Progress 02/26/13 to 02/12/18
Outputs
Progress Report 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. 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 or molecular responses to wounding in grasses and how these responses affect persistence, 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, identification and utilization of oxidative stress response factors may provide useful strategies for increasing tolerance 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 and over-expressed in a model grass. These transformed plants will be tested to determine if the over-expression of the homolog gene will improve the tolerance of the plant to different 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 Puccinia graminis, is a biotic stress that reduces the yield and 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 the presence weeds that can out-compete the agricultural crop for limited resources. 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. This is the final report for project 2072-21000-045-00D, which expired February 2018 and has been replaced by a new bridging project 2072-21000- 050-00D. For additional information, see the new project report. Endophytes are microorganisms that live symbiotically within plants and have been shown to improve plant tolerance to abiotic and biotic stresses. To address Sub-objective 1.1, a total of 172 fungal endophytes comprising 39 genera and 208 different bacterial endophytes comprised of 38 different genera/species were isolated and identified from plant tissues and seeds from eight different grass species found growing in saline environments and sandy soils along the Oregon Coast. Twenty-one of the bacterial isolates possessed an enzyme associated with improved plant growth under stress. Initial screening of endophytes in Lolium temulentum (Lt) indicated transmittance of endophytes into the seeds. However, despite Lt�s many advantageous traits as a model species for molecular studies in grasses, it was discovered not to be suitable for screening of endophytes for stress tolerance due to its robust growth. Long-term infection and establishment studies testing potential beneficial endophytes for their ability to improve growth or stress tolerance will be conducted in Lolium perenne. These novel endophytes continue to be investigated for their ability to improve growth and stress tolerance in the next project plan, along with endophytes that will be isolated from grasses found growing in arid regions of Eastern Oregon. Endophytes also provide a valuable source of potential natural products that may have utility in agricultural settings. During reinfection studies, ARS researchers in Corvallis, Oregon, discovered that one of the bacterial isolates released compound(s) that arrested the germination of grass seed, but did not disrupt the growth of established plants. The mode of action appears different from the previously identified bioherbicide 4-formylaminoixyvinylglycine. A total of 273 bacterial endophyte isolates were then screened for their ability to arrest germination of grass seed. Three isolates displayed strong inhibition, while seven others showed weak inhibition. These bacteria may secrete isolatable compounds that have utility to reduce emergence of weeds in agricultural production settings. Research conducted in support of Sub-objective 1.2 developed molecular resources to study abiotic stresses in forage and turf related grasses. In order to improve a plant's ability to respond to stress, it is critical to understand how a plant recognizes their environment. Grasses are routinely cut and grazed upon throughout their lifecycle. This research was the first to show that airborne, naturally produced, volatile compounds released by damaged grass plants activate stress signaling pathways. Plants exposed to green leaf volatiles (GLV) released after grasses are cut/damaged rapidly activated specific signaling cascades in adjacent undamaged plants. These same signaling cascades were also shown to be activated by other abiotic stresses in grasses. Gene expression analysis of Lolium plants exposed to GLV revealed over 10,000 differentially regulated genes one-hour after exposure. Expression analyses showed a direct correlation between activation of genes involved in the biosynthesis of stress hormones, growth and other stress related pathways by GLV and wounding. These results indicated that volatile compounds released from damaged plants (not only grasses) transiently prime the grass� response for the potential oncoming wound stress, and may have a dual role for inter- as well as intra-plant signaling. The identification of naturally produced compounds and their development for release/application in the grass production fields may have the potential to improve recovery/growth of grasses before/after cutting or grazing. Grass plants in the field are subjected to more than one stress at any given time; therefore, to improve a plant�s performance, the focus must be to improve the plant tolerance to multiple stresses. The lack of publicly available molecular resources has hindered progress towards understanding the molecular processes utilized by forage and turf grass species in mediating stress responses. To address Sub-objective 1.2, Lolium sp. stress transcriptomes for drought, fire, wounding, and salinity stress and exposure to GLVs were generated. This identified genes and pathways activated during different stresses. These transcriptomes provide critical data towards the development of a comprehensive public Lolium stress transcriptome database. This information will be utilized for the genetic improvement of grasses, leading to the development of approaches to increase adaptability, improve field performance and stand persistence of grasses in a variety of end-use environments. Research on genes controlling oxidative stress addresses Sub-objective 1. 2. Many stresses experienced by plants have an oxidative stress component associated with them, so characterizing the genetic and biochemical pathways that increase oxidative stress tolerance is useful for improving the tolerance of crops to multiple environmental stresses. A set of genes with increased expression in Brachypodium (Bd) when exposed to oxidative stress were identified for use as marker genes for evaluating oxidative stress levels in grasses. Overexpression of a salt-induced transcription factor in Bd increased tolerance to salt stress in this model grass species. A BZIP transcription factor was found to increase oxidative stress tolerance when overexpressed in Bd plants. These genes provide potential targets for screening germplasm or for genetic modification of commercial grasses to improve the plants� ability to survive a variety of abiotic stresses. Research was conducted to evaluate the efficacy of Pseudomonas fluorescens WH6 in field situations for control of grassy weeds. This research addresses Sub-objective 2.1. WH6 is a bacteria producing the novel herbicide 4-formylaminoixyvinylglycine (FVG). The presence of grassy weeds, particularly Poa annua, can have a negative impact on crop yields. Control of weeds predominantly relies on a limited number of chemical herbicides. The discovery and development of