| Summary: | In this study, the allelopathic properties of <i>Medicago sativa</i> on different weeds were investigated under in vitro conditions. The compounds involved in the autotoxicity of <i>M. sativa</i> were analyzed using high-performance liquid chromatography. The extracts of all concentrations inhibited the growth of the calluses of <i>Digitaria ciliaris, Chenopodium album</i>, <i>Amaranthus</i> <i>lividus, Portulaca oleracea</i>, and <i>Commelina communis</i>. Six allelopathic compounds in alfalfa were identified and quantified, and the most predominant phenolic compounds were salicylic acid and <i>p</i>-hydroxybenzoic acid. Various concentrations (10<sup>−2</sup>, 10<sup>−3</sup>, and 10<sup>−5</sup> M) of all the tested phenolic compounds exerted inhibitory effects on callus fresh weight. Rutin, salicylic acid, scopoletin, and quercetin significantly inhibited alfalfa seed germination. Of the seven identified saponins, medicagenic acid saponins exhibited the highest autotoxic effect and significantly lowered seed germination rate. Principal component analysis showed that the phenolic compounds and saponin composition significantly contributed to the different variables. The highly phytotoxic properties of the alfalfa-derived phenolic compounds and saponins indicate that these phytochemicals can be a potential source of bioherbicides.
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