大和 政秀

The mycorrhizal fungi in the roots of achlorophyllous Sciaphila japonica and S. tosaensis (Triuridaceae) were identified by molecular methods. The habitats of S. japonica were in a tree plantation of Japanese cypress, Chamaecyparis obtusa, and bamboo forests, and those of S. tosaensis were in a camellia forest and a bamboo forest. In the root cortical cells of both plants, aseptate hyphal coils were observed, which suggested the Paris-type arbuscular mycorrhiza (AM). A phylogenetic analysis based on a partial sequence of an AM fungal nuclear small subunit ribosomal RNA gene showed that the fungal DNA sequences of S. japonica were separated into three closely related clades. Those of S. tosaensis were separated into two clades, which were also closely related to each other. The AM fungi of S. japonica and S. tosaensis were completely separated in the phylogenetic tree even among those found in the same habitat, which suggests the high specificities in the plant-fungal partnerships. All the detected AM fungi in these plants belonged to Glomus-group A. Even though the habitats are in quite common environments, both plant species are known as endangered species in Japan. Such a definite specificity in AM symbioses seems to restrict the distribution of the myco-heterotrophic plants.

During taxonomic revision of genus Octaviania in Japan we examined herbarium and fresh specimens of O. columellifera and O. asterosperma sensu S. Yoshimi & Y. Doi With morphological and molecular techniques. These two species were identical in both macro- and micromorphological characters and were clearly different from the generally known O. asterosperma. The identity of the two species and their distinctness from O. asterosperma was further Supported by both nuclear large subunit and ITS rDNA phylogeny. The Molecular analyses also revealed that O. columellifera shares its lineage with the boletoid mushroom-forming Xerocomus chrysenteron complex and that it does not form a monophyletic clade with other Octaviania species. Our morphological reevaluation, including transmission electron microscopic observation of basidiospores, clarified the taxonomic boundary between O. columellifera and other Japanese Octaviania species. Accordingly we propose a new genus, Heliogaster, for O. columellifera with designation of the lectotype. We discuss phylogenetic relationships with Octaviania sensu stricto species and the closely related boletoid (pileate-stipitate) fungi, generic characters of Heliogaster and intraspecific phylogeny.

Epipogium aphyllum is a Eurasian achlorophyllous, mycoheterotrophic forest orchid. Due to its rarity, it is often protected, and its biology is poorly known. The identity and pattern of colonization of fungal associates providing carbon to this orchid have not been studied previously.Using samples from 34 individuals from 18 populations in Japan, Russia and France, the following were investigated: (a) colonization patterns of fungal associates of E. aphyllum by microscopy; (b) their identity by PCR amplification of nuclear ribosomal ITS carried out on rhizome fragments and hyphal pelotons.Microscopic investigations revealed that thick rhizomes were densely colonized by fungi bearing clamp-connections and dolipores, i.e. basidiomycetes. Molecular analysis identified Inocybe species as exclusive symbionts of 75 % of the plants investigated and, more rarely, other basidiomycetes (Hebeloma, Xerocomus, Lactarius, Thelephora species). Additionally, ascomycetes, probably endophytes or parasites, were sometimes present. Although E. aphyllum associates with diverse species from Inocybe subgenera Mallocybe and Inocybe sensu stricto, no evidence for cryptic speciation in E. aphyllum was found. Since basidiomycetes colonizing the orchid are ectomycorrhizal, surrounding trees are probably the ultimate carbon source. Accordingly, in one population, ectomycorrhizae sampled around an individual orchid revealed the same fungus on 11 center dot 2 % of tree roots investigated. Conversely, long, thin stolons bearing bulbils indicated active asexual multiplication, but these propagules were not colonized by fungi. These findings are discussed in the framework of ecology and evolution of mycoheterotrophy.

The community structure of arbuscular mycorrhizal (AM) fungi in the roots of drought-resistant trees, Moringa spp., was examined in semiarid regions in Madagascar and Uganda. Root samples were collected from 8 individuals of M. hildebrandtii and 2 individuals of M. drouhardii in Madagascar and from 21 individuals of M. oleifera in Uganda. Total DNA was extracted from the root samples, and partial nSSU rDNA of AM fungi was amplified using a universal eukaryotic primer NS31 and an AM fungalspecific primer AM1. The PCR products were cloned and divided by restriction fragment length polymorphism (RFLP) analysis with HinfI and RsaI. Some representatives in each RFLP types were sequenced, and a neighbor-joining phylogenetic analysis was conducted for the obtained sequences with analogous sequences of AM fungi. The RFLP and phylogenetic analyses showed that AM fungi closely related to Glomus intraradices or G. sinuosum were detected in many samples. The AM fungal groups frequently detected in the Moringa spp. might be widely distributed species in semiarid environments.

