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United States Department of Agriculture

Agricultural Research Service


Location: Plant Science Research

2010 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 was made on all project objectives and subobjectives. Improved germplasm with increased in vitro neutral detergent fiber digestibility (IVNDFD) is needed for bioenergy production. Alfalfa populations selected for increased or decreased IVNDFD were sampled from trials at two locations and tested for differences in livestock digestibility and potential ethanol production. Also, 26 alfalfa clones differing in cell wall composition were tested for differences in potential ethanol production. Hybrid and synthetic alfalfa populations were tested for forage yield potential at two Upper Midwest locations. The project is on track for development of improved plant materials. Utilization of plant biomass for bioenergy requires accurate prediction of ethanol yield. Data for the diverse set of alfalfa stem samples used for development of NIRS calibrations to predict ethanol yield showed large variation among samples; however, ethanol yield was not related to lignin concentration even though earlier work indicated a very strong relationship. To identify the genes involved in cell wall development in alfalfa, a protocol was developed that optimized the detection of gene expression using the Medicago GeneChip and resulted in a two-fold increase in the number of differentially expressed genes measured in lines that differ in cellulose and lignin. Constructs were made for further characterization of two genes that may regulate cell wall composition. Alfalfa transformants are being generated to test the role of these genes in regulating cell wall composition. Using a bioinformatic approach, DNA sequences needed for gene regulation were identified and their regulatory proteins isolated through DNA binding assays. Gene promoters were identified that are specific to vascular cells. An in-depth analysis of genes expressed in barrel medic and alfalfa roots in response to phosphorus (P) and nitrogen (N) deficiency was completed. Depending upon the species, more than 1,220 genes were responsive to either P or N deficiency. A plantacyanin gene was highly expressed in all stress conditions. Gene classes over-represented in both P and N deficiency included: cell wall, lipid metabolism, secondary metabolism, and signal transduction. Phosphorus stress induced expression of genes classified as related to biotic stress and reactive oxygen generation, suggesting overlap with responses to disease. Phosphorus deficiency appeared to have greater influence on transporter gene expression than did N deficiency. Commercial and experimental varieties of alfalfa were tested for resistance to brown root rot. Also, evaluation of genetic variation in the pathogen indicates regional differences in populations, which should be considered when developing new disease-resistant varieties. A new disease (Mycoleptodiscus crown and root rot) was identified causing significant losses in commercial alfalfa fields. Experiments were initiated to test different crop rotations and fungicides for reducing the disease.

4. Accomplishments
1. Corn stover for bioenergy. Corn stover is the single-most abundant crop residue biomass resource, and increasing ethanol yield from stover would improve the profitability of cellulosic ethanol systems. Relationships among grain yield and cellulosic ethanol traits were examined by ARS and University of Minnesota scientists at St. Paul, Minnesota, and genetic markers for cell wall traits important for cellulosic ethanol production were identified. All the cellulosic ethanol traits (cellulose, lignin, and glucose release) had moderate to high heritability and were not negatively related with grain yield, and genetic markers were found for all cell wall traits. The results indicate that it should be possible to breed corn for improved stover cellulosic ethanol traits while continuing to select for increased grain yield, and that marker-assisted selection could be used to speed corn stover improvement. Simultaneous improvements in corn yield and stover quality will allow significant increases in overall ethanol yield and net energy efficiency of biofuel production using corn.

2. Novel disease resistance genes identified in alfalfa. Diseases affecting alfalfa leaves significantly reduced crop yields, but stacking resistance genes for individual diseases is not practical. ARS researchers in St. Paul, Minnesota, with University of Minnesota colleagues, identified several genes expressed in response to two foliar diseases in the model plant, barrel medic, that were not previously associated with disease resistance. Knocking-out expression of either of two genes in alfalfa caused plants to become highly susceptible to a foliar disease, evidence that these genes are critical for disease resistance. These genes can be used as markers to identify disease-resistant plants and used to develop alfalfa cultivars with resistance to multiple foliar pathogens.

3. Low ferulate corn mutant. Corn silage is among the most important feedstuff for dairy and beef cattle; however, the digestibility of the fiber in the stover fraction is poor. Cross-linking of fiber has been shown to reduce fiber digestibility in grasses. University of Minnesota scientists discovered a mutant in corn to reduce cross-linking in stover, and the mutation was shown by ARS scientists in St. Paul, Minnesota, to improve digestibility of corn stover in a laboratory test. If the mutated gene can be isolated, fiber digestibility of corn silage and all other grasses could be improved.

5. Significant Activities that Support Special Target Populations
With high fertilizer prices, farms with cash-flow problems need to know how to reduce application rate without reducing income. We conducted 10 site-years of research on the potassium (K) needs of alfalfa during its last year of production. We found that alfalfa did not produce higher yields or forage quality with added K on soils with medium K supply, in contrast to current recommendations for application of 50 to 115 kg K per hectare. It appears from these results that farmers who reduce or eliminate K applications in the final season of alfalfa production can save up to $130 per hectare without reducing forage yield or quality.

Review Publications
Schipanski, M.E., Drinkwater, L.E., Russelle, M.P. 2010. Understanding the Variability in Soybean Nitrogen Fixation across Agroecosystems. Plant and Soil Journal. 329(1-2):379-397.

Yang, S.H., Xu, W.W., Tesfaye, M., Lamb, J.F., Jung, H.G., Samac, D.A., Vance, C.P., Gronwald, J.W. 2009. Single-Feature Polymorphism Discovery in the Transcriptome of Tetraploid Alfalfa. The Plant Genome. 2(3):224-232.

Braun, L.C., Gillman, J.H., Russelle, M.P. 2009. Fertilizer Nitrogen Timing and Uptake Efficiency of Hybrid Hazelnuts in the Upper Midwest, USA. HortScience. 44(6):1688-1693.

Jung, H.G., Phillips, R.L. 2010. Putative Seedling Ferulate Ester (sfe) Maize Mutant: Morphology, Biomass Yield, and Stover Cell Wall Composition and Rumen Degradability. Crop Science. 50(1):403-418.

Lewis, M.F., Lorenzana, R.E., Jung, H.G., Bernardo, R. 2010. Potential for Simultaneous Improvement of Corn Grain Yield and Stover Quality for Cellulosic Ethanol. Crop Science. 50(2):516-523.

Lorenzana, R.E., Friskop-Lewis, M., Jung, H.G., Bernardo, R. 2010. Quantitative Trait Loci and Trait Correlations for Maize Stover Cell Wall Composition and Glucose Release for Cellulosic Ethanol. Crop Science. 50(2):541-553.

Yang, S.H., Xu, W., Tesfaye, M., Lamb, J.F., Jung, H.G., VandenBosch, K.A., Vance, C.P., Gronwald, J.W. 2010. Transcript Profiling of Two Alfalfa Genotypes with Contrasting Cell Wall Composition in Stems Using a Cross-Species Platform: Optimizing Analysis by Masking Biased Probes. Biomed Central (BMC) Genomics. 11:323. Available:

Schlatter, D.C., Samac, D.A., Tesfaye, M., Kinkel, L.L. 2010. Rapid and Specific Method for Evaluating Streptomyces Competitive Dynamics in Complex Soil Communities. Applied and Environmental Microbiology. 76(6):2009-2012.

Digman, M.F., Shinners, K.J., Casler, M.D., Dien, B.S., Hatfield, R.D., Jung, H.G., Muck, R.E., Weimer, P.J. 2010. Optimizing On-farm Pretreatment of Perennial Grasses for Fuel Ethanol Production. Bioresource Technology. 101:5305-5314.

Last Modified: 06/23/2017
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