Taxonomic, phylogenetic and functional diversity of root-associated fungi in bromeliads: effects of host identity, life forms and nutritional modes.

Bromeliads represent a major component of neotropical forests and encompass a considerable diversity of life forms and nutritional modes. Bromeliads explore highly stressful habitats and root-associated fungi may play a crucial role in this, but the driving factors and variations in root-associated fungi remain largely unknown. We explored root-associated fungal communities in 17 bromeliad species and their variations linked to host identity, life forms and nutritional modes by using ITS1 gene-based high-throughput sequencing and by characterizing fungal functional guilds. We found a dual association of mycorrhizal and nonmycorrhizal fungi. The different species, life forms and nutritional modes among bromeliad hosts had fungal communities that differ in their taxonomic and functional composition. Specifically, roots of epiphytic bromeliads had more endophytic fungi and dark septate endophytes and fewer mycorrhizal fungi than terrestrial bromeliads and lithophytes. Our results contribute to a fundamental knowledge base on different fungal groups in previously undescribed Bromeliaceae. The diverse root-associated fungal communities in bromeliads may enhance plant fitness in both stressful and nutrient-poor environments and may give more flexibility to the plants to adapt to changing environmental conditions.

Vermicompost addition influences symbiotic fungi communities associated with leek cultivated in metal-rich soils.

In the context of urban agriculture, where soils are frequently contaminated with metal(loid)s (TM), we studied the influence of vermicompost amendments on symbiotic fungal communities associated with plants grown in two metal-rich soils. Leek (Allium porrum L.) plants were grown with or without vermicompost in two metal-rich soils characterized by either geogenic or anthropogenic TM sources, to assess the influence of pollutant origin on soil-plant transfer. Fungal communities associated with the leek roots were identified by high throughput Illumina MiSeq and TM contents were measured using mass spectrometry. Vermicompost addition led to a dramatic change in the fungal community with a loss of diversity in the two tested soils. This effect could partially explain the changes in metal transfer at the soil-AMF-plant interface. Our results suggest being careful while using composts when growing edibles in contaminated soils. More generally, this study highlights the need for further research in the field of fungal communities to refine practical recommendations to gardeners. Graphical abstract.

Opportunities and risks of biofertilization for leek production in urban areas: Influence on both fungal diversity and human bioaccessibility of inorganic pollutants.

The influence of biofertilization with arbuscular mycorrhizal fungi (AMF) on trace metal and metalloids (TM) - Pb, Cd and Sb - uptake by leek (Allium porrum L.) grown in contaminated soils was investigated. The effect of biofertilization on human bioaccessibility of the TM in the plants was also examined. Leek were cultivated in one soil with geogenic TM sources and one soil with anthropogenic TM, to assess the influence of pollutant origin on soil-plant transfer. Leek were grown for six months on these contaminated soils, with and without a local AMF based biofertilizer. Fungal communities associated with leek roots were identified by high throughput sequencing (illumina Miseq®) metagenomic analysis. The TM compartmentation was studied using electron microscopy in plants tissues. In all the soils, biofertilization generated a loss of diversity favoring the AM fungal species Rhizophagus irregularis, which could explain the observed modification of metal transfer at the soil-AMF-plant interface. The human bioaccessibility of Sb increased in biofertilized treatments. Consequently, this latter result highlights a potential health risk of the use of this fertilization technique on contaminated soil since further field investigation is performed to better understand the mechanisms governing (1) the effect of AMF on TM bioaccessibility and (2) the evolution of AMF communities in contaminated soils.

Influence of arbuscular mycorrhizal fungi on antimony phyto-uptake and compartmentation in vegetables cultivated in urban gardens.

1. CONTEXT: Urban areas are often contaminated with various forms of persistent metal (loid) and emerging contaminants such as antimony (Sb). Thus, in the context of urban agriculture where sustainable practices such as biofertilizers application (arbuscular mycorrhizal fungi, AMF) could improve nutrient transfer from the soil to the vegetables, the effect of AMF on metal (loid) mobility and human bioaccessibility is still poorly known. 2. METHODS: The role of AMF in Sb uptake by lettuce and carrot grown in artificial substrate spiked with different Sb chemical species was investigated. Plants were grown under hydroponic conditions and half of the treatments received a concentrated spore solution to obtain mycorrhized and non-mycorrhized plants. Three weeks before harvest, plants were exposed to 10 mg.L-1 of either Sb2O3 or KSbO-tartrate (KSb). 3. RESULTS: The presence of AMF significantly increased its accumulation in carrots (all organs) with higher accumulation in roots. In lettuce, accumulation appeared to be dependent on the Sb chemical species. Moreover, it was observed for the first time that AMF changed the human bioaccessible fraction of Sb in edible organs. 4. IMPLICATIONS: The present results highlight a possible risk of Sb transfer from soil to edible plants cultivated in soil naturally containing AMF propagules, or when AMF are added as biofertilizers. After validating the influence of soil environment and AMF on Sb behavior in the field, these results should be considered in health risk assessments.