naturally occurring bioherbicides provides an environmentally friendly alternative to these chemical based herbicides. Field experiments using WH6 did not show substantial weed reduction. Either the bacteria populations could not be maintained at a high enough level in the soil or did not produce enough FVG to be effective. A series of greenhouse experiments were designed to test if the addition of adjuvants to bacterial growth media would increase survival of the bacteria. These studies indicated that adjuvants and media modifications increased bacterial survival, but were not sufficient to increase the efficacy of weed control with single applications in soil. Soil flat experiments indicated that repeated sprays of WH6 controlled 44% of the P. annua population. Stem rust of perennial ryegrass grown for seed is a devastating airborne fungal disease that can reduce yields by as much as 98%. Resistant cultivars could reduce reliance on chemical control of the disease and molecular markers linked to resistance genes aids the breeding of resistant cultivars by public and private breeding programs. This project developed a genetic map and identified quantitative trait loci (QTL) in a cross between resistant and susceptible genotypes selected from the cultivar Kingston (Sub-objective 2.2). The Kingston map included markers from other Lolium maps and the Lolium genome zipper, linking our map to genetic resources being created by other researchers. Progeny from the cross of Kingston parents were evaluated for stem rust resistance in greenhouse experiments. The data was used to locate three regions on the ryegrass genome, termed QTL, that control stem rust resistance against two different pathotypes of the fungus. These results indicate that combining resistance QTLs may be necessary to achieve resistance to multiple diverse strains of stem rust. Associated molecular markers flanking and within the QTLs were identified. This is the first step towards developing linked markers useful for marker assisted breeding. Progeny of resistant plants were crossed with public forage perennial ryegrass cultivars. Selections derived from Kingston in field experiments in six Willamette Valley environments have expressed high levels of rust resistance, but not immunity. In recent years there has been a rise in areas of die-off in grass seed production fields in the Willamette Valley of Oregon, where roughly 40% of the world�s grass seed is grown. Symptoms of plants in and around these areas suggested the presence of viral infections. Virus surveys of grass seed production fields were conducted throughout the Willamette Valley, which confirmed the presence of Barley Yellow Dwarf Virus, Cereal Yellow Dwarf Virus and Cocksfoot Mottle Virus, and these viruses were found associated with areas of field die off and may also contribute to lower seed yields. Mitigation strategies to lessen disease impact, such as spraying for insects (vectors of viruses) and cleaning of equipment between fields to reduce spread of viruses have been recommended. As a result of these surveys, field trials were initiated to determine virus susceptibility of different grass species and varieties, and the effect of these viruses on stand persistence. Accomplishments 01 First report of cocksfoot mottle virus infecting orchardgrass in forage production fields in California. Orchardgrass (Dactylis glomerata L.) is a cool season perennial grass commonly used in pastures, and also grown for hay and silage. Cocksfoot mottle virus (CfMV), a disease known to occur outside of the U.S. and in seed production fields in Oregon, causes stand decline of orchardgrass resulting in reduced yield and shortened stand-life. In April of 2017, several forage growers in Northern California were experiencing extensive die off in their fields and ARS researchers in Corvallis, Oregon, found CfMV to be present in all �diseased� samples tested from these Northern California fields. This research demonstrates the spread of an important plant disease within the U.S. and the identification of CfMV is the first step towards effective management in afflicted regions. Since there are currently no pesticide treatments available to prevent the infection, growers are encouraged to look for symptoms of CfMV in early spring so they can prevent mechanical transmission of the CfMV by harvesting clean fields first, or by cleaning machinery thoroughly between fields to limit the spread of the disease. Furthermore, when growers replant fields they should consider using cultivars that are more resistant to CfMV. 02 Exposure to green leaf volatiles released from damaged plant tissue prime grasses for wound stress. Grasses are routinely cut and grazed upon throughout their lifecycle. ARS researchers in Corvallis, Oregon, discovered that grass plants exposed to green leaf volatiles (GLV) released from cut or damaged grasses for just one minute rapidly activated specific signaling cascades (three minutes after exposure) in adjacent undamaged plants. Fifteen different commercially available plant volatiles (alcohols, aldehydes, ketones and acetates) were tested and all 15 volatile compounds were able to activate these same signaling cascades. Gene expression studies of Lolium plants exposed to GLV identified over 10,000 differentially regulated genes involved in the biosynthesis of stress hormones, growth and other stress-related pathways one-hour post exposure, and all genes tested were also activated by wounding. These results, coupled with previously-collected data, indicated that volatile compounds released from damaged plants (not only grasses) transiently prime the grass� response for the potential oncoming wound stress, and may have a dual role for inter- as well as intra-plant signaling. The identification of naturally produced compounds and their use in grass production fields may improve plant growth before and after cutting or grazing. 03 Identification of regulatory elements that increase oxidative stress tolerance in model grass. Many stresses experienced by plants have an oxidative stress component associated with them, so characterizing the genetic and biochemical pathways that increase oxidative stress tolerance will be useful for improving the tolerance of crops to multiple environmental stresses. ARS scientists in Corvallis, Oregon, determined that a transcription factor, when over-expressed, increases resistance to oxidative stress and identified the key transcription factor�s potential downstream gene targets and DNA binding sites. Transcription factors are genes that regulate the expression of other genes. An expression analysis of these plants found that the most highly enriched class of genes were involved in regulating zinc levels in plants. Zinc is known to be a very important micronutrient in both animal and plants, and zinc deficiency or zinc excess can lead to oxidative stress. The identified genes provide potential targets for screening germplasm or for genetic modification of commercial grasses to improve the plants� ability to survive a variety of abiotic stresses.
Impacts
(N/A)
Publications
- Dombrowski, J.E., Martin, R.C. 2018. Activation of MAP kinases by green leaf volatiles in grasses. BMC Research Notes. 11(1):79.
