Stefano Ghignone

Périgord black truffle genome uncovers evolutionary origins and mechanisms of symbiosis

The Périgord black truffle (Tuber melanosporum Vittad.) and the Piedmont white truffle dominate today’s truffle market. The hypogeous fruiting body of T. melanosporum is a gastronomic delicacy produced by an ectomycorrhizal symbiont endemic to calcareous soils in southern Europe. The worldwide demand for this truffle has fuelled intense efforts at cultivation. Identification of processes that condition and trigger fruit body and symbiosis formation, ultimately leading to efficient crop production, will be facilitated by a thorough analysis of truffle genomic traits. In the ectomycorrhizal Laccaria bicolor, the expansion of gene families may have acted as a ‘symbiosis toolbox’. This feature may however reflect evolution of this particular taxon and not a general trait shared by all ectomycorrhizal species. To get a better understanding of the biology and evolution of the ectomycorrhizal symbiosis, we report here the sequence of the haploid genome of T. melanosporum, which at ∼125 megabases is the largest and most complex fungal genome sequenced so far. This expansion results from a proliferation of transposable elements accounting for ∼58% of the genome. In contrast, this genome only contains ∼7,500 protein-coding genes with very rare multigene families. It lacks large sets of carbohydrate cleaving enzymes, but a few of them involved in degradation of plant cell walls are induced in symbiotic tissues. The latter feature and the upregulation of genes encoding for lipases and multicopper oxidases suggest that T. melanosporum degrades its host cell walls during colonization. Symbiosis induces an increased expression of carbohydrate and amino acid transporters in both L. bicolor and T. melanosporum, but the comparison of genomic traits in the two ectomycorrhizal fungi showed that genetic predispositions for symbiosis—‘the symbiosis toolbox’—evolved along different ways in ascomycetes and basidiomycetes.

Inefficient photosynthesis in the Mediterranean orchid Limodorum abortivum is mirrored by specific association to ectomycorrhizal Russulaceae

Among European Neottieae, Limodorum abortivum is a common Mediterranean orchid. It forms small populations with a patchy distribution in woodlands, and is characterized by much reduced leaves, suggesting a partial mycoheterotrophy. We have investigated both the photosynthetic abilities of L. abortivum adult plants and the diversity of mycorrhizal fungi in Limodorum plants growing in different environments and plant communities (coniferous and broadleaf forests) over a wide geographical and altitudinal range. Despite the presence of photosynthetic pigments, CO2 fixation was found to be insufficient to compensate for respiration in adult plants. Fungal diversity was assessed by morphological and molecular methods in L. abortivum as well as in the related rare species Limodorum trabutianum and Limodorum brulloi. Phylogenetic analyses of the fungal internal transcribed spacer (ITS) sequences, obtained from root samples of about 80 plants, revealed a tendency to associate predominantly with fungal symbionts of the genus Russula. Based on sequence similarities with known species, most root endophytes could be ascribed to the species complex encompassing Russula delica, Russula chloroides, and Russula brevipes. Few sequences clustered in separate groups nested within Russula, a genus of ectomycorrhizal fungi. The morphotypes of ectomycorrhizal root tips of surrounding trees yielded sequences similar or identical to those obtained from L. abortivum. These results demonstrate that Limodorum species with inefficient photosynthesis specifically associate with ectomycorrhizal fungi, and appear to have adopted a nutrition strategy similar to that known from achlorophyllous orchids.

