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United States Department of Agriculture

Agricultural Research Service


Location: Plant Science Research

2009 Annual Report

1a. Objectives (from AD-416)
Objective 1: Develop germplasm and determine genetic and biological processes that regulate forage use for bioenergy and livestock production. Sub-objective 1.1. Identify genes and breeding strategies to be used for alfalfa improvement. Sub-objective 1.2. Improve energy availability from forages by modifying genetic, metabolic, and developmental processes that control cell wall synthesis and breakdown. Sub-objective 1.3. Identify and utilize mechanisms to improve nutrient uptake in Medicago spp. Objective 2: Develop and evaluate crop management strategies to increase use of perennial forages for livestock and bioenergy, and to protect the environment. Sub-objective 2.1. Develop management practices and systems to optimize alfalfa composition and biomass yield for the efficient production of liquid fuels and syngas. Sub-objective 2.2. Evaluate strategies to reduce root and foliar disease in alfalfa. Sub-objective 2.3. Develop and test management strategies to expand the role of alfalfa and other perennial forages in protecting water quality.

1b. Approach (from AD-416)
Alfalfa is the backbone of sustainable practices for producing crops and animals while protecting water and soil resources. However, use of alfalfa is not always maximized due to several limitations, and new germplasm and management systems are needed for biofuel production. The objectives of this project are to: (1) develop germplasm and determine genetic and biological processes that regulate forage use for bioenergy and livestock production, and (2) develop and evaluate crop management strategies to increase use of perennial forages for livestock and bioenergy, and to protect the environment. Alfalfa germplasm with greater stem in vitro neutral detergent fiber digestibility (IVNDFD) will be developed through selection and breeding utilizing near-infrared reflectance spectroscopy. Populations will be assessed for changes in cell wall chemistry and biofuel conversion under conventional and biomass management systems. Breeding strategies will be evaluated to increase yield potential. Yield will be evaluated in replicated field trials in multiple locations. Populations with highest yield will be evaluated for total forage yield under conventional and biomass management systems. Management methods for reducing diseases that impact yield and persistence will be assessed. The effect of glyphosate on foliar diseases (rust, powdery mildew, anthracnose, spring black stem) will be evaluated in Roundup Ready alfalfa. Alfalfa cultivars will be screened for resistance to brown root rot and the role of crop debris in pathogen survival and increase will be measured. The potential for green manures and traffic tolerance to reduce crown rot will be evaluated. Genes for disease resistance, cell wall biosynthesis, and nutrient uptake will be isolated to provide new knowledge on the genetic underpinnings of these traits and markers for plant improvement. Transcript profiling will be done to identify candidate genes involved in these traits. The function of specific genes will be investigated through detailed transcript expression studies, investigating promoter activity, biochemical assays, over-expression, and gene knock down. Alfalfa germplasm with greater capacity to remove soil nitrate will be developed by recurrent selection using bromide as a nitrate analog. Populations will be evaluated in multiple field locations for bromide and nitrate uptake. Removal of nitrogen from soil by alfalfa will be tested at the field scale in replicated plots in multiple locations. Forage samples will be analyzed for dry matter and N yield; topsoil will be measured for total N and C. Removal of nitrate from irrigation water via phytofiltration will be tested on a field scale. Replicated plots will be irrigated at normal, high, and intermediate rates and the total nitrate leached determined. Biocurtain strips along tile drainage sites will be managed using conventional and biomass systems along side a corn-soybean rotation. Inorganic N will be measured throughout the growing season in tile drainage water. Fulfilling the objectives will help meet the emerging needs of the nation in livestock and bioenergy production and environmental protection.

