Location: Forage-animal Production Research
2023 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
Sub-objective 1.A. Assess the genetic and physiological basis for endophyte transmission, stability and enhanced plant stress tolerance. Experiments were completed but the results were not as expected. Endophyte seed transmission was found to be 50% or less and expression of the auto fluorescent proteins (AFP) constructs in the ovaries was very low. Expression of the AFP constructs in the ovaries does not appear to be the same as observed in for the expression of these genes by regular molecular techniques. This is especially the case for the fusion of the red fluorescent AFP marker where no expression was observed. These results suggest that the AFPs might be detrimental for the fungal growth. Further studies are needed to determine if this is the case.
Sub-objective 1.B. Assess plant performance of tall fescue clones harboring non-toxic endophytes under field stress conditions. The analyses of the half-sib family field plants is continuing, but a year behind schedule as described in previous annual reports.
Sub-objective 1.C. Assess relationship between pasture botanical composition and local climate. The studies monitoring species change over time have been completed and showed an expected shift from C3 to C4 species during the warmer months of the growing season, but this shift was most dramatic under drought conditions and made worse by overgrazing. Drought conditions confounded with less-than-ideal management strategies caused estimated dry matter yield to decline significantly over the study period. A manuscript of the data is being prepared.
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. Quantified 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. Due to personnel leaving during this time, this work cannot be completed.
Sub-objective 2.B. Determine the effects of animal-transformed isoflavone metabolites on greenhouse gas production and soil health. Trace gas emissions from urine-amended soils decreased with the addition of biochanin A (BCA) compared to urine from animals fed no BCA, though urinary nitrogen excretion and soil microbial communities were unaffected, showing that BCA may mediate soil enzyme relationships or microbial activities that govern trace gas emissions from urine excreta patches.
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. Extractions and separation of extracts were resumed, using new solvent systems. The new separations yielded one antimicrobial zone, potentially simplifying the work remaining to be done to purify the antimicrobial compound.
Sub-objective 2.D. Assess effects of suppressing isoflavone biosynthetic genes altering clover metabolite profiles. This project has been completed, the results published, as reported in last year’s annual report.
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. Fructans were profiled in tall fescue and timothy harvested in April, June, August, and October of two years. Species differences in amounts of long and short fructans were determined. Resulting manuscript has been submitted and is under review.
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. Sample collection and analysis was completed, and manuscript has been submitted for publication.
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. Samples have been collected and majority of analysis was done prior to critical vacancy, but since primary investigator (PI) has left final analysis is not possible without expertise from the PI, thus it cannot be completed until similar expertise is available.
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. Analysis of two years of field trials demonstrated significant improvement in red clover 2,4-D tolerance in the field. These results have been published. Gene expression analysis has been completed and the results are being written up for journal submission.
Sub-objective 4.B. Explore drought tolerance of red clover under abiotic stress conditions. Forage biomass and isoflavone analysis has been completed. Gene expression analysis by RNA-sequencing has been completed and analysis of the data is underway for manuscript preparation.
Sub-objective 4.C. Explore the interaction of red clover root phenotypes and interactions with soil rhizobia using gene knockouts. Analysis of gene expression of nodulation has been completed and a manuscript published describing the red clover expression in response to rhizobium infection and colonization. Transformation experiments using CRISPR/Cas9 vectors to knockout genes that regulate red clover rhizobium symbiosis have been done. The expected knockout in the genes targeted has been successful, although most of the knockout plants are hemizygous requiring intercrossing to recover homozygous knockout plants in order to screen for rhizobium interactions. Crossing is in progress.
Accomplishments
1. Developed a method to characterize the fine structure of fiber in cool-season grasses for future monitoring of fiber fermentation by ruminants. Fiber in cool-season grasses is comprised of different carbohydrates, including a group of carbohydrates called arabinoxylans. In grains, the structure of arabinoxylans has been investigated because of their potential role in human nutrition and food processing, but arabinoxylans in cool-season grasses have not been investigated despite a potential role in ruminant nutrition. ARS scientists in Lexington, Kentucky, and University of Kentucky collaborators developed a method to isolate, separate, and compare arabinoxylan components of grasses. The method was validated with perennial ryegrass, Kentucky bluegrass, tall fescue, and timothy, all of which differed in arabinoxylan fingerprints created by carbohydrate profiles, carbohydrate concentrations, and concentrations of other chemicals (phenolics) bound to the carbohydrates. Perennial ryegrass, Kentucky bluegrass, tall fescue, and timothy each had a unique fingerprint of arabinoxylan components. The method lays the groundwork for future studies determining if some of the components of cool season forage fiber may affect fermentation, and hence ruminant performance, through their structure.
