Identification of the Pol Gene as a Species-Specific Diagnostic Marker for Qualitative and Quantitative PCR Detection of Tricholoma matsutake.

Tricholoma matsutake is a rare, precious, and wild edible fungus that could not be cultivated artificially until now. This situation has given way to the introduction of fake T. matsutake commodities to the mushroom market. Among the methods used to detect food adulteration, amplification of species-specific diagnostic marker is particularly important and accurate. In this study, the Pol gene is reported as a species-specific diagnostic marker to identify three T. matsutake varieties and 10 other types of edible mushrooms through qualitative and quantitative PCR. The PCR results did not reveal variations in the amplified region, and the detection limits of qualitative and quantitative PCR were found to be 8 ng and 32 pg, respectively. Southern blot showed that the Pol gene exists as a single copy in the T. matsutake genome. The method that produced the purest DNA of T. matsutake in this study was also determined, and the high-concentration salt precipitation method was confirmed to be the most suitable among the methods tested. The assay proposed in this work is applicable not only to the detection of raw materials but also to the examination of processed products containing T. matsutake.

Diversity and taxonomy of Tricholoma species from Yunnan, China, and notes on species from Europe and North America.

Although taxonomic knowledge on Tricholoma (Agaricales, Basidiomycota) is fairly comprehensive in northwest Europe, knowledge of the global diversity and distribution of Tricholoma spp. is still sparse. In this study, the diversity and distribution of some Tricholoma spp. are analyzed by morphological and molecular methods based on 70 collections from Yunnan, China, 45 from central Europe, 32 from Colorado, USA, 9 from Japan, and 3 from Ukraine. A Holarctic distribution is suggested for several species, based on collections and nuc rDNA internal transcribed spacer ITS1-5.8S-ITS2 (ITS) sequences. Six species new to science are formally described from Yunnan: five in existing sections, Tricholoma forteflavescens, T. olivaceoluteolum, T. melleum, T. olivaceum, and T. sinoportentosum, and one, T. muscarioides, in the newly described section Muscaria alongside several previously described species. Additional putatively new species cannot be formally described because they lack sufficient material. Tricholoma foliicola is recognized as a species of the genus Gerhardtia.

North American matsutake: names clarified and a new species described.

Tricholoma matsutake, known widely as "matsutake," has great commercial and cultural significance in Japan. Because Japanese production is insufficient to meet the high domestic demand, morphologically similar mushrooms, thought by many to belong to T. magnivelare, are imported from western North America. However, molecular data produced since the early 2000s have indicated that more than one species of matsutake occur in North America and this raises the question of correct naming for the different species. To address this question, we assessed the phylogenetic diversity within North American matsutake based on nuc rDNA ITS1-5.8S-ITS2 (internal transcribed spacer [ITS] barcode) sequences, including newly obtained sequences from the type collections for Agaricus ponderosus and Armillaria arenicola, and morphological characters. Our results agree with earlier indications that three matsutake species occur in North America and allow us to clarify the correct application of names-T. magnivelare from the eastern USA and Canada, T. murrillianum from the western USA and Canada, and T. mesoamericanum from Mexico, newly described here. The existence of the three North American species is further supported by the results of evolutionary divergence analysis, geographical distributions, and morphological characters.

Genetic variability and bottleneck detection of four Tricholoma matsutake populations from northeastern and southwestern China.

The excessive commercial collection of matsutake mushrooms can lead to extreme reduction of population size, which may cause genetic bottleneck and decrease genetic diversity of Tricholoma matsutake. Here, six polymorphic microsatellite loci markers were used to examine the genetic diversity of four natural T. matsutake populations from two main producing regions of China. The minimum combinations of four loci were able to discriminate total 86 sampled individuals with distinctive multilocus genotypes. Our analysis of molecular variance (AMOVA) revealed that about 80% and 20% of the overall genetic variation were respectively partitioned within and among populations. The principal-coordinate analyses (PCA) distinguished the four tested populations into three genetic clusters, each of which was correlated with respective endemic host plants on a geographical basis. The AMOVA, PCA and pairwise population FST estimates consistently displayed the same genetic divergence patterns and spatial structure of T. matsutake mediated by host plants in China. The significant heterozygosity excesses demonstrated that a recent genetic bottleneck occurred in each population tested. The complementary M-ratio test indicated past genetic bottleneck events over longer periods. Only four individuals were identified as putative first generation migrants within northeastern China, which implies restricted interpopulation gene flow in T. matsutake. We discuss that the significant genetic differentiation among populations of T. matsutake is most likely a function of host adaptation, host specificity, genetic bottleneck, limited dispersal and habitat fragmentation.

The host ranges of conifer-associated Tricholoma matsutake, Fagaceae-associated T. bakamatsutake and T. fulvocastaneum are wider in vitro than in nature.

Tricholoma matsutake is the most commercially important edible mushroom in pine forests in Japan. Tricholoma bakamatsutake and T. fulvocastaneum, species closely related to T. matsutake, occur in Fagaceae forests. We examined ectomycorrhizal (EM) formation by these Tricholoma species by in vitro synthesis among seven strains (two of T. matsutake, four of T. bakamatsutake, one of T. fulvocastaneum) and axenic plants of pine (Pinus densiflora) and oak (Quercus serrata, Q. phillyraeoides). All strains, except for one of T. matsutake, formed EM associations with both pine and oak. Plant growth and mycelial development were differently affected by EM formation depending on the plant-fungus combination.