Exploring fungus-plant N transfer in a tripartite ant-plant-fungus mutualism.

Background and Aims: The plant Hirtella physophora, the ant Allomerus decemarticulatus and a fungus, Trimmatostroma sp., form a tripartite association. The ants manipulate both the plant trichomes and the fungus to build galleries under the stems of their host plant used to capture prey. In addition to its structural role, the fungus also improves nutrient uptake by the host plant. But it still remains unclear whether the fungus plays an indirect or a direct role in transferring nutrients to the plant. This study aimed to trace the transfer of N from the fungus to the plant's stem tissue. Methods: Optical microscopy and transmission electron microscopy (TEM) were used to investigate the presence of fungal hyphae in the stem tissues. Then, a 15N-labelling experiment was combined with a nanoscale secondary-ion mass spectrometry (NanoSIMS 50) isotopic imaging approach to trace the movement of added 15N from the fungus to plant tissues. Key Results: The TEM images clearly showed hyphae inside the stem tissue in the cellular compartment. Also, fungal hyphae were seen perforating the wall of the parenchyma cell. The 15N provisioning of the fungus in the galleries resulted in significant enrichment of the 15N signature of the plant's leaves 1 d after the 15N-labelling solution was deposited on the fungus-bearing trap. Finally, NanoSIMS imaging proved that nitrogen was transferred biotrophically from the fungus to the stem tissue. Conclusions: This study provides evidence that the fungi are connected endophytically to an ant-plant system and actively transfer nitrogen from 15N-labelling solution to the plant's stem tissues. Overall, this study underlines how complex the trophic structure of ant-plant interactions is due to the presence of the fungus and provides insight into the possibly important nutritional aspects and tradeoffs involved in myrmecophyte-ant mutualisms.

Assessment of Ustilago maydis as a fungal model for root infection studies.

Ustilago maydis is a fungus infecting aerial parts of maize to form smutted galls. Due to its interest as a genetic tool in plant pathology, we evaluated its ability to penetrate into plant roots. The fungus can penetrate between epidermic root cells, forming inter and intracellular pseudohyphae. Root infection didn't provoke gall formation on the maize lines tested, and targeted PCR detection showed that U. maydis, unlike the other maize smut fungus Sporisorium reilianum, has a weak aptitude to grow from the roots up to the aerial part of maize. We also observed that U. maydis can infect Medicago truncatula hairy roots as an alternative host. This plant species is a model host to study root symbiosis, and this pathosystem can provide new insights on root-microbe interactions. Considering that U. maydis could be a soil fungus, we tested its responsiveness to GR24, a strigolactone analogue. Strigolactones are root exuded molecules which activate mitochondrial metabolism of arbuscular mycorrhizal (AM) fungi. Physiologic and molecular analysis revealed that GR24 also increases cell respiration of U. maydis. This result points out that strigolactones could have an incidence on several rhizospheric microbes. These data provide evidences that the biotrophic pathogen U. maydis has to be considered for studying root infection.

Antimony bioavailability: knowledge and research perspectives for sustainable agricultures.

The increasing interest in urban agriculture highlights the crucial question of crop quality. The main objectives for environmental sustainability are a decrease in chemical inputs, a reduction in the level of pollutants, and an improvement in the soil's biological activity. Among inorganic pollutants emitted by vehicle traffic and some industrial processes in urban areas, antimony (Sb) is observed on a global scale. While this metalloid is known to be potentially toxic, it can transfer from the soil or the atmosphere to plants, and accumulate in their edible parts. Urban agriculture is developing worldwide, and could therefore increasingly expose populations to Sb. The objective of this review was in consequences to gather and interpret actual knowledge of Sb uptake and bioaccumulation by crops, to reveal investigative fields on which to focus. While there is still no legal maximal value for Sb in plants and soils, light has to be shed on its accumulation and the factors affecting it. A relative absence of data exists about the role of soil flora and fauna in the transfer, speciation and compartmentation of Sb in vegetables. Moreover, little information exists on Sb ecotoxicity for terrestrial ecosystems. A human risk assessment has finally been reviewed, with particular focus on Sb bioaccessibility.