- Boyle, N., Kesoju, S., Greene, S.L., Martin, R.C., Walsh, D. 2016. Migratory beekeeping practices contribute insignificantly to transgenic pollen flow among fields of alfalfa produced for seed. Journal of Economic Entomology. 110(1):6�12. doi: 10.1093/jee/tow243.
|
Progress 10/01/16 to 09/30/17
Outputs
Progress Report 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. 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 or molecular responses to wounding in grasses and how these responses affect persistence, 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, identification and utilization of oxidative stress response factors may provide useful strategies for increasing tolerance 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 and over-expressed in a model grass. These transformed 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 Puccinia graminis, is a biotic stress that reduces the yield and 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 the presence weeds that can out-compete the agricultural crop for limited resources. 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. Progress was made in the evaluation of novel fungal and bacterial endophytes isolated from native grasses from saline environments at the Oregon Coast (sub-Obj. 1.1). Four bacterial isolates were selected for in- depth studies and five different methods were tested to introduce them into a model grass species. The resulting endophyte-plant combinations were tested for their tolerance of salinity stress. Preliminary results indicated that one of the bacterial strains may improve growth under long- term exposure to salinity stress. Infection of 142 fungal isolates in a model grass are currently being evaluated; these plants are being tested for improved growth characteristics under long-term salinity stress exposure. These initial studies for reinfection and establishment were conducted in the model grass Lolium temulentum, due to its ability to harbor endophytes, its short growth cycle, and its single long day transition to flowering, an important characteristic to determine if the endophytes are transmitted into the seed. Selected endophytes will be transitioned into Lolium perenne (Lp) for long-term infection and establishment studies in a commercially relevant grass species. We are currently in the process of identifying and acquiring salt sensitive Lp cultivars for testing. Endophytes can also provide a valuable source of potential natural products that may have utility in agricultural settings. During reinfection studies, we discovered that one of our bacterial isolates released a compound that arrested the germination of grass seed, but did not disrupt the growth of established plants. The mode of action appears different from the previously identified 4- formylaminoixyvinylglycine (FVG) compound (a germination arrest factor, GAF). This new compound may have utility as a means to reduce emergence of weeds in established plantings. The isolation and identification of the compound is ongoing, as well as the screening of our bacterial collection for additional isolates that can arrest seed germination. Progress was made toward the identification of genes and signaling components involved in abiotic stress responses (Sub-Obj. 1.2). The lack of publicly available molecular resources has hindered progress towards understanding the molecular processes utilized by forage and turf grass species in mediating abiotic and biotic stresses. Information derived from gene discovery and identification of molecular mechanisms related to abiotic stress tolerance are essential for developing approaches and molecular resources to improve growth, yield and persistence in grasses. A genomic approach is currently being utilized to annotate gene sequences, identify signaling networks, control elements and downstream gene clusters in order to dissect the plant�s response to different stresses and to determine common and shared molecular resources used by the plant to mediate stress responses. Continued progress has been made in the analysis and development of a public Lolium transcriptome stress database, that includes salinity, wounding, and fire stresses as well as Lolium exposed to green leaf volatiles (GLV) released after tissue damage. Two additional stress transcriptomes for drought and hypoxia (submergence/ flooding) stress are planned. This public database will enable stakeholders and researchers worldwide to identify genes involved in tolerance to multiple abiotic stresses for the genetic improvement of grasses, leading to increased adaptability, improved field performance and stand persistence of forage and turf related grasses in a variety of end-use environments. Continued progress has been made in the analysis of plant transcription factors (TF) for their role in mediating oxidative stress (Sub-Obj. 1.2). Many stresses experienced by plants have an oxidative stress component associated with them, so characterizing the genetic and biochemical pathways that increase oxidative stress resistance will be useful in improving the resistance of crops to many environmental stresses. Homozygous transgenic lines over-expressing five different TFs were generated. Preliminary analysis of TF over-expressing plants determined that they had improved oxidative stress tolerance. These results support the utility of this class of genes/proteins to improve stress tolerance. Further experiments are being conducted to understand the mode of action and the specific pathways or genes controlled by these TFs. Wild type and transgenic plants over-expressing a TF were exposed to short-term high salt stress, long-term salt stress and a control (no salt stress) treatment; complementary DNA (cDNA) expression libraries were generated and sequenced from the plants. The results from these experiments are expected to provide critical insight into the types of genes activated by the over-expression of the TF and how they mediate the stress response. Furthermore, detailed molecular analyses of a transgenic TF plant have identified specific TF binding regions in the genome that control expression of the targeted genes. The identification of these regions in the genome may have the potential to be valuable genetic markers that may indicate germplasm with broad tolerance to different stresses. Additionally, Brachypodium transgenic lines over-expressing a catalase gene have been generated. The over-expression of this gene has the potential to reduce oxidative stress in plants and will be a valuable molecular resource to dissect stress signaling events in grasses. Researchers at USDA-ARS Corvallis Oregon were the first to show that naturally produced volatile airborne compounds, Green Leaf Volatiles (GLV) , released by cut grass plants activated stress related signaling pathways. The activation of these signaling cascades by GLVs were found to occur very rapidly in undamaged plants, requiring only one minute of exposure to GLVs. Analysis of 14 commercially available GLV compounds showed that the majority activate these signaling cascades, but vary in their pattern/timing of activation and intensity. In order to determine what genes these signaling cascades are activating, cDNA expression libraries in undamaged plants exposed to GLV were generated and sequenced. Preliminary transcriptome analysis showed the up-regulation of biosynthetic enzymes associated with production of stress related hormones jasmonic acid and ethylene occurred within 1-2 hours of exposure. Since the wounding/cutting of grasses appears to stimulate growth and release volatile signals, new wound expression libraries were generated and sequenced for direct comparison to the GLV expression libraries. The comparison of these two transcriptomes will provide valuable information as to the molecular basis of the signal response network, identify the genes activated and determine what biochemical/physiological processes these GLVs control/influence. Recently there has been a rise in areas of die off in grass seed production fields in the Willamette Valley, where over 40% of the world's grass seed is produced. Symptoms of plants in and around these areas suggested the presence of