大和 政秀

Abstract Due to the loss of photosynthetic ability during evolution, some plant species rely on mycorrhizal fungi for their carbon source, and this nutritional strategy is known as mycoheterotrophy. Mycoheterotrophic plants forming Paris-type arbuscular mycorrhizas (AM) exhibit two distinctive mycorrhizal features: degeneration of fungal materials and specialization towards particular fungal lineages. To explore the possibility that some understory AM plants show partial mycoheterotrophy, i.e., both photosynthetic and mycoheterotrophic nutritional strategies, we investigated 13 green herbaceous plant species collected from five Japanese temperate forests. Following microscopic observation, degenerated hyphal coils were observed in four species: two Colchicaceae species, Disporum sessile and Disporum smilacinum, and two Gentianaceae species, Gentiana scabra and Swertia japonica. Through amplicon sequencing, however, we found that all examined plant species exhibited no specificity toward AM fungi. Several AM fungi were consistently found across most sites and all plant species studied. Because previous studies reported the detection of these AM fungi from various tree species in Japanese temperate forests, our findings suggest the presence of ubiquitous AM fungi in forest ecosystems. If the understory plants showing fungal degeneration exhibit partial mycoheterotrophy, they may obtain carbon compounds indirectly from a wide range of surrounding plants utilizing such ubiquitous AM fungi.

Gentiana zollingeri is an annual photosynthetic plant that employs a mycoheterotrophic growth strategy during its underground seedling stage (initial mycoheterotrophy). Notably, the morphological characteristics of its flowering shoots, such as shoot size, leaf size, and leaf color, are highly variable, and it was hypothesized that these variations may be linked to nutritional mode. The morphological characteristics of G. zollingeri individuals were thus investigated alongside environmental factors, 13C abundance, and diversity of colonizing arbuscular mycorrhizal (AM) fungi. The majority of G. zollingeri flowering individuals were found to exhibit a high affinity for the specific AM fungi that exclusively colonize roots of the mycoheterotrophic seedlings, while other phylogenetically diverse AM fungi could also be detected. The leaves to shoot dry weight ratio (leaf ratio) was negatively correlated with the canopy openness in the habitat, suggesting that leaf development is impeded in sunny conditions. Furthermore, the shoot weight of G. zollingeri was positively correlated with leaf 13C abundance. Given that 13C enrichment can provide indirect evidence of mycoheterotrophy in AM plants, the results suggest that the utilization of carbon obtained through mycoheterotrophy, at least during the underground seedling stage, is crucial for G. zollingeri.

Abstract Orchids parasitically depend on external nutrients from mycorrhizal fungi for seed germination. Previous findings suggest that orchids utilize a genetic system of mutualistic arbuscular mycorrhizal (AM) symbiosis, in which the plant hormone gibberellin (GA) negatively affects fungal colonization and development, to establish parasitic symbiosis. Although GA generally promotes seed germination in photosynthetic plants, previous studies have reported low sensitivity of GA in seed germination of mycoheterotrophic orchids where mycorrhizal symbiosis occurs concurrently. To elucidate the connecting mechanisms of orchid seed germination and mycorrhizal symbiosis at the molecular level, we investigated the effect of GA on a hyacinth orchid (Bletilla striata) seed germination and mycorrhizal symbiosis using asymbiotic and symbiotic germination methods. Additionally, we compared the transcriptome profiles between asymbiotically and symbiotically germinated seeds. Exogenous GA negatively affected seed germination and fungal colonization, and endogenous bioactive GA was actively converted to the inactive form during seed germination. Transcriptome analysis showed that B. striata shared many of the induced genes between asymbiotically and symbiotically germinated seeds, including GA metabolism- and signaling-related genes and AM-specific marker homologs. Our study suggests that orchids have evolved in a manner that they do not use bioactive GA as a positive regulator of seed germination and instead autoactivate the mycorrhizal symbiosis pathway through GA inactivation to accept the fungal partner immediately during seed germination.

