Molecular identification of Tuber magnatum ectomycorrhizae in the field.

Tuber ectomycorrhizae in a Tuber magnatum "truffière", located in Central Italy, were studied using molecular methods. Specifically, RFLP-ITS analyses, ITS sequencing and specific probes hybridization were used to identify 335 Tuber-like ectomycorrhizal morphotypes. Molecular identification was possible even when distinct morphological characteristics were lacking. For the first time, T. magnatum ectomycorrhizae and other coexisting Tuber species collected in the field were analysed using molecular tools for unambiguous identification. Although the "truffière" under investigation yields good harvests of T. magnatum fruiting bodies, the percentage of T. magnatum ectomycorrhizae found was very low (less than 4.4% of the 335 root tips analysed), whereas the percentages of Tuber maculatum and Tuber rufum were considerably higher (48.9% and 19.0%, respectively).

Solid-phase microextraction gas chromatography/mass spectrometry: a new method for species identification of truffles.

This study describes a rapid method to identify different truffle species by analysis of their volatile compound fraction using static headspace solid-phase microextraction gas chromatography/mass spectrometry. The volatile organic compounds (VOCs) were extracted using a new 2-cm 50/30 microm DVB/CAR/PDMS fiber placed for 10 min in the headspace of the truffle sample with the vial maintained at 20 degrees C (in a thermostatically controlled analysis room). The mass spectra of the VOC chromatograms were represented as 'fingerprints' of the analysed samples. Next, stepwise factorial discriminant analysis afforded a limited number of characteristic fragment ions that allowed a classification of the truffle species studied. This new method provides an effective approach to rapid quality control and identification of truffle species by analysis of their volatile fraction. Moreover, this method offers the advantage of minimizing thermal, mechanical, and chemical modifications of the truffles, thereby reducing the risk of analytical artifacts.

New evidence for bacterial diversity in the ascoma of the ectomycorrhizal fungus Tuber borchii Vittad.

The microbial community associated with ascocarps of the ectomycorrhizal fungus Tuber borchii Vittad. was studied by both cultivation and direct extraction of bacterial 16S rRNA gene (rDNA) sequence approaches. The inner part of six T. borchii ascoma collected in North-Central Italy was used to establish a bacterial culture collection and to extract the total genomic DNA to obtain a library of 16S rDNAs representative of the truffle bacterial community. Most of the isolates were affiliated to the gamma-Proteobacteria, mainly Fluorescent pseudomonads; some isolates were members of the Bacteroidetes group and Gram-positive bacteria, mostly Bacillaceae. The majority of the clones from the library were alpha-Proteobacteria showing significant similarity values, of greater than 97%, with members of the Sinorhizobium/Ensifer Group, Rhizobium and Bradyrhizobium spp. not previously identified as Tuber-associated bacteria. Only a few bacterial strains belonging to this bacterial subclass were found in the culture collection and isolated on a medium specific for Rhizobium-like organisms. A few clones were members of the beta- and gamma-Proteobacteria; as well as low and high G+C Gram-positive bacteria. Our findings clearly indicate that a dual approach increases the information obtained on the structural composition of a truffle bacterial community as compared to that derived via cultivation or direct recovery of 16S rDNA sequences alone.

Morpho-functional characterization of neuronal cells at different stages of maturation in granule cell layer of adult rat dentate gyrus.

Neurogenesis occurs throughout adult life in dentate gyrus of mammal hippocampus. Therefore, neurons at different stages of electrophysiological and morphological maturation and showing various, if any, synaptic inputs co-exist in the adult granule cell layer, as occurs during dentate gyrus development. The knowledge of functional properties of new neurons throughout their maturation can contribute to understanding their role in the hippocampal function. In this study electrophysiological and morphological features of granule layer cells, characterized as immature or mature neurons, without and with synaptic input, were comparatively described in adult rats. The patch-clamp technique was used to perform electrophysiological recordings, the occurrence of synaptic input evoked by medial perforant pathway stimulation was investigated and synaptic input was characterized. Cells were then identified and morphologically described via detection of biocytin injected through the patch pipette. The neuronal phenotype of recorded cells was assessed by immunohistochemistry and single-cell RT-PCR. Cells with very low capacitance, high input resistance, depolarized resting membrane potential and without synaptic activity were found exclusively at the border of the GCL facing hilus; this type of cell expressed the class III beta-tubulin neuronal marker (mRNA and protein) and did not express a glial marker. Immature neuronal cells with progressively increasing capacitance, decreasing input resistance and resting membrane potential getting more hyperpolarized showed only depolarizing GABAergic synaptic input at first and then also glutamatergic synaptic input. Finally, cells showing electrophysiological, synaptic, and morphological features of mature granule, expressing the mature neuron marker NeuN, were identified.