Unraveling the Life Cycle of Nyssopsora cedrelae: A Study of Rust Diseases on Aralia elata and Toona sinensis.

Rust disease poses a major threat to global agriculture and forestry. It is caused by types of Pucciniales, which often require alternate hosts for their life cycles. Nyssopsora cedrelae was previously identified as a rust pathogen on Toona sinensis in East and Southeast Asia. Although this species had been reported to be autoecious, completing its life cycle solely on T. sinensis, we hypothesized that it has a heteroecious life cycle, requiring an alternate host, since the spermogonial and aecial stages on Aralia elata, a plant native to East Asia, are frequently observed around the same area where N. cedrelae causes rust disease on T. sinensis. Upon collecting rust samples from both A. elata and T. sinensis, we confirmed that the rust species from both tree species exhibited matching internal transcribed spacer (ITS), large subunit (LSU) rDNA, and cytochrome oxidase subunit III (CO3) mtDNA sequences. Through cross-inoculations, we verified that aeciospores from A. elata produced a uredinial stage on T. sinensis. This study is the first report to clarify A. elata as an alternate host for N. cedrelae, thus providing initial evidence that the Nyssopsora species exhibits a heteroecious life cycle.

First Report of the Rust Fungus Melampsora ferrinii on Salix babylonica in China and a New Spermogonial and Aecial Host, Corydalis bungeana.

The occurrence of rust fungi on Corydalis bungeana Turcz. and Salix babylonica L. were found in same area of Hebei Province, China from 2022 to 2023. The life cycle connection of these rust fungi was suspected because Peng et al. (2022) reported the life cycle of Melampsora ferrinii Toome & Aime by inoculations, producing spermogonia and aecia on Corydalis species, and uredinia on S. babylonica. The morphology of the uredinial and telial stages on S. babylonica collected in the field was identical with the description of M. ferrinii by Toome and Aime (2015), and its identity was confirmed by phylogenetic analyses using the method of Ji et al. (2020) (LSU-PP087777, ITS-PP091274; Similarity with M. ferrinii: LSU-100%, ITS-99.85%). To confirm the life cycle of this rust fungus, inoculations were conducted on C. bungeana with basidiospores obtained from the teliospores on fallen leaves of Salix babylonica. The fallen leaves producing basidiospores were cut into small pieces (ca. 5 mm2) and placed on healthy leaves of C. bungeana. The inoculated plants were kept in a moist plastic box in darkness at 15-20℃ for 2 days and then transferred to the floor near windows at about 15-20℃ for observations. Ten days after inoculations small yellow spots of spermogonia appeared on the upper surface of the leaves of C. bungeana. About 7 days later, pale yellow aecia with aeciospores were produced mainly on the under surface of the leaves and petioles. The morphology of rust fungus on C. bungeana collected from the fields and obtained by inoculations was identical with the description by Peng et al. (2022). Phylogenetic analyses also showed that a specimen on C. bungeana collected from the field (LSU-OR607838, ITS-OR612063) were included into the same clade of M. ferrinii (Similarity: LSU-100 %, ITS-99.85). Based on morphology, inoculations and DNA sequence analyses, the rust fungi on C. bungeana and S. babylonica are identified as different stages of life cycle of M. ferrinii. This rust fungus has been reported to produce spermogonia and aecia on C. acuminata Franch., C. edulis Maxim. and C. racemosa (Thunb.) Pers. in China (Peng et al. 2022), and uredinia and telia on S. babylonica in USA, Argentina and Iran (Toome and Aime 2015, Abbasi et al. 2024), and on Salix sp. in Chile (Zapata 2016). Therefore, C. bungeana is a new host for M. ferrinii, and its field occurrence on S. babylonica is reported for the first time in China although Peng et al. (2022) reported successful results in its inoculations to S. babylonica in China. This report contributes to the control of rust diseases caused by this species. Specimens used in this experiment were deposited in the Fungal Herbarium of the Jilin Agricultural University, Changchun, China (HMJAU) and sequences newly analyzed were deposited in GenBank.

Phylogeny and taxonomy of three species of Blastospora (Pucciniales) from East Asia.

