Phytotoxic allelochemicals from roots and root exudates of Trifolium pratense.

Trifolium pratense, a widespread legume forage plant, is reported to exhibit phytotoxic activity on other plants, but the active metabolites have not been clarified so far. A bioassay-guided fractionation of the root extracts led to the isolation of five isoflavonoids, which were elucidated by spectroscopic analysis. All of the purified compounds observably showed phytotoxic activities against Arabidopsis thaliana. Moreover, the inhibitory effects were concentration-dependent. The furan ring linked at C-4 and C-2' positions by an oxygen atom and a 1,3-dioxolane at C-4' and C-5' positions are considered to be critical factors for the phytotoxic activity. The concentrations of (6aR,11aR)-maackiain and (6aR,11aR)-trifolirhizin, concluded to be allelochemicals from soil around plants of T. pratense, were determined by HPLC and LC-MS to be 4.12 and 2.37 mu g/g, respectively. These allelochemicals, which showed remarkable activities against the weed Poa annua may play an important role in assisting the widespread occurrence of T. pratense in nature.