Genetic and phylogeographic structures of the symbiotic fungus Tuber magnatum

The quality and market price of truffles vary with the species and, traditionally, the place of origin. The premium species Tuber magnatum produces white truffles and has a patchy distribution restricted to Italy and some Balkan areas. We used polymorphic microsatellites to evaluate 316 specimens grouped into 26 populations sampled across the species' geographic range to determine if natural populations of T. magnatum are genetically differentiated. We found that the southernmost and the northwesternmost populations were significantly differentiated from the rest of the populations. The simple sequence repeat data also could be used to make inferences about the postglacial T. magnatum expansion pattern. This study is the first to identify a genetic and phylogeographic structure in T. magnatum. The presence of a genetic structure can be of practical interest in tracing truffle populations according to their geographic origin for marketing strategies. Evidence for extensive outcrossing in field populations of T. magnatum also is provided for the first time.     

Genetic and Phylogeographic Structures of the Symbiotic Fungus Tuber magnatum

The quality and market price of truffles vary with the species and, traditionally, the place of origin. The premium species Tuber magnatum produces white truffles and has a patchy distribution restricted to Italy and some Balkan areas. We used polymorphic microsatellites to evaluate 316 specimens grouped into 26 populations sampled across the species' geographic range to determine if natural populations of T. magnatum are genetically differentiated. We found that the southernmost and the northwesternmost populations were significantly differentiated from the rest of the populations. The simple sequence repeat data also could be used to make inferences about the postglacial T. magnatum expansion pattern. This study is the first to identify a genetic and phylogeographic structure in T. magnatum. The presence of a genetic structure can be of practical interest in tracing truffle populations according to their geographic origin for marketing strategies. Evidence for extensive outcrossing in field populations of T. magnatum also is provided for the first time.     

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).     

New evidence for nitrogen fixation within the Italian white truffle Tuber magnatum

Diversity of nitrogen-fixing bacteria and the nitrogen-fixation activity was investigated in Tuber magnatum, the most well-known prized species of Italian white truffle. Degenerate PCR primers were applied to amplify the nitrogenase gene nifH from T. magnatum ascomata at different stages of maturation. Putative amino acid sequences revealed mainly the presence of Alphaproteobacteria belonging to Bradyrhizobium spp. and expression of nifH genes from Bradyrhizobia was detected. The nitrogenase activity evaluated by acetylene reduction assay was 0.5-7.5μmolC(2)H(4)h(-1)g(-1), comparable with early nodules of legumes associated with specific nitrogen-fixing bacteria. This is the first demonstration of nitrogenase expression gene and activity within truffle.     

Specific PCR-primers as a reliable tool for the detection of white truffles in mycorrhizal roots

During their symbiotic phase, white truffles are barely distinguishable morphologically, and molecular probes are needed for their identification. Here we report the design of species-specific primers for two white truffles ( Tuber magnatum and T. borchii ) on the basis of their ITS sequence. Their efficiency has been successfully tested on fruit bodies of the same species, many related and unrelated fungal species, as well as mycorrhizal roots in direct, nested and multiplex PCR experiments. They have allowed us to identify T. magnatum mycorrhizas for the first time.     

Molecular and morphological characterization of Tuber magnatum mycorrhizas in a long-term survey

Tuber magnatum Pico is an ectomycorrhizal fungus whose mycorrhizas can be barely distinguished morphologically from those of other related white truffles. Here we describe the use of specific primers based on the T. magnatum ITS sequence for screening mycorrhizas from a large number of growth chambers, greenhouse and nursery samples taken in a long-term survey. This molecular identification technique enabled a new morphological characterization to be set up for T. magnatum mycorrhizas.     