Three species of the rust fungus genus Blastospora, Bl. betulae, Bl. itoana, and Bl. smilacis, have been reported in East Asia. Although their morphological characteristics and life cycles have been investigated, their phylogenetic positions have not been clarified sufficiently. Phylogenetic analysis showed that these three species were included into Zaghouaniaceae of Pucciniales. However, Bl. betulae was phylogenetically distinct from Bl. itoana and Bl. smilacis and different from other genera. Based on this result, and applying recent International Code of Nomenclature decisions/recommendations/requirements, Botryosorus, gen. nov., and Bo. deformans,, comb. nov., were applied for Bl. betulae. Two new combinations, Bl. radiata for Bl. itoana and Bl. makinoi for Bl. smilacis, were also applied. Their host plants and distribution were described based on literature records. Zaghouania yunnanensis, comb. nov., was proposed for Cystopsora yunnanensis as a result of this analysis.

First report of rust caused by Puccinia paullula on Monstera deliciosa and M. adansonii in Japan.

Monstera deliciosa Liebm. and M. adansonii Schott. (Araceae) have been cultivated for the commercial production in Hachijo Island, Tokyo, Japan, since the 1950s. A rust disease producing uredinial sori was found on the leaves of M. deliciosa and M. adansonii in the fields on Hachijo Island in February 2021. Symptoms were first observed on the leaf surfaces as small chlorotic spots, which expanded and became brown to reddish-brown, and produced uredinia with abundant urediniospores. The disease occurred on the whole island including the farm land, symptom incidence ranged from 5 to 30%. To confirm the pathogenicity of this rust fungus, ten plant species of Araceae (M. deliciosa, M. adansonii, Alocasia macrorrhizos, Anthurium andreauum, Dieffenbachia maculata, Epipremnum mirabile, E. pinnatum, Philodendron scandens, Spathiphyllum sp., Syngonium podophyllum) were inoculated with urediniospores obtained from infected samples on M. deliciosa and M. adansonii. Urediniospores were suspended in distilled water (1 × 106 conidia/ml) and sprayed on healthy plants. The inoculated plants were kept in a dark chamber at about 25°C for 48 h, and then transferred to a greenhouse. After 40 days, uredinia were reproduced only on M. deliciosa and M. adansonii. Morphological characteristics of the urediniospores obtained from inoculated monstera plants matched those obtained from the field plants and used as inoculum, thus fulfilling Koch's postulates. No symptoms were observed on the other plant species inoculated. For identification of the rust fungus, dry specimens obtained from both naturally infected plants and inoculated plants were used for morphological observations. Urediniospores with pedicels emerging from the stomata were densely aggregated, globose, and 24.8-29.3 µm (n = 30). Their walls were brown, echinulate, and 1.4-2.2 µm thick. Telia were found in the specimens collected in July and August. The teliospores with pedicels emerging from the stomata were two-celled, ellipsoid, and 21.3-27.5 × 19.5-23.4 µm. Their walls were pale yellow, smooth, and 1.0-1.9 µm thick. Morphological differences among the specimens on M. deliciosa and M. adansonii were not observed. These morphological characteristics were consistent with the description of P. paullula (Sydow and Sydow 1913). For molecular identification, the large subunit (LSU) rDNA and cytochrome oxidase III (cox3) were amplified. LRust1R, LR3, and LR5 (Vilgalys and Hester 1990) primers were used for sequencing of LSU region. Cox3_F (5'-GTTCAGTATGTTATTTTAACG-3') and cox3_R (5'-ATAGGAATAGTCAAACAACATC-3') primers were constructed here based on the P. paullula sequences (KX999927) for the cox3 region. The sequences were deposited in the GenBank as accession numbers OK509070, OK509071, LC663719 and LC663720. BLAST analysis showed that LSU and cox3 sequences shared 98.8% (902/913 nts) and 100% (632/632 nts) identities with P. paullula, KX999886 and KX999927 (Marin-Felix et al. 2017). This is the first report of a rust disease of M. deliciosa and M. adansonii caused by P. paululla in Japan. The rust fungi has been reported as the pathogen on the species of Amorphophallus, Rhaphidophora and Monstera (Sydow and Sydow 1913, Shaw 1991, Chen et al. 2009). The occurrence of this disease should be monitored because it can reduced yield and commercial value of monstera plants. All the specimens used in the experiments were deposited in the National Museum of Nature and Science, Japan (TNS-F82068-82077).

Puccinia caricis-smilacis, a new caricicolous rust fungus producing spermogonial and aecial stages on Smilax china in Japan.

Puccinia caricis-smilacis, a new species of caricicolous rust fungus was described based on morphological characteristics and nuclear rDNA sequences from Japan. The heteroecious life cycle of P. caricis-smilacis was elucidated via field observations, inoculation experiments and molecular analyses. This rust fungus produces uredinial and telial stages on Carex fibrillosa, and spermogonial and aecial stages on Smilax china.

