Final Report for GNC12-159
Project Information
Perennial ryegrass seed, produced for sale as turf and forage grass seed, is grown in rural agricultural areas of northern Minnesota and contributes $15-20 million to rural economies annually. As a perennial, perennial ryegrass has many environmental benefits in a crop rotation including decreased erosion, reduced leaching, high organic matter production, and greater habitat for wildlife due to fewer tillage operations. From a farmers standpoint, perennial ryegrass is a desirable crop rotation option because it is profitable, tolerant of diverse weather conditions, requires less labor than annual crops, and harvest occurs earlier than for annual crops so labor requirements are spread throughout the season. Minnesota farmers have indicated that crown and stem rust pathogens are a severe issue in seed production fields (causing up to 80% yield reduction) and that new cultivars that are resistant to crown and stem rust pathogens would be desirable. Currently the only way to control rust pathogens is to spray fungicides which are costly and harmful to human and environmental health. The goal of the proposed research is to develop a method for rapid, and accurate selection for resistance to rust pathogens in perennial ryegrass germplasm based on plant chemical compounds (a “metabolic fingerprint”) associated with rust resistance. Termed metabolomics-assisted breeding, this technique will lead to faster rust resistant cultivar development, ultimately reducing fungicide use and making perennial ryegrass seed production a more profitable, marketable and sustainable option for farmers in rural communities in northern Minnesota as well as the Pacific Northwest and Canada.
Introduction:
Stem rust (Puccinia graminis) and crown rust (Puccinia coronata), the causal agents of rust diseases in Minnesota, are serious fungal diseases of most agriculturally important grass species and currently affect all 72,000 hectares of perennial ryegrass seed production within the United States. Rust pathogens also affect perennial ryegrass when grown as a turfgrass and can reduce turf quality and lead to increased water and nutrient use (4). Significant efforts have been made to combat rust pathogens in small grains but little attention has been given to rust in perennial ryegrass.
Perennial ryegrass seed produced for sale as turf and forage grass is grown in rural agricultural areas of northern Minnesota, Oregon, and Canada. Currently there are around 60,000 acres of perennial ryegrass seed production in northern Minnesota as well as 120,000 and 25,000 acres in Oregon, and Canada respectively. A perennial ryegrass seed crop is under-seeded in a spring wheat crop or planted into spring wheat stubble in the fall, then overwinters as a living cover crop and is harvested for seed the following summer. This double cropping approach reduces the number of field tillage operations required by farmers, protects against leaching and erosion, and spreads work load throughout the year because seed harvest occurs during summer before other annual crops are harvested (1, 11). Perennial ryegrass as a seed crop, along with the associated processing and distributing businesses, contribute significantly to rural economies (valued at $15-20 million in Minnesota). If demand for seed from European and Asian markets continues to grow and seed production remains sustainable and profitable compared to annual-type agricultural crops, perennial ryegrass acreage in Minnesota could increase to over 150,000 acres.
Although perennial ryegrass has been a desirable crop rotation option, Minnesota farmers have indicated that rust pathogens are a severe issue in seed production fields (causing up to 80% yield reduction) (19). Fungicides (costing $125 per acre per application) are routinely applied to obtain adequate seed yields suggesting that rust resistant cultivars would improve sustainability of the crop (15, 16). Through a Minnesota Rapid Agricultural Response grant we have been successful in conducting some phenotypic screening for rust resistant perennial ryegrass germplasm (13). Phenotypic screening however, can be time consuming, costly, and unpredictable do to pathogen and environment unpredictability (12). In addition we need a way to select for rust resistance in the absence of the actual pathogen in order to be able to select for other traits simultaneously and speed cultivar development. Metabolomics-assisted breeding has recently emerged a technique to select for desired traits based on chemical profiles that have been previously correlated with a trait of interest (5).
