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Protoplast fusion technology originated from cell-cell fusion technology in the 1950s and was initially applied to animal cells. The advantage of applying it in the field of edible fungi breeding is that compared with conventional natural selection and hybrid breeding methods, it could more effectively overcome certain incompatibility obstacles to achieve distant hybridization, and more purposefully select parents to obtain new varieties with outstanding characteristics. For these reasons protoplast fusion technology is becoming of great significance for the improvement of edible fungi strains, the breeding of new varieties, and the taming of difficult-to-cultivate species. At present, relatively stable and capable-of-fruiting fusants are limited to several genus and species, and using protoplast fusion for distant hybridization requires new breakthroughs overcoming vegetative incompatibilities. Technically, the acquisition and identification of fusants could also be improved to a more controllable approach. This article focuses on the longitudinal discussion of the role of protoplast fusion technology in the genetic breeding of edible fungi. It explains: i) the preparation and regeneration of protoplasts in the process, ii) the specific methods of fusion, iii) the identification, mutagenesis, and rejuvenation of fusants, and iv) possible impacts of two incompatible systems. Certain application practice cases are also introduced to summarize its promotion prospects.

Truffles of the Tuber genus (Pezizales, Ascomycetes) are among the most valuable and expensive foods, but their shelf life is limited to 7–10 days when stored at 4 °C. Alternative preservation methods have been proposed to extend their shelf life, though they may alter certain quality parameters. Recently, a hypogeal display case equipped with an ultrasonic humidity system (HDC) was developed, extending the shelf life to 2–3 weeks, depending on the truffle species. This study assesses the efficacy of HDC in preserving Tuber melanosporum and Tuber borchii ascomata over 16 days, using quantitative magnetic resonance imaging (QMRI) to monitor water content and other parameters. Sixteen T. melanosporum and six T. borchii ascomata were stored at 4 °C in an HDC or a static fridge (SF) as controls. QMRI confirmed that T. borchii has a shorter shelf life than T. melanosporum under all conditions. HDC reduced the rate of shrinkage, water, and mass loss in both species. Additionally, the Apparent Diffusion Coefficient (ADC), longitudinal relaxation time (T1), and transverse relaxation time (T2), which reflect molecular changes, decreased more slowly in HDC than SF. QMRI proves useful for studying water-rich samples and assessing truffle preservation technologies. Further optimization of this method for industrial use is needed.

Abstract Background Plastic is one of the most widely used materials worldwide in various fields, including packaging and agriculture. Its large quantities require proper disposal and for this reason more and more attention is paid to the issue of degrading plastic. Thanks to the production of non-specific enzymes, fungi are able to attack complex and recalcitrant xenobiotics such as plastics. In recent years, several spectroscopic methods were used to study the plastic degradation ability of different fungal species. Among these, Fourier transform infrared (FT-IR) and FT-Raman spectroscopy techniques are the most used. Surface-enhanced Raman scattering (SERS) spectroscopy is a powerful technique which uses metal nanoparticles (NPs) to enhance the Raman signal of molecules adsorbed on the NPs surface. In this work, the isolation of different fungi from field-collected plastic debris and the ability of these isolates to growth and colonizing the low-density polyethylene (LDPE) were explored by using scanning electron microscope (SEM), attenuated total reflectance-Fourier transform infrared (ATR-FTIR) and SERS spectroscopies. Results Forty-seven fungal isolates belonging to 10 genera were obtained; among them only 11 were able to grow and colonize the LDPE film. However, after 90 days trial, only one isolate of Cladosporium cladosporioides (Clc/1) was able to carry out the initial degradation of the LDPE film. In particular, based on SEM observations, small cavities and depressed areas of circular shape were visible in the treated samples. Additionally, ATR-FTIR, normal Raman and SERS analyses supported the structural changes observed via SEM. Notably, ATR-FTIR and normal Raman spectra showed a significant decrease in the relative intensity of the methylene group bands. Similarly, the SERS spectra of LDPE after the fungal attack, confirmed the decrease of methylene groups bands and the appearance of other bands referring to LDPE polyphenolic admixtures. Conclusions These results suggest that Cladosporium cladosporioides Clc/1 is able to carry out an initial degradation of LDPE. Moreover, combining ATR-FTIR, Raman and SERS spectroscopies with SEM observations, the early stages of LDPE degradation can be explored without any sample pretreatment. Graphical Abstract

