D.R., Spiegal, S.A., Swain, H., Unrine, J., Webb, P., White, K.E., Wilson, H., Witthaus, L.M. 2022. P-FLUX: A phosphorus budget dataset spanning diverse agricultural production systems in the United States and Canada. Journal of Environmental Quality. 51:451–461. https://doi.org/10.1002/jeq2.20351.
Baker, J.M., Feyereisen, G.W., Albrecht, K.A., Gamble, J.D. 2022. A perennial living mulch substantially increases infiltration in row crop systems. Journal of Soil and Water Conservation. 77(2):212-220. https://doi.org/10.2489/jswc.2022.00080.
Mowers, R.P., Bucciarelli, B., Cao, Y., Samac, D.A., Xu, Z. 2022. Good statistical practices in agronomy using categorical data analysis, with alfalfa examples having poisson and binomial underlying distributions. Crops. 2(2):154-171. https://doi.org/10.3390/crops2020012.
Giles, J.M., Tordsen, C.L., Rebstock, T.R., Bucciarelli, B., Samac, D.A., Sathoff, A.E. 2022. Management strategies and distribution of Aphanomyces root rot of alfalfa (Medicago sativa), a continuing threat to forage production in the United States. Plant Pathology. 71(6): 1231-1240. https://doi.org/10.1111/ppa.13563.
Heuschele, D.J., Furuta, D., Smith, K.P., Marchetto, P. 2022. Capturing high resolution plant movement in the field. Integrative & Comparative Biology. icac075. https://doi.org/10.1093/icb/icac075.
Pinson, S.R., Heuschele, D.J., Edwards, J., Jackson, A.K., Sharma, S., Barnaby, J.Y. 2022. Relationships among arsenic-related traits, including grain arsenic concentration and rice straighthead resistance, as revealed by genome-wide association. Frontiers in Genetics. https://doi.org/10.3389/fgene.2021.787767.
Schlatter, D.C., Hansen, J.C., Carlson, B.R., Leslie, I.N., Huggins, D.R., Paulitz, T.C. 2022. Are microbial communities indicators of soil health in a dryland wheat cropping system? Applied Soil Ecology. 170. Article 104302. https://doi.org/10.1016/j.apsoil.2021.104302.
Reardon, C.L., Klein, A.M., Melle, C.J., Hagerty, C.H., Klarer, E.R., Machado, S., Paulitz, T.C., Pritchett, L., Schlatter, D.C., Wuest, S.B. 2022. Enzyme activities distinguish long-term fertilizer effects under different soil storage methods. Applied Soil Ecology. 177. Article 104518. https://doi.org/10.1016/j.apsoil.2022.104518.