The mycorrhizal fungi of Stigmatodactylus sikokianus (Orchidaceae) were isolated and identified to be nearly related to Sebacina spp. in Sebacinaceae (Basidiomycota) by a neighbor-joining phylogenetic analysis based on the sequences of the ITS region of nuclear rDNA. In spite of the geographically separated samplings, high sequence similarity was found among the obtained DNA sequences, which suggested that S. sikokianus might be highly specialized to the group of fungi. It is known that Sebacina spp. are saprobes or ectomycorrhiza-forming fungi. The mycorrhizal fungi of S. sikokianus were regarded to be saprobic from the environment of their habitats.

We have identified Crassocephalum crepidioides (Benth.) S. Moore (Compositae) as a cadmium (Cd)-accumulator plant in a heavy-metal polluted environment. In soil polluted with Cd, 5.7-17.5 mg kg(-1) Cd, concentrations in the above-ground plant tissues were measured as 14.6-78.6 mg kg(-1) with transfer factors in the above-ground plant tissues (concentration in above-ground tissues/soil concentration) of 1.5-6.0. No other toxic heavy metals or plant micronutrients were found to have accumulated into the above-ground plant tissues. In a hydroponic culture with 1 mu mol L(-1) Cd added to Hoagland's nutrient solution, Cd concentration in the above-ground plant tissues was 121.0 mg kg(-1), with a transfer factor of more than 1000. In a pot culture carried out for 9 weeks in a greenhouse, the highest Cd concentration in the above-ground plant tissues, 121.2 mg kg(-1), was found in a treatment with 5 mg kg(-1) Cd, whereas the highest Cd content in an above-ground plant tissue, 106.1 mu g, was found in a treatment with 2 mg kg(-1) Cd. These results clearly showed that C. crepidioides is a Cd accumulator. In all samples, the Cd concentration in the above-ground plant tissues was higher than that in the roots. The results obtained in the present study show that this plant has a strong potential for use in phytoremediation in farm fields contaminated with Cd.

Community of arbuscular mycorrhizal (AM) fungi in a coastal vegetation on Okinawa island in Japan was examined. A sampling plot was established in a colony of Ipomoea pes-caprae (Convolvulaceae) on the beach in Tamagusuku, Okinawa Pref, in which eight root samples of I. pes-caprae and three root samples each of Vigna marina (Leguminosae) and Paspalum distichum (Poaceae) were collected. Partial 18S rDNA of AM fungi was amplified from the root samples by polymerase chain reaction (PCR) with primers NS31 and AM1. Restriction fragment length polymorphism analysis with HinfI and RsaI for cloned PCR products revealed that two types of Glomus sp., type A and type B, were dominant in the colony. Among them, the fungi of type A were especially dominant near the edge of the colony facing the sea. A phylogenetic analysis showed that the AM fungi of type B are closely related to Glomus intraradices and those of type A are nearly related to type B. From the sequence data, it was also found that type A was further divided into two types, type A1 and A2. One representative strain each of the three types, type A1, A2, and B, propagated from single spore each, was examined for the growth of sorghum (Sorghum bicolor) at three different salinity levels, 0, 100, and 200 mM NaCl. At the non-salt-treated condition, the type B fungus was the most effective on shoot growth enhancement of the host plant, whereas at the salt-treated conditions, the type A2 fungus was the most effective. An efficient suppression of Na+ stop translocation into the shoot by the examined AM fungi was found. These results suggested that the AM fungi dominant near the sea are adapted to salt-stressed environment to alleviate the salt stress of host plants.

A mycorrhizal fungus, ME1-1, isolated from an achlorophyllous orchid, Epipogium roseum, was identified as Coprinellus disseminatus (= Coprinus disseminatus) based on characteristics of basidiomata that were artificially induced. Spawn of ME1-1 cultivated on a medium that consisted of sawdust and wheat, sandwiched between two mats of volcanic soils, which was incubated at 20.0 degrees +/- 0.5 degrees C in 80.0%+/- 0.5% relative humidity in the dark. The basidiomata were formed on the soil after 2 months. Morphological and anatomical characteristics of the basidioma mostly accorded with those of Coprinellus disseminatus. We therefore concluded that C. disseminatus is one of the mycobionts of E. roseum.

