Tuber Aestivum Research Articles

Tuber aestivum and Tuber uncinatum: two morphotypes or two species?

Tuber spp. are ectomycorrhizal fungi that establish symbioses with shrubs and trees. Because of their different smell and taste, Tuber uncinatum and Tuber aestivum are two truffle morphotypes with a different market value, but whether or not T. uncinatum and T. aestivum are different taxa is still an open debate among mycologists. In order to identify molecular keys characterizing both T. aestivum and T. uncinatum morphotypes, ITS/RFLPs analyses were carried out on a large collection of samples from all over Italy and from other European countries, followed by a study of the phylogenesis of ITS, β-tubulin and EF 1-α genes, on representative samples. The present study provides compelling evidence that: (i) T. uncinatum and T. aestivum belong to the same species, (ii) neither morphotype presents a specific molecular fingerprint, but they may even share identical alleles at any of the loci analysed; (iii) T. aestivum is most likely under a selfing reproductive mode. Our findings suggest that ecological, rather than genetic causes may account for differences in sporal morphology, taste and smell between T. aestivum and T. uncinatum truffles.

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.

The population of the hypogeous fungus Tuber aestivum syn. T. uncinatum on the island of Gotland

The aim of our study was to examine the genetic variation within Tuber aestivum on the Baltic island of Gotland, Sweden. Variation in such a limited geographical area should help illuminate the dispersal abilities of T. aestivum. Knowledge of the genetic variation in this northern outpost could also be useful in the selection of inoculum for the establishment of truffle orchards. Genetic structure and homogeneity of the population were studied using principal component and parsimony analyses of randomly amplified polymorphic DNA data. Our inventories showed that T. aestivum is abundantly distributed in suitable habitats on Gotland. The genetic variation observed suggests sexual reproduction and slow dispersal on the island. It is possible that the present population was established from one introduction, which may be due to ability to survive in this habitat rather than to rare colonising events. The T. aestivum population on Gotland may be an ecotype adapted to the climate and soil conditions on the island.

Truffle aroma characterization by headspace solid-phase microextraction

In the present study, a headspace solid-phase microextraction (HS-SPME) combined to gas chromatography–mass spectrometry (GC–MS) has been used to fully characterize aroma of truffles of different species. A fiber of medium polarity (for flavors) was used to avoid discrimination towards very non-polar and polar volatile compounds. In a previous work, extraction conditions were optimized by means of an experimental design leading to the following conditions that were used in the present study: extraction temperature, 53 °C; extraction time, 13.6 min; and equilibrium time, 5 min. A comparison among different truffles species has been established in terms of qualitative and quantitative differences on volatile composition. By using the optimal extraction conditions and GC–MS it was possible to identify 89 compounds in two different truffle species such as Tuber aestivum and Tuber melanosporum. An attempt has been made in order to be able to determine the influence of different geographical origins on the aroma fraction of such fungi.

Genetic variability of Tuber uncinatum and its relatedness to other black truffles.

Genetic variability is one of the major survival strategies developed by symbiotic fungi. We focused on the ectomycorrhizal fungus Tuber uncinatum Chatin that produces edible ascomata. In order to understand the degree of its variability and its relatedness to another morphologically-similar truffle, T. aestivum Vittad., ascomata of T. uncinatum were collected from a single natural truffle-ground located in the north of Italy and compared with samples from other Italian sites, as well as with T. aestivum ascomata from other European regions. We used multi-locus approaches, such as microsatellite-primed PCR (polymerase chain reaction), and single locus markers, such as mitochondrial and nuclear ribosomal DNA on 30 samples. The results demonstrate that the level of genetic polymorphism among isolates of T. uncinatum was higher than in other Tuber species, like T. melanosporum. Neighbour-joining analyses were carried out on a binary data matrix on 12 ascomata of T. uncinatum and T. aestivum, and on 15 internal transcribed spacer (ITS) sequences of these species and 5 from other Tuber species. Taken together, they clustered T. uncinatum and T. aestivum in two separate groups. The mitochondrial rDNA primers, NMS1 and NMS2, were not able to differentiate morphologically related and unrelated truffles. Moreover, a pair of primers, intentionally designed to differentiate isolates of T. aestivum and T. uncinatum from other Tuber species, successfully amplified DNA from all the samples of T. aestivum and T. uncinatum considered in our analysis. In conclusion, different molecular approaches separate T. aestivum and T. uncinatum according to their spore reticulum and their taste and smell.

Truffle aroma analysis by headspace solid phase microextraction.

