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.     

Self/nonself recognition in Tuber melanosporum is not mediated by a heterokaryon incompatibility system

Vegetative incompatibility is a widespread phenomenon in filamentous ascomycetes, which limits formation of viable heterokaryons. Whether this phenomenon plays a role in maintaining the homokaryotic state of the hyphae during the vegetative growth of Tuber spp. Gene expression, polymorphism analysis as well as targeted in vitro experiments allowed us to test whether a heterokaryon incompatibility (HI) system operates in Tuber melanosporum. HI is controlled by different genetic systems, often involving HET domain genes and their partners whose interaction can trigger a cell death reaction. Putative homologues to HI-related genes previously characterized in Neurospora crassa and Podospora anserina were identified in the T. melanosporum genome. However, only two HET domain genes were found. In many other ascomycetes HET domains have been found within different genes including some members of the NWD (NACHT and WD-repeat associated domains) gene family of P. anserina. More than 50 NWD homologues were found in T. melanosporum but none of these contain a HET domain. All these T. melanosporum paralogs showed a conserved gene organization similar to the microexon genes only recently characterized in Schistosoma mansoni. Expression data of the annotated HI-like genes along with low allelic polymorphism suggest that they have cellular functions unrelated to HI. Moreover, morphological analyses did not provide evidence for HI reactions between pairs of genetically different T. melanosporum strains. Thus, the maintenance of the genetic integrity during the vegetative growth of this species likely depends on mechanisms that act before hyphal fusion.     

Calcareous amendments to soils to eradicate Tuber brumale from T. melanosporum cultivations: a multivariate statistical approach

Calcareous amendments are being used in Tuber melanosporum truffle plantations in attempts to eradicate Tuber brumale. However, there are no studies available which provide soil analysis and statistical data on this topic. We studied 77 soil samples to compare the values for carbonates, pH and total organic carbon in T. brumale truffières with the values for T. melanosporum truffières on contaminated farms and in natural areas. Statistical analyses indicate that the concentrations of active carbonate and total carbonate in the soil are significantly higher in T. brumale truffières than in T. melanosporum truffières, but that there are no significant differences in pH and total organic carbon. We conclude that liming would not suppress T. brumale ectomycorrhizas in contaminated T. melanosporum farms, and calcareous amendments do not therefore seem be a means of eradicating T. brumale in these farms.     

Cultivation of Tuber melanosporum in firebreaks: Short-term persistence of the fungus and effect of seedling age and soil treatment

Wildfires are a major threat to Mediterranean forests. Firebreaks are built as a prevention measure, but require a periodic and expensive maintenance. Cultivating the ectomycorrhizal mushroom Tuber melanosporum Vitt. in firebreaks could reduce costs and improve their sustainability. But firebreaks are built on forest soil, considered nonoptimum for T. melanosporum cultivation. A pot experiment was used to study the persistence of T. melanosporum in firebreak soils in the short term, as a first step to assess the viability of these plantations. The influence of seedlings, soil heating, and liming was also tested. During the 2 y after plantation, T. melanosporum mycorrhizas increased their number, showing its ability to proliferate. Percent root colonisation by native fungi importantly increased from month 12 to 22; although T. melanosporum remained dominant, with a colonisation level similar to those in standard truffle plantations. The age of seedlings at the time of planting influenced T. melanosporum proliferation, supporting a key role for nursery seedling quality in the performance of young plantations. Heating the soil before planting reduced the richness of native fungi, suggesting that this could increase plantation success. The results tend to support the viability of T. melanosporum cultivation in firebreaks, and encourage experimental field plantations.     

Genetic structure of <Emphasis Type="Italic">Tuber mesentericum</Emphasis> Vitt. based on polymorphisms at the ribosomal DNA ITS

Tuber mesentericum fruit bodies are in increasing demand on the food market and are an important economic resource for southern Italy, their major production area. Because molecular studies on this truffle species are very scarce, we analyzed ITS1 and ITS2 nucleotide variability of 126 ascocarps of T. mesentericum collected in different European areas, mainly southern Italy. The results of haplotype distribution, analysis of molecular variance, and spatial analysis of molecular variance analyses show strong genetic structuring of the samples collected in the different geographic areas, confirmed by parsimony and distance analyses. In particular, the Italian samples seem to be a well-distinguished group.     

