Hitoshi Murata

Tricholoma matsutake and its related species are ectomycorrhizal Agaricomycetes that produce prized mushrooms collectively called “ matsutake”. These mushrooms are mainly distributed in the Northern Hemisphere. In this review, we describe both the pioneering work and recent advances in our understanding of the distribution and evolution of matsutake, with a special focus on genomic studies. Deoxyribonucleic acid (DNA) sequence analyses revealed that Tricholoma caligatum was the earliest species within this group, followed by Tricholoma fulvocastaneum, the cluster with Tricholoma ilkkae, Tricholoma dulciolens, and Tricholoma bakamatsutake, and finally the cluster with Tricholoma murrillianum, Tricholoma mesoamericanum, Tricholoma anatolicum, and Tricholoma matsutake. Although analyses based on mobile DNAs and whole-genome sequences revealed a similar clustering pattern, there are distinct differences in the distribution of mobile DNAs and genomic structure of Tricholoma bakamatsutake and Tricholoma matsutake. Furthermore, repetitive DNA can be used as markers to distinguish among strains and populations of Tricholoma matsutake from different geographical regions, including identifying dispersals of basidiospores. “Telomere-to-telomere genome sequencing” analyses unearthed that both Tricholoma matsutake and Tricholoma bakamatsutake underwent explosive multiplications of retrotransposons within their genomes but with different mobile DNA elements expanded between them. Contrastingly, the structure of the mating loci between Tricholoma matsutake and Tricholoma bakamatsutake is highly conserved.

Tricholoma matsutake is an ectomycorrhizal agaricomycete that produces the prized matsutake mushroom in conifer forests. Here, we isolated mutants generated by the argon-ion beam irradiation of T. matsutake NBRC 33136, designated as Ar 5002 and Ar 5012, which are defective in β-1,4-endoglucanase activity, that promoted the above- and below-ground growth of the symbiotic partner Pinus densiflora in vitro, compared with the control. Pine seedlings associated with Ar 5002, compared with NBRC 33136, had significantly higher above-ground dry mass and significantly greater plant growth, while those associated with Ar 5012 had significantly higher above- and below-ground dry mass. There were no significant differences between the numbers of mycorrhizal root-tips synthesized by the wild-type and mutants, indicating that β-1,4-endoglucanase did not influence the mycorrhization of T. matsutake. In seedlings inoculated with Ar 5002- and Ar 5012, the Hartig nets resembled those of seedlings inoculated with NBRC 33136. The data suggest that T. matsutake lacking β-1,4-endoglucanase activity confer beneficial effects on the growth of P. densiflora. Further studies are necessary to determine whether the symbiotic relationship could be strengthened by such a mutated fungal trait.

Tricholoma matsutake is an ectomycorrhizal agaricomycete that produces the prized mushroom “matsutake” in Pinaceae forests. Currently, there are no available cultivars or cultivation methods that produce fruiting bodies. Heavy-ion beams, which induce mutations through double-stranded DNA breaks, have been used widely for plant breeding. In the present study, we examined whether heavy-ion beams could be useful in isolating T. matsutake mutants. An argon-ion beam gave a suitable lethality curve in relation to irradiation doses, accelerating killing at 100–150 Gy. Argon-ion beam irradiation of the agar plate cultures yielded several transient mutants whose colony morphologies differed from that of the wild-type strain at the first screening, but which did not persist following culture transfer. It also generated a mutant whose phenotype remained stable after repeated culture transfers. The stable pleiotropic mutant not only exhibited a different colony morphology to the wild type, but also showed increased degradation of dye-linked water-insoluble amylose and cellulose substrates. Thus, heavy-ion beams may be useful for isolating mutants of T. matsutake, although precautions may be required to maintain the mutants, without phenotypic reversion, during repetitive culture of their mycelia.

Tricholoma matsutake is an ectomycorrhizal basidiomycete that associates with Pinaceae in the Northern Hemisphere and produces prized “matsutake” mushrooms. We questioned whether the symbiont could associate with a birch that is an early-successional species in boreal, cool-temperate, or subalpine forests. In the present study, we demonstrated that T. matsutake can form typical ectomycorrhizas with Betula platyphylla var. japonica; the associations included a Hartig net and a thin but distinct fungal sheath, as well as the rhizospheric mycelial aggregate “shiro” that is required for fruiting in nature. The in vitro shiro also emitted a characteristic aroma. This is the first report of an ectomycorrhizal formation between T. matsutake and a deciduous broad-leaved tree in the boreal or cool-temperate zones that T. matsutake naturally inhabits.

