No abstract

The effects of two arbuscular mycorrhizal fungi (AMF) (Glomus mosseae and G. claroideum) and a pathogenic fungus (Pythium ultimum) on the production of eight flavonoids in roots of two white clover (Trifolium repens L.) cultivars were evaluated. Quantification of AM and pathogenic fungi in the roots showed that the AM symbiosis significantly reduced P. ultimum biomass and in some cases prevented infection. The flavonoid productions in clover roots varied depending on the presence of beneficial and/or pathogenic fungi, fungal isolate or plant cultivar. Only plants colonized with G. claroideum showed detectable concentrations of either coumestrol or kaempferol (cultivar-dependant). In addition, inoculation with G. claroideum resulted in significantly higher concentrations of coumestrol in cv. Sonja and medicarpin in cv. Milo. A low production of coumestrol and kaempferol in mycorrhizal plants may be G. mosseae-specific. Only the concentrations of formononetin and daidzein increased in clover roots in response to infection with P. ultimum. These flavonoids are supposedly stress metabolites, synthesized or produced from glycosides in response to pathogen infection. However, the presence of one or both AMF significantly lowered the formononetin and daidzein concentrations, and overruled the inductive effect of P. ultimum. Therefore the antagonistic action of AM against the pathogen must take place through another mechanism.

To exploit biologically active compounds from white clover (Trifolium repens L.) for suppressing weeds and soil-borne diseases, either as isolated products (biopesticides) or through cultivars with enhanced production of these compounds, the biologically active compounds must be identified, plant content measured, and their fate in soil known. The present review summarizes the published knowledge needed for such exploitation; providing essential information on structure and concentration of flavonols, flavones, condensed tannins, isoflavones, isoflavanones, pterocarpans, coumestans, cyanogenic glucosides, and saponins in healthy and stressed white clover plants. Various stresses and particular cultivars affect the concentrations of several of the compounds. Information on biological effects and the degradation/transformation of these compounds in plants or by microorganisms is available. There is no information on the degradation pathway in soil, the mechanisms of exudation and leaching of compounds from plants, and soil sorption properties of the compounds. The clover soil fatigue problem is increasing in grasslands and causes problems especially in organic farming. Research efforts focused on biological elements of clover soil fatigue have not explained it, and the influence of secondary metabolites has not been investigated. There are few investigations into the interaction between beneficial fungi/fungal-diseases and the occurrence of biologically active secondary metabolites in white clover plants. Such studies are critical to better understand beneficial fungi and pathogens.

In the present study, a range of benzoxazinoid compounds and phenolic acids, all known to be allelochemicals of rye, were identified and quantified in 13 rye cultivars grown at three different localities. Plant samples were collected in the spring at the time when an autumn-sown rye cover crop would be incorporated into the soil. Significant variations in content among shoots and roots were seen for all of the secondary metabolites, with non-methoxy-substituted benzoxazinoids (BX) dominating the shoots, whereas comparable levels were found in the concentrations of BX and methoxy-substituted benzoxazinoids (MBX) in the roots. This distribution of compounds may indicate different biosynthetic pathways and/or different mechanisms of action of these compounds. Concentrations not only depended on plant part, but also on the geographical location-with differences in contents of up to a factor of 5. These differences can probably be attributed to differences in growing conditions. The variation among cultivars was similar to that among geographical localities, with differences within localities of up to a factor of 7 in the shoots and a factor of 14 in the roots. In roots, the contents of the four phenolic acids and the benzoxazinoid 6-methoxybenzoxazolin-2-one (MBOA) were correlated. In shoots, the contents of the two benzoic acids were correlated with each other, whereas the two cinnamic acids were correlated with MBOA and several other benzoxazinoids. The lack of correlation between MBOA and all other benzoxazinoids in the roots of rye might indicate that a hitherto unknown synthetic pathway exists for MBOA. The genes responsible for the synthesis of some of the benzoxazinoids have never been identified, and further gene expression studies are required to assess the observed correlation between the concentration of these compounds and other benzoxazinoids for which the responsible genes are known. The present study revealed a potential for breeding rye cultivars with a high content of biologically active secondary metabolites. However, growing conditions significantly influenced the level of these compounds.

In the present study, the major flavonoids of white clover (Trifolium repens L.) cv. Sonja were extracted, isolated and identified. The major flavonoids in leaves and stems were the four flavonol glycosides: kaempferol-3-O-{Xyl(1→2)-Gal} (kaempferol-Xyl-Gal), kaempferol-3-O-{Rha(1→6)-[Xyl(1→2)]-Gal} (kaempferol-Rha-Xyl-Gal), quercetin-3-O-{Xyl(1→2)-Gal} (quercetin-Xyl-Gal), and quercetin-3-O-{Rha(1→6)-[Xyl(1→2)]-Gal} (quercetin-Rha-Xyl-Gal). Quercetin-Rha-Xyl-Gal has never been reported before and kaempferol-Rha-Xyl-Gal has not previously been identified in clover aerial parts. Concentrations of those compounds, together with aglyconic flavonoids previously described in white clover, as well as their glycosides, were quantified in leaves/stems and flowers of four white clover cvs Rabani, Klondike, Ramona and Aran using tandem mass spectrometry. There were significant differences in flavonoid concentrations in the two plant parts, with the highest concentrations of most aglycones in flowers and the highest concentrations of most glycosides in leaves/stems. This distribution of compounds may indicate different ways of storage and/or different mechanisms of action of the compounds. The cultivars were selected for genetic diversity, which resulted in distinctly different amounts of flavonoids in the plants. Concentrations of 17 of 24 compounds varied significantly-for some compounds up to a factor of 10-among cultivars. Total flavonoid concentrations in flowers did not vary greatly among cultivars, at 28.9-35.8 mmol/g dry material (DM). In contrast, in leaves/stems, the cvs Rabani and Klondike had lower concentrations of most flavonoids (total concentrations 10.0 and 12.7 mmol/g DM , respectively) compared to cvs Aran and Ramona (32.3 and 22.1 mmol/g DM , respectively). There is a potential for breeding/selection of cultivars with targeted concentrations of particular flavonoids.

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.