Skip to main content
ARS Home » Midwest Area » Lexington, Kentucky » Forage-animal Production Research » Research » Research Project #436088

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

Location: Forage-animal Production Research

2022 Annual Report

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. Objective 2: Improve pasture sustainability and enhance animal nutrition, health and performance by exploring and manipulating plant secondary metabolites. 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 Objective 4. Improve the contribution of red clover to pasture quality by enhancing stress resistance and root interactions with rhizobium. Objective 5: Assemble and integrate current hemp related data and collect new data in collaboration with University partners to support hemp production modeling efforts at ARS Corvallis, OR. (NP215 C3, PS3B) Objective 6: Perform research to support the use of hemp and hemp residual biomass as a livestock feed, including exploration of possible benefits of compounds produced by hemp for animal production. (NP215 C4, PS4C)

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
Sub-objective 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 the fungal endophyte Epichloë coenophiala and have been introduced into endophyte-free tall fescue seedings. Plants are currently being grown in order to visualize transmission in the seeds, but not many have flowered due to difficulty getting plants with the introduced E. coenophiala strains mature enough to subject to vernalization treatment. This work is ongoing. Vectors to knock down key regulatory E. coenophiala genes were 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 have been introduced into endophyte-free tall fescue seedings. The three plants were vernalized over winter to induce flowering. The plants were then tested again for endophyte, and each contained little or none. Seeds were harvested from the plants and 36–40 seeds from each were examined for viable endophyte, but none was found. We conclude that constitutive reactive oxygen species (ROS) generation by the endophyte NoxA complex probably compromises symbiosis and seed-transmission, possibly by reducing colonization of young vegetative and reproductive tissues. Sub-objective 1.B. Assess plant performance of tall fescue clones harboring non-toxic endophytes under field stress conditions. 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-sequencing technology. Following delays in processing samples for RNA-seq, a subset of the first year wild-type and non-endophyte harboring plants have been completed and analysis of the gene expression under the different conditions is currently underway. Analysis of the different alkaloid levels under the different environmental conditions did not show any major differences between treatments, but significant changes over years. Sub-objective 1.C. Assess relationship between pasture botanical composition and local climate. Data for botanical composition, using five different methods, has been collected in all the proposed pastures. An extension publication which will help producers estimate pasture mass using a rising plate meter is under review. The work looking at changes over time in warm season encroachment and comparing the different methods of botanical analysis has suggested a decline in the warm season grasses in the pastures sampled compared to the previous year sampling. However, we should highlight that rainfall was above average since the last update. Preliminary analysis has shown that the visual estimation method is subjective and could be biased compared to the others and tends to overestimate the species with more leaf area. In addition, sod forming grasses tend to be overestimated in comparison to bunch type grasses. Sub-objective 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. Field-cured red clover samples were collected, mini-bales prepared and drying of these are in progress for isoflavone concentration analysis. Due to personnel leaving during this time, the isoflavone concentration analysis has not been completed. Sub-objective 2.B. Determine the effects of animal-transformed isoflavone metabolites on greenhouse gas production and soil health. Three sets of incubations, where excreta were applied to soil and trace gas fluxes were measured in 2020, 2021, and 2022. The metabolite analysis for the previous year was completed once COVID restrictions were lifted. Statistical analysis of these datasets is ongoing, and a manuscript is in preparation. Sub-objective 2.C. Explore properties of cool-season grass plant secondary metabolites with the potential to benefit ruminant health and performance, based on activity towards rumen microorganisms. Inhibitory fractions were separated by liquid chromatography with mass spectrometric detection. Fractions contained too many compounds, at too low concentrations, for identification. Sub-objective 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, and additional analysis on overall gene expression was documented in our published manuscript in a peer reviewed journal. Sub-objective 3.A. Improve understanding of the relationship between forage fructans and the efficiency of rumen fermentation to gain tools for enhancing health and performance. Analysis of fructan profiles in timothy and tall fescue is underway. Fermentation studies will be performed on selected samples after key times of profile changes are identified. Sub-objective 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. We generated an extensive and extremely useful set of data from the Year 1 samples tracking the changes in the forage cell wall (monosaccharide composition of the cell wall carbohydrates, the degree of substitution on the arabinoxylan backbones, phenolic acid contents, and lignin levels) over the growing season for five cool-season forages. Samples were collected at four different timepoints over the season (April, June, August, October) and analyzed in quadruplicate. Lignin and para-coumaric acid levels were higher in the summer months, and the degree of substitution on the arabinoxylan backbone increased dramatically over the growing season. These fully- characterized samples will be subjected to fermentation by rumen microorganisms in the next stage of the project. Sub-objective 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. Both equine and bovine animal trials were conducted, and samples collected. All subsequent microbial and nutrient leaching analyses are complete. Sub-objective 4.A. Explore the genetic basis and genome-wide gene expression of 2,4-D resistance in 2,4-D tolerant red clover lines. An analysis of two years of field trials on the project has been completed and the data is being written up for submission to a refereed journal. Analysis of gene expression in the UK2014 resistant and the 2,4-D sensitive red clover cultivar (Kenland) before and after 2,4-D treatment has not identified a particular cytochrome P450 candidate that would be responsible for the tolerance, but changes in expression of a number of different P450’s have been identified and are being further analyzed. While significant improvement in red clover 2,4-D tolerance has been achieved, additional tolerance would be desirable both from a commercial standpoint and from the standpoint of aiding in the identification of the physiological nature for the tolerance. Sub-objective 4.B. Explore drought tolerance of red clover under abiotic stress conditions. Forage biomass analysis has been done on the samples collected and these have been submitted for isoflavone analysis but not yet completed. Samples were collected at four different time points for gene expression analysis by RNA-seq. RNA isolation for RNA-seq analysis was delayed due COVID restrictions, but has been initiated. Subobjective 4.C. Explore the interaction of red clover root phenotypes and interactions with soil rhizobia using gene knockouts. Transformation experiments using the vectors planned for the CRISPR/Cas9 experiments were initiated and initial transgenic plants are being screened for potential rhizobia using gene knockouts. Transformation experiments using the vectors planned for the CRISPR/Cas9 experiments were initiated and initial transgenic plants are being screened for potential knockouts in the genes that regulate red clover rhizobium symbiosis. Preliminary results suggest that the expected knockout in the genes targeted has been successful, although most of the putative knockout plants appear to be heterozygous, thus requiring crossing in order to recover homozygous mutant plants.