Chemical compounds within plants (particularly secondary metabolites) are the end products of complex biochemical pathways that are regulated by genes and are often used directly as stress mediation or plant defense mechanisms making them closely associated to plant phenotypes (3, 9). Some plant produced chemicals can also stimulate growth of rust pathogen structures involved in the infection process (7, 20). The potential for using secondary metabolites as biomarkers for selecting desired traits in plants has been demonstrated in arabidopsis, grapes, wheat, and conifer (14, 6, 8, 17). Selecting for quantitative rust resistance in a recurrent selection program is a viable and desirable option for perennial ryegrass considering a lack of research on the genetics of resistance in this crop (12, 5). Our research, studying relationships between perennial ryegrass-produced chemicals and rust pathogens, is a significant step towards succesful implementation of a metabolomics-assisted selection strategy for selecting durable, quantitative rust resistance and faster development of rust resistant perennial ryegrass cultivars.
Objective 1: Quantify and verify levels of durable, quantitative resistance to crown and stem rust in an advanced perennial ryegrass breeding population using a controlled environment.
Objective 2: Determine metabolic fingerprints associated with crown rust resistance.
The long term goal of this research is to develop a method for rapid, and accurate selection of resistance to rust pathogens in perennial ryegrass germplasm based on plant chemical compounds (a “metabolic fingerprint”) associated with rust resistance. Termed metabolomics-assisted breeding, this technique will lead to faster cultivar development, ultimately reducing fungicide use and making perennial ryegrass seed production a more profitable, marketable and sustainable option for farmers in rural communities in northern Minnesota as well as the Pacific Northwest and Canada.
Short-term outcomes:
1) Thorough analysis and verification of rust resistance variability of important perennial ryegrass germplasm in our breeding program.
2) First development of metabolomics-assisted selection techniques for high-throughput, accurate, and dependable crown rust resistance screening in a plant breeding program.
3) Improved knowledge of the biological basis for plant resistance to rust pathogens plus greater farmer and researcher understanding of ryegrass and pathogen interactions.
Intermediate-term outcomes:
1) Rust resistant perennial ryegrass lines identified in this study will be incorporated into our breeding program.
2) This research will result in our breeding program having a fast, reliable metabolomics-assisted selection method to supplement often unpredictable phenotypic screening methods leading to faster delivery rust resistant cultivars to farmers.
3) The crown rust resistance selection model will be adapted for stem rust resistance selection.
Long-term outcomes:
1) New crown and stem rust resistant cultivars will make perennial ryegrass seed crops a more profitable and environmentally sustainable crop rotation option for farmers in northern Minnesota.
2) Seed from rust resistant cultivars will be more marketable to end users and will result in more environmentally sustainable turfgrasses.
3) Our methods could be used as a model for metabolomics-assisted selection in other important agricultural crops or for other traits in perennial ryegrass.
Cooperators
Research
OBJECTIVE 1
Plant germplasm as of project start:
Prior to the start of this project our group began screening a population of 50 perennial ryegrass lines from our breeding program for resistance to crown rust at the University of Minnesota’s agricultural experiment fields in St. Paul, MN. The 50 lines, which were developed by repeatedly backcrossing a spreading type perennial ryegrass from our perennial ryegrass breeding program to high quality germplasm from Rutgers University, have previously been selected for horizontal growth, good turf quality and winterhardiness. To summarize, beginning in the fall of 2008, 59 perennial ryegrass lines were planted into soybean stubble with 4 replications for each line and 20 plants representing each line per replication. The 59 lines were rated for crown rust disease due to natural infection on three dates during the summer of 2009. Area under the disease progress curve was calculated for each line and the 12 most resistant and 12 most susceptible based on the AUDPC value were selected for further evaluation. Within each selected line the most resistant or most susceptible plant was selected from each replication and clonally propagated in the greenhouse for use in later screening. In the fall of 2009, remnant seed, which was the seed used to plant the nursery in 2008, was used to establish a second nursery similar to the one planted in 2009 with a main difference being that it included six of the most resistant lines, 7 of the most susceptible, and 12 lines identified as having medium resistance in summer of 2009. The nursery planted in fall of 2009 contained a total of 25 of the original 59 perennial ryegrass lines and each line was represented by 6 plants in each of the four replications. Crown rust disease severity was rated for this nursery three times during the summer of 2010 using a modified Horsefall-Barret scale where 1= no rust, 2 = <10%, 3 = 11-25%, 4 = 26-40%, 5 = 41-60%, 6 = 61-70%, 7 = 71-80% 8 = 81-90% 9 = >90%, and 10 = 100% coverage of rust pustules (Helgeson et al., 1998). Crown rust ratings from the rating date at peak severity were used to make selections. A total of 14 lines ranked consistently in the resistant, medium resistance, or susceptible categories (4, 5, and 5 lines respectively). From these 14 lines, the most resistant plant, most susceptible plant, or median plant was selected from each line in each rep and each plant was clonally propagated in the greenhouse for future screening. In the fall of 2010, clones from each of the selected 14 lines demonstrating consistent crown rust resistance over two growing seasons were planted into a field nursery to verify resistance rankings and obtain a more robust data set. The 14 lines were screened in four replications using a randomized complete block design. Each line was represented by eight clones which included the four clones selected from the 2009 screening experiment for each line, and the four clones selected from the 2010 screening experiment for each line. Each replication of a line in the nursery planted in 2010 contained an identical set of plants (genotypes) obtained through clonal propagation. The nursery was rated for crown rust disease severity in the summer of 2011 using the modified Horsefall-Barret scale. A set of plants identical to those found in the field nursery screened in 2011 was maintained in the greenhouse and beginning in the summer of 2012 this material was screened for crown rust resistance in the greenhouse via artificial inoculation.