Species diversity, taxonomy, and phylogeny of Gymnosporangium in China.

Gymnosporangium is a group of plant fungal pathogens that cause rust diseases on many economically important fruit trees. Most Gymnosporangium are heteroecious and demicyclic, producing four morphologically diverse spore stages on two taxonomically unrelated host plants, the Cupressaceae and Rosaceae. The complex life cycle and heteroecism make it difficult to investigate the species within Gymnosporangium. To determine the taxonomy, phylogeny, and species diversity of Gymnosporangium in China, a large collection of 672 specimens were analyzed using a combination of morphological observations and phylogenetic analyses. In total, 27 Gymnosporangium species from China are documented here, including 22 known species, one new combination, one new record, and three new species. The study also documents a novel aeciospore surface structure with an irregular surface that is described here as "surfy."

Application of Toxigenic Alternaria oxytropis to Soybeans and its Effect on Swainsonine Detection in Different Environments.

Alternaria oxytropis is an endophytic fungus of locoweeds that synthesizes swainsonine toxin. In this work, we evaluated the effect of A. oxytropis on soybean seedlings and quantified swainsonine in different culture conditions. Soybean (Glycine max) seeds were co-cultured with A. oxytropis (at different concentrations of mycelial suspensions) in agar media and soil culture, and swainsonine was assayed using LC-MS/MS. The results showed evidence that A. oxytropis infected soybean seedlings produced detectable swainsonine in agar culture while the toxin was undetectable or below the detection limit (0.006% of swainsonine dry weight) in soil media even at higher concentrations of the fungus. These results suggest that swainsonine detection is highly dependent on culture conditions and that soybeans co-cultured with A. oxytropis in soil could potentially be used to limit toxin production.

The enigmatic Mixia osmundae revisited: a systematic review including new distributional data and recent advances in its phylogeny and phylogenomics.

The enigmatic basidiomycete genus Mixia includes intracellular parasites of Osmunda and Osmundastrum ferns. Here, the authors review the systematic and phylogenetic history of M. osmundae, originally known as Taphrina osmundae, and provide new data from investigations of specimens of Osmunda japonica collected in Yunnan Province, China, which we determine to be conspecific with M. osmundae. In addition, Taphrina higginsii, a parasite on fronds of Osmundastrum cinnamomeum described from Georgia, USA, was confirmed to be phenotypically identical with M. osmundae. The name T. higginsii is lectotypified with a Mix specimen. Collections examined to date document four localities for M. osmundae: Japan (Honshu and Kyushu), Taiwan (Taichung), USA (Georgia), and China (Yunnan), and host-parasite relationships with the old extant ferns Osmunda japonica and its relatives and with Osmundastrum cinnamomeum. The phylogenetic placement of M. osmundae on the fungal tree of life, its evolutionary implications, and recent advances in the phylogenomics of this fungus are briefly reviewed and discussed.

The effect of Hypomyces perniciosus on the mycelia and basidiomes of Agaricus bisporus.

Hypomyces perniciosus has been reported as a destructive pathogen of Agaricus bisporus. Previous research suggested that the pathogenesis may not only be perpetuated by H. perniciosus, but also by bacteria. Clarification of the interaction between A. bisporus and H. perniciosus is a prerequisite for the development of effective control measures against wet bubble disease. Here, the effects of H. perniciosus on A. bisporus mycelia are examined in dual culture on agar media and in open-ended test tubes. During disease development, the putative causal agents and cytology of wet bubble-diseased mushrooms were followed microscopically. The interaction between H. perniciosus and the basidiome of A. bisporus was also studied using dual-cultured H. perniciosus and basidiome tissues. Dual-cultured mycelia from both fungi showed that growth continued even after contact was made, without any observable antagonistic lines or cytoplasmic changes of A. bisporus mycelia. Hypomyces perniciosus could be isolated from diseased basidiomes any time after inoculation, but bacteria were only recovered after the basidiomes of A. bisporus had been killed by H. perniciosus. Dual culture of the basidiome tissue of A. bisporus and H. perniciosus on agar media established that H. perniciosus can independently and rapidly degrade the basidiomes of A. bisporus. We conclude that H. perniciosus has no pathogenic activity on the mycelial stage of A. bisporus, but it can destroy A. bisporus basidiomes in the absence of bacteria. Wet bubble disease is evidently not caused by bacteria, but by the fungus, although bacteria likely participate in the disease after invasion by the fungus.

