Diversity of Endophytic and Pathogenic Fungi of Saffron (Crocus sativus) Plants from Cultivation Sites in Italy

Crocus sativus is an important crop for the production of saffron and bioactive compounds. Plant endophytic fungi are a source of secondary metabolites additional to those produced by the plant itself. We analysed the biodiversity of endophytic fungi present in corms, stems, leaves, tepals, and stigmas of C. sativus from ten Italian sites; furthermore, we isolated putative pathogenic fungi from rotten plants. We used an in vitro isolation approach followed by molecular analysis of the internal transcribed spacer (ITS rDNA) region. We obtained 165 strains belonging to 39 OTUs, spreading over 26 genera and 29 species. Dark septate endophytes of the genus Cadophora and the species Talaromyces pinophilus dominated in corms, while Alternaria alternata, Epicoccum spp., T. pinophilus, Mucor fragilis, and Stemphylium vesicarium dominated in other tissues. The most frequently isolated pathogens were Fusarium oxysporum and Rhizopus oryzae. Endophytic communities significantly differed among tissues and life stages, whereas differences among cultivation sites were not statistically supported. Several endophytes were hypothesized to have changing trophic modes and/or to be latent pathogens in C. sativus. All strains were conserved ex-situ for future bioactivity tests and production of metabolites

Exploring new biodiversity hotspots in Tuber aestivum natural distribution

Tuber aestivum is associated with a wide range of tree species and is the most widespread edible truffle. Its capability of colonizing many different areas along with a long reaping period, increased in the last decades scientific studies and commercial interest. A number of large- and fine-scale population genetics studies have been conducted to explore its genetic diversity and genetic structure. Recent investigations based on ITS sequence analyses revealed a strong phylogenetic pattern and a higher level of genetic diversity in Turkish and southern European populations than in northern European populations, suggesting that Turkey and southern Europe may have acted as glacial refugia for this species. Despite these findings, the southernmost distributional range of the species is not yet fully explored. With the aim of evaluating other potential diversity hotspots in this study we expand previous investigation, extending the sampling to other southernmost natural T. aestivum areas, such as Moroccan mountainous regions (Middle Atlas), Serbia and several Spanish regions. The ongoing analyses based on haplotypes alignment of the ITS region of the rDNA, reveal as preliminary result a pronounced genetic variability among Moroccan T. aestivum populations, suggesting a possible phylogeographic differentiation also in these new areas of investigation. Additional analyses are underway to better trace natural populations of T. aestivum according to their geographic origin. These aims could be critical both for truffle industry interest in selecting well-adapted inoculum for the production of mycorrhizal seedlings or traceability of truffles provenance, and for the establishment of programs devoted to biodiversity conservation

Diversity of Endophytic and Pathogenic Fungi of Saffron (Crocus sativus) Plants from Cultivation Sites in Italy

Crocus sativus is an important crop for the production of saffron and bioactive compounds. Plant endophytic fungi are a source of secondary metabolites additional to those produced by the plant itself. We analysed the biodiversity of endophytic fungi present in corms, stems, leaves, tepals, and stigmas of C. sativus from ten Italian sites; furthermore, we isolated putative pathogenic fungi from rotten plants. We used an in vitro isolation approach followed by molecular analysis of the internal transcribed spacer (ITS rDNA) region. We obtained 165 strains belonging to 39 OTUs, spreading over 26 genera and 29 species. Dark septate endophytes of the genus Cadophora and the species Talaromyces pinophilus dominated in corms, while Alternaria alternata, Epicoccum spp., T. pinophilus, Mucor fragilis, and Stemphylium vesicarium dominated in other tissues. The most frequently isolated pathogens were Fusarium oxysporum and Rhizopus oryzae. Endophytic communities significantly differed among tissues and life stages, whereas differences among cultivation sites were not statistically supported. Several endophytes were hypothesized to have changing trophic modes and/or to be latent pathogens in C. sativus. All strains were conserved ex-situ for future bioactivity tests and production of metabolites

Tuber melanosporum: mating type distribution in a natural plantation and dynamics of strains of different mating types on the roots of nursery-inoculated host plants

International audienceP>In light of the recent finding that Tuber melanosporum, the ectomycorrhizal ascomycete that produces the most highly prized black truffles, is a heterothallic species, we monitored the spatial distribution of strains with opposite mating types (MAT) in a natural truffle ground and followed strain dynamics in artificially inoculated host plants grown under controlled conditions. In a natural truffle ground, ectomycorrhizas (ECMs), soil samples and fruit bodies were sampled and genotyped to determine mating types. Simple sequence repeat (SSR) markers were also used to fingerprint ECMs and fruit bodies. The ECMs from nursery-inoculated host plants were analysed for mating type at 6 months and 19 months post-inoculation. In open-field conditions, all ECMs from the same sampling site showed an identical mating type and an identical haploid genotype, based on SSR analysis. Interestingly, the gleba of fruit bodies always demonstrated the same genotype as the surrounding ECMs. Although root tips from nursery-grown plants initially developed ECMs of both mating types, a dominance of ECMs of the same MAT were found after several months. The present study deepens our understanding of the vegetative and sexual propagation modes of T. melanosporum. These results are highly relevant for truffle cultivation

