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Rhododendron ripense Makino is a Japanese endemic species of evergreen azaleas, belonging to Ericaceae family. The species has been widely utilised for breeding for 400 years due to its high ornamental value and ability to adapt to different environmental conditions, leading to the creation of several varieties for ornamental purposes. The Rhododendron genus is also known to establish root association with soil fungi. Among the root-associated fungi, the mycorrhizal ones represent a very important group. Ericoid mycorrhizae indeed can act as biostimulants for plants, favouring the nutrient uptake in exchange of carbon, and are able to alleviate certain environmental stresses, facilitating the establishment, survival and persistence of hosting plants in variable habitats. In this work the diversity of root-associated cultivable fungi was thus investigated in R. ripense in the native range of the species. Two sites have been chosen for the sampling, one above and one below the Obara dam constructed on the Hiikawa River, Shimane Prefecture (Japan). The sampling has included bulk soil and hairy roots. The culturable fungi were isolated and molecularly identified through the sequencing of the ITS and 28S DNA regions. In this work, it is reported a snapshot of the biodiversity of the culturable fungi isolated in two years (2012 and 2015) from R. ripense roots in the two natural sites.

As obligate biotrophs colonizing the roots of most land plants, arbuscular mycorrhizal fungi (AMF) are crucial members of the rhizosphere microbiota, being able to increase plant fitness through mineral nutrition.Some AMF species are at the center of a network which involves plants, fungi and bacteria, since they possess obligate endobacteria inside their cytoplasm (Bonfante and Desirò, 2017, in press). These bacteriashow reduced genomes and nutritional dependence on the fungal host (1,2), but functional analyses based on OMICs approaches suggested that their presence deeply impacts AMF host physiology (3,4). However, genomics AMF data are available only for Rhizophagus irregularis (5,6), which does not possess endobacteria. Here, we present the draft genome sequence of Gigaspora margarita BEG34, which lives associated with the endobacterium Candidatus Glomeribacter gigasporarum. By discriminating the genomic scaffolds on the basis of their GC content, we were able to exclude the bacterial and mitochondrial sequences from the dataset, and estimated the nuclear genome size of G. margarita ~785Mb, almost 6 times bigger than R. irregularis. While scanning for transposable elements and centromeric/telomeric regions, we discoveredthat ~80% of G.margarita consists of repeated regions. Also, some nuclear scaffolds have eukaryotic GC content but show homology with bacterial sequences. Further investigations will shed light on this complex genome, offering a new tool to better understand AMF lifestyle. 1-Ghignone et al., 2012, The ISME Journal, 6(1), pp. 136-145. 2-Torres-Cortés et al., Proceedings of the National Academy of Sciences, 112(25), pp.7785-7790. 3-Salvioli et al., 2016, The ISME Journal, 10(1), pp. 130-144 4-Vannini et al., 2016, New Phytologist, 211(1), pp. 265-275. 5-Tisserant et al., 2013, Proceedings of the National Academy of Sciences, 110(50), pp. 20117-20122. 6-Lin et al., 2014, PLoS Genetics, 10(1), p.e1004078.

As very ancient and widespread in nature, arbuscular mycorrhizal fungi (AMF) are key com-ponents of the plant microbiota. Recent publication of genomic and transcriptomic data shed light on the biology and evolution of AMF. The genetic complexity of AMF is increased by the presence of endobacteria which live inside many AMF1. A further component of such genetic complexity is the virome associated to AMF, whose knowledge is still very limited2, 3. Here, by exploiting recently published transcriptomic data4 we describe the virome associated to Gi-gaspora margarita. Viral RNA-dependent RNA polymerases (RdRp) are markers of my-coviruses with an RNA genome. A Blast search for RdRp sequences allowed us to identify four mitoviruses (so called because they replicate in mitochondria), one narnavirus (Ourmia-like), one Giardia-like virus, and two sequences related to Fusarium mycoviruses. Northern blot and RT-PCR assays on RNA from germinating spores confirmed the authenticity of all the se-quences with the exception of the Fusarium-related ones. All the mitoviruses are replicative and functional since both positive strand and negative strand RNA are present. Interestingly, in all the four mitoviral sequences 100% of tryptophan residues are encoded by the TGG co-don: apparently a functional RdRp can be translated both in cytosol and mitochondria. In ad-dition we report, for the first time, DNA fragments corresponding to mitovirus sequences as-sociated to the presence of viral RNA: mitoviral DNA sequences are not integrated in the ge-nome, but likely exist as extrachromosomal genetic units. Phylogenetic analyses show evidence of co-evolution of mitoviruses with their AMF hosts