New data on ectomycorrhizae and soils of the Chinese truffles Tuber pseudoexcavatum and Tuber indicum, and their impact on truffle cultivation

Chinese truffles serve as a good complement to the market for Tuber melanosporum (Périgord black truffle). However, Chinese truffles could be introduced accidentally or fraudulently into the plantations of Mediterranean truffles, and they could have a negative effect on truffle production and natural ecosystems. The study of Tuber species from China which are commercialized in Europe began 14 years ago. Tuber pseudoexcavatum was proposed as a new species, and this has been validated by some authors based on molecular and phylogenetic studies. We synthesize their ectomycorrhizae using samples from the type collection, and we compare T. pseudoexcavatum and Tuber indicum ectomycorrhizae. The ectomycorrhizae of these species have a morphology which is related to the ectomycorrhizae of T. melanosporum. We provide useful information for the rapid screening of the above-mentioned Chinese truffles ectomycorrhizae, for the quality control of commercial plants mycorrhized with Tuber. Moreover, we analyze the soil tolerance and the host plant affinity of T. pseudoexcavatum and T. indicum, in order to assess the capacity of both Chinese truffles to penetrate T. melanosporum plantations and habitats.     

Molecular species of ceramides from the ascomycete truffle Tuber indicum

The ceramide fractions were isolated from the chloroform/methanolic extractable of the fruiting bodies of Tuber indicum and separated into three kinds of molecular species TI-1, TI-2, and TI-3 by normal and reverse phase silica gel-column chromatography. By means of (1)H NMR and (13)C NMR spectroscopy, fast atom bombardment mass spectrometry (FAB-MS), and chemical degradation experiment, their component sphingoid base for TI-1 and TI-2 was uniformly (2S,3S,4R)-2-amino-1,3,4-octadecantriol, while the sphingoid of TI-3 was d-erythro-sphingosine, and their structures have been determined unequivocally to be (2S,2'R,3S,4R)-2-(2'-d-hydroxyalkanoylamino) octadecane-1,3,4-triol, the fatty acid composition of which consists of 2-hydroxydocosanoic, 2-hydroxytetracosanoic, and 2-hydroxytricosanoic acids (from major to minor); (2S,3S,4R)-2-(alkanoylamino)octadecane-1,3,4-triol, the fatty acid composition of which is unusual and consists of docosanoic, hexadecanoic, tricosanoic, octadecanoic and nonadecanoic acids (from major to minor); and (2S,3R,4E)-2-(alkanoylamino)-4-octadecene-1,3-diol, the component fatty acids of which were hexadecanoic (predominant) and octadecanoic acids, respectively.     

A new pair of primers designed for amplification of the ITS region in Tuber species

The alignment of the 28S gene of several species of Pezizales allowed to select two pairs of primers able to amplify the internal transcribed spacer region of ribosomal DNA in mycorrhizal fungi, such as truffles. The higher yield of the amplification product demonstrates a better annealing of the new primers to the rDNA, as compared to the universal primers internal transcribed spacer 1 and internal transcribed spacer 4. Therefore, the new primers can be used as an easier and more sensitive tool for the identification of truffle species in any stage of their life cycle, including the mycorrhizal phase.     

Influence of pH on in vitro mycelial growth in three Japanese truffle species: Tuber japonicum,T. himalayense,and T. longispinosum

Truffle species indigenous to Japan include a white-colored truffle Tuber japonicum and two black-colored truffles, T. himalayense and T. longispinosum. As the fruiting bodies of these Tuber species are promising edible, studies on the artificial cultivation for these truffles are currently underway in Japan. In the present study, we investigated the influence of pH on in vitro mycelial growth in these Tuber species to determine the optimal pH conditions for the cultivation of these truffles. Mycelia of five strains from each species were cultured in modified Melin–Norkrans liquid medium at different pH values. Tuber japonicum grew well at pH 5.0 and 6.0, whereas T. himalayense and T. longispinosum grew well at pH 7.0. This results suggest that the optimal pH for mycelial growth varies among Tuber species. The growth data collected in this study can be used to design optimal pH conditions for artificial cultivation of these Japanese truffles.     

Seasonal fungal diets of small mammals in an Australian temperate forest ecosystem

Australian temperate forests support a high diversity of truffle-like fungi, and a rich assortment of mammals that feed upon them. We sampled seasonal diets of four sympatric mammals (two rodents, two marsupials) in an eastern Australian wet sclerophyll forest and identified all dietary fungi. Fifty-two different spore types were found in diets, most of which were from truffle-like fungi. All mammals consumed fungi, but occurrence of fungi and the variety of taxa in the diets peaked in winter and spring. Bush rats (Rattus fuscipes) were significantly more mycophagous than other mammals sampled in terms of proportion of scats containing fungi, number of taxa per sample, and overall diversity of dietary fungi. Most fungal taxa were eaten only occasionally, but a few truffle-like taxa dominated diets and appear to be staple food for the small mammal community. Our work supports the view that mycophagous mammals are important for maintenance of ecosystem health through their spore dispersal abilities.