Morphological analyses of three glomoid spore-producing fungi suggested that two of them were undescribed species of Glomeraceae (phylum Glomeromycota), and the third differed slightly from Dominikia glomerocarpica and Epigeocarpum crypticum, recently described in Glomeraceae. The first two fungi originated from the Mediterranean Sea sand dunes of the Peloponnese, Greece, and the third was originally found in a tree plantation in Yokohama City, Japan. Phylogenetic analyses of sequences of the 45S nuc rDNA region and the RPB1 gene showed that (i) the three fungi belonged to Glomeraceae; (ii) the first two represented a new genus, here described as Complexispora gen. nov. with C. multistratosa sp. nov. and C. mediterranea sp. nov. and (iii) the third enlarged the monospecific genus Epigeocarpum, as E. japonicum sp. nov.

Abstract. Kaewgrajang T, Yamato M, Polamart T, Sangwanit U. 2023. A comparison between the ectomycorrhizal fungal communities associated with the natural and plantation populations of Dipterocarpus alatus. Biodiversitas 24: 2088-2098. Dipterocarpus alatus Roxb. is a native tree species of Southeast Asian countries and one of the most economically important dipterocarp species. We investigated the ectomycorrhizal (ECM) fungi associated with D. alatus using molecular identifications on putative fruiting bodies and ECM roots collected from three natural forests and 10 D. alatus plantations. In this investigation, we aimed to report the ECM fungal communities of D. alatus in natural forests and plantations. Combining the results of fruiting bodies and ECM roots, 82 taxa belonging to 12 families were identified as ECM fungi. The ECM fungal community comprised many rare species and a few frequently found species. The Sebacina genus was found the most frequently in natural forests and plantations. The ECM fungal diversity was higher in plantations compared to natural forests. However, the frequently occurring ECM fungal taxa were not different between the natural forests and plantations. The result suggests that ECM fungal communities could be maintained in D. alatus plantations even if a reduction occurs in the natural population of dipterocarp forest.

Gentiana zollingeri (Gentianaceae) is an initial mycoheterotrophic plant that depends on a specific group of arbuscular mycorrhizal (AM) fungi for carbon source during underground growth after seed germination. In this study, a mycorrhizal fungus dominant in mycohetero-trophic seedlings of G. zollingeri was successfully isolated from a soil core collected from a point close to a flowering G. zollingeri. The AM fungal isolate was identified as conspecific or closely related to Dominikia aurea (Glomeraceae) by spore morphology and molecular phylogeny. Basic Local Alignment Search Tool (BLAST) searches against the MaarjAM database showed that the nuclear small subunit ri-bosomal DNA sequences of the isolate matched the AM fungal sequences obtained from a wide range of plants in various ecosystems, including several mycoheterotrophs. Thus, it is suggested that the AM fungal isolate is one of the cheating susceptible AM fungi. Further-more, the sequences corresponded to those of a group of AM fungi dominantly detected in Japanese temperate forests. Accordingly, there is a possibility that mycoheterotrophic plants, including seedlings of G. zollingeri, may target AM fungi with a wide host range and ubiquitous distribution.

Some arbuscular mycorrhizal (AM) fungal species known to form sporocarps (i.e., aggregations of spores) are polyphyletic in two orders, Glomerales and Diversisporales. Spore clusters (sporocarp-like structures) often formed in pot cultures or in vitro conditions are supposed to be clonal populations, while sporocarps in natural habitats with a fungal peridium are morphologically similar to those of epigeous sexual (zygosporic) sporocarps of Endogone species. Thus, in this study, we explored the genetics of sporocarpic spores of two AM fungi with a view to possibilities of clonal or sexual reproduction during sporocarps formation. To examine these possibilities, we investigated single-nucleotide polymorphisms (SNPs) in reduced genomic libraries of spores isolated from sporocarps molecularly identified as Rhizophagus irregularis and Diversispora epigaea. In addition, partial sequences of the MAT locus HD2 gene of R. irregularis were phylogenetically analyzed to determine the nuclear status of the spores. We found that most SNPs were shared among the spores isolated from each sporocarp in both species. Furthermore, all HD2 sequences from spores isolated from three R. irregularis sporocarps were identical. These results indicate that those sporocarps comprise clonal spores. Therefore, sporocarps with clonal spores may have different functions than sexual reproduction, such as massive spore production or spore dispersal via mycophagy.

