Bacteria of the genus Rhodopseudomonas (Bradyrhizobiaceae): obligate symbionts in mycelial cultures of the black truffles Tuber melanosporum and Tuber brumale.

BACKGROUND: This work aimed at characterizing 12 isolates of the genus Tuber including Tuber melanosporum (11 isolates) and Tuber brumale (one isolate). This was done using internal transcribed spacer (ITS) sequences, confirming their origin. RESULTS: Analysis of their mating type revealed that both MAT1-1 and MAT1-2 exist within these isolates (with 3 and 8 of each, respectively). We observed that each of these cultures was consistently associated with one bacterium that was intimately linked to fungal growth. These bacterial associates failed to grow in the absence of fungus. We extracted DNA from bacterial colonies in the margin of mycelium and sequenced a nearly complete 16S rDNA gene and a partial ITS fragment. We found they all belonged to the genus Rhodopseudomonas, fitting within different phylogenetic clusters. No relationships were evidenced between bacterial and fungal strains or mating types. Rhodopseudomonas being a sister genus to Bradyrhizobium, we tested the nodulation ability of these bacteria on a promiscuously nodulating legume (Acacia mangium), without success. We failed to identify any nifH genes among these isolates, using two different sets of primers. CONCLUSIONS: While the mechanisms of interaction between Tuber and Rhodopseudomonas remain to be elucidated, their interdependency for in vitro growth seems a novel feature of this fungus.

Phylogeny and phylogeography of the Tuber brumale aggr.

The Tuber brumale (winter truffle) is a black truffle reported from most European countries, belonging to the Melanosporum group. Its significance in the economy is ambivalent as the winter truffle has been shown to be a frequent contaminant species in the orchards of the Perigord truffle and occasionally in those of the summer truffle, yet owing to its delicate fragrance, its trade is worthy of note. The phylogeny and phylogeography of economically important truffles are relatively well-explored; however, no thorough research has been published on these aspects of the winter truffle. Therefore, here, we report the first phylogeographic analyses based on samples representing the entire distribution of the species. ITS sequences were used in this survey for haplotype and coalescent analyses, while phylogenetic analyses were based on the ITS, LSU and PKC loci. According to all loci, the samples clustered into two big clades imply the existence of two phylogenetic species. Based on our results, one of these appears to be endemic to the Carpathian Basin. In the other more widespread species, two main phylogeographic groups can be distinguished that show east-west separation with a zone of overlap in the Carpathian Basin, suggesting that they survived the latest glacial period in separate refugia.

Morphological characterization of mycorrhizae formed between three Terfezia species (desert truffles) and several Cistaceae and Aleppo pine.

Six Cistaceae species, Helianthemum ledifolium, Helianthemum lippii, Fumana procumbens, Cistus albidus, Cistus incanus, Cistus salvifolius, and Pinus halepensis (Aleppo pine) were inoculated with three mycorrhizal desert truffles, Terfezia leptoderma, Terfezia boudieri, and Terfezia claveryi under greenhouse conditions, on soil originating from desert truffle natural habitat in Algeria. The syntheses have led to the formation of typical endomycorrhizae in annual Cistaceae (H. ledifolium) and perennial ones (H. lippii and F. procumbens) and an ectomycorrhiza with a less developed sheath in Cistus species and Aleppo pine. These results demonstrate the plasticity of Terfezia species to form different mycorrhizal types. The formation of an endomycorrhiza with H. ledifolium and F. procumbens and a sheathing ectomycorrhiza with P. halepensis inoculated by T. leptoderma in in vivo culture conditions was obtained for the first time.

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 approximately 125 megabases is the largest and most complex fungal genome sequenced so far. This expansion results from a proliferation of transposable elements accounting for approximately 58% of the genome. In contrast, this genome only contains approximately 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.

Tuber aestivum (syn. T. uncinatum) biotopes and their history on Gotland, Sweden.

This study aimed at testing the hypothesis that the genetically distinct Tuber aestivum population on the island of Gotland, Sweden, is adapted to habitats different from French T. aestivum populations. The soil structure, soil chemistry, bedrock, climate, vegetation and host tree continuity of 18 T. aestivum sites on Gotland were analysed and compared with data from France. We conclude that T. aestivum can grow in soils with a broad soil structure range and that no striking differences in soil chemistry were found. No T. aestivum indicator plants other than the host trees were found, but the host tree continuity on the T. aestivum sites on Gotland was more than 300 yr. If the T. aestivum population on Gotland constitutes an ecotype it is rather an adaptation to the colder and drier climate on Gotland. Selecting local T. aestivum inoculum for truffle orchards in northern Europe could be important for successful truffle production.

Polymorphism at the ribosomal DNA ITS and its relation to postglacial re-colonization routes of the Perigord truffle Tuber melanosporum.

• Glaciations and postglacial migrations are major factors responsible for the present patterns of genetic variation we see in natural populations in Europe. For ectomycorrhizal fungi, escape from refugia can only follow range expansion by their specific hosts. • To infer phylogeographic relationships within Tuber melanosporum, sequences of internal transcribed spacers (ITS) and the 5.8S coding region of the ribosomal DNA repeat were obtained for 188 individuals sampled over the entire distribution of this species in France, and in north-western Italy and north-eastern Spain. • Ten distinct ITS haplotypes were distinguished, mapped and treated using F- and NST -statistics and nested clade (NCA) analyses. They showed a significant genetic differentiation between regional populations. NCA revealed a geographical association of ITS haplotypes, an old fragmentation into two major groups of populations, which likely colonized regions on different sides of the French Central Massif. • This re-colonization pattern is reminiscent of the one observed for host trees of the Perigord truffle, such as oaks and hazelnut trees. This suggests that host postglacial expansion was one of the major factors that shaped the mycobiont population structure.