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United States Department of Agriculture

Agricultural Research Service


Location: Forage-animal Production Research

2010 Annual Report

1a. Objectives (from AD-416)
The long-term goal of this project is to improve competitiveness and sustainability of forage-based enterprises in the transition zone of the Eastern half of the United States of America. Over the next five years we will focus on the following objectives as determined by stakeholder input and ARS National Program 215 (Rangeland, Pastures and Forages) approval. Objective 1. Improve persistence, productivity, and quality of forage grasses and legumes for use in the transition zone of the Eastern and Midwestern U.S. Subobjective 1.A. Determine relative interactions between tall fescue (TF) and endophytes involved in regulating plant growth, forage quality, and persistence under a variety of environmental conditions. Subobjective 1.B. Identify, characterize, and manipulate useful traits (chemical, physical, genetic) to develop forages that are: biologically diverse, tolerant of biotic and abiotic stresses, competitive, high quality for animal production, persistent, and easy to establish and maintain. Objective 2. Improve animal and forage productivity on forage-based systems through optimal combinations of forages, supplementation and grazing systems tailored to animal needs and environmental conditions. Subobjective 2.A. Evaluate forage production characteristics relevant to plant and animal performance on pastures containing new novel endophyte-infected (EI) TFs. Subobjective 2.B. Develop a forage system for the upper transition zone that utilizes warm season perennial grasses to improve animal performance and profitability. Subobjective 2.C. Identify the abiotic/biotic components and mechanisms of the plant-animal-environment interface impacting pasture production and environmental quality factors.

1b. Approach (from AD-416)
Forage systems provide low-cost feed, conserve soil and water resources, and mitigate man’s impact on the environment. Limited basic biological information exists on how plant and/or fungal metabolites affect forage plant quality, persistence, and production. Even less information exists on the cross-talk mechanism between tall fescue (the predominant forage of the transition zone) and its endophyte. Furthermore, little is known about the impact that forage and/or fungal metabolites have on their pasture ecosystems. Gaps in our current knowledge are hindering researchers’ abilities to predict and select best combinations of forages and management systems for use by various forage-animal production enterprises. Aiding researchers to develop new forage varieties, forage systems, and management recommendations will require an improved understanding of both metabolite (plant and fungal) profiles and their biological functions at the molecular and organismal levels. Such understanding of metabolites, molecular mechanisms, and whole-organism responses, and of their impact on plant quality, persistence, and production, is necessary for improving sustainability of forage-based enterprises. This Project Plan, through the development and utilization of cutting-edge technologies and real-world testing, proposes to decipher the complex interactions within the animal-plant-environment interface. In order to accomplish this task and improve sustainability of forage-based enterprises, the following two objectives are proposed: 1) Improve persistence, productivity, and quality of forage grasses and legumes for use in the transition zone of the Eastern and Midwestern United States; and 2) Improve animal and forage productivity on forage-based systems through optimal combinations of forages, supplementation, and grazing systems tailored to animal needs and environmental conditions. These objectives are focused on the predominant forage of the transition zone, tall fescue, as well as on its alternatives and companion species. Accomplishing these objectives will improve sustainability of forage-based enterprises through improved forages, forage management and systems, and basic understanding of the plant/fungal metabolite effects on forage plant persistence and production, as well as consequent effects on the structure and function of pasture ecosystems.

3. Progress Report
Tall fescue expressed sequence tags were assembled resulting in the identification of approximately 39,000 gene sequences. A method for extracting low polarity soluble phenolics has been developed and used successfully to extract compounds from red clover with antimicrobial activity. Over 12,500 clover Messenger Ribo Nucleic Acid (mRNA) have been sequenced using 454 technology with approximately 60% identified as novel clover sequences and are expected to yield novel flowering time homologues. The gene expression of rice and tall fescue Protein-L-Isoaspartate-Methyltransferase have been confirmed. Additional progress information is available in the Annual Reports for the Congressionally mandated Specific Cooperative Agreements (6440-32630-001-06S, Continuation of Improved Forage Livestock Production Systems; 6440-32630-001-03S, Improved Forage Livestock Production) that is partially funded by this project. Likewise, relevant information is available in the sister project (6440-32630-001-00D, Optimizing the Biology of the Animal-Plant Interface for Improved Sustainability of Forage-based Animal Enterprises) to this project.

