Exploring Metabolite Variations in Annual Ryegrass Seeds to Understand Associated Annual Ryegrass Toxicity (ARGT)

Sucharita Basavarajappa

Annual ryegrass is a forage crop grown in many countries due to its high nutritional value, palatability, productivity, and adaptability to different soil conditions. However, an often-fatal poisoning of livestock called Annual Ryegrass Toxicity (ARGT) happens by consuming this grass infected by the bacterium Rathayibacter toxicus. This bacterium is carried into the grass by the nematode Anguina funesta. In Western Australia and South Australia, thousands of cattle and sheep deaths were recorded from 1968 to 2000 due to ARGT. Since 2005, it has been mandatory to test hay to be exported for the presence of corynetoxins. Therefore, further understanding of causative agents and annual ryegrass through metabolomic study is important. This study was undertaken in three phases of investigation. The first phase is to find similarities or differences among the ryegrass seeds from different geographical locations and harvest years in Western Australia. This evaluated the impact of the environment and time on metabolites. A variety of statistical methods, including univariate and multivariate data analysis, were performed. The results are promising, with metabolites forming clusters with the same year of harvest and region. A few metabolites like 10(E),12(Z)-conjugated linoleic acid, 1,2-Benzenedicarboxylic acid, and bis(2-methylpropyl) ester were expressed in 2020, 3-Tetradecanol, Ergost-5-en-3-ol, acetate, (3β,24R)- in 2022 and cis-10-Heptadecenoic acid, Phytol in 2024 were expressed in 2024, proving unique fingerprinting for each harvest year. In addition, a few of the metabolites, viz 1-hexacosanol, 11,14-Eicosadienoic acid, methyl ester, and isopentyl 3-hydroxy-2-methylenebutanoate, were expressed in regions closer in distance, confirming the greater influence of plant environment on the chemical fingerprint of plants. The second phase was to study metabolites from infected galls and germinating ryegrass seeds. In coinfected galls, linoleic acid ethyl ester was identified, and cis-10-Heptadecenoic acid, Ethyl Oleate, and Octadecanoic acid, ethyl ester, were identified in nematode-infected galls alone. These metabolites could be biomarkers for coinfected galls and nematode galls, respectively. The first two phases used the solvent extraction method with gas chromatography-mass spectrometry (GC-MS). The third phase was studied in two parts, first, via physical assay to understand the nematode attraction. The second phase involved investigating the volatile organic compounds (VOCs) produced by bacteria when nematodes and germinating seeds were present. Physical attraction assay results agreed with a previous study that the in vitro setting is not favourable for nematode Anguina spp movement. In the volatile organic compounds study, 1-heptanol, 4-methyl-Benzenemethanol, 2,5-dimethyl-Benzaldehyde were recorded in the presence of nematodes, which is a novel discovery. This study provided new biomarkers and insight into ARGT, demonstrating that the techniques of solvent extraction and Head Space Solid Phase Micro Extraction (HS SPME), along with GC-MS, were valid for differentially expressed metabolites. Additionally, this study is pivotal since no previous studies have reported nematode volatiles related to ARGT.

Exploring Metabolite Variations in Annual Ryegrass Seeds to Understand Associated Annual Ryegrass Toxicity (ARGT)

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