Asymbiotic seedling propagation and introduction of seedlings into a natural habitat were achieved for Cephalanthera falcata. For immature seeds collected 65 days after pollination, high germination rate (av. 50%) was achieved on Hyponex agar medium plates. Root development occurred in about 10% of the protocorms 5 months after seed sowing. Rooted protocorms were transferred to a culture bottle containing 100 ml of the Hyponex agar medium and incubated continually. In about 30% of the transferred individuals, shoot height reached 1.5-2 cm 8 months after the transfer. After acclimatization in wet vermiculite at 4 degrees C for 6 months, 135 individuals were planted in a natural stand of C. falcata in mid February 2001. Shoot appearance rate was 44.4% at the first year and flowering was noted in some plants. At the fifth year, shoots with an average height of 21.6 cm still appeared in four plants, and flowering was noted in three of them. Colonization of mycorrhizal fungi was examined in two of them as well as one wild plant, in which the mycorrhizal fungi were identified to be in Thelephoraceae or Russulaceae. These fungi are known to form ectomycorrhiza with trees, and thus a tripartnership symbiosis consisting of C. falcata, mycorrhizal fungi and trees was suggested. The involvement of ectomycorrhizal fungi might be the reason for the low survival rate in the field experiment, because the distribution of ectomycorrhizal fungi relevant to this orchid is assumed to be uneven. The possibility of introducing artificially propagated orchids into natural habitats was discussed.

Mycorrhizal fungi were isolated from the non-photosynthetic orchid Chamaegastrodia sikokiana and identified as members of Ceratobasidiaceae by phylogenetic analysis of the internal transcribed spacer (ITS) region of ribosomal deoxyribonucleic acid. The ITS sequences were similar among geographically separated samples obtained from Mt. Kiyosumi in Chiba Prefecture and Mt. Yokokura in Kochi Prefecture. One of the isolated fungi, KI1-2, formed ectomycorrhiza on seedlings of Abies firma in pot culture, suggesting that tripartite symbiosis exists among C. sikokiana, mycorrhizal fungi, and A. firma in nature, and carbon compounds are supplied from A. firma to C. sikokiana through the hyphae of the mycorrhizal fungi. To our knowledge, this is the second study to suggest the involvement of Ceratobasidiaceae fungi in tripartite symbiosis with achlorophyllous orchids and photosynthetic host plants.

We have achieved the symbiotic cultivation of an apparently achlorophyllous orchid, Epipogium roseum Lindl., with a mycorrhizal fungus isolated from an underground organ of this orchid. Although the seed germination rate was extremely low, subsequent growth from protocorm to flowering was induced in a medium containing volcanic soils and sawdust. Stolons elongated from each protocorm, and rhizomes were formed at certain intervals on the stolons. Some of the rhizomes developed into a coralloid form, and tubers were formed from the coralloid rhizomes. The coralloid rhizomes degenerated concurrently with maturation of the tubers. Six months after seed sowing, around 80 tubers were produced from a single protocorm. An inflorescence appeared from each of the large tubers, and the process to flowering was observed in one of these. Consequently, the developmental processes from seed to flowering in E. roseum was clearly revealed in this study.

Charred bark of Acacia mangium (bark charcoal), which is made of wood waste from pulp production, was applied as soil amendment for the cultivation of maize, cowpea and peanut to examine its effects on crop yield and soil chemical properties in South Sumatra, Indonesia. The yields of maize and peanut significantly increased after the application of bark charcoal under a fertilized condition in an infertile soil environment. In addition, increases in the root amount and colonization rate of arbuscular mycorrhizal (AM) fungi after bark charcoal application were also observed in maize. In general, the application of bark charcoal induced changes in soil chemical properties by increasing the pH value, total N and available P2O5 contents, cation exchange capacity, amounts of exchangeable cations and base saturation, and by decreasing the content of exchangeable Al3+. The amelioration of the soil chemical properties could be effective in highly weathered infertile tropical soils. The application of charcoal in agriculture is expected to lead to the formation of a carbon sink in soil and to increase crop yield because it has been suggested that charcoal is highly resistant to abiotic and biotic degradation, even in a soil environment.

A community of arbuscular mycorrhizal (AM) fungi was investigated in a warm-temperate deciduous broad-leaved forest using a molecular analysis method. Root samples were obtained from the forest, and DNA was extracted from the samples. Partial 18S rDNA of AM fungi were amplified from the extracted DNA by polymerase chain reaction using a universal eukaryotic primer NS31 and an AM fungal-specific primer AM1. After cloning the PCR products, 394 clones were obtained in total, which were divided into five types by restriction fragment length polymorphism (RFLP) with HinfI, RsaI, and Hsp92II. More than 20% of the clones were randomly selected from each RFLP type and sequenced. Phylogenetic analysis showed that all the obtained clones belonged to Glomus but could not be identified at species level. Topsoil of the forest containing plant roots was inoculated to nonmycorrhizal seedlings of indigenous woody plants, Rhus javanica var. roxburghii and Clethra barvinervis, to introduce the community of AM fungi into the seedlings. Among these five RFLP types, four types were detected from both seedlings, which indicates that the AM fungal community in the forest root samples was introduced at least partly into the seedlings. Meanwhile, an additional four types that were not found in the forest root samples were newly detected in the seedlings, these types were closely related to one another and close to G. fasciculatum or G. intraradices. It is expected that a community of indigenous diverse AM fungi could be introduced into target fields by planting these mycorrhizal seedlings. © The Mycological Society of Japan and Springer-Verlag 2005.