Tuber magnatum Pico, a species of limited geographical distribution: its genetic diversity inside and outside a truffle ground

The aim of this work was to clarify the genetic structure of the ectomycorrhizal fungus, Tuber magnatum Pico, in a natural truffle ground located in north Italy. Ascomata of this population of T. magnatum were collected over a period of up to 5 years. For comparative analysis, T. magnatum fruit bodies of different geographical origin were also considered. We used single locus markers, such as the variable region of ribosomal genes (ITS), the beta-tubulin gene and sequence-characterized amplified regions (SCAR), as tools to identify single-nucleotide polymorphisms (SNPs). On the basis of the molecular results, which were indirectly supported by a karyological analysis, a self-fertilization mechanism is suggested. A SCAR region was polymorphic within the samples of the truffle ground, leading to the identification of two genotypes. In addition, both the SCAR and the ITS proved to be polymorphic among samples coming from different geographical regions, revealing a genetic differentiation in T. magnatum.     

Tinea Capitis in the Paediatric Population in Milan, Italy: The Emergence of Trichophyton violaceum

Tinea capitis (TC) is the most common type of dermatophytosis in children. The epidemiology of TC depends on the geographical areas, and it changes over time. The aim of the study is to determine the incidence of TC and to identify the causative species in children observed at Dermatology Outpatient Department of the University of Milan, Italy, between January 2004 and December 2011. Four hundred and eighty-six children with suspected dermatomycosis were observed; TC was the most prevalent dermatomycoses with 86 cases. The most common isolated dermatophyte in scalp lesions was Trichophyton violaceum with 33 cases. The most recent epidemiological Italian studies still show zoophilic fungi as primary cause of TC. We are the first medical team in Italy to demonstrate a dominance of anthropophilic fungi, in particular T. violaceum.     

Morphological and molecular typing of the below-ground fungal community in a natural Tuber magnatum truffle-ground

The aims of the work were to elucidate the distribution of the ectomycorrhizal fungus Tuber magnatum Pico during its symbiotic stage, and to identify the root-associated fungi in a natural truffle-ground located in North Italy. Ectomycorrhizal root tips were harvested in the truffle ground, sorted in morphotypes and analyzed by ITS. Morphological and molecular analyses revealed that (i) T. magnatum mycorrhizae were rare and independent on the fruitbody productions and (ii) the dominant fungal species belonged to Thelephoraceae, followed by Tuberaceae and Sebacinaceae.     

Updated Italian checklist of Soldier Flies (Diptera,Stratiomyidae)

An updated checklist for Stratiomyidae of Italy is presented. Previous knowledge and information are put together in a comprehensive way, integrated also with results obtained by sampling with Malaise traps in some of the test areas of the LIFE+ project ManFor C.BD.At the time of writing, with 91 known species, the Italian fauna of Stratiomyidae is the richest in Europe. Neopachygaster meromelas (Dufour, 1841) and Zabrachia minutissima (Zetterstedt, 1838) are new to the Italian fauna. A comprehensive key to the European species of Chorisops Rondani, 1856 is given.     

Occurrence and diversity of bacterial communities in Tuber magnatum during truffle maturation

Tuber magnatum, an ascomycetous fungus and obligate ectomycorrhizal symbiont, forms hypogeous fruit bodies, commonly called Italian white truffles. The diversity of bacterial communities associated with T. magnatum truffles was investigated using culture-independent and -dependent 16S rRNA gene-based approaches. Eighteen truffles were classified in three groups, representing different degrees of ascocarp maturation, based on the percentage of asci containing mature spores. The culturable bacterial fraction was (4.17 +/- 1.61) x 10(7), (2.60 +/- 1.22) x 10(7) and (1.86 +/- 1.32) x 10(6) cfu g(-1) for immature, intermediate and mature ascocarps respectively. The total of bacteria count was two orders of magnitude higher than the cfu g(-1) count. Sequencing results from the clone library showed a significant presence of alpha-Proteobacteria (634 of the 771 total clones screened, c. 82%) affiliated with Sinorhizobium, Rhizobium and Bradyrhizobium spp. The bacterial culturable fraction was generally represented by gamma-Proteobacteria (210 of the 384 total strains isolated, c. 55%), which were mostly fluorescent pseudomonads. Fluorescent in situ hybridization confirmed that alpha-Proteobacteria (85.8%) were the predominant components of truffle bacterial communities with beta-Proteobacteria (1.5%), gamma-Proteobacteria (1.9%), Bacteroidetes (2.1%), Firmicutes (2.4%) and Actinobacteria (3%) only poorly represented. Molecular approaches made it possible to identify alpha-Proteobacteria as major constituents of a bacterial component associated with T. magnatum ascoma, independently from the degree of maturation.     

