Spatial distribution and ecological variation of re-discovered German truffle habitats

Several truffle species (Tuber spp.) are highly prized by chefs and gourmets with some commanding prices of up to €9.000 kg^?1 on international markets. Their ecological drivers and geographical patterns, however, often remain a puzzle. Truffle species in Germany are classified as Very Rare or even Extinct on the national Red Lists, while historical literature described their sporadic existence. Here we present evidence of seven Tuber species (T. aestivum, T. brumale, T. excavatum, T. fulgens, T. macrosporum, T. mesentericum, T. rufum), discovered at 121 sites in Southwest Germany. The valuable Burgundy truffle (T. aestivum) occurred at 116 sites. An unexpected abundance of Tuber spp. associated with 13 potential host plants along wide ecological gradients in a region far outside the traditional Mediterranean truffle foci in France, Italy and Spain, is likely indicative of possible responses to climate change, and also suggests ample truffle cultivation potential north of the Alpine arc.     

The Role of the Microbiome of Truffles in Aroma Formation: a Meta-Analysis Approach

Truffles (Tuber spp.) are ascomycete subterraneous fungi that form ectomycorrhizas in a symbiotic relationship with plant roots. Their fruiting bodies are appreciated for their distinctive aroma, which might be partially derived from microbes. Indeed, truffle fruiting bodies are colonized by a diverse microbial community made up of bacteria, yeasts, guest filamentous fungi, and viruses. The aim of this minireview is two-fold. First, the current knowledge on the microbial community composition of truffles has been synthesized to highlight similarities and differences among four truffle (Tuber) species (T. magnatum, T. melanosporum, T. aestivum, and T. borchii) at various stages of their life cycle. Second, the potential role of the microbiome in truffle aroma formation has been addressed for the same four species. Our results suggest that on one hand, odorants, which are common to many truffle species, might be of mixed truffle and microbial origin, while on the other hand, less common odorants might be derived from microbes only. They also highlight that bacteria, the dominant group in the microbiome of the truffle, might also be the most important contributors to truffle aroma not only in T. borchii, as already demonstrated, but also in T. magnatum, T. aestivum, and T. melanosporum.     

Report on a New Truffle Species,Tuber koreanum sp. nov., from Korea

The truffle and ectomycorrhizal roots formed by Tuber sp. were collected from the rhizosphere of Quercus aliena in Korea. The morphological characteristics of the ascoma, and molecular phylogenetic analysis using sequences from the internal transcribed spacer (ITS) and large subunit (LSU) of ribosomal DNA, translation elongation factor 1-alpha (TEF), and RNA polymerase second largest subunit (RPB2) regions confirmed the distinct morphology of the truffle. This truffle belongs to a monophyletic clade among the other Tuber species in the phylogeny. This study describes the truffle, Tuber koreanum, as a new species reported from Korea.     

Mycorrhization of Quercus spp. with Tuber huidongense and T. himalayense Collected in Korea

Fungi of the genus Tuber are ectomycorrhizal fungi that form a symbiotic relationship mainly with oak and hazel trees. Tuber spp. exhibit a highly selective host plant preference; thus, for cultivation purposes it is important to select an appropriate host plant for successful mycorrhization. In addition, as mycorrhizal characteristics differ according to Tuber spp., it is necessary to understand the differences in mycorrhizae according to the fungal species. Tuber huidongense and Tuber himalayense were recently discovered in Korea; therefore, we used spore suspensions from these two species to inoculate two species of oak trees, Quercus acutissima and Quercus dentata, to compare colonization rates and morphologies of the mycorrhizae. The colonization rates demonstrated that the different Tuber spp. favored different host plant species. In addition, unique morphological and anatomical characteristics were observed for T. huidongense and T. himalayense depending on the host species. These findings can lead to new economically important agricultural activities related to truffle cultivation in Korea.     