Evolution of the plant-microbe symbiotic 'toolkit'.

Beneficial associations between plants and arbuscular mycorrhizal fungi play a major role in terrestrial environments and in the sustainability of agroecosystems. Proteins, microRNAs, and small molecules have been identified in model angiosperms as required for the establishment of arbuscular mycorrhizal associations and define a symbiotic 'toolkit' used for other interactions such as the rhizobia-legume symbiosis. Based on recent studies, we propose an evolutionary framework for this toolkit. Some components appeared recently in angiosperms, whereas others are highly conserved even in land plants unable to form arbuscular mycorrhizal associations. The exciting finding that some components pre-date the appearance of arbuscular mycorrhizal fungi suggests the existence of unknown roles for this toolkit and even the possibility of symbiotic associations in charophyte green algae.

Origin of strigolactones in the green lineage.

The aims of this study were to investigate the appearance of strigolactones in the green lineage and to determine the primitive function of these molecules. We measured the strigolactone content of several isolated liverworts, mosses, charophyte and chlorophyte green algae using a sensitive biological assay and LC-MS/MS analyses. In parallel, sequence comparison of strigolactone-related genes and phylogenetic analyses were performed using available genomic data and newly sequenced expressed sequence tags. The primitive function of strigolactones was determined by exogenous application of the synthetic strigolactone analog, GR24, and by mutant phenotyping. Liverworts, the most basal Embryophytes and Charales, one of the closest green algal relatives to Embryophytes, produce strigolactones, whereas several other species of green algae do not. We showed that GR24 stimulates rhizoid elongation of Charales, liverworts and mosses, and rescues the phenotype of the strigolactone-deficient Ppccd8 mutant of Physcomitrella patens. These findings demonstrate that the first function of strigolactones was not to promote arbuscular mycorrhizal symbiosis. Rather, they suggest that the strigolactones appeared earlier in the streptophyte lineage to control rhizoid elongation. They may have been conserved in basal Embryophytes for this role and then recruited for the stimulation of colonization by glomeromycotan fungi.

Strigolactones affect lateral root formation and root-hair elongation in Arabidopsis.

Strigolactones (SLs) have been proposed as a new group of plant hormones, inhibiting shoot branching, and as signaling molecules for plant interactions. Here, we present evidence for effects of SLs on root development. The analysis of mutants flawed in SLs synthesis or signaling suggested that the absence of SLs enhances lateral root formation. In accordance, roots grown in the presence of GR24, a synthetic bioactive SL, showed reduced number of lateral roots in WT and in max3-11 and max4-1 mutants, deficient in SL synthesis. The GR24-induced reduction in lateral roots was not apparent in the SL signaling mutant max2-1. Moreover, GR24 led to increased root-hair length in WT and in max3-11 and max4-1 mutants, but not in max2-1. SLs effect on lateral root formation and root-hair elongation may suggest a role for SLs in the regulation of root development; perhaps, as a response to growth conditions.

Role of mitochondria in the response of arbuscular mycorrhizal fungi to strigolactones.

The plant signals strigolactones activate seed germination of the parasitic weeds (Striga and Orobanche), growth of arbuscular mycorrhizal (AM) fungi and have recently been described as a new class of plant hormones that inhibit shoot branching. In AM fungi, the synthetic strigolactone analogue GR24 rapidly stimulates mitochondrial metabolism (within minutes) and biogenesis (within one hour). New gene expression, more active nuclear division and cell proliferation occur later (within days). By using pharmacological approaches to inhibit the mitochondrial ATP synthesis, various steps of the respiratory chain and the mitochondrial protein translation, we further describe the mechanisms underlying the mitochondrial response to GR24. We show with SHAM and KCN inhibition treatments that the respiratory chain of Gigaspora rosea is branched and includes an alternative oxydase. The two electron transports can be used for GR24 activation of hyphal branching but only the alternative one is used for spore germination. By using the inhibitors Oligomycin, Rotenone, Antimycine A and KCN, we show that indirect (proton pumping) and direct inhibition of ATP synthase does not completely abolish the activation of hyphal branching by GR24. However, hyphal branching was totally inhibited with the suppression of mitochondrial biogenesis, confirming the essential role played by mitochondria to amplify the strigolactone response of AM fungi.

GR24, a synthetic analog of strigolactones, stimulates the mitosis and growth of the arbuscular mycorrhizal fungus Gigaspora rosea by boosting its energy metabolism.