Resolving the Melampsora epitea complex.

Melampsora epitea (Pucciniales, Basidiomycota) has been widely known as the main rust pathogen on willows, but its species boundary has been extremely confusing. Melampsora epitea was often confused with several morphologically similar species, such as M. arctica, M. humilis, and M. epiphylla. Recent DNA-based studies using specimens from North America and Europe suggested that M. epitea was a complex of several phylogenetic species. In this study, we examined the species boundary of M. epitea based on rDNA phylogeny and the general mixed Yule coalescent (GMYC) model from 534 specimens. Twelve distinct species corresponding to 10 known species and 2 new species were recognized from East Asian specimens. We also evaluated the association of morphological characteristics, geographic origins, and host ranges with phylogenetic relationships. Our results revealed that several previously overlooked morphological characters in uredinial and telial stages were informative in delimiting M. epitea and allied species. Two novel species, M. salicis-futurae and M. salicis-triandrae, are described and compared with closely related species. A lectotype for M. epitea and epitypes for M. epitea, M. epiphylla, and M. humilis are designated to stabilize the use of names.

[The development of Agaricus bisporus wet bubble disease and the nuclear phase of pathogen].

Objective: We studied the dynamic nuclear behavior of Hypomyces perniciosus on axenic culture and its disease progression after infection on different growth stages of Agaricus bisporus. Methods: Infection process was initiated by inoculating different stages of A. bisporus fruit body, and different depths of compost and casing soil with H. perniciosus. Disease progression was studied by observing symptoms on the fruit body using light microscopy and scanning electron microscopy. The nuclear behavior of H. perniciosus was determined by observation using fluorescence light microscopy after binding of DNA specific fluorochrome dye (DAPI:4, 6-Diamidino-2-phenylindole dihydrochloride) to the nuclei. Results: Inoculating H. perniciosus on different depths of compost and casing soil resulted in different disease rate as follows:on the surface of casing soil>in the center of casing soil>between the casing soil and the compost>in the center of compost. H. perniciosus can infect any stage of fruit body development, when young primordial (up to 3 mm) was infected, large, irregular and tumorous fungal masses were formed. H. perniciosus directly penetrated A. bisporus without the formation of appressorium-like structures. The germination of the conidia led to a necrotic brown lesion symptom on A. bisporus at the beginning stages of disease development. The mycelium of A. bisporus plasmolysed, hydropically degenerated, cytoplasmolysed, emptied of mycelium cytosol and eventual death as the disease advanced. H. perniciosus produced two types of conidia. Group I conidia had no septa, colorless and smooth containing one nucleus. Group II didymoconidium had septa, containing two nuclei, separated by septa. The first round of mitosis occurred in conidia with no nucleus in the germinal tube. Another kind of asexual spore for thicker cell wall wart convex chlamydospore, chlamydospore had two cells. The upper cell had two nuclear while the basal cell had one or two nuclear, when germinated, it produced one or two germinal tubes. The number of nuclear in the germinal tube was irregular, usually contained 0 to 2 nuclear. Conclusion: H. perniciosus can infect any part of the A. bisporus fruit body and can cause tremendous cytology changed. If we perform single spore isolation to do genetic analysis, one must isolate conidia with no septa.

Integrating a Numerical Taxonomic Method and Molecular Phylogeny for Species Delimitation of Melampsora Species (Melampsoraceae, Pucciniales) on Willows in China.

The species in genus Melampsora are the causal agents of leaf rust diseases on willows in natural habitats and plantations. However, the classification and recognition of species diversity are challenging because morphological characteristics are scant and morphological variation in Melampsora on willows has not been thoroughly evaluated. Thus, the taxonomy of Melampsora species on willows remains confused, especially in China where 31 species were reported based on either European or Japanese taxonomic systems. To clarify the species boundaries of Melampsora species on willows in China, we tested two approaches for species delimitation inferred from morphological and molecular variations. Morphological species boundaries were determined based on numerical taxonomic analyses of morphological characteristics in the uredinial and telial stages by cluster analysis and one-way analysis of variance. Phylogenetic species boundaries were delineated based on the generalized mixed Yule-coalescent (GMYC) model analysis of the sequences of the internal transcribed spacer (ITS1 and ITS2) regions including the 5.8S and D1/D2 regions of the large nuclear subunit of the ribosomal RNA gene. Numerical taxonomic analyses of 14 morphological characteristics recognized in the uredinial-telial stages revealed 22 morphological species, whereas the GMYC results recovered 29 phylogenetic species. In total, 17 morphological species were in concordance with the phylogenetic species and 5 morphological species were in concordance with 12 phylogenetic species. Both the morphological and molecular data supported 14 morphological characteristics, including 5 newly recognized characteristics and 9 traditionally emphasized characteristics, as effective for the differentiation of Melampsora species on willows in China. Based on the concordance and discordance of the two species delimitation approaches, we concluded that integrative taxonomy by using both morphological and molecular variations was an effective approach for delimitating Melampsora species on willows in China.