As obligate biotrophs that colonize the roots of most land plants, arbuscular mycorrhizal fungi (AMF) are important members of the plant microbiota, since they are able to enhance host's nutrient acquisition. Some AMF possess endobacteria inside their cytoplasm, giving rise to a special type of endosymbiosis. Bacterial genome sequencing revealed a reduced genome and a dependence on the fungal host (Ghignone et al 2012, Torres et al..2015). Among them, Candidatus Glomeribacter gigasporarum (CaGg) has a positive influence on the physiology of its fungal host, the AMF Gigaspora margarita, rising its bioenergetic potential and enhancing its capability in detoxifying endogenous reactive oxygen species (Salvioli et al., 2016). Since a proteomic analysis revealed some changes in plant proteins which are markers of stress situations (Vannini et al, 2016), the purpose of this study was to verify whether the bacterial positive impact on the fungal physiology may be transmitted to the host plant. In order to test whether mycorrhizal plants were affected at transcriptional level depending on the endobacterial-fungal association, we performed an RNA-seq analysis of Lotus japonicus roots colonized by both the B+ (containing CaGg) and B- (without CaGg) G. margarita lines after 28 days. As expected, only a few genes (112) resulted to be differentially expressed: among them plant genes involved in hormones signaling, flavonoid biosynthesis and mineral exchange resulted to be sensitive to the presence or absence of the endobacterium inside the AMF. Interestingly, some genes involved in defense response to pathogens, such as PR10, were up-regulated in the B- colonized roots. The results suggest that endobacteria living inside the AM fungus may -directly or indirectly- influence the host plant responses.

Arbuscular mycorrhizal fungi (AMF) are crucial drivers of plant evolution: as obligate biotrophs, they have a key role in plant health. Some AMF possess endobacteria, whose genome sequencing revealed a reduced genome and a dependence on fungal host. We previously combined transcriptomics and cell biology to demonstrate that Candidatus Glomeribacter gigasporarum (CaGg) positively influences the physiology of its fungal host, Gigaspora margarita. We demonstrated how CaGg rises host's bioenergetic potential in terms of ATP production, enhancing its capability in detoxifying endogenous reactive oxygen species (Salvioli et al., 2016). Starting from these results we wondered whether such a positive impact of the bacterial presence on the fungal physiology may be transmitted to the host plant. With this aim, we performed an RNA-seq analysis of Lotus japonicus roots colonized by both the B+ (containing CaGg) and B- (without CaGg) G. margarita lines after 28 days. As expected, only a few genes (112) resulted to be differentially expressed: among them plant genes involved in hormones signaling, flavonoid biosynthesis and mineral exchange resulted to be sensitive to the presence or absence of the endobacterium inside the AMF. Interestingly, some genes involved in defense response to pathogens, such as PR10, were up-regulated in the B- colonized roots. The results suggest that the intracellular fungal microbiota may influence plant responses.