A review of research on Chinese <Emphasis Type="Italic">Tuber</Emphasis> species

Truffles are abundant in some regions of China. Nevertheless, it was not until the 1980s that Tuber species were discovered by Chinese mycologists. In recent years, international truffle markets have shown an increasing interest in the import of Chinese Tuber. These truffles serve as a complement to European truffles due to their lower prices and their greater availability in a deficit market. However, Chinese truffles have been the subject of fraudulent commercial practices, and these could have a negative effect on truffle culture. These concerns have been behind numerous recent studies designed to obtain detailed information about Chinese Tuber species. Unfortunately, many of these studies are not published in English, and are dispersed throughout the specific local or national bibliography and proceedings of specialized truffle conferences. In view of the need to expand current knowledge of Chinese Tuber species, we present a comprehensive summary of the taxonomy, ecology, mycorrhizae, genetics, biochemistry, and cultivation of Chinese Tuber species. We also provide a synthetic taxonomy and morphological characterization of 16 Chinese Tuber species in order to assist in their verification and monitoring.     

Cultivation of Mediterranean species of Tuber (Tuberaceae) in British Columbia,Canada

Based on an assessment of soil and climatic conditions in British Columbia (BC), the Truffle Association of British Columbia (TABC) determined that the cultivation of Mediterranean Tuber melanosporum and Tuber aestivum might be possible in the warmer parts of the province. With the cooperation of independent truffle growers, TABC assessed the colonization of host tree roots collected from eight truffle orchards planted 2–7 years earlier using morphological and molecular criteria. Both Tuber species persisted on the roots of inoculated trees in six of the eight truffle orchards studied. The identity of Tuber ectomycorrhizas that had been characterized morphologically as differing from those of T. melanosporum and T. aestivum were determined using DNA sequence analysis to belong to three species of truffles native to the Pacific Northwest. One of those species, Tuber anniae, had been previously reported from BC, but the other two, Tuber menseri nom. prov. and Tuber beyerlei, are reported here from BC for the first time. Recently, production of three Périgord black truffles in one truffle orchard and one Burgundy truffle in another orchard demonstrates that these truffles are able to fruit in BC.     

Molecular and morphological identification of truffle-producing <Emphasis Type="Italic">Tuber</Emphasis> species in New Zealand

Molecular and morphological techniques were used to examine New Zealand ascomycetous truffle (Tuber spp.) samples deposited in the Plant & Food Research and Landcare Research Fungi Herbarium collections. Truffles have been found on the roots of many Northern Hemisphere tree species growing in New Zealand, but not on indigenous plant species. Comparisons of ribosomal DNA sequences proved to be a simple and rapid method to identify the Tuber species. Tuber maculatum was by far the predominant species in New Zealand, and was distributed throughout the country. A single truffle sample from Christchurch was identified as T. rufum. Two other groups of truffle samples from Pinus spp. were closely related to anonymous Northern Hemisphere Tuber sequences. Ascocarps with these sequences have not previously been described. Specific primers for the PCR detection of these Pinus isolates were developed. None of these Tuber species accidentally introduced to New Zealand is of economic value.     

Historical Biogeography and Diversification of Truffles in the Tuberaceae and Their Newly Identified Southern Hemisphere Sister Lineage

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.     

Morphological,mycorrhizal and molecular characterization of Finnish truffles belonging to the Tuber anniae species-complex

The truffle species Tuber anniae was originally described from the U.S. Pacific Northwest and is purported to be uncommon. Here, we report for the first time on the fruiting of closely related taxa in Baltic Rim countries. These truffles were found in a forest dominated by Scots pine in eastern Finland. Mycorrhizal analyses confirmed its symbiosis with Pinus sylvestris. Morphological observations of ascomata and mycorrhizae, and phylogenetic analyses confirmed that these white truffles belong within the group of Tuber puberulum (i.e., Puberulum clade). Further, they group in Clades II and III of the T. anniae species-complex. With the inclusion of sequences from GenBank we are able to demonstrate that the previously unnamed environmental clade (Clade II) has been found as ectomycorrhiza in symbiosis with pine, birch, oak, aspen and even orchids in Europe. Thus, the T. anniae species-complex as a whole (and two of the three clades within) exhibit considerable geographic disjuncts: Northwestern North America and the Baltic Rim of Europe. Clade II, which was collected in agricultural soils in Finland and along roadsides in Alaska, may also be adapted for colonization into new habitats. This may help to explain its presence in New Zealand (where Tuber is not native), which most likely resulted from human-mediated dispersal of these fungi through forestry or the nursery trade. Based on our results, we hypothesize that management practices such as organic and lime amendments, along with aeration, are beneficial to the fruiting of T. anniae. Further research is needed to determine the edibility of these species and whether commercial markets can be developed.     

