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ARS Home » Midwest Area » St. Paul, Minnesota » Plant Science Research » Research » Research Project #424185

Research Project: Enhanced Alfalfa Germplasm and Genomic Resources for Yield, Quality, and Environmental Protection

Location: Plant Science Research

2018 Annual Report

Objective 1: Develop strategies and tools for improving alfalfa yields, disease resistance, and nutrient cycling efficiency. Sub-objective 1.1: Identify and compare breeding strategies for alfalfa yield improvement. Sub-objective 1.2: Identify strategies and develop tools for reducing losses from diseases. Sub-objective 1.3: Develop strategies to produce alfalfa germplasm with improved herbage yield and nutrient cycling (phosphorus (P) and potassium (K)) in pure stands and in mixtures with forage grasses. Sub-objective 1.4: Develop a gene expression atlas for two divergent alfalfa gene pools (Medicago sativa subsp. sativa and M. sativa subsp. falcata), identify organ-specific genes, and mine sequences for gene pool diversity (SNPs and SSRs). Objective 2: Develop forage germplasm with modified cell-wall structure and chemistry to improve digestibility, and evaluate impacts on livestock and bioenergy productivity. Sub-objective 2.1: Assess alfalfa germplasm altered for in vitro neutral detergent fiber digestibility (IVNDFD) for forage yield and energy availability for livestock production and biofuel conversion efficiency. Sub-objective 2.2: Identify genetic, metabolic, and developmental processes in alfalfa stems that regulate cell wall composition and energy availability. Objective 3: Develop alfalfa germplasm and crop rotation management systems to improve nitrogen cycling and carbon sequestration. Sub-objective 3.1: Identify and utilize mechanisms to improve nutrient uptake in alfalfa. Sub-objective 3.2: Identify and characterize rhizosphere soil microbes that promote carbon sequestration and improve the agronomic and environmental benefits from crop rotation. Sub-objective 3.3: Measure and predict N credits for second-year corn grown after alfalfa to improve N management and reduce N losses.

To increase yield potential, the contribution of heterosis to yield potential and the effectiveness of selecting for high yield per stem as a yield component in alfalfa will be assessed. Synthetic populations, semi-hybrid populations, parent populations, and two commercial varieties will be established in replicated field trials and total annual forage yield will be evaluated for at least two production years. We will develop DNA markers to increase disease resistance and measure diversity in pathogen populations. A bulked segregant analysis will be done using populations segregating for resistance to Aphanomyces root rot. Histochemical and gene expression studies will be used to gain an understanding of the infection process and mechanisms of resistance in resistant and susceptible plants. Simple sequence repeat (SSR) markers will be identified in the Verticillium albo-atrum genome sequence and tested for polymorphisms on field isolates and standard strains. Plant responses to these strains will be measured with the standard disease severity scale and the amount of pathogen present determined by the qPCR method. Plants with the lowest amount of pathogen present will be retained, intermated, and progeny tested for Verticillium wilt resistance. DNA markers will also be developed from a gene expression atlas for two divergent alfalfa gene pools using transcripts from leaves, roots, nodules, flowers, and elongating and post-elongation stem internodes. To develop alfalfa germplasm with improved herbage yield and nutrient cycling, germplasm differing in root system architecture will be examined in replicated field experiments to determine: P and/or K uptake capacity under low and adequate soil nutrient levels; symbiosis with arbuscular mycorrhizal (AM) fungi; and prevalence of root and foliar diseases. Alfalfa germplasm selected for in vitro neutral detergent fiber digestibility (IVNDFD) and original parents will be evaluated in replicated field trials for forage quality traits, gain from selection, and heritability estimates. Replicated sward plot field trials will be used to determine forage yield and the best crop management methods for germplasm selected for IVNDFD. Energy availability for livestock and biofuel conversion in the harvested forage will be determined by near infrared reflectance spectroscopy. To improve alfalfa stem cellulose content, a comparison of miRNA profiles in elongating stem and post-elongation stem internodes will be used to identify miRNAs that play key roles in the development of secondary xylem. The microbial communities in the rhizosphere that influence plant growth and carbon sequestration will be characterized using culture-dependent and metagenomics approaches. Field tests will determine whether selection for nitrate uptake alters yields of alfalfa-grass mixtures. On-farm field experiments will be established at 10 locations to improve predictions of whether nitrogen contributed by alfalfa to subsequent corn crops will improve farm profit and reduce nitrogen losses.

