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United States Department of Agriculture

Agricultural Research Service

Research Project: Improvement of Biotic and Abiotic Stress Tolerance in Cool Season Grasses

Location: Forage Seed and Cereal Research

2013 Annual Report


1a.Objectives (from AD-416):
The overall goal of this research is to reduce the impact of abiotic and biotic stresses on the quality and productivity of grasses that serve as the basis for livestock production, turf, and bioenergy. Objective 1: Improve grass germplasm by developing molecular and biological resources that can be applied to reduce the impact of abiotic stresses on the productivity of grasses used for livestock production, turf and bioenergy.

Sub-objective 1.1: Isolate and identify endophytes from native grasses from saline environments at the Oregon Coast to find novel endophytes that can be utilized to improve persistence and yield of forage (Festuca sp.), turf (Lolium sp.), and bioenergy (switchgrass and Miscanthus sp.) grasses along with cereals (rice and wheat) in environments with substandard water quality.

Sub-objective 1.2: Identify genes and signaling components involved in abiotic stress responses in the model grasses Lolium temulentum and Brachypodium to evaluate their utility for improving the performance of turf, forage, and bioenergy grasses in diverse end-use environments. Objective 2: Reduce the impact of weeds and stem rust, two biotic stresses that negatively impact seed production and the utilization of grasses for livestock production, turf, and bioenergy.

Sub-objective 2.1: Conduct field trials to evaluate the efficacy of a bioherbicide in reducing the germination of cheatgrass, medusahead, and annual bluegrass weeds in seed production fields and rangeland.

Sub-objective 2.2: Identify mapping and marker tools that enable the selection of perennial ryegrass (Lolium perenne) germplasm with resistance to stem rust.


1b.Approach (from AD-416):
Foundational and applied research will be conducted to reduce the impact of abiotic and biotic stresses on the quality and productivity of grasses that serve as the basis for livestock production, turf, and bioenergy. Objective 1 focuses on the improvement of grass germplasm by developing molecular and biological resources that can be applied to reduce the impact of abiotic stresses on the productivity of grasses. It has been shown that various types of endophytes can enhance a plant’s ability to tolerant stress. Research will be conducted to isolate and identify novel bacterial or fungal endophytes from native grasses growing in saline environments along the Oregon Coast. These endophytes will be isolated from plant tissues and seeds, identified, characterized, and reintroduced into a model grass species that is salt sensitive, in order to determine if the novel endophytes are able to improve the plant’s ability to grow when subjected to salt stress. There is limited knowledge on the perception ormolecular responses to wounding in grasses and how these responses affectpersistence, the regrowth of new tissues and the long-term nutritional quality of the grass. Research will be focused on identifying wound inducible genes, locally and systemically, and the signals involved in wounding in forage and turf related grasses. Since most stresses have an oxidative stress component, identificationand utilization of oxidative stress response factors may provide useful strategies for increasingtolerance to multiple stresses in grasses. Using bioinformatics approaches, homologs of transcription factors that have been shown to be involved in mediating the oxidative stress responses in eukaryotic systems will be identified. Homologs of the genes will be identified,isolated from grass species andover-expressed in a model grass. Thesetransformed plants will be tested to determine if the over-expression of the homolog gene will improve the tolerance of the plant to different of abiotic stresses. Research conducted in Objective 2 will reduce the impact of weeds and stem rust, two biotic stresses that negatively impact seed production and the utilization of grasses for livestock production, turf, and bioenergy. Stem rust, caused by Pucciniagraminis, is a biotic stress that reduces the yieldand quality of grasslands where perennial ryegrass is produced.Whole-plant inoculation and molecular genetic methods will be used to identify and map genetic markers associated with resistance to the stem rust pathogen. The identification and development of mapping and marker tools will enable the selection of perennial ryegrass germplasm with resistance to stem rust. Another form of stress that seriously impacts the quality and utility of grasslands is thepresence weeds that can out-compete the agricultural crop for limitedresources. Soil application of live pseudomonads which produce a previously identified bioherbicide will tested in two different environments to determine its ability reduce the germination of grassy weeds in field trials.


3.Progress Report:
This project started in February of 2013 and continues research from expired project, 5358-21000-039-00D, "Improvement of Seed and End-Use Quality of Cool Season Grasses". The discovery of novel beneficial endophytes could improve yield and persistence, as well as increase the adaptability of these grasses to multiple stresses encountered in various end use environments. A Plant Tissue Collection Permit from the Oregon Department of Parks and Recreation was submitted and approved. The initial collection trip yielded seed from 8 different grass species along the Oregon Coast. Putative bacterial/fungal endophytes were isolated from the collected seed and from plants generated from the seed. Additional collection trips for plant tissue samples from various sites along the mid-Oregon coastal region and the subsequent isolation of potential endophytes from the collected plant tissues are currently underway. Identified and tested Lolium temulenum (salt sensitive and salt tolerant) plants lines that will be used in endophyte testing and evaluation. Oxidative stress and damage is a component common to most abiotic stresses. Therefore, improving the tolerance to oxidative stress can potentially improve the tolerance to multiple stresses. Fourteen genes coding for proteins involved in the regulation of oxidative stress have been identified from Brachypodium and Lolium databases. Five of these regulatory genes have been shown to increase their expression during oxidative stress. These genes are currently being cloned and will be transformed into the model grass Brachypodium. The transgenic plants expressing these genes will then be evaluated for their ability to increase tolerance to various abiotic stresses. In order to evaluate these transgenic plants, target genes for these TF involved in the oxidative stress responses need to be identified. Eight target genes have been identified and were shown to increase their expression level during oxidative stress. In addition to these genes, assays have been developed to assess tissue viability and measure levels of reactive oxygen species for future use in evaluation of the transgenic plants. Another stress grasses are subjected to in end-use environments is wounding. Gene sequence information, associated with wounding stress, was generated from plant tissues subjected to wounding and from plant tissues on the adjacent systemic non-wounded tiller, in the model grass Lolium temulentum. Stem rust is the most significant disease problem for seed production crops of perennial ryegrass and tall fescue. After identifying 3 regions on the chromosome for stem rust resistance in Lolium, we added several additional markers to the genetic map. These markers include some from other Lolium maps, thus linking our map to those being created by other researchers to investigate resistance to other diseases and stresses. We also identified a marker associated with stem rust reaction in the model grass Brachypodium, from work in 5358-22000-038-00D. Progeny from resistant x susceptible plant lines have been produced and tested for resistance. These progeny will be used to test candidate genetic markers for use in breeding.


Review Publications
Martin, R.C., Glover-Cutter, K.M., Martin, R.R., Dombrowski, J.E. 2013. Virus induced gene silencing in Lolium temulentum. Plant Cell Tissue And Organ Culture. 113:163-171.

Lee, X., Azevedo, M.D., Armstrong, D.J., Banowetz, G.M., Reimmann, C. 2013. The Pseudomonas aeruginosa oxyvinylglycine L-2-amino-4-methoxy-trans-3-butenoic acid inhibits growth of Erwinia amylovora and acts as a weak seed germination-arrest factor. Environmental Microbiology Reports. 5:83-89.

Last Modified: 9/29/2014
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