Ectomycorrhizal Basidiomycete Tricholoma Matsutake Research Articles

The ectomycorrhizal basidiomycete Tricholoma matsutake associates with the root tissues of the model tree Populus tremula × tremuloides in vitro

The ectomycorrhizal basidiomycete Tricholoma matsutake associates with the root tissues of the model tree Populus tremula × tremuloides in vitro

Estimation of novel colony establishment and persistence of the ectomycorrhizal basidiomycete Tricholoma matsutake in a Pinus densiflora forest

Abstract Tricholoma matsutake is an economically valuable fungus in Japan. Its fruit bodies occur in colonies established in natural pine forests. We traced colony establishment processes of T. matsutake in 2001 and 2010 by using microsatellite markers. T. matsutake genets persisted at least for a decade. Comparing colonies found in 2001 and 2010, five novel colonies were observed in 2010, where the organic layer was removed or pine trees were regenerated less than 50 y ago. The novel colonies were genotyped, and all of them were different from colonies found in 2001, indicating that these colonies were established by dispersed basidiospores. Novel genet and candidate parent genets were located in the same ridge, and the mean geographical distance between a novel genet and a parent genet was 19.59 ± 14.73 m, indicating that basidiospores may colonize more easily on roots of hosts near parental basidiocarps. In conclusion, sufficient basidiospore supply and organic layer removal or pine tree regeneration were essential factors for colony establishment of T. matsutake.

Monokaryotic hyphae germinated from a single spore of the ectomycorrhizal basidiomycete Tricholoma matsutake

We document here that monosporous isolates of the ectomycorrhizal basidiomycete Tricholoma matsutake were initially uni- or bi-nucleate. During pure culture, however, many uninucleate isolates became multinucleate. While the parent strain had two patterns of single nucleotide polymorphisms (SNPs) within its single-copy DNA, 19 of 20 monosporous isolates exhibited one of the two parent SNPs, and an isolate that was binucleate upon germination had both, indicating the former were monokaryotic and the latter dikaryotic. Of those, two isolates carrying SNPs different from one another have been predominantly uninucleate for 9 years. These isolates may be useful in genetics/breeding of “matsutake” mushrooms.

Root endophyte interaction between ectomycorrhizal basidiomycete Tricholoma matsutake and arbuscular mycorrhizal tree Cedrela odorata, allowing in vitro synthesis of rhizospheric “shiro”

The ectomycorrhizal basidiomycete Tricholoma matsutake associates with members of the Pinaceae such as Pinus densiflora (red pine), forming a rhizospheric colony or "shiro," which produces the prized "matsutake" mushroom. We investigated whether the host specificity of T. matsutake to conifers is innately determined using somatic plants of Cedrela odorata, a tropical broad-leaved tree (Meliaceae) that naturally harbors arbuscular mycorrhizal fungi. We found that T. matsutake could form in vitro shiro with C. odorata 140days after inoculation, as with P. densiflora. The shiro was typically aromatic like that of P. densiflora. However, this was a root endophytic interaction unlike the mycorrhizal association with P. densiflora. Infected plants had epidermal tissues and thick exodermal tissues outside the inner cortex. The mycelial sheath surrounded the outside of the epidermis, and the hyphae penetrated into intra- and intercellular spaces, often forming hyphal bundles or a pseudoparenchymatous organization. However, the hyphae grew only in the direction of vascular bundles and did not form Hartig nets. Tricholoma fulvocastaneum or "false matsutake" naturally associates with Fagaceae and was also able to associate with C. odorata as a root endophyte. With T. matsutake, C. odorata generated a number of roots and showed greatly enhanced vigor, while with T. fulvocastaneum, it generated a smaller number of roots and showed somewhat lesser vigor. We argue that the host-plant specificity of ectomycorrhizal matsutake is not innately determined, and that somatic arbuscular mycorrhizal plants have a great potential to form mutualistic relationships with ectomycorrhizal fungi.