Mating Type Locus of Chinese Black Truffles Reveals Heterothallism and the Presence of Cryptic Species within the T. indicum Species Complex

Tuber spp. are filamentous ascomycetes which establish symbiosis with the roots of trees and shrub species. By virtue of this symbiosis they produce hypogeous ascocarps, known as truffles. Filamentous ascomycetes can reproduce by homothallism or heterothallism depending on the structure and organization of their mating type locus. The first mating type locus in a truffle species has been recently characterized in Tuber melanosporum and it has been shown that this fungus, endemic in Europe, is heterothallic. The availability of sequence information for T. melanosporum mating type genes is seminal to cloning their orthologs from other Tuber species and assessing their reproductive mode. Here we report on the organization of the mating type region in T. indicum, the black truffle species present in Asia, which is the closest relative to T. melanosporum and is characterized by an high level of morphological and genetic variability. The present study shows that T. indicum is also heterothallic. Examination of Asiatic black truffles belonging to different genetic classes, sorted according to the sequence polymorphism of the internal transcribed spacer rDNA region, has revealed sequence variations and rearrangements in both coding and non-coding regions of the mating type locus, to suggest the existence of cryptic species within the T. indicum complex. The presence of transposable elements within or linked to the mating type region suggests a role of these elements in generating the genotypic diversity present among T. indicum strains. Overall, comparative analyses of the mating type locus have thus allowed us to tackle taxonomical and phylogenetic issues within black truffles and make inferences about the evolution of T. melanosporum-T. indicum lineage. Our results are not only of fundamental but also of applied relevance as T. indicum produces edible fruit bodies that are imported also into Europe and thus may represent a biological threat for T. melanosporum.     

Morphological and molecular evidence support a new truffle, <Emphasis Type="Italic">Tuber lannaense</Emphasis>, from Thailand

A new truffle species, Tuber lannaense, is described based on collections from northern Thailand. This species is characterized by yellow-brown to brown ascomata with dark brown gleba and ellipsoid to narrow ellipsoid spores with spiny reticulum. It grows in mycorrhizal association with Betula alnoides and Carpinus poilanei. Tuber lannaense is similar to T. huidongense, but differs in the thinner outer peridium layer. Molecular phylogenetic analysis of the internal transcribed spacers (ITS1?+?ITS2) and large subunit of nuclear ribosomal DNA, as well as genetic distance analysis of ITS1?+?ITS2, support T. lannaense as being distinct from other Tuber species. Moreover, based on genetic distance analysis, we consider that T. furfuraceum and T. huidongense, which have previously been synonymized, are different species.     

Influence of pH on in vitro mycelial growth in three Japanese truffle species: Tuber japonicum,T. himalayense,and T. longispinosum

Truffle species indigenous to Japan include a white-colored truffle Tuber japonicum and two black-colored truffles, T. himalayense and T. longispinosum. As the fruiting bodies of these Tuber species are promising edible, studies on the artificial cultivation for these truffles are currently underway in Japan. In the present study, we investigated the influence of pH on in vitro mycelial growth in these Tuber species to determine the optimal pH conditions for the cultivation of these truffles. Mycelia of five strains from each species were cultured in modified Melin–Norkrans liquid medium at different pH values. Tuber japonicum grew well at pH 5.0 and 6.0, whereas T. himalayense and T. longispinosum grew well at pH 7.0. This results suggest that the optimal pH for mycelial growth varies among Tuber species. The growth data collected in this study can be used to design optimal pH conditions for artificial cultivation of these Japanese truffles.     

