Detection of the edible ectomycorrhizal fungus Lyophyllum shimeji colonising seedlings of cultivated conifer species in New Zealand

Lyophyllum shimeji is an edible ectomycorrhizal fungus that is widely distributed in East Asia and also present in the northern regions of Europe. In Japan, L. shimeji is a culinary delicacy, considered amongst all edible mushrooms to have the best taste and to be second only to Tricholoma matsutake in price. Traditionally, fruiting bodies of L. shimeji have been collected from the wild but fruiting of L. shimeji is now relatively uncommon and cannot keep up with increasing consumer demand. As a result, methods for its cultivation are being developed for commercial production in Japan and other countries. In this work, techniques were developed to cultivate L. shimeji on coniferous seedlings using a pure culture inoculum. They resulted in successful mycorrhization of Pinus pinaster and Picea abies in only 8 to 10 months. As ectomycorrhizae of L. shimeji are difficult to identify morphologically, mycorrhization was confirmed using an L. shimeji-specific PCR diagnostic, which was designed following a phylogenetic analysis of the Lyophyllum section Difformia using DNA sequences of the internal transcribed spacer (ITS), intergenic spacer (IGS) and elongation factor 1-α (EF1-α) gene. L. shimeji is a member of the Lyophyllum decastes complex in section Difformia, which also includes Lyophyllum fumosum and L. decastes. This analysis confirmed the separation of L. shimeji from closely related Lyophyllum spp. and enabled its unambiguous detection using an IGS-based PCR diagnostic. This is the first report of successful mycorrhization of L. shimeji on P. pinaster and P. abies and provides an opportunity for its commercial cultivation on conifers in New Zealand.     

Phylogenetic position of the ectomycorrhizal basidiomycete Tricholoma dulciolens in relation to species of Tricholoma that produce “matsutake” mushrooms

Tricholoma dulciolens is an ectomycorrhizal basidiomycete that produces “matsutake”-like mushrooms in association with Picea abies in Fennoscandia. The phylogenetic position of T. dulciolens relative to matsutake species is, however, unknown. In the present study, we demonstrated that T. dulciolens is phylogenetically independent from T. matsutake and its allied species. In addition, herbarium specimens identified as T. caligatum from Abies alba forests in Spain were phylogenetically related to but distinct from T. dulciolens. Based on ribosomal internal transcribed spacer DNA sequence data, T. dulciolens appears to exist in North America, as well. This is the first phylogeny of T. dulciolens in Fennoscandia and its related species in southern Europe.     

Resistance to and uptake of heavy metals in mushrooms

Resistance levels to heavy metals in 21 mushrooms obtained mainly from Japan, but also from Thailand, were tested. The mushrooms were 7 Pleurotus species, 2 Pycnoporus and Pholiota species and one each of Agrocybe, Cryptoporus, Coriolus, Inonotus, Lampteromyces, Grifola, Flammulina, Lyophyllum, Agaricus, and Polyporus species. The Pleurotus species strains showed higher resistance to the heavy metals, copper, cadmium, zinc, nickel, cobalt, mercury than the other species. Pleurotus ostreatus exhibited the highest resistance to all these heavy metals. Pholiota species, Flammulina veltipes, Lyophyllum ulmarium, Agaricus bisporus and Polyporus arcularius were rather sensitive to all the metals tested. The accumulation of copper, zinc and cadmium in Pleurotus ostreatus was studied. The uptake of heavy metals into the mycelia of P. ostreatus increased proportionally to an increasing concentration of these metals in the medium.     

Two new deer truffle species,Elaphomyces marmoratus and Elaphomyces fuscus spp. nov., from a secondary forest in Japan

We describe two new species of deer truffle, Elaphomyces marmoratus and E. fuscus spp. nov., collected from a secondary forest dominated by Quercus serrata, in Tokyo, Japan. Both species have morphological similarities to E. muricatus and E. granulatus; however, the former has a gleba without a purplish color and the latter has reticulated ascospores more closely resembling E. asperulus. Molecular phylogenetic analyses also support the assignment of these specimens as new species. ITS rDNA homologies with known species were low (<91% and <97%). In addition, phylogenetic trees using neighbor-joining and maximum likelihood methods showed that the sequences of the two new species each formed a monophyletic group within section Elaphomyces with bootstrap support of 99%. Analyses of ectomycorrhizal roots collected concurrently with the ascomata revealed that E. marmoratus is associated with at least Pinus densiflora, while E. fuscus is associated with at least Q. serrata.     