The ascomycetes in the genus Morchella (commonly called morels) have a considerable economic and biotechnological value for their culinary and medicinal proprieties. However, their biotechnological interest is not limited to the fruiting body cultivation, but also in mycelial production to obtain bioactive compounds and other biotech products. In order to better exploit the biotechnological potentialities of morels it is necessary to improve the knowledge on their biology and mycelial characteristics. In this paper morphological and growth characteristics of mycelia, as well as biomass formation of Italian collections of five edible medicinal Morchella species [M. esculenta (L.) Pers., M. dunalii Boud., M. importuna M. Kuo, O'Donnell & T.J. Volk, M. disparilis Loizides & P.-A. Moreau and M. purpurascens (Krombh. ex Boud.) Jacquet.] are presented.

Tuber magnatum Picco is the most expensive of the truffles and a great deal of research has been carried out in an attempt to solve the mysteries of its ecology and biology. However, considerable work remains to be done particularly on those secrets of its life cycle that remain a mystery. It is known that T. magnatum is heterothallic, but it has yet to be determined how fertilization occurs between the two strains of different mating types. It is also known that the white truffle is an ectomycorrhizal fungus, and its mycorrhizas can be produced in greenhouses, but then they seem to disappear in the field. The role of other soil microorganisms, fungi and bacteria, on its soil mycelial development and fructification is intriguing but is far from being completely understood. All these uncertainties have made the cultivation of T. magnatum extremely difficult and only recently have we had the scientific proofs that it is possible. Even so, many questions remain unanswered and the management practices of T. magnatum plantations are still to be better defined to also enable the taming of this truffle.

Abstract Background Mushrooms have always been considered an important source of food and biologically active compounds with several medicinal properties. In recent years, different methods were used to study the quality and chemical composition of mushrooms. Among these, Fourier transform infrared (FT-IR) and FT-Raman spectroscopy techniques have been successfully applied to identify different mushroom species. However, the structural biomolecule components existing in the mycelium or in the fruiting bodies may produce strong fluorescence emission that overlaps the Raman radiation, thus avoiding their analyses by Raman. SERS spectroscopy is a powerful technique which uses metal nanoparticles (NPs) to enhance the Raman signal of molecules adsorbed on the NPs surface. In addition, SERS is able to quench the macromolecule florescence. In this work, we have employed silver nanoparticles in order to get mushroom fingerprints based on SERS as quick procedure to analyze and identify different chemical compounds from the fruiting bodies of six edible and/or medicinal mushrooms: Lentinula edodes, Ganoderma lucidum, Pleurotus cornucopiae, Pleurotus ostreatus, Tuber aestivum and Tuber magnatum. Results SERS analyses performed directly on fruiting body fragments produced characteristic spectra for each species. One group of mushrooms (L. edodes, G. lucidum, T. aestivum and T. magnatum) was dominated by the bands of nucleic acids; and the other one (P. cornucopiae and P. ostreatus), by the bands of pigments such as melanins; carotenoids; azafilones; polyketides; and flavonoids located in the cell wall. Additionally, bands corresponding to cell wall polysaccharides, particularly chitosan and 1,3-β D-glucan, were identified in the extracts of P. cornucopiae, P. ostreatus and L. edodes. No signal of cell wall polysaccharides was found in G. lucidum extract. Raman mapping of the analyzed samples was useful in tracking the spatial distribution of the marker bands. Moreover, the principal component analysis (PCA) carried out on the acquired SERS spectra, allows to discriminate the analyzed mushroom species. Conclusions The SERS technique has the ability to generate a strong Raman signal from mushroom fruiting bodies using Ag-NPs deposited directly on intact, untreated mushroom tissues. Using this methodology, commonly applied laboratory time-consuming methods can be avoided or bypassed as well as analysis time can be reduced. Graphical Abstract