Metagenomic analysis of fungal communities inhabiting the fairy ring zone of Tricholoma matsutake.

Tricholoma matsutake, an ectomycorrhiza that has mutual relationships with the rootlet of Pinus denisflora, forms a fruiting body that serves as a valuable food in Asia. However, the artificial culture of this fungus has not been successful. Soil fungi, including T. matsutake, coexist with many other microorganisms and plants; therefore, complex microbial communities have an influence on the fruiting body formation of T. matsutake. Here, we report on the structures of fungal communities associated with the fairy ring of T. matsutake through the pyrosequencing method. Soil samples were collected inside the fairy ring zone, in the fairy ring zone, and outside the fairy ring zone. A total of 37,125 sequencing reads were obtained and 728 to 1,962 operational taxonomic units (OTUs) were observed in the sampling zones. The fairy ring zone had the lowest OTUs and the lowest fungal diversity of all sampling zones. The number of OTUs and fungal taxa inside and outside the fairy ring zone was, respectively, about 2 times and 1.5 times higher than the fairy ring. Taxonomic analysis showed that each sampling zone has different fungal communities. In particular, out of 209 genera total, 6 genera in the fairy ring zone, such as Hemimycena, were uniquely present and 31 genera, such as Mycena, Boletopsis, and Repetophragma, were specifically absent. The results of metagenomic analysis based on the pyrosequencing indicate a decrease of fungal communities in the fairy ring zone and changes of fungal communities depending on the fairy ring growth of T. matsutake.

A molecular contribution to the assessment of the Tricholoma equestre species complex.

In recent years, interest in the Tricholoma equestre species complex has increased because of several cases of severe and sometimes fatal rhabdomyolysis reported in France and Poland. These occurred after repeated consumption of large portions of T. equestre sporophores during consecutive meals, despite the fact that this species is renowned as a tasty edible wild mushroom. The T. equestre species complex includes three ectomycorrhizal species Tricholoma flavovirens (Pers.) S. Lundell, Tricholoma auratum (Paulet) Gillet, and T. equestre (L.) P. Kummer. All these species produce sporophores with intense yellow gills but are difficult to distinguish by morphological analyses at both the macroscopic and microscopic levels. In T. equestre, two additional varieties are recognized: T. equestre var. populinum (Christensen & Noordeloos) associated with Populus sp. and/or Betula sp. trees and sometimes recognized as Tricholoma frondosae (Kalamees & Shchukin) and T. equestre var. pallidifolia characterized by pale to white gills, frequently recognized as Tricholoma joachimii (Bon & Riva). To explore the taxonomic (species delimitation), ecological, and geographical extent and limits of the T. equestre species complex, we have carried out a molecular comparison of worldwide strains belonging to this complex by using sequences of two molecular markers: the internal transcript spacer (ITS)1/5.8S/ITS2 region of the nuclear ribosomal unit and the 5' part of the mitochondrial cox1 gene. Phylogenetic analyses support the placement of European T. equestre, T. flavovirens, and T. auratum strains as representatives of a single species. This species appears associated with various conifers trees, depending on the geographic origin (Pinus pinaster for T. auratum, Pinus sylvestris or Abies alba for T. equestre and T. flavovirens). However, in the context of a single T. equestre species, the geographical location could lead to the characterization of sub-species or varieties, as suggested by the gathering of the four Asian (Japanese) T. auratum strains in a strongly supported distinct phylogenetic clade. Moreover, our analysis strongly argues for considering T. joachimii and the synonymised T. equestre var. pallidifolia as two representatives of a different species not belonging to the T. equestre group. This species would be phylogenetically related to the Tricholoma columbetta species with which they share white gills. Similarly, the phylogenetic analysis of the molecular data and the lack of gene flow between the strains associated with broad-leaved trees and those of the T. equestre complex, rather argues for two distinct species depending on the ecological niche: T. frondosae under broad-leaved trees and T. equestre under conifers.

Mobile DNA distributions refine the phylogeny of "matsutake" mushrooms, Tricholoma sect. Caligata.