An experimental design has been used to optimize the extraction of volatile compounds from summer truffle aroma (Tuber aestivum) by using headspace solid phase microextraction. The extracted compounds have been analyzed by gas chromatography with a flame ionization detector and by gas chromatography-mass spectrometry (GC-MS). In an attempt to develop an objective method to fully characterize truffle aroma, a fiber of medium polarity (for flavors) was used to avoid discrimination toward very nonpolar and polar volatile compounds. To optimize the extraction conditions, a response surface experimental design was applied considering three factors such as extraction temperature, equilibrium time, and extraction time. From the statistical analysis of the experimental design, it was possible to determine that the most important factor influencing the abundance of aroma compounds was the extraction temperature. Optimal extraction temperature was established at approximately 50 degrees C. By using GC-MS, it was possible to identify 37 compounds, most of them previously described as responsible for truffle aroma.

Morphological and molecular characterization of mycelia of some Tuber species in pure culture.

• Pure cultures of Tuber maculatum , Tuber melanosporum , Tuber aestivum , Tuber macrosporum , Tuber rufum and Tuber brumale were isolated and characterized by morphological and molecular methods. • The Tuber mycelia were isolated from fruit bodies and molecular identification was performed using specific primers, restriction fragment length polymorphism and/or sequence analyses of the ITS region. • The species grew between 1.1mmwk -1 and 14mmwk -1 on the selected medium. The mycelium of different Tuber species showed several common morphological features such as hyphal anastomoses, vesicle formation and hyphal aggregation. Differences were found in the frequency of these morphological features and in the hyphal pattern. The isolated mycelia also showed differences in the hyphal branch angle, septal distance, hyphal diameter and rate of growth of the hyphae. • This result opens the possibility of using pure mycelial cultures of Tuber spp. for experimental purposes and for the commercial production of infected truffle plants.

Molecular tools for the identification of Tuber melanosporum in agroindustry.

Tuber melanosporum Vitt., Tuber magnatum Pico, and Tuber uncinatum Chat. can be differentiated by their morphological characters. Fraud problems have arisen recently with the importation to Europe of truffles from China. T. melanosporum is morphologically very close, but distinct from the Chinese species [Tuber indicum (Cooke and Massee) and T. himalayense BC (Zhang and Winter)]. We have optimized molecular tools to unequivocally identify T. melanosporum. DNA extraction from ascocarps of black truffles is not straightforward. Problems to obtain pure DNA are due to high contents of phenolic compounds, melanine, and various polymers (proteins, polysaccharides, etc). These compounds coprecipitate with the DNA during extraction and strongly inhibit the PCR reaction. We have developed an efficient and reliable protocol for DNA extraction from truffle ascocarps. It was used successfully for DNA extraction from mycorrhizal root tips as well as from canned preparations of T. melanosporum. Several approaches to identify T. melanosporum by PCR were developed. Two specific primers for T. melanosporum were designed after comparison of the ITS region of this species with those of three Chinese fungi. They proved to be efficient to specifically detect the presence of T. melanosporum by PCR. The mycorrhizal status of trees inoculated with T. melanosporum but unable to produce truffles was confirmed in a single-step PCR reaction. A multiplex PCR approach was also developed with three sets of primers (including a specific one for Chinese truffles) to detect, in one PCR reaction, the presence of any other Tuber species mixed with T. melanosporum ascocarps. This optimized protocol, in association with the specific primers we designed, is applicable to quality control in the truffle industry from the production stages to final commercial products.

Biochemical, electrophoretic and immunohistochemical aspects of malate dehydrogenase in truffles (Ascomycotina)

The malate dehydrogenase (MDH; EC 1.1.1.37; L-malate-NAD +-oxidoreductase) activities of truffles of the genus Tuber ( Tuber melanosporum Vittad., Tuber brumale Vittad., Tuber aestivum Vittad., Tuber magnatum Pico, Tuber rufum Pico) have been characterized with regard to the K m and V max values in the direct and reverse reactions. The isoelectrofocusing has revealed bands showing p I values ranging from pH 5.85 to 7.8. The MDH of T. melanosporum has been partially purified by hydroxyapatite treatment, DEAE-cellulose and Sephadex G-75 columns. With the partially purified T. melanosporum MDH activity polyclonal anti- T. melanosporum MDH antibodies have been prepared and used to localize MDH in the mycorrhizae and ascocarps of T. melanosporum. These antibodies inhibit T. melanosporum MDH activity as well as that of T. magnatum but not that of rabbit liver; this supports the specificity of the MDH antibodies used to localize MDH in truffle tissues.

Biochemical, electrophoretic and immunohistochemical aspects of malate dehydrogenase in truffles (Ascomycotina).