4. Accomplishments
1. Red Clover Produces Beneficial Antimicrobials. Ruminal proteolysis and subsequent amino acid degradation represent considerable economic loss in ruminant production. ARS scientists in Lexington, Kentucky, understanding that hyper ammonia-producing bacteria are largely responsible for reduced protein utilization by ruminants and that these bacteria are susceptible to control by some plant secondary metabolites, pursued a study to determine if red clover (a common and high quality forage legume) produced beneficial antimicrobials capable of improving nitrogen utilization in the rumen. Specifically they wanted to determine if any of the soluble phenolics in red clover possessed antimicrobial activity against the bovine hyper ammonia-producing bacteria, Clostridium sticklandii SR. High performance liquid chromatography analysis revealed that clover tissues were rich in the isoflavonoids (soluble phenolics), formononetin and biochanin A. Biochanin A and crude clover extracts inhibited the growth of Clostridium (C.) sticklandii. However, the compound formononetin had no effect. These results indicate that clover phenolic compounds may have a role in preventing amino acid fermentation and thus, may provide a natural alternative to ionophore treatments in improving nitrogen utilization in ruminants.

2. Drought Stress Tolerance in Tall Fescue. Tall fescue plants symbiotic with the endophytic fungus, Neotyphodium coenophialum, have better survivability under stresses such as drought than do plants lacking the endophyte. University of Kentucky researchers in collaboration with ARS scientists in Lexington, KY, and Athens, GA, used pairs of identical plant genotypes with the endophyte (E+) and without it (E-) in time courses of withholding water for 0-5 days to simulate drought. Recovery from drought and changes in levels of free sugars, free sugar alcohols, and free amino acids were measured. In two experiments with one plant genotype, recovery upon rewatering was significantly greater for E+ than E- plants after 2 or 3 days of withholding water. Differences in metabolites were evident one day before establishing the difference in recovery, when shoot and root levels of glucose, fructose and proline were 2 to 3-fold higher in E+ than E-. In an experiment with a second plant genotype, similar but less dramatic effects of endophyte on recovery were observed, and the endophyte effects on metabolites were more evident in roots than in shoots. Roots of E+ exhibited higher levels of free fructose (56 µmol/g dw) compared to E- (37 µmol/g dw). Levels of free glutamine and asparagine were 3-fold higher in E+ roots than E- roots. These results demonstrate that the endophyte affects levels of free osmolytes that help protect plants against drought stress and indicates that managers should consider use of endophyte containing plants in drought prone areas.

3. Loline Alkaloid Production and Pest Control. Neotyphodium species that are fungal symbionts (endophytes) of meadow fescue protect the grass from insects by producing loline alkaloids. Past reports indicate that insect damage or clipping increases loline levels dramatically. Although this helps protect the plant, it is possible that loline production may deplete levels of amino acids that are precursors of lolines, but also essential for protein. University of Kentucky researchers in collaboration with ARS scientists in Lexington, KY, and Athens, GA, examined levels of loline alkaloids and free amino acids in younger versus older leaf tissues. In plants with loline-producing endophyte, lolines were 2-5-fold higher in younger tissues compared to older tissues. Interestingly, in endophyte-free plants or plants with endophytes that do not produce lolines, certain free amino acids such as proline, glutamine and asparagine were much higher in younger versus older tissues. However, when a loline-producing endophyte was present, levels of these free amino acids were greatly depleted. Thus, substrate (amino acid) availability determines levels of loline alkaloids produced in young tissues of the grass plant. Further, because lolines are produced from amino acids that are typically mobilized to growing plant tissues, lolines can be synthesized in greater amounts in young tissues. Therefore, lolines help to defend tissues that are too young to have substantial physical defenses thereby providing the plant a competitive advantage during regrowth and establishment.

4. Ergot Alkaloid Longevity in Herbicide Killed Tall Fescue. The degradation of ergot alkaloids to a point below toxicity for grazing livestock, especially the horse, in tall fescue monocultures or in mixed is unknown. University of Kentucky researchers under contract with the ARS location in Lexington, Kentucky, determined the degradation rate of these alkaloids after killing with imazapic and glyphosate. Treating with these herbicides stopped dry weight accumulation within 2 weeks and by 4 weeks alkaloid accumulation began to decrease. After 12 weeks the ergot alkaloids were still above 200 ppb. Therefore, a minimum of 12 weeks should elapse prior to any animal reentry.