The genome of the obligate endobacterium of an AM fungus reveals an interphylum network of nutritional interactions

As obligate symbionts of most land plants, arbuscular mycorrhizal fungi (AMF) have a crucial role in ecosystems, but to date, in the absence of genomic data, their adaptive biology remains elusive. In addition, endobacteria are found in their cytoplasm, the role of which is unknown. In order to investigate the function of the Gram-negative Candidatus Glomeribacter gigasporarum, an endobacterium of the AMF Gigaspora margarita, we sequenced its genome, leading to an ∼1.72-Mb assembly. Phylogenetic analyses placed Ca. G. gigasporarum in the Burkholderiaceae whereas metabolic network analyses clustered it with insect endobacteria. This positioning of Ca. G. gigasporarum among different bacterial classes reveals that it has undergone convergent evolution to adapt itself to intracellular lifestyle. The genome annotation of this mycorrhizal-fungal endobacterium has revealed an unexpected genetic mosaic where typical determinants of symbiotic, pathogenic and free-living bacteria are integrated in a reduced genome. Ca. G. gigasporarum is an aerobic microbe that depends on its host for carbon, phosphorus and nitrogen supply; it also expresses type II and type III secretion systems and synthesizes vitamin B12, antibiotics- and toxin-resistance molecules, which may contribute to the fungal hosts ecological fitness. 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. Our work represents the first step towards unraveling a complex network of interphylum interactions, which is expected to have a previously unrecognized ecological impact.

Symbiosis with an endobacterium increases the fitness of a mycorrhizal fungus, raising its bioenergetic potential

Arbuscular mycorrhizal fungi (AMF) occur in the rhizosphere and in plant tissues as obligate symbionts, having key roles in plant evolution and nutrition. AMF possess endobacteria, and genome sequencing of the endobacterium Candidatus Glomeribacter gigasporarum revealed a reduced genome and a dependence on the fungal host. To understand the effect of bacteria on fungal fitness, we used next-generation sequencing to analyse the transcriptional profile of Gigaspora margarita in the presence and in the absence of its endobacterium. Genomic data on AMF are limited; therefore, we first generated a gene catalogue for G. margarita. Transcriptome analysis revealed that the endobacterium has a stronger effect on the pre-symbiotic phase of the fungus. Coupling transcriptomics with cell biology and physiological approaches, we demonstrate that the bacterium increases the fungal sporulation success, raises the fungal bioenergetic capacity, increasing ATP production, and eliciting mechanisms to detoxify reactive oxygen species. By using TAT peptide to translocate the bioluminescent calcium reporter aequorin, we demonstrated that the line with endobacteria had a lower basal intracellular calcium concentration than the cured line. Lastly, the bacteria seem to enhance the fungal responsiveness to strigolactones, the plant molecules that AMF perceive as branching factors. Although the endobacterium exacts a nutritional cost on the AMF, endobacterial symbiosis improves the fungal ecological fitness by priming mitochondrial metabolic pathways and giving the AMF more tools to face environmental stresses. Thus, we hypothesise that, as described for the human microbiota, endobacteria may increase AMF innate immunity.

Navigating the labyrinth: a guide to sequence‐based, community ecology of arbuscular mycorrhizal fungi

Data generated from next generation sequencing (NGS) will soon comprise the majority of information about arbuscular mycorrhizal fungal (AMF) communities. Although these approaches give deeper insight, analysing NGS data involves decisions that can significantly affect results and conclusions. This is particularly true for AMF community studies, because much remains to be known about their basic biology and genetics. During a workshop in 2013, representatives from seven research groups using NGS for AMF community ecology gathered to discuss common challenges and directions for future research. Our goal was to improve the quality and accessibility of NGS data for the AMF research community. Discussions spanned sampling design, sample preservation, sequencing, bioinformatics and data archiving. With concrete examples we demonstrated how different approaches can significantly alter analysis outcomes. Failure to consider the consequences of these decisions may compound bias introduced at each step along the workflow. The products of these discussions have been summarized in this paper in order to serve as a guide for any researcher undertaking NGS sequencing of AMF communities.

