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Dark septate endophytes (DSEs) are often trace element (TE)-tolerant fungi and are abundant in TE-polluted environments. The production of melanin, a black polymer found in cell walls, was hypothesized by several authors to play a role in the TE tolerance of DSEs. To test this hypothesis, we established a series of experiments using albino strains and melanin inhibitors and examined the responses to Cd and Zn. Six DSEs belonging to genera Cadophora sp., Leptodontidium sp. and Phialophora mustea, were evaluated. The strains mainly produced 1,8-dihydroxynaphthalene (DHN) melanin whereas 3,4-dihydroxyphenylalanin melanin was also synthetized. Cd and Zn decreased melanin synthesis in most of the strains. A reduction in melanin concentration in hyphae through the use of tricyclazole, an inhibitor of DHN-melanin synthesis, did not reduce the tolerance of the strains to Cd and Zn. Similarly, albino mutants of Leptodontidium sp. were not more sensitive to Cd and Zn than the WT strain. Moreover, tricyclazole-treated colonies accumulated less Cd but more Zn compared to untreated colonies. The Cd and Zn contents of Leptodontidium albino strains were variable and similar to that of the WT. The results suggest that melanin production is not an important functional trait that contributes to Cd and Zn tolerance, but might contribute to Cd accumulation.

National audience

International audience; The presence of dark septate endophytes (DSEs) or arbuscular mycorrhizal fungi (AMF) in plant roots and their effects on plant fitness have been extensively described. However, little is known about their interactions when they are simultaneously colonizing a plant root, especially in trace element (TE)-polluted soils. We therefore investigated the effects of Cadophora sp. and Funneliformis mosseae on ryegrass (Lolium perenne) growth and element uptake in a Cd/Zn/Pb-polluted soil. The experiment included four treatments, i.e., inoculation with Cadophora sp., inoculation with F. mosseae, co-inoculation with Cadophora sp. and F. mosseae, and no inoculation. Ryegrass biomass and shoot Na, P, K, and Mg concentrations significantly increased following AMF inoculation as compared to non-inoculated controls. Similarly, DSE inoculation increased shoot Na concentration, whereas dual inoculation significantly decreased shoot Cd concentration. Moreover, oxidative stress determined by ryegrass leaf malondialdehyde concentration was alleviated both in the AMF and dual inoculation treatments. We used quantitative PCR and microscope observations to quantify colonization rates. They demonstrated that DSEs had no effect on AMF colonization, while AMF colonization slightly decreased DSE frequency. We also monitored fluorescein diacetate (FDA) hydrolysis and alkaline phosphatase (AP) activity in the rhizosphere soils. FDA hydrolysis remained unchanged in the three inoculated treatments, but AMF colonization increased AP activity and P mobility in the soil whereas DSE colonization did not alter AP activity. In this experiment, we unveiled the interactions between two ecologically important fungal groups likely to occur in roots which involved a decrease of oxidative stress and Cd accumulation in shoots. These results open promising perspectives on the fungal-based phytomanagement of TE-contaminated sites by the production of uncontaminated and marketable plant biomass.

Rare earth elements (REEs) are now considered emerging pollutants in the environment. Phytolacca americana, an REE hyperaccumulating plant, has been proposed for the remediation of REE-contaminated soils. However, there is no REE-related information for other Phytolacca species. Here, we examined five species (P. americana, P. acinosa, P. clavigera, P. bogotensis, and P. icosandra) for their response to REEs. REE accumulation and fractionation traits both occurred on the same order of magnitude among the five species. Heavy REEs were preferentially transferred to leaves relative to light REEs. Regardless of the species, lateral root length and chlorophyll content decreased under REE exposure, and lateral roots and foliar anthocyanins increased. However, plants did not experience or only slightly experienced oxidative stress. Finally, REE exposure strongly modulated the ionome of roots and, to a lesser extent, that of leaves, with a negative correlation between REE and Mn contents. In conclusion, our study provides new data on the response of several Phytolacca species to REEs. Moreover, we highlighted that the REE accumulation trait was conserved among Phytolacca species. Thus, we provide valuable information for the phytoremediation of REE-contaminated sites since the most appropriate Phytolacca species could be selected depending on the climatic/pedological area to be remediated.

