Location: Forage-animal Production Research
2020 Annual Report
Objectives
Objective 1: Improve the management and use of tall fescue as forage through improved understanding of interactions among tall fescue, endophytes (harmful and beneficial), and climate.
Subobjective 1.A. Assess the genetic and physiological basis for endophyte transmission, stability and enhanced plant stress tolerance.
Subobjective 1.B. Assess plant performance of tall fescue clones harboring non-toxic endophytes under field stress conditions.
Subobjective 1.C. Assess relationship between pasture botanical composition and local climate.
Objective 2: Improve pasture sustainability and enhance animal nutrition, health and performance by exploring and manipulating plant secondary metabolites.
Subobjective 2.A. Determine stability of isoflavones in red clover during the process of cutting and drying for storage by quantifying the variability of isoflavone concentration in fresh through field-cured red clover hay stored under a) ambient conditions under cover and b) climate-controlled storage conditions. Quantify isoflavone degradation kinetics in fresh material, and as a function of drying conditions. The effect of drying conditions on the isoflavone degradation kinetics during subsequent storage will also be quantified.
Subobjective 2.B. Determine the effects of animal-transformed isoflavone metabolites on greenhouse gas production and soil health.
Subobjective 2.C. Explore properties of C3 grass plant secondary metabolites with the potential to benefit ruminant health and performance, based on activity towards rumen microorganisms.
Subobjective 2.D. Assess effects of suppressing isoflavone biosynthetic genes altering clover metabolite profiles.
Objective 3: Improve forage production and management by exploring and manipulating ruminant and non-ruminant gastrointestinal microbiology and manipulating interactions between plant primary and secondary metabolites and the digestive tracts
Subobjective 3.A. Improve understanding of the relationship between forage fructans and the efficiency of rumen fermentation to gain tools for enhancing health and performance.
Subobjective 3.B. Optimize digestive fermentation by ruminants and non-ruminants through improved understanding of the relationship between grass structural polymers and the efficiency of fermentation.
Subobjective 3.C. Determine the effect of site of fermentation, as consequence of digestive tract differences between ruminant and hindgut fermenters, on subsequent manure nutrient leaching potential in animals fed a grass/legume forage only diet or a grass/legume forage diet supplemented with grain.
Objective 4. Improve the contribution of red clover to pasture quality by enhancing stress resistance and root interactions with rhizobium.
Subobjective 4.A. Explore the genetic basis and genome-wide gene expression of 2,4-D resistance in 2,4-D tolerant red clover lines.
Subobjective 4.B. Explore drought tolerance of red clover under abiotic stress conditions.
Subobjective 4.C. Explore the interaction of red clover root phenotypes and interactions with soil rhizobia using gene knockouts.
Approach
Experiments conducted to determine the changes in endophyte gene expression during infection of the ovary by comparing expression inflorescence primordial & ovary tissues to vegetative tissues, the lemma & palea of young florets & pseudostems (Ob. 1A). Experiments conducted to determine the effect of reactive oxygen species (ROS) during endophyte colonization of host ovaries & ovules using fluorescent tagged proteins to monitor expression (Obj 1A). Determine compatibility of 8 non-toxic producing endophyte strains with the Continental tall fescue variety by following endophyte transmission under field conditions using seed staining & immunoblot approaches (Obj 1A). Evaluate the effect of stress on the transmission of different endophyte strains under heat stress (Obj 1B). Growth, seed set & alkaloid production of different endophyte strains under stress conditions in the field will be conducted. Assess the relationship between pasture botanical composition & the ratio of cool season & warm season grasses of on-farm experiments during four years & correlate with changes observed from satellite imagery over longer time to provide producers with a measure of the change from cool season to warm season for the transition zone (Obj 1C).
