High-throughput sequencing analysis of the rhizosphere arbuscular mycorrhizal fungi (AMF) community composition associated with Ferula sinkiangensis.

BACKGROUND: Ferula sinkiangensis is an increasingly endangered medicinal plant. Arbuscular mycorrhiza fungi (AMF) are symbiotic microorganisms that live in the soil wherein they enhance nutrient uptake, stress resistance, and pathogen defense in host plants. While such AMF have the potential to contribute to the cultivation of Ferula sinkiangensis, the composition of AMF communities associated with Ferula sinkiangensis and the relationship between these fungi and other pertinent abiotic factors still remains to be clarified. RESULTS: Herein, we collected rhizosphere and surrounding soil samples at a range of depths (0-20, 20-40, and 40-60 cm) and a range of slope positions (bottom, middle, top). These samples were then subjected to analyses of soil physicochemical properties and high-throughput sequencing (Illumina MiSeq). We determined that Glomus and Diversispora species were highly enriched in all samples. We further found that AMF diversity and richness varied significantly as a function of slope position, with this variation primarily being tied to differences in relative Glomus and Diversispora abundance. In contrast, no significant relationship was observed between soil depth and overall AMF composition, although some AMF species were found to be sensitive to soil depth. Many factors significantly affected AMF community composition, including organic matter content, total nitrogen, total potassium, ammonium nitrogen, nitrate nitrogen, available potassium, total dissolvable salt levels, pH, soil water content, and slope position. We further determined that Shannon diversity index values in these communities were positively correlated with total phosphorus, nitrate-nitrogen levels, and pH values (P < 0.05), whereas total phosphorus, total dissolvable salt levels, and pH were positively correlated with Chao1 values (P < 0.05). CONCLUSION: In summary, our data revealed that Glomus and Diversispora are key AMF genera found within Ferula sinkiangensis rhizosphere soil. These fungi are closely associated with specific environmental and soil physicochemical properties, and these soil sample properties also differed significantly as a function of slope position (P < 0.05). Together, our results provide new insights regarding the relationship between AMF species and Ferula sinkiangensis, offering a theoretical basis for further studies of their development.

Glomus ibericum, Septoglomus mediterraneum, and Funneliformis pilosus, three new species of arbuscular mycorrhizal fungi.

Three new arbuscular mycorrhizal fungal species-Glomus ibericum, Septoglomus mediterraneum, and Funneliformis pilosus-are described and illustrated. In the field, the three species were associated with roots of Ammophila arenaria (Poaceae), Elymus farctus (Poaceae), Otanthus maritimus (Asteraceae), and Echinophora spinosa (Apiaceae) colonizing maritime dunes located along the Mediterranean coast in eastern Spain. The novelty of these species is supported by morphological, molecular, and phylogenetic analyses. Single-species cultures of S. mediterraneum and F. pilosus were obtained using Trifolium repens as a host plant, both forming arbuscular mycorrhizae, whereas single-species cultures from G. ibericum could not be obtained. Spores of G. ibericum usually occur in sporocarps, rarely singly in soil or inside roots. In contrast, S. mediterraneum only forms single spores in soil and F. pilosus occurs in sporocarps and singly in soil or inside roots. The respective small subunit, internal transcribed spacer, and large subunit (SSU-ITS1-5.8S-ITS2-LSU) nrDNA sequences placed the new species in the genera Glomus, Septoglomus, and Funneliformis, all of them separated from previously described species.

Biotic and abiotic factors shape arbuscular mycorrhizal fungal communities associated with the roots of the widespread fern Botrychium lunaria (Ophioglossaceae).