Dark septate endophytes (DSEs) are widely distributed worldwide and can promote plant growth. Therefore, they are considered potentially important plant allies, especially in stressful environments. Previous studies have reported that DSEs cohabit roots with other microorganisms such as ectomycorrhizal (ECM), endophytic and pathogenic fungi/oomycetes. However, interactions between different DSE species have not yet been reported, and studies on the interactions between DSEs and other fungi are scarce. Using a simple and reproducible pairwise growth assay in vitro, we studied the synergistic/antagonistic interactions between eight DSEs, two ECM fungi and three root pathogens. Most of the DSE/DSE outcomes were neutral. Interestingly, we identified several DSE strains acting in synergy with other strains, as well as strains that could potentially act as biocontrol agents. Notably, three metal-tolerant DSE strains, namely, Cadophora sp., Leptodontidium sp. and Phialophora mustea, could decrease the growth of the root phytopathogens Pythium intermedium, Phytophthora citricola and Heterobasidion annosum. The present data are discussed in the general context of the use of fungal consortia as inocula in the tree-based phytomanagement of marginal lands.

In the soil-dwelling organism Pseudomonas putida KT2440, the rare earth element (REE)-utilizing and pyrroloquinoline quinone (PQQ)-dependent ethanol dehydrogenase PedH is part of a periplasmic oxidation system that is vital for growth on various alcoholic volatiles. Expression of PedH and its Ca2+-dependent counterpart PedE is inversely regulated in response to lanthanide (Ln3+) bioavailability, a mechanism termed the REE-switch. In the present study, we demonstrate that copper, zinc, and in particular, iron availability influences this regulation in a pyoverdine-independent manner by increasing the minimal Ln3+ concentration required for the REE-switch to occur by several orders of magnitude. A combined genetic- and physiological approach reveals that an ABC-type transporter system encoded by the gene cluster pedA1A2BC is essential for efficient growth with low (nanomolar) Ln3+ concentrations. In the absence of pedA1A2BC, a ~100-fold higher La3+ concentration is needed for PedH-dependent growth but not for the ability to repress growth based on PedE activity. From these results, we conclude that cytoplasmic uptake of lanthanides through PedA1A2BC is essential to facilitate REE-dependent growth under environmental conditions with poor REE bioavailability. Our data further suggest that the La3+/Fe3+ ratio impacts the REE-switch through the mismetallation of putative La3+-binding proteins, such as the sensor histidine kinase PedS2, in the presence of high iron concentrations. As such, this study provides an example for the complexity of bacteria-metal interactions and highlights the importance of medium compositions when studying physiological traits in vitro in particular in regards to REE-dependent phenomena.

International audience

Dark septate endophytes (DSEs) are abundant in stressful environments, including trace element (TE)-enriched soils. However, knowledge about the effects of DSEs on plant growth in such soils is poor compared to the well-known mycorrhizal fungi. The aim of this work was to evaluate the effects of three DSE strains isolated from TE-contaminated soils on the growth and mineral nutrition of Betula pendula and Populus tremula x alba grown on two contrasting TE-polluted soils. The three DSEs evenly colonized the two plant species in both soils. Nevertheless, plant responses to DSE inoculation varied from neutral to beneficial depending on soil properties. Depending on fungal strain and plant species, different factors seemed to contribute to plant growth promotion. Phialophora mustea Pr27 and Leptodontidium Pr30 decreased lipid peroxidation in birch shoots. Chlorophyll, K, and P concentrations increased in the shoots of Leptodontidium Pr30-inoculated trees, whereas Cd concentration decreased in Cadophora Fe06-inoculated birch. The absence of a general DSE-mediated plant growth-promoting behavior could represent a limiting factor for a generic use of DSEs in the tree-based phytomanagement of TE-contaminated soils. Our results suggest that the selection of strains adapted to particular edaphic conditions should not be overlooked within the framework of phytomanagement.

