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United States Department of Agriculture

Agricultural Research Service


Location: Plant Science Research

2011 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 in developing germplasm and determining genetic and biological processes that regulate forage use for bioenergy and livestock production. Sample collection from alfalfa populations selected for increased or decreased forage digestibility was completed from trials at two locations and tested for differences in livestock digestibility and potential ethanol production. Samples were collected from field experiments comparing selected alfalfa populations for differences in N-uptake capacity. Next generation sequencing technology was used to identify genes expressed in two alfalfa lines that differ in stem cell wall composition. A large number of simple sequence DNA repeats were identified that differed between the two lines that may be useful as markers for selection and breeding. An enzyme that plays an important role in cell wall biosynthesis in plants was downregulated in transgenic alfalfa and the effect on cell composition was measured. Gene promoters that are active in developing stems undergoing lignification and secondary cell wall synthesis were cloned and activity is being evaluated in transgenic alfalfa. A field test was established to evaluate resistance to brown root rot in progeny from resistant parents. The chromosome of a bacterial pathogen of alfalfa was sequenced and gene identification initiated. Two genes previously identified through expression analysis as involved in acclimation to N and P acquisition and use were silenced in barrel medic and resulted in aberrant root development. Progress was also made on developing and testing management practices to expand the role of alfalfa and other forages for use as biofuel feedstocks and in protecting water quality. Corn stover was analyzed for cell wall carbohydrate and lignin composition to determine the impact of N fertilizer application rates and alfalfa as a preceding crop on cellulosic ethanol conversion potential. Research on 10 Minnesota farms was completed that measured the need for potassium in the last year of alfalfa, the carryover of excess potassium to the next corn crop, and the amount of fertilizer nitrogen needed for high yield of the corn crop grown after alfalfa. There was no yield or quality improvement from potassium fertilizer when alfalfa was grown on medium-testing soils, and relatively little of the excess potassium carried over for the next corn crop. Results have been summarized and transferred to more than 1,000 farmers, farm advisors, State and Federal agricultural agency personnel, and other scientists.

1. Improving alfalfa yield through hybrid vigor. In contrast to other field crops, yields of harvested alfalfa have not increased appreciably during the past 30 years. Hybrid plants produced from crossing parents with different genetic backgrounds often have greater yields in other crop species, but hybrid vigor for yield in alfalfa has not been tested. ARS scientists at St. Paul, Minnesota, and Madison, Wisconsin, found that the parents that produced the highest yielding progeny differed depending on whether the cross was made within the alfalfa species or as a hybrid between alfalfa and the closely related species, yellow flowered alfalfa. These results suggest that the highest yielding hybrid will not necessarily result from crossing the highest yielding individuals from each species. Thus, selection of parents to successfully increase yields based on hybrid vigor will need to be based on performing test crosses between the two species and measuring yield of the resulting hybrids. This research demonstrated that although yield increases occur in alfalfa from hybrid vigor, additional technologies such as genetic markers will be needed to assess test crosses to take advantage of hybrid vigor to improve yield in alfalfa.

2. Cell walls impact corn borer resistance without reducing digestibility by cattle. European and Mediterranean corn borers are important economic pests of corn production in both the U.S. and Europe and are typically controlled using pesticides or genetic modification technology. Because both of these control methods are controversial, resistance using conventional breeding for natural resistance to corn borers is being investigated. ARS researchers at St. Paul, Minnesota, in collaboration with scientists at the Mision Biologica de Galicia in Pontevedra, Spain, found that corn inbred lines selected in Spain for resistance to both corn borers have more cell wall material in stalk tissues and these cell walls contained higher concentrations of cross linking structures, which made the stalks more difficult for corn borers to tunnel through. Although some forms of cell wall cross linking has been shown to reduce digestibility of corn stalks by cattle, the specific alteration related to corn borer resistance did not impact digestibility. These results indicate that breeding for specific cell wall traits is a viable alternative for controlling corn borer damage and may not adversely impact digestibility of corn stover by cattle.