Crown rust inoculum as of project start:
Significant collections of crown rust uredineospores were made between 2009 and 2012. All uredineospores were collected off of perennial ryegrass leaf tissue and identified by spore morphology. Crown rust isolates used for artificial inoculation in growth chamber experiments were collected from the environments listed in Table 1. Crown rust uredineospores were collected by scraping spores off the leaf surface with the edge of a gelatin capsule and letting spores fall into the capsule. Gelatin capsules containing spores were then placed in a sealed plastic bag and chilled on ice until they were placed in a desiccators with the plastic bag un-sealed for four days. Following desiccation the gelatin capsules containing uredineospores were place inside a sealed air free plastic bag at -80 C until needed.
Crown rust resistance ranking verification:
Fourteen perennial ryegrass lines from our breeding program which demonstrated consistent crown rust resistance rankings over 3 years of field screening trials were further screened under controlled conditions in the growth camber to verify resistance rankings and develop a robust data set for use in further development of a metablomics assisted selection model. Clones (described in the Plant germplasm section) from all plants used in the 2011 field crown rust screening nursery were maintained in 6.4 x 6.4 x 8.9 cm plastic form pots in the greenhouse. The pots containing plants from the 14 lines were arranged in a randomized complete block design with four replications and individual plants from a line were grouped into whole plots. Leaf tissue was sampled for metabolomics analysis (see Objective 2) and then each line was inoculated with a bulked sample of previously collected crown rust isolates. For inoculation, uredineospores were first heat shocked at 45 C for 15 minutes then re-hydrated at ambient temperature (25 C) and humidity for 3 hours. Spores were then mixed with Soltrol 170 isoparaffin and sprayed onto the perennial ryegrass plants with a siphon feed spray nozzle at a rate of 0.0268 mg per pot. Following application of spores the Soltrol 170 isoparaffin was allowed to evaporate for 1 hour and then plants were placed in a dew chamber where they were continually misted for 1 hour. Following the initial 1 hour misting period the plants were then misted for two minutes every hour for 14 hours. After 14 hours the mist was stopped and the dew chamber door was opened for four hours to facilitate drying of the leaf surface. The plants were then transferred into a growth chamber where they were maintained at 25 C daytime temperature and 20 C nighttime temperature with a photoperiod of 14 hours. Relative humidity in the growth chamber was maintained near 80%.
Crown rust severity was rated after 21 days using the same rating scale that was used in the field rust screening nursery. One plant was selected that had disease severity representative of each number of the rating scale and all ratings were based on comparisons with the representative plants. Means for each line were computed and then the growth chamber and field 2011 data were subject to the PROC CORR procedure in SAS to determine the Spearman rank-order correlation between the field and growth chamber data.
Stem rust resistance screening:
Stem rust uredineospores were collected from infected perennial ryegrass in the summer of 2012 and 2013 in St. Paul, MN at the University of Minnesota Agricultural Experiment Station. Uredineospores were also collected in 2012 at Roseau, MN at the University of Minnesota’s Magnusson research farm and in numerous grower fields throughout northern Minnesota. Stem rust spores were collected and stored in the same manner as the crown rust spores with the exception that spores were collected from stem tissue.
