Characterization of the reproductive mode and life cycle of the whitish truffle <Emphasis Type="Italic">T. borchii</Emphasis>

Truffles are the fruiting structures of ascomycetes in the genus Tuber. Because of their economic importance, truffles have been cultivated for many years using artificially inoculated host plants. Nevertheless, the life cycle and reproductive mode of Tuber spp. are still poorly understood. In filamentous ascomycetes, sexual reproduction is genetically controlled by the mating-type (MAT) locus. Among Tuber spp., the MAT locus has been recently characterized in the black truffles Tuber melanosporum and Tuber indicum. Here, by using sequence information derived from these species and from a Tuber borchii expressed sequence tag (EST) showing similarity to the mat1 gene of Alternaria brassicicola, we embarked on a chromosome-walking procedure to sequence the complete MAT region of T. borchii. This fungus produces highly commercialized whitish truffles and represents a model species for addressing basic questions concerning the life cycle of Tuber spp. We show that T. borchii is heterothallic, as its MAT locus is organized into two idiomorphs, each harbored by different mycelial strains. The alignment of the MAT locus from black truffles and T. borchii reveals that extensive sequence rearrangements and inversions occurred between these species. Moreover, by coupling mating-type analyses to karyological observation, we show that mycelia isolated from ascocarps and mycorrhizae are formed by homokaryotic hyphae.     

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.     

Identification of Internal Transcribed Spacer Sequence Motifs in Truffles: a First Step toward Their DNA Bar Coding

This work presents DNA sequence motifs from the internal transcribed spacer (ITS) of the nuclear rRNA repeat unit which are useful for the identification of five European and Asiatic truffles (Tuber magnatum, T. melanosporum, T. indicum, T. aestivum, and T. mesentericum). Truffles are edible mycorrhizal ascomycetes that show similar morphological characteristics but that have distinct organoleptic and economic values. A total of 36 out of 46 ITS1 or ITS2 sequence motifs have allowed an accurate in silico distinction of the five truffles to be made (i.e., by pattern matching and/or BLAST analysis on downloaded GenBank sequences and directly against GenBank databases). The motifs considered the intraspecific genetic variability of each species, including rare haplotypes, and assigned their respective species from either the ascocarps or ectomycorrhizas. The data indicate that short ITS1 or ITS2 motifs (≤50 bp in size) can be considered promising tools for truffle species identification. A dot blot hybridization analysis of T. magnatum and T. melanosporum compared with other close relatives or distant lineages allowed at least one highly specific motif to be identified for each species. These results were confirmed in a blind test which included new field isolates. The current work has provided a reliable new tool for a truffle oligonucleotide bar code and identification in ecological and evolutionary studies.     

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.     

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.     

Typing Tuber melanosporum and Chinese black truffle species by molecular markers

Morphologically similar species such as Chinese black truffles and Tuber melanosporum were typed by restriction length polymorphism of internal transcribed spacers. This analysis together with sequence comparison revealed the presence of high genetic variability among fruit bodies collected in China. Selection of primer pairs allowed the internal transcribed spacer region of both Chinese truffles and T. melanosporum to be specifically amplified.     

Ascoma genotyping and mating type analyses of mycorrhizas and soil mycelia of Tuber borchii in a truffle orchard established by mycelial inoculated plants

Tuber borchii (the Bianchetto truffle) is a heterothallic Ascomycete living in symbiotic association with trees and shrubs. Maternal and paternal genotype dynamics have already been studied for the black truffles Tuber melanosporum and Tuber aestivum but not yet for T. borchii. In this study, we analysed maternal and paternal genotypes in the first truffle orchard realized with plants inoculated with five different T. borchii mycelia. Our aims were to test the persistence of the inoculated mycelia, if maternal and/or paternal genotypes correspond to inoculated mycelia and to assess the hermaphroditism of T. borchii. The mating type of each isolate as well as those of mycorrhizas, ascomata and extraradical soil mycelia was determined. Moreover, simple sequence repeat (SSR) profiles of maternal and paternal genotypes were assessed in 18 fruiting bodies to investigate the sexual behaviour of this truffle. The maternal genotypes of the fruiting bodies corresponded to those of the inoculated mycelia with only two exceptions. This confirmed that the inoculated mycelia persisted 9 years after plantation. As regards paternal partner, only two had the same genotype as those of the inoculated mycelia, suggesting hermaphroditism. Most of the new paternal genotypes originated from a recombination of those of inoculated mycelia.     

Identification and bioactivity of the growth inhibitors in Tuber spp. methanolic extracts

The methanolic extracts of fresh fruiting bodies of Tuber melanosporum, T. aestivum, T. magnatum and T. borchii were evaluated for their free fatty acids (FFAs) content to test their contribution to truffle allelopathic activity. Linoleic acid was the major FFA detected in all truffle species. In addition to linoleic acid, oleic acid and palmitic acid were the other abundant FFAs in the Tuber spp. extracts. Looking at the total production, T. melanosporum and T. aestivum synthesized significantly higher amounts of all identified FFA than T. magnatum and T. borchii. The allelopathic activity, based on the specific activity (EC₅₀) of the identified compounds, was evaluated in the growth of Arabidopsis thaliana, Lotus corniculatus, Melica ciliata and Silene vulgaris. Linoleic acid had the lowest specific activity and palmitic acid had the highest. The black truffle extracts had a higher inhibitory activity than white truffles, and this could be explained by the high relative amount of FFAs in them.     

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.     

