Location: Forage-animal Production Research
2024 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.
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)
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
This is the final report for this project which terminated in February 2024. See the report for the replacement project, 5042-21500-001-000-D, “Increasing Sustainability of Forage Production in the Mid-South Agroecosystems” for additional information.
Objective 1: Tall fescue is a highly adaptable forage, pasture and turf grass that is grown in over 14 M ha in the eastern half of the United States and in other temperate regions of the world. A significant factor in adaptability, productivity and stand persistence under stress conditions is in part due to the presence of an intercellular seed-transmissible endophytic fungus Epichloë coenophiala especially during stress. How the endophyte senses and responds to stress when the host plant is subjected to stress was investigated by monitoring gene expression of E. coenophiala, and closely related non-toxic producing endophytes, when tall fescue plants were subjected to acute water-deficit stress. Plants harboring different endophyte strains were grown in the greenhouse, then half were deprived of water for 48 hours and the other half remained under the regular watering regime. RNA was isolated from different plant tissues and whole transcriptome sequencing (mRNA-seq) was conducted to identify genes that were differentially expressed under the stress treatment. Our results showed that the fungal response to drought involved gene-expression changes in similar pathways that have been documented for plants under stress. Increased expression of genes involved in oxidative stress response, oxygen radical detoxification, heat shock, cellular transport and carbohydrate metabolism pathways were observed. The magnitude of fungal gene responses during stress depended 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 persistence.
Objective 1: Epichloë endophytes have been shown to produce a number of alkaloid compounds only in planta, some which are beneficial in repelling insects, while others are toxic to animals resulting in production losses for animal producers. The goal of this work was to monitor the level of the ergot and loline alkaloid accumulation in individual plants to determine the plant genotype contribution to alkaloid levels. The experimental design consisted of sixteen tall fescue Kentucky 31 plants harboring the common toxic endophyte in a space planted replicated trial over the course of three years. Our results demonstrated that while changes in the alkaloid concentrations were observed over the three years due to differences in yearly weather conditions, the overall relative plant/endophyte combination levels tended to remain the same relative to other plant/endophyte combinations in the field over the course of the study. Additionally, we observed that overall levels of the ergot and loline alkaloid accumulation did not vary in the same manner over the three years. Since the E. coenophiala endophyte was the same genotype in all the clones, these results indicate that it is the plant genetics that is responsible for determining the levels of the different alkaloids, and suggests that the signal(s) from the plant to the endophyte may not be the same for the different alkaloid production. Future work is needed to identify these signals and how the plant and fungus communicate to produce the different alkaloid compounds that aid in the fitness of this grass. Work on the different non-toxic alkaloids produced by the endophyte, and how these may be insect and nematodes deterrents, is being addressed on the new project plan.
Objective 2: Plant phenolic compounds are natural products that are sometimes antimicrobial. ARS scientists in Lexington, KY, with University of Kentucky faculty, studied the total phenolic content and phenolic profiles of five cool-season grasses. 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 hyper-ammonia-producing bacteria.
Objective 2: Red clover produces a number of isoflavonoid compounds that have come into the forefront of biomedical and agricultural research due to their potential for medicinal and antimicrobial applications. In addition, one of the postulated roles for isoflavones in soybeans is that they function as a signal molecule exuded from the roots in attracting nitrogen fixing rhizobia. In order to study the role, and effects of lack of, isoflavonoids in red clover, CRISPR/Cas9 technology was used to knock out the function of a key enzyme in the biosynthesis of isoflavones, isoflavone synthase, and gene expression profiles were compared between the mutant and wild-type plants by RNA-seq technology. Mutant plants, carrying a deletion in the isoflavone synthase gene, had significantly reduced levels of the isoflavones formononetin, biochanin A and genistein. However, no differences in the number of nodules were observed on the wild-type and mutant roots when inoculated with rhizobium suggesting that these isoflavones are not the signaling molecule for the interaction of the rhizobium bacteria with red clover. However, when gene expression profiles were compared between the mutant and wild-type plants, it was observed that genes involved in defense response were more highly expressed in the mutant plants. This result suggests that the exuded isoflavones may aid to function in the control of harmful bacteria in the root zone and illustrates the putative role for the production of these compounds in legumes.