5. Tall Fescue Experimental Sampling Protocols. Many pastures are sampled for alkaloid content of endophyte-infected tall fescue plants. Sampling time and plant tissue to sample impacts the results obtained. University of Kentucky researchers in collaboration with the ARS location in Lexington, Kentucky, determined that for reproductive plants the inflorescence had the highest level of ergopeptide alkaloids present followed in descending order of the culm (stem), flag leaf, and second leaf. For vegetative leaves the basal portion of the leaf blade had higher ergopeptide alkaloid content than the terminal end. Pesudostem tissues were higher than blade tissue. Therefore, when sampling one must get a representative sample of forage that would be expected to be consumed by the grazing animal. For plant research and pasture management decision this means a representative sample of all the tissue present in the sward.

6. Alkaloid Accumulation Following Repeated Harvests. Forage management may impact alkaloid accumulation in the tall fescue. University of Kentucky researchers in collaboration with the ARS location in Lexington, Kentucky, investigated how simulated grazing of vegetation above 10 cm changes alkaloid accumulation in the forage. Initial sampling was done at 3 week intervals beginning at 3 weeks for 12 weeks and regrowth tissue every 3 weeks for an additional 12 weeks. Alkaloid levels increased in regrowth tissue at all sampling times for the initial 3 and 6 week sampling. With initial harvests at 9 and 12 weeks the later harvest at 24 and 27 weeks after seeding had decreased alkaloid accumulation. The amount of grazing or harvests will increase alkaloid accumulation of alkaloids in the forage and thus, impact the consuming animal. Understanding these patterns will aid in developing appropriate grazing strategies for managing potential fescue toxicosis cases.

7. Growth Regulator Impact on Ergopeptide Alkaloid Accumulation. Frequent harvesting of tall fescue causes an increase in alkaloid accumulation. University of Kentucky researchers in collaboration with the ARS location in Lexington, Kentucky, investigated the impact of a hormone on alkaloid production. Methyl jasmonate (a hormone) was used to simulate clipping the sward of tall fescue. For the initial harvest or methyl jasmonate treatment at 4 weeks the methyl jasmonate treatment increased ergopeptide alkaloids significantly more than the clipping when measured 3 weeks later. Methyl jasmonate treatment at 3 week intervals continued to increase alkaloid content more than clipping, at least to 13 weeks. These results indicate that we must understand the mobilization of plant reserves for use by the endophyte for biosynthesis of the alkaloids. This could potentially be a means of reducing toxic ergot alkaloids in a grazing sward, thereby reducing incidence of fescue toxicosis.

8. Incidence of Arthropod Pests and Natural Enemies in Tall Fescue Pastures Expressing Modified Alkaloid Profiles. Grass breeders are putting novel endophytes with attenuated alkaloid production into new or existing cultivars, but their use could alter the balance between arthropod pests and their natural enemies, depending on their alkaloid sensitivity. University of Kentucky researchers in collaboration with ARS scientists in Lexington, KY, collected vacuum samples in 2008 and 2009 and sorted samples for herbivores (leaf- and planthoppers, caterpillars, wireworms, grasshoppers, and crickets, and natural enemies including spiders, predatory beetles, and parasitic wasps. There were few differences between pastures planted in endophytic or endophyte-free grasses. Novel endophytes with attenuated alkaloid production may be planted for improved livestock performance without increasing risk of pest outbreaks and loss of natural enemies.

