Effects of fungal endophytes and arbuscular mycorrhizal fungi on growth of Echium vulgare and alkannin/shikonin and their derivatives production in roots.

Endophytic fungi as well as arbuscular mycorrhizal fungi (AMF) are known to stimulate plant growth and production of secondary metabolites in medicinal plants. Here, 10 endophytic fungi isolated from roots of wild Alkanna tinctoria plants and 5 AMF purchased from the Glomeromycota in vitro collection were evaluated, during two successive three-month greenhouse experiments, on the growth of Echium vulgare and alkannin/shikonin and their derivatives (A/Sd) production in the roots. Some of the endophytic fungi tested significantly increased plant growth parameters as compared to the control: Cladosporium allicinum, Cadophora sp., Clonostachys sp., Trichoderma hispanicum and Leptosphaeria ladina increased root volume, Plectosphaerella sp. And T. hispanicum root fresh weight and root water retention and T. hispanicum plant water retention. However, none of these fungi impacted A/Sd production. Conversely, none of the AMF strains tested impacted plant growth parameters, but those inoculated with Rhizophagus intraradices MUCL 49410 had a significantly higher concentration of alkannin/shikonin (A/S), acetyl-A/S, ß,ß- dimethylacryl-A/S, isovaleryl-A/S and total A/Sd, compared to the control plants. Further studies are needed to investigate the mechanisms involved in the production of A/Sd in plants associated to specific endophytic fungi/AMF and on the cultivation conditions required for optimal production of these compounds.

Arbuscular mycorrhizal fungi impact the production of alkannin/shikonin and their derivatives in Alkanna tinctoria Tausch. grown in semi-hydroponic and pot cultivation systems.

Introduction: Alkanna 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. Methods: Here, 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. Results: In 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. Discussion: Overall, 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.

Metabolite Production in Alkanna tinctoria Links Plant Development with the Recruitment of Individual Members of Microbiome Thriving at the Root-Soil Interface.

Plants are naturally associated with diverse microbial communities, which play significant roles in plant performance, such as growth promotion or fending off pathogens. The roots of Alkanna tinctoria L. are rich in naphthoquinones, particularly the medicinally used enantiomers alkannin and shikonin and their derivatives. Former studies already have shown that microorganisms may modulate plant metabolism. To further investigate the potential interaction between A. tinctoria and associated microorganisms, we performed a greenhouse experiment in which A. tinctoria plants were grown in the presence of three distinct soil microbiomes. At four defined plant developmental stages, we made an in-depth assessment of bacterial and fungal root-associated microbiomes as well as all extracted primary and secondary metabolite content of root material. Our results showed that the plant developmental stage was the most important driver influencing the plant metabolite content, revealing peak contents of alkannin/shikonin derivatives at the fruiting stage. Plant root microbial diversity was influenced both by bulk soil origin and to a small extent by the developmental stage. The performed correlation analyses and cooccurrence networks on the measured metabolite content and the abundance of individual bacterial and fungal taxa suggested a dynamic and at times positive or negative relationship between root-associated microorganisms and root metabolism. In particular, the bacterial genera Labrys and Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium as well as four species of the fungal genus Penicillium were found to be positively correlated with higher content of alkannins. IMPORTANCE 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. A detailed bacterial and fungal microbiome and metabolome analysis of A. tinctoria roots revealed that the plant developmental stage influenced root metabolite production, whereas soil inoculants from three different geographical origins in which plants were grown shaped root-associated microbiota. Metabolomes of plant roots of the same developmental stage across different soils were highly similar, pinpointing to plant maturity as the primary driver of secondary metabolite production. Correlation and network analyses identified bacterial and fungal taxa showing a positive relationship between root-associated microorganisms and root metabolism. In particular, the bacterial genera Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium and Labrys as well as the fungal species of genus Penicillium were found to be positively correlated with higher content of alkannins.

The Metabolic Profile of Anchusa officinalis L. Differs According to Its Associated Arbuscular Mycorrhizal Fungi.

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.

Arbuscular mycorrhizal fungi and production of secondary metabolites in medicinal plants.

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.

The Arbuscular Mycorrhizal Fungus Rhizophagus irregularis MUCL 41833 Modulates Metabolites Production of Anchusa officinalis L. Under Semi-Hydroponic Cultivation.

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.

The Mycorrhizal Donor Plant (MDP) In Vitro Culture System for the Efficient Colonization of Whole Plants.