Characterization of the glycoside hydrolase family 15 glucoamylase gene from the ectomycorrhizal basidiomycete Tricholoma matsutake

The gene encoding the glycoside hydrolase family 15 glucoamylase (TmGlu1) in the ectomycorrhizal fungus Tricholoma matsutake was cloned and characterized. After the culture of T. matsutake mycelia in media containing different forms of starch as a carbon source, increased extracellular glucoamylase activity in the culture medium and a correspondingly higher transcriptional level of TmGlu1 in mycelia were detected, particularly in amylose- supplemented medium, when compared with those in the glucose medium. These results suggest that starch, especially amylose, affects the transcription of TmGlu1 and downstream glucoamylase activity, which is directly related to starch utilization. Similar results were obtained when compound forms of starch were used to culture mycelia. Glucoamylase genes from saprophytic and ectomycorrhizal fungi formed a single clade. The observed inducibility of TmGlu1 and lack of distinct phylogenetic differences among glucoamylase genes of saprophytic and ectomycorrhizal fungi suggest that glucoamylase may relate to some common functions in these two types of fungi.

Agrobacterium-mediated transformation of the ectomycorrhizal basidiomycete Tricholoma matsutake that produces commercially valuable fruit bodies, matsutake

Using agroinfection with a T-DNA vector carrying a hygromycin resistance marker, the recombinants were generated for the first time from the ectomycorrhizal basidiomycete Tricholoma matsutake, which produces commercially valuable fruit bodies, matsutake, during association with Pinus sp. plants. The transformation system may be useful in the genetic analysis of T. matsutake.

Intra- and inter-specific variations in the copy number of two types of retrotransposons from the ectomycorrhizal basidiomycete Tricholoma matsutake

To explore intra- and inter-specific variations of the ectomycorrhizal basidiomycete Tricholoma matsutake that produces the fruit body "matsutake", we carried out real-time PCR analysis based on two types of retrotransposons, one designated marY1, which resembles a retrovirus carrying the long terminal repeat (LTR) and the other marY2N, which resembles mRNA carrying the polyadenylated tail. Calculation based on the average genome size of homobasidiomycetes (34 Mbp) shows that ca. 5.5% of the total genome of T. matsutake isolated from Asia is made up of these retrotransposons, whereas they occupy ca. 1.4% in the isolates from Morocco, ca. 0.8% in isolates from Mexico, and ca. 0.5% in Tricholoma magnivelare, the species which produces "American matsutake". Other Tricholoma spp. that produce fruit bodies similar to those of T. matsutake, such as T. bakamatsutake, T. fulvocastaneum, and T. robustum, carry them in the region less than 0.05% of their total genome. Copy number of LTR of marY1 is consistently and markedly higher than that of the coding regions of marY1 and marY2N. Data suggest that retrotransposons are deeply involved in evolution of the ectomycorrhizal symbiont.

Genetic mosaics in the massive persisting rhizosphere colony “shiro” of the ectomycorrhizal basidiomycete Tricholoma matsutake

The ectomycorrhizal basidiomycete Tricholoma matsutake produces commercially valuable fruit bodies "matsutake" on a massive persisting rhizosphere aggregate of mycelia and mycorrhizas called "shiro." Using inter-retrotransposon amplified polymorphism analysis, we attempted to explore the potential diversity within the population of T. matsutake isolated from small Pinus densiflora woodlands located in various parts of Japan. In general, random phylogenetic relationship was noted among T. matsutake tested. The population from each limited sampling area was highly heterogeneous. Even some isolates from fruit bodies produced in the same shiro and those from spores in the same fruit bodies were found to be genetically diverse, indicating the occurrence of genetic mosaics in shiro. In a mosaic shiro, heterologous genets produced their fruit bodies concurrently. Data suggested that the dispersal of spores through sexual reproduction may have been more prevalent than generally accepted in T. matsutake to bring mosaicism and coordination of heterologous genets within the shiro. Implementation of management taking such diversity into consideration is urgently needed for the restoration of devastated matsutake fields in Japan. Exploration of individual clones in mosaic fungal resources that promote colonization and fruit body production is necessary for it.

