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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

2014 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 aims to increase alfalfa yields and utilization for livestock and cellulosic biomass and to amplify alfalfa’s environmental services by contributing new knowledge from genes to fields. Progress was made on all project objectives. Under Objective 1 progress was made on developing strategies and tools for improving alfalfa yields, disease resistance, and nutrient cycling efficiency. The second year of data were collected from field trials in Minnesota and Wisconsin to evaluate forage yield potential between semi-hybrid and synthetic populations as well as populations selected for yield per stem. The initial goals for developing DNA markers for accelerating breeding for disease resistance were accomplished. Over 60 alfalfa plant introductions from the National Plant Germplasm System were screened for resistance to three races of the root pathogen Aphanomyces euteiches. Two lines that showed race non-specific resistance were characterized further and compared to cultivars with race-specific resistance. Race-specific resistance prevents pathogen infection by means of a rapid hypersensitive response while race non-specific resistance allows the pathogen to enter the outer cells of the stem, but not the inner portion. Additionally, resistant and susceptible plants were selected from two cultivars with race-specific resistance and a susceptible cultivar. A method to validate resistance in plants from vegetative cuttings was developed as well as a method to rescue susceptible plants after inoculation with the pathogen. Selected plants from race-specific and race non-specific lines will be used for genetic and genomic analysis to identify markers associated with resistance. Bacterial stem blight of alfalfa is a disease likely to increase in frequency due to climate change. In collaboration with scientists at the University of Exeter, the genome of the pathogen was sequenced and the genes present identified. Based in these analyses, the alfalfa pathogen is most closely related to pathogens of beet and pear in the same species complex; however, the alfalfa pathogen caused only mild symptoms on these plants. A standardized test was developed for screening plants for resistance to bacterial stem blight and 16 cultivars have been tested. Cultivars with greater fall dormancy were found to have a higher percentage of resistant plants. Under Objective 2 progress was made in developing forage germplasm with modified cell wall structure and chemistry to improve livestock and bioenergy productivity. Final field samples from experiments evaluating selection for in vitro neutral detergent fiber digestibly (IVNDFD) and its impact on livestock nutrition and biofuel conversion efficiency were assayed for forage quality traits and data sets are ready for statistical analyses. The first production year samples were harvested from field trials to investigate whether a near infrared spectroscopy (NIRS) selection methodology used to create alfalfa populations that differ in stem IVNDFD can be used to develop alfalfa with increased whole forage IVNDFD. The effect of over-expression of sucrose synthase, an important enzyme in energy metabolism, on growth of alfalfa was examined. In contrast to what was expected, sucrose synthase levels decreased due to a phenomenon called gene silencing in which an inserted gene expressed at high levels can turn off similar genes. Plant growth was reduced primarily because nodules on the roots of plants containing the extra sucrose synthase gene exhibited premature senescence. The results demonstrated the critical role of sucrose synthase in nitrogen fixation in alfalfa root nodules. Progress was made in developing genomic resources for alfalfa. Modern alfalfa cultivars are the result of hybridization of Medicago sativa sub-species sativa and M. sativa sub-species falcata. In collaboration with scientists at the Noble Foundation, the first atlas of expressed genes in alfalfa was developed. Expressed gene sequences, gene sequence polymorphisms, the level of expression of each gene in roots, leaves, stems, and flowers, and the function of each gene was determined and assembled into a database for use by alfalfa breeders and geneticists. Under Objective 3 progress was accomplished in developing alfalfa germplasm and crop management systems to improve nitrogen cycling and carbon sequestration. Samples from an intensive four-year project to determine the performance of alfalfa selected for high or low nitrate uptake capacity have been analyzed and data sets are ready for statistical analyses. The first steps in characterizing the microbial communities associated with alfalfa roots were carried out. Total DNA was obtained from soil samples around alfalfa roots from three types of alfalfa grown in three locations under differing soil fertility, diagnostic sequences were amplified to be able to identify the bacteria and fungi present, and the DNA submitted for sequencing. In parallel, bacteria were isolated from soil samples and initial characterization of each community was done. Due to the rapid completion of work on Subobjective 3.3 to measure and predict nitrogen credits for second-year corn grown after alfalfa to improve nitrogen management and reduce nitrogen losses, further research is underway to validate the results obtained. First, development of a database is nearly complete for use in devising prediction equations for nitrogen response in second-year corn after alfalfa. Second, new field trials on Minnesota farms have been established to acquire validation data for first-year corn after alfalfa on fine-textured soils. Third, data from an unpublished multi-location trial that compared nitrogen requirements for first-year corn after alfalfa of different stand ages have been collated and statistically analyzed and a technical manuscript is being prepared. Lastly, samples were collected from long-term field trials in the region to evaluate whether a particular soil test method helps predict fertilizer nitrogen response in corn following alfalfa.