International audience; Dark septate endophytes (DSEs) are melanin-enriched ascomycetous fungi that are abundant in stressed environments. However, little is known about their physiology and metabolism, and DSE genes have not been functionally characterized yet. Therefore developing molecular genetic tools to investigate the biological function of genes of interest in DSEs is of major significance. We investigated Agrobacterium tumefaciens-mediated transformation (ATMT) efficiency in eight DSE strains belonging to Cadophora sp., Cadophora malorum, Leptodontidium sp., Phialophora mustea, and Cladosporium cladosporioides. ATMT efficiency was DSE-dependent and ranged from 0.6% to 125 %. The transformants were mitotically stable and expressed the gfp marker. The patterns of transformant appearance as a function of time were diverse and also strain-dependent. We further focused on the effect of mycelium ageing on ATMT efficiency. Leptodontidium sp. Me07, Leptodontidium sp. Pr30, and C. cladosporioides CBS 101367 were significantly more transformed using fifteen-day-old mycelium (44.5, 6.9, and 1.1%, respectively) as compared to two-day-old mycelium (121, 28.7, and 25.1, respectively), whereas P. mustea Pr29 was more transformed using young mycelium (21.5% compared to 5.3% for the old mycelium). Finally, we focused on the effect of melanin content on ATMT efficiency. Melanin content in mycelium ranged from 0.9 to 3.2 mg per gram of dry weight. Tricyclazole, an inhibitor of melanin biosynthesis, negatively modulated melanin content, while copper, an inducer of melanin biosynthesis, positively modulated them. However there was no correlation between hyphal melanin content and ATMT efficiency.

International audience

International audience

International audience

International audience; This study aimed to isolate, identify, and character-ise metal-tolerant fungi colonising poplar roots at a metal-contaminated phytoremediation site. Poplar roots were colonised by arbuscular mycorrhizal, ectomycorrhizal, and endophytic fungi, and the species were determined by ITS molecular analyses. Eight different isolates were successfully isolated into pure culture. Three isolates belonging to the Helotiales (P02, P06) and the Serendipita vermifera species (P04) were highly tolerant to metals (Cd, Zn, Pb, and Cu) compared to the mycorrhizal Hebeloma isolates. The three isolates degraded complex carbohydrates, such as xylan and cellulose, indicating that they could partially degrade root cell walls and penetrate into cells. This hypothesis was confirmed by further in vitro re-synthesis experiments, which showed that the three isolates colonised root tissues of poplar plantlets whereas two of them formed microsclerotia-like structures. Taken together, these results suggest an endophytic lifestyle of these isolates. This is the first evidence of S. vermifera as a root endophyte of poplar. A new endophytic putative species belonging to the Helotiales and closely related to Leohumicola is also reported. Interestingly, and when compared to mock-inoculated plants, both P06 and P04 isolates increased the number of root tips of inoculated poplar plantlets in vitro. Moreover, the S. vermifera P04 isolate also increased the shoot biomass. The results are discussed in relation to the potential use of endophytic strains for tree-based phytoremediation of metal-contaminated sites.