The Gram-negative bacterium Candidatus Glomeribacter gigasporarum is an obligate endobacterium living inside the cytoplasm of the arbuscular mycorrhizal fungus Gigaspora margarita. We sequenced its genome, leading to a 1.8-Mb final assembly. Phylogenetic analyses placed Ca. G. gigasporarum among Burkholderiaceae, whereas metabolic network analyses clustered it with insect endobacteria, revealing that it has undergone convergent evolution to adapt to an intracellular lifestyle. Ca. G. gigasporarum has an extreme dependence on its host for nutrients and energy, whereas the fungal host is itself an obligate biotroph that relies on a photosynthetic plant. It is thus surprising that, notwithstanding its reduced genome, the endobacterium possesses genes related to the production of secondary metabolites. It retains indeed the whole biosynthetic pathway for vitamin B12, as well as two large genes coding for nonribosomal peptide synthase and polyketide syntase. These features may contribute to the fungal host"s ecological fitness. Ca. G. gigasporarum proliferates inside a fungal phagosome/vacuole, suggesting that its communication with the fungal host should be mediated by some, still unknown, molecular determinants. A few sequences that are likely to code for putative secreted effectors were identified, and their potential role in regulating the bacterial-fungal interaction is being to be further investigated. Lastly, many toxin-antitoxin (TA) modules were found in the Ca. G. gigasporarum genome, and their expression was monitored over the fungal life cycles' stages. For one of them the toxic activity was demonstrated heterologously. These data open the question whether TA systems might play a role in the functioning of such fungal-bacterial symbiosis.

Root-associated microbiota play a major role in shaping plant physiology under diverse environmental conditions. Such an impact may have systemic outcomes, also influencing traits of agronomic relevance. In particular, arbuscular mycorrhizae (AM) improve mineral nutrition and fortify plants against biotic and abiotic stresses. We took advantage of next-generation sequencing to study interactions between Solanum lycopersicum (tomato) and its root-associated microbiota. Two agricultural soils (RO and AL) containing diverse microbiota and with different biotic and abiotic features were considered. The RO and AL soils were conducive and suppressive, respectively, to the pathogen Fusarium oxysporum f. sp. lycopersici (FOL). A steamed, peat-moss soil was used as a control. Two tomato genotypes (FOL-resistant and susceptible) were grown in microcosms containing the three soils without pathogen inoculation. After three months, roots were sampled, AM colonization was assessed, and the transcriptome was analyzed by RNAseq. Morphological observations indicated that AM structures were present in the roots from RO soil, while their presence was severely reduced in AL soil. RNAseq analysis indicates that the two soils, with their microbiota, shape the root transcriptome differently than the steamed soil. Moreover, the soil type was shown to cause a more relevant impact on gene expression than plant genotype, that is, AL soil activated metabolic pathways dealing with plant-defense irrespective of genetic background. However, comparing the profiles of the two genotypes, distinct sets of transcripts involved in plant-pathogen signaling emerged among the differentially expressed genes, suggesting that genotype is the second parameter that impacts the transcript profile interacting with the soil. Finally, a meta-transcriptome reconstruction confirmed the presence and activity of fungal communities strictly associated with the roots in both soils. Overall, our data sheds light on tomato responses to complex natural microbiota and suggest a strong interplay occurring between soil biotic and abiotic features, microbiota diversity, and genotype in tuning plant gene expression.

For more than 400 million years, arbuscular mycorrhizal fungi (AMF) have formed symbiotic associations with most land plants. The obligate plant-symbiotic AMF hosts the uncultivable Mollicutes-related endobacteria (MRE). Mollicutes-related endobacteria could be detected in all major evolutionary AMF lineages studied, but their function in arbuscular mycorrhiza (AM) is unknown. 16S rRNA gene sequences of MRE fall in a monophyletic clade in the Mollicutes and are highly polymorphic within a single AMF spore. Thus, MRE represent an unnamed, diverse, but monophyletic higher bacterial taxon. They probably evolved endosymbiotically for more than 400 million years. To uncover putative roles of these widespread MRE and to shed light on MRE evolution, we sequenced the genome of the MRE living in Dentiscutata heterogama called DhMRE. Multilocus phylogenetic analyses showed that DhMRE belong to a previously unknown lineage that is sister to a Mycoplasma clade. In addition, the draft annotation indicated an extremely reduced metabolic capacity, a strict dependency on the fungal host, and a large number of unique proteins. Strikingly, several DhMRE proteins show similarity to nuclear encoded proteins of AMF, indicating trans-kingdom gene transfer between MRE and AMF. The DhMRE genome encodes many proteins with domains predominantly described for eukaryotic proteins involved in bacteria-host interactions. Moreover, we found evidence for gene transfer of MRE with fungi of the Mucuromycotina, raising new questions about the role of MRE in the early evolution of fungus-plant associations and the AM symbiosis.

