Development of Metabolomics-Assisted Selection Methods for Improving Rust Resistance in Perennial Ryegrass.

 

Stem rust (Puccinia graminis subsp. graminicola) and crown rust (Puccinia coronata f.sp. lolii) are fungal pathogens of perennial ryegrass that can reduce seed yields up to 70%, reduce forage quality, and reduce turfgrass aesthetic quality and performance.  Fast and reliable methods for selecting perennial ryegrass germplasm with durable quantitative resistance to rust pathogens are urgently needed in order to meet the demands of seed producers and end users.  Metabolomics-based techniques can identify metabolic biomarkers associated with disease resistance in plants and can supplement unreliable phenotypic screening.  The goal of this research was to develop methods for extraction and identification of metabolic biomarkers in perennial ryegrass and to identify rust resistant and susceptible perennial ryegrass germplasm for further biomarker identification.  Fast and efficient methods for grinding, extracting, and analyzing a broad spectrum of compounds from perennial ryegrass leaf tissue on a large scale have been demonstrated using a UPLC-MS approach.  Over 900 metabolic features could be detected via UPLC-MS-TOF when 40 mg of perennial ryegrass tissue was extracted with 400µl of 90 percent aqueous methanol, followed by drying and reconstituting in 200µl 0.1 percent formic acid.  Using UPLC we were able to detect a large number of compounds using a nine minute gradient and 5µl injection volume which is critical for the high throughput analysis required for disease screening. 

Thirty nine perennial ryegrass breeding lines were screened for crown rust resistance under field conditions in St. Paul during the 2009 growing season and were divided into resistant (R), moderately resistant (M) and susceptible (S) groups.  A subset of these breeding lines (15 percent most R, M and S) were  planted in Roseau, MN where they will be screened for stem rust resistance and in St. Paul, MN where crown rust resistance was verified during the 2010 growing season.  Five lines demonstrated good resistance levels and five lines demonstrated susceptibility to crown rust consistently in both years of screening in a St. Paul field nursery.  The R, M and S perennial ryegrass groups will be used as a plant material for the identification if metabolic biomarkers via UPLC-MS.  Metabolic biomarkers associated with rust resistance can potentially be used to select for rust resistance in the absence of pathogen pressure potentially making screening for disease resistance faster and less reliant on environmental factors.