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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

2019 Annual Report

1a. Objectives (from AD-416):
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.

1b. Approach (from AD-416):
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).

3. Progress Report:
Gene expression profiles of the tall fescue endophyte comparing vegetative and inflorescence and ovary tissues was conducted using Next-Gen sequencing technology. Gene expression profiles of the tall fescue under stress was conducted using Next-Gen sequencing technology. Seed from different tall fescue genotypes harboring different non-toxic endophyte strains was collected and will be used for studies on seed transmission to verify tall fescue endophyte compatibility. CRISPR-Cas vectors to target the Isoflavone Synthase (IFS) genes have been constructed and being utilized in transformation experiments to knock out IFS gene expression. CRISPR-Cas vectors to target the Cleavage and Polyadenylation Specificity Factor (CPSF) 30 subunit has been constructed and being utilized in transformation experiments to knock out CPSF30 gene expression. Expression of genes in 2,4-D resistant and susceptible red clover genotypes have analyzed using Next-Gen technology and identification of 2,4-D metabolizing genes that would potentially play a role in the tolerance have been identified. Identification of these differences in protein levels or protein sequence should aid breeders in developing technologies to select for natural variants that tolerate 2,4-D for incorporation in variety development. RNA-seq libraries for nodule specific expression have been prepared and the 450 red clover genes that encode small Nodule-specific Cysteine Rich (NCR) proteins that interact with rhizobium bacterium have identified.

4. Accomplishments
1. Gene expression of the tall fescue endophyte, Epichloe coenophiala, in inflorescences and ovary tissues. Using Next-Gen RNA-seq technology comparison of gene expression profiles of the tall fescue endophyte in vegetative and inflorescence and ovary tissues was done. The results demonstrated that the endophyte gene expression pattern shifts to allow for colonization of the ovary tissues. This is critical for the endophyte to be successfully transmitted through the seed ensuring the transmission and persistence of the endophyte in tall fescue. This work provides the first look at the molecular interaction between the tall fescue and its endophyte, whereby novel, or non-toxic, endophyte strains can be tested to ensure that they are also successfully transmitted and maintained in order that renovated pastures remain non-toxic for producers. This information is critical to producers in order to maintain tall fescue pastures devoid of the endophyte that will cause fescue toxicosis.