Arbuscular mycorrhizal fungi (AMF) play central roles in terrestrial ecosystems by interacting with both above and belowground communities as well as by influencing edaphic properties. The AMF communities associated with the roots of the fern Botrychium lunaria (Ophioglossaceae) were sampled in four transects at 2400 m a.s.l. in the Swiss Alps and analyzed using metabarcoding. Members of five Glomeromycota genera were identified across the 71 samples. Our analyses revealed the existence of a core microbiome composed of four abundant Glomus operational taxonomic units (OTUs), as well as a low OTU turnover between samples. The AMF communities were not spatially structured, which contrasts with most studies on AMF associated with angiosperms. pH, microbial connectivity and humus cover significantly shaped AMF beta diversity but only explained a minor fraction of variation in beta diversity. AMF OTUs associations were found to be significant by both cohesion and co-occurrence analyses, suggesting a role for fungus-fungus interactions in AMF community assembly. In particular, OTU co-occurrences were more frequent between different genera than among the same genus, rising the hypothesis of functional complementarity among the AMF associated to B. lunaria. Altogether, our results provide new insights into the ecology of fern symbionts in alpine grasslands.

Effect of floristic composition and configuration on plant root mycobiota: a landscape transposition at a small scale.

Fungal communities in the root endosphere are heterogeneous at fine scale. The passenger hypothesis assumes that this heterogeneity is driven by host plant distribution. Plant composition and host plant configuration should then influence root fungal assemblages. We used a large-scale experimental design of 25 mixtures of grassland plants. We sampled Brachypodium pinnatum in each mesocosm, and used amplicon mass-sequencing to analyze the endospheric mycobiota. We used plant distribution maps to assess plant species richness and evenness (heterogeneity of composition), and patch size and the degree of isolation of B. pinnatum (heterogeneity of configuration) on fungal community assembly. The Glomeromycotina community in B. pinnatum roots was not related to either floristic heterogeneity or productivity. For Ascomycota, the composition of operational taxonomic units (OTUs) was driven by plant evenness while OTU richness decreased with plant richness. For Basidiomycota, richness increased with host plant aggregation and connectivity. Plant productivity influenced Ascomycota, inducing a shift in OTU composition and decreasing evenness. Plant heterogeneity modified root mycobiota, with potential direct (i.e. host preference) and indirect (i.e. adaptations to abiotic conditions driven by plant occurrence over time) effects. Plant communities can be envisioned as microlandscapes consisting of a variety of fungal niches.

In Vivo Modulation of Arbuscular Mycorrhizal Symbiosis and Soil Quality by Fungal P Solubilizers.

Phosphorus (P) is an essential nutrient with low bioavailability in soils for plant growth. The use of P solubilization fungi (PSF) has arisen as an eco-friendly strategy to increase this nutrient's bioavailability. The effect of PSF inoculation and its combination with P-transporting organisms (arbuscular mycorrhizal fungi, AMF) on plant growth has been previously studied. However, these studies did not evaluate the combined effect of PSF and AMF inoculation on plant growth, symbiosis, and soil quality. Therefore, the aim of this study is to assess the impact of PSF on the AMF-wheat symbiosis establishment and efficiency, considering the effect on plant growth and soil quality. We performed a greenhouse experiment with wheat under different treatments (+/-AMF: Rhizophagus irregularis; +/-PSF strains: Talaromyces flavus, T. helicus L7B, T. helicus N24, and T. diversus) and measured plant growth, AMF root colonization, symbiotic efficiency, and soil quality indicators. No interaction between PSF and R. irregularis was found in wheat growth, showcasing that their combination is not better than single inoculation. T. helicus strains did not interfere with the AMF-wheat symbiosis establishment, while T. diversus and T. flavus decreased it. The symbiotic efficiency was increased by T. flavus and T. helicus N24, and unchanged with T. helicus L7B and T. diversus inoculation. The soil quality indicators were higher with microbial co-inoculation, particularly the alkaline phosphatases parameter, showing the beneficial role of fungi in soil. This work highlights the importance of microbial interactions in the rhizosphere for crop sustainability and soil quality improvement, assessing the effects of PSF on AMF-wheat symbiosis.

Septoglomus mexicanum, a new species of arbuscular mycorrhizal fungi from semiarid regions in Mexico.