viral infection. Identification and detection of newly emerging diseases and finding approaches to mitigate disease is critical to maintain this important agronomic production system. Virus surveys were conducted in grass seed production fields throughout the Willamette Valley, and the presence of Barley Yellow Dwarf Virus, Cereal Yellow Dwarf Virus and Cocksfoot Mottle Virus were confirmed using an enzyme-linked immunosorbent assay (ELISA). These viruses were found associated with areas of field die off and may contribute to lower seed yields. Field trials of 100 different cultivars/varieties (tall fescue, fine fescue, orchardgrass and perennial ryegrass) have been planted to determine virus susceptibility of different grass species and varieties and the effect of these viruses on stand persistence. In perennial grass seed crops, the economic loss due to weeds is substantial and the indirect cost of weed management is even more considerable. Therefore, novel herbicides are desperately needed to control annual seed germination in perennial cropping systems. A novel herbicide, 4-formylaminoixyvinylglycine (FVG) controls the germination of weedy grasses without damaging perennial crops. Progress towards understanding the behavior of FVG was made in support of Sub-Obj. 2.1. Previously we observed that the ability of FVG-producing bacteria to inhibit germination of Poa annua was significantly hindered in soil systems because very little FVG is produced in situ. Therefore, we focused our efforts on increasing FVG production by manipulating the expression of biosynthetic genes and regulatory proteins that are responsible for FVG biosynthesis. Our results indicated that overexpression of regulatory genes decreased production of FVG, subsequent RNA sequencing (RNA-seq) analysis of mutants of these genes indicated that expression of these genes are coordinately regulated, which likely explains these results. Further efforts to manipulate the specific gene cluster are focused on overexpression of biosynthetic enzymes and using RNA-seq analysis to identify feedback pathways that inhibit FVG production. Genetic disease resistance is the best long-term approach for managing stem rust, which is the most significant disease problem for seed production of perennial ryegrass and tall fescue. Despite a critical vacancy, progress has been made in the evaluation of genetic markers associated with stem rust resistance and field evaluation of perennial ryegrass (Lolium perenne) germplasm for resistance to stem rust (Sub-Obj. 2.2). Accomplishments 01 Discovery of new emerging viral diseases in grass seed production fields. Roughly 40% of the world�s grass seed comes from the Willamette Valley Oregon and this production is critical for sustaining forage, turf and grassland systems throughout the U.S. and worldwide. Recently there has been a rise in areas of plant die off in grass seed production fields and plant symptoms suggested the presence of viral infection. ARS scientists in Corvallis, Oregon conducted virus surveys of grass seed production fields, which confirmed the presence of Barley Yellow Dwarf Virus, Cereal Yellow Dwarf Virus and Cocksfoot Mottle Virus throughout the Willamette Valley. These viruses were found associated with areas of field die off and may also contribute to lower seed yields. Mitigation strategies to lessen disease impact, such as spraying for aphids and cleaning of equipment between fields to reduce spread of viruses have been recommended. This study has led to the initiation of field trials to determine virus susceptibility of different grass species and varieties and their effect on stand persistence. 02 Fostering coexistence: industry-driven research on gene flow in genetically engineered alfalfa. Alfalfa is the world�s most important forage crop. Genetically engineered (GE) glyphosate resistant (GR) alfalfa was deregulated in 2011 and it is essential to minimize GE contamination in alfalfa grown for GE-sensitive markets. Studies conducted by scientists at USDA-ARS Fort Collins, Colorado, Corvallis, Oregon, and Washington State University found that isolating honey bee hives for eight hours after bees foraged in GE fields was sufficient to have a negligible incidence of GE contamination in non-GE fields. Efforts to minimize seed spillage and to eradicate feral plants are recommended to minimize transgene dispersal. Preliminary results indicate the distances between GE and non-GE seed fields need to be 330, 602 or 2441 meters to maintain percent contamination below 0.9, 0.5, and 0.1%, respectively in Walla Walla County, Washington, though this may vary between locations and with different pollinators. This study yielded valuable insights on potential approaches for minimizing transgene flow into alfalfa grown for GE-sensitive markets. 03 Identified pathotype-specific stem rust resistance QTLs in perennial ryegrass. Stem rust of perennial ryegrass grown for seed is a devastating airborne fungal disease that can reduce yields by as much as 98%. Resistant cultivars could reduce reliance on chemical control of the disease and molecular markers linked to resistance genes can aid the breeding of resistant cultivars by public and private breeding programs. ARS researchers in Corvallis, Oregon located three regions of the ryegrass genome controlling stem rust resistance against two different pathotypes of the fungi. These regions are referred to as quantitative trait loci (QTL) and contain the gene or gene clusters responsible for resistance. In some cases, QTLs conferred resistance to a specific pathotype; this indicates that combining resistance QTL may be necessary to achieve resistance to multiple diverse strains of stem rust. Molecular markers flanking and within the QTLs were identified and are the first step towards developing markers useful for marker assisted breeding. 04 Identification of novel compounds that can improve plant stress tolerance and growth. The discovery of environmentally safe new compounds that can improve growth characteristics, yield or stress tolerance will improve the productivity and economic viability of grass related producers and consumers. Researchers in Corvallis, Oregon were the first to show that airborne naturally produced volatile compounds released by grass plants activate stress related signaling pathways. Plants exposed to Green Leaf Volatiles (GLV) released after grasses are cut/damaged rapidly activated signaling proteins/cascades (three minutes after exposure) in adjacent undamaged plants. These signaling proteins/cascades are also activated by various abiotic stresses (fire, herbicides, salinity, wounding, and heat) in grasses. These findings initiated genomic studies to identify the genes activated and to determine what biochemical/physiological processes these GLVs control/ influence. The identification of specific naturally produced compounds and their development for release/application in the grass fields may have the potential to improve stress, growth or yield of grasses. 05 Genome sequencing of gray garden slug Deroceras reticulatum to facilitate RNAi approaches for slug control. Slugs are a major pest of grass seed production fields and new methods for controlling slugs are needed. Until recently, sequence information was very limited for terrestrial slug species. The development of the gray garden slug transcriptome is an essential first step towards developing an RNA interference (RNAi) approach for slug control. ARS scientists in Corvallis, Oregon, in collaboration with Oregon State University, sequenced and annotated the mitochondrial genome and transcriptome of the gray garden slug. 40,000 transcripts were annotated and most annotated transcripts were similar to those found in California sea slug and freshwater snail. This molecular database provides essential information for identifying potential target genes for exploring RNAi- based control method for slugs.
Impacts
(N/A)
Publications
- Greene, S.L., Kesoju, S., Martin, R.C., Kramer, M.H. 2015. Occurrence of transgenic feral alfalfa (Medicago sativa subsp. sativa L.) in alfalfa seed production areas in the United States. PLoS One. 10(12):e0143296. doi:10.1371/journal.pone.0143296.
