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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

2021 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
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 introduced into endophyte-free tall fescue seedings. Seedling survival and E. coenophiala stability is currently being verified. 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 have been introduced into endophyte-free tall fescue seedings. Seedling survival and E. coenophiala stability is currently being verified. 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 and wild-type toxic strains are being evaluated following stress treatment in the greenhouse. These plants have been transferred to the field following delays due to maximum telework stand-down to monitor transmission in seeds. 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 and alkaloid analysis completed. Subobjective 1.C. Assess relationship between pasture botanical composition and local climate. Pasture sampling from our six project farms occurred twice during the summer/fall of the 2020 growing season. Data were collected on 16 farm-field locations in Tennessee and Kentucky. Purchase of satellite imagery was delayed in order buy all imagery at one time, thus saving money. 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. Field-cured red clover samples were collected, mini-bales prepared and drying of these are in progress for isoflavone concentration analysis. Personnel have turned over during this time and samples will not be collected this growing season. However, the experiment is still on schedule. Subobjective 2.B. Determine the effects of animal-transformed isoflavone metabolites on greenhouse gas production and soil health. Two incubations have been performed to date although data analysis of the second incubation has not yet been done. In the first incubation, ammonia (NH3) emissions from soil amended with urine from lambs receiving the high biochanin A dose were significantly lower than the control during the first week of the incubation. No significant differences were found in fluxes of carbon dioxide, methane, or nitrous oxide though both methane and nitrous oxide showed similar trends to NH3 (lower in biochanin A fed animals than controls). Biochemical analysis of urine samples has not been done due to restrictions on Agricultural Research Service laboratory access to non-Agricultural Research Service personnel. 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 were being scaled up prior to the pandemic 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, and additional analysis on overall gene expression was documented. The results have been published in a peer reviewed journal. 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. Samples from Year 1 were freeze-dried, and university collaborators have ground them. Field sampling from Year 2 plots has begun. A fermentation experiment is being analyzed in order to finalize a method for monitoring fructan disappearance during fermentation. 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. Relevant analysis parameters were determined and optimized if necessary (enzymatic hydrolysis conditions; high-performance anion-exchange chromatography separation gradient, internal standard, retention time, and relevant response factors; concentration range; limit of detection; and limit of quantification), and the method was tested on insoluble cell wall material isolated from four cool-season forage species (timothy, perennial rye, tall fescue, and bluegrass). The arabinoxylan profiles of these species were dominated by unbranched regions, which corroborated the very low arabinose/xylose ratio observed in these species. 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. One set of animal trials has been completed but the second set had to be postponed. That is reflected in sample analysis, though for the majority of the samples taken, analyses have been completed and should be able to be completed as scheduled when COVID restrictions are lifted. 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. An analysis of two years of field trials on the project has been completed and the date is being written up and to be submitted 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 in cytochrome P450 candidate that would be responsible for the tolerance, but changes in expression of a number of different P450’s has 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 to aid in the identification of the physiological nature for the tolerance. Subobjective 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. Subobjective 4.C. Explore the interaction of red clover root phenotypes and interactions with soil rhizobia using gene knockouts. Laboratory work for transformation experiments using the vectors planned for the CRISPR/Cas9 experiments were not initiated and milestones will need to be adjusted back minimally one year.

1. Analysis of red clover isoflavone levels and gene expression in a CRISPR/Cas9 knockout of the Isoflavone Synthase 1 (IFS1) gene. Red clover is a high-quality forage legume well suited for grazing and hay production and produces a number of isoflavonoid compounds that have the potential for medicinal and antimicrobial applications. Like other legumes, it harbors nitrogen fixing bacteria and does not need nitrogen fertilizer, which means nitrogen fertilizers can be reduced if red clover is used in pastures. One of the postulated roles for isoflavones in soybeans is that they function as a signal molecule exuded from the roots to attract nitrogen fixing bacteria. In order to study the role of isoflavonoids in red clover, CRISPR/Cas9 technology was used by ARS scientists in Lexington, Kentucky, along with University of Kentucky collaborators, to knock out the function of a key enzyme in the biosynthesis of isoflavones, isoflavone synthase, and gene expression profiles were compared between the CRISPR/Cas9 mutant and wild-type plants. Mutant plants carrying a deletion in the isoflavone synthase gene had reduced levels of the isoflavones. No differences in the number of nitrogen fixing nodules were observed between the wild-type and CRISPR isoflavone synthase knockout mutants. Interestingly, gene expression profiles showed that defense response genes were more highly expressed in the mutant plants suggesting that other bacterial in the root zone might be causing harm. Thus, while it appears that the production of these isoflavones, are not involved in attracting rhizobium, but may function as an anti-microbial agent to repel harmful bacteria in the root zone.

2. Prediction of ethanol-soluble carbohydrate concentrations of cool-season grasses by near-infrared reflectance spectroscopy, and evaluation of effects of cultivar and management. Short-chain fructans from chicory have been implicated in changes in the microbial community and chemical composition of the equine hindgut. Laminitis, an extremely painful hoof condition triggered by diet, is often coincident with gastrointestinal disorders. Hence, rapid quantification of ethanol-soluble carbohydrates (ESCs) in pastures may help to make grazing management decisions to reduce laminitis and improve horse welfare. ARS scientists in Lexington, Kentucky, with University of Kentucky collaborators, used wet chemistry and near-infrared spectroscopy (NIRS) to predict ESC concentrations of cool-season grasses with 95% accuracy. The method was used for rapid determination of ESCs of repeatedly defoliated grasses in central Kentucky, and the effects of cultivar and management on ESCs were studied. ESC concentrations were generally higher in the afternoon than in the morning. Concentrations tended to be highest in cultivars of perennial ryegrass or tall fescue, and lowest in cultivars of orchardgrass. The equation will expedite quantifying ESCs in cool-season grass pastures, and the results of the study may help to guide grazing management decisions for horses.

Review Publications
Kramer, K.J., Kagan, I., Lawrence, L.M., Smith, S.R. 2021. Ethanol-soluble carbohydrates of cool-season grasses: prediction of concentration by near-infrared reflectance spectroscopy (NIRS) and evaluation of effects of cultivar and management. Journal of Equine Veterinary Science. 101. Article 103421.
Dinkins, R.D., Hancock, J., Coe, B.L., May, J.B., Goodman, J.P., Bass, W.T., Liu, J., Fan, Y., Zheng, Q., Zhu, H. 2021. Isoflavone levels, nodulation and gene expression profiles of a CRISPR/Cas9 deletion mutant in the isoflavone synthase gene of red clover. Plant Cell Reports. 40(3):517-528.
Kagan, I. 2021. Soluble phenolic compounds of perennial ryegrass (Lolium perenne L.): potential effects on animal performance, and challenges in determining profiles and concentrations. Animal Feed Science and Technology. 277. Article 114960.
Pan, L., Cui, S., Dinkins, R.D., Jiang, Y. 2021. Plant growth, ion accumulation and antioxidant enzymes of endophyte-infected and endophyte-free tall fescue to salinity stress. Acta Physiologiae Plantarum. 43. Article 95.
Liu, J., Yu, X., Qin, Q., Dinkins, R.D., Zhu, H. 2020. The impacts of domestication and breeding on nitrogen fixation symbiosis in legumes. Frontiers in Genetics. 11. Article 00973.