Two perennial ryegrass breeding nurseries, one in Roseau, MN and one in St. Paul, MN were screened for stem rust resistance during the summer of 2012. Fifteen lines of a spreading type perennial ryegrass, having a similar background to the material screened for crown rust resistance, were planted in crossing nurseries using a randomized complete block design with five replications. Each line within a rep was represented by 10 individual plants. Stem rust was not present in the Roseau nursery however the St. Paul nursery was rated for stem rust severity. Stem rust severity was visually rated by estimating the percentage of stem and spike tissue covered in stem rust on each plant and assigning a value from the modified Horsefall-Barret scale described above. Means for each line were calculated and the two most resistant or susceptible plants within each rep for the three most resistant and three most susceptible lines were selected for clonal propagation in the greenhouse.
OBJECTIVE 2
Plant tissue harvest and extraction:
Leaf tissue was sampled from all lines that were screened for crown rust resistance in the greenhouse just prior to artificial inoculation and growth chamber screening with the intent of getting a snapshot of the metabolome at the time of disease infection. The first fully expanded leaf was harvested from each plant representing a line within a replication. Tissue from each line within a rep was bulked, placed in a 1.5 ml eppendorf tube, sealed and immediately quenched in liquid N until frozen. Samples were then stored at -80 C until further processing. After 1 month at -80 C, samples were lyophilized and then again stored at -80 C until needed.
To prepare extracts for metabolomics analysis, the lyophilized tissue samples were ground in 1.5 ml eppendorf tubes with two tungsten beads using a bead mill (Spex Sample Prep 2010 GenoGrinder) set to 1100 revolutions per minute, for 15 minutes. Approximately 16.82 mg (+/- 5%) of ground leaf tissue was weighed into a new low-retention 1.5 ml tube for each sample (this is approximately equivalent to 40mg fresh weight for our samples), and the exact weight recorded. Two clean tungsten carbide beads were then added to the tube along with 800 ?l of a 90% methanol (Chromasolv®, Sigma-Aldrich) and 10% double distilled water solution. The tubes were then placed in the bead mill for 30 minutes at a speed of 800 revolutions per minute. Samples were then centrifuged at 14,000 revolutions per minute for one minute. Using low retention pipette tips, 400 ?l of the supernatant was removed and placed in another low-retention 1.5 ml eppendorf tube where it was evaporated using a speed-vac. The remaining supernatant was stored at -80 C for future analysis. Once dried the sample was reconstituted in 100 ?l of 15% acetonitrile (Chromasolv® Plus, Sigma-Aldrich) and 85% double distilled water by shaking in the bead mill with one tungsten bead for 10 minutes. The tube containing the sample was then sonicated for 5 minutes. To prepare for HPLC analysis the sample was centrifuged for 20 minutes at 14,000 revolutions per minute and 50 ?l was pipette into a glass HPLC vial insert. A composite sample of all lines was also created by bulking small amounts of each extract. The remaining sample was stored at -80 C for future analysis.
High Performance Liquid Chromatography
Chromatography was performed using a Thermo Scientific Dionex UltiMate 3000 HPLC, and injecting 2 ?l directly onto a Acquity UPLC BEH C18 column (2.1 x 100 mm, 1.7 µm particle size, Phenomenex SecurityGuard Ultra guard column), equilibrated in 90% solvent A (0.1% aqueous solution of formic acid), 10% solvent B (Chromasolv® Plus acetonitrile containing 0.1% formic acid). Prior to injection samples were stored in the autosampler at 10 C. The column temperature was maintained at 40 C. Compounds were eluted from the column using a constant flow rate of 400 ?l per minute and linearly increasing the concentration of solvent B beginning 1 minute after injection from 10% to 98% over 26.5 minutes. The concentration of solvent B was maintained at 98% for 5 minutes and then re-equilibrated in 90% solvent A and 10% solvent B for 2 minutes for a total run time of 34.5 minutes.
