Gérard Chevalier

Typing truffle species by PCR amplification of the ribosomal DNA spacers

Variation within the internal transcribed spacer (ITS) and the intergenic spacer (IGS) of the ribosomal RNA genes of European species of Tuber was examined by polymerase chain reaction (PCR) and coupled restriction fragment length polymorphism (RFLP) analysis. Ribosomal DNA spacers were successfully amplified from mycelium, ectomycorrhiza, and fruit bodies of a wide range of truffle species (Tuber aesfivum, T. albidum, T. brumale, T. excavarnm, T. ferrugineum, T. magnatum, T. melanosporum, T. rufum and T. uncinatum). Interspecific variation in the length and number of restriction sites of the amplified ITS and IGS was observed and most truffles could thus be reliably distinguished by PCR-RFLP. In contrast, the degree of intraspecific rONA variation observed was low within T. melanosporum, but sufficient to discriminate several isolates. These results demonstrate that the PCR-RFLP analysis of rONA spacers provides an efficient alternative for typing pure fungal cultures and fruit bodies for the food industry and a versatile tool for strain fingerprinting of ectomycorrhizas in ecosystems. The commercial inoculation of oak and hazel tree seedlings with ecologically adapted truffles is presently being developed in France and Italy (Chevalier & Grente, 1979; Bencivenga, 1982). Although many experiments have shown that truffle inoculation is beneficial in terms of fruit-body production, results have been somewhat erratic on outplanting sites where indigenous truffles and ectomycorrhizal fungi communities are large and competitive. How this natural population could interact with introduced truffle isolates needs to be determined to select potential candidates for large scale inoculation programs. This requires tracking the environmental fate of the introduced truffles to determine their survival, growth, and dissemination within a microbial community. The identification of mycorrhizas and fruit bodies of truffles, based on morphological features, is reliable for most species. However, the ectomycorrhizas of several morphological species, such as Tuber aestivum Vittad. and T. uncinatum Ch., T. aibidum Pico and T. magnatum Pico, are morphologically similar, but only T. uncinatum, T. aestivum and T. magnatum are gastronomically and thus economically valuable (Chevalier et al., 1986). Biophysical (Papa et ai. 1987; Papa & Polimeni, 1990) and biochemical (protein patterns, isoenzyme electrophoresis, scent composition) (Dupre & Chevalier, 1991; Dupre et ai., 1992; Mouches et ai., 1981; Pacioni et al., 1989; Pacioni & Pompini, 1989; Palenzona et al., 1990) techniques have been

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.

Différenciation des truffes européennes par l'analyse des isoenzymes

Summary Electrophoretic analysis of isozymes was used to characterize and identify some Tuber species. Ascocarps, mycelial cultures and mycorrhizas were studied using 5 isozyme systems (MDH, GPI, NADP-GDH, SOD, ACP). Electrophoresis was carried out on starch gels, Polyacrylamide gels and agarose-polyacryiamide gels. All systems (except SOD) showed a broad intraspecific variability, among ascocarps. However, SOD was homogeneous for a species but heterogeneous between different species. This system can be used to elaborate a biochemical identification key. The statistical analysis indicated a close similarity between some taxa (T. aestivum-T. uncinaturm) but distinguished others (T. brumale-T. moschatum). MDH, GPI, NADP-GDH and SOD patterns for ascocarps and mycelial cultures are similar. NADP-GDH was the only isozyme which could be detected in ectomycorrhizas.

The European Desert Truffles

In the northern hemisphere, the terfez or ‘desert truffles’ (the genera Mattirolomyces, Terfezia, Tirmania and Picoa) are not the privilege of North African countries, nor of the Middle East and the Arabic Peninsula. There are desert truffles (terfez) in Europe. The country with the largest number of species is Spain and then comes Italy. The most common terfez of commercial interest are Mattirolomyces terfezioides (formerly Terfezia terfezioides) in Hungary and T. arenaria, T. leptoderma and T. olbiensis and T. boudieri and T. claveryi in Spain and Italy. The latter two are more adapted to desert conditions and are not found frequently. Tirmania nivea, still more adapted to the arid zones, is rare (south of Spain, Sicilia). For each species of terfez, geographic distribution and habitat are described. The different factors in the terfez distribution in Europe are analysed (climatic and edaphic conditions, host plants). The commercial interest of some species is discussed.

Molecular Identification of Tuber Species and Isolates by PCR-Based Techniques

Morphological features of fruit bodies and ectomycorrhizas allow the identification of most truffle species. However, economically important truffles (e.g. Tuber aestivum / T. uncinatum; T. borchii/ T. magnatum ) are difficult to distinguish using these criteria. It is therefore required, when morphological traits are unsufficient for discriminating, to call for additional markers (e.g. isozymes and DNA). DNA polymorphisms in truffle genomes have therefore been used to generate molecular markers by PCR amplification of specific regions and by random amplification of polymorphic DNA (RAPD). Amplification of the internal transcribed spacer (ITS) and intergenic spacer (IGS) of the ribosomal DNA combined with endonuclease digestion yielded unique diagnostic patterns for most European truffle species. However, rDNA polymorphism was not high enough to distinguish T. aestivum and T. uncinatum. This approach revealed a low intraspecific variation within T. melanosporum. The PCR amplification of the rDNA spacers can be combined with RFLP, allowing truffle species to be typed in less than a day and is most immediately applicable to molecular epidemiology. DNA polymorphism has been further assessed by RAPD technique. The high interspecific genomic variability already observed for most truffle species using PCR-RFLP was confirmed. In addition, large intraspecific variations have been found in all species, except T. melanosporum and T. brumale. Molecular techniques provide an efficient tool, on the one hand for typing fruiting bodies for the food industry, mycelium for patenting purposes as well as mycorrhiza for checking the inoculated seedlings and on the other hand for tracking the fate of mycorrhizal seedlings within truffieres and forest ecosystems.

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

BackgroundThis 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.ResultsAnalysis 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.ConclusionsWhile 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.