- Dombrowski, J.E., Hollenbeck, V.G., Martin, R.C. 2017. Isolation and identification of bacterial endophytes from grasses along the Oregon coast. American Journal of Plant Sciences. 8(3):574.
- Ahn, S., Martin, R.C., Rao, S., Choi, M.Y. 2017. Neuropeptides predicted from the transcriptome analysis of the gray garden slug Deroceras reticulatum. Peptides. 93:51-63. doi: 10.1016/j.peptides.2017.05.005.
- Ahn, S., Martin, R.C., Rao, S., Choi, M.Y. 2017. The complete mitochondrial genome of the gray garden slug Deroceras reticulatum (Gastropoda: Pulmonata: Stylommatophora). Mitochondrial DNA Part B. 2(1) :255-256. doi: 10.1080/23802359.2017.1318677.
- Gilmore, B.S., Martin, R.C., Dombrowski, J.E., Martin, R.R., Mosier, N.J., Berger, M.C. 2017. Incidence of viruses in fescue (Festuca sp.) seed production fields in the Willamette Valley in 2016. Seed Production Research at Oregon State University. 153:5-8.
- Gilmore, B.S., Martin, R.C., Dombrowski, J.E., Alderman, S.C., Martin, R.R. , Mosier, N.J., Sullivan, C.S., Anderson, N.P., Hoffman, G., Guy, P.L. 2017. Virus incidence in orchardgrass (Dactylis glomerata L.) seed production fields in the Willamette Valley. Crop, Forage & Turfgrass Management. doi: 10.2134/cftm2016.12.0087.
- Okrent, R.A., Trippe, K.M., Maselko, M.B., Manning, V. 2017. Functional analysis of a biosynthetic cluster essential for production of 4- formylaminooxyvinylglycine, a germination-arrest factor from Pseudomonas fluorescens WH6. Microbiology. 163(2):207-217.
- Okrent, R.A., Manning, V., Trippe, K.M. 2017. Draft genome sequences of seven 4-Formylaminooxyvinylglycine producers belonging to the Pseudomonas fluorescens species complex. Genome Announcements. 5(18):e00277-17.
|
Progress 10/01/15 to 09/30/16
Outputs
Progress Report 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. 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 or molecular responses to wounding in grasses and how these responses affect persistence, 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, identification and utilization of oxidative stress response factors may provide useful strategies for increasing tolerance 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 and over-expressed in a model grass. These transformed 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 Puccinia graminis, is a biotic stress that reduces the yield and 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 the presence weeds that can out-compete the agricultural crop for limited resources. 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. Progress was made in the isolation and identification of endophytes from native grasses from saline environments at the Oregon Coast (sub- Objective 1.1). The discovery and development of novel beneficial endophytes can facilitate the adaptability of grasses and other plants to changing environments and stresses. With the current negative public perception of Genetically Modified Organisms (GMOs), the utilization of a naturally occurring endophyte is a more natural approach to improve stress tolerance in a variety of crop species. ARS scientists isolated and identified 111 fungal endophytes comprising 39 genera and 133 different bacterial endophytes comprised of 37 different genus/species of which 94 were unique, from eight different species of grasses exposed to saline environments in sandy soils along the Oregon Coast. Fourteen of the isolates were found to have ACC deaminase activity. During stress, plants often produce ethylene, which inhibits root growth and retards the overall growth of the plant. Recent studies have shown that bacteria displaying ACC deaminase activity have been shown to promote plant growth by lowering plant ethylene levels. Inoculation methods to introduce isolated endophytes back into plants were tested. Four bacterial and four fungal isolates have been selected for initial studies and are currently being introduced into a model grass species and will be tested for their ability to improve salinity stress. With water resources diminishing throughout the U.S. and other parts of the world due to climate change, innovative approaches such as the discovery and utilization of novel endophytes will be necessary to improve water stress tolerance in crops and will be critical for food security. These newly discovered endophytes have the potential to improve the adaptability of grasses and other crop species for production in less than ideal environments with limited water resources. Continued progress was made in the identification of genes and signaling components involved in abiotic stress responses (Sub-Objective 1.2). Stress signaling in forage and turf related grasses is not very well understood. The wounding/cutting of grasses appears to stimulate growth and release volatile signals. Identifying the underlying molecular components such as signaling molecules could lead to approaches to improve growth and yield in pastures and production fields. ARS researchers in Corvallis, Oregon were the first group to report that green leaf volatiles (GLV) can activate MAP Kinase signaling cascades in any plant species. We are currently investigating 14 commercially available individual compounds of GLVs found to be release upon wounding in grasses, to determine their ability to activate these signaling cascades. Preliminary results indicate that most of the individual components have the ability to activate these signaling cascades, but vary in the pattern/timing of activation and intensity. Furthermore, it was also discovered that wound inducible GLVs from a completely different species can activate these same pathways, in this case GLVs released from wounded tomato (dicot) leaves can activate these signaling cascades in Lolium (monocot) plants. We are currently investigating the effects of concentration and length of exposure on activation. An expression library for plants exposed to GLVs was generated and the analysis of the transcriptome is being conducted to identify the genes that GLVs activate and their roles in the plant. These findings have stimulated research in Europe and in the U.S. towards identifying volatile signals released as a result of other types of stress that also activate these same signaling pathways in other crop species. Long-term benefits of this research could be the potential development of field-applied compounds that could influence growth and yield. Information derived from gene discovery and identification of molecular mechanisms related to abiotic stress tolerance are essential for developing approaches and molecular resources to improve growth, yield and