Tuber borchii is a precious truffle, which forms ectomycorrhizal associations with several coniferous and broadleaf trees, arbutoid mycorrhizas with the strawberry tree and recently it was found also to form orchid mycorrhizas with Epipactis atrorubens. In this work, we investigated the formation of T. borchii mycorrhizas in other orchid species (Anacamptis morio, Cephalanthera longifolia, Limodorum abortivum, Ophrys sphegodes, Orchis purpurea). To this aim the orchids growing in three T. borchii production areas located in Emilia Romagna region (Italy) were collected and the presence of T. borchii mycorrhizas was investigated by morphological and molecular approaches. For T. borchii molecular identification, specific primers were used by a nested PCR approach. Tuber borchii was identified in 2 out of 6 samples of A. morio, 3 out of 4 samples of C. longifolia, and 2 out 6 samples of O. sphegodes. The ecological and biological roles of the orchid association in the truffle life cycle are discussed.

Tuber melanosporum is an ascomycete that forms ectomycorrhizal (ECM) symbioses with a wide range of host plants, producing edible fruiting bodies with high economic value. The quality of seedlings in the early symbiotic stage is important for successful truffle cultivation. Numerous bacterial species have been reported to take part in the truffle biological cycle and influence the establishment of roots symbiosis in plant hosts and the development of the carpophore. In this work, three different bacteria formulations were co-inoculated in Quercus ilex L. seedlings two months after T. melanosporum inoculation. At four months of bacterial application, the T. melanosporum ECM root tip rate of colonization and bacterial presence were assessed using both morphological and molecular techniques. A 2.5-fold increase in ECM colonization rate was found in the presence of Pseudomonas sp. compared to the seedlings inoculated only with T. melanosporum. The same treatment caused reduced plant growth either for the aerial and root part. Meanwhile, the ECM colonization combined with Bradyrhizobium sp. and Pseudomonas sp. + Bradyrhizobium sp. reduced the relative density of fibrous roots (nutrient absorption). Our work suggests that the role of bacteria in the early symbiotic stages of ECM colonization involves both the mycorrhizal symbiosis rate and plant root development processes, both essential for improve the quality of truffle-inoculated seedlings produced in commercial nurseries.

From the early 1800s science virtually ignored the cultivation of edible mycorrhizal mushrooms other than the true truffles. The drought was finally broken by Nicole Poitou when she cultivated Lactarius deliciosus and Suillus granulatus in the Institut National de la Recherche Agronomique’s laboratories at Pont-de-la-Maye, France, in the mid-1970s. However, another 20 years were to pass before Yun Wang working at Invermay Agricultural Centre near Dunedin, New Zealand, was able to begin the routine cultivation of L. deliciosus. The purpose of this paper is to document the history of the cultivation of L. deliciosus in New Zealand since its introduction in the late 1990s, its spread back to Europe and its subsequent introduction into Yunnan, Tibet and Sichuan starting about 2014.

Inside commercial batches of T. aestivum imported from Iran to Italy the ascomata of six Tuber spp. (Tuber borchii, Tuber brumale, Tuber macrosporum, Tuber rufum f. lucidum, Tuber excavatum and Tuber fulgens) were found and described. Sixteen specimens were analyzed; most of them were completely immature probably because truffles in Iran are harvested by digging all the soil forest without using trained dogs. The morphology of the Iranian ascomata and their spore dimensions are similar to those of the European ascomata and phylogenetic analyses based on their ITS sequences placed them inside the European clades of Tuber. In Iran, truffles are found in the northern part of the country with the highest production in the Hyrcanian region (between Guilan and Golestan provinces). To date, Iran seems to be the most Eastern location where European species of Tuber are found.