Tuber latisporum sp. nov. and related taxa, based on morphology and DNA sequence data

A new species of white truffles, Tuber latisporum, is described from southwestern China. This species can be distinguished from other species in the genus by its white and pubescent ascomata, pseudoparenchymatous peridium and broadly ellipsoid ascospores with alveolate reticulum. Molecular phylogenetic analyses of this and morphologically similar species using ITS sequences demonstrated that it is significantly different from other white truffles such as T. borchii, T. puberulum, T. zhongdianense, T. liui, T. dryophilum, T. magnatum, T. rapaeodorum, T. foetidum and T. maculatum. Data from ITS sequences indicate that white and black truffles are not monophyletic lineages.     

Soil analysis reveals the presence of an extended mycelial network in a Tuber magnatum truffle-ground

Truffles are hypogeous ectomycorrhizal fungi. They belong to the genus Tuber and are currently considered a hot spot in fungal biology due to their ecological and economic relevance. Among all the species, Tuber magnatum is the most appreciated because of its special taste and aroma. The aim of this work was to set up a protocol to detect T. magnatum in soil and to assess its distribution in a natural truffle-ground. We used the β-tubulin gene as a marker to identify T. magnatum in the soil. This gene allowed us to trace the distribution of the fungus over the entire truffle-ground. Tuber magnatum was found, in one case, 100 m from the productive host plant. This study highlights that T. magnatum mycelium is more widespread than can be inferred from the distribution of truffles and ectomycorrhizas. Interestingly, a new haplotype – never described from fruiting body material – was identified. The specific detection of T. magnatum in the soil will allow to unravel the ecology of this fungus, following its mycelial network. Moreover, this new tool may have practical importance in projects aimed to increase large-scale truffle production, checking for T. magnatum persistence in plantations.     

Phylogenetic relationships between European and Chinese truffles based on parsimony and distance analysis of ITS sequences

Phylogenetic relationships among truffle species from Europe and China were investigated through parsimony analysis of the ITS sequences. Three major clades were obtained among the species analysed. The so-called white truffles appeared polyphyletic since Tuber magnatum was grouped with brown truffles and not with the other white species (T. maculatum, T. borchii, T. dryophilum, T. puberulum). The black truffles investigated in this study, T. brumale, T. melanosporum, T. indicum and T. himalayense, were grouped in an independent clade. The Périgord black truffle T. melanosporum and the Chinese black truffles T. indicum and T. himalayense, were very closely related. The delimitation of these species was estimated by a distance analysis on several isolates collected from different geographic areas. In spite of intraspecific variations of the internal transcribed spacers (ITS) sequences, T. melanosporum and the Chinese black truffles can be unambiguously attributed to distinct taxa.     

Multi-locus microsatellite analysis supports the hypothesis of an autochthonous focus of Echinococcus multilocularis in northern Italy

Echinococcus multilocularis is characterised by a wide geographical distribution, encompassing three continents (North America, Asia and Europe) yet very low genetic variability is documented. Recently, this parasite has been detected in red foxes (Vulpes vulpes) circulating in an Alpine region of Italy, close to Austria. This finding raised the question as to whether an autochthonous cycle exists in Italy or whether the infected foxes originated from the neighbouring regions of Austria. Studies have shown that multi-locus microsatellite analysis can identify genomic regions carrying mutations that result in a local adaptation. We used a tandem repeated multi-locus microsatellite (EmsB) to evaluate the genetic differences amongst adult worms of E. multilocularis collected in Italy, worms from neighbouring Austria and from other European and extra-European countries. Fluorescent PCR was performed on a panel of E. multilocularis samples to assess intra-specific polymorphism. The analysis revealed four closed genotypes for Italian samples of E. multilocularis which were unique compared with the other 25 genotypes from Europe and the five genotypes from Alaska. An analysis in the Alpine watershed, comparing Italian adult worms with those from neighbouring areas in Austria, showed a unique cluster for Italian samples. This result supports the hypothesis of the presence of an autochthonous cycle of E. multilocularis in Italy. EmsB can be useful for ‘tracking’ the source of infection of this zoonotic parasite and developing appropriate measures for preventing or reducing the risk of human alveolar echinococcosis.     