¹³7Cs concentrations in foliose lichens within Tsukuba-city as a reflection of radioactive fallout from the Fukushima Dai-ichi Nuclear Power Plant accident.

(137)Cs concentrations in ten species of foliose lichens collected within Tsukuba-city in August 2013 ranged from 1.7 to 35 kBq/kg. The relationships between (137)Cs in two dominant species, Dirinaria applanata and Physcia orientalis, and the air dose rate (µSv/h) at the sampling sites were investigated. (137)Cs in P. orientalis measured about 1 year after the Fukushima nuclear accident was correlated (r(2) = 0.80) more closely with the air dose rate than those measured after about 2 years (r(2) = 0.65), possibly demonstrating its continued value as a biomonitor to reflect ambient fall-out levels. In contrast, those of Dirinaria applanata were not correlated with the air dose rate in either year.

Lack of cross-resistance to a novel succinate dehydrogenase inhibitor, fluopyram, in highly boscalid-resistant isolates of Corynespora cassiicola and Podosphaera xanthii.

BACKGROUND: Recently in Japan, isolates resistant to boscalid, a succinate dehydrogenase inhibitor (SDHI), have been detected in Corynespora cassiicola (Burk. & Curt.) Wei and Podosphaera xanthii (Castaggne) Braun & Shishkoff, the pathogens causing Corynespora leaf spot and powdery mildew disease on cucumber, respectively. Resistant isolates of C. cassiicola are widely distributed and represent a serious problem in disease control at present. Novel SDHI fungicides, including fluopyram, are now under development. RESULTS: The growth of very highly boscalid-resistant, highly resistant and sensitive isolates of C. cassiicola was strongly suppressed on fluopyram-amended YBA agar medium. Although boscalid and another SDHI, penthiopyrad, hardly controlled Corynespora leaf spot and powdery mildew on cucumber plants when very highly or highly boscalid-resistant isolates were employed for inoculation, fluopyram still exhibited excellent control efficacy against these resistant isolates as well as sensitive isolates of C. cassiicola and P. xanthii. CONCLUSION: Differential sensitivity to boscalid, penthiopyrad and fluopyram, clearly found in these two important pathogens of cucumber, may indicate involvement of a slightly distinct site of action for fluopyram from the two other SDHIs. This finding may lead to the discovery of unique SDHIs in the future.

Proteome analysis of probenazole-effect in rice-bacterial blight interactions.

To study the effect of probenazole on the induced systemic resistance mechanism of rice-bacterial interaction, a proteomic approach was applied. Oryza sativa cv. Java 14 seedlings were treated with probenazole, followed by inoculation with compatible (Xo7435) and incompatible (T7174) races of Xanthomonas oryzae pv. oryzae. Cytosolic proteins were fractionated from leaf blades, separated by two-dimensional polyacrylamide gel electrophoresis. Pathogenesis-related protein 5 (PR5) was significantly induced with probenazole treatment followed by inoculation with T7174 or Xo7435. The sense PR5 transgenic rice plants were more highly resistant than the susceptible vector control against Xo7435. These results indicate that probenazole strongly induces PR5 in the interaction between rice and X. oryzae pv. oryzae, and might be involved in the resistance mechanism of rice against bacterial blight.

Phylogenetic relationships of Polyporus and morphologically allied genera.