Progress Report
This project terminated in 2018. The project successfully developed strategies and tools for improving alfalfa yields, disease resistance, and nutrient cycling efficiency. The National Agricultural Statistics Service reports of alfalfa yields for the past 20 years have been stagnant across the US. However, there is considerable uncertainty about reported yields of alfalfa and other perennial forages because unlike commodity crops, silage and dry hay crops often are not weighed, so yields are rough estimates. In addition, there is concern that perennial forages are not managed for optimum yield. Yield information was collected from small plot field trials, from measurements of whole-field yields in Wisconsin, and from reports by forage extension agronomists in several states. Top alfalfa growers produce yields two to three times larger than reports from average growers. This yield gap is many times larger than yield gaps reported for other crops. Although the reasons for the alfalfa yield gap probably vary among farmers, knowing what can be produced may help them improve their crop management to achieve those higher yields. Selection for yield in alfalfa has focused on aboveground plant traits, largely ignoring the potential contribution of the root system to improve yield. Previous research found that alfalfa plants with a highly branched root system supported greater forage yields than plants with a typical root system; however, selection required a minimum of 20 weeks to identify plants with the branching root phenotype. A method to identify plants with a strong taproot or strong branch roots after only 2 weeks of growth was developed. Several candidate genes were identified that were associated with the branching root phenotype. One of the reasons that gains in alfalfa forage yield have lagged behind those of annual grain and seed crops is the lack of tools for crop improvement. To partially fill this gap, the first comprehensive guide to the genes in the crop was developed. A website was developed for accessing tissue-specific genes, identifying gene expression differences among tissues, retrieving gene sequences, and developing DNA markers for specific genes. Genes involved in cold tolerance, nutritional quality, antioxidant production, and nitrogen fixation were identified. This database increased the availability of alfalfa gene sequences nine-fold and will be an essential tool used by plant breeders for alfalfa improvement. Root and crown diseases are a major impediment to achieving highly productive alfalfa stands. Failure of cultivars with disease resistance had been observed suggesting the presence of new or emerging diseases. Surveys conducted in commercial alfalfa production fields found that fields frequently cropped to alfalfa had highly aggressive strains of the Aphanomyces root rot pathogen not controlled by current resistant varieties and had isolates of Pythium causing seed rot and damping off that were not controlled by current seed treatments. Resistant plants were identified and the mechanism for resistance to Aphanomyces root rot was found to involve rapid responses of infected root cells that limits the penetration of the pathogen followed by production of chemical barriers in the root that prevent further infection. The chromosomal location of disease resistance genes, DNA sequences associated with resistance, and candidate resistance genes were determined. The DNA markers may be used to accelerate selection of resistant plants and make selection of plants with broad resistance to the pathogen more accurate. Selection for plants with resistance to Pythium seed rot and damping off was successful and increased resistance significantly. Bacterial wilt of alfalfa occurs throughout most alfalfa growing regions worldwide and for this reason the pathogen is considered a phytosanitary risk for international seed movement. The lack of a complete genome sequence for the pathogen causing this disease has hampered progress in understanding pathogen biology and developing tests to detect the pathogen. The complete genome sequence was obtained from three strains of the bacterium using two sequencing methods, resulting in very high quality sequence information. Economic damage of alfalfa fields by late spring and early fall frosts have increased in the past several years and in some locations increased sensitivity to frost was associated with herbicide application. Damaged alfalfa was infected by the bacterial stem blight pathogen, which as the ability to increase temperatures at which frost damage occurs. The genome of the pathogen was sequenced and comparisons with other bacterial genomes found that it is closely related to bacteria infecting pear and beet. Methods were