Stability of isoflavones in storage evaluated by sampling fresh & field-cured (hay) material over time & under different drying & storage conditions (Obj 2A). Excreta from lambs or steers fed isoflavones or hops beta-acids evaluated for greenhouse gas emission (Obj 2B). Bioassay-guided fractionation applied to extracts of phenolic compounds from Lolium perenne to identify specific metabolites inhibiting ruminal hyper-ammonium-producing bacteria (Obj 2C). Isoflavone concentrations & profiles evaluated in clovers mutated in the isoflavone biosynthetic pathway (Obj 2D). Fructan concentrations & profiles determined in several cool-season grasses, & effects on growth of various ruminal bacteria (both mixed & pure cultures) assessed (Obj 3A). Lignin & arabinoxylan extracted from those cool-season grasses & a warm-season grass (Obj 3B). Effects of lignin & arabinoxylan profiles & concentrations on ruminal & equine hindgut bacteria characterized (Obj 3B). Mineral leaching compared from feces of steers & horses fed hay or grain diets (Obj 3C). Mineral leaching compared from feces of horses fed hay with a low or high fructan content (Obj 3C).
Characterize the mode of action for 2-4D resistance in red clover using a whole genome transcription approach to identifying differences between susceptible & resistant germplasm (Obj 4A). Characterize red clover growth parameters, N-fixation & whole genome transcription as affected by heat stress under field conditions (Obj 4B). Gene knock-out experiments will be conducted using the CRISPR/Cas9 system to genes known to affect root morphology & interaction with rhizobium explore interaction of red clover with different rhizobial strains (Obj 4C). Alternate polyadelynation will be evaluated to determine how alternative RNA processing that results in different protein products affects nodulation & nitrogen fixing efficiency (Obj 4C).
Progress Report
Subobjective 1.A. - Assess the genetic and physiological basis for endophyte transmission, stability and enhanced plant stress tolerance. Vectors with two auto fluorescent protein (AFP) genes fused to highly expressed promoters were constructed, transformed into Epichloë coenophiala and are currently being verified that these are stable in the fungus. The transformed E. coenophiala will be used as markers to monitor the transmission of the endophyte through tall fescue seed. Vectors to knock down key regulatory E. coenophiala genes have been constructed to monitor stress effects and fungal stability in tall fescue under stress conditions. These vectors have been transformed into E. coenophiala and the transformed fungi are being introduced into tall fescue plants.
Subobjective 1.B. - Assess plant performance of tall fescue clones harboring non-toxic endophytes under field stress conditions. Transmission and stability of several novel (non-toxic) E. coenophiala endophyte strains have been determined by seed stain microscopy. Pseudostems of tall fescue plants harboring common- and non-toxic endophytes that were grown under different environmental conditions in the field have been harvested at different intervals for gene expression analysis using RNA-seq technology. Seed from these plants were also harvested for alkaloid analysis.
Subobjective 1.C. - Assess relationship between pasture botanical composition and local climate. Baselines for botanical composition and alkaloid composition and levels on selected producer pastures have been determined. Satellite images have been purchased are being analyzed to distinguish between dormant and growing vegetation corresponding to cool and warm season grasses in the selected areas in order to determine validity of the time intervals and periods for analysis. Drone images have been taken and will be used to determine the baseline.
Subobjective 2.B. - Determine the effects of animal-transformed isoflavone metabolites on greenhouse gas production and soil health. Excreta and urine from lambs fed different amounts of biochanin A have been obtained from our sister NP101 Project (Hypothesis 3.C.1), and these have been applied to soil. Greenhouse gas production (carbon dioxide, methane, nitrous oxide, and ammonia) from the tested soil is being measured.
Subobjective 2.C. - Explore properties of C3 grass plant secondary metabolites with the potential to benefit ruminant health and performance, based on activity towards rumen microorganisms. Four fractions from an extract of perennial ryegrass inhibited a ruminal hyper-ammonia-producing bacterium that interferes with assimilation of protein in ruminants. Purification methods are being scaled up for identification of the bioactive molecules (potential alternatives to growth-promoting feed antimicrobials).
Subobjective 2.D. - Assess effects of suppressing isoflavone biosynthetic genes altering clover metabolite profiles. Red clover plants, transformed with CRISPR/Cas9 technology, were obtained that had a deletion in a key gene of the isoflavone biosynthetic pathway. Lower isoflavone levels were observed in the transformed CRISPR/Cas9 plants, thus providing a tool to investigate the role of red clover isoflavones on plant/bacterial and fungal interactions, and red clover plant growth and development.
