Overview of molecular methods for quick identification of Tuber species

Truffles represent a resource of great value all over the world. The various species are widespread and occupy various habitats. Methods to identify their value in terms of marketing, cultivation, and the ecological–forestal field are needed. The traditional classification of Tuber species is based on their morphology; nevertheless, the macroscopic and microscopic characteristics of the fruiting body, spores, and ectomycorrhizae may be absent or insufficiently discriminating. This problem is amplified when dealing with valuable truffle species, which are often replaced by truffles with lower organoleptic qualities and commercial value. Today, molecular biology can help to correctly identify them through several widely used methods that are fast, specific, and sensitive. Molecular methods are mostly PCR-based, with primers designed on various targets, genes, or regions widely variable between species but sufficiently conserved within the same species. The DNA regions analyzed for identifying truffles include ribosomal internal transcribed spacer (ITS), ribosomal large subunit (LSU), β-tubulin, and EF-α elongation factor. This review summarizes the main and most reliable DNA-based molecular methods for identifying Tuber species. The methods are listed schematically and clearly for quick consultation and use in all areas of study in which monitoring, and verification of the species is essential

In vitro interactions between Bradyrhizobium spp. and Tuber magnatum mycelium

Tuber magnatum is the most expensive truffle, but its large-scale cultivation is still a challenge compared to other valuable Tuber species. T. magnatum mycelium has never been grown profitably until now, which has led to difficulties to studying it in vitro. This study describes beneficial interactions between T. magnatum mycelium and never before described bradyrhizobia, which allows the in vitro growth of T. magnatum mycelium. Three T. magnatum strains were co-isolated on modified Woody Plant Medium (mWPM) with aerobic bacteria and characterised through microscopic observations. The difficulties of growing alone both partners, bacteria and T. magnatum mycelium, on mWPM demonstrated the reciprocal dependency. Three bacterial isolates for each T. magnatum strain were obtained and molecularly characterised by sequencing the 16S rRNA, glnII, recA and nifH genes. Phylogenetic analyses showed that all nine bacterial strains were distributed among five subclades included in a new monophyletic lineage belonging to the Bradyrhizobium genus within the Bradyrhizobium jicamae supergroup. The nifH genes were detected in all bacterial isolates, suggesting nitrogen-fixing capacities. This is the first report of consistent T. magnatum mycelium growth in vitro conditions. It has important implications for the development of new technologies in white truffle cultivation and for further studies on T. magnatum biology and genetics

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

<p>Abstract</p> <p>Background</p> <p>Small GTPases of the Rho family function as tightly regulated molecular switches that govern important cellular functions in eukaryotes. Several families of regulatory proteins control their activation cycle and subcellular localization. Members of the guanine nucleotide dissociation inhibitor (GDI) family sequester Rho GTPases from the plasma membrane and keep them in an inactive form.</p> <p>Results</p> <p>We report on the characterization the RhoGDI homolog of <it>Tuber borchii </it>Vittad., an ascomycetous ectomycorrhizal fungus. The Tb<it>gdi </it>gene is present in two copies in the <it>T. borchii </it>genome. The predicted amino acid sequence shows high similarity to other known RhoGDIs. Real time PCR analyses revealed an increased expression of Tb<it>gdi </it>during the phase preparative to the symbiosis instauration, in particular after stimulation with root exudates extracts, that correlates with expression of Tb<it>cdc42</it>. In a translocation assay TbRhoGDI was able to solubilize TbCdc42 from membranes. Surprisingly, TbRhoGDI appeared not to interact with <it>S. cerevisiae </it>Cdc42, precluding the use of yeast as a surrogate model for functional studies. To study the role of TbRhoGDI we performed complementation experiments using a RhoGDI null strain of <it>Dictyostelium discoideum</it>, a model organism where the roles of Rho signaling pathways are well established. For comparison, complementation with mammalian RhoGDI1 and LyGDI was also studied in the null strain. Although interacting with Rac1 isoforms, TbRhoGDI was not able to revert the defects of the <it>D. discoideum </it>RhoGDI null strain, but displayed an additional negative effect on the cAMP-stimulated actin polymerization response.</p> <p>Conclusion</p> <p>T. borchii expresses a functional RhoGDI homolog that appears as an important modulator of cytoskeleton reorganization during polarized apical growth that antecedes symbiosis instauration. The specificity of TbRhoGDI actions was underscored by its inability to elicit a growth defect in <it>S. cerevisiae </it>or to compensate the loss of a <it>D. discoideum </it>RhoGDI. Knowledge of the cell signaling at the basis of cytoskeleton reorganization of ectomycorrhizal fungi is essential for improvements in the production of mycorrhized plant seedlings used in timberland extension programs and fruit body production.</p