1. Developed method to monitor the change in fructan in simulated ruminal fermentations. Fructans, which are chains of the sugar fructose, are produced by cool-season grasses. Some chains are very long, and some are short (fewer than 10 units long). Little is known about their effects on the performance of ruminants grazing those grasses. To determine if they have effects on ruminant performance, a method is needed for monitoring their disappearance in the rumen. ARS researchers in Lexington, Kentucky, analyzed the fructans in orchardgrass and timothy before and during fermentation by rumen microorganisms in the laboratory. After two hours of fermentation, longer fructans began to disappear, while shorter fructans increased. After five hours of fermentation, most fructans had disappeared. The results suggest that in the rumen, longer fructans are cleaved to shorter fructans prior to being utilized. The work provides a method for monitoring changes in fructans during utilization by ruminants, which is a first step to determining their effects on ruminant performance.

2. Compared metabolism of fructan and cellulose by gut microbiota. Fructan and cellulose are both considered fiber by nutritionists, but gut microbes utilize them differently, potentially leading to various health outcomes for animals consuming these different forms of fiber. ARS researchers in Lexington, Kentucky, gave labeled fructan or cellulose to an uncultivated mixture of bacteria and other microorganisms that were harvested from mice. The labeled fiber types revealed a great diversity of products made by the bacteria. In general, more compounds were made from fructan than from cellulose. Some of the products were associated with acids stress response in the bacteria, which is consistent with highly fermentable feeds causing acid-related health problems in ruminants and horses. The results are a first step to understanding different health outcomes from consuming different types of fiber.

3. Screened for genes that allow tall fescue to survive water stress conditions. Tall fescue is a popular pasture and turf grass particularly known for its drought resistance and persistence. However, during summer drought periods, and over-winter, the above-ground tissues die and plant regrowth emerges from the crown. ARS researchers in Lexington, Kentucky, used a genomic approach to characterize the gene expression in different tissues during water stress to characterize the molecular mechanisms involved in stress survival and persistence. Gene expression was monitored in leaves, pseudostems (tillers), crown and roots. Gene expression between the different tissues across stressed and unstressed plants was compared. Genes encoding proteins involved stress response pathways, such as photosynthesis, sugar metabolism, phytohormone biosynthesis and signaling were observed in all tissues subjected to stress. However, a few genes were differentially expressed primarily in crown tissues and meristems. These results suggest that regulation of these genes in the crown may aid in the survival of the meristem thereby allowing for continued plant survival. These results should allow for selection molecular markers in screening for resistant genotypes.

4. Conducted a molecular analysis on the tall fescue endophyte response to water stress. Epichloë coenophiala is a seed- borne fungal symbiont (endophyte) of tall fescue and has been documented to confer better persistence to tall fescue plants than plants lacking the endophyte, especially under stress conditions. How the endophyte senses and responds to stress when the host plant is subjected to stress has not been previously characterized. Thus, this work was done to investigate the effects on gene expression of E. coenophiala, and closely related non-toxic producing endophytes, when tall fescue plants were subjected to acute water stress. ARS researchers in Lexington, Kentucky, showed that the fungal response to drought involved gene-expression changes in oxidative stress response, oxygen radical detoxification, heat shock, cellular transport and carbohydrate metabolism pathways. These genes are similar to those that we have previously identified as affected in the plant during water stress. The magnitude of fungal gene responses during stress was depended on, and varied with, the host plant and endophyte strain. Our results suggest that Epichloë fungi, along with their host plants, cooperate to regulate stress responses or to separately activate stress response mechanisms, that when combined together for mutual protection, results in better plant stress tolerance. This mutual cooperation, leading increased persistence, has been one of the hallmarks of why tall fescue has become a dominant forage species in the United States transition zone.

Review Publications
Chakrabarti, M., Nagabhyru, P., Schardl, C.L., Dinkins, R.D. 2022. Differential gene expression in tall fescue tissues in response to water deficit. The Plant Genome. 15:e20199.
Kagan, I., Harlow, B.E., Flythe, M.D. 2022. A chromatographic method to monitor fructan catabolism in two cool-season grasses fermented by mixed bovine ruminal microbiota. Journal of the Science of Food and Agriculture Report. 2:264-271.
Kagan, I. 2022. Water- and ethanol-soluble carbohydrates of temperate grass pastures: a review of factors affecting concentration and composition. Journal of Equine Veterinary Science. 110. Article 103866.
Nagabhyru, P., Dinkins, R.D., Schardl, C.L. 2022. Transcriptome analysis of Epichloë strains in tall fescue in response to drought stress. Mycologia. 114(4):697-712.
Deng, P., Valentino, T., Flythe, M.D., Moseley, H., Leachman, J., Morris, A., Henning, B. 2021. Untargeted stable isotope probing of gut microbiota metabolome using 13C-labeled dietary fibers. Journal of Proteome Research. 20(5):2904-2913.