persistence in grasses. A Lolium spp. stress transcriptome is currently being generated to compliment the developmental transcriptome recently released by the international community in 2015. Previously we generated a composite salt stress gene-expression library from Lolium that was sequenced and annotated, resulting in 6800 distinct genes. Additionally, gene expression libraries were generated and sequenced for related abiotic stresses in Lolium from exposure to fire, wounding and green leaf volatiles. These combined expression libraries are being analyzed to create a Lolium stress transcriptome. This stress transcriptome will provide a valuable publicly available resource for researchers worldwide working on abiotic stresses in grasses and related species. Many stresses experienced by plants have an oxidative stress component associated with them, so characterizing the genetic and biochemical pathways that increase oxidative stress resistance will be useful in improving the resistance of crops to many environmental stresses. Transcription factors (TF) have the potential to modulate multiple stress response pathways. Bioinformatic analysis identified seven different candidate plant transcription factors (TF) for their role in mediating oxidative stress. These seven TFs, some in multiple configurations, have been transformed into a model grass and multiple transgenic lines generated. These various lines have been screened for expression levels and homologous lines are being generated. Preliminary analysis of heterozygous lines for four different TF indicated that the plants over- expressing the gene lead to improved oxidative stress tolerance. These results support the utility of this class of genes/proteins to improve stress tolerance. Additionally, detailed molecular analyses are ongoing to determine specific TF binding regions in the genome that control expression of target genes. These TFs have the potential to provide an important molecular tool for identification of specific molecular markers in breeding programs as well as direct modification in other plant species to improve stress tolerance in grasses and other crop species. Genetic resistance to disease is the best long-term approach to managing stem rust, which is the most significant disease problem for seed production of perennial ryegrass and tall fescue. Limited progress was made in the selection of perennial ryegrass (Lolium perenne) germplasm with resistance to stem rust (Sub-Objective 2.2) due to a critical vacancy and weather conditions that were not conducive/favorable for stem rust this past growing season preventing evaluations of resistance/ susceptibility of test populations in the field plots. Therefore an additional year of field evaluation of our test populations for rust resistance/susceptibility will be required. Furthermore, analysis of potential molecular markers appear to show that they are more specific to individual plant populations and may not be transferable between different populations, such as between turf and forage grass populations. Analysis is on-going. The critical vacancy related to this project will delay the validation of the molecular markers and the generation of stem rust resistant germplasm. The development of stem rust molecular markers and/or resistant germplasm will be a valuable resource for grass breeders nationwide. Maintenance of grasslands to provide high quality pastures, rangelands and turfs also depends on controlling weeds that reduce the quality of forage and turf value, and that reduce export value of U.S. grass seed. Progress was made in the evaluation of the efficacy of Pseudomonas fluorescens WH6, producing the bioherbicide GAF (germination arrest factor), to control grassy weeds in a field setting (Sub-Objective 2.1). Initial field trials did not show substantial weed reduction, potentially due to diminished survival of the GAF producing microbe in different soil types and varied environmental conditions. Research is being conducted to investigate amendments that can improve survivability and increase effective microbe populations in different soil compositions, thereby increasing GAF efficacy in field settings. A series of greenhouse experiments were designed to test if the addition of adjuvants to bacterial growth media would increase survival of the bacteria by preventing osmotic or acid-induced stresses. Several adjuvants were evaluated as osmotic protectants in both greenhouse and laboratory experiments. Medium modification including pH adjustments and amino acid additions were also tested to determine if changing these conditions would increase bacterial survival. Laboratory based evaluations indicated that adjuvants and media modifications did increase bacterial survival and prevented osmotic stress, however they were not sufficient to increase the efficacy of weed control with single applications in soil. Experiments are currently underway to determine if the number and/or the timing of these applications of the GAF-producing bacteria in test plots can result in effective weed control. The development of effective approaches to deliver this naturally occurring herbicide in production fields would lead to lower production costs and improved yield and quality of seed production in grasses and other crop species. Accomplishments 01 Discovery of novel plant endophytes for improved stress tolerance. Endophytes are microorganisms that live symbiotically within plants and may improve plant tolerance to abiotic and biotic stresses. ARS researchers in Corvallis, Oregon isolated and identified 111 fungal endophytes comprising 39 genera and 133 different bacterial endophytes comprised of 37 different genus/species of which 94 were unique isolates. The endophytes were collected from eight different grass species growing in saline environments and sandy soils along the Oregon Coast. Fourteen of the bacterial isolates possessed ACC deaminase activity, an enzyme associated with improved plant growth under stress. These newly discovered endophytes have the potential to improve the health and productivity of grasses and other crop species grown in marginal or stressful conditions.
Impacts
(N/A)
Publications
- Martin, R.C., Dombrowski, J.E. 2015. Isolation and identification of fungal endophytes from grasses on the Oregon coast. American Journal of Plant Sciences. 6:3216-3230.
- Alderman, S.C., Martin, R.C., Gilmore, B.S., Martin, R.R., Hoffman, G.D., Sullivan, C.S., Anderson, N.P. 2016. First report of cocksfoot mottle virus infecting Dactylis glomerata in Oregon and the United States. Plant Disease. 100(5):1030.