The Generic Genome Browser (GBrowse) is a simple but highly confgurable web-based genome browser. GBrowse consists in a combination of database, interactive web pages and a rich set of utilities for manipulating and displaying annotations on genomes. The Gbrowse platform has been largely used as visualization tool for most of the model organisms. This tutorial shows how to configure a GBrowse genome browser installation to display the mitochondrial genome annotation of the Arbuscular Mycorrhizal Fungus Gigaspora margarita BEG34.

Diversity of arbuscular mycorrhizal fungi (AMF) in soil is considered as one of the major factors contributing to the maintenance of plant biodiversity and to grassland ecosystem functioning. It is therefore of great interest to understand how AMF communities respond to disturbances expected with climate change, such as drought. Thus far, however, the impact of drought on AMF diversity in soil has been largely overlooked. By means of a tag-assisted MiSeq (Illumina) sequencing approach, we addressed changes in the composition of soil AMF communities in the field-based mesocosm experiment by de Vries et al. (this meeting), after imposing an experimental drought. Approx. 800,000 AMF 18S amplicons (ca. 420 bp-long) obtained from soil samples collected under different plant assemblages at four time-points (before, at the end, and two and eight weeks after ending the drought treatment) were analyzed. The AMF diversity covered by the primers used was (almost) saturated in most samples (per sample Good's coverage estimator ranging 0.873-1.000). 97% sequence identity OTUs were identified as Virtual Taxa (VTX) using a customized reference repository, derived from the MaarjAM database (http://maarjam.botany.ut.ee/). Most (80%) of the 60 VTX retrieved following subsampling at even sampling depth were unaffected by the treatment. However, sequence abundances of 12 VTX significantly increased or decreased at either the end and eight weeks after drought (METASTATS, 1000 permutations). At both time points, VTX exhibiting a significant increase in the treated samples belonged to the Glomeraceae or Claroideoglomaceae (Glomerales), whereas VTX exhibiting a decrease belonged to the Acaulosporaceae or Gigasporaceae (Diversisporales). This finding suggests a certain degree of phylogenetic conservatism in the response of AMF to drought disturbance.

The current wealth of studies on the plant microbiota reflects the widespread interest in this topic, since root-microbe interactions are not only crucial for better understanding plant growth and health, but also for sustainable crop production in a changing world. To date, such studies focus almost exclusively on the bacterial dimension of the root-associated microbiota. However, given the unique biological and ecological features of fungi, recruitment of the fungal microbiota may deviate from the relationships documented for bacteria. For instance, due to the mycelial growth form, the distinction among the different rhizosphere compartments (root surface/tissues vs rhizosphere soil) may be less clear-cut for fungi than for bacteria. Within the framework of a project combining meta-barcoding and systems biology to infer the principles underlying the interactions between tomato and its root mycobiota (Mycoplant), we addressed the relative importance of different factors (soil type, cultivar-specificity, abundant pathogen presence) shaping this fungal microbiota. Approx. 900 000 ITS2 and EF1-alpha amplicons obtained from the roots of four tomato cultivars grown in two different soils under greenhouse conditions were analyzed. Tomato genotypes either susceptible (Moneymaker, Cuore di Bue) or resistant (Heinz; Battito) to Fusarium oxysporum f. sp. lycopersici were used; the two resistant cultivars had been grown either in the presence or in the absence of the pathogen. Rhizosphere and bulk soil samples were also analyzed. Both soil type and plant genotype were significant drivers of the structure of root mycobiota, the magnitude of the impact of the two factors being comparable in some cases. Inoculation with the pathogen also significantly affected the communities associated with the resistant cultivars.

Relazione scentifica finale del progetto "V2P2 Repository" relativo al contributo MIUR L.6/2000 per la diffusione della cultura scientifica.