We previously reported that Tricholoma matsutake and Tricholoma fulvocastaneum, ectomycorrhizal basidiomycetes that associate with Pinaceae and Fagaceae, respectively, in the Northern Hemisphere, could interact in vitro as a root endophyte of somatic plants of Cedrela odorata (Meliaceae), which naturally harbors arbuscular mycorrhizal fungi in South America, to form a characteristic rhizospheric colony or “shiro”. We questioned whether this phenomenon could have occurred because of plant–microbe interactions between geographically separated species that never encounter one another in nature. In the present study, we document that these fungi formed root endophyte interactions and shiro within 140 days of inoculation with somatic plants of Prunus speciosa (=Cerasus speciosa, Rosaceae), a wild cherry tree that naturally harbors arbuscular mycorrhizal fungi in Japan. Compared with C. odorata, infected P. speciosa plants had less mycelial sheath surrounding the exodermis, and the older the roots, especially main roots, the more hyphae penetrated. In addition, a large number of juvenile roots were not associated with hyphae. We concluded that such root endophyte interactions were not events isolated to the interactions between exotic plants and microbes but could occur generally in vitro. Our pure culture system with a somatic plant allowed these fungi to express symbiosis-related phenotypes that varied with the plant host; these traits are innately programmed but suppressed in nature and could be useful in genetic analyses of plant–fungal symbiosis.

“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.

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 140 days 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.

A novel bacterium, designated strain OX-01^T, was isolated from acidic soil, taxonomically investigated and identified as an agent that catabolizes (+)-catechin into taxifolin. Strain OX-01^T is a Gram-reaction-negative, aerobic, non-sporulating, non-motile and rod-shaped bacterium. 16S rRNA gene sequence analysis identified this strain as a member of the genus Burkholderia and occupying a phylogenetic position closest to, but clearly distinct from, Burkholderia sacchari. Strain OX-01^T does not have any nif genes, which are required for N₂-fixation, in its genome, a feature that is similar to B. sacchari, which lacks nifH, but is distinct from the N₂-fixing features of many other phylogenetically related taxa, such as Burkholderia ferrariae, B. heleia, B. mimosarum, B. nodosa, B. silvatlantica, B. tropica and B. unamae. Strain OX-01^T has the following chemotaxonomic characteristics: the major ubiquinone is Q-8, the DNA G+C content is 64 mol% and the major fatty acids are C_16 : 0, C_17 : 0 cyclo and C_18 : 1 ω7c. It also has a unique profile of carbohydrate utilization among other species of the genus Burkholderia. The strain cannot assimilate many pentoses, hexoses and oligosaccharides, whereas it can catabolize (+)-catechin and its putative aromatic derivatives, such as 4-hydroxy-3-methoxycinnamic acid, protocatechuic acid, p-hydroxybenzoic acid, trans-p-coumaric acid and vanillic acid. Based on its morphological, physiological and chemotaxonomic characteristics, together with DNA–DNA relatedness values and 16S rRNA gene sequence comparison data, we show that strain OX-O1^T represents a novel species of the genus Burkholderia, for which the name Burkholderia oxyphila sp. nov. is proposed. The type strain is OX-01^T (=NBRC 105797^T =DSM 22550^T).

The ectomycorrhizal basidiomycete Tricholoma matsutake produces commercially valuable fruit bodies, matsutake , in forests. Here we report a PCR system targeting retroelement integration sites to differentiate among individual Asian isolates of T. matsutake based on their geographical origins, such as Japan, the area of South Korea through North Korea, the northeastern provinces of China, and the area of the southwestern provinces of China through Bhutan. The overall misjudgment rate of the analytical system was approximately 5% based on 95 samples of T. matsutake examined including those from cultures and from commodities. We also provide evidence that T. matsutake isolates grown throughout the Far East, including the northeastern provinces of China, are closely related to each other while distinct from those in the area of the southwestern provinces of China through Bhutan. The method allows us to trace back geographical origins of Asian matsutake , thus contributing to food safety, appropriate tariffs, and proper price setting.

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.