Tuber borchii is an edible ectomycorrhizal mushroom of considerable economic value. Its cultivation has become popular in recent years, but there are few studies on the factors affecting its productivity. In this work, the ascoma production and the ectomycorrhizal (ECM) community of a T. borchii plantation, established in an intensive farming area where this truffle is not naturally present, were studied. Tuber borchii production drastically declined from 2016 to 2021, and ascomata of other Tuber species (T. maculatum and T. rufum) were found from 2017. Molecular characterization of ectomycorrhizae carried out in 2016 identified 21 ECM fungal species, of which T. maculatum (22%) and Tomentella coerulea (19%) were the most abundant. Tuber borchii ectomycorrizae (16%) were almost entirely confined to the fruiting points. The diversity and structure of the ECM community on Pinus pinea were significantly different from those observed on hardwood trees. The obtained results suggest that T. maculatum (a native of the study site) tends to replace T. borchii through a mechanism of competitive exclusion. Although T. borchii cultivation is possible in suboptimal environments, particular care should be taken to limit competition with ECM fungi more suitable for local conditions.

In this work we used an inductive and deductive approach to produce the map of suitability for the spontaneous growth of Tuber magnatum (white truffle) (SSGT) in Emilia-Romagna region (northern Italy). This map was produced in order to identify the environments where appropriate actions should be applied to protect this threatened truffle species.The steps used to define the map were: 1) surveying and mapping the actual productive areas (APAs) in two provinces of Emilia-Romagna (Bologna and Modena); 2) identification of some morphological, climatic, vegetational and pedological properties related to T. magnatum development; 3) production of digital maps representing the value (classified) that each property assumes in discrete land portions; 4) overlapping the map of APAs with each of the thematic maps and selection of the properties showing the strongest relationships with the presence of T. magnatum; 5) creating the map of SSGT, covering the whole hilly area of Emilia Romagna region; 6) assessing the reliability of the SSGT map, by overlapping the APAs map and the areas of the provinces of Parma and Piacenza where the mycelium of T. magnatum was found using species-specific primers.The relationships found by means of the inductive approach (comparison between selected properties and observed frequency of truffle) demonstrated its effectiveness in predicting deductively the areas with distinct suitability for truffle.

In the last decades, the number of studies about the valorization of agricultural by-products has strongly increased due to the growing focus on the circular economy. In this context, mushroom cultivation is raising attention for its nutritional properties and adaptability to different growing conditions. The goal of this project is, therefore, to create a link between two production chains, valorizing the solid anaerobic digestate fraction from the biogas plant as a potential substrate for Pleurotus ostreatus full-scale cultivation. A preliminary in vitro trial shows that the mycelia can colonize at the same rate in both the conventional substrate, which was wheat straw added with sunflower cake and the experimental ones supplemented with corn digestate. The most important and innovative part of the work was full-scale cultivation. Quantitative analyses demonstrate that P. ostreatus is capable of growing on the conventional substrate as well as on the substrate containing 15% of digestate without reducing the mushroom harvest. Even better, digestate seems to stimulate a precocious and more homogeneous production. Chemical and spectroscopic analyses on the fruiting bodies confirm the results; in fact, mushrooms show a comparable structural composition, highlighted with FTIR and SERS spectroscopic evaluations. Moreover, the nutrient content did not change due to the addition of digestate. These promising results demonstrate that anaerobic digestate can become a resource for P. ostreatus cultivation without production losses and maintaining the same qualitative characteristics.

Morchella species are known as famous and prized edible fungi due to their culinary flavor and medicinal properties. The asomata are collected throughout the temperate regions of the northern hemisphere. Morchella spp. taxonomy has long been debated as a result of the high phenotypic plasticity charaterizing the genus. Most morels are considered saprobic but some species has been reported to interact with roots of many plant species forming different types of associations. In Emilia-Romagna (Italy), morels became a part of the culinary tradition, especially for the populations of the coastal areas. This work aimed to describe and identify the ascomata collected on the white dune habitat as well as to verify the extent of the interaction with plant species growing in the same area. All ascomata collected since 2001 shared a similar morphology with a range of variability mainly due to the harvesting period. Ascomata collected in 2017 were grouped within the Mes-17 clade, in the Esculenta group, based on their ITS rDNA sequences. Nine different plant species were found to be connected with morel ascomata through characteristic sand-mycelial structures never described before. The amount of the ascomata collected on the sand dunes demonstrates that the study area is particularly suitable for morel fruiting. Therefore, the protection of the dune system is imperative for preserving Morchella genetic diversity within the local population.

