Masahide Yamato

Effects of the application of charred bark of Acacia mangium on the yield of maize, cowpea and peanut, and soil chemical properties in South Sumatra, Indonesia

Abstract 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.

Isolation and identification of mycorrhizal fungi associating with an achlorophyllous plant, Epipogium roseum (Orchidaceae)

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.

Morphological types of arbuscular mycorrhizal fungi in roots of weeds on vacant land.

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.

Ectomycorrhizal Inocybe species associate with the mycoheterotrophic orchid Epipogium aphyllum but not its asexual propagules.

BACKGROUND AND AIMS 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. METHODS 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. RESULTS AND CONCLUSIONS 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 11.2 % 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.

Mixotrophy of Platanthera minor, an orchid associated with ectomycorrhiza‐forming Ceratobasidiaceae fungi

• We investigated the fungal symbionts and carbon nutrition of a Japanese forest photosynthetic orchid, Platanthera minor, whose ecology suggests a mixotrophic syndrome, that is, a mycorrhizal association with ectomycorrhiza (ECM)-forming fungi and partial exploitation of fungal carbon. • We performed molecular identification of symbionts by PCR amplifications of the fungal ribosomal DNA on hyphal coils extracted from P. minor roots. We tested for a (13)C and (15)N enrichment characteristic of mixotrophic plants. We also tested the ectomycorrhizal abilities of orchid symbionts using a new protocol of direct inoculation of hyphal coils onto roots of Pinus densiflora seedlings. • In phylogenetic analyses, most isolated fungi were close to ECM-forming Ceratobasidiaceae clades previously detected from a few fully heterotrophic orchids or environmental ectomycorrhiza surveys. The direct inoculation of fungal coils of these fungi resulted in ectomycorrhiza formation on P. densiflora seedlings. Stable isotope analyses indicated mixotrophic nutrition of P. minor, with fungal carbon contributing from 50% to 65%. • This is the first evidence of photosynthetic orchids associated with ectomycorrhizal Ceratobasidiaceae taxa, confirming the evolution of mixotrophy in the Orchideae orchid tribe, and of ectomycorrhizal abilities in the Ceratobasidiaceae. Our new ectomycorrhiza formation technique may enhance the study of unculturable orchid mycorrhizal fungi.

Ceratobasidiaceae mycorrhizal fungi isolated from nonphotosynthetic orchid Chamaegastrodia sikokiana

Mycorrhizal fungi were isolated from the nonphotosynthetic 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.

Developmental processes of achlorophyllous orchid, Epipogium roseum: from seed germination to flowering under symbiotic cultivation with mycorrhizal fungus.

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.

Identification of a mycorrhizal fungus in the roots of achlorophyllous Sciaphila tosaensis Makino (Triuridaceae)

Abstract 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 asa 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.

Specific arbuscular mycorrhizal fungi associated with non-photosynthetic Petrosavia sakuraii (Petrosaviaceae).

Mycorrhizal fungi in roots of the achlorophyllous Petrosavia sakuraii (Petrosaviaceae) were identified by molecular methods. Habitats examined were plantations of the Japanese cypress Chamaecyparis obtusa in Honshu, an evergreen broad-leaved forest in Amami Island in Japan and a mixed deciduous and evergreen forest in China. Aseptate hyphal coils were observed in root cortical cells of P. sakuraii, suggesting Paris-type arbuscular mycorrhiza (AM). Furthermore, hyphal coils that had degenerated to amorphous clumps were found in various layers of the root cortex. Despite extensive sampling of P. sakuraii from various sites in Japan and China, most of the obtained AM fungal sequences of the nuclear small subunit ribosomal RNA gene were nearly identical and phylogenetic analysis revealed that they formed a single clade in the Glomus group A lineage. This suggests that the symbiotic relationship is highly specific. AM fungi of P. sakuraii were phylogenetically different from those previously detected in the roots of some mycoheterotrophic plants. In a habitat in C. obtusa plantation, approximately half of the AM fungi detected in roots of C. obtusa surrounding P. sakuraii belonged to the same clade as that of P. sakuraii. This indicates that particular AM fungi are selected by P. sakuraii from diverse indigenous AM fungi. The same AM fungi can colonize both plant species, and photosynthates of C. obtusa may be supplied to P. sakuraii through a shared AM fungal mycelial network. Although C. obtusa plantations are widely distributed throughout Japan, P. petrosavia is a rare plant species, probably because of its high specificity towards particular AM fungi.

Taxonomic reconsideration of a sequestrate fungus, Octaviania columellifera, with the proposal of a new genus, Heliogaster, and its phylogenetic relationships in the Boletales

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.