1. A mineral seed treatment for control of seedling diseases of alfalfa suitable for organic production systems. The most common fungicide used on alfalfa seed does not protect against all soil-borne diseases and cannot be used in organic production systems. ARS researchers at St. Paul, Minnesota found that a novel mineral seed treatment using aluminosilicate (natural zeolite), which is allowed under the National Organic Plan (NOP) Rule 205.203(d)(2), gave significantly greater control of major seedling diseases of alfalfa than the Apron XL seed treatment. The mineral treatment resulted in excellent control of multiple races of the pathogen causing Aphanomyces root rot for which Apron XL is ineffective. The mineral seed treatment resulted in a similar or greater percentage of protected plants than the Apron XL treatment in field soils with a range of disease pressure. The mineral treatment had no effect on symbiotic bacteria needed for nitrogen fixation. These experiments indicate that the zeolite mineral seed treatment is a promising new means of controlling seedling diseases in conventional and organic alfalfa production systems.

2. Knowing your goal is the key to getting there. There is considerable uncertainty about reported yields of alfalfa and other perennial forages because unlike commodity crops (like grains), silage and dry hay crops are not often weighed, so yields are rough estimates. In addition, there is concern that perennial forages are not managed for optimum yield. In this research, an ARS scientist in Saint Paul, Minnesota, collected yield data from small plot field trials in several states to test alfalfa variety performance from measurements of whole-field yields in Wisconsin and from reports by forage extension agronomists in several states. It appears that 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.

3. Stepping back provides better perspective. For over 50 years, small field trials in the US and Canada have shown that the first year of corn grown after alfalfa rarely needs fertilizer nitrogen to produce optimum yields. To determine how often corn actually needs more fertilizer after alfalfa on non-sandy soil, an ARS scientist in Saint Paul, Minnesota and University of Minnesota scientists compiled the largest dataset ever published on this crop rotation. They found that first-year corn needed 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, and the timing of alfalfa termination could separate the fields that respond to nitrogen from those that do not and these measurements also help to determine how much fertilizer nitrogen to apply. Field experiments are in progress to determine if these measurements can be used to predict nitrogen response on farmer's fields.

4. Improving nutritive value of alfalfa for livestock. 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, ARS scientists at St. Paul, Minnesota tested alfalfa lines shown previously to have a range of fiber contents 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. These results show that plants with greater digestibility can be identified from field grown plants independent of growth conditions and provide plant breeders with the tests necessary to identify superior plants with high nutritive value.

5. The view from space gives new insight into field management. Alfalfa is a perennial forage crop that provides benefits to the crops that follow it. It promotes higher soil nitrogen supply, so less fertilizer is needed; it improves soil condition and root penetration, so the next crops can send roots deeper and so more rainfall enters the soil and less runs off; and it lowers weed and pest populations that would have reduced yield of the next crop. However, alfalfa can overly dry the soil, resulting in lower yield of the next crop and may expose the soil to water or wind erosion. 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. An ARS scientist in Saint Paul, Minnesota and University of Minnesota scientists produced the first assessment of alfalfa stand length and identification of the first and second crops grown after alfalfa termination in the Upper Midwest from annual maps produced by the National Agricultural Statistics Service. 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.

Review Publications
Yost, M.A., Morris, T.F., Russelle, M.P., Coulter, J.A. 2014. Second-year corn after alfalfa often requires no fertilizer nitrogen. Agronomy Journal. 106(2):659-669.
Griffis, T.J., Lee, X., Baker, J.M., Russelle, M.P., Xhang, X., Venterea, R.T., Millet, D.B. 2013. Reconciling the differences between top-down and bottom-up estimates of nitrous oxide emissions for the US corn belt. Global Biogeochemical Cycles. 27:746-754.
Samac, D.A., Allen, S., Witte, D., Miller, D., Peterson, J. 2014. First report of race 2 of Colletotrichum trifolii causing anthracnose on alfalfa (Medicago sativa) in Wisconsin. Plant Disease. 98(6):843.
Samac, D.A., Halfman, B., Jensen, B., Brietenbach, F., Behnken, L., Willbur, J.F., Undersander, D., Blonde, G., Lamb, J.F. 2013. Evaluating Headline fungicide on alfalfa production and sensitivity of pathogens to pyraclostrobin. Plant Health Progress. DOI: 10.1094/PHP-2013-0917-01-RS.
Yost, M.A., Russelle, M.P., Coulter, J.A. 2014. Field-specific fertilizer nitrogen requirements for first-year corn following alfalfa. Agronomy Journal. 106(2):645-658.
Lamb, J.F., Jung, H.G., Riday, H. 2014. Growth environment, harvest management and germplasm impacts on potential ethanol and crude protein yield in alfalfa. Biomass and Bioenergy. 63:114-125.
Lamb, J.F., Jung, H.G., Samac, D.A. 2014. Environmental variability and/or stability of stem fiber content and digestibility in alfalfa. Crop Science. DOI: 10.2135.cropsci2014.04.0323.
Russelle, M.P. 2013. The alfalfa yield gap: A review of the evidence. Forage and Grazinglands. DOI: 10.1094/FG2013-0002-RV.
Samac, D.A., Willbur, J.F., Behnken, L., Brietenbach, F., Blonde, G., Halfmann, B., Jensen, B., Sheaffer, C.C. 2014. First report of Stemphylium globuliferum causing Stemphylium Leaf Spot on alfalfa (Medicago sativa) in the U.S. Plant Disease. 98(7):993. Available: