Identification of Tuber magnatum Pico DNA markers by RAPD analysis

Summary 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.     

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

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).     

A rapid mini-prep method for isolation of ectomycorrhizal DNA from Tuber species

A rapid procedure has been developed to isolate DNA from the ectomycorrhizae of Tuber spp. for use in PCR experiments. The method described is fast and sensitive and can overcome the amplification problems that can arise in the presence of inhibitors. For this reason it can be used to type ectomycorrhizae even starting from a single root tip and make mycorrhizae identification much more rapid.     

Molecular characterisation of the small GTPase CDC42 in the ectomycorrhizal fungus Tuber borchii Vittad.

Summary. The small GTPase CDC42 is ubiquitously expressed in eukaryotes, where it participates in the regulation of the cytoskeleton and a wide range of cellular processes, including cytokinesis, gene expression, cell cycle progression, apoptosis, and tumorigenesis. As very little is known on the molecular level about mycorrhizal morphogenesis and development and these events depend on a tightly regulated reorganisation of the cytoskeleton network in filamentous fungi, we focused on the molecular characterisation of the cdc42 gene in Tuber borchii Vittad., an ascomycetous hypogeous fungus forming ectomycorrhizae. The entire gene was isolated from a T. borchii cDNA library and Southern blot analyses showed that only one copy of cdc42 is present in the T. borchii genome. The predicted amino acid sequence is very similar to those of other known small GTPases and the similar domain structures suggest a similar function. Real-time PCR analyses revealed an increased expression of Tbcdc42 during the phase preparative to the instauration of symbiosis, in particular after stimulation with root exudate extracts. Immunolocalisation experiments revealed an accumulation of CDC42 in the apical tips of the growing hyphae. When a constitutively active Tbcdc42 mutant was expressed in Saccharomyces cerevisiae, morphological changes typical of pseudohyphal growth were observed. Our results suggest a fundamental role of CDC42 in cell polarity development in T. borchii. Correspondence and reprints: Istituto di Chimica Biologica “G. Fornaini”, Università degli Studi di Urbino, Via Saffi 2, 61029 Urbino, Italy.     

Bacteria-produced ferric exopolysaccharide nanoparticles as iron delivery system for truffles (Tuber borchii)

Applied Microbiology and Biotechnology - Iron exopolysaccharide nanoparticles were biogenerated during ferric citrate fermentation by Klebsiella oxytoca DSM 29614. Before investigating their...     

Microbial and pigment profile of the reddish patch occurring within Tuber magnatum ascomata

Tuber magnatum Pico, the delectable white truffle, is the most prized truffle species. In this study, we examined the reddish pigmentation that frequently occurs in T. magnatum ascomata for the presence of pigment-producing bacteria.The inner part of the reddish-pigmented region of three T. magnatum ascomata collected in North–Central Italy was analysed. This reddish part was used to establish a bacterial culture collection and to extract the total genomic DNA in order to obtain a library of 16S rRNA genes representative of the bacterial community. The molecular approach revealed limited microbial diversity within the reddish-pigmented regions compared to the wider range of bacterial species commonly found at the same maturation stage and season in T. magnatum ascomata. The pigmented regions showed a prevalence of specific bacterial species belonging to α-, β- and γ- Proteobacteria, Actinobacteria and Firmicutes. From the tandem mass spectrometry analysis of the extracted pigment, four compounds were identified: i) bixin, ii) β-carotene, iii) cis-1-glycosyl-apo-8′- lycopene and iv) the fucoxanthin. Carotenoid producing species such as Microbacterium and Chryseobacterium emerged as the most likely cause of the peculiar reddish pigment production. Indeed, our findings suggest that the peculiar reddish pigment might be produced by these bacterial species.     

A macrophage-derived factor different from interleukin 1 and able to induce interferon-gamma and lymphoproliferation in resting T lymphocytes

A novel monokine different from interleukin 1 (IL-1) is secreted from human or murine macrophages stimulated with galactose oxidase, a well-characterized enzyme able to induce marked polyclonal activation of lymphocytes. This monokine, preliminarly termed macrophage-derived blastogenic factor (MBF), stimulates resting T lymphocytes to produce interferon (IFN)-gamma and to proliferate. The apparent MW of MBF ranges between 29,000 and 35,000, although some active fractions show smaller MW ranging from 2000 to 7000, as demonstrated by gel filtration. The biological relationship between MBF and IL-1 was examined and it was found that MBF (1) is able to induce IFN-gamma production and proliferation by T lymphocytes in the absence of other inducers, (2) is not able to activate PHA-stimulated C3H mouse thymocytes, (3) is not able to induce production of IFN-beta by fibroblast cultures, (4) is resistant to proteolytic enzymes, and (5) is not neutralized by antibodies to IL-1. MBF was released in the macrophage supernatants in two waves after the oxidation process, namely, after 15 min and 2.5 hr, and each wave was capable of inducing lymphocyte activation at dilutions up to 1:32. Because the oxidation of galactose residues on cell surface structures is considered a general feature of lymphocyte activation whatever the inducer, it seems that MBF may be a mediator involved in mitogenic activation of T cells leading to IFN-gamma production and proliferation.     

Importance of sucking stimulation in lactation: ultrastructural aspects of mammary glands in the rat

The Authors refer the results of a study concerning the mammary glands of lactating rats. We made use of rats Wistar subdivided in three groups. Group A: each rat lactates 8 pups. Group B: each rat lactates 4 pups. 8 days before delivery we provided to hide the nipples of a half of breasts with sutures. Group C: They do not lactate. In the 6th day of suckling, after a period of intense suction, we removed the mammary glands. This material was processed for electronic microscopy. Morphological aspects obtained show a difference in development of cytoplasmatic organs specially manifest in R.E.R. and Golgian apparatus.     

Characterization and complementation of a Fus3/Kss1 type MAPK from Tuber borchii, TBMK

Mitogen-activated protein kinases (MAPK) are used by organisms to transduce extra cellular signals from the environment in cellular events such as proliferation and differentiation. In the present study, we have characterized the first MAPK from the ectomycorrhizal fungus Tuber borchii (TBMK) which belongs to the YERK1 (yeast extra cellular regulated kinase) subfamily. TBMK is present as a single copy in the genome and the codified protein was phosphorylated during the interaction with the host plant, Tilia americana. Complementation studies showed that TBMK restores pheromone signaling in Saccharomyces cerevisiae and partially restores invasive growth of Fusarium oxysporum that lack the fmk1 gene. This suggests a protein kinase activity and its involvement in the infection processes. Hence, TBMK could play an important role during the pre-symbiotic phase of T. borchii with its host plant in the modulation of genes necessary for the establishment of symbiosis leading to the synthesis of functional ectomycorrhizae.     

Selection of a set of specific primers for the identification of Tuber rufum: a truffle species with high genetic variability

Tuber rufum is a truffle widely distributed throughout Europe, which forms mycorrhizal associations with numerous species of broadleaf and coniferous trees. The possibility of T. rufum contamination in commercial truffle-infected plants makes its detection important. To facilitate the identification of T. rufum from mycorrhiza and fruitbodies, species-specific primers were designed and tested. To overcome the high intraspecific genetic variability within the internal transcribed spacer (ITS) regions of T. rufum, as demonstrated by phylogenetic analysis, two forward primers, Ru1f and Ru2f, located on the ITS1 region were designed to be used in concert with the reverse primer ITS4. Only T. rufum was amplified with this primer combination, while DNA of Tuber magnatum, Tuber brumale, Tuber maculatum, Tuber borchii, Tuber excavatum and Tuber melanosporum was not. These primers give a specific amplicon ranging between 566 and 572 bp and are able to discriminate between T. rufum, T. borchii and T. magnatum in multiplex PCR. In addition, T. rufum-specific amplicons were obtained from both spore suspensions and mycorrhiza by direct PCR. Tuber rufum mycorrhiza obtained in the greenhouse using mycelial inoculation techniques had morphological features similar to those of other species of Tuber, stressing the importance of molecular tools for their identification.