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United States Department of Agriculture

Agricultural Research Service

Research Project: IMPROVEMENT OF SEED AND END-USE QUALITY OF COOL SEASON GRASSES
2012 Annual Report


1a.Objectives (from AD-416):
Provide the seed industry with improved grass germplasm and management practices that reduce the impact of salinity, weeds, and diseases on seed quality and profitability. Identify bioactive compounds and genes that mediate the response of Lolium and Festuca to salinity related stress. Develop approaches to characterize, select and utilize components of host genetic resistance to pathotypes of the stem rust pathogen in Lolium. Determine whether a small molecular weight bioherbicide, produced by selected naturally occurring soil rhizobacteria (Pseudomonas sp.), that inhibits the germination of Poa annua can reduce the impact of this weed on seed production and turf quality. Develop molecular methods and tools that facilitate germplasm improvement for diverse uses. Develop a means to improve gene stability and minimize escape of transgenes in forage and turf grasses (Lolium sp.). Characterize genetics of host resistance to stem rust, and develop molecular markers for stem rust resistance in Lolium. Identify candidate genes that can be exploited to increase biomass of cool-season grass plants (Lolium sp.) to improve forage quality and source material for bioenergy production.


1b.Approach (from AD-416):
Conduct complex basic and applied research to improve the production and utilization of seed and grain from forage and turf seed cropping systems which include wheat. Traditional breeding and molecular genetics will be used to identify the genetic basis for stem rust resistance, factors that impact seed quality, flowering, and abiotic stress tolerance. Develop molecular and traditional approaches useful for altering plant developmental pathways and plant structures, and enhancing forage quality in end-use environments that differ from the seed-producing region. Bioherbicides that reduce weed presence in seed production and turf environments will be identified and characterized to enable commercialization of new products.


3.Progress Report:
Stress tolerance, disease resistance and effective weed control in forage and turf grass seed production systems are needed to ensure a sustainable supply of these crops for global markets. Unfortunately, genetic information and molecular resources needed to improve forage and turf grass systems are lacking. An approach to disrupt specific gene function in forage and turf grass species has been demonstrated and is being tested in grasses. Transformation systems for Brachypodium and Lolium have been established. A series of genes expressed in response to salt stress was sequenced and annotated and genes that regulate the expression of specific DNA sequences in response to oxidative stress have been identified.

Forage and turf grasses are repeatedly cut or grazed upon by animals, but there is little information on the physiological and molecular responses to repeated wounding in these grasses. We identified an intermediate signaling protein and pathway that is activated when grasses are wounded or cut, and in response to heat and salt stress. We sequenced a wound expression library from Lolium temulentum and showed that specific gene sequences are activated by wounding both locally and in the systemic unwounded tiller of the grass plant. This research will enable further development of genetic approaches to improve yield and persistence of forage grasses used for livestock and bioenergy.

ARS scientists at Corvallis, Oregon, and Oregon State University showed that the Germination Arrest Factor (GAF), a bioherbicide produced by the soil bacterium Pseudomonas fluorescens also inhibits the growth of Erwinia amylovora, the bacterium that causes fire blight in apple and pear crops. This discovery demonstrated that GAF has utility as a biological control agent. The growth inhibition also provides a convenient means for detecting the presence of GAF in bacterial cultures. Genetic regions in Pseudomonas fluorescens that regulate GAF biosynthesis were identified, an important step in developing bacteria that produce commercially significant quantities of the bioherbicide.

Genetic resistance is the best long-term approach to managing stem rust, the most significant disease problem for seed production crops of perennial ryegrass and tall fescue. Resistance has been difficult to incorporate due to the lack of information about nature and inheritance of resistance genes in these out-crossing species. We determined that there are genes for partial resistance that result in a greatly reduced "infection efficiency" and that this resistance is heritable. We also discerned the approximate location of these resistance genes on two chromosomes of perennial ryegrass, and detected nearby sections of DNA that might be useful as markers for plants carrying the resistance genes in future plant breeding efforts to enhance resistance.