Objective 3: The fiber in cool-season grasses consists partly of arabinoxylans, a class of sugars that help to determine the structure of plant cell walls. Arabinoxylans (AX) exist in different lengths (different numbers of sugars linked together), and different AX have different amounts of phenolic compounds (natural products that can be antioxidants) bound to them. These structural differences among AX are likely to affect how grasses are digested by grazing animals, but too little is known about the structures of AX in cool-season grasses to consider AX when choosing feed for forage animals. The purpose of this study was to develop a method to profile AX structures in different cool-season forage grasses (timothy, perennial ryegrass, tall fescue, and Kentucky bluegrass. Individual sugars in the cell walls of the grasses were also measured, as were some of the phenolic compounds bound to AX. The four grasses differed in the amounts of simple sugars recovered from the cell walls, suggesting differences in cell wall structure. More small chains of sugars were recovered from perennial ryegrass cell walls than from the cell walls of timothy, Kentucky bluegrass, or tall fescue. The method that was developed is ideally suited to monitor structural changes of AX in forages as a result of plant breeding, pasture management, and fermentation of plant material.
Objective 3: Cool-season grasses contain chains of fructose (a simple carbohydrate) of varying length, called fructans. In the rumen of cattle, bacteria can break down fructans to fructose, thus providing a source of energy for the animal. Little is known about the process of fructan breakdown. Mixed bovine ruminal microbes were incubated with milled timothy and orchardgrass, and water-soluble carbohydrates (fructans, monosaccharides, and disaccharides) were profiled over time to monitor their disappearance. Timothy and orchardgrass contained different initial amounts of glucose and sucrose, and the extent of disappearance of these sugars differed between orchardgrass and timothy solutions, although the same bacteria fermented both. Results suggested that bacteria in the rumen of cattle convert long-chain fructans into shorter ones before breaking them down completely to fructose.
Objective 4: Red clover is an important forage and pasture legume grown throughout temperate regions and is a major contributor of biological fixed nitrogen because of its ability to fix atmospheric nitrogen. Red clover has not been a model legume primarily due to self-incompatibility and the associated high heterozygosity despite its relatively small genome. Thus, it has not been a significant contributor in molecular or genetic studies and basic information on red clover legume/rhizobium symbiosis is lacking. Using recently developed red clover genomic resources and RNA sequencing expression analysis, we described the genes that are expressed in nodule forming roots. As has been observed for other species, red clover has many of the same genes involved in rhizobium symbiosis that are conserved with those of other legumes, although some of the genes are red clover-specific with little or no similarity with other legumes. These include the nodule-specific cysteine rich proteins (NCRs) that while they have common features, lack sequence similarity with other legumes and likely regulate the specific plant/ rhizobium symbiont strain interactions. With the knowledge of the expression of these genes and genetic variation in red clover, breeding for increased nitrogen fixation efficiency by selection of genotypes with increased rhizobium specificity should be feasible. Using these resources the genes involved in secondary compound production, rhizobium interaction and genes and pathways affecting persistence, forage yield and quality, all of which will facilitate genetic improvement and red clover cultivar development. Work on genes that involved in Rhizobium interaction and stress tolerance is continuing in the new Project Plan.
