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Hop (Humulus lupulus L.) is a worldwide distributed crop, a perennial, dioecious and climbing species classified in the family Cannabaceae. The female hop flower (hop cone) is used in brewing to impart flavors (bitterness and aroma) and to preserve beer durability. Hop has also long been used in traditional medicine, especially as a sedative and antimicrobial agent. Traditionally, a few commercial varieties are used for brewing. Wild hops are distributed throughout the Northern Hemisphere and molecular phylogeny has shown that European, Asian and North American hops are clearly distinct. Modern hop varieties are characterized by limited genetic variation and wild germplasm is needed to provide new genetic resources for breeding. In addition, the characterization of wild Italian hops may also allow the production of beers based on local ingredients. To evaluate the intraspecific diversity of wild hop in Umbria region, several plants have been collected from 20 different locations. The DNA extracted from these plants has been genotyped with microsatellite markers. Wild accessions will also be evaluated for their aromatic profile and bitter acids composition. Some selected plants will be used for surveying microbreweries to evaluate which hop accessions are the most interesting for beer production. Moreover, a characterization of the hop endophytic fungi is in progress to identify fungal species producing bioactive compounds.

The growth of trees in forests is promoted by root colonization by ectomycorrhizal fungi, which assist the trees in nutrient uptake. Most ectomycorrhizal fungi belong to Basidiomycota and Ascomyceta and some of them provide also non-wood products of forest such as Tuber spp.. These are ecomycorrhizal Ascomycota producing edible fructifications, called truffles upon fertilization between strains of opposite mating type. Truffle formation faces important bottlenecks: the initiation of the sexual reproduction and the growth of ascocarps during a period of several months. It is therefore critical to better understand the mechanisms leading to sexual reproduction such as recognition between compatible strains. Using genomic resources the genes involved in Tuber melanosporum (the Pe?rigord black truffle) sexual reproduction have been characterized but their role cannot be verified by genetic approach due to the absence of genetic tools. More generally, genetic tools are lacking for many ecologically and economically important Ascomycota for which genomic resources are or will be soon available. Belonging to the Pezizomycete class, the saprophytic Ascomycete Ascobolus immersus is closely related to T. melanosporum, also a Pezizomycete. A. immersus has been used as model organism for decades: it is easy to grow in vitro and its sexual cycle can be completed within two weeks in lab conditions. To propagate itself, as truffle species do, this heterothallic fungus relies on sexual reproduction only. Thus, when two strains of opposite mating type (MAT) encounter each other, female gametes are fertilized by male gametes of the opposite MAT, which leads to the building of the fruiting body. Germination of the produced ascospores gives rise to new generations of fungal individuals. In addition, A. immersus can be efficiently transformed and using reverse genetic strategy, several of its genes have already been knocked-out. The aim of our project is to use A. immersus as a test tube to address fundamental questions for other Ascomycete species such as truffles. To start with, we will focus on deciphering the truffle genetic mechanisms involved in strains recognition for sexual reproduction. Recently the first genetic transformation of A. immersus with promoters of mating type genes from T. melanosporum that drive the expression of the GFP gene was realized to gain functional evidence on these regulatory sequences. Overall, the use of A. immersus as test tube should lead to the development of new tools to address fundamental issues on life cycle and reproductive biology of un-culturable ascomycetes or ascomycetes that cannot be efficiently transformed.

The toxicity vs. edibility of some Amanita spp. is a long-lasting debate among mycologists. Morphological, phytochemical and toxicological tests can hardly distinguish between A. ovoidea and A. proxima, raising the question whether or not they belong to the same species. Here, we performed for the first time molecular analyses of fungal samples displaying either Amanita ovoidea or A. proxima morphotype, with the aim to shed lights into their phylogenetic relationships. In agreement with Bulliard's and Dumée's first descriptions, we show that A. ovoidea and A. proxima belong to different genetic clusters and, likely, are member of two different species or species complexes. Further investigations employing additional Amanita samples are needed to get a closer look into the phylogenesis of this genus.

The article reports the new results regarding the truffle biology; in particular, the sexual reproduction of the Tuber melanosporum and the possible applications of these basic researches are mentioned.

Typing truffle species by morphological markers is often problematic. Molecular approaches can now be employed to differentiate among truffle species with similar morphological features. Coupling molecular markers to the host plant inoculation process has recently allowed scientists to produce mycorrhizae of T.magnatum under controlled condition and describe ex novo their morphology to avoid species mischaracterization. The combination of morphological and molecular analyses is essential to certify plants inoculated with truffle species and promote a successful cultivation of these fungi.

The last several years have witnessed a leap forward in our understanding of the life cycle and the reproductive biology of truffles . The sequencing of Tuber melanosporum genome has permitted the identification of the MAT locus, which is the genomic region controlling the reproduction mode of fungi . Since T. melanosporum strains harbor either the MAT1-1-1 or MAT 1-2-1 gene at this locus, we concluded that this fungus is heterothallic . The availability of sequence information for T. melanosporum MAT genes gives way to cloning their orthologs in other truffle species. Among these, the Asiatic species T. indicum is the closest relative of T. melanosporum, and produces black truffles of inferior value with respect to the Périgord truffle. The high rate of polymorphism at morphological and molecular traits within black truffles from Asia prompted several authors to claim the presence of several other species in addition to T. indicum . Here, benefiting from the information and genomic tools relative to T. melanosporum MAT genes, we aimed to: a) corroborate the occurrence of a mating type dependent spatial segregation of T. melanosporum strains on host plants and b) clone the orthologs of these genes from T. indicum. With respect to the first point we collected ectomycorrhizas and soil samples from four T. melanosporum grounds located in central Italy and screened them with MAT specific primer pairs. These results lend further support to our contention of a spatial segregation, likely due to a competitive exclusion between genotypes, of strains of opposite mating type on their hosts. Concerning T. indicum, here we show that it is also a heterothallic species since its MAT locus is organized in two idiomorphs harbored by different strains . By virtue of their high evolutionary rates, the MAT genes are good candidates for taxonomic and phylogenetic analyses [10]. On these grounds, the comparative analyses of MAT locus from several Chinese specimen point to the presence of at least two cryptic species within the T. indicum complex and support the hypothesis of vicariance for explaining the allopatric isolation of T. melanosporum from T. indicum populations in Europe and Asia, respectively. Notwithstanding, all T. indicum MAT1-1-1 and MAT1-1-2 genes show a high rate of similarity with their orthologs from T. melanosporum. The evidence that T. indicum is not only the closest relative to T. melanosporum, but it is also heterothallic is pragmatically very relevant. Because T. indicum and T. melanosporum could be sexually compatible, the deliberate or accidental introduction of T. indicum in Europe may represent a serious ecological threat for the indigenous populations of T. melanosporum. Tools and strategies that span from innovative host plant mycorrhization techniques to methods for certifying species identity and mating type of truffle strains will be discussed. These instruments are fundamental to preserve T. melanosporum biodiversity from erosion and counteract gamete limitation that likely occurs in T. melanosporum stands, as a important consequence of the spatial segregation of strains with different mating types.

Tuber melanosporum Vittad. is the symbiotic ascomycete that produces the Périgord truffle, one of the most expensive foods worldwide. Although basic research on symbiotic ascomycetes lags behind that on symbiotic basidiomycetes, some progress has been made in the last few years on key questions concerning the biology and ecology of Tuber spp. In particular, the recent release of the T. melanosporum genome was a breakthrough that helped improve our understanding of the reproductive biology of this species. It also provided insight into the gene repertoires underlying the symbiotic relationship with host plants and the production of the distinctive truffle aroma. Genome mining has provided mycologists with a plethora of microsatellite markers that may be highly informative for population genetics studies. These recent advances in T. melanosporum research are discussed here in the context of fundamental research questions and the cultivation and marketing of truffle species.

Previous studies have shown that within the T. aestiuvum species-complex, specimen can show a continuum of morphological traits between the T. uncinatum and the T. aestivum morphotypes and different ITS/RFLP patterns (Paolocci et al. 2004). T aestivum, differently from T. melanosporum and T. magnatum whose geographical ranges are confined around the Mediterranean area and surroundings, is distributed all over Europe. Natural T. melanosporum and T. magnatum populations from the southernmost areas of their ranges show higher levels of genetic diversity than those from northern sites (Murat et al. 2004; Rubini et al. 2005; Riccioni et al. 2008). As it is likely that these species experienced a population bottleneck during the last ice age, these data have been interpreted to mean that refugia for T. magnatum and T. melanosporum were located in the southernmost areas of their original ranges to spread northward as the ice receded. Whether T. aestivum also experienced a population bottleneck during the last ice age is currently unknown. A detailed analysis of distribution and extent of genetic variability within and among its southernmost populations would certainly help to address this crucial point and likely mark these truffles according to their origin. To this end, we started collecting truffles from different natural sites located in the Anatolian peninsula (Turkey). Here we present preliminary results on biodiversity of T. aestivum samples from this region by means of ITS sequencing analysis. These preliminary analyses let us to argue that T. aestivum-T. uncinatum truffles from Anatolian peninsula could be typed according to their provenance and that they likely display specific signatures to be differentiated from European populations. The ongoing sequencing of ITS from additional Anatolian samples and alignment with sequences of T. aestivum truffles from all over Europe will provide us with more data to verify these hypotheses.