The screening of morphological and growth characteristics of mycelial collections of medicinal bracket fungus Ganoderma adspersum (Agaricomycetes, Polyporales) of different geographical origins (Armenia, Georgia, Iran) on malt-extract agar (MEA) and potato-dextrose agar (PDA) media for 6 days, as well as in submerged culture (malt-extract, ME, 200 rt min) for 14 days at different temperature (25, 30, 35, 38 °C) was performed. Species-specific mycelial macro- and micromorphological characteristics, such as white, cottony-felt, later chamois, leathery, creamy-lemon-yellowish fast-growing colonies, round-shaped hyphal clamps, hyaline, round-shaped, smooth chlamydospore-like swellings and brownish cuticular cells, numerous tetrahedral crystals were described in agar and submerged cultures. The formation of small, dense and smooth pellets, as well as hyphal swellings during submerged growth was observed. The favorable growth temperature for studied collections of G. adspersum was 25-30 °C. The revealed cultural characteristics will be used for taxonomic identification and quality control of mycelial cultures during their biotechnological cultivation.

Recently, the cultivation of hazel is undergoing a large expansion. Italy is the world’s second largest producer of hazelnuts, with a production of around 98,530 tons in 2019. The processing of hazelnuts produces large amounts of waste, especially woody pericarps, due to the cracking process, generally used for domestic heating, causing air pollution. The high lignin content present in the pericarps makes them a suitable substrate for the cultivation of edible and medicinal mushrooms. To this aim, Ganoderma lucidum, Lentinula edodes, and Pleurotus cornucopiae were grown and cultivated on different hazelnut-shell-based substrates: Hazelnut Shell (HS), Hazelnut Shell and Wheat Straw (HS-WS), and Wheat Straw mixed with Beech Chips (WS-BC) as control. In vitro mycelial grow rate, the degradation capacity of the lignocellulosic fraction, the biological efficiency, and the qualitative differences between mushrooms growing on different substrates by using Attenuated Total Reflectance–Fourier transform infrared (ATR-FTIR) spectroscopy were investigated. Our results suggested the ability of G. lucidum, L. edodes, and P. cornucopiae to grow and decay the lignocellulosic fraction of HS. Cultivation trials showed a similar biological efficiency but a different Fruiting Body Production (FBP) in the presence of HS with respect to the control. ATR-FTIR analysis provided a chemical insight for the examined fruiting bodies, and differences were found among the substrates studied. These results provide attractive perspectives both for more sustainable management and for the improvement of mushroom cultivation efficiency.

The first record of Densocarpa crocea (Quél.) Healy & M.E. Sm in Italy is reported. The specimen was morphologically described and the ITS sequence was deposited in GenBank.

Mushroom community in artificial Pinus nigra forest in Amiata mount (Tuscany, Italy) was described. 3220 fruit bodies belonging to 106 different species were found during five surveys from autumn 2014 to spring 2015. The biodiversity indices and the dominance–diversity curves indicate a discrete fungal diversity with the dominance of few species. Edible mushrooms such as Hydnum repandum, Lactarius deliciosus, L. sanguifluus, Suillus granulatus S. luteus and truffles (Tuber aestivum, T. borchii and T. macrosporum) were found in the study area. These fungi are traditionally harvested and are an important complementary economical source for local population. Considering the economic importance of these non-wood forest products, forest management should play major attention to their safeguard.