Septoglomus mexicanum is here described as a new species of arbuscular mycorrhizal fungi (AMF; Glomeromycota) based on morphological and phylogenetic analyses. It was isolated from rhizospheric soil of two endemic Mexican legumes: Prosopis laevigata and Mimosa luisana, which grow in semiarid regions of central Mexico. Septoglomus mexicanum is characterized by forming globose spores of (154.5-)202.8(-228.9) µm diam and a spore wall consisting of four layers (SWL1-SWL4): outer wall layer (SWL1) hyaline, evanescent, (1.7-)3.2(-4.3) µm thick; SWL2 laminate and smooth, orange to reddish orange, (3.1-)4.5(-6.1) µm thick; SWL3 laminate, smooth, reddish orange to reddish brown, (4.1-)5.1(-5.7) µm thick; and SWL4 hyaline, semiflexible, (0.93-)1.2(-1.4) µm thick. None of the spore wall layers stain with Melzer's reagent. The subtending hypha has a color from yellowish to golden and presents a septum on spore base. Septoglomus mexicanum can be distinguished from all other Septoglomus species by spore size and color, by spore wall structure (four layers), and by color change of the subtending hypha. Phylogenetic analysis based on the AMF extended DNA barcode covering a 1.5-kb fragment of the small subunit (SSU), internal transcribed spacer region (ITS1-5.8S-ITS2), and the large subunit (LSU) of rRNA genes places S. mexicanum in the genus Septoglomus, separated from other described Septoglomus species, especially S. turnauae, with whom it could be confused morphologically. All available sequences in public databases suggest that this new fungal species has not yet been previously detected. Thus, there are currently 149 Glomeromycota species registered in Mexico, representing 47.4% of the known species worldwide.

Rhizoglomus dalpeae, R. maiae, and R. silesianum, new species.

We examined three arbuscular mycorrhizal fungi (AMF; phylum Glomeromycota) producing glomoid spores. The mode of formation and morphology of these spores suggested that they represent undescribed species in the genus Rhizoglomus of the family Glomeraceae. Subsequent morphological studies of the spores and molecular phylogenetic analyses of sequences of the nuc rDNA small subunit (18S), internal transcribed spacer (ITS1-5.8S-ITS2 = ITS), and large subunit (28S) region (= 18S-ITS-28S) confirmed the suggestion and indicated that the fungi strongly differ from all previously described Rhizoglomus species with known DNA barcodes. Consequently, the fungi were described here as new species: R. dalpeae, R. maiae, and R. silesianum. Two of these species lived hypogeously in the field in habitats subjected to strong environmental stresses. Rhizoglomus dalpeae originated from an inselberg located within Guineo-Sudanian transition savanna zone in Benin, West Africa, where the temperature of the inselberg rock during a 5-mo drought ranges from 40 to 60 C. Rhizoglomus silesianum originated from a coal mine spoil heap in Poland, whose substrate is extremely poor in nutrients, has unfavorable texture, and may heat up to 50 C. By contrast, R. maiae was found in more favorable habitat conditions. It produced an epigeous cluster of spores among shrubs growing in a tropical humid reserve in Brazil. Moreover, the compatibility of phylogenies of species of the family Glomeraceae reconstructed from analyses of sequences of 18S-ITS-28S and the largest subunit of RNA polymerase II (RPB1) gene was discussed.

Archaeospora ecuadoriana sp. nov. from a mountainous biodiversity hotspot area in Ecuador, and transfer of Palaeospora spainiae to Archaeospora, as A. spainiae comb. nov.