Subobjective 3.A. - Improve understanding of the relationship between forage fructans and the efficiency of rumen fermentation to gain tools for enhancing health and performance. Fructans of orchardgrass and timothy, fermented in the laboratory by mixed rumen bacteria for different amounts of time, have been separated to determine how quickly they disappear during fermentation and which sizes of fructan chains disappear first. Forages from the first of a 2-year study are being harvested to continue work under Subobjectives 3A and 3B.
Subobjective 3.B. - Optimize digestive fermentation by ruminants and non-ruminants through improved understanding of the relationship between grass structural polymers and the efficiency of fermentation. A method to separate oligosaccharides created after enzymatic digestion of arabinoxylans (structural carbohydrates) of grasses has been developed, and some parameters have been optimized. The esterified acetyl group content of the insoluble cell wall material, a factor which influences forage digestibility, was quantified by a proton nuclear magnetic resonance (NMR)- based method. Monosaccharides of cell wall carbohydrates have been profiled, and bound phenolic acids quantified, from grasses collected in a preliminary study.
Subobjective 4.A. - Explore the genetic basis and genome-wide gene expression of 2,4-D resistance in 2,4-D tolerant red clover lines. Comparison between 2,4-D resistant and susceptible red clover lines using RNA-seq technology has allowed for the identification of a subset of genes that affect the auxin biosynthesis and degradation pathways were found to be expressed differently between the resistant and susceptible lines. However, no specific gene was identified that would be a candidate for the degradation of 2,4-D in the resistant line based on differential expression levels, suggesting that the 2,4-D resistance is not due to a difference in expression of a particular gene.
Subobjective 4.B. - Explore drought tolerance of red clover under abiotic stress conditions. Red clover plants grown under different environmental conditions in the field were harvested for RNA-seq and isoflavone analysis.
Subobjective 4.C. - Explore the interaction of red clover root phenotypes and interactions with soil rhizobia using gene knockouts. Vector construction using the CRISPR/Cas9 technology to knock out genes involved in rhizobium symbiosis is ongoing and transformation of red clover was initiated using vectors that have been confirmed. Alternative polyadenylation sites and alternative splicing of genes critical to red clover and rhizobium symbiosis have been identified using RNA-seq technology.
Accomplishments
1. Effects of genetics and management on grass water-soluble carbohydrates, and prediction of concentrations. Water-soluble carbohydrates (sugars and fructose polymers) of cool-season grasses can provide energy for grazing ruminants but may exacerbate the risk of equine pasture-associated laminitis, an extremely painful hoof condition triggered by diet and often coincident with gastrointestinal disorders. ARS scientists in Lexington, Kentucky, with University of Kentucky collaborators, used near-infrared reflectance spectroscopy and wet chemistry to build an equation predicting the amount of water-soluble carbohydrates in grasses with 90% accuracy. The equation was used to study effects of season and management on water-soluble carbohydrates of selected cool-season grasses. Afternoon increases in water-soluble carbohydrates, and higher levels in perennial ryegrass and tall fescue than in orchardgrass, were observed across a growing season. This is the first such study done in Kentucky. The equation will facilitate future analyses, and the information gained may help producers manage grazing in order to mitigate equine pasture-associated laminitis or optimize ruminant performance.
2. Measurement of water-soluble carbohydrates in red and white clovers. Clovers are often present in cool-season grass pastures, but relatively little is known about how much they contribute to the amount of water-soluble carbohydrates ingested by grazing animals. They may provide energy for ruminants but may exacerbate the risk of equine pasture-associated laminitis, an extremely painful hoof condition triggered by diet and often coincident with gastrointestinal disorders. ARS scientists in Lexington, Kentucky, with University of Kentucky collaborators, quantified water-soluble carbohydrates in repeatedly mowed red and white clovers at different times of day and year. Clovers were 8 to 10% water-soluble carbohydrate (freeze-dried matter basis). Preliminary starch analyses indicated that clovers were 2 to 6% starch (also a source of energy or a risk factor for equine pasture-associated laminitis), depending on time of day and year. This is the first such study done in Kentucky. The information may help producers to make pasture or grazing management decisions for horses or ruminants.