Cortinarius pseudofallax (Cortinariaceae,Agaricales), the first records from the Iberian Peninsula and Fennoscandia,and taxonomic notes on the C. parvannulatus/cedriolens group

Diverse collections of Cortinarius pseudofallax from several European countries were studied using morphological characters and ITS1-5.8S-ITS2 genes from sequence data. They were compared with some close species such as C. cedriolens, C. neofallax and C. parvannulatus. All these taxa share some features like a light brown pileus, a stipe provided with a more or less developed whitish veil, small and well-ornamented spores and, above all, a typical strong and perfumed acidulous smell; in addition. the phylogenetic tree built with the ITS gene sequences of these vouchers showed that they clustered in a well-supported clade, here treated as section Parvuli. Our specimens of C. pseudofallax are morphologically described and illustrated with pictures of fresh specimens and basidiospores seen under SEM. A discussion of their taxonomic relationships with another group of similar species belonging to the C. decipiens sensu lato complex is presented.     

Morphological and molecular identification of helminths of the greater bulldog bat Noctilio leporinus (Quiroptera: Noctilionidae) from Campeche,Mexico

Surveys on parasites of bats from the Americas have been conducted, but information on helminths is still scarce, especially in the Neotropical region. In Mexico, there are species of bats that lack of a record for helminth species, such as members of the family Noctilionidae. The present study describes for the first time the helminths of Noctilio leporinus in Campeche, Mexico. In 2017, six specimens of N. leporinus were studied for helminths. The species identification of helminths was based on morphological studies and molecular analysis of fragments of the 28S rDNA. All bat specimens were infected for at least one helminth species. Three helminth taxa were identified: the trematode Pygidiopsis macrostomum, and the nematodes Tricholeiperia cf. proencai, and Heligmonellidae gen. sp. The morphological identification of P. macrostomum was confirmed by sequence analysis of 28S rDNA gene. The phylogeny of P. macrostomum grouped our sequence with other sequences of the same species collected in Brazil. The phylogenetic tree of Heligmonellidae gen. sp. indicated that the helminth belongs to clade formed by the species Odilia bainae, Nippostrongylus magnus and Nippostrongylus brasiliensis of the family Heligmonellidae. The phylogenetic analysis of the 28S sequences of T. cf. proencai did not show any similarity or close affinity with nematodes from which that gene has been sequenced to date. The findings of the present study increase the number of helminth species parasitizing bats in Mexico.     

Favolaschia in remnants of the Atlantic Forest,Brazil

The examination of recent collections of Favolaschia from remnants of the Atlantic Forest, Brazil, resulted in the identification of F. aurantiaca, F. cinnabarina and F. luteoaurantiaca sp. nov. Internal transcribed spacer (ITS) sequences obtained from these collections were introduced into the previously published phylogenetic tree of the genus to assess the position of these species within the Favolaschia clade. A maximum likelihood analysis generated a phylogenetic tree with a better resolution, especially for the clade that contains species belonging to section Favolaschia subsection Auriscalpium, where the three specimens collected in Brazil also were clustered in.     

Species diversity of <Emphasis Type="Italic">Pseudocercospora</Emphasis> from Far East Asia

This study reflects on the monophyly of, and species diversity within, the genus Pseudocercospora in Far East Asia. Morphological characteristics and phylogenetic analyses of Pseudocercospora species were based on type specimens and ex-type cultures, which were collected from Japan and Taiwan. A phylogenetic tree was generated from multi-locus DNA sequence data of the internal transcribed spacer regions of the nrDNA cistron (ITS), partial actin (actA), and partial translation elongation factor 1-alpha (tef1), as well as the partial DNA-directed RNA polymerase II second largest subunit (rpb2). Based on these results, Pseudocercospora amelanchieris on Amelanchier and Ps. iwakiensis on Ilex were newly described from Japan, and a further 22 types (incl. two neo-, five lecto-, and 15 epitypes), were designated. The genus Pseudocercospora as presently circumscribed was found to be monophyletic, while the secondary barcodes, actA, tef1, and rpb2 were shown to be well suited to delimitate species within the genus.     

