Arbuscular mycorrhizal fungi as tool in pharmaceutical and cosmeceutical industry for the enhanced production of secondary metabolites of Anchusa officinalis

Arbuscular mycorrhizal fungi (AMF) are biotrophe microorganisms that establish a symbiotic association with host-plants. These plant-dependent microorganisms have already shown their strong effects concerning plant growth promotion and were reported to stimulate the secondary metabolites (SM) production of their host-plant. In this study, Anchusa officinalis plants, well-known for their therapeutic properties, were analyzed to assess the AMF effects on SM production. Two different experiments were developed on a semi-hydroponic system to compare treated A. officinalis plants with different AMF. In the first investigation, plants were inoculated with Rhizophagus irregularis, cultivated for 30 days and harvested two times. Concerning the second experiment, four different strains of AMF (R. clarus, R. intraradices, R. irregularis and Glomus aggretum) were tested separately on plants and one sampling was performed after one week. Roots and shoots were separated, lyophilized, ground and extracted using ultrasounds with EtOAc and MeOH (35:65 v/v) at 25°C. After centrifugation, the supernatants were removed and evaporated to dryness. Samples were analyzed with UHPLC-HRMS as well as HPTLC and HPLC-DAD-ELSD. Preliminary results of a targeted metabolomic analysis, showed that the concentration of main compound, rosmarinic acid, present in all the treated plants had no statistically difference from the controls. However, a discernable up-regulation and down-regulation of specific minor SMs in colonized plants was observed, suggesting that the aforementioned AMF affect specific biosynthetic pathways. Further experiments and analyses is needed but the cultivation of medicinal plants with AMF looks a promising way to enhance bioactive metabolites with applications in pharmaceutical and cosmeceutical industry

Direct transfer of zinc between plants is channelled by common mycorrhizal network of arbuscular mycorrhizal fungi and evidenced by changes in expression of zinc transporter genes in fungus and plant

The role that common mycorrhizal networks (CMNs) play in plant‐to‐plant transfer of zinc (Zn) has not yet been investigated, despite the proved functions of arbuscular mycorrhizal fungi (AMF) in crop Zn acquisition. Here, two autotrophic Medicago truncatula plants were linked by a CMN formed by Rhizophagus irregularis. Plants were grown in vitro in physically separated compartments (Donor‐C and Receiver‐C) and their connection ensured only by CMN. A symbiosis‐defective mutant of M. truncatula was used as control in Receiver‐C. Plants in both compartments were grown on Zn‐free medium, and only the leaves of the donor plants were Zn fertilized. A direct transfer of Zn was demonstrated from donor leaves to receiver shoots mediated by CMN. Direct transfer of Zn was supported by changes in the expression of fungal genes, RiZRT1 and RiZnT1, and plant gene MtZIP2 in roots and MtNAS1 in roots and shoots of the receiver plants. Moreover, Zn transfer was supported by the change in expression of MtZIP14 gene in AM fungal colonized roots. This work is the first evidence of a direct Zn transfer from a donor to a receiver plant via CMN, and of a triggering of transcriptional regulation of fungal‐plant genes involved in Zn transport‐related processes

Cutting-edge analytical technologies for the comprehensive metabolic profiling of Alkanna tinctoria roots cultured in greenhouse conditions