The mycorrhizal donor plant (MDP) in vitro culture system allows the fast and homogeneous colonization of a wide range of photosynthetically active plants. Here we detailed the setup of the system and its potential applications for basic studies as well as mass production and applied purposes.

Increased Silicon Acquisition in Bananas Colonized by Rhizophagus irregularis MUCL 41833 Reduces the Incidence of Pseudocercospora fijiensis.

This work aimed to test the hypothesis that the combination of arbuscular mycorrhizal fungi (AMF) and accumulation of silicon (Si) in banana plants via its uptake and transport by the fungus reduces the incidence of Black Leaf Steak Disease (BLSD) caused by Pseudocercospora fijiensis. Methods: A pot experiment was conducted to compare BLSD symptoms on leaves of banana plants colonized or not by the AMF Rhizophagus irregularis MUCL 41833 and exposed or not to Si added to the growth substrate. Results: A marked increase in plant growth parameters (i.e., pseudostem diameter and height, leaf surface area, shoot, root and total dry weight) as well as accumulation of Si, P, and Ca were noticed in the AMF-colonized banana plants in presence as well as in absence of Si added to the growth substrate. Similarly Si addition to the substrate increased plant growth parameters. Leave symptoms caused by the pathogen were observed in all the treatments but were reduced in presence of AMF as well as in presence of Si added to the growth substrate. The more drastic reduction was noticed in the AMF-colonized plants with Si added to the growth substrate. The Severity Index as well as Area Under Disease Progress Curve were considerably decreased both at 21 (∼48% and 48%, respectively) and 35 days (∼21% and ∼32%, respectively) after inoculation of the pathogen as compared with non-AMF-colonized plants in absence of Si added to the substrate. Conclusion: Our findings revealed that AMF-colonized banana plants grown in a subs-trate supplemented with Si were less impacted by P. fijiensis than non-colonized plants grown without Si added to the growth substrate. The combination of AMF-colonized banana plants (during the weaning phase or in vitro) with the application of Si to soil seems thus a thoughtful option to mitigate the impact of BLSD in bananas, although such strategy needs first to be evaluated under field conditions to appraise its real potential.

Cryopreservation of arbuscular mycorrhizal fungi from root organ and plant cultures.

Long-term maintenance of arbuscular mycorrhizal fungi (AMF) by in vitro or in vivo subcultivation is often expensive and time-consuming and could present the risk of contaminations and possibly morphological, physiological, and genetic variations over time. Recently, in vitro produced AMF isolates belonging to the genus Rhizophagus were successfully cryopreserved at -130 °C following encapsulation-drying. Here, this method was tested on 12 single species cultures belonging to six different genera (i.e., Rhizophagus, Glomus, Claroideoglomus, Septoglomus, Paraglomus, and Gigaspora) produced in vitro or in vivo. Their viability was estimated, after 1 month of cryopreservation at -130 °C, by the percentage of potentially infective beads (i.e., the percentage of beads that contained at least one germinated propagule) for the in vitro produced species and the percentage of infective beads (i.e., the percentage of beads that contained at least one propagule able to colonize a new host plant in pot culture) for the in vivo produced species. With the exception of Gigaspora sp. MUCL 52331 and Septoglomus constrictus PER 7.2, no significant differences were observed in the viability of the single species cultures before and after cryopreservation. These results, thus, demonstrated the suitability of the cryopreservation method by encapsulation-drying for AMF species belonging to different genera and produced in vitro or in vivo. This method opens the door to the long-term preservation at ultra-low temperature of a large number of AMF species and for the preservation of species that are still recalcitrant to in vitro cultivation.

Maintenance and preservation of ectomycorrhizal and arbuscular mycorrhizal fungi.

Short- to long-term preservation of mycorrhizal fungi is essential for their in-depth study and, in the case of culture collections, for safeguarding their biodiversity. Many different maintenance/preservation methods have been developed in the last decades, from soil- and substrate-based maintenance to preservation methods that reduce (e.g., storage under water) or arrest (e.g., cryopreservation) growth and metabolism; all have advantages and disadvantages. In this review, the principal methods developed so far for ectomycorrhizal and arbuscular mycorrhizal fungi are reported and described given their distinct biology/ecology/evolutionary history. Factors that are the most important for their storage are presented and a protocol proposed which is applicable, although not generalizable, for the long-term preservation at ultra-low temperature of a large panel of these organisms. For ECM fungi, isolates should be grown on membranes or directly in cryovials until the late stationary growth phase. The recommended cryopreservation conditions are: a cryoprotectant of 10% glycerol, applied 1-2 h prior to cryopreservation, a slow cooling rate (1 °C min(-1)) until storage below -130 °C, and fast thawing by direct plunging in a water bath at 35-37 °C. For AMF, propagules (i.e., spores/colonized root pieces) isolated from cultures in the late or stationary phase of growth should be used and incorporated in a carrier (i.e., soil or alginate beads), preferably dried, before cryopreservation. For in vitro-cultured isolates, 0.5 M trehalose should be used as cryoprotectant, while isolates produced in vivo can be preserved in dried soil without cryoprotectant. A fast cryopreservation cooling rate should be used (direct immersion in liquid nitrogen or freezing at temperatures below -130 °C), as well as fast thawing by direct immersion in a water bath at 35 °C.