Identification of Fusarium oxysporum f. sp. basilici Isolated from Soil, Basil Seed, and Plants by RAPD Analysis

Fifty-two isolates of Fusarium oxysporum, obtained from infected basil plants, seed, flower residues, and soil from different growing areas in Italy and Israel, were analyzed by random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR), coupled to a DNA extraction protocol from colonies grown on Fusarium-selective medium. In a pathogenicity assay, 35 isolates caused 32 to 92% disease on seedlings of the highly susceptible basil cultivar Fine verde, while 17 isolates were nonpathogenic on basil. Thirty of the F. oxysporum f. sp. basilici isolates obtained from soil or wilted plants gave identical amplification patterns using 31 different random primers. All tested primers allowed clear differentiation of F. oxysporum f. sp. basilici from representatives of other formae speciales and from nonpathogenic strains of F. oxysporum. RAPD profiles obtained from DNA of isolates extracted directly from cultures grown on Fusarium selective medium were identical to those obtained from DNA extracted from lyophilized mycelia.

Mosaic genome of endobacteria in arbuscular mycorrhizal fungi: Transkingdom gene transfer in an ancient mycoplasma-fungus association.

Significance Obligate plant-symbiotic, arbuscular mycorrhizal fungi (AMF) are major drivers of terrestrial ecosystems and host enigmatic Mollicutes-related endobacteria (MRE) in their cytoplasm. The genome analysis of a MRE living in the AMF Dentiscutata heterogama revealed it to represent a previously unidentified bacterial lineage of Mycoplasma-related species. DhMRE shows strongly reduced metabolic capacity and underwent trans-kingdom gene transfer: its genome codes for an arsenal of eukaryotic-like putative effector proteins, with nuclear encoded homologues in AMF and Mortierella. The MRE-fungus (-plant) association probably evolved in ancestors of Glomeromycota and Mucoromycotina. This calls for a targeted search for ancient effector proteins that play crucial roles in the MRE interaction with fungal hosts, and putatively also with plants. For more than 450 million years, arbuscular mycorrhizal fungi (AMF) have formed intimate, mutualistic symbioses with the vast majority of land plants and are major drivers in almost all terrestrial ecosystems. The obligate plant-symbiotic AMF host additional symbionts, so-called Mollicutes-related endobacteria (MRE). To uncover putative functional roles of these widespread but yet enigmatic MRE, we sequenced the genome of DhMRE living in the AMF Dentiscutata heterogama. Multilocus phylogenetic analyses showed that MRE form a previously unidentified lineage sister to the hominis group of Mycoplasma species. DhMRE possesses a strongly reduced metabolic capacity with 55% of the proteins having unknown function, which reflects unique adaptations to an intracellular lifestyle. We found evidence for transkingdom gene transfer between MRE and their AMF host. At least 27 annotated DhMRE proteins show similarities to nuclear-encoded proteins of the AMF Rhizophagus irregularis, which itself lacks MRE. Nuclear-encoded homologs could moreover be identified for another AMF, Gigaspora margarita, and surprisingly, also the non-AMF Mortierella verticillata. Our data indicate a possible origin of the MRE-fungus association in ancestors of the Glomeromycota and Mucoromycotina. The DhMRE genome encodes an arsenal of putative regulatory proteins with eukaryotic-like domains, some of them encoded in putative genomic islands. MRE are highly interesting candidates to study the evolution and interactions between an ancient, obligate endosymbiotic prokaryote with its obligate plant-symbiotic fungal host. Our data moreover may be used for further targeted searches for ancient effector-like proteins that may be key components in the regulation of the arbuscular mycorrhiza symbiosis.