The use of plants containing naphthoquinone derivatives Alkannins & Shikonins (A/S) by humans dates back to ancient times. In recent decades, the use of A/S has seen a resurgence and A/S have risen to a pivotal role as pharmaceutical and cosmeceutical agents, since they possess strong wound healing, antimicrobial, anti-inflammatory, tissue regenerative and antitumor properties. It is thus crucial to enhance the biosynthesis of bioactive A/S in Alkanna tinctoria plants, that naturally produce high amounts of these metabolites [1]. In the frame of “MICROMETABOLITE” EU H2020 project, we have optimized a workflow for the metabolic profiling of A. tinctoria roots, cultured in the greenhouse from plants obtained by in vivo shoot cuttings. A fast and reliable extraction procedure was achieved for comprehensive profiling and identification of A/S and other metabolites biosynthesized in the roots. The aim of this work was to determine the growth stage with peak A/S production, while simultaneously obtaining additional information on the root metabolome. A combination of UHPLC-HRMS and NMR was used for metabolite identification, HPLC was utilized for reliable quantitation of A/S and the extracts were subjected to chiral HPLC analysis [2] for determination of the enantiomeric A/S ratio. Different A/S derivatives and other metabolites were identified in plant roots using UHPLC-HRMS and NMR. Six A/S derivatives and total A/S were quantified using HPLC-DAD. From six vegetation stages of A. tinctoria grown under greenhouse conditions, fruiting period was found to peak A/S production (1% wt/wt of root), while the enantiomeric alkannin/shikonin ratio remained constant (93.7%)

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. © 2013 Springer-Verlag Berlin Heidelberg

In vitro colonization of date palm plants by Rhizophagus irregularis during the rooting stage

The use of in vitro culture of date palm plants Phoenix dactylifera, associated with arbuscular mycorrhizal (AM) fungi is a novel approach for the production of bio-fortified plants that are free of pathogens. Here, we report, for the first time, the in vitro mycorrhization of in vitro date palm plants using the AM fungus Rhizophagus irregularis MUCL 41833. Date Plants were used in an in vitro cultured system that consisted of a root compartment (RC) containing germinated seeds of Barrel Clover, Medicago truncatula, and spores of Rhizophagus irregularis as a mycorrhizal donor, and a hyphal compartment (HC) with a barrier separating the RC from the HC. In vitro cultured date palm plants, at the two-leaf stage, were placed in the HC section of the culture plate that after 6 weeks contained an active growing extraradical mycelium network of the fungus. Roots of the date palm became colonized after 10 weeks and hyphae, vesicles, spores and arbuscules, were detected. No differences were noticed in above-ground parameters between mycorrhized and non-mycorrhized plants, in which there was no fungus in the HC. However, the total root length was significantly higher and secondary and tertiary roots were significantly more numerous, in the mycorrhized plants. It is hypothesized that these differences are related to stimulating molecules released by the profuse extraradical mycelium of the fungus growing in close contact with the palm root system. Root colonization percentages were of the same order as those reported in pots cultures of the date palm plants. This work opens the door for the large-scale in vitro mycorrhization of date palm plants, potentially better adapted to acclimatization phase and possibly to the field

Arctic arbuscular mycorrhizal spore community and viability after storage in cold conditions

Arbuscular mycorrhizal fungi (AMF) form proba- bly the most widespread symbiosis on earth and are found across all ecosystems including the Arctic regions. In the Arctic, the prevalent harsh cold conditions experienced by both host plants and fungi may have selected for AMF species with long-surviving spores, the principal means for dispersal and survival. However, basic knowledge about their viability is lacking. AMF spore assembly from two Arctic sites was examined in soil samples collected across an 11-year period and stored at −20 °C for up to 10 years. AMF spore viability and ability to colonize plants were investigated in the green- house using Plantago lanceolata. It was predicted that Arctic AMF spores would survive in cold conditions for several years, with an expected decrease in viability over time as suggested by other experiments with temperate material. Results show that even though the two study sites differed in AMF spore density, the relative abundance of spore morphotypes was rather similar across sites and years. Furthermore, spore viability over time was site-dependent as it decreased only in one site. Although spores were viable, only a very small proportion of hosts and roots became colonized in the greenhouse even 21 months after inoculation. Taken together, these results suggest a certain site-dependent variability in AMF spore communities and the ability of Arctic AMF spores to remain viable after a long-term storage in cold conditions. The lack of host colonization in the green- house may be related to the inability to overcome spore dormancy under these conditions