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.

Medicinal plants are an important source of therapeutic compounds used in the treatment of many diseases since ancient times. Interestingly, they form associations with numerous microorganisms developing as endophytes or symbionts in different parts of the plants. Within the soil, arbuscular mycorrhizal fungi (AMF) are the most prevalent symbiotic microorganisms forming associations with more than 70% of vascular plants. In the last decade, a number of studies have reported the positive effects of AMF on improving the production and accumulation of important active compounds in medicinal plants.In this work, we reviewed the literature on the effects of AMF on the production of secondary metabolites in medicinal plants. The major findings are as follows: AMF impact the production of secondary metabolites either directly by increasing plant biomass or indirectly by stimulating secondary metabolite biosynthetic pathways. The magnitude of the impact differs depending on the plant genotype, the AMF strain, and the environmental context (e.g., light, time of harvesting). Different methods of cultivation are used for the production of secondary metabolites by medicinal plants (e.g., greenhouse, aeroponics, hydroponics, in vitro and hairy root cultures) which also are compatible with AMF. In conclusion, the inoculation of medicinal plants with AMF is a real avenue for increasing the quantity and quality of secondary metabolites of pharmacological, medical, and cosmetic interest.

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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IntroductionAlkanna tinctoria Tausch. is a medicinal plant well-known to produce important therapeutic compounds, such as alkannin/shikonin and their derivatives (A/Sd). It associates with arbuscular mycorrhizal fungi (AMF), which are known, amongst others beneficial effects, to modulate the plant secondary metabolites (SMs) biosynthesis. However, to the best of our knowledge, no study on the effects of AMF strains on the growth and production of A/Sd in A. tinctoria has been reported in the literature.MethodsHere, three experiments were conducted. In Experiment 1, plants were associated with the GINCO strain Rhizophagus irregularis MUCL 41833 and, in Experiment 2, with two strains of GINCO (R. irregularis MUCL 41833 and Rhizophagus aggregatus MUCL 49408) and two native strains isolated from wild growing A. tinctoria (R. irregularis and Septoglomus viscosum) and were grown in a semi-hydroponic (S-H) cultivation system. Plants were harvested after 9 and 37 days in Experiment 1 and 9 days in Experiment 2. In Experiment 3, plants were associated with the two native AMF strains and with R. irregularis MUCL 41833 and were grown for 85 days in pots under greenhouse conditions. Quantification and identification of A/Sd were performed by HPLC-PDA and by HPLC-HRMS/MS, respectively. LePGT1, LePGT2, and GHQH genes involved in the A/Sd biosynthesis were analyzed through RT-qPCR.ResultsIn Experiment 1, no significant differences were noticed in the production of A/Sd. Conversely, in Experiments 2 and 3, plants associated with the native AMF R. irregularis had the highest content of total A/Sd expressed as shikonin equivalent. In Experiment 1, a significantly higher relative expression of both LePGT1 and LePGT2 was observed in plants inoculated with R. irregularis MUCL 41833 compared with control plants after 37 days in the S-H cultivation system. Similarly, a significantly higher relative expression of LePGT2 in plants inoculated with R. irregularis MUCL 41833 was noticed after 9 versus 37 days in the S-H cultivation system. In Experiment 2, a significant lower relative expression of LePGT2 was observed in native AMF R. irregularis inoculated plants compared to the control.DiscussionOverall, our study showed that the native R. irregularis strain increased A/Sd production in A. tinctoria regardless of the growing system used, further suggesting that the inoculation of native/best performing AMF is a promising method to improve the production of important SMs.