Quantification of extraradical mycelium of Tuber melanosporum in soils from truffle orchards in northern Spain

Quantification of extraradical mycelium of black truffle (Tuber melanosporum) has been carried out in a natural truffle ground and in seven truffle orchards (around 20 years old) established in Tierra Estella and Valdorba sites, within the natural distribution area of the black truffles in Navarre (northern Spain). Specific primers and a Taqman® probe were designed to perform real-time PCR with DNA extracted from soil samples. Amplification of T. melanosporum DNA was obtained from 131 out of the 160 soil samples. The detection limit of the technique was 1.48 μg mycelium/g of soil. The extraradical mycelium biomass detected in the soil from the natural truffle ground was significantly greater (up to ten times higher) than the mycelium biomass detected in any of the orchards. Soil from productive, nonirrigated orchards in the Tierra Estella site contained significantly more extraradical mycelium than the rest of orchards irrigated, productive of T. brumale, or nonproductive. The comparison of soil mycelium biomass in nonirrigated evergreen oak orchards in both sites showed significantly more mycelium biomass in the Tierra Estella site. This study is the first attempt to quantify extraradical mycelium of T. melanosporum in the soil using Taqman® probes. The obtained quantitative results are of special interest to evaluate the fungal response to cultural treatments and to monitor the dynamics of the extraradical mycelium of T. melanosporum in the soil.     

Five years investigation of female and male genotypes in périgord black truffle (Tuber melanosporum Vittad.) revealed contrasted reproduction strategies

The Périgord black truffle (Tuber melanosporum Vittad.) is a heterothallic ascomycete that establishes ectomycorrhizal symbiosis with trees and shrubs. Small‐scale genetic structures of female genotypes in truffle orchards are known, but it has not yet been studied in male genotypes. In this study, our aim was to characterize the small‐scale genetic structure of both male and female genotypes over five years in an orchard to better understand the T. melanosporum sexual reproduction strategy, male genotype dynamics, and origins. Two‐hundred forty‐one ascocarps, 475 ectomycorrhizas, and 20 soil cores were harvested and genotyped using microsatellites and mating type genes. Isolation by distance analysis revealed pronounced small‐scale genetic structures for both female and male genotypes. The genotypic diversity was higher for male than female genotypes with numerous small size genotypes suggesting an important turnover due to ascospore recruitment. Larger and perennial female and male genotypes were also detected. Only three genotypes (1.5%) were found as both female and male genotypes (hermaphrodites) while most were detected only as female or male genotype (dioecy). Our results suggest that germinating ascospores act as male genotypes, but we also proposed that soil mycelium could be a reservoir of male genotypes.     

Tuber melanosporum: mating type distribution in a natural plantation and dynamics of strains of different mating types on the roots of nursery-inoculated host plants

? In light of the recent finding that Tuber melanosporum, the ectomycorrhizal ascomycete that produces the most highly prized black truffles, is a heterothallic species, we monitored the spatial distribution of strains with opposite mating types (MAT) in a natural truffle ground and followed strain dynamics in artificially inoculated host plants grown under controlled conditions. ? In a natural truffle ground, ectomycorrhizas (ECMs), soil samples and fruit bodies were sampled and genotyped to determine mating types. Simple sequence repeat (SSR) markers were also used to fingerprint ECMs and fruit bodies. The ECMs from nursery-inoculated host plants were analysed for mating type at 6 months and 19 months post-inoculation. ? In open-field conditions, all ECMs from the same sampling site showed an identical mating type and an identical haploid genotype, based on SSR analysis. Interestingly, the gleba of fruit bodies always demonstrated the same genotype as the surrounding ECMs. Although root tips from nursery-grown plants initially developed ECMs of both mating types, a dominance of ECMs of the same MAT were found after several months. ? The present study deepens our understanding of the vegetative and sexual propagation modes of T. melanosporum. These results are highly relevant for truffle cultivation.     

Olfactory orientation of the truffle beetle, Leiodes cinnamomea

Although the truffle beetle, Leiodes cinnamomea, inflicts substantial damage to the ripe stage of fruiting bodies of the economically important black truffle (Tuber melanosporum), it is not attracted by ripe truffle odours. Rather, male beetles are attracted to infested truffles only in the presence of female beetles, suggesting that the former employ a pheromone to locate truffles over short distances. In contrast, female beetles show no attraction to infested or uninfested truffles, suggesting that they employ other cues, possibly linked to odours emitted by truffles prior to the ripe stage. We hypothesize that the chemical composition of truffle volatiles changes over the life of the truffle fruiting body, being attractive to insects early on and to mammals just prior to decomposition.     

Receptivity of cloned hazels to artificial ectomycorrhizal infection by Tuber melanosporum and symbiotic competitors

 The cultivation of the black truffle, Tuber melanosporum (Vitt.), is based upon plantation of seedlings artificially infected with Tuber. At the present time, neither the host-plant nor the fungal material used for planting truffle orchards are genetically well defined. Cloning the host-plant was performed as a first step in improvement. A clone of hazel (Corylus avellana L.) was artificially infected with T. melanosporum. The heterogeneity of the root volume was not completely suppressed by cloning but very extensive growth unfavorable to Tuber colonization rarely occurred. The root system was highly receptive to ectomycorrhizal fungi. The percentage of roots infected by T. melanosporum reached a higher level on cloned hazels than uncloned seedlings. The relation between Tuber and other symbionts appeared to depend on the morphology of the root system. T. melanosporum spread more easily on medium-sized root systems whereas the other symbionts (Scleroderma sp., Cenococcum sp., Pulvinula globifera) developed in parallel to the root volume. The practical potential of this system is discussed. Accepted: 4 June 1995     

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.     

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.     

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.