A new glomeromycotan fungus, Archaeospora ecuadoriana sp. nov., was found in the south Ecuadorian mountain rainforest region, a global plant biodiversity hotspot. It was cultivated as single spore isolate originating from nursery-grown native tree seedlings inoculated with mixed soil from pristine forest and agricultural fields. The new species is known from the Loja area, southern Ecuador, at about 2100 m above mean sea level (mamsl) and has been detected in potato roots from an Andean region in Peru at 2658 mamsl by previous molecular data. The fungus forms small, colourless to frosted white, mainly globose spores, averaging 61 × 60 µm, formed singly or very rarely in clusters. There is no reaction to Melzer's reagent, other than a slight unspecific overall yellow iodine staining. The spores are very similar to those of Archaeospora trappei and A. schenckii. However, molecular phylogenetic analysis shows the species to be clearly separate from all other described Archaeospora species. The analysis of the available Archaeospora sequence data shows that sequences of Palaeospora spainiae, of the monospecific genus Palaeospora, cluster within the genus Archaeospora. Palaeospora therefore is synonymised with Archaeospora and P. spainiae is transferred to Archaeospora, as A. spainiae comb. nov.

Evolutionary history and genetic diversity study of heat-shock protein 60 of Rhizophagus irregularis.

Despite the ubiquitous occurrence of heat-shock protein 60 (Hsp60) and their role in maintenance of cell activity and integrity, this protein remains poorly characterized in many of the symbiotic soil mycorrhizal fungi such as Rhizophagus irregularis. Thus, in the current study, an attempt has been made to elucidate the evolutionary history, time of divergence followed by estimation of population genetic parameters of hsp60 using R. irregularis as a model organism. Sequence alignment reported here identified several close homologues for hsp60 (gene) and Hsp60 (protein) from diverse taxa, while the output from protein-based phylogenetic tree indicates that mitochondrial Hsp60 of R. irregularis shares close evolutionary relationship with classical α-proteobacteria. This is perhaps the first line of evidence elucidating the likelihood of hsp60 from fungal taxa sharing a close evolutionary relationship with classical α-proteobacteria as a common ancestor. Comprehensive analysis of mitochondrial hsp60 from selected fungal taxa from the evolutionary point of view explains the possibility of gene duplication and or horizontal gene transfer of this gene across various fungal species. Synteny relationships and population genetics credibly explain high genetic variability associated with fungal hsp60 presumably brought by random genetic recombination events. The results presented here also confirm a high level of genetic differentiation of hsp60 among all the three fungal populations analysed. In this context, the outcome of the current study, basedon computational approach, stands as a testimony for explaining the possibility of increased genetic differentiation experienced by hsp60 of R. irregularis.

Is a mixture of arbuscular mycorrhizal fungi better for plant growth than single-species inoculants?

Inoculation of arbuscular mycorrhizal fungi (AMF) as plant growth promoters has mostly been conducted using single-species inoculum. In this study, we investigated whether co-inoculation of different native AMF species induced an improvement of plant growth in an ultramafic soil. We analyzed the effects of six species of AMF from a New Caledonian ultramafic soil on plant growth and nutrition, using mono-inoculations and mixtures comprising different numbers of AMF species, in a greenhouse experiment. The endemic Metrosideros laurifolia was used as a host plant. Our results suggest that, when the plant faced multiple abiotic stress factors (nutrient deficiencies and high concentrations of different heavy metals), co-inoculation of AMF belonging to different families was more efficient than mono-inoculation in improving biomass, mineral nutrition, Ca/Mg ratio, and tolerance to heavy metals of plants in ultramafic soil. This performance suggested functional complementarity between distantly related AMF. Our findings will have important implications for restoration ecology and mycorrhizal biotechnology applied to ultramafic soils.

Fungal Elevational Rapoport pattern from a High Mountain in Japan.

Little is known of how fungal distribution ranges vary with elevation. We studied fungal diversity and community composition from 740 to 2940 m above sea level on Mt. Norikura, Japan, sequencing the ITS2 region. There was a clear trend, repeated across each of the fungal phyla (Basidiomycota, Ascomycota, Zygomycota, Chytridomycota and Glomeromycota), and across the whole fungal community combined, towards an increased elevational range of higher elevation OTUs, conforming to the elevational Rapoport pattern. It appears that fungi from higher elevation environments are more generalized ecologically, at least in terms of climate-related gradients. These findings add to the picture from latitudinal studies of fungal ranges, which also suggest that the classic Rapoport Rule (broader ranges at higher latitudes) applies on a geographical scale. However, there was no mid-elevation maximum in diversity in any of the phyla studied, and different diversity trends for the different phyla, when different diversity indices were used. In terms of functional guilds, on Norikura there were trends towards increased saprotrophism (Zygomycota), symbiotrophism (Basidiomycota), symbiotrophism and saprotrophism (Ascomycota) and pathotrophism (Chytridiomycota) with elevation. The causes of each of these trends require further investigation from an ecological and evolutionary viewpoint.

