Inoculation of Indigenous Arbuscular Mycorrhizal Fungi as a Strategy for the Recovery of Long-Term Heavy Metal-Contaminated Soils in a Mine-Spill Area.

Symbiotic associations with arbuscular mycorrhizal fungi (AMF) offer an effective indirect mechanism to reduce heavy metal (HM) stress; however, it is still not clear which AMF species are more efficient as bioremediating agents. We selected different species of AMF: Rhizoglomus custos (Custos); Rhizoglomus sp. (Aznalcollar); and Rhizophagus irregularis (Intraradices), in order to study their inoculation in wheat grown in two soils contaminated with two levels of HMs; we tested the phytoprotection potential of the different AMF symbioses, as well as the physiological responses of the plants to HM stress. Plants inoculated with indigenous Aznalcollar fungus exhibited higher levels of accumulation, mainly in the shoots of most of the HM analyzed in heavily contaminated soil. However, the plants inoculated with the non-indigenous Custos and Intraradices showed depletion of some of the HM. In the less-contaminated soil, the Custos and Intraradices fungi exhibited the greatest bioaccumulation capacity. Interestingly, soil enzymatic activity and the enzymatic antioxidant systems of the plant increased in all AMF treatments tested in the soils with both degrees of contamination. Our results highlight the different AMF strategies with similar effectiveness, whereby Aznalcollar improves phytoremediation, while both Custos and Intraradices enhance the bioprotection of wheat in HM-contaminated environments.

MtCOPT2 is a Cu+ transporter specifically expressed in Medicago truncatula mycorrhizal roots.

Arbuscular mycorrhizal fungi are critical participants in plant nutrition in natural ecosystems and in sustainable agriculture. A large proportion of the phosphorus, nitrogen, sulfur, and transition metal elements that the host plant requires are obtained from the soil by the fungal mycelium and released at the arbuscules in exchange for photosynthates. While many of the plant transporters responsible for obtaining macronutrients at the periarbuscular space have been characterized, the identities of those mediating transition metal uptake remain unknown. In this work, MtCOPT2 has been identified as the only member of the copper transporter family COPT in the model legume Medicago truncatula to be specifically expressed in mycorrhizal roots. Fusing a C-terminal GFP tag to MtCOPT2 expressed under its own promoter showed a distribution pattern that corresponds with arbuscule distribution in the roots. When expressed in tobacco leaves, MtCOPT2-GFP co-localizes with a plasma membrane marker. MtCOPT2 is intimately related to the rhizobial nodule-specific MtCOPT1, which is suggestive of a shared evolutionary lineage that links transition metal nutrition in the two main root endosymbioses in legumes.

Expression analysis of the first arbuscular mycorrhizal fungi aquaporin described reveals concerted gene expression between salt-stressed and nonstressed mycelium.

Roots of most plants in nature are colonized by arbuscular mycorrhizal (AM) fungi. Among the beneficial effects of this symbiosis to the host plant is the transport of water by the AM mycelium from inaccessible soil water resources to host roots. Here, an aquaporin (water channel) gene from an AM fungus (Glomus intraradices), which was named GintAQP1, is reported for the first time. From experiments in different colonized host roots growing under several environmental conditions, it seems that GintAQP1 gene expression is regulated in a compensatory way regarding host root aquaporin expression. At the same time, from in vitro experiments, it was shown that a signaling communication between NaCl-treated mycelium and untreated mycelium took place in order to regulate gene expression of both GintAQP1 and host root aquaporins. This communication could be involved in the transport of water from osmotically favorable growing mycelium or host roots to salt-stressed tissues.

Identification of a gene from the arbuscular mycorrhizal fungus Glomus intraradices encoding for a 14-3-3 protein that is up-regulated by drought stress during the AM symbiosis.

In the present study, a 14-3-3 protein-encoding gene from Glomus intraradices has been identified after differential hybridization of a cDNA library constructed from the fungus growing in vitro and subjected to drought stress by addition of 25% PEG 6000. Subsequently, we have studied its expression pattern under drought stress in vitro and also when forming natural symbioses with different host plants. The results obtained suggest that Gi14-3-3 gene may be involved in the protection that the arbuscular mycorrhizal (AM) symbiosis confers to the host plant against drought stress. Our findings provide new evidences that the contribution of AM fungi to the enhanced drought tolerance of the host plant can be mediated by a group of proteins (the 14-3-3) that regulate both signaling pathways and also effector proteins involved in the final plant responses.