The malate dehydrogenase (MDH; EC 1.1.1.37; L-malate-NAD(+)-oxidoreductase) activities of truffles of the genus Tuber (Tuber melanosporum Vittad., Tuber brumale Vittad., Tuber aestivum Vittad., Tuber magnatum Pico, Tuber rufum Pico) have been characterized with regard to the K(m) and V(max) values in the direct and reverse reactions. The isoelectrofocusing has revealed bands showing pI values ranging from pH 5.85 to 7.8. The MDH of T. melanosporum has been partially purified by hydroxyapatite treatment, DEAE-cellulose and Sephadex G-75 columns. With the partially purified T. melanosporum MDH activity polyclonal anti-T. melanosporum MDH antibodies have been prepared and used to localize MDH in the mycorrhizae and ascocarps of T. melanosporum. These antibodies inhibit T. melanosporum MDH activity as well as that of T. magnatum but not that of rabbit liver; this supports the specificity of the MDH antibodies used to localize MDH in truffle tissues.

Ploidy and chromosomal number in Tuber aestivum

Chromosome number is generally considered to be the most accurate and direct measure of ploidy. In fungi, however, counting of chromosomes is difficult and inaccurate because of their small size. To date truffles have been characterized using molecular approaches (analysis of DNA and PCR-based techniques) in order to find out differences among species, but not by a cytogenetical approach. Although the small size of truffle chromosomes is of some hindrance for a cytogenetic study, in the present work Tuber aestivum chromosome counts were determined in metaphase configurations from haploid nuclei of ascospores by aceto-orcein staining. Nuclear chromosome number and topography were also evaluated by propidium iodide DNA staining using confocal microscopy. Tuber aestivum was found to possess a basic number of 5 or 6 chromosomes, medium length ≤0.95 μm. The karyology of ascospores during their developmental stages was also investigated.

Ploidy and chromosomal number inTuber aestivum

Chromosome number is generally considered to be the most accurate and direct measure of ploidy. In fungi, however, counting of chromosomes is difficult and inaccurate because of their small size. To date truffles have been characterized using molecular approaches (analysis of DNA and PCR-based techniques) in order to find out differences among species, but not by a cytogenetical approach. Although the small size of truffle chromosomes is of some hindrance for a cytogenetic study, in the present work Tuber aestivum chromosome counts were determined in metaphase configurations from haploid nuclei of ascospores by aceto-orcein staining. Nuclear chromosome number and topography were also evaluated by propidium iodide DNA staining using confocal microscopy. Tuber aestivum was found to possess a basic number of 5 or 6 chromosomes, medium length ≤0.95 μm. The karyology of ascospores during their developmental stages was also investigated.

The Volatile Organic Compounds of Tuber uncinatum from Middle Italy

The volatile organic compounds of Tuber uncinatum and their headspace variations during storage are determined by purge and trap methodology. The main component was 2-methyl butanol, but 2,4-dithiapentane, previously found in more appreciated species of truffles, 2-thiapentane and 2-thiα-3(Z)-pentene, which were present in lower amounts, appear to be the impact flavor compounds.

Grouping and identification of Tuber species using RAPD markers

Mycorrhizal fungi of the genus Tuber are classified by morphological characters that allow differentiation of most species. However, some economically important species are difficult to differentiate on morphological grounds. When morphological traits are not sufficient to discriminate between taxa, other markers are needed. Genetic variation of fruit bodies of 12 Tuber taxa was studied by the random amplified polymorphic DNA (RAPD) technique. High interspecific variability was observed between most species. Moreover, important infraspecific variation occurred in all species, except Tuber brumale s.L, Tuber melanosporum, and Tuber magnatum. Southern hybridization patterns of RAPD products of the various species were used to confirm the data. Relationships among Tuber species were determined by cluster analyses. UPGMA analyses revealed several main clusters and a low genetic similarity between taxa. These results indicate that RAPD and polymerase chain reaction are useful for analysing genetic variation within Tuber species. Most species can be identified by differences in their amplified DNA profiles. However, the two pairs of closely related taxa Tuber aestivum – Tuber uncinatum and Tuber brumale var. brumale – Tuber brumale var. moschatum did not appear to differ genotypically. Key words: Tuber, RAPD, Southern, UPGMA, inter- and infra-specific variability.

Physiological and textural response of truffles during low-temperature storage

SummaryTruffles (Tuber aestivum Vitt.) were stored at 0°C, 5°C and 10°C for 40 d, and ethylene, carbon dioxide and ethane production was measured. No difference was observed between samples kept at 0°C and 5°C while at 10°C truffles underwent decay and produced high amounts of ethylene, ethane and carbon dioxide. When truffles were stored in either low oxygen (1%) or high CO2 (60%) at 5°C and 10°C, ethylene was better controlled by high CO2 than low O2. Even the weight loss of truffles treated in high CO2 was reduced. Studying the rheological characteristics by using TPA (Texture Profile Analysis), we observed that truffles stored in high CO2 at 5°C kept values of hardness, gumminess and chewiness similar to those of fresh samples. Frozen-thawed samples, as a commercial comparison, showed completely altered texture. The shape of TPA peaks are useful for the definition of truffle pulp texture. Carbon dioxide treatment at 5°C could be a good technique for storing fresh truffles because truffles keep their s...

