Anchusa officinalis is recognized for its therapeutic properties, which are attributed to the production of different metabolites. This plant interacts with various microorganisms, including the root symbiotic arbuscular mycorrhizal fungi (AMF). Whether these fungi play a role in the metabolism of A. officinalis is unknown. In the present study, two independent experiments, associating A. officinalis with the AMF Rhizophagus irregularis MUCL 41833, were conducted in a semi-hydroponic (S-H) cultivation system. The experiments were intended to investigate the primary and secondary metabolites (PMs and SMs, respectively) content of shoots, roots, and exudates of mycorrhized (M) and non-mycorrhized (NM) plants grown 9 (Exp. 1) or 30 (Exp. 2) days in the S-H cultivation system. Differences in the PMs and SMs were evaluated by an untargeted ultrahigh-performance liquid chromatography high-resolution mass spectrometry metabolomics approach combined with multivariate data analysis. Differences in metabolite production were shown in Exp. 1. Volcano-plots analysis revealed a strong upregulation of 10 PMs and 23 SMs. Conversely, in Exp. 2, no significant differences in PMs and SMs were found in shoots or roots between M and NM plants whereas the coumarin scoparone and the furanocoumarin byakangelicin, accumulated in the exudates of the M plants. In Exp. 1, we noticed an enhanced production of PMs, including organic acids and amino acids, with the potential to act as precursors of other amino acids and as building blocks for the production of macromolecules. Similarly, SMs production was significantly affected in Exp 1. In particular, the phenolic compounds derived from the phenylpropanoid pathway. Fifteen di-, tri-, and tetra-meric C6-C3 derivatives of caffeic acid were induced mainly in the roots of M plants, while four oleanane-types saponins were accumulated in the shoots of M plants. Two new salvianolic acid B derivatives and one new rosmarinic acid derivative, all presenting a common substitution pattern (methylation at C-9”' and C-9' and hydroxylation at C-8), were detected in the roots of M plants. The accumulation of diverse compounds observed in colonized plants suggested that AMF have the potential to affect specific plant biosynthetic pathways.

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Previous studies have shown that individual, isolated microorganisms may influence secondary metabolism of plants and induce or stimulate the production of medicinally relevant secondary metabolism. Here, we analyzed the microbiome-metabolome linkage of the medicinal plant Alkanna tinctoria , which is known to produce valuable compounds, particularly the naphthoquinones alkannin and shikonin and their derivatives.

Anchusa officinalis (L.) interacts with various microorganisms including arbuscular mycorrhizal fungi (AMF). Recently, the AMF Rhizophagus irregularis MUCL 41833 has been shown to modulate the metabolome of A. officinalis. However, little information is available on the impact that different AMF species may have on primary and secondary plant metabolites. In this study, four AMF species belonging to the genus Rhizophagus (R. irregularis MUCL 41833, R. intraradices MUCL 49410, R. clarus MUCL 46238, R. aggregatus MUCL 49408), were evaluated for their potential to modulate A. officinalis metabolome under controlled semi-hydroponic cultivation conditions. An untargeted metabolomic analysis was performed using UHPLC-HRMS followed by a multivariate data analysis. Forty-two compounds were reported to be highly modulated in relation to the different AMF associations. Among them, six new secondary metabolites were tentatively identified including two acetyl- and four malonyl- phenylpropanoid and saponin derivatives, all presenting a common substitution at position C-6 of the glycosidic moiety. In addition, an enhanced accumulation of primary and secondary metabolites was observed for R. irregularis and R. intraradices, showing a stronger effect on A. officinalis metabolome compared to R. clarus and R. aggregatus. Therefore, our data suggest that different AMF species may specifically modulate A. officinalis metabolite production.

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