"Matsutake" mushrooms are formed by several species of Tricholoma sect. Caligata distributed across the northern hemisphere. A phylogenetic analysis of matsutake based on virtually neutral mutations in DNA sequences resolved robust relationships among Tricholoma anatolicum, Tricholoma bakamatsutake, Tricholoma magnivelare, Tricholoma matsutake, and Tricholoma sp. from Mexico (=Tricholoma sp. Mex). However, relationships among these matsutake and other species, such as Tricholoma caligatum and Tricholoma fulvocastaneum, were ambiguous. We, therefore, analyzed genomic copy numbers of σ marY1 , marY1, and marY2N retrotransposons by comparing them with the single-copy mobile DNA megB1 using real-time polymerase chain reaction (PCR) to clarify matsutake phylogeny. We also examined types of megB1-associated domains, composed of a number of poly (A) and poly (T) reminiscent of RNA-derived DNA elements among these species. Both datasets resolved two distinct groups, one composed of T. bakamatsutake, T. fulvocastaneum, and T. caligatum that could have diverged earlier and the other comprising T. magnivelare, Tricholoma sp. Mex, T. anatolicum, and T. matsutake that could have evolved later. In the first group, T. caligatum was the closest to the second group, followed by T. fulvocastaneum and T. bakamatsutake. Within the second group, T. magnivelare was clearly differentiated from the other species. The data suggest that matsutake underwent substantial evolution between the first group, mostly composed of Fagaceae symbionts, and the second group, comprised only of Pinaceae symbionts, but diverged little within each groups. Mobile DNA markers could be useful in resolving difficult phylogenies due to, for example, closely spaced speciation events.

Phylogenetic relationship and species delimitation of matsutake and allied species based on multilocus phylogeny and haplotype analyses.

Tricholoma matsutake (S. Ito & S. Imai) Singer and its allied species are referred to as matsutake worldwide and are the most economically important edible mushrooms in Japan. They are widely distributed in the northern hemisphere and established an ectomycorrhizal relationship with conifer and broadleaf trees. To clarify relationships among T. matsutake and its allies, and to delimit phylogenetic species, we analyzed multilocus datasets (ITS, megB1, tef, gpd) with samples that were correctly identified based on morphological characteristics. Phylogenetic analyses clearly identified four major groups: matsutake, T. bakamatsutake, T. fulvocastaneum and T. caligatum; the latter three species were outside the matsutake group. The haplotype analyses and median-joining haplotype network analyses showed that the matsutake group included four closely related but clearly distinct taxa (T. matsutake, T. anatolicum, Tricholoma sp. from Mexico and T. magnivelare) from different geographical regions; these were considered to be distinct phylogenetic species.

Phylogenetic species delimitation in ectomycorrhizal fungi and implications for barcoding: the case of the Tricholoma scalpturatum complex (Basidiomycota).

Population studies have revealed that the fungal ectomycorrhizal morphospecies Tricholoma scalpturatum consists of at least two genetically distinct groups that occur sympatrically in several geographical areas. This discovery prompted us to examine species boundaries and relationships between members formerly assigned to T. scalpturatum and allied taxa using phylogenetic analyses. Sequence data were obtained from three nuclear DNA regions [internal transcribed spacer (ITS), gpd and tef], from 101 carpophores collected over a large geographical range in Western Europe, and some reference sequences from public databases. The ITS was also tested for its applicability as DNA barcode for species delimitation. Four highly supported phylogenetic clades were detected. The two previously detected genetic groups of T. scalpturatum were assigned to the phylospecies Tricholoma argyraceum and T. scalpturatum. The two remaining clades were referred to as Tricholoma cingulatum and Tricholoma inocybeoides. Unexpectedly, T. cingulatum showed an accelerated rate of evolution that we attributed to narrow host specialization. This study also reveals recombinant ITS sequences in T. inocybeoides, suggesting a hybrid origin. The ITS was a useful tool for the determination of species boundaries: the mean value of intraspecific genetic distances in the entire ITS region (including 5.8S rDNA) was <0.2%, whereas interspecific divergence estimates ranged from 1.78% to 4.22%. Apart from giving insights into the evolution of the T. scalpturatum complex, this study contributes to the establishment of a library of taxonomically verified voucher specimens, an a posteriori correlation between phenotype and genotype, and DNA barcoding of ectomycorrhizal fungi.

High sexual reproduction and limited contemporary dispersal in the ectomycorrhizal fungus Tricholoma scalpturatum: new insights from population genetics and spatial autocorrelation analysis.

Dispersal and establishment are fundamental processes influencing the response of species to environmental changes, and the long-term persistence of populations. A previous study on the symbiotic ectomycorrhizal fungus Tricholoma scalpturatum revealed strong genetic differentiations between populations in Western Europe, suggesting restricted dispersal for this wind-dispersed cosmopolitan fungus. Two distinct genetic groups (genetic groups 1 and 2), co-occurring in some locations, were also identified and could correspond to cryptic species. In the present work, we examine the reproductive strategy and dispersal biology of the two T. scalpturatum's genetic groups. Variable molecular markers (intersimple sequence repeats and intergenic spacer 2-restriction fragment length polymorphisms) and spatial autocorrelation analyses were used to examine fine-scale patterns (< 140 m) of genetic structure, in an effort to determine the physical scale at which genetic structure exists. A total of 473 fruit bodies were mapped and collected over 3 years from two plots located in the south of France, including 219 and 254 samples from group 1 and group 2, respectively. High genetic diversity and the presence of numerous small genets were observed in both groups. Autocorrelation analyses revealed significant positive spatial genetic structures of genets at close distances (up to few metres for both groups). Mantel tests confirmed this isolation-by-distance pattern. These results clearly demonstrate high sexual reproduction and spatial structuring of genets at very small geographical scales in this wind-dispersed ectomycorrhizal fungal species, a pattern consistent with restricted contemporary dispersal of spores.