Accomplishments
1. Established a basis for 2,4-D tolerance in red clover. Incorporation of red clover into grass pastures offers a number of benefits. However, the lack of a selective herbicide that does not injure the red clover presents a considerable limitation to the management of broadleaf weed species in interseeded clover-grass pastures. The standard for broadleaf weed control in pastures is 2,4-D. To investigate its effect on a novel red clover line that is 2,4-D tolerant, ARS scientists in Lexington, Kentucky, with University of Kentucky collaborators, employed a transcriptome analysis. The objectives of this study were to: (1) determine if the increased 2,4-D tolerance in the UK2014 line was reflected in a change in overall gene expression and/or a quicker recovery response following 2,4-D treatment; (2) identify genes, that were expressed or induced by 2,4-D, that could be the basis for the increased 2,4-D tolerance. While no specific gene was identified that would be a candidate in the degradation of 2,4-D in the resistant line, the experiment identified several genes that can be used as markers for breeding tolerance. These results corroborated previous findings suggesting that 2,4-D tolerance is due to multiple genes. Additionally, using the RNA-seq sequencing for the experiment, we identified genetic changes in several genes that are postulated to be key mediators of 2,4-D metabolism. Further study will be needed to determine if these differences in these proteins are the cause of the increased tolerance in the red clover UK2014 line.
2. Profiling of selected water-soluble carbohydrates of tall fescue and timothy for information on their potential benefits for ruminal fermentation. Fructans are chains of fructose found in cool-season grasses. The length of the fructan chains has been found to affect their fermentation in vitro by both ruminants and horses. ARS scientists at Lexington, Kentucky, in collaboration with a University of Kentucky researcher, profiled fructans in tall fescue and timothy harvested from spring to fall. On most harvest dates, tall fescue had more short-chain fructan than timothy, and timothy had more long-chain fructan than tall fescue. Ruminants may ferment these types of fructans differently, thus obtaining different nutritional benefits. The study is a first step to determining if grasses with different types of fructans may differ in the nutritional benefits provided to ruminants.
Review Publications
Kagan, I., Davis, B.E., Schendel, R.R. 2023. Seasonal and species variation in raffinose, short-chain fructan, and long-chain fructan accumulation in tall fescue (Festuca arundinacea Schreb.) and timothy (Phleum pratense L.) grown in Central Kentucky. Grass and Forage Science. 78(4):536-546. https://doi.org/10.1111/gfs.12633.
McGrail, R.K., Carlisle, E.A., Nelson, J.A., Dinkins, R.D., McCulley, R.L. 2024. Tall fescue and endophyte genetics influence vertical transmission and seed characteristics under climate change scenarios. Phytobiomes Journal. https://doi.org/10.1094/PBIOMES-09-23-0102-R.
de Araujo, L.P., Barrett, M., Dinkins, R.D. 2024. Comparative gene expression following 2,4-D treatment in two Red Clover (Trifolium pratense L.) populations with differential tolerance to the herbicide. Agronomy. 14(6). Article 1198. https://doi.org/10.3390/agronomy14061198.
Lakes, J.E., Ferrell, J.L., Flythe, M.D. 2023. Growth and metabolism of Clostridioides difficile in hungate-style media. Applied Microbiology. 4(1):85-95. https://doi.org/10.3390/applmicrobiol4010006.
Parker, J.L., Page, A., Jacob, O., Stanton, T., Davis, B.E., Flythe, M.D., Adam, E.N. 2024. Equine fecal microbiota response to short term antibiotic administration. Journal of Equine Veterinary Science. 133. https://doi.org/10.1016/j.jevs.2023.104993.
Pyles, M., Agbana, M., Hayes, S., Flythe, M.D., Lawrence, L. 2023. The establishment of fibrolytic bacteria in the foal gastrointestinal tract is related to the occurrence of coprophagy by foals. Animals. 13(17):2718. https://doi.org/10.3390/ani13172718.
Lakes, J.E., Ferrell, J.L., Berhow, M.A., Flythe, M.D. 2024. Antimicrobial effects of cannabidiol on select agriculturally important Clostridia. Anaerobe. 87. Article 102843. https://doi.org/10.1016/j.anaerobe.2024.102843.
