2009 Annual Report
1a.Objectives (from AD-416)
The long-term goal of this project is to improve competitiveness and sustainability of forage-based enterprises in the transition zone of the Eastern half of the United States of America. Over the next five years we will focus on the following objectives as determined by stakeholder input and ARS National Program 215 (Rangeland, Pastures and Forages) approval.
Objective 1. Improve persistence, productivity, and quality of forage grasses and legumes for use in the transition zone of the Eastern and Midwestern U.S.
Subobjective 1.A. Determine relative interactions between tall fescue (TF) and endophytes involved in regulating plant growth, forage quality, and persistence under a variety of environmental conditions.
Subobjective 1.B. Identify, characterize, and manipulate useful traits (chemical, physical, genetic) to develop forages that are: biologically diverse, tolerant of biotic and abiotic stresses, competitive, high quality for animal production, persistent, and easy to establish and maintain.
Objective 2. Improve animal and forage productivity on forage-based systems through optimal combinations of forages, supplementation and grazing systems tailored to animal needs and environmental conditions.
Subobjective 2.A. Evaluate forage production characteristics relevant to plant and animal performance on pastures containing new novel endophyte-infected (EI) TFs.
Subobjective 2.B. Develop a forage system for the upper transition zone that utilizes warm season perennial grasses to improve animal performance and profitability.
Subobjective 2.C. Identify the abiotic/biotic components and mechanisms of the plant-animal-environment interface impacting pasture production and environmental quality factors.
1b.Approach (from AD-416)
Forage systems provide low-cost feed, conserve soil and water resources, and mitigate man’s impact on the environment. Limited basic biological information exists on how plant and/or fungal metabolites affect forage plant quality, persistence, and production. Even less information exists on the cross-talk mechanism between tall fescue (the predominant forage of the transition zone) and its endophyte. Furthermore, little is known about the impact that forage and/or fungal metabolites have on their pasture ecosystems. Gaps in our current knowledge are hindering researchers’ abilities to predict and select best combinations of forages and management systems for use by various forage-animal production enterprises. Aiding researchers to develop new forage varieties, forage systems, and management recommendations will require an improved understanding of both metabolite (plant and fungal) profiles and their biological functions at the molecular and organismal levels. Such understanding of metabolites, molecular mechanisms, and whole-organism responses, and of their impact on plant quality, persistence, and production, is necessary for improving sustainability of forage-based enterprises. This Project Plan, through the development and utilization of cutting-edge technologies and real-world testing, proposes to decipher the complex interactions within the animal-plant-environment interface. In order to accomplish this task and improve sustainability of forage-based enterprises, the following two objectives are proposed:.
1)Improve persistence, productivity, and quality of forage grasses and legumes for use in the transition zone of the Eastern and Midwestern United States; and.
2)Improve animal and forage productivity on forage-based systems through optimal combinations of forages, supplementation, and grazing systems tailored to animal needs and environmental conditions. These objectives are focused on the predominant forage of the transition zone, tall fescue, as well as on its alternatives and companion species. Accomplishing these objectives will improve sustainability of forage-based enterprises through improved forages, forage management and systems, and basic understanding of the plant/fungal metabolite effects on forage plant persistence and production, as well as consequent effects on the structure and function of pasture ecosystems.
Research conducted under this project improves forage management and ecosystem stability through enhanced understanding of plant-endophyte associations, impacts of endophytes on pasture ecology, secondary/fungal metabolite functions within forages, and the utilization of alternative forages and forage systems.
Additional validation of our carbohydrate extraction and analysis method for the assessment of sugars and fructans in cold stressed forage-grasses has been conducted. The method has been used to successfully complete the profiling of a large forage sample set obtained from a Virginia Polytechnic Institute and State University project concerning grass induced laminitis in horses. Data are currently being analyzed. Additionally, approximately 127,000 expressed sequence tags (ESTs) have been sequenced from meadow fescue (Festuca pratensis) and tall fescue (Lolium arundinaceum = Schedonorus arundinaceus = Festuca arundinacea) endophyte (Neotyphodium coenophialum) infected tissues. The fungal sequences, based on the Epichloe festucea genome sequence, were filtered out and the remaining sequences assembled together with Festuca and Lolium spp. sequences in the National Center for Biotechnology Information (NCBI) database. This work has resulted in assembly of 39,517 plant unigenes. These sequences will serve as the basis for our Lolium/fescue microarray development.