Tuber melanosporum is an ectomycorrhizal ascomycete producing edible fruiting bodies, known as black truffles. For their distinctive taste and aroma the truffles produced by different Tu-ber spp. are appreciated worldwide. Therefore these fungi are of considerable economic importance and their market is steadily expanding. The demand for truffles greatly exceeds their natural production that has been largely dropped over the last dec-ades. Thus, the presence of these products in the market depends largely on their cultivation. How-ever, attempts of truffle cultivation by outplanting nursery-produced mycorrhizal plants have been not always, and not for all species, successful. Yet, most of the ecological and biological aspects that govern truffle life cycle such as their reproduction mode, are still under debate, since these fungi are neither amenable for in vitro cultivation nor for mating ex-periments under controlled conditions. Thus, many basic aspects concerning these fungal species have not been fully elucidated yet and truffle sex is a long lasting dilemma for mycologists.Mating in fungal species occurs either through a heterothallic (self-sterile) or homothallic (self-fertile) mechanism. In heterothallic species, mating takes places between two sexually compatible strains that are morphologically identical and are distinguished genetically by mating type. In homothallic species, each strain can mate itself.1 Mating type in the As-comycota is typically bipolar, determined by two possible DNA sequences at the mating-type locus (MAT). The alternative sequences at MAT locus are not alleles in the classic sense because they lack significant sequence similarity and encode different transcriptional regulators. The term idiomorph is used to describe this unusual genetic organization, which is common among all MAT loci from heter-othallic ascomycetes investigated to date. The two alternative MAT idiomorphs encode dif-ferent transcription factors that permit sexual com-patibility: the two key mating type genes conserved in all filamentous ascomycetes encode regulatory proteins with a high mobility group (HMG) or a ?-box DNA-binding domain in MAT1-2 and MAT1-1 idiomorphs, respectively. Until recently, the prevalent view was that truffles strictly self, since genetic analyses had never detect-ed heterozygotic profiles in their putatively diploid/dikaryiotic structures. By using highly informative molecular markers such as SSR and SNPs, we have recently provided evidence for the prevalence of the haploid phase in the truffle life cycle and for the occurrence of outcrossing in both T. melanosporumand Tuber magnatum.2-4Despite this new finding, however, the hypothesis of selfing is far from being settled.Here we took advantage of the recently pub-lished T. melanosporum genome,5 and by using a combination of bioinformatics and genome-based approach, we attempted to clone and characterized the second mating idiomorph in T. melano-sporum

Vengono desctritti i principali metodi di analisi molecolare applicati all'identificazione di ascocarpi e micorrize di Tuber.

Tubers spp. are fungi that establish symbiosis with several trees and shrubs. Some of these fungal species produce edible ascomata, also known as truffles, which are highly appreciated for their taste and odour. We isolated and characterized eight polymorphic microsatellite loci from Tuber magnatum, the finest white truffle species, and assessed their variability in 370 individuals collected from all over the species range of distribution. Although two to 18 alleles per locus were found, no heterozygous individuals were observed. The availability of simple sequence repeat loci provides valuable tools for assessment of the genetic structure and population dynamics in this species.

Tuber spp. Are ectomycorrhizal fungi that establish symbioses with shrubs and trees. Because of their different smell and taste, Tuber uncinatum and Tuber aestivum are two truffle morphotypes with a different market value, but whether or not T. uncinatum and T. aestivum, are different taxa is still an open debate among mycologists. In order to identify molecular keys characterizing both T. aestivum and T. uncinatum morphotypes, ITS/RFLPs analyses were carried out on a large collection of samples from all over Italy and from other European countries, followed by a study of the phylogenesis of ITS, ß-tubulin and EF 1-± genes, on representative samples. The present study provides compelling evidence that: (i) T. uncinatum and T. aestivum belong to the same species, (ii) neither morphotype presents a specific molecular fingerprints, but they may even share identical alleles at any of the loci analysed; (iii) T. aestivum is most likely under a selfing reproductive mode. Our findings suggest that ecological, rather than genetic causes may account for differences in sporal morphology, taste and smell between T. aestivum and T. uncinatum truffles.

Vengono descritti vari metodi per l'identificazione molecolare di ascocarpi emicorrize di Tuber.