A comprehensive metabolite profiling of germinated, healthy, and bacterial and nematode infected ryegrass seeds to manage annual ryegrass toxicity (ARGT)

Annual ryegrass toxicity (ARGT), a fatal toxicity to grazing animals, is caused by ingestion of bacteria ( Rathayibacter toxicus ) infected ryegrass ( Lolium rigidum ) seeds. This toxicity mainly prevailed in Australia, specifically in Western Australia and South Australia. Many approaches have been used to manage the occurrence of ARGT, but due to limited utilisation, no effective approach is in practice. Therefore, this study explored comprehensive metabolites change in healthy, bacterial, and nematode infected and various stages (2nd day, 4th day, 8th day, and 2-leaf stage) of germination of ryegrass seeds. To screen the potential biomarkers for detecting the early infestation. Thirty-two metabolites were identified from acetonitrile extracts of all seven samples analysed by Gas Chromatography-Mass spectrometry. E-7-tetradecenol and tetradecanoic acid were identified in healthy and germinated seeds. On the other hand, 1-butanamine, N-butyl, was detected exclusively at the 2-leaf stage and was absent throughout the 2nd-8th day germination stages. In contrast, campesterol was present at all germination stages, showing an increasing trend, nearly doubling from the 2nd day to the 2-leaf stage. Interestingly, n-hexadecanoic acid and γ-sitosterol were found in all categories. γ-Sitosterol is essential for their nematodes development but cannot be synthesized by them. Similarly, linoleic acid ethyl ester, detected in co-infected bacteria and nematode galls, could be an early diagnostic marker for infestation. A deeper understanding of metabolomics, as depicted in the paper, can aid in better management and prevention of ARGT in the future. • This study explored metabolite changes in nematode and bacteria infected ryegrass seeds along with healthy and germinated seeds. • A total of 32 metabolites were identified, n-hexadecanoic acid and ϒ-sitosterol were found in all categories, and linoleic acid ethyl ester was detected in co-infected galls. • Compounds like cis-10-Heptadecenoic acid, Ethyl Oleate, and Octadecanoic acid, ethyl ester were expressed in nematode galls alone. • The identified chemical biomarkers could be used to develop an early diagnostic tool for detecting the infestation.