3. Evaluating corn germplasm for biofuel potential. Production of biofuels from corn stover, the dry plant material remaining after grain harvest, will contribute an estimated one-third of the 30 billion gallons of cellulosic ethanol set out in the U.S. Renewable Fuels Standards. To meet this goal, corn stover is needed with a greater potential ethanol yield. Therefore, geneticists around the world are evaluating corn germplasm to identify lines with improved performance. ARS scientists at St. Paul, Minnesota, in collaboration with University of Minnesota scientists demonstrated that contrary to some reports, the laboratory method commonly used for estimating how well cattle digest cellulose, the major component in cell walls in corn stover, is not reliable for predicting the conversion potential of cellulose to ethanol using industrial processes. Corn geneticists must use different evaluation methods modeled more closely on industrial conversion processes to successfully identify superior corn germplasm for improved biofuel production.

4. First alfalfa gene index assembled. Alfalfa offers considerable potential as a bioenergy crop that would reduce the Nation's dependence on foreign oil imports. Two major components of alfalfa stems are cellulose, a sugar molecule that is easily converted to ethanol, and lignin, a cross-linking molecule that interferes with conversion of cellulose to ethanol. Increasing cellulose and decreasing lignin in cell walls would increase the value of alfalfa as a bioenergy crop; however, knowledge of the genes that regulate the amount of cellulose and lignin in alfalfa cell walls is limited. Utilizing a recently developed high throughput DNA sequencing technology, ARS scientists at St. Paul, Minnesota, conducted an in-depth analysis of the genes active during cell wall development and assembled the first alfalfa gene index that identifies a majority of alfalfa genes. Several genes associated with the regulation of lignin and cellulose biosynthesis were identified. Plant breeders and molecular biologists can use the alfalfa gene index and genes identified to improve alfalfa as a bioenergy crop as evidenced by the publication being accessed nearly 3,000 times since its publication in April 2011.

5. Validating the legume nitrogen credit in high-yield environments. Many farmers are reluctant to follow State recommendations to reduce fertilizer nitrogen rates on crops that follow legumes, such as alfalfa. Although their reasons for this reluctance are numerous, some have argued that more nitrogen is needed as corn yields rise. Results of this grant-funded research on 10 farms by ARS scientists and colleagues in St. Paul, Minnesota, however, supported current State recommendations that no additional nitrogen fertilizer is needed on the first corn crop after a good stand of alfalfa is terminated and incorporated into the soil. These results should be applied to situations with relatively normal weather conditions because wetter-than-normal conditions in spring may cause nitrogen losses that may need to be replaced by later fertilizer applications. If farmers followed these recommendations on the estimated 250,000 acres of alfalfa that are rotated to corn each year in Minnesota, they would save about $40 per acre in fertilizer cost. Furthermore, these lower fertilizer rates would reduce total nitrogen application to Minnesota cropland by about 10,000 fewer tons annually, thereby reducing losses of nitrogen to air and water.

6. Fertilizer nitrogen needs of a new bio-oil crop. Improved cropping systems are needed to increase agricultural productivity, profitability, and environmental sustainability. Hybrid hazelnut is a perennial shrub being developed as a specialty crop to produce nuts for human and bio-oil uses. It also provides good habitat, may improve soil quality, and could be grown as an intercrop with legumes such as alfalfa. Research by ARS scientists and University colleagues in St. Paul, Minnesota, determined that hybrid hazelnut required very little additional nitrogen during the first couple of years after transplanting. Furthermore, they showed that leaf tissue nitrogen concentration of 1.9% was the threshold between too little and enough nitrogen. This is the first report of fertilizer nitrogen requirements of this new crop and provides guidance to farmers to be more profitable and reduce nitrogen losses to air and water.

Review Publications
Russelle, M.P., Kelley, D.W., Birr, A.S., Tiffany, D.G. 2011. Spatial ramifications of crop selection: water quality and biomass energy. In: Clay, D.E., Shanahan, J.F., editors. GIS Applications in Agriculture. Volume 2. Nutrient Management for Energy Efficiency. Boca Raton, FL: CRC Press. p. 395-424.

Barros-Rios, J., Malvar, R.A., Jung, H.G., Santiago, R. 2011. Cell wall composition as a maize defense mechanism against corn borers. Phytochemistry. 72(4-5):365-371.