Phylogeny of the Pluteaceae (Agaricales, Basidiomycota): taxonomy and character evolution

The phylogeny of the genera traditionally classified in the family Pluteaceae (Agaricales, Basidiomycota) was investigated using molecular data from nuclear ribosomal genes (nSSU, ITS, nLSU) and consequences for taxonomy and character evolution were evaluated. The genus Volvariella is polyphyletic, as most of its representatives fall outside the Pluteoid clade and shows affinities to some hygrophoroid genera (Camarophyllus, Cantharocybe). Volvariella gloiocephala and allies are placed in a different clade, which represents the sister group of Pluteus, and a new generic name, Volvopluteus, is proposed to accommodate these taxa. Characters such as basidiospore size and pileipellis structure can be used to separate Pluteus, Volvariella and Volvopluteus. The genus Pluteus is monophyletic and includes species with partial veil traditionally classified in the genus Chamaeota. The evolution of morphological features used in the infrageneric taxonomy of the genus, such as metuloid cystidia and pileipellis structure, was analyzed. Agreement between the molecular phylogeny and morphological subdivision of Pluteus is, generally speaking, good, though some rearrangements are necessary: (i) species with non-metuloid pleurocystidia and pileipellis as a cutis are placed either in sect. Celluloderma, together with the species characterized by a hymenidermal pipeipellis, or in sect. Pluteus, with the metuloid bearing species; (ii) subdivision of sect. Celluloderma according to the presence/absence of cystidioid elements in the pileipellis is not supported by molecular data.

Species recognition in Pluteus and Volvopluteus (Pluteaceae, Agaricales): morphology, geography and phylogeny

The phylogeny of several species-complexes of the genera Pluteus and Volvopluteus (Agaricales, Basidiomycota) was investigated using molecular data (ITS) and the consequences for taxonomy, nomenclature and morphological species recognition in these groups were evaluated. Conflicts between morphological and molecular delimitation were detected in sect. Pluteus, especially for taxa in the cervinus-petasatus clade with clamp-connections or white basidiocarps. Some species of sect. Celluloderma are apparently widely distributed in Europe, North America and Asia, either with (P. aurantiorugosus, P. chrysophlebius, P. fenzlii, P. phlebophorus) or without (P. romellii) molecular differentiation in collections from different continents. A lectotype and a supporting epitype are designated for Pluteus cervinus, the type species of the genus. The name Pluteus chrysophlebius is accepted as the correct name for the species in sect. Celluloderma, also known under the names P. admirabilis and P. chrysophaeus. A lectotype is designated for the latter. Pluteus saupei and Pluteus heteromarginatus, from the USA, P. castri, from Russia and Japan, and Volvopluteus asiaticus, from Japan, are described as new. A complete description and a new name, Pluteus losulus, are given for the African P. cervinus var. ealaensis. The American Volvopluteus michiganensis is described in detail. Taxonomic comments and a morphology-based key to all known species of Volvopluteus are provided.

Development of specific PCR primers for identification and detection of Rhizopycnis vagum

Rhizopycnis vagum is a recently described coelomycete known to belong to the complex of root rot pathogens contributing to vine decline of cucurbits in several parts of the world. However, the fungus has also been reported to infect tomato, and as an endophytic associate of mycorrhizal roots of wild, asymptomatic Pinus halepensis and Rosmarinus officinalis plants in Italy. To accelerate epidemiological and ecological investigations on this fungus, a PCR primer pair was developed. Primers Rv1-F and Rv1-R were designed, based on alignment of internal transcribed spacer (ITS) sequences (ITS1-5.8S-ITS2), which amplified a 396-bp fragment from all R. vagum isolates tested, including isolates pathogenic to melons and endophytic isolates from mycorrhizae. Specificity of the primer pair was verified both in silico (BLAST searches using each primer string as a query) and in PCR assays, where the primers failed to amplify DNA from any isolate of fungi taxonomically related to R. vagum (e.g. Massarina walkeri and Stagonospora spp.) and other vine decline and common soilborne pathogens (e.g. Monasporascus cannonballus, Acremonium cucurbitacearum, Fusarium spp. and Rhizoctonia solani). Under optimum conditions, detectable amplification of the specific sequence required 0.05 pg of target DNA. Amplification of the expected 369-bp fragment was also obtained from DNA root extracts of nearly asymptomatic Cucumis melo plants inoculated with R. vagum under greenhouse conditions.