Targeted analysis of flavonoids associated with weed suppression in selected annual pasture legumes

Pasture weeds incur an estimated cost of AU$ 2.2 billion per year in management expenses and yield loss to Australian livestock producers. The use of herbicides for pasture weeds management is common, but the prevalence of herbicide resistance amongst populations of some major weeds to a range of chemicals is adding to the economic burden. Therefore, the choice of competitive pastures/cultivars along with optimal agronomic practices can provide one mean to reduce reliance on herbicides. Several novel self-regenerating annual pasture legumes originating from temperate to Mediterranean regions of Europe and Northern Africa were selectively improved for release in Australia. However, limited knowledge exists regarding their competitive or allelopathic ability to suppress weeds. Pasture legumes adapted to the moderate rainfall zone in central NSW include Biserrula pelecinus L. (biserrula), Ornithopus sativus L. (French serradella), Ornithopus compressus L. (yellow serradella), Trifolium glanduliferum L. (gland clover), Trifolium spumosum L. (bladder clover) and Trifolium vesiculosum Savi. (arrowleaf clover). Results of field trials conducted at several locations demonstrated the significant weed suppressive potential of arrowleaf clover, biserrula and yellow serradella against common annual broadleaf and grass weeds. Multivariate data analysis suggested that mechanisms other than resource competition were associated with weed suppression in case of both yellow serradella and biserrula. Legumes are known to produce a diverse assortment of bioactive secondary metabolites including phytotoxic flavonoids present as allelochemicals in the plant rhizosphere. In our search for those bioactive secondary metabolites associated with weed suppression, we adopted a targeted metabolic profiling approach using ultra high pressure liquid chromatography ( UPLC, Agilent 1290 infinity) coupled with quadrupole time-of-flight (Agilent 6530) and triple quadrupole (Agilent 6470) mass spectrometry followed by chemometric analysis. Compound identification was further confirmed by comparing results obtained with flavonoids and phenolic standards in our personal compound database and library (PCDL). Visual examination of UPLC/MS chromatograms confirmed the presence of numerous bioactive flavonoids in pasture legume extracts and purified phytotoxic fractions following flash column. Principal component analysis revealed considerable species dependent variations in the metabolic profiling of selected pasture legumes. Regression analysis showed a strong correlation between bioactivity and abundance of key bioactive flavonoids such as quercetin, kaempferol and p-coumaric acid in selected pasture legumes.