Correlation between pathogenicity and molecular characteristics in the willow leaf rusts <Emphasis Type="Italic">Melampsora epitea</Emphasis> and <Emphasis Type="Italic">M. humilis</Emphasis> in Japan

 We studied the correlation between pathogenicity and restriction fragment length polymorphism (RFLP) type, which was determined by polymerase chain reaction-based RFLP analysis of the internal transcribed spacer regions of ribosomal DNA, in the willow leaf rust fungi Melampsora epitea and M. humilis. Eighteen clones of eight Salix species were inoculated with urediniospores from seven collections of the two rust species. M. epitea and M. humilis (RFLP type-5 collections) were pathogenic to six to eight Salix species. RFLP type-7 collections of M. epitea were pathogenic to only two Salix species. The taxonomic relationships of the two rust species are discussed. Received: December 11, 2002 / Accepted: February 17, 2003 RID="*" ID="*" Contribution no. 179, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan Acknowledgments We thank K. Katsuya, former professor at the University of Tsukuba, for his encouragement in this study. We are also grateful to M. Yashima, Botanical Garden, University of Tohoku, for his assistance in collecting materials and to R. Suzuki, University of Tsukuba, for providing a rust isolate.     

<Emphasis Type="Italic">Exobasidium dubium</Emphasis> and <Emphasis Type="Italic">E. miyabei</Emphasis> sp. nov. causing Exobasidium leaf blisters on <Emphasis Type="Italic">Rhododendron </Emphasis>spp. in Japan

 Two Exobasidium species causing Exobasidium leaf blister on Rhododendron spp. are described. An Exobasidium leaf blister on Rhododendron yedoense var. yedoense f. yedoense has been recognized in Hokkaido Prefecture, Japan, since the first report was issued in 1950. The causal fungus is identified with Exobasidium dubium from the morphology of its hymenial structure and mode of germination of the basidiospores. Another Exobasidium leaf blister on Rhododendron dauricum has been observed in Hokkaido Prefecture, Japan. In comparison with morphology based on hymenial structure and mode of germination of the basidiospores of the 100 validly described taxa, this fungus differs from those known taxa in the size of basidia and basidiospores, the numbers of sterigmata and septa of basidiospores, and the mode of germination of basidiospores. Thus, a new species, Exobasidium miyabei, is established and illustrated. Received: February 13, 2002 / Accepted: September 25, 2002  Present address: National Institute of Agrobiological Sciences, Tsukuba 305-8602, Japan Acknowledgments We profoundly appreciate the cooperation of Dr. V. Melnik in providing Russian papers and Dr. L. Vasilyeva for translating them into English. We thank Prof. H. Takahashi for loaning the materials in the Herbarium of the Hokkaido University Museum and Dr. W. Abe, Graduate School of Science, University of Hokkaido, for his kind help with the sampling of R. dauricum in Teshikaga, Hokkaido Prefecture. This study was supported in part by a Grant-in-Aid for Scientific Research (B) (No. 13460019), Japan Society for the Promotion of Science (JSPS). Contribution No. 171, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba. Correspondence to:M. Kakishima     

Formation of structures resembling ericoid mycorrhizas by the root endophytic fungus Heteroconium chaetospira within roots of Rhododendron obtusum var. kaempferi

A resynthesis study was conducted to clarify the relationship between the root endophyte, Heteroconium chaetospira and the ericaceous plant, Rhododendron obtusum var. kaempferi. The host plant roots were recovered 2 months after inoculation, and the infection process and colonization pattern of the fungus were observed under a microscope. The hyphae of H. chaetospira developed structures resembling ericoid mycorrhizas, such as hyphal coils within the host epidermal cells. These structures were morphologically the same as previously reported ericoid mycorrhizal structures. The frequencies of hyphal coils within the epidermal cells of host roots ranged from 13 to 20%. H. chaetospira did not promote or reduce host plant growth. This is the first reported study that H. chaetospira is able to form structures resembling mycorrhizas within the roots of ericaceous plants.     

Fungal coil formation ofRhizoctonia repens in seedlings ofGaleola septentrionalis (orchidaceae)

Protocorms or protocorms with roots of an achlorophyllous orchidGaleola septentrionalis were inoculated with isolates ofRhizoctonia repens, R. solani, andRhizoctonia spp. The seedlings were infected with eight of twelve isolates ofR. repens. Fungal coils were formed in the cells, which was suggestive of a symbiotic association. The other isolates caused soft rot or no infection to the protocorms or the protocorms with a root. Contribution No. 97, Laboratories of Plant Pathology and Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Tsukuba, Ibaraki 305, Japan.     