Variability of nuclear and mitochondrial ribosomal DNA of a truffle species ( Tuber aestivum)

The intraspecific genetic variability of Tuber aestivum was studied using molecular markers at various geographical scales. We used the polymerase chain reaction (PCR) coupled with restriction fragment length polymorphism (RFLP) analysis to examine the variation of the nuclear and mitochondrial ribosomal DNA (rDNA). RFLPs were found in the nuclear internal transcribed spacer (ITS) and three alleles were detected in the six populations analysed. No variability was found in mitochondrial rDNA. We found, in a very few cases, that truffles sharing different ITS genotypes could be present within a single symbiotic tree.

Translocation and metabolism of phosphate following absorption by the ascocarps of Tuber melanosporum and T. aestivum

The capacity of the tufts of hyphae which sprout from the scales of the peridium of the ascocarps of Tuber aestivum and Tuber melanosporum to absorb phosphate has been confirmed using labelled phosphate. The detailed distribution of the tracer was determined from autoradiographs and these and the corresponding photographs were studied using image analysis. The aim of this investigation was to identify the conduction pathways of absorbed [32P]phosphate; these were found to be the fertile veins of the gleba, and in particular the sterile hyphae adjacent to the paraphyses. Some of the compound was metabolised rapidly by the ascocarp. These results provide additional evidence that the ascocarp may become nutritionally autonomous at some stage in its development. In addition, the role of the tufts of hyphae in the physiology of the ascocarp suggests a link between its mode of growth and its external form. The distribution of absorbed phosphate was heterogeneous even after a 24 h incubation period. The similarity of the distribution profiles of the isotope across fertile veins, both with and without incubation, suggests that translocation continues throughout the incubation period, and that metabolised phosphate may be remobilised.

Mode of the absorption of water and nutrients by ascocarps of Tuber melanosporum and Tuber aestivum: a radioactive tracer technique

A simple technique has been developed to study the absorption capacity of the tufts of mycelial filaments, or hyphae, observed on both black and white truffles Tuber melanosporum and Tuber aestivum. These hyphae sprout from the tops of certain scales on the peridium of Tuber. They colonize the surrounding soil and could therefore exploit the nutrients from a large volume of soil. The ability to absorb organic and inorganic nutrients from a source other than the host is a necessary condition for the hypothesis we wished to test, namely that at some stage in its development the truffle ascocarp becomes autonomous. The absorption was demonstrated using autoradiography and liquid scintillation counting. We found that the tufts of hyphae are water permeable (3H2O). In addition, they absorb a sugar ([14C]mannose) and an anion ([32P]phosphate). An analysis of our results shows that the spatial distribution of [32P]phosphate is independent of that of tritiated water; the transport of phosphate is therefore not determined by water flux. The quantity and redistribution of the tracer depended on the application site, directly onto the peridium or on a mycelial tuft. The tufts increase the absorption of the tracer and influence its internal redistribution towards accumulation zones. Key words: ascocarp, Tuber melanosporum, Tuber aestivum, absorption, truffle nutrition.

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 aestivum, T. albidum, T. brumale, T. excavatum, 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 rDNA variation observed was low within T. melanosporum, but sufficient to discriminate several isolates. These results demonstrate that the PCR-RFLP analysis of rDNA 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.

L'interface plante—champignon dans les ectomycorhizes de truffes: étude ultrastructurale et microanalyses par perte d'énergie des électrons et par rayons X

Summary The confrontation between Tuber uncinatum or Tuber magnatum and Corylus avellana or Quercus robur roots leads to ultrastructural modifications. In root, three simultaneous process show an epidermic cell differentiation: radial cell growth, increase of vacuolar volume and cytoplasmic changes. The root cell evolution precedes the Hartig net formation. The interface established between both partners is characterised by a lot of fungal and root plasmalemmasomas. Contacts between plasmalemma and endoplasmic reticulum are frequent. The matrix that joins walls of both symbiots is composed of three constituents: (i) outer mucilaginous wall of fungus; (ii) granular areas which apparently come from cell wall degradation at the time of Hartig net installation; (iii) a lot of fibrilla that establishes bridges between both partners walls. Hartig net establishment brings about the isolation of cells and stops symplasmic transferts. Microlocalisations of calcium and phosphorus are realized in the different parts...