The phylogenetic position of Tuber glabrum sp. nov. and T. sinomonosporum nom. nov., two Paradoxa-like truffle species from China

Based on morphological and molecular phylogenetic analysis, the new species Tuber glabrum is described from China, and the new name T. sinomonosporum is proposed for Paradoxa sinensis. Tuber glabrum is similar to T. gigantosporum but differs by its glabrous ascomata. The ITS sequences of the two Paradoxa-like truffle species, which invariably have one-spored asci, fall into the lineage of Tuber and cluster in a distinct clade together with the European T. macrosporum and the North American T. canaliculatum.     

Two new truffles species,Tuber alboumbilicum and Tuber pseudobrumale from China

Two new truffles species, Tuber alboumbilicum and Tuber pseudobrumale are described based on collections from Southwest China. T. alboumbilicum is characterized by its white umbilicate ascomata. T. pseudobrumale is similar to T. pseudoexcavatum, but differs in spinulose ascospores. Molecular analysis also supported T. alboumbilicum and T. pseudobrumale as two new species. T. alboumbilicum grows in mycorrhizal association with Tsuga chinensis at elevations of 2,710 m. T. pseudobrumale grows in mycorrhizal association with Pinus yunnanensis in limestone soils, and has a pleasant aroma and is good edible.     

Mycorrhizal detection of native and non-native truffles in a historic arboretum and the discovery of a new North American species, <Emphasis Type="Italic">Tuber arnoldianum</Emphasis> sp. nov.

During a study comparing the ectomycorrhizal root communities in a native forest with those at the Arnold Arboretum in Massachusetts (USA), the European species Tuber borchii was detected on the roots of a native red oak in the arboretum over two successive years. Since T. borchii is an economically important edible truffle native to Europe, we conducted a search of other roots in the arboretum to determine the extent of colonization. We also wanted to determine whether other non-native Tuber species had been inadvertently introduced into this 140-year-old Arboretum because many trees were imported into the site with intact soil and roots prior to the 1921 USDA ban on these horticultural practices in the USA. While T. borchii was not found on other trees, seven other native and exotic Tuber species were detected. Among the North American Tuber species detected from ectomycorrhizae, we also collected ascomata of a previously unknown species described here as Tuber arnoldianum. This new species was found colonizing both native and non-native tree roots. Other ectomycorrhizal taxa that were detected included basidiomycetes in the genera Amanita, Russula, Tomentella, and ascomycetes belonging to Pachyphlodes, Helvella, Genea, and Trichophaea. We clarify the phylogenetic relationships of each of the Tuber species detected in this study, and we discuss their distribution on both native and non-native host trees.     

Truffle diversity (<Emphasis Type="Italic">Tuber</Emphasis>, Tuberaceae) in British Columbia

To improve baseline data for the developing truffle industry in British Columbia, we compiled existing Tuber species sequences from published and unpublished studies and generated new ITS sequences for truffles belonging to Tuber collected in the province. In doing so, we obtained evidence that 13 species of Tuber occur in the province, including six introduced and seven native species, two of which are putative undescribed species. Of the native species, the Tuber anniae species complex is widely distributed in the province while Tuber beyerlei appears to be much more restricted in distribution. Four of the introduced species have commercial value (Tuber melanosporum, Tuber aestivum, Tuber brumale, and Tuber borchii) as do two of the native species (Tuber gibbosum and Tuber oregonense). Focused sampling on likely tree hosts, both hardwood and Pinaceae species, as well as in currently unexplored parts of the province seems likely to expand our knowledge of the diversity and distribution of Tuber species in British Columbia.     

