Determination of the Effect of Co-cultivation on the Production and Root Exudation of Flavonoids in Four Legume Species Using LC–MS/MS Analysis

[Image: see text] Flavonoids play a key role in the regulation of plant–plant and plant–microbe interactions, and factors determining their release have been investigated in most of the common forage legumes. However, little is known about the response of flavonoid production and release to co-cultivation with other crop species. This study investigated alterations in the concentration of flavonoids in plant tissues and root exudates in four legumes [alfalfa (Medicago sativa L.), black medic (Medicago polymorpha L.), crimson clover (Trifolium incarnatum L.), and subterranean clover (Trifolium subterraneum L.)] co-cultivated with durum wheat [Triticum turgidum subsp. durum (Desf.) Husn.]. For this purpose, we carried out two experiments in a greenhouse, one with glass beads as growth media for root exudate extraction and one with soil as growth media for flavonoid detection in shoot and root biomass, using LC–MS/MS analysis. This study revealed that interspecific competition with wheat negatively affected legume growth and led to a significant reduction in shoot and root biomass compared with the same legume species grown in monoculture. In contrast, the concentration of flavonoids significantly increased both in legume biomass and in root exudates. Changes in flavonoid concentration involved daidzein, genistein, medicarpin, and formononetin, which have been found to be involved in legume nodulation and regulation of plant–plant interaction. We hypothesize that legumes responded to the co-cultivation with wheat by promoting nodulation and increasing exudation of allelopathic compounds, respectively, to compensate for the lack of nutrients caused by the presence of wheat in the cultivation system and to reduce the competitiveness of neighboring plants. Future studies should elucidate the bioactivity of flavonoid compounds in cereal-legume co-cultivation systems and their specific role in the nodulation process and inter-specific plant interactions such as potential effects on weeds.