developed for identifying resistant alfalfa plants, cultivars with moderate numbers of resistant plants were identified, and gene transcripts in resistant and susceptible plants quantified. Fuller characterization of the pathogen, disease resistance mechanisms, and mapping of disease resistance genes is ongoing. Many plant diseases are caused by a complex of multiple pathogens that include several bacteria and fungi. Crown rot of alfalfa is caused by such a complex of pathogens and is found in all alfalfa stands more than 2 years old, resulting in yield losses and decreased stand life. Antimicrobial peptides were identified with activity against crown rot pathogens and when expressed in alfalfa plants provide enhanced resistance to several crown rot pathogens. Top performing plants are being used in crosses with adapted alfalfa germplasm for future field-testing. The project successfully developed forage germplasm with modified cell-wall structure and chemistry to improve digestibility. High concentrations of poorly digestible fiber in alfalfa forage limits feed intake and energy availability in dairy and beef production systems. Nutritive quality in alfalfa forage is affected by environmental growth conditions, hampering efforts to breed alfalfa with improved nutritional value. Focusing on the fiber-rich stem portion of the plant, alfalfa lines shown previously to have a range of fiber contents were tested in 12 growing environments to identify specific traits that could be used for selecting superior plants. Three traits, total fiber content in stems, total lignin content measured as a proportion of the fiber content, and degradation of stems after 96 hours in an assay simulating digestion by cattle were found to be the most consistent in the different environments. Plants with the highest and lowest degradation after 96 hours were intercrossed and the resulting populations evaluated in two locations over two production years. The selection method resulted in populations with greater fiber digestibility and reduced amounts of total lignin in plant stems at longer harvest intervals. After seed increases, germplasm will be released for use by the alfalfa breeding community. The project successfully developed crop rotation management systems to improve nitrogen cycling. For over 50 years, small field trials in the U.S. and Canada have shown that the first year of corn grown after alfalfa rarely needs fertilizer nitrogen to produce optimum yields. A large dataset was analyzed to determine how often corn actually needs more fertilizer after alfalfa. First-year corn needs extra fertilizer nitrogen about 20% of the time on silty soils and about one-half the time on clayey soils. A few simple measurements, rainfall and temperature, the age of the alfalfa stand, and the timing of alfalfa termination, separated the fields that respond to nitrogen. Results from 28 on-farm trials validated the small field trials and found that about 50% of the time alfalfa provides all the nitrogen needed for both the first and second crops of corn, saving about $160 per acre and reducing the amount of fossil fuel needed to produce corn. The decision to rotate from alfalfa to the next crop relies on several factors, but there has been no quantitative information about how long alfalfa stands are kept nor which crops are grown afterward. The first assessment of alfalfa stand length and identification of the first and second crops grown after alfalfa termination in the Upper Midwest was determined from annual maps produced from satellite images. The resulting maps and graphs provide researchers, Extension educators, and agricultural professionals information that will help them design education and research programs to solve problems specific to each area. Alfalfa provides benefits to the next crops but annual crops have replaced alfalfa in many rotations. To increase alfalfa utilization, a sustainable protein for aquaculture was developed from alfalfa. The growing demand for fish and seafood products is accelerating development of aquaculture nationwide and alternative feed ingredients are needed to meet these demands. A protein concentrate made from alfalfa foliage was found to be a suitable replacement for fishmeal in the diet for yellow perch and rainbow trout. Several methods were tested for producing the protein concentrate from leaves of a biomass type of alfalfa and a simple heat treatment after juicing was found to result in the highest yield of protein concentrate. Alfalfa stems, the press cake resulting from leaf juicing, and the de-proteinized juice have potential as additional value-added products in biorefining of alfalfa. High value products derived from alfalfa will increase the value of the crop and farm gate revenue.