2. Identified red clover nodulation specific genes as targets to improve nitrogen fixation. Red clover is an important forage crop and serves as a major contributor of nitrogen in pastures due to nitrogen-fixation when associated with nodule forming Rhizobium bacteria. Using recently developed red clover genomic resources, ARS scientists at Lexington, Kentucky, conducted a study to identify genes that are expressed only in nodule forming roots. Many of the genes involved in rhizobium symbiosis were conserved when compared to other legumes, although we identified over 450 genes that are red clover-specific with little or no sequence similarity with genes in other legumes. The latter included genes that would encode for proteins that are similar to nodule-specific cysteine rich proteins (NCRs) that are postulated to regulate the plant / rhizobium interaction. This knowledge can be used to aid in breeding for selection of rhizobium strain interactions that are specific with red clover, thereby increasing nitrogen fixation efficiency.
3. Tall fescue genotype controls of fescue toxicosis alkaloid production. Tall fescue is a highly adaptable forage grass in part due to the presence of an endophytic fungus, Epichloë coenophiala, that has been reported to provide tolerance to stress environments. A feature of these endophytes is that they produce alkaloids when associated with tall fescue. Some of the alkaloids are beneficial in repelling insects, while others are toxic to animals resulting in production losses for animal producers. ARS scientists in Lexington, Kentucky, monitored the level of alkaloid accumulation in individual plants to determine how the plant genotype contributes to alkaloid levels. Changes in the alkaloid concentrations were observed over the course of three years under different yearly weather conditions. While weather induced changes were observed, the rankings of alkaloid accumulation differences between genotypes tended to remain the same over the course of the study. These results suggest that the plant genetic background is responsible for signaling the endophyte to produce the different levels of alkaloids. This work provides a working background to aid in future molecular breeding to identify factors that are responsible for the regulation of alkaloid levels while simultaneously maintaining the positive aspects of the symbiosis that provides for tall fescue stress tolerance.
4. Fusarium head blight in hemp fields across Kentucky. As hemp becomes established as a commodity in the U.S., continued cultivation demands a greater understanding of the pathogens that affect the portions of the plant with livestock feed potential, such as flowers and grain. Several Fusarium species, including F. graminearum, that are known to produce mycotoxins, have been confirmed to cause disease on hemp. University of Kentucky scientists, through a Non-Assistance Cooperative Agreement with ARS researchers from Lexington, Kentucky, isolated Fusarium species from harvested and stored hemp floral material from seven Kentucky producers from 2019 and 2020 harvests. At least 12 different species of Fusarium were isolated. Understanding of Fusarium species that affect hemp, and the mycotoxins and mycotoxin concentrations produced by the different Fusarium species, will facilitate making better management recommendations and developing testing protocols to ensure safety of hemp as livestock feed.
Review Publications
Joyce, G.E., Kagan, I., Flythe, M.D., Davis, B.E., Schendel, R.R. 2023. Profiling of cool-season forage arabinoxylans via a validated HPAEC-PAD method. Frontiers in Plant Science. 14. Article 1116995. https://doi.org/10.3389/fpls.2023.1116995.
Dinkins, R.D., Coe, B.L., Phillips, T.D., Ji, H. 2023. Accumulation of alkaloids in different tall fescue KY31 clones harboring the common toxic Epichloë coenophiala endophyte under field conditions. Agronomy. 13(2). Article 356. https://doi.org/10.3390/agronomy13020356.
Liu, S., Coyne, M.S., Grove, J.H., Flythe, M.D. 2023. Nitrite oxidizing bacteria, Nitrobacter and Nitrospira, are differently influenced by season, fertilizer, and tillage in long-term maize culture. Applied Soil Ecology. 177. Article 104530. https://doi.org/10.1016/j.apsoil.2022.104530.
Dinkins, R.D., Hancock, J.A., Bickhart, D.M., Sullivan, M.L., Zhu, H. 2022. Expression and variation of the genes involved in rhizobium nodulation in red clover. Plants. 11(21). Article 2888. https://doi.org/10.3390/plants11212888.
Araujo, L.P., Burke, T., Dinkins, R.D., Barrett, M. 2022. Field performance of a red clover germplasm selected for increased tolerance to 2,4-D. Weed Technology. 36(6):831-837. https://doi.org/10.1017/wet.2022.89.
Hansen, T.L., Fowler, A.L., Davis, B.E., Hayes, S.H., Crum, A., Lawrence, L.M. 2021. Modeling digesta retention in horses fed high or low neutral detergent fiber concentration forages. Livestock Science. 250. Article 104592. https://doi.org/10.1016/j.livsci.2021.104592.