4.Accomplishments
1. GAF inhibits the growth of the bacterium that causes fire blight. Biological properties and the genetic basis for biosynthesis of the Germination Arrest Factor (GAF) produced by the soil bacterium Pseudomonas fluorescens are only partially understood. ARS scientists at Corvallis, Oregon, worked with Oregon State University collaborators and demonstrated that GAF also exhibits a rather specific antimicrobial activity against Erwinia amylovora, the pathogen that causes fire blight in apples and pears. This group also demonstrated that certain amino acids reverse GAF inhibition of bacterial growth, an observation which suggests that GAF affects the activity of aminotransferases, enzymes that are involved in plant nitrogen metabolism. This accomplishment may have commercial applications for GAF and structurally similar compounds because aminovinlyglycine, a compound similar to GAF also inhibited the growth of the fire blight pathogen.

2. Abiotic stress signaling. Forage and turf grasses are utilized in diverse environments, which expose them to a variety of abiotic stresses. However, very little is known concerning the perception or molecular responses to these various stresses. ARS scientists at Corvallis, Oregon, discovered that plants subjected to either heat, salt or wound stress, but not cold, rapidly activated a MAP kinase signaling cascade in the model grass species Lolium temulentum. Additionally, if plants were subjected to heat and cold stress prior to wounding, the wound activation of this signaling protein was significantly inhibited. This research provides an important first step towards understanding molecular mechanisms utilized by grasses in response to environmental stresses and how they are perceived. In the long term, this information will lead to improvements and increases in the yield, sustainability, and the quality of grasses used as a feedstock for livestock and biofuels in different end-use environments.

3. Salinity stress signaling. Soil salinity is one of the major abiotic stresses responsible for reduced crop productivity worldwide and the salinization of arable land has dramatically increased over the last few decades. Consequently, as land becomes less amenable for conventional agriculture, plants grown on marginal soils will be exposed to higher levels of soil salinity. ARS scientists at Corvallis, Oregon, identified a gene coding for a transcription factor, a gene which influences the expression of specific DNA sequences (FaZnF) in tall fescue exposed to salt stress. This transcription factor was cloned and analyzed for expression under different abiotic stress conditions. Transgenic cell lines over-expressing this transcription factor gene showed increased expression of known stress tolerance genes. This work provides evidence that this transcription factor is involved in the regulation of at least two pathways initiated during the salt stress response; furthering our understanding of the mechanisms of cellular action during stress that is applicable to commercial crops worldwide.

4. Resistance to stem rust mapped to three chromosomes in perennial ryegrass. Genetic improvement of ryegrass for stem rust resistance has been hampered by a lack of information about the diversity and location of resistance genes in this grass. ARS researchers in Corvallis Oregon, determined that there is an all-or-none type of resistance gene located on chromosome 7, and effective partial resistance genes located on chromosomes 1 and 6, for stem rust in perennial ryegrass. These results lay the groundwork for developing genetic tools ("markers") to locate stem rust resistance genes. Technology based on these markers will allow breeders to produce rust-resistant varieties of ryegrass, saving growers the expense and environmental cost of fungicide treatment. Information about the location of rust resistance genes in grasses will also add to the knowledge base for finding rust resistance genes in cereal grasses such as wheat, which is currently threatened with a new strain of stem rust worldwide.


Review Publications
Halgren, A.B., Azevedo, M.D., Mills, D.I., Armstrong, D., Thimmaiah, M., Mcphail, K., Banowetz, G.M. 2011. Selective inhibition of Erwinia amylovora by the herbicidally-active Germination-Arrest Factor (GAF) produced by Pseudomonas bacteria. Journal of Applied Microbiology. 111: 949-959.

Halgren, A.B., Banowetz, G.M. 2012. Life cycle expression analysis of three cell wall degradation-related genes in ethylene-treated grass. Plant Growth Regulation. 66: 167-177.

Dombrowski, J.E., Martin, R.C. 2012. Abiotic stresses activate a MAPkinase in the model grass species Lolium temulentum L. Journal of Plant Physiology. 169: 915-919.

Last Modified: 10/25/2014
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