|
Progress 10/01/14 to 09/30/15
Outputs
Progress Report 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. 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 or molecular responses to wounding in grasses and how these responses affect persistence, 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, identification and utilization of oxidative stress response factors may provide useful strategies for increasing tolerance 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 and over-expressed in a model grass. These transformed 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 Puccinia graminis, is a biotic stress that reduces the yield and 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 the presence weeds that can out-compete the agricultural crop for limited resources. 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. Progress was made in the isolation and identification of endophytes from native grasses from saline environments at the Oregon Coast (sub- Objective 1.1). Endophytes (microorganisms that live inside grasses) have the potential to improve crop yield and increase the adaptability of grasses to multiple stresses encountered in the environment. Previously we isolated and identified over 150 bacterial and 70 fungal putative endophytes from grass plants growing in coastal high salt areas. We are currently evaluating each of these endophytes, (i.e. eliminating potential pathogens and identifying the presence of signature biochemical pathways) to select for isolates that are good candidates to test for their ability to improve plant performance under salinity stress. We are also in the process of isolating bacterial and fungal endophytes from seeds. Beneficial endophytes have the potential to improve plant growth under biotic and abiotic stresses, not only in grasses but also in other crop species, without the need for genetically modifying the plants. Progress was made in the identification of genes and signaling components involved in abiotic stress responses (Sub-Objective 1.2). Many stresses experienced by plants have an oxidative stress component associated with them, so characterizing the genetic and biochemical pathways that increase oxidative stress resistance will be useful in improving the resistance of crops to many environmental stresses. Initial analysis of a specific transcription factor (TF) transformed into a model grass demonstrated that the over-expression of this protein in grass plants increased the oxidative stress resistance and cell viability. This demonstrated the utility of this class of proteins to improve stress tolerance. Four additional TFs have been identified as having potential involvement in mediating oxidative stress and are currently being transformed into the model grass Brachypodium distachyon. Plants overexpressing these TF genes will be evaluated to determine if they have increased tolerance to various abiotic stresses. This research has also identified a panel of other genes that are over-expressed when exposed to oxidative stress. These genes provide a potential screening target for either germplasm selection or genetic modification of commercial grass crops to improve the plants� ability to survive abiotic stresses. Unlike most cereal grasses, forage and turf grasses are often repeatedly grazed by animals or mechanically cut. Currently there is very little information available on the wound response in forage and turf grass species and its affect on plant persistence and yield. Progress was made on identifying genes activated by wounding in grasses. Sequences derived from wound cDNA expression libraries of the model grass Lolium temulentum have been assembled and gene sequences identified. Expression analysis of selected genes have been initiated. In FY14 we discovered that volatiles released from cut leaves of grass (Lolium spp.) were found to activate a class of signal relaying proteins (mitogen activated protein kinase: MAPK) in L. temulentum. MAPKs are important signaling proteins that mediate the plant�s response to stress. This was the first report detailing the activation of MAPKs by volatiles obtained from green leaves in any plant species. We have initiated studies to investigate what specific component of these green leaf volatiles is activating these signaling cascades. The investigation into the role of green leaf volatiles in wound signaling, the genes they activate and their affects on growth will be conducted in the next phase of the research. Long-term benefits of this research would be the potential development of field applied compounds that could influence growth and yield. Reduced water resources due to climate change have resulted in the increased risk of fires in grasslands. Grasses are slow to recover after these fires, which can lead to erosion issues. Very little is known on the molecular response or the effect of fire on grasses. In our evaluation of the effects of various abiotic stresses on grasses, we observed that when plants were exposed to fire, MAPK signaling proteins were activated not only locally in damaged tissues, but also in adjacent undamaged tillers. We generated and sequenced cDNA libraries to identify the genes that were activated by fire. These studies have the potential to yield information on how grasses respond to fire and may provide insights on approaches to improve the persistence and recovery of grasses after exposure to fire. Significant progress was made in the selection of perennial ryegrass (Lolium perenne) germplasm with resistance to stem rust (Sub-Objective 2. 2). Stem rust is the most significant disease problem for perennial ryegrass and tall fescue crops grown for seed production. Individual plants of L. perenne that displayed improved resistance to one pathotype of Puccinia graminis subsp. graminicola (stem rust pathogen) were identified. The resistant plants were screened using markers putatively associated with resistance. We are in the process of sequencing these marker regions in the resistant plants in order to validate their utility in screening for resistance. In addition, the plants were tested for resistance to the crown rust pathogen, in collaboration with Swiss geneticists to investigate their resistance to stem and crown rusts. Progeny of crosses between our resistant plants and advanced breeding lines of turf-type perennial ryegrass from the Rutgers breeding program were screened, and the selected progeny were returned to Rutgers for their continued evaluation and incorporation into turf lines. Progeny of our resistant plants crossed with a well-adapted public forage type were selected based on greenhouse tests, and the selections were successfully tested in field plots with a high level of naturally-occurring infection. These turf and forage selections will be useful in creating improved germplasm to be used by Lolium breeders nationwide. Progress was made in the evaluation of the efficacy of Pseudomonas fluorescens WH6, producing the bioherbicide GAF (germination arrest factor), to control grassy weeds in a field setting (Sub-Objective 2.1). The presence of grassy weeds can have a negative impact on crop yields. Control of weeds predominantly relies on a limited number of chemical herbicides. The discovery and development of naturally occurring bioherbicides provides an environmentally friendly alternative to these chemical based herbicides. Our initial field trials identified conditions that can reduce the effectiveness of the P. fluorescens to control weeds. These findings have substantially improved our ability to evaluate the commercial potential of P. fluorescens. Data collected in FY15 from previously established field plots indicated that when a single application of P. fluorescens was applied, it did not substantially control annual bluegrass populations in the following spring or fall. However we observed that seed of the weedy grass species annual bluegrass did not begin to germinate for at least 10 days post application, and that germination randomly occurred over a two-month period. Therefore a single application of P. fluorescens WH6 was unable to control P. annua germination because it is unlikely that the bacterium was present over the two-month germination period. In FY15, we redesigned our application protocols to account for these observations, and applied weekly sprays of WH6 to perennial ryegrass plots and to soil flats each overlaid with P. annua seeds. Data collected from the soil flats indicated that repeated sprays of WH6 controlled 44% of the P. annua population. On-going greenhouse experiments are being conducted to evaluate the ability of adjuvants and media additions to increase the rate of effective weed control. The results of these evaluations will be implemented in applications in FY16 field trials. Accomplishments 01 Resistance to stem rust in Lolium perenne. Stem rust is the most significant disease problem for perennial ryegrass and tall fescue crops grown for seed production. A critical component to addressing this problem is the identification of germplasm with resistance to the disease. ARS researchers in Corvallis, Oregon, have identified individual plants of Lolium perenne displaying improved resistance to one pathotype of Puccinia graminis subsp. graminicola (stem rust pathogen). Progeny of the resistant plants were crossed with advanced breeding lines of turf-type perennial ryegrass and sent to collaborators at Rutgers University for evaluation. Selected progeny of our resistant plants crossed with well-adapted public forage types were successfully tested in field plots with a high level of naturally- occurring infection. These turf and forage selections will be very useful in creating improved germplasm resistant to stem rust to be used by Lolium breeders nationwide.