Carlos J. ALONSO GUTIÉRREZ, El evolucionismo y otros mitos. La crisis del paradigma darwinista, EUNSA («Astrolabio»), Pamplona 2004, 445 pp., 15 x 22, ISBN 84-313-2167-9. [RESEÑA]

. The blot was probed with a 207 bp long radioactively labeled probe. Tbappears to be a two-copy gene.<p><b>Copyright information:</b></p><p>Taken from "Molecular and functional characterization of a Rho GDP dissociation inhibitor in the filamentous fungus "</p><p>http://www.biomedcentral.com/1471-2180/8/57</p><p>BMC Microbiology 2008;8():57-57.</p><p>Published online 9 Apr 2008</p><p>PMCID:PMC2362126.</p><p></p

Périgord black truffle genome uncovers evolutionary origins and mechanisms of symbiosis

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

Overview of molecular methods for quick identification of Tuber species

Truffles represent a resource of great value all over the world. The various species are widespread and occupy various habitats. Methods to identify their value in terms of marketing, cultivation, and the ecological–forestal field are needed. The traditional classification of Tuber species is based on their morphology; nevertheless, the macroscopic and microscopic characteristics of the fruiting body, spores, and ectomycorrhizae may be absent or insufficiently discriminating. This problem is amplified when dealing with valuable truffle species, which are often replaced by truffles with lower organoleptic qualities and commercial value. Today, molecular biology can help to correctly identify them through several widely used methods that are fast, specific, and sensitive. Molecular methods are mostly PCR-based, with primers designed on various targets, genes, or regions widely variable between species but sufficiently conserved within the same species. The DNA regions analyzed for identifying truffles include ribosomal internal transcribed spacer (ITS), ribosomal large subunit (LSU), β-tubulin, and EF-α elongation factor. This review summarizes the main and most reliable DNA-based molecular methods for identifying Tuber species. The methods are listed schematically and clearly for quick consultation and use in all areas of study in which monitoring, and verification of the species is essential

GTPases in Hyphal Growth

Hyphal growth is a fundamental event in filamentous fungi, in that they grow by means of apical expansion of tube-shaped cells (hyphae). This event requires vesicular trafficking, reorganization of the cytoskeleton, construction of the cell wall. The underlying molecular mechanism it is complex and diverse. GTPases play important roles as molecular switches, passing from the active-GTP bound state to the inactive-GDP bound state. These proteins turn various pathways on or off and are crucial regulators of hyphal growth, septation, cell wall synthesis and the exocytic and endocytic pathway. In particular, the roles of Ras, Arf, Rab, Rho have been deepened

Enhancement of food production quality: the truffle case

The central role of quality agri-food products in the promotion of a given territory is actually widely recognized by both the economic and marketing literature and the stakeholders involved in the enhancement process of rural systems. On this basis, this work analyzes one of the finest Italian agri-food products: the truffle. This work tries to point out the main problems characterizing the current regulatory framework, the trade and the production of the Italian truffle sector, emphasizing their causes, consequences and possible solutions