Interactions between the arbuscular mycorrhizal (AM) fungus Glomus intraradices and nontransformed tomato roots of either wild-type or AM-defective phenotypes in monoxenic cultures.

Monoxenic symbioses between the arbuscular mycorrhizal (AM) fungus Glomus intraradices and two nontransformed tomato root organ cultures (ROCs) were established. Wild-type tomato ROC from cultivar "RioGrande 76R" was employed as a control for mycorrhizal colonization and compared with its mutant line (rmc), which exhibits a highly reduced mycorrhizal colonization (rmc) phenotype. Structural features of the two root lines were similar when grown either in soil or under in vitro conditions, indicating that neither monoxenic culturing nor the rmc mutation affected root development or behavior. Colonization by G. intraradices in monoxenic culture of the wild-type line was low (<10%) but supported extensive development of extraradical mycelium, branched absorbing structures, and spores. The reduced colonization of rmc under monoxenic conditions (0.6%) was similar to that observed previously in soil. Extraradical development of runner hyphae was low and proportional to internal colonization. Few spores were produced. These results might suggest that carbon transfer may be modified in the rmc mutant. Our results support the usefulness of monoxenically obtained mycorrhizas for investigation of AM colonization and intraradical symbiotic functioning.

Competition and substrate colonization strategies of three polyxenically grown arbuscular mycorrhizal fungi.

Intra- and extraradical colonization competition and hyphal interactions among arbuscular mycorrhizal fungi (AMF) Glomus intraradices, Glomus proliferum and Gigaspora margarita were investigated in two in vitro experimental systems. AMF were polyxenically cultured with a Ri T-DNA transformed carrot root organ culture (ROC) in either big Petri plates containing three culture compartments and a common hyphal compartment (i.e. an independent host root for each AMF) or two by two in the culture compartment of regular bicompartmented Petri dishes (i.e. a common host root and a common hyphal compartment). Maps of the extraradical mycelial development of the three AMF were obtained. Two distinct substrate colonization strategies (Glomus-type and Gigaspora-type) were identified, reflecting intrinsic differences among AMF genera/families. Our data reveal a general lack of antagonism between the isolates when extraradical hyphae explore and exploit the substrate outside the root influence zone; however certain growth restrictions were imposed by Gi. margarita extraradical mycelium when developing near the host root and by G. proliferum intraradical hyphae. This work highlights once more the appropriateness of AM in vitro culture systems to perform in vivo studies on the biology of this symbiosis and opens new avenues to the formulation of in vitro AMF inoculants.

Differential morphogenesis of the extraradical mycelium of an arbuscular mycorrhizal fungus grown monoxenically on spatially heterogeneous culture media.

A new in vitro experimental system was developed to study the morphogenesis of discrete regions of a single extraradical mycelium of the arbuscular mycorrhizal (AM) fungus Glomus intraradices, growing simultaneously in six different agar-based media. The media were (i) unamended water agar (WA), (ii) WA+PO(4)(3-) (PO(4)(3-)), (iii) WA+NO(3)(-) (NO(3)(-)), (iv) WA+NH(4)(+) (NH(4)(+)), (v) WA+NH(4)(+)+MES (NH(4)(+)+MES) and (vi) minimal medium (M, complete nutrients). Each medium was amended with the pH indicator bromocresol purple. The extraradical mycelium of the fungus showed between-treatment differences in morphogenesis, architecture, formation of branched absorbing structures (BAS) and sporulation. Extraradical hyphae that developed in WA or PO(4)(3-) compartments exhibited an economic development pattern, in which runner hyphae radially extended the external colony. Extraradical hyphal growth in the NO(3)(-) compartments was characterized by increased formation of runner hyphae, BAS and spores and an alkalinization of the medium. In the two NH(4)(+)-amended media (NH(4)(+), NH(4)(+)+MES), sporulation was suppressed and considerable morphological changes were noted. These results show the plasticity of G. intraradices that lets it efficiently exploit an heterogeneous substrate.