The identity of mycorrhizal fungi associated with the achlorophyllous orchid Epipogium roseum was investigated by DNA analysis. The fungi were isolated from each coiled hypha (peloton), and the ITS region of nuclear rDNA was sequenced. Phylogenetic analysis based on the neighbor-joining method showed that all the isolates clustered with fungi belonging to Psathyrella or Coprinus in Coprinaceae. Those fungi are known as saprobes, using dead organic materials for a nutritive source. Large colonies of this orchid were frequently found around tree stumps or fallen logs. In such colonies, these decaying wood materials would be used as a large and persistent carbon source for the growth of this orchid. This is the first report of Coprinaceae as an orchid mycorrhizal fungi. © The Mycological Society of Japan and Springer-Verlag 2005.

Morphological types of arbuscular mycorrhizas (AM) in pioneer woody plants were examined in an oil palm farm 1 year after reclamation. In total, 18 plant species belonging to 15 genera in 9 families were examined, and the Arum type was found in 12 species belonging to 9 genera in 5 families. In contrast, the Paris type was found in 5 species belonging to 5 genera in 4 families. The number of plant species with Arum-type AM was always higher than that with Paris-type AM in all four sampling plots. All the examined species in Euphorbiaceae, most of which are fast-growing pioneer trees and were dominant in forming the canopy in secondary forests, were found to have Arum-type AM. These results suggest that Arum-type colonization is beneficial for fast-growing woody plant species. © The Mycological Society of Japan and Springer-Verlag Tokyo 2005.

Morphological types of arbuscular mycorrhizal (AM) fungi in weeds of vacant land were examined in spring and autumn. In total, 33 plant species belonging to 28 genera in 13 families were examined. The number of plant species with Arum-type AM was higher than those with Paris- or intermediate types in both seasons. Thus, Arum-type colonization may be beneficial for fast-growing plant species on vacant land. There was a strong relationship between plant identity and AM morphological type, as the colonization types were mostly distinguished at the plant family level.

Morphological types of arbuscular mycorrhizal (AM) fungi in roots of understorey plants were examined in three different Japanese deciduous broad-leaved forests. In total, 43 species belonging to 33 genera from 27 families were examined for the morphological types of AM. The number of flowering plant species having Paris-type AM was greater than those having Arum-type AM in each plot. This tendency was more prominent in herbaceous plants than woody plants with nine species having Paris-type associations among ten herbaceous plant species examined. Therefore, it is suggested from the ecological point of view that Paris-type associations could be advantageous for the herbaceous understorey plants growing slowly in these environments. The influence of plant identity on the morphological types of AM was also discussed by arranging the plants examined with the morphological types in a current plant phylogeny scheme. In this study, some new records on the morphological types of AM in some new plant families were obtained including the first report of a typical Arum-type AM in gymnosperms.

The identity of a mycorrhizal fungus in the roots of achlorophyllous Sciaphila tosaensis was investigated by DNA analysis and examination of the morphology of the association. The morphological features of the mycorrhizal fungus, i.e. aseptate hyphal coils, vesicles, arbuscule-like branching, and degenerate coils were similar to those previously reported for other achlorophyllous plants. Spore-like propagules identified as a glomalean fungus were propagated from root pieces of S. tosaensis in pot culture using alfalfa as the host trap plant. A PCR product was obtained from colonized root of S. tosaensis using the taxon-specific primers, VANS1 and VAGLO. Sequence analysis of the DNA fragment showed it to be almost identical to other Glomus species. Although it has been reported many times that the morphology of mycorrhizal fungi in achlorophyllous plants is quite similar to that of arbuscular mycorrhizal fungi, this is the first report of the isolation and identification of such a fungus itself.

Plasmids pRt032 and pRKR9032, both carrying a 14 kb HindIII fragment from Rhizobium leguminosarum bv. trifolii ANU843 that harbours genes essential for clover modulation, were transferred by conjugation to Rhizobium fredii USDA192 which nodulates soybean roots. The transconjugant strains NA102 and YA101 induced nodules on clover roots in addition to soybean roots. This fact indicates that conjugal transfer of the nodulation genes of R. l. bv. trifolii to R. fredii USDA192 extended the host range of R. fredii USDA192 to clover, the host of R. l. bv. trifolii. However, the clover nodules induced by the transconjugants were small, whitish and nitrogen fixation was not detected. Moreover nitrogen fixation activity also could not be detected in the soybean nodules induced by the transconjugant strain NA102. The Nod factors produced by transconjugants in the presence of flavonoids such as apigenin and genistein differed from those of their recipient strains. These results indicate that the unusual Nod factors may cause the induction of ineffective nodules. © 1997 Taylor & Francis Group, LLC.