Agricultural intensification reduces microbial network complexity and the abundance of keystone taxa in roots.

Root-associated microbes play a key role in plant performance and productivity, making them important players in agroecosystems. So far, very few studies have assessed the impact of different farming systems on the root microbiota and it is still unclear whether agricultural intensification influences the structure and complexity of microbial communities. We investigated the impact of conventional, no-till, and organic farming on wheat root fungal communities using PacBio SMRT sequencing on samples collected from 60 farmlands in Switzerland. Organic farming harbored a much more complex fungal network with significantly higher connectivity than conventional and no-till farming systems. The abundance of keystone taxa was the highest under organic farming where agricultural intensification was the lowest. We also found a strong negative association (R2 = 0.366; P < 0.0001) between agricultural intensification and root fungal network connectivity. The occurrence of keystone taxa was best explained by soil phosphorus levels, bulk density, pH, and mycorrhizal colonization. The majority of keystone taxa are known to form arbuscular mycorrhizal associations with plants and belong to the orders Glomerales, Paraglomerales, and Diversisporales. Supporting this, the abundance of mycorrhizal fungi in roots and soils was also significantly higher under organic farming. To our knowledge, this is the first study to report mycorrhizal keystone taxa for agroecosystems, and we demonstrate that agricultural intensification reduces network complexity and the abundance of keystone taxa in the root microbiome.

The soil microbial community of turf: linear and nonlinear changes of taxa and N-cycling gene abundances over a century-long turf development.

Turf, consisting of closely spaced grasses and the subtending soil, is a unique ecosystem subject to intense management. Yet soil organic matter accumulates quickly and reaches equilibrium after 20 to 50 years. Resource availability is an important driver of species richness and theoretically their relationship is expected to be unimodal. In this work, we examined the effects of turf development (i.e. a 1, 15, 20 and 109 year-old chronosequence) on microbial taxon richness, community composition, and abundances of genes putatively involved in N cycling through 16S rRNA gene and ITS region amplicon sequencing. Microbial alpha-diversity remained relatively stable although soil organic C and N increased by up to 3-fold over a century-long turf development. However, both bacterial and fungal community compositions changed substantially from those in the previous land use, pine stands and along turf development. Youngest turf was closer to the oldest turf than to middle-aged ones, specifically for bacterial community. Microbial changes to resource availability were also taxonomically specific. The relative abundance of Proteobacteria was independent of resource availability; Nitrospirae increased monotonically, and Bacteroidetes, Actinobacteria and Glomeromycota varied curvilinearly. However, abundances of most taxa from the phylum to operational taxonomic unit level and N-cycling genes varied nonlinearly with turf development.

Biogeography of arbuscular mycorrhizal fungi (Glomeromycota): a phylogenetic perspective on species distribution patterns.