1. Methods for accurately identifying soil microbial communities. The majority of soil microbes important for soil health, nutrient cycling and carbon sequestration cannot be cultured on artificial laboratory media but can be inventoried using high throughput sequencing of total DNA from soil samples. However, information on the number of samples and replicates that are needed to accurately represent the complete assemblage and influence of sampling method on results had not been investigated previously. ARS scientists in Saint Paul, Minnesota and University of Minnesota colleagues demonstrated that collecting several replicated individual samples or creating a composite sample may be most suitable for microbial community studies in agricultural soils. Experiments were also done to investigate the effect of diluting DNA samples prior to sequencing on estimates of soil fungal diversity. Absolute quantification of DNA prior to sequencing to ensure that the target sequence exceeds the sequencing depth of the sample was found to eliminate bottlenecks to identification of sequences found at a low frequency. Accurate and economical inventories of soil microbes will enable researchers and producers to develop approaches for achieving healthy soils for sustainable crop production.

2. Selection enhances resistance to seed rot and damping off in alfalfa. Rapid, vigorous, and uniform seed germination is key to establishing a productive, resilient, and long-lasting alfalfa stand. However, seeds are often attacked and killed by pathogens in soil, which impairs water and nutrient uptake of adult plants, or are attacked and killed (damped off) after germination. Disease resistance would protect seeds and seedlings and provide longer control to these pathogens, many of which are not well controlled by current fungicide seed treatments. Alfalfa seeds from three different genetic backgrounds were selected by ARS scientists in Saint Paul, Minnesota to identify seedlings with resistance to a highly aggressive seed rot and damping off pathogen, then the resistant plants were intercrossed to produce seeds. Approximately one-third of the plant lines tested were found to have at least 50% of the seeds resistant to the highly aggressive strain of the pathogen, a 10-fold increase in the percentage of resistant seeds and seedlings, and to have similar or higher resistance to additional strains of the pathogen. These results demonstrate that rapid progress can be made for enhancing resistance to seed rot and damping off pathogens of alfalfa for improving disease resistance in alfalfa varieties.

3. Identification of more competitive nitrogen fixing bacteria for use in alfalfa production. Most alfalfa seed is treated with symbiotic bacteria prior to planting to ensure the formation of nitrogen-fixing nodules on roots. Improving nitrogen fixation would reduce reliance on synthetic fertilizers, but establishment of superior bacterial strains is hampered by competition by indigenous, less effective bacteria. ARS scientists in Saint Paul, Minnesota and University of Minnesota colleagues developed methods for identifying the origin of bacteria in root nodules in two field sites that had not been in alfalfa cultivation for over 30 years. All bacteria in nodules originated from soil rather than from seed inoculation and were genetically diverse. However, approximately one-third of the bacterial strains in nodules had a gene involved in transfer of bacterial proteins to plant cells, which appears to accelerate nodulation, potentially making these strains more competitive in forming root nodules. This gene gives researchers a marker to rapidly identify more competitive strains that would be more effective as seed inoculants. Increasing nitrogen fixation and the amount of nitrogen available to alfalfa plants will increase crop yields without increasing costs for crop production.

Review Publications
Cao, Y., Miller, S.S., Dornbusch, M.R., Castle, S., Lenz, P., Ferguson, J., Sadowsky, M.J., Nelson, M.S., Klatt, C.G., Samac, D.A. 2018. Widespread occurrence of Sinorhizobium meliloti strains with a type IV secretion system. Symbiosis. 75(2):81-91.
Berg, L.E., Miller, S.S., Dornbusch, M.R., Samac, D.A. 2017. Seed rot and damping-off of alfalfa in Minnesota caused by Pythium and Fusarium species. Plant Disease. 101:1860-1867.
Grev, A.M., Wells, M., Samac, D.A., Martinson, K.L., Sheaffer, C.C. 2017. Forage accumulation and nutritive value of reduced lignin and reference alfalfa cultivars. Agronomy Journal. 109:2749-2761.
Lu, Y., Ishimaru, C.A., Glazebrook, J., Samac, D.A. 2018. Comparative genomic analyses of Clavibacter michiganensis subsp. insidiosus and aggressiveness on Medicago truncatula. Phytopathology. 108:172-185.
Peterson, J.J., Samac, D.A., Grau, C.R. 2018. First report of Fusarium wilt of alfalfa caused by Fusarium oxysporum f. sp. medicaginis in Wisconsin. Plant Disease. 102:447.
Ao, S., Bucciarelli, B., Dornbusch, M.R., Miller, S.S., Samac, D.A. 2018. First report of alfalfa (Medicago sativa L.) seed rot, seedling root rot, and damping off caused by Pythium spp. in Sudanese soil. Plant Disease. 102(5):1043.
Tahir, M.N., Holland, C., Samac, D.A., Mollov, D.S. 2017. First report of 16SrII subgroup D phytoplasma in alfalfa in Sudan. Plant Disease. https://doi:10.1094/PDIS-04-17-0603-PDN.
Nemchinov, L.G., Shao, J.Y., Lee, M., Postnikova, O.A., Samac, D.A. 2017. Resistant and susceptible responses in alfalfa (Medicago sativa) to bacterial stem blight caused by Pseudomonas syringae pv. syringae. PLoS One. 12(12):e0189781.