Mass spectrometry:
A Thermo Scientific Q Exactive™ hybrid quadrupole-Orbitrap mass spectrometer for high resolution and accurate mass was operated using electrospray ionization in positive and negative polarity switching mode. The resolving power was 35,000. Other parameters were IT = 200ms, AGC Target = 1,000,000, and full scan mode was used with a mass range of 215 m/z to 2000 m/z. Conditions at the source for both ionization polarities were: spray voltage = 3.80 kV, sheath gas = 50 arbitrary units, auxillary gas = 20 arbitrary units, sweep gas = 0 arbitrary units, heater temperature = 300 C, and capillary temperature = 350 C. Prior to running samples the sweep cone and capillary tube were cleaned and the instrument was calibrated using the recommended Thermo Scientific calibration solutions specifically designed for positive and negative polarity modes. Solvent blanks were run before each biological replicate as well as after every fourth unknown sample. A quality control standard of known composition and accurate mass was run before each biological replicate, once during, and once after to insure consistent and accurate performance of the mass spectrometer.
Chromatogram alignment, component detection, and data analysis:
Chromatographic alignment, background subtraction, and feature detection were conducted using Thermo SIEVE™ 2.0 software. This software performed data reduction by grouping isotopes and adducts into main components and also allowed for removing components (features) with greater than 40% covariance within a plant line. The feature table output from SIEVE was further subject to partial least squares (PLSDA) using GeneData Analyst software. Components which were most important for explaining differences between resistant and susceptible groups were determined and pairwise comparisons were used to compare the differences in abundance of the components determined to be important.
Short-term outcomes:
1) Thorough analysis and verification of rust resistance variability of important perennial ryegrass germplasm in our breeding program.
We have succesfully verified and quantified crown rust resistance levels of 14 perennial ryegrass lines (Figure 1). Crown rust severity ratings ranged from 1.5 to 3.6 in the field and from 1.1 to 3.9 in the growth chamber (Figure 1.) We observed lower standard error in the growth chamber indicating that this data may be more reliable than field data for use in developing a training set for identifying potential biomarkers. However the Spearman rank-order correlation coefficient between the field and growth chamber data was significant at 0.78 indicating that the field and growth chamber data are highly correlated. It was concluded that this data will be adequate phenotype data for use in a supervised multivariate analysis for detecting secondary metabolite biomarkers.
2) First development of metabolomics-assisted selection techniques for high-throughput, accurate, and dependable crown rust resistance screening in a plant breeding program.
Our preliminary data analysis indicates that the high pressure liquid chromatography and mass spectrometry methods that we have developed can successfully identify a wide range of compounds in perennial ryegrass that differentiate perennial ryegrass lines based on rust resistance levels alone. A loadings and scores plot from our PLSDA analysis (Figure 2) indicates larger metabolic variation between resistant and susceptible lines than within resistant or susceptible lines. Component axis 1 in Figure 2 separated the resistant and suscepible lines and explained 77.4% of the overall variation. We detected over 900 features in our samples using positive ionization mode and around 370 were determined to be reliable features based on covariance levels. Of these around 15 features had a VIP level of greater that 0.6 (Table 2)and many of them were detected at significantly higher or lower abundance in resistant vs. susceptible lines. A full analysis of all lines using both positive and negative ionization data still needs to be conducted.
3) Improved knowledge of the biological basis for plant resistance to rust pathogens plus greater farmer and researcher understanding of ryegrass and pathogen interactions.
To date we have not made conclusions on the biological basis for rust resistance but as we finish our analysis and begin to identify important compounds via MS/MS analysis we will be able to make further conclusions.
Intermediate-term outcomes:
1) Rust resistant perennial ryegrass lines identified in this study will be incorporated into our breeding program.
Data from screening trials has been taken into account when making selections using related germplasm. In addition we are begining to try to develop lines which have both crown and stem rust resistance with the material screened in this study being a potential source of resistance.
2) This research will result in our breeding program having a fast, reliable metabolomics-assisted selection method to supplement often unpredictable phenotypic screening methods leading to faster delivery rust resistant cultivars to farmers.
We have made significant strides toward implementing a metabolomics-assisted selection technique into our perennial ryegrass breeding program. As the final analysis of data from this study is completed we will begin to screen more of our perennial ryegrass germplasm for crown rust resistance with this technique.