Information on the biogeography of arbuscular mycorrhizal fungi (AMF) is important because this group of obligately symbiotic soil microbes is a ubiquitous and functionally critical component of terrestrial ecosystems. In this paper, we utilize a biogeography database summarizing data on AMF species distribution linked to geographic and environmental conditions to describe global distribution patterns and interpret these patterns within a phylogenetic perspective. The data were obtained from accessions in living culture collections (INVAM, CICG), species descriptions, and other published literature from 1960 to 2012. The database contains 7105 records, 6396 of them from 768 published papers and the remaining 709 from culture accessions. Glomeromycotan species were recorded in all seven continents, 87 countries, 11 biogeographical realms, and 14 biomes. The distribution of families differed among climatic zones and continents, but they, together with all genera, appear to be cosmopolitan. Distribution of AMF species shows a slight decrease from low to high latitudes, but this decrease is steeper in the southern than in the northern hemisphere. A total of 189 species is shared between ancient supercontinents Gondwana and Laurasia and 78 species are common to all climatic zones. Ninety-five species (43% of the total) have known cosmopolitan distribution, including members of all genera except Redeckera. Some species have disjunct distribution and 26% of species have been registered from only one continent. Data on AMF distribution challenge the "Everything is everywhere" hypothesis in favor of the "moderate endemicity model" for species distribution. Data from this study provide a foundation to formulate and test hypotheses of biogeographic patterns and processes in Glomeromycota.

Plant host habitat and root exudates shape fungal diversity.

The rhizospheric microbiome is clearly affected by plant species and certain of their functional traits. These functional traits allow plants to adapt to their environmental conditions by acquiring or conserving nutrients, thus defining different ecological resource-use plant strategies. In the present study, we investigated whether plants with one of the two nutrient-use strategies (conservative versus exploitative) could influence fungal communities involved in soil organic matter degradation and root exudate assimilation, as well as those colonizing root tissues. We applied a DNA-based, stable-isotope probing (DNA-SIP) approach to four grass species distributed along a gradient of plant nutrient resource strategies, ranging from conservative to exploitative species, and analyzed their associated mycobiota composition using a fungal internal transcribed spacer (ITS) and Glomeromycotina 18S rRNA gene metabarcoding approach. Our results demonstrated that fungal taxa associated with exploitative and conservative plants could be separated into two general categories according to their location: generalists, which are broadly distributed among plants from each strategy and represent the core mycobiota of soil organic matter degraders, root exudate consumers in the root-adhering soil, and root colonizers; and specialists, which are locally abundant in one species and more specifically involved in soil organic matter degradation or root exudate assimilation on the root-adhering soil and the root tissues. Interestingly, for arbuscular mycorrhizal fungi analysis, all plant roots were mainly colonized by Glomus species, whereas an increased diversity of Glomeromycotina genera was observed for the exploitative plant species Dactylis glomerata.

The genome of Rhizophagus clarus HR1 reveals a common genetic basis for auxotrophy among arbuscular mycorrhizal fungi.

BACKGROUND: Mycorrhizal symbiosis is one of the most fundamental types of mutualistic plant-microbe interaction. Among the many classes of mycorrhizae, the arbuscular mycorrhizae have the most general symbiotic style and the longest history. However, the genomes of arbuscular mycorrhizal (AM) fungi are not well characterized due to difficulties in cultivation and genetic analysis. In this study, we sequenced the genome of the AM fungus Rhizophagus clarus HR1, compared the sequence with the genome sequence of the model species R. irregularis, and checked for missing genes that encode enzymes in metabolic pathways related to their obligate biotrophy. RESULTS: In the genome of R. clarus, we confirmed the absence of cytosolic fatty acid synthase (FAS), whereas all mitochondrial FAS components were present. A KEGG pathway map identified the absence of genes encoding enzymes for several other metabolic pathways in the two AM fungi, including thiamine biosynthesis and the conversion of vitamin B6 derivatives. We also found that a large proportion of the genes encoding glucose-producing polysaccharide hydrolases, that are present even in ectomycorrhizal fungi, also appear to be absent in AM fungi. CONCLUSIONS: In this study, we found several new genes that are absent from the genomes of AM fungi in addition to the genes previously identified as missing. Missing genes for enzymes in primary metabolic pathways imply that AM fungi may have a higher dependency on host plants than other biotrophic fungi. These missing metabolic pathways provide a genetic basis to explore the physiological characteristics and auxotrophy of AM fungi.

A new genus, Planticonsortium (Mucoromycotina), and new combination (P. tenue), for the fine root endophyte, Glomus tenue (basionym Rhizophagus tenuis).