CNR IVV and CNR IPP Institutes have been dealing with NGS DATA (Next-gen sequencing) for years, but, until 2012, no CNR-based high-performance server was available. Powerful multi-core servers, clusters and virtual cloud-based machines represent are essential to address the computational demands of NGS. CNR CERIS Institute built up a server for such bio-applications and the joint effort amongst, IVV, IPP and CERIS put the basis for a new bioinformatics facility in the CNR Turin Research Area.

Il recente sviluppo di strumenti molecolari e tecniche di sequenziamento di nuova generazione (NGS) hanno rivoluzionato gli studi di ecologia microbica, fornendo nuove prospettive per investigare sistemi complessi, come quelli creati dalle radici delle piante. In particolare, le radici di riso (Oryza sativa), rappresentano una nicchia per diversi microrganismi del suolo che sono fortemente influenzati dai sistemi di gestione agronomica, come condizioni di aerobiosi rispetto a condizioni di anaerobiosi. L' obiettivo del nostro progetto è lo studio del microbioma associato al suolo e alla radice di Oryza sativa coltivato in queste due diverse condizioni. Le radici di riso e il circostante terreno rizosferico sono stati oggetto di campionamento a tre fasi di sviluppo delle piante (accestimento, formazione della pannocchia e stadio di maturazione lattea) e in due diverse condizioni di gestione agronomica (sommersione e asciutta). La complessità delle comunità microbiche associate all'ambiente risaia è stata stimata mediante analisi dei profili ARISA (Automated Ribosomal Intergenic Spacer Analysis) nelle diverse condizioni sperimentali rappresentate dalle radici e dalla rizosfera in condizioni di crescita aerobiche e anaerobiche. È stato inoltre applicato un approccio di pirosequenziamento 454 basato su metabarcoding di regioni iper-variabile dei geni rRNA delle comunità archeali e batteriche (16S), fungine micorrizico arbuscolari (18S) e Dikarya (ITS). Sono state create otto librerie di ampliconi per un totale di 559K reads (220.7Mb). I dati di sequenziamento e i metadata dei campioni oggetto di analisi sono stati elaborati utilizzando la pipeline del software QIIME (Quantitative Insigths Into Microbial Ecology, Caporaso et al. 2010). Le sequenze che hanno passato gli standard di qualità imposti sono state raggruppate in OTUs (Unità Tassonomiche Operative) al 3% di dissimilarità. L' identificazione tassonomica delle sequenze rappresentative delle OTUs per Batteri e Archaea (16S) è stata ottenuta tramite il Ribosomal Database Project (RDP, Cole et al. 2009). Per la componente fungina si è resa necessaria la creazione di due archivi di riferimento personalizzati e appositamente sviluppati per superare i limiti e gli errori presenti nelle banche dati normalmente utilizzati dalla comunità scientifica: il primo si riferisce alle sequenze ITS (FunITSinnova) derivato da UNITE (ITS; Abarenkov et al.,2010b; Tedersoo et al., 2011) e integrato ad hoc, il secondo considera le sequenze 18S relative agli AMF, ed è derivato dal web-based database MaarjAM (Opik et al. 2010). Il lavoro offre molti aspetti di novità in quanto considera le dinamiche microbiche sia della componente procariotica che di quella fungina. L'analisi dei dati di sequenziamento ottenuti dall'ambiente risaia rivelano differenze statisticamente significative del microbioma del riso coltivato in aerobiosi rispetto a condizioni di anaerobiosi. Analisi di NGS permettono pertanto di determinare quali siano le principali forze che modellano la struttura, i rapporti e la composizione delle diverse comunità microbiche associate al riso. Referenze Abarenkov K, Nilsson RH, Larsson K-H, Alexander IJ, Eberhardt U, Erland S,, Hoiland K, Kjøller R, Larsson E, Pennanen T et al. 2010b. New Phytologist 186: 281-285. Caporaso, J.G.; Kuczynski, J.; Stombaugh, J.; Bittinger, K.; Bushman, F.D.; Costello, E.K.; Fierer, N.; Gonzalez Pena, A.; Goodrich, J.K.; Gordon, J.I.; et al. (2010). Nature Methods 7, 335-336. Cole JR, Wang Q, Cardenas E, Fish J, Chai B, Farris RJ, Kulam-Syed-Mohideen AS, McGarrell DM, Marsh T, Garrity GM, Tiedje JM. Nucleic Acids Res.37:D141-5. Öpik, M., Vanatoa, A., Vanatoa, E., Moora, M., Davison, J., Kalwij, J.M., Reier, Ü., Zobel, M. (2010). New Phytologist 188: 223-241. Tedersoo L, Abarenkov K, Nilsson RH, Schussler A, Grelet GA, Kohout P, Oja J, Bonito GM, Veldre V, Jairus T et al. 2011. PLoS ONE 6: e24940.