Diversity of root-associated fungal endophytes in Rhododendron fortunei in subtropical forests of China

To investigate the diversity of root endophytes in Rhododendron fortunei, fungal strains were isolated from the hair roots of plants from four habitats in subtropical forests of China. In total, 220 slow-growing fungal isolates were isolated from the hair roots of R. fortunei. The isolates were initially grouped into 17 types based on the results of internal transcribed spacer-restriction fragment length polymorphism (ITS-RFLP) analysis. ITS sequences were obtained for representative isolates from each RFLP type and compared phylogenetically with known sequences of ericoid mycorrhizal endophytes and selected ascomycetes or basidiomycetes. Based on phylogenetic analysis of the ITS sequences in GenBank, 15 RFLP types were confirmed as ascomycetes, and two as basidiomycetes; nine of these were shown to be ericoid mycorrhizal endophytes in experimental cultures. The only common endophytes of R. fortunei were identified as Oidiodendron maius at four sites, although the isolation frequency (3–65%) differed sharply according to habitat. Phialocephala fortinii strains were isolated most abundantly from two habitats which related to the more acidic soil and pine mixed forests. A number of less common mycorrhizal RFLP types were isolated from R. fortunei at three, two, or one of the sites. Most of these appeared to have strong affinities for some unidentified root endophytes from Ericaceae hosts in Australian forests. We concluded that the endophyte population isolated from R. fortunei is composed mainly of ascomycete, as well as a few basidiomycete strains. In addition, one basidiomycete strain was confirmed as a putative ericoid mycorrhizal fungus.     

The mycorrhiza of <Emphasis Type="Italic">Schizocodon soldanelloides</Emphasis> var. <Emphasis Type="Italic">magnus</Emphasis> (Diapensiaceae) is regarded as ericoid mycorrhiza from its structure and fungal identities

Structure and fungal identities were examined in the mycorrhizal roots of Schizocodon soldanelloides var. magnus (Diapensiaceae) to determine the mycorrhizal category. Previous studies had suggested the mycorrhizae of Diapensiaceae could be categorized as ericoid, but the mycorrhizal fungi have never been identified. The diameter of the fine lateral roots, in which coiled hyphae were found in epidermal cells, was mostly less than 100 μm. Molecular analyses identified the fungal isolates to be Helotiales and Oidiodendron. From the structure and fungal identities, we confirmed that the mycorrhiza of S. soldanelloides is an ericoid mycorrhiza.     

Mycorrhizal association of isolates from sporocarps and ectomycorrhizas withPinus densiflora seedlings

Thirty-two isolates from sporocarps of 27 species of macromycetes, 43 isolates from ectomycorrhizas ofPinus densiflora (Japanese red pine) and 1 isolate from an ectomycorrhiza ofQuercus myrsinaefolia were tested for the ability to form mycorrhizas withP. densiflora seedlings in glass tubes. Ten isolates from sporocarps ofHebeloma sp.,Laccaria bicolor, Lactarius chrysorrheus, Suillus granulatus, Scleroderma areolatum, Russula mariae andR. nigricans had formed ectomycorrhizas by 8 months after transplantation. Twenty isolates taken from mycorrhizas including ofCenococcum geophilum, R. mariae andR. nigricans formed ectomycorrhizas. The synthesized mycorrhizas were classified based on morphological characteristics such as hyphal arrangement of their fungal sheath, and appearance of cystidia and emanating hyphae. Twenty-one mycorrhizal types were recognized.Contribution No. 122, Laboratories of Plant Pathology and Mycology, Institute of Agriculture and Forestry, University of Tsukuba.     

Mycorrhizal differences between genuine roots and tuberous roots of adult plants ofSpiranthes sinensis var.amoena (Orchidaceae)

Genuine roots ofSpiranthes sinensis var.amoena were infected with the mycorrhizal fungusRhizoctonia repens immediately after root formation in autumn. Infection by the mycorrhizal fungus extended, reaching a maximum the following early summer. The amount of living mycorrhizal fungus in the genuine roots dramatically declined in the flowering season, and then the roots decomposed. Tuberous roots were formed in spring. Mycorrhizas were limited to local infections and did not spread along the roots. The infection level of living mycorrhizal fungus in the tuberous roots was less than in the genuine roots throughout the year. The amount of dead fungal coils in the tuberous roots increased as the tuberous roots aged. The mycorrhizal characteritics of tuberous roots ofS. sinensis var.amoena were totally different from those of genuine roots although the tuberous roots morphologically resembled the genuine roots. Contribution No. 96, Laboratories of Plant Pathology and Mycology, Institute of Agriculture and Forestry, University of Tsukuba.     

