Using non-targeted direct analysis in real time-mass spectrometry (DART-MS) to discriminate seeds based on endogenous or exogenous chemicals

Forage seeds are a highly traded agricultural commodity, and are a primary mechanism for introducing improved plant genetics into farm systems. Maintaining quality control and quality assurance of forage seeds are therefore high priority. Direct Analysis in Real-Time Mass Spectrometry (DART-MS) and a biomarker discovery toolkit comprising Receiver-Operator Characteristic (ROC) curve analysis, t-test and Extracted Ion Chronograms (XICs), were used to discriminate global metabolic profiles of intact forage seeds based on either endogenously present or exogenously applied chemicals. Using this system, perennial ryegrass (Lolium perenne L.) seed were differentiated based on the presence or absence of the herbicide diuron. This approach also detected oxylipins, a class of endogenous antimicrobial compounds, on seed coats of white clover (Trifolium repens L.) and alfalfa (Medicago sativa L.). Presence of oxylipins was considered to confer toxicity towards symbiotic, nitrogen-fixing rhizobia which are routinely coated on legume seeds before planting. Washing seeds with water reduced oxylipin content, which may have contributed to enhanced survival of nitrogen-fixing rhizobia. This is the first report of automatic introduction of intact seeds in the DART ion source, and detecting oxylipins using DART-MS. Apart from providing scope to investigate legume-rhizobia symbiosis further in the context of oxylipins, the results presented here will enable future studies aimed at classification of seeds based on chemicals bound to the seed coat, thereby offering an efficient screening device for industry.