Antonella Amicucci

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

Cultivation of edible ectomycorrhizal mushrooms

The edible mycorrhizal mushrooms include some of the worlds most expensive foods and have a global market measured in US

Identification of ectomycorrhizal fungi of the genus Tuber by species‐specific ITS primers

billions. Despite this, few have been cultivated with any degree of success, and certainly not in volumes that are likely to reverse the catastrophic declines in production that have occurred over the past 100 years. The main obstacles to their cultivation are their need to be associated with a host plant to successfully grow and fruit, contamination with other ectomycorrhizal fungi both before and after the establishment of plantations, and a general lack of understanding of each mushrooms trophic relationships, and biotic, edaphic and climatic requirements.

Geographical traceability of Italian white truffle (Tuber magnatum Pico) by the analysis of volatile organic compounds

This study reports PCR-based techniques for a reliable molecular identification of five species of white truffles: Tuber magnatum Pico, T. borchii Vittad., T. maculatum Vittad., T. dryophilum Berk. & Br. and T. puberulum Berk. & Br. The sequences of the ITS region of several Tuber spp. were analysed and a pair of primers was designed for each species under study. The selected pairs of specific primers can be used for simple, rapid, reliable and unambiguous identification during the three developmental phases of the truffle life cycle: fruitbody, mycelium and ectomycorrhiza.

Simultaneous high-performance capillary electrophoretic determination of reduced and oxidized glutathione in red blood cells in the femtomole range

Results are presented that were obtained on the geographic traceability of the white truffle Tuber magnatum Pico. Solid-phase microextraction coupled to gas chromatography/mass spectrometry (SPME-GC/MS) was employed to characterize the volatile profile of T. magnatum white truffle produced in seven geographical areas of Italy. The main components of the volatile fraction were identified using SPME-GC/MS. Significant differences in the proportion of volatile constituents from truffles of different geographical areas were detected. The results suggest that, besides genetic factors, environmental conditions influence the formation of volatile organic compounds. The mass spectra of the volatile fraction of the samples were used as fingerprints to characterize the geographical origin. Next, stepwise factorial discriminant analysis afforded a limited number of characteristic fragment ions that allowed a geographical classification of the truffles studied.

Identification of ectomycorrhizae from Tuber species by rflp analysis of the its region

This paper describes a high-performance capillary electrophoretic (HPCE) method which allows a quick, simultaneous and quantitative determination of reduced (GSH) and oxidized (GSSG) glutathione in mammalian red blood cells using a Supelco-bonded hydrophilic phase capillary CElect-P150. The extraction procedure of GSH and GSSG from erythrocytes using Microcon-10 membranes is very simple and allows a correct evaluation of these compounds present in the red blood cells. Furthermore, the HPCE method does not require removal of the excess N-ethylmaleimide used to block the glutathione in its reduced state, making the simultaneous evaluation of GSH and GSSG possible in a very short time (ca. 4 min), with a sensitivity at femtomole level.

Identification of Tuber magnatum Pico DNA markers by RAPD analysis

SummaryPolymorphisms of a ribosomal DNA region (ITS) have been analysed using a specific pair of primers, in order to type fruitbodies and ectomycorrhizae of different truffle species. The identification of ectomycorrhizae was obtained by digestion of the PCR products using restriction enzymes. The results show that the strategy used is both suitable and sensitive to characterize the symbiotic fungi from few mycorrhized root tips.

Use of sequence characterised amplified region and RAPD markers in the identification of the white truffle Tuber magnatum Pico

Different species of truffle were studied in order to identify species-specific markers. The isolation of two Tuber magnatum Pico markers is reported. One of these could be used as a probe in dot blot hybridization, allowing the development of a rapid test able to identify Tuber magnatum species.

Hyphal and cytoskeleton polarization in Tuber melanosporum: a genomic and cellular analysis.

Isolates of white truffles were identified as Tuber magnatum Pico species using a pair of primers selected from a sequence characterised amplified region (SCAR) and a specific random amplified polymorphic DNA (RAPD) marker. The present study reveals that PCR-fragment-pattern polymorphisms, the construction of probes and couples of primers from one or more of these polymorphic fragments may provide a useful and rapid tool for identifying species of ectomycorrhizal fungi in addition to conventional methods (morphological parameters).

Molecular and functional characterization of a Rho GDP dissociation inhibitor in the filamentous fungus Tuber borchii

Filamentous polarized growth involves a series of events including polarization of the cytoskeleton to selected growth sites, and the transport of secretory vesicles containing the components required for growth. The availability of fungal genome sequences has recently led to the identification of a large number of proteins involved in these processes. We have explored the Tuber melanosporum genome sequence by searching for homologs of genes known to play crucial roles in the morphogenesis and cell polarity of yeasts and filamentous fungi. One hundred and forty-nine genes have been identified and functionally grouped according to the deduced amino acid sequences (44 genes involved in cell polarity/morphogenesis, 39 belonging to the actin cytoskeleton and 66 involved in membrane dynamics, septation and exocytosis). A detailed gene annotation has shown that most components of the cell polarity machinery, morphogenesis and cytoskeleton found in yeasts and filamentous fungi are conserved, although the degree of similarity varies from strong to weak. Microscopic analysis of quick-frozen truffle hyphae detected the characteristic subcellular components of the hyphal tip in septate filamentous fungi, while transcript profiles revealed a moderately variable pattern during the biological cycle.