Fossil records of subsection Pinus (genus Pinus,Pinaceae) from the Cenozoic in Japan

Extant pines of subsection Pinus (section Pinus, genus Pinus, Pinaceae) are predominantly distributed in Eastern Asia. However, the extent of diversification in the section has yet to be fully clarified. We reviewed fossil records of subsection Pinus from Japan and collected permineralized materials, in which anatomical details are preserved for better understanding of the diversification. Our results suggest that this subsection appeared in Japan no earlier than the Middle Eocene, with extant species (i.e., Pinus densiflora and Pinus thunbergii) appearing around the beginning of the Pleistocene. Pinus fujiii (Early Miocene to Early Pleistocene) is inferred to have a close affinity to P. thunbergii based on the medial arrangement of its leaf resin canals. Additionally, P. fujiii has a similar cone morphology to those of extant species living in China, bridging the morphological gap between P. thunbergii and Chinese relatives of P. thunbergii as inferred by molecular phylogenetic analyses. Our results also suggest that taxonomic revisions of Pinus miocenica and Pinus oligolepis are required among the Japanese fossil species reported to date.     

Hygrophorus yukishiro sp. nov., a new vernal edible mushroom from Nagano Prefecture,Japan

A novel basidiomycetous species, Hygrophorus yukishiro, is described from Japan. The basidiomata of this fungus have been referred to as “yuki-shiro”, “yuki-take”, and “yuki-wari”, and used as an edible mushroom in the central region of Nagano, Japan. Hygrophorus yukishiro fruits in the early spring season under oak-dominated stands. Morphological and phylogenetic analyses of the internal transcribed spacer region of the nuclear ribosomal RNA gene indicated that H. yukishiro is a sister species of H. arbustivus and belongs to section Hygrophorus, subsect. Fulventes. Hygrophorus yukishiro is similar in its external morphology and fruiting phenology to H. roseobrunneus, H. leucophaeo-ilicis, and H. roseodiscoideus, but differs in the color and texture of its pileus surface and microscopic characteristics.     

Genetic diversity and phylogenetic relationships of the endangered species Vaccinium sieboldii and Vaccinium ciliatum (Ericaceae)

The edible berry species Vaccinium sieboldii, Vaccinium ciliatum, and Vaccinium oldhami are deciduous shrubs found growing at low altitudes in Japan. They are classified in the same section. Of these, the former two species are listed as endangered species. The present status of these two species was surveyed. V. sieboldii was found in a very limited range, 65 km wide, in central Japan. Many plants are thought to have been lost because of habitat destruction in the past 50 years. Loss of pine forests from pine-wilt disease is thought to be a major threat to this species. In contrast, V. ciliatum was found over a range of more than 300 km in western Japan. Habitats of both species were heavily fragmented. The genetic diversity and phylogenetic relationships of the three species were studied using nine simple-sequence repeat loci. V. sieboldii and V. ciliatum have a close genetic relationship, although their present growth ranges are widely separated. Despite its limited growth range and the limited number of remaining plants, the genetic diversity of V. sieboldii is similar to that of another endangered species, V. ciliatum, and that of the widely grown species, V. oldhami. Seven populations of the former two species were used to create population-based dendrograms. They were separated into three groups: three populations of V. sieboldii, two mountainous populations of V. ciliatum, and two coastal populations of V. ciliatum. The separation was supported by high bootstrap values. Isolation factors of the two population groups in V. ciliatum are discussed.     

Aspergillus yunnanensis,a new and rare species in the Aspergillus section Jani

Aspergillus is a monophyletic genus comprising the subgenera Aspergillus, Circumdati, Cremei, Fumigati, Nidulantes and Polypaecilum. The subgenus Circumdati contains many economically important species and mycotoxin producers. Section Jani was recently introduced with morphological and molecular support. In the present study, two strains isolated from farmland soil were assigned in section Jani based on multi-locus phylogenetic analyses but showed low similarity with existing species. Further morphological observation found they had wider vesicles and conidia connections which were different from the known species. Based on phylogenetic and morphological data, Aspergillus yunnanensis was introduced as the third species in section Jani. Members in section Jani are rarely distributed, this is the first report of this section in China.     

Russula velenovskyi new to Japan,with phylogenetic implications of Russula species between Japanese subalpine forests and Northern Europe

To compare morphological characters and phylogenetic placement between Japanese and European Russula, 32 specimens of 12 species were collected from Japanese subalpine forests and Northern Europe. Several sequences of nrDNA ITS region (ITS) of these Russula species were obtained. High homological similarities were shown between ITS sequences of several Russula samples collected from Japanese subalpine forests, Europe and North America. These facts show distribution of the same Russula species among these areas. From morphological observations and phylogenetic analyses, two same Russula species, R. velenovskyi, and R. decolorans are found in Japan, Europe and North America. Of these, R. velenovskyi collected from Mt. Fuji, Mt. Nyukasa and Mt. Tateshina in mountainous area of central Honshu is reported as a new Japanese record.     

