White clover is frequently used as a leguminous cover crop, serving as green manure, and is also included with grasses in cattle feed mixtures. Numerous biological effects reported for clover cultivation have been attributed to the production of bioactive secondary metabolites. Thus far the presence in soil of bioactive secondary metabolites from clover has received limited attention. In this paper we examine for the first time the release of flavonoids both from field-grown white clover and from soil-incorporated white clover plants of flavonoids, as analyzed by LC-MS/MS. The dominant flavonoid aglycones were formononetin, medicarpin, and kaempferol. Soil-incorporated white clover plants generated high concentrations of the glycosides kaempferol-Rha-Xyl-Gal and quercetin-Xyl-Gal. Substantial amounts of kaempferol persisted in the soil for days while the other compounds were degraded faster. These compounds should be considered in future studies of soil fatigue, allelopathic activity, and possible environmental risks from extended clover cultivation. 1. Introduction Sustainable agricultural practices include the use of leguminous plants to supply the soil with nitrogen in a crop rotation strategy to limit the use of agrochemicals. The leguminous crop white clover is frequently used as a cover crop serving as green manure and is also included in mixtures with grasses for cattle feed. Legumes that are used as cover crops or incorporated into the soil may also function as a supplement or even replacement for pesticides. Several studies have reported suppressive effects of white clover on weed pressure and diseases or unexplained failure of oversown grasses in swards dominated by clover [1–12]. Secondary metabolites have been suspected of causing these effects, and basic knowledge on the release, active or passive, of biologically active secondary metabolites from white clover is therefore much needed. Such knowledge would aid an evaluation of the importance of secondary metabolites in biological effects from white clover. The objective of this study was therefore to clarify the pattern of both flavonoid release from field-grown white clover and their leaching into soils amended with white clover plant material. 2. Materials and Methods 2.1. Origin of White Clover and Sampling Whole plants of the white clover cultivar Klondike were collected in June 2008 along with soil to a depth of 15？cm from an organically grown field in Flakkebjerg, Denmark (soil A). The soil was carefully removed from the plant material. To determine the flavonoid concentration and the
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