3. Phenolic compounds of cool-season grasses. Plant phenolic compounds are natural products that are sometimes antimicrobial. Some selectively inhibit gut bacteria contributing to gastrointestinal problems of grazing animals, thus even improving the efficiency of digestion by beneficial gut microorganisms. ARS scientists in Lexington, Kentucky, with University of Kentucky collaborators, studied the total phenolic content and phenolic profiles of five cool-season grasses to determine potential benefits to grazing horses or ruminants. The grasses differed in amounts and types of phenolic compounds present. Two phenolic compounds with known antimicrobial activity were present in the grasses studied. The results of this study are informing current research on cool-season grass phenolic compounds that inhibit ruminal ammonia-producing bacteria, thereby improving the efficiency of ruminant digestion.
4. Gene expression analysis of tall fescue harboring different endophyte strains show variable response to water stress. Tall fescue is one of the most abundant cultivated pasture grasses in the United States, with exceptional stress tolerances partly attributed to effects of its symbiotic fungal endophyte, Epichloë coenophiala. Unfortunately, ergot alkaloids produced by common toxic E. coenophiala (CTE) strains have negative effects on livestock. In contrast, novel non-toxic E. coenophiala (NTE) strains lack ergot alkaloids, but may provide insect deterrence or other fitness benefits to tall fescue. ARS scientists in Lexington, Kentucky, along with university collaborators in Kentucky, Oklahoma and Texas, analyzed gene expression profiles of tall fescue clones harboring the CTE and two NTE strains in response to water stress. The results suggested subtle and variable effects of the different endophyte strains on tall fescue gene expression. Also, where the endophyte confers protection, its effects on plant gene expression may help prime the plant for resistance, thereby allowing for better survival in future stress events. This information should aid breeding programs during selection of plant genotypes and endophyte strains for new variety releases.
Review Publications
Kramer, K.J., Kagan, I., Lawrence, L.M., Goff, B.M., Smith, S. 2020. Water-soluble carbohydrates of cool-season grasses: prediction of concentrations by near-infrared reflectance spectroscopy and evaluation of effects of genetics, management and environment. Journal of Equine Veterinary Science. 90:103014. https://doi.org/10.1016/j.jevs.2020.103014.
Kagan, I., Goodman, J.P., Seman, D.H., Lawrence, L.M., Smith, S. 2019. Effects of harvest date, sampling time, and cultivar on total phenolic concentrations, water-soluble carbohydrate concentrations, and phenolic profiles of selected cool-season grasses in central Kentucky. Journal of Equine Veterinary Science. 76:86-93. https://doi.org/10.1016/j.jevs.2019.05.005.
Kagan, I., Anderson, M.L., Kramer, K., Seman, D.H., Lawrence, L.M., Smith, S. 2019. Seasonal and diurnal variation in water-soluble carbohydrate concentrations of repeatedly defoliated red and white clovers in central Kentucky. Journal of Equine Veterinary Science. 84:102858. https://doi.org/10.1016/j.jevs.2019.102858.
Slaughter, L.C., Nelson, J.A., Carlisle, E., Bourguignon, M., Dinkins, R.D., Phillips, T.D., McCulley, R.L. 2019. Tall fescue and E. coenophiala genetics influence root-associated soil fungi in a temperate grassland. Frontiers in Microbiology. 10:2380. https://www.frontiersin.org/articles/10.3389/fmicb.2019.02380/full.
Azzouz-Olden, F., Hunt, A.G., Dinkins, R.D. 2020. Transcriptome analysis of drought-tolerant sorghum genotype SC56 in response to water stress reveals an oxidative stress defense strategy. Journal of Molecular Biology. 47:3291-3303. https://doi.org/10.1007/s11033-020-05396-5.
Dinkins, R.D., Nagabhyru, P., Young, C., West, C., Schardl, C.L. 2019. Transcriptome analysis and differential expression in tall fescue harboring different endophyte strains in response to water deficit. The Plant Genome. 12(2):180071.