Polyporus accommodates morphologically heterogeneous species and is divided into six infrageneric groups based on macromorphological characters. On the other hand allied genera have macro- and microscopic characters similar to those of Polyporus. The phylogenetic relationships of Polyporus and allied genera were established from sequences of RNA polymerase II second largest subunit (RPB2), nuclear ribosomal large subunit (nucLSU) and mitochondrial ATPase subunit 6 (ATP6). The molecular phylogenetic trees confirmed that Polyporus is a polyphyletic genus and recognized six major clades (1-6) containing species of Polyporus and several allied genera. Among the clades one contained three infrageneric groups of Polyporus and two allied genera, Datronia and Pseudofavolus while one other contained group Polyporellus and Lentinus. Five of the six major clades contained species belonging to a single infrageneric group, Favolus, Melanopus, Polyporellus or Polyporus. This suggests that morphological characters used to define these groups have phylogenetic significance and reveals the need for a taxonomic revision of Polyporus and its allied genera.

Proteomic analysis of jasmonic acid-regulated proteins in rice leaf blades.

Jasmonates play a critical role in plant defense against pathogens through regulation of the expression of defense-related genes. To study the role of jasmonic acid (JA) in the rice self-defense mechanism, a proteomic approach was applied. When 3-week-old rice cv. Java 14 was treated with 100 microM JA for 3 days, numerous necrotic brown spots were observed on the leaf blade. Three-week-old rice was treated with JA and proteins from cytosolic and membrane fractions of leaf blade were separated by two-dimensional polyacrylamide gel electrophoresis. A total of 305 proteins were detected in both cytosolic and membrane fractions. When rice plant was treated with 100 microM JA for 2 days, 12 proteins were up-regulated and 2 proteins were down-regulated. Out of them, 8 proteins were changed in dose dependence manner, while 4 proteins were changed in a time course manner. Among them, pathogenesis-related protein 5 (PR5) and probenazole inducible protein 1 (PBZ1) were significantly induced by 100 microM JA for 2 days. These results suggest that PR5 and PBZ1 are important proteins expressed down-stream of JA signals in rice cv. Java 14.

Proteomic analysis of bacterial-blight defense-responsive proteins in rice leaf blades.

Plants exhibit resistance against incompatible pathogens, via localized and systemic responses as part of an integrated defense mechanism. To study the compatible and incompatible interactions between rice and bacteria, a proteomic approach was applied. Rice cv. Java 14 seedlings were inoculated with compatible (Xo7435) and incompatible (T7174) races of Xanthomonas oryzae pv. oryzae (Xoo). Cytosolic and membrane proteins were fractionated from the leaf blades and separated by 2-D PAGE. From 366 proteins analyzed, 20 were differentially expressed in response to bacterial inoculation. These proteins were categorized into classes related to energy (30%), metabolism (20%), and defense (20%). Among the 20 proteins, ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (RuBisCO LSU) was fragmented into two smaller proteins by T7174 and Xo7435 inoculation. Treatment with jasmonic acid (JA), a signaling molecule in plant defense responses, changed the level of protein accumulation for 5 of the 20 proteins. Thaumatin-like protein and probenazole-inducible protein (PBZ) were commonly up-regulated by T7174 and Xo7435 inoculation and JA treatment. These results suggest that synthesis of the defense-related thaumatin-like protein and PBZ are stimulated by JA in the defense response pathway of rice against bacterial blight.

Thysanophora penicillioides includes multiple genetically diverged groups that coexist respectively in Abies mariesii forests in Japan.

We investigated intraspecific diversity and genetic structures of a saprotrophic fungus--Thysanophora penicillioides--based on sequences of nuclear ribosomal internal transcribed spacer (ITS) in 15 discontinuous Abies mariesii forests of Japan. In such a well-defined morphological species, numerous unexpected ITS variations were revealed: 12 ITS sequence types detected in 254 isolates collected from 15 local populations were classified into five ITS sequence groups. Maximally, four ITS groups consisted of seven ITS types coexisting in one population. However, group 1 was dominant with approximately 65%; in particular, one haplotype, 1a, was most dominant with approximately 60% in respective populations. Therefore, few differences were recognized in genetic structure among local populations, implying that the gene flow of each lineage of the fungus occurs among local populations without geographic limitations. However, minor haplotypes in some ITS groups were found only in restricted areas, suggesting that they might expand steadily from their places of origin to neighboring A. mariesii forests. Aggregating sequence data of seven European strains and four North American strains from various substrates to those of Japanese strains, 18 ITS sequence types and 28 variable sites were recognized. They were clustered into nine lineages by phylogenetic analyses of the beta-tubulin and combined ITS and beta-tubulin datasets. According to phylogenetic species recognition by the concordance of genealogies, respective lineages correspond to phylogenetic species. Plural phylogenetic species coexist in a local population in an A. mariesii forest in Japan.