Morphological and molecular phylogenetic analysis of <Emphasis Type="Italic">Melampsora</Emphasis> species on poplars in China

Many species of Melampsora on Populus have been reported in China, based on morphological characteristics of both uredial and telial states, and on host species, but their morphology and taxonomy are still poorly defined. In this study, 196 specimens representing Melampsora species on poplars and collected from various areas of China were used for morphological observations. The morphological characteristics of urediniospores and teliospores were examined with light and scanning electron microscopy. The specimens could be classified into five groups based on their morphology. For the sequencing of the nuclear large subunit rDNA (D1/D2), 5.8S rDNA and their internal transcribed spacers, ITS1 and ITS2 region, 54 specimens were selected from the specimens used in morphological observations. These specimens were separated into six clades by phylogenetic analyses of the D1/D2 and ITS regions. Correlations among morphological groups and phylogenetic clades based on these results suggest a revision of these species. In particular, no evidence to discriminate specimens of M. acedioides, M. magnusiana, and M. rostrupii was found from either morphological characteristics or sequence analysis.Contribution no. 185 Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan     

Multiple origins of sequestrate basidiomes within Entoloma inferred from molecular phylogenetic analyses

The genus Entoloma comprises diverse trophic modes and basidiome morphologies. Although Entoloma includes some sequestrate species, their origins are not clearly understood in relation to phylogenetic position and trophic status. In this study, we collected 34 sequestrate Entoloma specimens in Japan over a 9-y period. Their identities and phylogenetic positions were determined by molecular analyses using three nuclear loci [internal transcribed spacer (ITS) and large subunit (LSU) regions of rDNA and RNA polymerase II second LSU (rpb2) gene]. Based on species delimitation of 97 % sequence matches in the ITS region, which is a suitable region for species-level identification of higher fungi, we identified four sequestrate Entoloma species. Molecular phylogenetic analyses that included all related sequences in the International Nucleotide Sequence Database revealed that the four sequestrate Entoloma species belonged to two major phylogroups. One of the phylogroups was Inocephalus–Cyanula, which is composed only of saprotrophic species. Three of the Japanese sequestrate species, as well as three previously known sequestrate species from other regions, fell into at least two independent clades in this phylogroup, indicating multiple origins of sequestrate forms within this saprotrophic lineage. Another phylogroup, Rhodopolioid, was also shown to include a sequestrate species for the first time. Because the Rhodopolioid phylogroup is composed exclusively of mycorrhizal species (ectomycorrhizal and tuberculate mycorrhizal species), the sequestrate form may also have evolved from a mycorrhizal ancestor. Our results suggest that sequestrate basidiomes have evolved multiple times, irrespective of their trophic status in Entoloma. Finally, based on molecular and morphological characteristics, here we describe two new sequestrate Entoloma species, i.e., Entoloma prismaticum Sasaki, Kinoshita et Nara, sp. nov. and Entoloma hypogaeum Sasaki, Kinoshita et Nara, sp. nov.     

Four new species of Entoloma s.l. (Agaricales) from southern China

Four new Entoloma s.l. species (E. azureosquamulosum, E. caeruleoflavum, E. hainanense, and E. subtenuicystidiatum) are described from southern China. E. azureosquamulosum fits well within the section Rhamphocystotae, E. caeruleoflavum belongs to the section Entoloma, E. hainanense is placed in the section Calliderma, and E. subtenuicystidiatum is a species in the section Cyanula. Preliminary phylogenetic analyses for the four new species are provided based on ITS and LSU sequences in this paper.     

Japanese species of the Longibrachiatum Clade of Trichoderma

The Longibrachiatum Clade of the genus Trichoderma in Japan was examined, among which two new species and three new records are herewith reported. The new species, T. tsugarense and T. kunigamense were isolated from a bed log (cultivation of Lentinula edodes) and volcanic ash soil, respectively. These species are distinguished from closely related species by growth and morphological characteristics and in phylogenetic analysis. Additional species new to Japan were T. ghanense, T. parareesei and T. sinense. The significance of their distribution is discussed. Most species of the Longibrachiatum Clade are tropical rather than temperate in distribution. Their in vitro optimum growth tends to be >35 °C but the optimum temperature for some Japanese species was lower. Some species are endophytes of temperate plant species, some of which are endemic in Japan.