A new Macrolepiota taxon from Italy, M. rhodosperma var. velicopia, is described and illustrated based on morphological and ITS rDNA data. It is characterized by a well-developed volva and abundant, evident velar remnants on the pileus, a stipe with a minutely squamulose covering, and very thick-walled elements in the pileipellis. A discussion on its taxonomic position within Macrolepiota and notes on closely related taxa are provided. DNA sequence analyses support the new taxon within the variability of M. fuliginosa sensu Vellinga, a non-volvate taxon that differs from Barla’s original sense of M. fuliginosa. As Barla did not indicate a holotype in his protologue of Lepiota procera var. fuliginosa and there are no extant original herbarium specimens, Barla’s Fig. 5/PL. 9 (from Les champignons des Alpes maritimes) is selected as a lectotype, and a recent collection from Liguria (Italy) is chosen as the epitype. A recent collection from Sardinia is chosen as epitype for L. permixta. Finally, M. fuliginosa and M. permixta are reduced in rank to forms of M. procera, based on their morphology.

Many AMF host endobacteria in their cytoplasm. To elucidate the role of the Gram negative endobacterium Candidatus Glomeribacter gigasporarum (CaGg), we sequenced its genome using a metagenomic approach which combined Sanger sequencing of fosmid clones from a Gigaspora margarita (the host) library with a 454 pyrosequencing of an enriched- endobacterial fraction. The final assembly led to 35 contigs, totalling 1.72 Mb, consisting of a chromosome and three plasmids. The genome features of CaCg place it in the Burkolderiaceae group, while metabolic networks analysis clustered CaCg with insect endobacteria, mirroring its obligate intracellular life-style. CaCg resembles an aerobic microbe, with no fermentative and limited energy-production capabilities via glycolysis and phosphate-pentose pathways. It depends upon its fungal host for C, P and N supply; its ability to synthesize amino acids is limited suggesting that most amino acids are imported from its host. In summary, the bacterial genome data of the first endosymbiont's endosymbiont revealed a novel context of intimate symbiosis between bacteria and fungi. Since the CaGg fungal host is itself an obligate biotroph dependent on its photosynthetic host, our work uncovers a network of nutritional/evolutionary interactions between plants, AM fungi and endobacteria.

Metallothioneins are low molecular weight polypeptides, present in most eukaryotic phyla, with role in metal homeostasis and detoxification. We previously reported the identification and the characterization of a metallothionein gene (GmarMT1) from the arbuscular mycorrhizal fungus Gigaspora margarita. Here, we have used real-time quantitative RT-PCR to show that GmarMT1 expression was turned off during the symbiotic fungal growth in the hexose-rich mycorrhizal apoplast, whereas transcripts were abundant during all other fungal growth stages, when products of lipid breakdown and the glyoxylate cycle feed carbohydrate-consuming pathways. In support of a nutritional regulation of GmarMT1 expression, we show that transcriptional activity of GmarMT1 promoter in yeast was strongly induced by glucose starvation (up to 20 times the basal level). We speculate that GmarMT1-encoded protein, with its proved metal binding ability, could regulate the homeostasis of zinc, a fundamental cofactor involved in C metabolism regulation and glucose repression.