3) The crown rust resistance selection model will be adapted for stem rust resistance selection.
This remains a goal and this project has made significant progress in identifying a set of perennial ryegrass germplasm that can be used as a training set for selecting metabolic biomarkers to be used in metabolomics-assisted selection for identifying stem rust resistant plants.
Long-term outcomes:
1) New crown and stem rust resistant cultivars will make perennial ryegrass seed crops a more profitable and environmentally sustainable crop rotation option for farmers in northern Minnesota.
Due to the short time frame of this study and the long time frame required for release of new cultivars we have not yet observed this outcome however rust resistant varieties remain a desirable product for farmers.
2) Seed from rust resistant cultivars will be more marketable to end users and will result in more environmentally sustainable turfgrasses.
This remains a projected long term outcome.
3) Our methods could be used as a model for metabolomics-assisted selection in other important agricultural crops or for other traits in perennial ryegrass.
Our program has hired a post-doctoral researcher to use similar methods to the one developed here to identify metabolic biomarkers associated with cold or freezing tolerance in perennial ryegrass which will further improve sustainability of perennial ryegrass turfgrass and seed production in our region.
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- Table 2. Retention times, M/Z values, and variable importance in predictiction values for 370 features detected in positive ionization mode.
- Figure 1. Crown rust screening data and line selection strategy.
- Figure 2. Loadings and scores plot showing separation of the two most crown rust resistant and two most susceptible perennial ryegrass lines along principle component axis 1.
Educational & Outreach Activities
Participation Summary:
Outreach:
1) A presentation discussing the status and importance of this research was given to perennial ryegrass growers during a field day at the University of Minnesota Magnusson Research Farm during the summer of 2012.
2) Created a freely available online presentation discussion this project for the 2012 Minneosta Turf and Grounds Foundation and University of Minnesota Virtual Field Day (Link: http://turf.umn.edu/eric-koeritz-ryegrass-improvement-studies/).
Publications:
1) Koeritz, E., A. Hegeman, E. Watkins, and N. Ehlke. 2012. Improving perennial ryegrass seed production in northern Minnesota: Developing Metabolomics-assisted selection techniques for crown and stem rust resistant cultivar development. 2012 Annual Meeting Abstracts [ASA/CSSA/SSSA].
2) A scientific manuscript for publication in a peer reviewed journal is currently being constructed with data from this project.
Project Outcomes
The development of a metabolomics-assisted selection strategy is a very complicated and time consuming process. Our rust resistance phenotyping data gave us a solid foundation to proceed with beginning to correlate metabolic biomarkers with crown rust resistance which is crucial. Successfully differentiating between rust resistant and susceptible germplasm based on secondary metabolites gives us confidence that we can apply this technique to other traits in our breeding program. The perennial ryegrass seed producers have given partial funding to develop a fast and accurate metabolomics assisted screening method for identifying more winter-hardy perennial ryerass.
Economic Analysis
Due to the more long-term nature of plant breeding we did not conduct an economic analysis for this project. However, we anticipate that improving rust resistance in perennial ryegrass could will reduce the need for applying expensive fungicides and will reduce seed yield losses for farmers thus improving the economic viability of the crop.
Farmer Adoption
To date the outcomes have been largely scientific but the results are being applied to efforts to develop new perennial ryegrass varieties that are more sustainable and profitable. Farmers value the efforts to develop new,faster, and more efficient breeding strategies. Interest developed by this project has led to farmers financially contributing to additional projects applying metabolomics-assisted selection to improving winter-hardiness in perennial ryegrass.
Areas needing additional study
A more comprehensive analysis of the data developed through this project is currently underway. Once, completed these techniques will be tested on a perennial ryegrass population with known rust resistance levels and the accuracy of the strategy will be evaluated.
Future research should investigate which secondary metabolites are affected over time as rust pathogens attack the plant. This project was only able to focus on non-induced secondary metabolites.
Perennial ryegrass disease resistance and other traits can also be affected by endophytic fungi which are naturally present in many varieties. It could be important to investigate the impact of these endophytes on the metabolites associated with rust resistance that are identified by this work.