Numerous studies address the distribution and the diversity of dark septate endophytes (DSEs) in the literature, but little is known about their ecological role and their effect on host plants, especially in metal-polluted soils. Seven DSE strains belonging to Cadophora, Leptodontidium, Phialophora and Phialocephala were isolated from roots of poplar trees from metal-polluted sites. All strains developed on a wide range of carbohydrates, including cell-wall-related compounds. The strains evenly colonized birch, eucalyptus and ryegrass roots in re-synthesis experiments. Root and shoot growth promotion was observed and was both plant and strain dependent. Two Phialophora and Leptodontidium strains particularly improved plant growth. However, there was no correlation between the level of root colonization by DSEs and the intensity of growth promotion. All strains produced auxin and six also stimulated plant growth through the release of volatile organic compounds (VOCs). SPME-GC/MS analyses revealed four major VOCs emitted by Cadophora and Leptodontidium The strains exhibited growth at high concentrations of several metals. The ability of metal-resistant DSE strains to produce both soluble and volatile compounds for plant growth promotion indicates interesting microbial resources with high potential to support sustainable production of bioenergy crops within the context of the phytomanagement of metal-contaminated sites.

International audience; We developed an integrated bioremediation strategy that combines two poplar clones with rhizospheric microorganisms, exploiting the complex interactions evolved for the mutual benefit of both organisms, in which plant roots provide habitat, nutrients and exudates to microbial populations, whereas microbes facilitate mineral nutrition of plants The rationale behind combining tree species and mycorrhizal fungi resides in the fact that poplar species selected are fast growing, producing large amount of harvestable biomass and capable of forming arbuscular mycorrhizal and ectomycorrhizal symbiosis with fungi. Hosting different mycorrhizal types might be of functional importance for plant nutrition and has been shown to contribute to metal tolerance of host plants as fungi can reduce the metal uptake by the plant by sequestration, extracellular precipitation and biosorption to the cell walls. Indeed, a number of recent data support the hypothesis that mycorrhizal fungi play a filtering/sequestering role on plant roots enhancing root to shoot metal ratio and increasing survival rate in harsh conditions Implementations of large scale field trials of poplars (SRC) inoculated with symbiotic microorganisms have been set up. Three sites of about 1 ha have been selected on the basis of (i) contaminants pollution level/history, (ii) hydrological/soil conditions, (iii) ongoing soil remediation research. The first site is a former agricultural area contaminated for nearly a century by metals due to the deposit of waste waters from the city of Paris. The second site is an industrial tailing pond contaminated by mercury and arsenic. The third site is a former agricultural soil that received large amounts of metal-enriched sediments. Poplars were planted and inoculated or not with a consortium of mycorrhizal fungi. Mycorrhizal species were chosen from results of molecular analyses of the mycorrhizal diversity of poplar roots collected from several metal-polluted sites. Genomic analyses revealed a predominance of Glomus and Hebeloma species. Two year after planting, woody biomasses were estimated using dendrometric parameters. Mycorrhizal poplars produced significantly more biomass than non inoculated ones. Height, diameter, and basal area of inoculated trees varied among the three studied sites and were generally between 15 and 35% higher when compared to non inoculated trees. From these experimental sites, we have also developed a strategy for isolating fungal strains of interest. We have therefore isolated a set of ectomycorrhizal and endomycorrhizal isolates, as well as fungal endophytes from poplar roots. The isolated strains were first characterized and tested in metal tolerance assays and the most tolerant strains were used in a second set of experiments for their capacity to improve plant growth under controlled conditions. Four fungal strains were able to stimulate growth of their host plants. The two most promising strains, belonging to the group of dark-septate endophytes, were find to be tolerant to a set of metals and significantly improve plant growth. We also succeeded in isolating and producing in vitro large scale inocula of several endomycorrhizal isolates. The usefulness of symbiotic inocula for biomass production on metal-polluted sites, as well as their potential for improving the efficiency of phytotechnologies in the remediation process will be further discussed.

Microorganisms present in soils play a key role in soil fertility and in the regulation of biogeochemical cycles. New cultural practices are being studied to improve soil health while achieving satisfactory agronomic objectives.; Les microorganismes présents dans les sols jouent un rôle clé dans la fertilité des sols et participent à la régulation des cycles biogéochimiques. De nouvelles pratiques culturales sont étudiées pour améliorer la santé des sols tout en atteignant des objectifs agronomiques satisfaisants.