Mating Type Locus of Chinese Black Truffles Reveals Heterothallism and the Presence of Cryptic Species within the<i>T. indicum</i> Species Complex

<div><p><i>Tuber</i> spp. are filamentous ascomycetes which establish symbiosis with the roots of trees and shrub species. By virtue of this symbiosis they produce hypogeous ascocarps, known as truffles. Filamentous ascomycetes can reproduce by homothallism or heterothallism depending on the structure and organization of their mating type locus. The first mating type locus in a truffle species has been recently characterized in <i>Tuber melanosporum</i> and it has been shown that this fungus, endemic in Europe, is heterothallic. The availability of sequence information for <i>T. melanosporum</i> mating type genes is seminal to cloning their orthologs from other <i>Tuber</i> species and assessing their reproductive mode. Here we report on the organization of the mating type region in <i>T. indicum</i>, the black truffle species present in Asia, which is the closest relative to <i>T.</i><i>melanosporum</i> and is characterized by an high level of morphological and genetic variability. The present study shows that <i>T. indicum</i> is also heterothallic. Examination of Asiatic black truffles belonging to different genetic classes, sorted according to the sequence polymorphism of the internal transcribed spacer rDNA region, has revealed sequence variations and rearrangements in both coding and non-coding regions of the mating type locus, to suggest the existence of cryptic species within the <i>T. indicum</i> complex. The presence of transposable elements within or linked to the mating type region suggests a role of these elements in generating the genotypic diversity present among <i>T. indicum</i> strains. Overall, comparative analyses of the mating type locus have thus allowed us to tackle taxonomical and phylogenetic issues within black truffles and make inferences about the evolution of <i>T. melanosporum-T. indicum</i> lineage. Our results are not only of fundamental but also of applied relevance as <i>T. indicum</i> produces edible fruit bodies that are imported also into Europe and thus may represent a biological threat for <i>T. melanosporum</i>.</p></div

Amino acid alignment of <i>T. indicum</i> and <i>T. melanosporum MAT1-1-1</i> (A) and <i>MAT1-2-1</i> (B) deduced proteins.

<p>The HMG-box and α-box regions are underlined. The default ClustalX color code is used.</p

Amino acid alignment of <i>T. indicum Ti_orf3</i> encoded hypothetical protein.

<p>The ANK repeats are underlined.</p

Isolation and characterization of MAT genes in the symbiotic ascomycete Tuber melanosporum

International audienceThe genome of Tuber melanosporum has recently been sequenced. Here, we used this information to identify genes involved in the reproductive processes of this edible fungus. The sequenced strain (Mel28) possesses only one of the two master genes required for mating, that is, the gene that codes for the high mobility group (HMG) transcription factor (MAT1-2-1), whereas it lacks the gene that codes for the protein containing the alpha-box- domain (MAT1-1-1), suggesting that this fungus is heterothallic. A PCR-based approach was initially employed to screen truffles for the presence of the MAT1-2-1 gene and amplify the conserved regions flanking the mating type (MAT) locus. The MAT1-1-1 gene was finally identified using primers designed from the conserved regions of strains that lack the MAT1-2-1 gene. Mating type-specific primer pairs were developed to screen asci and gleba from truffles of different origins and to genotype single ascospores within the asci. These analyses provided definitive evidence that T. melanosporum is a heterothallic species with a MAT locus that is organized similarly to those of ancient fungal lineages. A greater understanding of the reproductive mechanisms that exist in Tuber spp. allows for optimization of truffle plantation management strategies

List of selected ascocarps analyzed for the mating type.

<p>List of selected ascocarps analyzed for the mating type.</p

Procédé moléculaire permettant d'identifier des gènes de type sexuel chez les espèces de truffes

The invention concerns a method for determining the Tuber species and fertility of a truffle sample, said method comprising: - identifying whether said truffle sample comprises a first nucleotide sequence associated with a first mating type idiomorph (MAT1-2); and - identifying whether said truffle sample comprises a second nucleotide sequence associated with a second mating type idiomorph (MAT1-1); the fertility of said truffle sample requiring the presence of both the first and the second nucleotide sequences.Cette invention concerne un procédé permettant de déterminer l'espèce Tuber et la fertilité d'un échantillon de truffe, ledit procédé comprenant les étapes consistant à : déterminer si ledit échantillon de truffe comprend ou non une première séquence de nucléotides associée à un premier idiomorphe de type sexuel (MAT1-2) ; et - à déterminer si ledit échantillon de truffe comprend ou non une seconde séquence de nucléotides associée à un second idiomorphe de type sexuel (MAT1-1) ; la fertilité dudit échantillon de truffe exigeant la présence à la fois de la première et de la seconde séquence de nucléotides