Chemical extractions of phenolics from red clover have been started and are being analyzed for biological activity via antimicrobial assays. Additionally, we have isolated two Protein-L-Isoaspartate Methyltransferase (EC 126.96.36.199; PIMT) forms from rice and tall fescue, and verified that PIMT1 undergoes alternative splicing in vivo, demonstrating alternative splicing in monocots. These sequences, along with putative alternative splicing products from PIMT2, were fused to Green Fluorescent Protein (GFP) and monitored in tobacco, barley and maize leaf cells. A T-DNA insert into the 5’UTR of PIMT1 in rice is also being evaluated to determine the phenotype from knocking out this gene. The lines homozygous for the T-DNA insert are presently being evaluated.
Regulation of Loline Alkaloid Production. Loline alkaloids, produced by Neotyphodium coenophialum in tall fescue and related endophytes in meadow fescue, help protect the plants from insect pests. Regulation of loline alkaloid levels in plants is poorly understood. Regulation of loline alkaloid levels in meadow fescue with Neotyphodium uncinatum and N. siegelii was investigated. After harvesting (clipping) leaf blades, loline alkaloids in regrowth reached 2-5-fold higher levels than in the initial harvest. This effect was attributable to tissue age, because young leaf blades had much higher levels of lolines than did older leaf blades. Interestingly, the fungal loline alkaloid biosynthesis (LOL) genes were not expressed more highly in regrowth over the initial harvest. However, the amino-acid precursors of loline alkaloids were much higher in younger than older leaf blades of E- plants, and were depleted in the E+ plants by molar amounts that approximated the loline-alkaloid levels. It was concluded that substrate availability rather than gene expression determined the loline-alkaloid levels. Understanding the relationship between free amino acids and alkaloids will facilitate plant breeding and management to control the alkaloid levels and optimize stands for their persistence and suitability as forage.
Generation of Tall Fescue Clone Pairs With and Without Endophyte. Because of the plant-to-plant genetic diversity of the self-incompatible forage grass, tall fescue, physiological and gene expression studies are best conducted on clones generated by propagation of vegetative ramets. To test effects of the seed-transmissible endophyte, Neotyphodium coenophialum, clone pairs need to be generated in which one clone possesses the endophyte and the other lacks it. Such clone pairs were not widely available. Plants of tall fescue cultivar Kentucky 31 infected with N. coenophialum, and other tall fescue plants with novel endophytes, were used. Each plant represented a distinct genotype. Plants were divided into ramets, and fungicide (propaconazole) treatment of some ramets of each plant generated endophyte-free clones. A total of 29 endophyte-infected and endophyte-free tall fescue clone pairs were generated. The clone pairs are a valuable resource now available to the research community for experiments to test endophyte effects on tall fescue physiology and gene expression under various conditions such as water deficit stress, grazing, nematode parasitism or insect feeding.
5.Significant Activities that Support Special Target Populations
University of Kentucky Pasture Management Meeting. Title: Pasture Weed Management After Two Years of Drought. Lexington, KY. This meeting was for small horse farms.
Zhang, D., Nagabhyru, P., Schardl, C. 2009. Regulation of a Chemical Defense Against Herbivory Produced by Symbiotic Fungi in Grass Plants. Plant Physiology. June 2009. 150:1072-1082.
Zhang, D., Stromberg, A.J., Spiering, M.J., Schardl, C.L. 2009. Coregulated Expression of Loline Alkaloid-Biosynthesis Genes in Neotyphodium Uncinatum Cultures. Fungal Genetics and Biology. 46:517-530.
Spiering, M.J., Faulkner, J.R., Zhang, D., Machado, C., Grossman, R.B., Schardl, C.L. 2008. Role of the LolP Cytochrome P450 Monooxygenase in Loline Alkaloid Biosynthesis. Fungal Genetics and Biology. 45:1307-1314.
Tavva, V.S., Dinkins, R.D., Collins, G.B., Palli, S. 2009. Ecdysone receptor-based gene switches for applications in plants. In: Smaghhe, G., editor. Ecdysone, Structures and Functions. Heidelberg, Germany: Springer Science. p. 505-531.
Jia, X., Wang, W., Ren, L., Chen, Q., Mendu, V., Willcut, B., Dinkins, R.D., Tang, X., Tang, G. 2009. Differential and Dynamic Regulation of miRNA398 in Response to ABA and Salt Stress in Populus tremula and Arabidopsis thaliana. Plant Mol Biol. 71:51-59.