9. Soil Organic Carbon Fractions Differ in Two Contrasting Tall Fescue Systems. The value of tall fescue (Festuca arundinacea Schreb.) for Carbon (C) sequestration in addition to forage production and soil conservation is of current interest. However, studies relating to the impacts of endophyte infected (E+) and endophyte free (E-) tall fescue on soil organic matter fractions are few. University of Kentucky researchers in collaboration with the ARS location in Lexington, Kentucky, examined how E+ and E- growth affected soil C fractions 4 years after establishment. Significant effects between E+ and E- fescue were sometimes observed for microbial biomass C (MBC), mineralizable C (Min C), C in micro-aggregates, and aggregate distribution, but not for total C, and particulate organic matter C (POM C). At 0-15 cm MBC (E+ 26% greater than E-), Min C (E+ 43% lower than E-), C associated with micro-aggregates (E+ 15% lower than E-), and micro-aggregates (46% more micro-aggregates in E+ than E-), were affected by endophyte infection, confirming the hypotheses that early changes in soil properties were reflected in labile C fractions and soil structure. Endophyte infection status in tall fescue has quantifiable effects on C sequestration and soil structure, achievable in a relatively short period that can be used to monitor conservation efforts and the consequences of pasture renovation strategies.

10. Earthworm Distribution in Endophyte-infected and Endophyte-free Tall Fescue. Anecdotal observations suggest that earthworms preferentially avoid endophyte-infected tall fescue relative to endophyte-free fescue. Because earthworm burrowing contributes to infiltration in pastures, this could influence groundwater quality. University of Kentucky researchers in collaboration with the ARS location in Lexington, Kentucky, extracted earthworms in a spatially discrete manner from a sinkhole containing endophyte-free and infected fescue. Earthworms appeared to preferentially avoid the wettest part of the sinkhole, which was the throat. There was a tendency for larger and fewer earthworms to be present in endophyte-infected fescue and smaller but more numerous earthworms to be present in endophyte-free fescue. Buffer zones that were fescue free had the greatest number of earthworms regardless of position in the sinkhole, and earthworms congregated in these buffers in contrast to the established fescue. Impact. Endophyte infection status had an effect on earthworm numbers and distribution and more significantly, the presence or absence of fescue affected the number of earthworms. Fescue, particularly endophyte infected fescue, may be a natural barrier to earthworm growth and activity in sinkhole environments.

Review Publications
Thakare, D., Kumudini, S., Dinkins, R.D. 2010. Expression of Flowering-Time Genes in Soybean E1 Near-isogenic Lines Under Short and Long Day Conditions. Planta (2010). 231:951-963.

Handayani, I.P., Coyne, M.S., Tokosh, R.S. 2010. Soil Organic Matter Fractions and Aggregate Distribution In Response to Tall Fescue Stands. International Journal of Soil, Sediment, and Water. 5:1-10.

Belesky, D.P., Ruckle, J.M., Bush, L.P. 2009. Microsite conditions influence nutritive value characteristics of a common tall fescue cultivar infected with either a native or a novel non-ergogenic endophyte. Environmental and Experimental Botany. 67:284-292.

Florea, S., Andreeva, K., Machado, C., Mirabito, P.M., Schardl, C.L. 2009. Elimination of Marker Genes from Transformed Filamentous Fungi by Unselected Transient Transfection with a Cre-expressing Plasmid. Fungal Genetics and Biology. 46:721-730.

Clement, S.L., Bradley, V.L., Elberson, L.R., Bragg, D.E., Phillips, T.D. 2009. Cereal Leaf Beetle Colonizes Grass Germplasm Nurseries and Impacts Seed Production Activities. Online. Forage and Grazinglands. doi:10.1094/FG-2009-1214-01-RS.

Mubiru, D.N., Coyne, M.S. 2009. Legume Cover Crops are More Beneficial than Natual Fallows in MInimally Tilled Ugandan Soils. Agronomy Journal. 101:644-652.

Handayani, I.P., Coyne, M.S., Phillips, T.D. 2010. Soil Organic Carbon Fractions Differ in Two Contrasting Tall Fescue Systems. Plant and Soil Journal. doi:10.1007/s11104-010-0352-z.

Siegrist, J.A., Mcculley, R.L., Bush, L.P., Phillips, T.D. 2010. Alkaloids May Not be Responsible for Endophyte Associated Reductions in Tall Fescue Decomposition Rates. Functional Ecology 2010. 24:460-468.

Omacini, M., Chaneton, E.J., Bush, L., Ghersa, C.M. 2009. A Fungal Endosymbiont Affects Host Plant Recruitment Through Seed- and Litter-mediated Mechanisms. Functional Ecology 2009. 23:1148-1156.

Last Modified: 05/26/2017
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