3. Progress Report
Progress in developing germplasm and determining genetic and biological processes that regulate forage use for bioenergy and livestock production include the following: 1) Eight populations selected for increased or decreased in vitro neutral detergent fiber digestibility were established in field trials at two locations along with two unselected populations. Material was harvested for testing differences in livestock digestibility and biofuel conversion efficiency. Also, 145 hybrid and synthetic alfalfa populations are under evaluation for forage yield potential at two Upper Midwest locations. 2) Genetic mapping of powdery mildew resistance in Medicago truncatula was done using 136 inbred lines. An additional 500 plants were screened with flanking markers to identify lines to fine map the gene. Several candidate resistance genes were characterized from resistant and susceptible lines. Transcript profiling identified differentially expressed genes between susceptible and resistant lines. 3) A protocol was developed that detected single-feature polymorphisms (SFPs) in genes of two alfalfa genotypes that differ in cell wall cellulose and lignin concentrations. More than 10,000 SFPs were detected. 4) Alfalfa transformants containing RNA interference constructs for genes involved in cell wall biosynthesis were screened to determine the level of gene down-regulation and three lines were selected for further analysis. 5) Putative promoter regions were isolated for lupin genes involved in phosphate uptake, tonneau 1, cytokinin oxidase, glycerophosphate phosphodiesterase, and auxin influx. Reporter gene activity showed that Medicago, lupin, and bean have similar phosphate signal transduction systems for the genes tested. Progress in developing and evaluating crop management strategies to increase use of perennial forages for livestock and bioenergy includes the following: 1) Twenty alfalfa stem samples were selected from six field studies for constructing a robust near infrared spectroscopy (NIRS) calibration for ethanol production. These samples were analyzed for cell wall composition and ethanol production. 2) A field evaluation of 26 cultivars identified those that persisted well under pressure from the fungal disease brown root rot. Rotation crops in which the pathogen decreased over time were identified. Key genes of the pathogen were sequenced to evaluate pathogen diversity within and across locations. 3) We completed the first year of field testing a new method of estimating soil N uptake and symbiotic nitrogen fixation by alfalfa. The first experiment on the effect of soil texture on phytofiltration efficiency was completed. We found that this method of using plants to remove excess nitrate from water is not effective when fine-textured soil horizons are thicker than about 1 foot.

4. Accomplishments
1. Genes to Improve Alfalfa as a Bioenergy Crop Identified Alfalfa, a nitrogen-fixing perennial forage widely grown in the U.S., offers considerable potential as a bioenergy crop. Identifying genes that control cell wall composition and content of alfalfa stems will advance the development of this non-food crop as a biofuel feedstock; however, an alfalfa GeneChip that can be used for measuring gene expression is not available. To overcome this limitation, scientists in the Plant Science Research Unit in St. Paul, MN, used the GeneChip developed for barrel medic, a close relative of alfalfa, to investigate gene expression in stems of alfalfa plants selected for cell wall polymers important in efficient conversion of lignocellulosic biomass to ethanol. A protocol was developed that increased the number of alfalfa genes detected on the barrel medic GeneChip. Numerous genes that regulate polymer biosynthesis in alfalfa were identified for the first time. The research demonstrated that the barrel medic GeneChip could be used successfully to identify genes for improving alfalfa as a bioenergy crop.

2. Management Strategies for Reducing Damage from the Fungal Disease Brown Root Rot of Alfalfa Brown root rot of alfalfa is a fungal disease widespread in the northern U.S. that causes winter kill of plants and significantly reduces stand life and forage yield. Management strategies to reduce damage are needed because chemical control is not possible. Collaborative research between ARS in St. Paul, MN, and University of Minnesota scientists found that corn and soybean residues increase pathogen populations while spring wheat, oat, and canola residues decrease pathogen populations. Multi-year field trials in Minnesota identified alfalfa varieties that persist in locations with high amounts of the brown root rot fungus, are well adapted to the region, and have high forage yield potential. Based on this research we recommend that alfalfa fields suffering crop loss from brown root rot can be brought back into high productivity by rotating crops with a spring-seeded small grain crop and by planting brown root rot tolerant varieties.

Review Publications
Gronwald, J.W., Jung, H.G., Litterer, L.A., Somers, D.A. 2009. Comparison of Post-Germination Mobilization of Cell Wall Polysaccharides and Non-Cell Wall Carbohydrates in Soybean (Glycine max L.) Cotyledons. Journal of the Science of Food and Agriculture. 89(11):1981-1986.

Gebeyaw, M.T., Samac, D.A., Lamb, J.F. 2008. Alfalfa. In: Kole, C., Hall, T.C., editors. A Compendium of Transgenic Crop Plants. Volume 3. Oxford, UK: Blackwell Publishing. p. 199-210.

Chandran, D., Sharopova, N., Vandenbosch, K.A., Garvin, D.F., Samac, D.A. 2008. Physiological and Molecular Characterization of Aluminum Resistance in Medicago truncatula. Biomed Central (BMC) Plant Biology. 8:89. Available:

Casler, M.D., Heaton, E., Shinners, K.J., Jung, H.G., Weimer, P.J., Liebig, M.A., Mitchell, R., Digman, M.F. 2009. Grasses and Legumes for Cellulosic Bioenergy. In: Wedin, W.F. and Fales, S.L., editors. Grassland: Quietness and Strength for a New American Agriculture. Madison, Wisconsin: ASA-CSSS-SSSA. p. 205-219.

Tesfaye, M., Yang, S.H., Lamb, J.F., Jung, H.G., Samac, D.A., Vance, C.P., Gronwald, J.W., Vandenbosch, K.A. 2009. Medicago truncatula as a Model for Dicot Cell Wall Development. BioEnergy Research. 2(1-2):59-76.

Last Modified: 06/25/2017
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