Life cycle of <Emphasis Type="Italic">Uromyces appendiculatus</Emphasis> var. <Emphasis Type="Italic">azukicola</Emphasis> on <Emphasis Type="Italic">Vigna angularis</Emphasis>

Field observations and inoculation experiments revealed that Uromyces appendiculatus var. azukicola has an autoecious and macrocyclic life cycle and produces spermogonia, aecia, uredinia, and telia on Vigna angularis var. angularis and V. angularis var. nipponensis. From inoculation experiments, it was suggested that this rust fungus has different host relationships from other varieties. Morphological examinations revealed that the characteristics of urediniospores and teliospores are different among varieties, although aeciospores are morphologically similar to each other.Contribution no. 182, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan     

Caeoma tsukubaense n. sp., a rhododendron rust fungus of Japan and southern Asia, and its relationship to Chrysomyxa rhododendri

A rust fungus found in Japan on Rhododendron kaempferi, R. kiusianum, and R. dauricum has previously been identified as Chrysomyxa rhododendri. Light and scanning electron microscopy of fresh and herbarium materials of the rust fungus, however, show that the spore surface morphology differs from the urediniospores of C. rhododendri, and the spores are slightly smaller. Furthermore, the DNA sequence of the 5′-end of the large subunit of ribosomal DNA differs from that of C. rhododendri by 3%. Telia have not been found; therefore, it is redescribed as a new anamorphic species, Caeoma tsukubaense. Several specimens from North Korea, Tibet, and Nepal bearing a similar rust fungus are also included in the species.Contribution no.193 from the Laboratory of Plant Parasitic Mycology, Graduate School of Life and Environmental Sciences, University of Tsukuba, Japan     

Three species of rust fungi (Uredinales) from Okinawa,Japan

Three species of rust fungi were reported based on the specimens collected in the islands of the Yaeyama group and Okinawa, Japan.Puccinia tarennicola onTarenna gracilipes andUredo daphniphylli onDaphniphyllum teijsmannii were described as new species.Villebrunea frutescens was added to the host plants ofUredo pipturi. Contribution No. 115, Laboratories of Plant Pathology and Mycology, Institute of Agriculture and Forestry, University of Tsukuba.     

Molecular diversity of fungi from ericoid mycorrhizal roots

In order to investigate the diversity of fungal endophytes in ericoid mycorrhizal roots, about 150 mycelia were isolated from surface-sterilized roots of 10 plants of Calluna vulgaris. Each mycelium was reinoculated to C. vulgaris seedlings under axenic conditions, and the phenotype of the plant-fungus association assessed by light and electron microscopy. Many isolates that were able in vitro to produce typical ericoid mycorrhizae did not form reproductive structures under our culture conditions, whereas others could be identified as belonging to the species Oidiodendron maius. Morphological and molecular analysis of the fungal isolates showed that the root system of a single plant of C. vulgaris is a complex mosaic of several populations of mycorrhizal and non mycorrhizal fungi. PCR-RFLP techniques, used to investigate the mycorrhizal endophytes, revealed up to four groups of fungi with different PCR-RFLP patterns of the ITS ribosomal region from a single plant. Some of the mycorrhizal fungi sharing the same PCR-RFLP pattern showed high degree of genetic polymorphism when analysed with the more sensitive RAPD technique; this technique may prove a useful tool to trace the spread of individual mycorrhizal mycelia, as it has allowed us to identify isolates with identical RAPD fingerprints on different plants.     

Diversity of culturable ericoid mycorrhizal fungi of Rhododendron decorum in Yunnan, China

The diversity of ericoid mycorrhizal fungi isolated from Rhododendron decorum Franch. in Yunnan, southwestern China, was examined for the first time. In total 300 hair-root samples were collected from 13 R. decorum individuals in two adjacent wild population sites and one cultivated population site. Two hundred eighteen slow-growing isolates were obtained; the ability of some to form ericoid mycorrhiza was tested in vitro. One hundred twenty-five isolates formed hyphal structures morphologically corresponding to ericoid mycorrhiza, and these were determined by morphological and molecular means to belong to 12 fungal species. There were hardly any differences in species among the three sampled populations. The sequences of several isolates were similar to those of Oidiodendron maius and ericoid mycorrhizal fungi from Helotiales, accounting respectively for 18.4% and 24.8% of the total culturable ericoid mycorrhizal fungi assemblage. Dark septate endophytes were detected in the sampled hair roots by microscopy.     