Highly polymorphic DNA markers to specify strains of the ectomycorrhizal basidiomycete Tricholoma matsutake based on sigmamarY1, the long terminal repeat of gypsy-type retroelement marY1.

The ectomycorrhizal basidiomycete Tricholoma matsutake produces commercially valuable fruit bodies--matsutake--in Pinus sp. forest. Here we report that PCR with outward facing primers designed based on sequences comprising sigma(marY1), the long terminal repeat of the gypsy-type retroelement marY1, specifies strains of T. matsutake. PCR with a primer based on the 22-bp sequence conserved at the 5'-end of sigma(marY1) conferred 73 reliable bands overall whose profiles depend upon strains of T. matsutake and T. magnivelare, the latter known as 'American matsutake'. This PCR system gave no detectable band in any other species of Tricholoma tested, including T. bakamatsutake and T. fulvocastaneum, symbionts closely related to T. matsutake, as well as a host plant, Pinus densiflora. Similarly, PCR with a set of primers based on 26-bp and 28-bp sequences at bp 48-73 and bp 281-308 of sigma(marY1), internal regions that are mutated in a variant of marY1, conferred 90 reliable bands only in strains of T. matsutake. Theoretically, PCR with the 22-bp primer would allow generation of 2(73), or 9.4x10(21), types of polymorphism, and PCR with a combination of 26- and 28-bp primers, 2(90), or 1.2x10(27) types. The probability of falsely specifying two different isolates as the same strain is <1/10(21). While polymorphisms conferred by the primer based on the 5' end of sigma(marY1) rather exhibit genetic conservation of a group of T. matsutake, those resulting from primers based on the internal sequences more clearly demonstrate intra-specific diversification. Both systems revealed that T. matsutake is divergent within the species. Ectomycorrhizas formed between P. densiflora and T. matsutake were identified by the PCR systems developed in the present study. This method, using sigma(marY1) as a genetic marker, is useful in analyzing the diversity of T. matsutake, monitoring the behavior of individual mycorrhizas, and specifying the ecological background of fruit bodies traded in markets.

MarY1, a member of the gypsy group of long terminal repeat retroelements from the ectomycorrhizal basidiomycete Tricholoma matsutake.

We cloned an intact copy of a long terminal repeat retroelement designated marY1 from the ectomycorrhizal basidiomycete Tricholoma matsutake. The reverse transcriptase domain is found in T. matsutake and Tricholoma magnivelare worldwide. This finding suggests that retroelements associate with ectomycorrhizal basidiomycetes and may be useful as genetic markers for identification, phylogenetic analysis, and mutagenesis of this fungal group.

Identification of repetitive sequences containing motifs of retrotransposons in the ectomycorrhizal basidiomycete Tricholoma matsutake

An ectomycorrhizal basidiomycete, Tricholoma matsutake, produces fruiting bodies known as matsutake mushrooms in Pinus sp. forests. Matsutake mushrooms are a commercially valuable edible fungi. By using designed oligonucleotides as primers and genomic DNA of T. matsutake strain Y1 as a template, 967 and 1375 bp nucleotides were amplified by the polymerase chain reaction (PCR). The fragments consist of repetitive sequences and are predicted to encode peptides highly similar to those of reverse transcriptase, RNase H and integrase of retrotransposons. Internal fragments of 967 bp and 1375 bp DNA hybridized to genomic digests of T. matsutake isolated from various locations, and a strain of Tricholoma magnivelare. No hybridization was detected in 9 other Tricholoma species and one species each of 3 other genera of basidiomycetes that form an ectomycorrhizal symbiosis with the Pinus sp. tested. Hybridization signals generated by a probe of the 967 bp fragment were much stronger than those of the 1375 bp fragment. Restriction fragment length polymorphism revealed that DNA segments containing the 967 bp fragments are highly conserved in strains of T. matsutake and T. magnivelare whereas those containing the 1375 bp fragments are markedly diversified between these species. PCR with primers used for cloning of these fragments and those designed based on internal complementary inverted repeats showed both conservation and differentiation of the repetitive sequences containing motifs of retrotransposons in T. matsutake and the closely related fungi T. magnivelare.