Lettuce cultivation under cover and/or in openfield is an economic interest in Pays de la Loire, France. However cryptogamic diseases and pest such as aphids are major constraint in market gardening production. In the current context of reducing the use of phytopharmaceutical products, soil pests are becoming a concern in market gardening. Alternative methods exist, but their partial and variable efficacity makes research and development of these methods against pests necessary for vegetable growers. In this study, plant extracts will be tested for their antifungal, biostimulant, anti-feeding, repellent and insecticidal properties.; La culture de la laitue sous abri et/ou en plein champ présente un intérêt économique en région des Pays de la Loire. Cependant, les maladies cryptogamiques ainsi que les ravageurs comme les pucerons constituent des contraintes majeures en production de cultures maraichères. Dans le contexte actuel de réduction de l’usage des produits phytopharmaceutiques, les bioagresseurs du sol deviennent préoccupants en maraîchage. Des méthodes alternatives existent, mais leurs efficacités partielles et variables rendent nécessaires la recherche et le développement sur ces méthodes contre ces bioagresseurs pour les maraichers. Dans cette étude, il sera question de tester des extraits végétaux en évaluant leur pouvoir antifongique, biostimulant, anti-appétant, répulsif et insecticide.

Dark septate endophytes are root-colonizing fungi without causing damages to host tissues to and presenting a high tolerance to heavy metals (HM). Their abundance in contaminated soils indicates that they could play an important role in tolerance of their host plants. However, the molecular mechanisms involved in this tolerance are still unknown. The general aim of this internship was to define the potential of DSE transformation and use this process to study the mechanisms of metal tolerance of DSE. Firstly, our study focused on the impact of melanin on metal tolerance. The presence of metals did not influence the content of melanin. Moreover, pigmentation of a given strain did not modify its tolerance. The potential of DSE transformation by Agrobacterium tumefaciens and optimization of the process (influence of the age of mycelium and melanin content) was studied in order to realize a mutant library of a specific strain. Strain Me07 displayed optimal transformation rate. The tested parameters did not change the frequency of mutant formation. A mutant library with Me07 strain was obtained and its screening on media containing different metals allowed to isolate a mutant of interest sensitive to zinc. The molecular analyse of this mutant will allow a better understanding of the cellular mechanisms involved in their tolerance to metal.; Les Dark septate endophytes sont des champignons colonisant les tissus racinaires de végétaux sans causer de dommages et présentant une tolérance accrue aux éléments traces métalliques (ETM). Leur abondance sur les sols pollués indique qu’ils pourraient jouer un rôle important sur la tolérance de leur plante-hôte à ces contaminants. Cependant, les mécanismes moléculaires mis en jeu dans cette tolérance ne sont pas connus. L’objectif général de ce stage a donc été de définir les potentiels d’agrotransformation des DSE et d’utiliser cette technique afin d’étudier les mécanismes cellulaires impliqués dans leur capacité de tolérances aux ETM. Dans un premier temps, l’étude a porté sur l’impact de la mélanine sur cette tolérance. La présence de métaux n’influencerait pas le taux de mélanine dans les hyphes et le niveau de pigmentation d’une souche donnée n’interviendrait pas dans sa tolérance aux ETM. L’étude du potentiel de transformation des DSE par Agrobacterium tumefaciens et l’optimisation de la transformation (âge du mycélium et mélanisation) ont été réalisées afin de créer une banque de mutants d’une souche spécifique. La souche Me07 présentait des taux de transformation optimaux. Les paramètres testés ne semblent pas modifier les fréquences d’apparition des mutants. Une banque de mutants de la souche Me07 a été réalisée et son criblage sur différents métaux a permis de mettre en évidence un mutant d’intérêt sensible au zinc. Son analyse moléculaire permettra une meilleure compréhension des mécanismes cellulaires impliqués dans leur tolérance aux métaux.