Vesicular-arbuscular mycorrhizal fungi in coastal dune plant communities I. Spore formation ofGlomus spp. predominates under a patch ofElymus mollis

Vesicular-arbuscular (VA) mycorrhizal fungi in pure patches of coastal dune plantsElymus mollis, Wedelia prostrata andZoysia macrostachya were examined for frequency of occurrence and number of spores of VA mycorrhizal fungi over one year. Six species in three genera of VA mycorrhizal fungi were recovered. Under a patch ofE. mollis, spores ofAcaulospora sp. 1,Glomus tortuosum, Glomus sp. 1,Glomus sp. 2 andScutellospora gregaria were recovered. Spores ofGlomus spp. were most common. In patches ofW. prostrata andZ. macrostachya spores ofAcaulospora sp. 1,G. tortuosum, Glomus sp. 1,Glomus sp. 2,S. gregaria andScutellospora sp. 1 were found.Contribution No. 112, Laboratories of Plant Pathology and Mycology, Institute of Agriculture and Forestry, University of Tsukuba.     

<Emphasis Type="Italic">Umbelopsis gibberispora</Emphasis> sp. nov. from Japanese leaf litter and a clarification of <Emphasis Type="Italic">Micromucor ramannianus </Emphasis>var. <Emphasis Type="Italic">angulisporus</Emphasis>

 Umbelopsis gibberispora is described as a new species in the genus Umbelopsis, Umbelopsidaceae, Mucorales. The species differs from others in this genus by ellipsoidal sporangiospores with unilaterally thickened walls. Phylogenetic analyses based on nuclear large subunit ribosomal DNA (nLSU rDNA) partial sequences suggest that U. gibberispora, U. swartii, and U. westeae form a clade together with the strains of Umbelopsis ramanniana. The ex-type strain of Micromucor ramannianus var. angulisporus is found to be very close to Umbelopsis vinacea, whereas other isolates identified under the former name in the sense of Linnemann fall in the U. ramanniana subclade. For these isolates, a new species, Umbelopsis angularis, is introduced. Phylogenetic relationships among Umbelopsis species are discussed related to their attributes of the sporangial wall and mature spore shapes. Received: August 27, 2002 / Accepted: March 11, 2003 Acknowledgments We thank Dr. Takashi Ohsono, Graduate School of Agriculture, Kyoto University, Japan, for providing the strain of U. gibberispora (CBS 109328). We also thank Dr. Wieland Meyer, University of Sydney, Australia for access to the phylogenetic tree based on ITS sequence data before publishing, and Dr. Richard C. Summerbell, Centraalbureau von Schimmelcultures, the Netherlands, for linguistic corrections.     

Fungal associations of Danish Calluna vulgaris roots with special reference to ericoid mycorrhiza

Fungi were isolated from young, serial-washed roots of Calluna sampled from a Danish heathland, Hjelm Hede. Of the 626 isolates, those that were dark, sterile and septate were divided into 13 morphological groups based on their appearance in culture on malt agar. Mycorrhizal synthesis in vitro showed that several groups formed typical ericoid mycorrhiza with seedlings of Calluna; these ericoid mycorrhizal fungi were morphologically similar to Hymenoscyphus ericae. The identities of the other dark, septate fungi are uncertain. Oidiodendron spp. were isolated in a very low frequency; these fungi also formed typical ericoid mycorrhiza. The Calluna root system on Hjelm Hede demonstrated a high morphological diversity among the associated dark, septate fungi suggesting that more than one fungus could coexist in the same host root system.     

Phylogenetic analyses of <Emphasis Type="Italic">Uromyces viciae-fabae</Emphasis> and its varieties on <Emphasis Type="Italic">Vicia</Emphasis>, <Emphasis Type="Italic">Lathyrus</Emphasis>, and <Emphasis Type="Italic">Pisum</Emphasis> in Japan

A pea rust fungus, Uromyces viciae-fabae, has been classified into two varieties, var. viciae-fabae and var. orobi, based on differences in urediniospore wall thickness and putative host specificity in Japan. In principal component analyses, morphological features of urediniospores and teliospores of 94 rust specimens from Vicia, Lathyrus, and Pisum did not show definite host-specific morphological groups. In molecular analyses, 23 Uromyces specimens from Vicia, Lathyrus, and Pisum formed a single genetic clade based on D1/D2 and ITS regions. Four isolates of U. viciae-fabae from V. cracca and V. unijuga could infect and sporulate on P. sativum. These results suggest that U. viciae-fabae populations on different host plants are not biologically differentiated into groups that can be recognized as varieties.Contribution no. 184, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan