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ARS Home » Midwest Area » Lexington, Kentucky » Forage-animal Production Research » Research » Research Project #434196

Research Project: Sustainable Forage-Based Production for the Mid-South Transition Zone

Location: Forage-animal Production Research

2018 Annual Report

Objective 1. Improve pasture productivity and animal performance while reducing fertilizer inputs on southeastern pastures by developing optimal combinations of forage legumes and grasses including controlling and replacing toxic endophyte-infected (EI) tall fescue (TF). Subobjective 1.A. Determine if clover in mixture with common toxic endophyte (CTE) TF can dilute ergot alkaloids (EA) in the diet to mitigate the effects that FTOX (fescue toxicosis) has on animal performance and wellbeing. Subobjective 1.B. Determine the effects of CTE TF seed head suppression on EA concentrations, animal performance and recovery from toxicosis. Objective 2. Improve forage production and utilization strategies by developing and applying a better understanding of how genetic and environmental factors affect metabolites in southeastern forage grasses and legumes. Subobjective 2.A. Determine metabolite changes in forage grasses and legumes due to genetics/phenotype and/or environmental factors and determine subsequent implications for forage production and utilization. Subobjective 2.B. Identify fractions of clover extracts, or pure compounds (e.g., biochanin A) having antimicrobial activity on selected rumen bacteria. Subobjective 2.C. Develop transgenic forage legumes to alter or knock out biochemical pathways to gain a better understanding of isoflavonoid biosynthesis. Objective 3. Improve the productivity, quality and persistence of forage grasses by developing and applying a better understanding of the interactive mechanisms between endophytes, host plants and environmental factors. Subobjective 3.A. Response of novel or non-toxic endophyte (NTE) strain combinations to stress. Subobjective 3.B. Examine the effects of endophyte on survival and regrowth in different Endophyte-Infected/Endophyte-Free (E+/E-) TF clone pairs after drought stress. Subobjective 3.C. Genome-wide analysis of transcription and RiboNucleic Acid (RNA) processing in the endophyte-plant system. Objective 4. Develop guidelines for managing animal-plant-soil characteristics to improve soil quality and reduce the risks of climatic variations on southeastern pasture productivity and animal performance. Subobjective 4.A. Determine how TF genotype interacts with both fungal endophyte presence/genotype and changes in climate to alter TF production, secondary metabolite concentrations, and overall fescue forage quality. Subobjective 4.B. Legume levels in TF pastures affect carbon sequestration after renovation. Subobjective 4.C. Water soluble Phosphorus (P) losses within pastures are spatially coincident with reduced carbon (C) storage.

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 the cross-talk mechanism between tall fescue (TF) (the predominant forage of the transition zone) and its fungal endophyte (symbiont – beneficial to TF plant), as well as how the plant and/or fungal metabolites affect forage quality, persistence, and production. 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 forage-animal production enterprises. Aiding researchers to develop new forage varieties, forage systems, and management recommendations for improving sustainability of forage-based enterprises will require an improved understanding of metabolite (plant and fungal) profiles and their biological functions at the molecular, cellular and organismal levels. This research project proposes to decipher the complex interactions within the animal-plant-environment interface; improve sustainability of forage-based enterprises through improved forages and forage systems management; and improve 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. The objectives of the proposal are focused on the predominant forage of the transition zone, TF, as well as on its companion species (e.g., red clover).

Progress Report
Quantification of two red and two white clover cultivars showed that non-structural carbohydrates accounted for 14-27% of the starch accumulation and thus suggesting that further analysis of starch accumulation in clover is warranted. Isoflavone synthase knockout lines using the CRISPR/cas9 system previously shown to affect only one of the chromosomes have been crossed to generate homozygous knockout plants for analysis of isoflavone content. Analysis of stress response of tall fescue plants harboring different strains of its symbiotic endophyte failed to show any effect on plant gene expression, however it does appear that the presence of the endophyte may prime plants that harbor the endophyte thus potentially aid in the recovery from stress. Tall fescue transcriptome assembly originating using polyA reads was finished and being used for analysis of differential mRNA processing in response to abiotic stress in endophyte-harboring and endophyte-free clones. Demonstrated that tissue specific alternate polyadenylation for a number of genes is similar to the model species Arabidopsis and rice suggesting a conserved functional role in higher plants.

1. Differential gene expression response of tall fescue endophyte-harboring and endophyte-free plants under stress. Tall fescue is the predominant forage in the eastern US and is important in U.S. agriculture as well as a turf for yards and athletic fields. A significant factor in the exceptional fitness of tall fescue is its seed-transmissible symbiont, the fungal endophyte Epichloë coenophiala. The most common E. coenophiala strains, called common-toxic endophyte (CTE) strains, found in tall fescue in North America produce ergot alkaloids and can cause episodes of “fescue toxicosis” to grazing livestock. Development and adoption tall fescue cultivars using non-mammal-toxic endophyte (NTE) strains, is currently underway but requires additional evaluation to assess whether the endophytes will contribute to abiotic stress tolerance in these novel symbiotic combinations. To evaluate this, ARS scientists in Lexington KY, in collaboration with University of Kentucky scientists, developed tall fescue clone combinations with and without different endophyte strains. Two tall fescue clone pairs harboring the common toxic endophyte (CTE) and two clone pairs harboring novel non-toxic endophytes (NTE) strains were subjected to stress and gene expression in relation to stress was evaluated using RNA-seq next generation sequencing. The results demonstrated that the endophyte appeared to have minimal affect plant gene expression upon stress. However, a few genes were differentially regulated when comparing clones with and without the endophyte. In conclusion, the results did not support the model that the endophyte has positive effects on stress tolerance but did suggest that presence of the endophyte may reduce expression of anti-fungal factors in the plant and may prepare the plant for stress or aid the plant during the recovery of stress. Additional studies are needed following stress treatments to determine how the presence of the endophyte might aid in the recovery from stress.

2. Genome-wide atlas of alternative polyadenylation in the forage legume red clover. Polyadenylation is a post-transcriptional processing step essential for the maturation of the majority of higher animal and plant messenger RNAs (mRNAs). A gene may contain multiple polyadenylation sites and differential usage of these sites gives rise to different distinct mRNAs with the potential to produce different protein products. This phenomenon is termed as ‘alternative polyadenylation’ (APA). There are significant implications of APA in numerous biological processes. Tissue specific occurrences of APA are known to play a regulatory role in humans and plants. Mis-regulation of APA has been attributed as the cause for several human diseases including thalassemia, thrombophilia, muscular dystrophy, and others. Our goal was to characterize tissue specific APA regulation in red clover by comparing expression in leaves, roots and flowers. ARS scientists in Lexington, Kentucky, in collaboration with University of Kentucky scientists, identified a number of genes that displayed APA in the different tissues, for example, genes involved in photosynthesis, those implicated in metabolite precursors and energy production were enriched in leaves. A comparison of genes displaying APA in red clover with APA studies done on the model plants Arabidopsis and rice suggest that there is conservation in APA in the genes observed, thus a conserved mechanistic role of the use of APA across plant species. The impact of this work is that it provides for a background that will allow for further characterization of the functional role of regulation of gene expression and production of different proteins in different tissues, and during development, in red clover.

Review Publications
Liu, S., Coyne, M.S., Grove, J.H., Flythe, M.D. 2018. Long-term tillage and nitrogen fertilization in maize influences the ammonia-oxidizing bacterial community. Applied Soil Ecology. 129:98-106.
Majee, M., Kumar, S., Kathare, P.K., Wu, S., Gingerich, D., Nayak, N.R., Salaita, L., Dinkins, R.D., Martin, K., Goodin, M., Dirk, L.M., Lloyd, T.D., Zhu, L., Chappell, J., Hunt, A.G., Vierstra, R., Huq, E., Downie, A.B. 2018. KELCH F-BOX protein positively influences Arabidopsis seed germination by targeting PHYTOCHROME-INTERACTING FACTOR1. Proceedings of the National Academy of Sciences.
Slaughter, L.C., Nelson, J.A., Carlisle, E., Bourguignon, M., Dinkins, R.D., Phillips, T.D., McCulley, R.L. 2018. Climate change and Epichloë coenophiala association modify belowground fungal symbioses of tall fescue host. Fungal Ecology. 31:37-46.
Kagan, I., Lawrence, L.M., Seman, D.H., Prince, K.J., Fowler, A.J., Smith, S. 2018. Effects of sampling time, cultivar, and methodology on water- and ethanol-soluble carbohydrate profiles of three cool-season grasses in Central Kentucky. Journal of Equine Veterinary Science. 61:99-107.
Paz, H.A., Hales Paxton, K.E., Wells, J., Kuehn, L.A., Freetly, H.C., Berry, E.D., Flythe, M.D., Spangler, M.L., Fernando, S. 2018. Rumen bacterial community structure impacts feed efficiency in beef cattle. Journal of Animal Science. 96(3):1045-1058.
Chakrabarti, M., Dinkins, R.D., Hunt, A.G. 2018. Genome-wide atlas of alternative polyadenylation in the forage legume red clover. Scientific Reports. 8:11379. 10.1038/s41598-018-29699-7.