The cultivation of truffles in the Southern Hemisphere began in New Zealand in the mid-1980s and the first fruiting bodies of Tuber melanosporum were harvested in Gisborne in 1993. Cultivation started in Australia soon after and the first truffles were found in Tasmania in 1999. Now there are more than 150 mostly small truffières in New Zealand, and approximatively 200-250 plantations in Australia some of which are very large. Most of the productive truffières have been established with Tuber melanosporum Vittad. infected plants but more recently fruiting bodies of Tuber borchii Vittad. and Tuber aestivum Vittad. have been harvested. Chile followed by Argentina and South Africa were the next to get involved and produced in 2009, 2014 and 2015 respectively. Over the past 10 years alternative techniques have been tried in Italy, China and New Zealand in attempts to control contaminating ectomycorrhizal fungi in the nursery – arguably the most important problem facing the cultivation of truffles worldwide.

In recent years, fungi have been recognized as producers of acetylcholinesterase (AChE) inhibitors, agents important for the prevention of Alzheimer’s disease (AD). This study aimed to examine the AChE inhibitory, the antioxidative and antibacterial activity of two different Schizophyllum commune strains that originated from Serbia (SRB) and Italy (IT). Submerged cultivation of grown mycelia (M) and fermentation broth (F) of ethanol (EtOH) and polysaccharide (PSH) extracts lasted for 7, 14, 21 and 28 days. For AChE activity Ellman method was performed, while for antioxidative activity, sevendifferent assays were conducted: DPPH, ABTS, FRAP, SOA, OH, NO together with total phenolic content. Antimicrobial screen, LC–MS/MS technique and FTIR measurements were performed. Different isolates exhibited different AChE activity, with PSH being the strongest (SRB, M, 28 days IC90 79.73 ± 26.34 µg/mL), while in EtOH extracts, IT stood out (F, 14 days, IC50 0.8 ± 0.6 µg/mL). PSH extracts (7 days) exhibit significant antioxidative activity (AO), opposite to EtOH extracts where 14 and 21days periods stood out. Only tw extracts showed antibacterial activity. Following LC–MS/MS analysis p-hydroxybenzoic and gallic acids were the most abundant phenolics. PSH extracts demonstrated remarkable results, making this study debut and introducing S. commune as a valuable resource of AChE inhibitors.

The current management practice of digestate from biogas plants involves its use for land application as a fertilizer. Nevertheless, the inadequate handling of digestate may cause environmental risks due to losses of ammonia, methane and nitrous oxide. Therefore, the key goals of digestate management are to maximize its value by developing new digestate products, reducing its dependency on soil application and the consequent air pollution. The high nitrogen and lignin content in solid digestate make it a suitable substrate for edible and medicinal mushroom cultivation. To this aim, the mycelial growth rate and degradation capacity of the lignocellulosic component from corn silage digestate, undigested wheat straw and their mixture were investigated on Cyclocybe aegerita, Coprinus comatus, Morchella importuna, Pleurotus cornucopiae and Pleurotus ostreatus. The structural modification of the substrates was performed by using attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy. Preliminary in vitro results demonstrated the ability of P. ostreatus, P. cornucopiae and M. importuna to grow and decay hemicellulose and lignin of digestate. Cultivation trials were carried out on C. aegerita, P. cornucopiae and P. ostreatus. Pleurotus ostreatus showed the highest biological efficiency and fruiting body production in the presence of the digestate; moreover, P. ostreatus and P. cornucopiae were able to degrade the lignin. These results provide attractive perspectives both for more sustainable digestate management and for the improvement of mushroom cultivation efficiency.

In this review the current status of truffle cultivation in Europe and outside Europe is reported. While the cultivation of Tuber melanosporum (Périgord black truffle), Tuber aestivum (summer or Burgundy truffle) and Tuber borchii (bianchetto truffle) gave good results, only the Italian white truffle (Tuber magnatum), which is the most expensive, has yet to be successfully cultivated. In future a revolutionary approach to truffle cultivation would be the application of mycelial inoculation techniques for producing Tuber infected plants which will allow to select the fungal strains adapted to specific climatic, edaphic conditions and hosts. The new insights which will be gained by the extensive Tuber genome sequencing programme will also help to improve truffle cultivation techniques.

