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United States Department of Agriculture

Agricultural Research Service


Location: Forage Seed and Cereal Research

2009 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. Formerly 5358-21000-012-00D and 5358-21000-018-00D(2/01), 5358-21000-028-00D. FY03 Program Increase $100,592. FY04 Program Increase $120,784. Add 1 SY. Formerly 5358-21000-032-00D (5/08).

3.Progress Report
Stress and disease resistance along with weed control in the forage and turf grass seed production systems are needed to ensure a sustainable supply of these crops. Unfortunately, genetic information pertinent to grasses and to naturally occurring organisms that can provide weed control is lacking. Scientists in this project identified regions of the DNA involved in the production of an herbicide by soil bacteria that occur naturally in seed production fields. Identification of these genetic regions provides critical information needed to produce sufficient quantities of the bioherbicide to enable commercialization to control weeds in cereal and seed crops and in turf.

In stress tolerance research, twenty grass accessions were screened for the capacity to germinate at various salt concentrations. Accessions with contrasting salt tolerance and salt sensitivities were identified that will be useful in future studies. Molecular tools for quantifying stress-associated gene expression in grasses were developed. Studies showing that the presence of endophyte in tall fescue did not significantly improve salt stress tolerance were completed. Advances in “high through-put” sequencing technologies have made portions of the project plan with respect to generation of subtractive hybridization libraries for salt stress obsolete. Accordingly, we altered our approach to produce a salt stress gene-expression library from a grass and generated a database that will be utilized in future gene expression studies to characterize molecular components associated with salinity stress tolerance. One approach to genetically improve stress tolerance and other traits is the introduction of novel genes into plants. The process requires regeneration of grass cell cultures into intact plants after new genes have been introduced. The capability to regenerate intact plants from cell cultures is not universal. Out of fifty grass accessions, two were found that had improved regeneration potential. Studies were initiated to develop molecular tools and grass cell suspension cultures to study gene stability. In addition, new vectors for delivering novel genes into grasses were constructed. A marker gene was successfully introduced into tall fescue cell cultures and trials were initiated to improve the efficiency of the process.

Stem rust continues to impact the yield and quality of perennial grasses. Basic information on the disease is needed to develop management practices and genetic resistance. A population of perennial ryegrass that demonstrated a genetic link to rust resistance was produced. Six purified cultures of the pathogen were isolated and tested against perennial ryegrass host plants that had variable resistance to rust. Data from these analyses were used to define regions on the chromosomes that are linked to stem rust resistance. In collaboration the Nobel Foundation (Norman, OK), genetic markers for this plant population were obtained and studies were initiated to construct a genetic map to define factors for rust resistance. This research provides the basis for improved survivability and yield in pasture and turf grass systems.

1. Genetic regions that regulate bioherbicide production identified. The ability to commercially produce sufficient quantities of a naturally occurring herbicide that is produced by soil bacteria is dependent on developing a chemical approach to synthesize the herbicide in the laboratory, or causing bacteria to produce large quantities when they are cultured. ARS scientists in Corvallis, OR and their collaborators at Oregon State University identified the genetic regions that regulate the natural production of the herbicide by soil bacteria. This accomplishment will enable the scientists to produce sufficient quantities of the herbicide to enable the isolation and identification of the chemical structure of the herbicide. The identification of the chemical structure of the herbicide will accelerate the development of an approach to commercialize this product to provide a new means to control weeds in grass and cereal crops.

2. Identified reference genes for accurately quantifying gene expression levels. Currently there are limited molecular resources publicly available for quantitative analysis of gene expression levels in forage and turf grass species. Reference genes were identified and developed by ARS scientists in Corvallis, OR specifically for accurately quantifying gene expression levels in ryegrasses (Lolium sp.) throughout plant development. These same genes can be used for gene expression evaluation during exposure to various abiotic stresses. The identification and development of these reference genes have enabled future gene expression studies that will lead to improved stress tolerance in forage and turf grasses.

3. Variants of the stem rust pathogen in ryegrass discovered. Stem rust is the most important disease problem in producing seed of cool-season grasses in the Northwest U.S. However, and no current commercial cultivar of perennial ryegrass has a level of rust resistance sufficient to avoid use of fungicides for disease control. Genetically purified cultures of the pathogen were isolated and tested against host plants with variable resistance to rust, ARS scientists in Corvallis, OR determined that there is genetic variation for pathogenicity for these different isolates of the pathogen in the different host genotypes. This information will enable scientists and plant breeders to more effectively define, discover and develop disease-resistant lines of this important seed crop.

Review Publications
Clement, S.L., Martin, R.C., Dombrowski, J.E., Elberson, L.R., Kynaston, M., Azevedo, M.D. 2008. Neotyphodium endophytes in tall fescue seed: infection frequencies after seed production and prolonged cold storage. Seed Science and Technology 36:710-720.

Banowetz, G.M., Azevedo, M.D., Armstrong, D.J., Halgren, A.B., Mills, D.I. 2008. Germination-Arrest Factor (GAF): Biological properties of a novel, naturally-occurring herbicide produced by selected isolates of rhizosphere bacteria. Biological Control.46:380-390.

Dombrowski, J.E., Martin, R.C. 2009. Evaluation of reference genes for quantitative RT-PCR in Lolium temulentum under abiotic stress. Plant Science. 176:390-396.

Banowetz, G.M., Azevedo, M.D., Armstrong, D., Mills, D. 2009. Germination Arrest Factor (GAF): 2. Physical and chemical properties of a novel, naturally-occurring herbicide produced by Pseudomonas fluorescens strain WH6.. Biological Control. 50:103-110.

Armstrong, D., Azevedo, M.D., Mills, D., Bailey, B., Russell, B., Groenig, A., Halgren, A.B., Banowetz, G.M., Mcphail, K. 2009. Germination-Arrest Factor (GAF): 3. Determination that the herbicidal activity of GAF is associated with a ninhydrin-reactive compound and counteracted by selected amino acids. Biological Control. 51:181-190.

Last Modified: 4/23/2014
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