Whitebird, R.R., Bliss, D.Z., Savik, K., Lowry, A., Jung, H.G. 2010. Comparing community and specialty provider-based recruitment in a randomized clinical trial: clinical trial in fecal incontinence. Research in Nursing and Health. 33(6):500-511.

Yang, S.H., Tu, Z., Foo, C., Xu, W., Lamb, J.F., Jung, H.G., Vance, C.P., Gronwald, J.W. 2011. Using RNA-Seq for gene identification, polymorphism detection and transcript profiling in two alfalfa genotypes with divergent cell wall composition in stems. Biomed Central (BMC) Genomics. 12:199. Available:

Braun, L.C., Gillman, J.H., Hoover, E.E., Russelle, M.P. 2011. Nitrogen fertilization for new plantings of hybrid hazelnuts in the Upper Midwest of the United States of America. Canadian Journal of Plant Science. 91(4):773-782.

Jung, H.G., Bernardo, R. 2011. Comparison of cell wall polysaccharide hydrolysis by a dilute acid/enzymatic saccharification process and rumen microorganisms. BioEnergy Research. Available:

Russelle, M.P., Lamb, J.F. 2011. Divergent alfalfa root system architecture is maintained across environment and nutrient supply. Agronomy Journal. 103(4):1115-1123.

Vogel, K.P., Dien, B.S., Jung, H.G., Casler, M.D., Masterson, S.D., Mitchell, R. 2011. Quantifying actual and theoretical ethanol yields for switchgrass strains using NIRS analyses. BioEnergy Research. 4(2):96-110. DOI: 10.1007/s12155-010-9104-4.

Samac, D.A., Penuela, S., Schnurr, J.A., Hunt, E.N., Foster-Hartnett, D., Vandenbosch, K.A., Gantt, J.S. 2011. Expression of coordinately regulated defense response genes and analysis of their role in disease resistance in Medicago truncatula. Molecular Plant Pathology. 12(8):786-798.

Shane, E.M., Endres, M.I., Russelle, M.P., Rosen, C.J., Johnson, D.G. 2011. Compost: a potential value-added product for dairy operations? Applied Engineering in Agriculture. 27(2):225-233.

Kuster, H., Becker, A., Samac, D.A., Tesfaye, M. 2006. Transcriptomics. In: The Medicago truncatula Handbook. The Samuel Roberts Noble Foundation, Ardmore, OK. Available:

Ellwood, S., Kamphuis, L., Pfaff, T., Oliver, R., Samac, D.A., Foster-Hartnett, D., Tivoli, B., Onfroy, C., Moussart, A. 2006. Inoculation and growth with foliar pathogenic fungi. In: The Medicago truncatula Handbook. The Samuel Roberts Noble Foundation, Ardmore, OK. Available:

Gronwald, J.W., Miller, S.S., Vance, C.P. 2008. Arabidopsis UDP-sugar pyrophosphorylase: evidence for two isoforms. Plant Physiology and Biochemistry. 46(12):1101-1105.

Pagliari, P.H., Rosen, C.J., Strock, J.S., Russelle, M.P. 2010. Phosphorus availability and early corn growth response in soil amended with turkey manure ash. Communications in Soil Science and Plant Analysis. 41(11):1369-1382.

Rock, K.P., Thelemann, R.T., Jung, H.G., Tschirner, U.W., Sheaffer, C.C., Johnson, G.A. 2009. Variation due to growth environment in alfalfa yield, cellulosic ethanol traits, and paper pulp characteristics. BioEnergy Research. 2(3):79-89.

Bliss, D.Z., Savik, K., Jung, H.G., Whitebird, R., Lowry, A. 2011. Symptoms associated with dietary fiber supplementation over time in individuals with fecal incontinence. Nursing Research. 60(3S):S58-S67.

Vance, C.P. 2011. Phosphorus as a critical macronutrient. In: Hawkesford, M., Barraclough, P., editors. The Molecular and Physiological Basis of Nutrient Use Efficiency in Crops. West Sussex, United Kingdom: John Wiley and Sons, Ltd. p. 229-261.

Last Modified: 4/18/2014
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