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.     

Mitochondrial DNA and microsatellite markers evidence a different pattern of hybridization in red-legged partridge (Alectoris rufa) populations from NW Italy

Genetic introgression with exotic genomes represents a major conservation concern for the red-legged partridge (Alectoris rufa, Phasianidae). In particular, massive releases of chukar partridges (Alectoris chukar) and/or red-legged × chukar partridge hybrids for hunting purposes have cast serious doubts on the Italian present-day occurrence of wild A. rufa populations not affected by introgressive hybridization. This study investigates the genetic structure of red-legged partridges populations in two ecologically different areas in Northern Italy. Analysis of maternal mitochondrial DNA and biparental microsatellite markers excluded the presence of hybridization in a typical agricultural habitat where hunting and release of reared birds are strictly banned. By contrast, signs of chukar introgression were detected in a perifluvial habitat unusual for the red-legged partridge in Italy. The present study documents the first red-legged partridge population with no genetic evidences of recent chukar introgression presently living in Italy. We recommend that urgent conservation actions are taken to preserve the genetic integrity of this population from the risk of hybridization with farm-reared birds and to support its long-term conservation.     

Analysis of STAT5A/AvaI Gene Polymorphism in Four Italian Cattle Breeds

The STAT5A/AvaI polymorphism was investigated with PCR-RFLP in a sample of 339 cattle belonging to four breeds: Italian Friesian, Jersey, Italian Brown, and Podolica reared in south Italy. All three possible genotypes for the C/T polymorphism were identified. In these breeds, PCR-RFLP showed the predominance of the TT genotype in Italian Brown and Jersey cows; in Podolica and Italian Friesian CT is the most frequent genotype. The frequency of the T allele ranged from 0.55 to 0.81 in the analyzed populations. The distribution of genotypic and allelic frequencies at this locus was significantly different among the four populations based on a χ² test (P < 0.001), suggesting that the molecular characteristics of the STAT5A gene could be significantly affected by the breed selection. Gene heterozygosity, gene homozygosity, effective allele number, fixation index, and polymorphism information content (PIC) were calculated. The observed heterozygosity, as well as the N _e and PIC values, indicates high genetic variability in the Podolica breed. Podolica could be considered an interesting reservoir of genetic diversity for a species under high selective pressure elsewhere.     

First detection of anti-Besnoitia spp. specific antibodies in horses and donkeys in Italy

Among Apicomplexa protozoa infecting equids, Besnoitia spp., Toxoplasma gondii and Neospora spp. represent important issues from a sanitary and zootechnical viewpoint. However, only scarce epidemiological data are available on the spread of the infections in horses and donkeys in Europe. Therefore, a serosurvey was planned to estimate the prevalence of these Sarcocystidae species in Italian equids. Serum samples from 268 horses and 18 donkeys raised in Italy were collected and serologically analyzed to detect anti-Besnoitia spp., anti-T. gondii and anti-Neospora spp. antibodies: an approach based on an initial screening by in-house ELISA followed by a confirmatory WB was used. Two horses (0.7%) and four donkeys (22.2%), showed antibodies anti-Besnoitia spp. Ten horses (3.7%) resulted positive to T. gondii and one of these (0.4%) was seropositive also to Neospora spp. This is the first detection of anti-Besnoitia spp. specific antibodies in Italian horses and donkeys. The study confirmed the circulation of Besnoitia spp. among equids in Europe. Low prevalence of T. gondii and Neospora spp. in horses raised in Italy was reported. Nevertheless, it is noteworthy to consider that consumption of horse meat could represent a source for human toxoplasmosis.