Reevaluation of the Life Cycle of Tuber magnatum

Tuber spp. are ectomycorrhizal ascomycetes that produce ascocarps known as truffles. Basic aspects of Tuber biology have yet to be fully elucidated. In particular, there are conflicting hypotheses concerning the mating system and the ploidy level of the mycorrhizal and truffle hyphae. We used polymorphic microsatellites to compare the allelic configurations of asci with those from the network of the surrounding hyphae in single Tuber magnatum truffles. We then used these truffles to inoculate host plants and evaluated the microsatellite configurations of the resulting mycorrhizal root tips. These analyses provide direct evidence that T. magnatum outcrosses and that its life cycle is predominantly haploid. In addition to its scientific significance, this basic understanding of the T. magnatum life cycle may have practical importance in developing strategies to obtain and select nursery-produced mycorrhizal plants as well as in the management of artificial plantations of this and other Tuber spp.     

Diversity of ectomycorrhizal Thelephoraceae in <Emphasis Type="Italic">Tuber melanosporum</Emphasis>-cultivated orchards of Northern Spain

Truffles are edible hypogeous ascomycetes highly appreciated worldwide, especially the black truffle (Tuber melanosporum Vittad.). In recent decades, the cultivation of the black truffle has expanded across the Mediterranean climate regions in and outside its native range. Members of the Thelephoraceae (Thelephorales, Agaricomycetes, Basidiomycota) are commonly found in truffle plantations, but their co-occurrence with Tuber species and other members of the fungal community has been scarcely reported. Thelephoraceae is one of the most represented families of the ectomycorrhizal fungal community in boreal and Mediterranean forests. To reveal the diversity of these fungi in T. melanosporum-cultivated plantations, ten orchards located in the Navarra region (Northern Spain) were surveyed for 2 years. Morphological and molecular approaches were used to detect and identify the Thelephoraceae ectomycorrhizas present in those plantations. Ten different mycorrhizal types were detected and described. Four of them were morphologically identified as Tomentella galzinii, Quercirhiza cumulosa, Q. squamosa, and T39 Thelephoraceae type. Molecular analyses revealed 4–6 operational taxonomic units (OTUs), depending on the nucleotide database used, but similarities remained under 95 % and no clear species assignments could be done. The results confirm the diversity and abundance of this fungal family in the ectomycorrhizal community of black truffle plantations, generally established in Mediterranean areas. The occurrence and relative abundance of Thelephoraceae ectomycorrhizas is discussed in relation to their possible influence on truffle production.     

Ectomycorrhizal fungal diversity in orchards of cultivated pecan (Carya illinoinensis; Juglandaceae)

Carya illinoinensis (pecan) belongs to the Juglandaceae (walnut family) and is a major economic nut crop in the southern USA. Although evidence suggests that some species in the Juglandaceae are ectomycorrhizal, investigations on their ectomycorrhizal fungal symbionts are quite limited. Here we assessed the ectomycorrhizal fungal diversity in cultivated orchards of C. illinoinensis. Five pecan orchards in southern Georgia, USA, were studied, three of which were known to fruit the native edible truffle species Tuber lyonii. We sequenced rDNA from single ectomycorrhizal root tips sampled from a total of 50 individual trees. Mycorrhizae were identified by ITS and LSU rDNA sequence-based methods. Forty-four distinct ectomycorrhizal taxa were detected. Sequestrate taxa including Tuber and Scleroderma were particularly abundant. The two most abundant sequence types belonged to T. lyonii (17%) and an undescribed Tuber species (~20%). Because of our interest in the ecology of T. lyonii, we also conducted greenhouse studies to determine whether this species would colonize and form ectomycorrhizae on roots of pecan, oak, or pine species endemic to the region. T. lyonii ectomycorrhizae were formed on pecan and oak seedlings, but not pine, when these were inoculated with spores. That oak and pecan seedling roots were receptive to truffle spores indicates that spore slurry inoculation could be a suitable method for commercial use and that, ecologically, T. lyonii may function as a pioneer ectomycorrhizal species for these hosts.     

Isolation and characterization of polymorphic microsatellite loci in white truffle (Tuber magnatum)

Tuber 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.