In 1977, the fine root endophyte, originally named Rhizophagus tenuis, was transferred into the genus Glomus as G. tenue, thus positioning the species with all other known arbuscular mycorrhizal fungi (Glomeromycota, Glomeromycotina). Recent molecular evidence, however, places it in a different subphylum, Mucoromycotina in the Mucoromycota. No suitable genus exists in the Mucoromycotina to accommodate G. tenue, so it is moved to Planticonsortium gen. nov. as P. tenue comb. nov.

Glomeromycotina: what is a species and why should we care?

A workshop at the recent International Conference on Mycorrhiza was focused on species recognition in Glomeromycotina and parts of their basic biology that define species. The workshop was motivated by the paradigm-shifting evidence derived from genomic data for sex and for the lack of heterokaryosis, and by published exchanges in Science that were based on different species concepts and have led to differing views of dispersal and endemism in these fungi. Although a lively discussion ensued, there was general agreement that species recognition in the group is in need of more attention, and that many basic assumptions about the biology of these important fungi including sexual or clonal reproduction, similarity or dissimilarity of nuclei within an individual, and species boundaries need to be re-examined and scrutinized with current techniques.

A population genomics approach shows widespread geographical distribution of cryptic genomic forms of the symbiotic fungus Rhizophagus irregularis.

Arbuscular mycorrhizal fungi (AMF; phylum Gomeromycota) associate with plants forming one of the most successful microbe-plant associations. The fungi promote plant diversity and have a potentially important role in global agriculture. Plant growth depends on both inter- and intra-specific variation in AMF. It was recently reported that an unusually large number of AMF taxa have an intercontinental distribution, suggesting long-distance gene flow for many AMF species, facilitated by either long-distance natural dispersal mechanisms or human-assisted dispersal. However, the intercontinental distribution of AMF species has been questioned because the use of very low-resolution markers may be unsuitable to detect genetic differences among geographically separated AMF, as seen with some other fungi. This has been untestable because of the lack of population genomic data, with high resolution, for any AMF taxa. Here we use phylogenetics and population genomics to test for intra-specific variation in Rhizophagus irregularis, an AMF species for which genome sequence information already exists. We used ddRAD sequencing to obtain thousands of markers distributed across the genomes of 81 R. irregularis isolates and related species. Based on 6 888 variable positions, we observed significant genetic divergence into four main genetic groups within R. irregularis, highlighting that previous studies have not captured underlying genetic variation. Despite considerable genetic divergence, surprisingly, the variation could not be explained by geographical origin, thus also supporting the hypothesis for at least one AMF species of widely dispersed AMF genotypes at an intercontinental scale. Such information is crucial for understanding AMF ecology, and how these fungi can be used in an environmentally safe way in distant locations.

New method for the identification of arbuscular mycorrhizal fungi by proteomic-based biotyping of spores using MALDI-TOF-MS.

Arbuscular mycorrhizal fungi (AMF, Glomeromycota) are mutualistic symbionts associated with majority of land plants. These fungi play an important role in plant growth, but their taxonomic identification remains a challenge for academic research, culture collections and inoculum producers who need to certify their products. Identification of these fungi was traditionally performed based on their spore morphology. DNA sequence data have successfully been used to study the evolutionary relationships of AMF, develop molecular identification tools and assess their diversity in the environment. However, these methods require considerable expertise and are not well-adapted for "routine" quality control of culture collections and inoculum production. Here, we show that Matrix-Assisted Laser Desorption Ionisation Time of Flight Mass Spectrometry proteomic-based biotyping is a highly efficient approach for AMF identification. Nineteen isolates belonging to fourteen species, seven genera and five families were clearly differentiated by MALDI biotyping at the species level, and intraspecific differentiation was achieved for the majority. AMF identification by MALDI biotyping could be highly useful, not only for research but also in agricultural and environmental applications. Fast, accurate and inexpensive molecular mass determination and the possibility of automation make MALDI-TOF-MS a real alternative to conventional morphological and molecular methods for AMF identification.