Cryopreservation of in vitro-produced Rhizophagus species has minor effects on their morphology, physiology, and genetic stability.

Cryogenic storage is considered to be the most convenient method to maintain phenotypic and genetic stability of organisms. A cryopreservation technique based on encapsulation-drying of in vitro-produced arbuscular mycorrhizal fungi has been developed at the Glomeromycota In Vitro Collection. In this study, we investigated fungal morphology (i.e., number and size of spores, number of branched absorbing structures (BAS), hyphal length, and number of anastomosis per hyphal length), activity of acid phosphatase and alkaline phosphatase in extraradical hyphae, and variation in amplified fragment length polymorphism (AFLP) profiles of in vitro-produced isolates of five Rhizophagus species maintained by cryopreservation for 6 months at -130 °C and compared to the same isolates preserved at 27 °C. Isolates were stable after 6 months cryopreservation. Comparing isolates, the number of BAS increased significantly in one isolate, and hyphal length decreased significantly in another isolate. No other morphological variable was impacted by the mode of preservation. Phosphatase activities in extraradical hyphae and AFLP profiles were not influenced by cryopreservation. These findings indicate that cryopreservation at -130 °C of encapsulated-dried and in vitro-produced Rhizophagus isolates (i.e., Rhizophagus irregularis, Rhizophagus fasciculatus, Rhizophagus diaphanous, and two undefined isolates) is a suitable alternative for their long-term preservation.

Preservation at ultra-low temperature of in vitro cultured arbuscular mycorrhizal fungi via encapsulation-drying.

At present, over 300 species of arbuscular mycorrhizal fungi (AMF) have been identified, most of which being stored in international collections. Their maintenance is mostly achieved in greenhouse via continuous culture on trap plants or in vitro in association with excised root organs. Both methods are work-intensive and for the former present the risk of unwanted contaminations. The in vitro root organ culture of AMF has become an alternative preventing contamination. Nevertheless, the risk for somaclonal variation during the sub-cultivation process cannot be excluded. A method for the long-term conservation that guarantees the stability of the biological material is thus highly demanded to preserve the microorganisms and their genetic stability. Here, 12 AMF isolates cultured in vitro in association with excised carrot roots were encapsulated in alginate beads and subsequently cryopreserved. Several protocols were tested taking into consideration culture age, alginate bead pre-drying, and rate of decrease in temperature. The viability of the AMF isolates was estimated by the percentage of potentially infective beads (%PIB) that measure the % of beads that contain at least one germinated propagule. Thermal behaviour of alginate beads was analysed by a differential thermal calorimeter before and after drying to estimate the frozen and unfrozen water during the cryopreservation process. It was shown that the spore damage was directly related to ice formation during cryopreservation. The encapsulation and culture age were also determinant parameters for the successful cryopreservation. Irrespective of the AMF isolate, the optimal procedure for cryopreservation comprised five steps: (1) the encapsulation of propagules (i.e. spores and mycorrhizal root pieces) isolated from 5m old cultures, (2) the incubation overnight in trehalose (0.5M), (3) the drying during 48h at 27°C, (4) the cryopreservation in the freezer at -130°C following a two-step decrease in temperature: a fast decrease (∼12°Cmin(-1)) from room temperature (+20°C) to -110°C followed by a slow decrease in temperature (∼1°Cmin(-1)) from -110°C to -130°C, and (5) the direct thawing in a water bath (+35°C). The % PIB was above 70 % for all the isolates and even above 95% for 11 out of the 12 isolates after several months of storage at ultra-low temperature. All the isolates kept their capacity to associate to an excised carrot root in vitro and to reproduce the fungal life cycle with the production of several hundreds to thousands of spores after 2m. This method opens the door for the long-term maintenance at ultra-low temperature of AMF isolates within international repositories.