Arbuscular mycorrhizal (AM) fungi are obligate biotrophs that participate in a highly beneficial root symbiosis with 80% of land plants. Strigolactones are trace molecules in plant root exudates that are perceived by AM fungi at subnanomolar concentrations. Within just a few hours, they were shown to stimulate fungal mitochondria, spore germination, and branching of germinating hyphae. In this study we show that treatment of Gigaspora rosea with a strigolactone analog (GR24) causes a rapid increase in the NADH concentration, the NADH dehydrogenase activity, and the ATP content of the fungal cell. This fully and rapidly (within minutes) activated oxidative metabolism does not require new gene expression. Up-regulation of the genes involved in mitochondrial metabolism and hyphal growth, and stimulation of the fungal mitotic activity, take place several days after this initial boost to the cellular energy of the fungus. Such a rapid and powerful action of GR24 on G. rosea cells suggests that strigolactones are important plant signals involved in switching AM fungi toward full germination and a presymbiotic state.

Strigolactones stimulate arbuscular mycorrhizal fungi by activating mitochondria.

The association of arbuscular mycorrhizal (AM) fungi with plant roots is the oldest and ecologically most important symbiotic relationship between higher plants and microorganisms, yet the mechanism by which these fungi detect the presence of a plant host is poorly understood. Previous studies have shown that roots secrete a branching factor (BF) that strongly stimulates branching of hyphae during germination of the spores of AM fungi. In the BF of Lotus, a strigolactone was found to be the active molecule. Strigolactones are known as germination stimulants of the parasitic plants Striga and Orobanche. In this paper, we show that the BF of a monocotyledonous plant, Sorghum, also contains a strigolactone. Strigolactones strongly and rapidly stimulated cell proliferation of the AM fungus Gigaspora rosea at concentrations as low as 10(-13) M. This effect was not found with other sesquiterperne lactones known as germination stimulants of parasitic weeds. Within 1 h of treatment, the density of mitochondria in the fungal cells increased, and their shape and movement changed dramatically. Strigolactones stimulated spore germination of two other phylogenetically distant AM fungi, Glomus intraradices and Gl. claroideum. This was also associated with a rapid increase of mitochondrial density and respiration as shown with Gl. intraradices. We conclude that strigolactones are important rhizospheric plant signals involved in stimulating both the pre-symbiotic growth of AM fungi and the germination of parasitic plants.

Root factors induce mitochondrial-related gene expression and fungal respiration during the developmental switch from asymbiosis to presymbiosis in the arbuscular mycorrhizal fungus Gigaspora rosea.

During spore germination, arbuscular mycorrhizal (AM) fungi show limited hyphal development in the absence of a host plant (asymbiotic). In the presence of root exudates, they switch to a new developmental stage (presymbiotic) characterized by extensive hyphal branching. Presymbiotic branching of the AM fungus Gigaspora rosea was induced in liquid medium by a semipurified exudate fraction from carrot (Daucus carota) root organ cultures. Changes in RNA accumulation patterns were monitored by differential display analysis. Differentially appearing cDNA fragments were cloned and further analyzed. Five cDNA fragments could be identified that show induced RNA accumulation 1 h after the addition of root exudate. Sequence similarities of two fragments to mammalian Nco4 and mitochondrial rRNA genes suggested that root exudates could influence fungal respiratory activity. To support this hypothesis, additional putative mitochondrial related-genes were shown to be induced by root exudates. These genes were identified after subtractive hybridization and putatively encode a pyruvate carboxylase and a mitochondrial ADP/ATP translocase. The gene GrosPyc1 for the pyruvate carboxylase was studied in more detail by cloning a cDNA and by quantifying its RNA accumulation. The hypothesis that respiratory activity of AM fungi is stimulated by root exudates was confirmed by physiological and cytological analyses in G. rosea and Glomus intraradices. Oxygen consumption and reducing activity of both fungi was induced after 3 and 2 h of exposition with the root factor, respectively, and the first respiration activation was detected in G. intraradices after approximately 90 min. In addition, changes in mitochondrial morphology, orientation, and overall biomass were detected in G. rosea after 4 h. In summary, the root-exuded factor rapidly induces the expression of certain fungal genes and, in turn, fungal respiratory activity before intense branching. This defines the developmental switch from asymbiosis to presymbiosis, first by gene activation (0.5-1 h), subsequently on the physiological level (1.5-3 h), and finally as a morphological response (after 5 h).