Impacts
(N/A)
Publications
- Glover-Cutter, K.M., Alderman, S.C., Dombrowski, J.E., Martin, R.C. 2014. Enhanced oxidative stress resistance through activation of a zinc deficiency transcription factor in Brachypodium distachyon. Plant Physiology. 166:1492-1505.
- Dombrowski, J.E., Martin, R.C.2014. Green leaf volatiles, fire and nonanoic acid activate MAPkinases in the model grass species Lolium temulentum. BMC Research Notes. 7:807.
- Hann, C.T., Bequette, C.J., Dombrowski, J.E., Stratmann, J.W. 2014. Methanol and ethanol modulate responses to danger- and microbe-associated molecular patterns. Frontiers in Plant Science. 5(550). doi: 10.3389/fpls. 2014.00550.
- Trippe, K.M., Okrent, R.A., Halgren, A.B., Azevedo, M.D., Chang, J.H., Mills, D.I., Maselko, M.B., Armstrong, D.J., Banowetz, G.M. 2014. Negative regulation of Germination-Arrest Factor (GAF) production in Pseudomonas fluorescens WH6 by a putative extracytoplasmic function sigma factor. Microbiology. 160(11):2432-2442.
|
Progress 10/01/13 to 09/30/14
Outputs
Progress Report 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. 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 or molecular responses to wounding in grasses and how these responses affect persistence, 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, identification and utilization of oxidative stress response factors may provide useful strategies for increasing tolerance 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 and over-expressed in a model grass. These transformed 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 Puccinia graminis, is a biotic stress that reduces the yield and 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 the presence weeds that can out-compete the agricultural crop for limited resources. 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. The discovery of beneficial microorganisms that live inside grasses (i.e., endophytes) has the potential to improve crop yield and increase the adaptability of grasses to multiple stresses encountered in the environment. Tissues from 36 different grass plants were collected from 4 different locations along the Oregon Coast. Over 150 bacteria and 70 fungi were isolated from the plant tissues collected. Genomic DNA was isolated from the isolates and sequencing primers are being designed and tested in order to identify the specific bacterial/fungal species isolated from these plants. Oxidative stress and damage is a component common to most abiotic stresses. Therefore improving the tolerance of plants to oxidative stress can improve the plants� tolerance to multiple stresses. Three genes encoding transcription factors (TF) believed to be involved in oxidative stress response have been cloned from the model grass Brachypodium distachyon and overexpressed in that plant species. Plants overexpressing these TF genes are currently being evaluated to determine if they have increased tolerance to various abiotic stresses. Wounding is another stress grasses are subjected to in the environment. Sequences derived from wound cDNA expression libraries of the model grass Lolium temulentum have been assembled and gene sequences identified. The sequence information generated from these plants will be used to select genes that will be further analyzed for wound gene expression in the next phase of the research. Additionally, volatiles released from cut leaves of grass (Lolium spp.) were found to activate a class of signal relaying proteins (mitogen activated protein kinase: MAPK) in L. temulentum. MAPKs are important signaling proteins that mediate the plant�s response to stress. This is the first report detailing the activation of MAPKs by volatiles obtained from green leaves in any plant species. Investigations into the role of green leaf volatiles in stress response of grasses are underway. Stem rust is the most significant disease problem for perennial ryegrass and tall fescue crops grown for seed production. The density of markers in the quantitative trait loci regions for stem rust resistance was increased in the genetic map of perennial ryegrass (Lolium perenne). These additions made the map more useful for the global Lolium genetics community by including markers that are common to several other published maps. We genotyped and phenotyped progeny from sibling crosses in our population of Lolium perenne, and found a correlation between marker alleles on two of the linkage groups and resistance to one of the stem rust pathotypes. In addition, progeny from crosses of resistant and susceptible plant lines have been produced and tested for resistance. These progeny are being used to test candidate genetic markers for use in breeding. Field trials are currently being conducted to assess the efficacy of the application of live bacteria producing a bioherbicide as a means to control emergence of grassy weeds. Research is underway to investigate factors likely to optimize survival of the bacteria, maintain/increase herbicide production in field settings, and investigate factors that influence treatment effectiveness. Accomplishments 01 Identification of genes mediating the resistance of a grass species to environmental stress. Strategies to increase the resistance of crops to environmental stress will become increasingly important for global food security as our population increases and our climate changes. Many stresses experienced by plants have an oxidative stress component associated with them, so characterizing the genetic and biochemical pathways that increase oxidative stress resistance will be useful in improving the resistance of crops to many environmental stresses. Researchers at the Forage Seed and Cereal Research Unit in Corvallis, Oregon, identified and characterized a zinc deficiency transcription factor that is associated with oxidative stress response in the grass Brachypodium distachyon. They also identified a panel of other genes that are over-expressed by B. distachyon when exposed to oxidative stress. These genes provide a potential screening target for either germplasm selection or genetic modification of commercial grass crops to improve the plants� ability to survive abiotic stresses.
Impacts
(N/A)
Publications
- Tran, N., Zielkel, R.A., Vining, O.B., Azevedo, M.D., Armstrong, D.J., Banowetz, G.M., Mcphail, K.L., Sikora, A.E. 2013. Development and application of a quantitative assay amenable for high-throughput screening to target the type II secretion system for new treatments against plant- pathogenic bacteria. Journal of Biomolecular Screening. 18(8):921-929.
- Gordon, S.P., Priest, H., Des Marais, D., Schackwitz, W., Figueroa, M., Martin, J., Bragg, J., Tyler, L., Lee, C., Bryant, D., Wang, W., Messing, J., Manzaneda, A., Barry, K., Garvin, D.F., Budak, H., Tuna, M., Mitchell- Olds, T., Pfender, W.F., Juenger, T., Mockler, T., Vogel, J.P. 2014. Genome diversity in Brachypodium distachyon: deep sequencing of highly diverse inbred lines. Plant Journal. 79:361-374.
|
Progress 10/01/12 to 09/30/13
Outputs
Progress Report 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. 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. 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.
Impacts
(N/A)
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.
|
|