Wild boars (Sus scrofa L.) are well-known for soil disturbance in natural and cultivated truffières but their role in spore dispersal is poorly investigated. In the present work we studied the occurrence of hypogeous fungal spores in faecal contents of 14 wild boars randomly hunted in “Parco dei Gessi e Calanchi dell’Abbadessa” Regional Park (North of Italy) where truffle production has been previously investigated for three years. Six methods for spore analysis in faeces were compared and the suspension of faeces in ZnSO4 (70%) solution resulted to be the most reliable. Hypogeous fungal spores, including Tuber magnatum and Tuber aestivum spores, were detected in 9 animals. This result suggests that the detection of fungal spores in faeces of wild boars may provide information on the presence of hypogeous fungi in an area. However, the poor abundance of spores suggests that the wild boar can be considered an opportunistic mycophagist, ingesting truffles only occasionally, as a seasonal source of food. Considering the magnitude of wild boar movements during seasonal migrations, it is possible to speculate that they play a key role in truffle long distance dispersal.

Tuber magnatum produces the world's most expensive truffle. This fungus produces very rare ectomycorrhizas which are difficult or even impossible to detect in the field. A "real-time" PCR assay was recently developed to quantify and to track T. magnatum mycelium in soil. Here, this technique was used to investigate the spatial distribution of T. magnatum extra-radical mycelium in soil productive patches and its dynamic across seasons. This study was carried out in four different natural T. magnatum truffle grounds located in different Italian regions. During the fruiting seasons, the amount of T. magnatum mycelium was significantly higher around the fruiting points and decreased going farther away from them. Moreover, T. magnatum mycelium inside the productive patches underwent seasonal fluctuations. In early spring, the amount of T. magnatum mycelium was significantly higher than in summer. In summer, probably due to the hot and dry season, T. magnatum mycelium significantly decreased, whereas in autumn it increased again and was concentrated at the putative fruiting points. These results give new insights on T. magnatum ecology and are useful to plan the most appropriate sampling strategy for evaluating the management of a truffle ground.

Truffles have evolved from epigeous (aboveground) ancestors in nearly every major lineage of fleshy fungi. Because accelerated rates of morphological evolution accompany the transition to the truffle form, closely related epigeous ancestors remain unknown for most truffle lineages. This is the case for the quintessential truffle genus Tuber, which includes species with socio-economic importance and esteemed culinary attributes. Ecologically, Tuber spp. form obligate mycorrhizal symbioses with diverse species of plant hosts including pines, oaks, poplars, orchids, and commercially important trees such as hazelnut and pecan. Unfortunately, limited geographic sampling and inconclusive phylogenetic relationships have obscured our understanding of their origin, biogeography, and diversification. To address this problem, we present a global sampling of Tuberaceae based on DNA sequence data from four loci for phylogenetic inference and molecular dating. Our well-resolved Tuberaceae phylogeny shows high levels of regional and continental endemism. We also identify a previously unknown epigeous member of the Tuberaceae--the South American cup-fungus Nothojafnea thaxteri (E.K. Cash) Gamundí. Phylogenetic resolution was further improved through the inclusion of a previously unrecognized Southern hemisphere sister group of the Tuberaceae. This morphologically diverse assemblage of species includes truffle (e.g. Gymnohydnotrya spp.) and non-truffle forms that are endemic to Australia and South America. Southern hemisphere taxa appear to have diverged more recently than the Northern hemisphere lineages. Our analysis of the Tuberaceae suggests that Tuber evolved from an epigeous ancestor. Molecular dating estimates Tuberaceae divergence in the late Jurassic (~156 million years ago), with subsequent radiations in the Cretaceous and Paleogene. Intra-continental diversification, limited long-distance dispersal, and ecological adaptations help to explain patterns of truffle evolution and biodiversity.