Livestock grazing of drylands supports millions of livelihoods worldwide; however, overgrazing causes severe biodiversity loss and degrades ecosystem functions. Understanding the effects of grazing intensity, aridity, and soil microbes is required for sustainable management. Here, we focused on arbuscular mycorrhizal (AM) fungi in soils and roots and examined their relationship with ecosystem multifunctionality under livestock grazing of Mongolian grasslands with different aridity levels. Above- and below-ground plant biomass were used to measure ecosystem-service multifunctionality, excluding annual plants that are unfavorable for ecosystem function and rangeland management. In both mountain forest steppe (mean annual precipitation = 207.7 mm) and steppe–desert steppe transition zone (mean annual precipitation = 128.4 mm), ecosystem multifunctionality was decreased by livestock grazing, as indicated by palatable plant shoot biomass. In line with the multifunctionality, the dominant plant species, Stipa krylovii, and AM plant biomass were decreased by livestock grazing. The Berger–Parker dominance index of soil AM fungi and Simpson's diversity had a positive and negative relationship with multifunctionality, respectively, indicating that dominant AM fungi rather than AM fungal diversity in soil are key for maintaining multifunctionality. Root and soil AM fungal communities were significantly different with dominance of Rhizophagus operational taxonomic units (OTUs) in roots and Claroideoglomus OTUs in soil. However, the similarity between soil and root AM fungal communities increased with livestock grazing. Correlation analysis between palatable plant biomass and relative abundance of dominant AM fungal OTUs identified indicator virtual taxa that decreased (VTX00325 in roots, and VTX00165, VTX00214, and VTX00222 in soil) or increased (VTX00100, VTX00130, and VTX00295 in soil) with grazing intensity. The OTUs that were increased by grazing may be r-selected, grazing-tolerant AM fungi that accept a low C supply and supply few nutrients to plants. Our results suggest that grassland management focusing on ecosystem-service multifunctionality via control of AM plants, including S. krylovii and their co-dominant dominant AM fungi, in ungrazed grasslands are key for sustainable rangeland management in Mongolia.

We found mycoheterotrophic seedling growth (initial mycoheterotrophy) of Gentiana zollingeri, a spring-flowering photosynthetic species of Gentianaceae family. Small seeds (about 300 µm in length) were buried in a habitat by using seed packets, and development of the subterranean seedlings to form shoots, more than 3 cm in length, was observed in symbiosis with arbuscular mycorrhizal (AM) fungi in the dark (i.e., underground of a field). Hyphal coils and their degenerations were observed in the root cortical cells of the subterranean seedlings as well as those of adult plants. Among the mycobionts identified on the basis of partial small subunit rDNA sequences, it was found that AM fungi of a lineage in Glomeraceae dominantly colonized, and the AM fungi were also dominant in adult individuals of G. zollingeri in three habitats separated one another by 17.2, 34.7, and 49.6 km. Though initial mycoheterotrophy in symbioses with AM fungi has been observed in some pteridophytes, this is the first study to demonstrate this type of symbiosis in a photosynthetic seed plant. The mycoheterotrophy means that an energy distribution occurs through the hyphal bridges of AM fungi among different photosynthetic seed plants, which may be important in constructing plant species diversity in some ecosystems.

Most green orchids form mycorrhizal associations with rhizoctonia fungi, a polyphyletic group including Serendipitaceae, Ceratobasidiaceae, and Tulasnellaceae. Although accumulating evidence indicated that partial mycoheterotrophy occurs in such so-called rhizoctonia-associated orchids, it remains unclear how much nutrition rhizoctonia-associated orchids obtain via mycoheterotrophic relationships. We investigated the physiological ecology of green and albino individuals of a rhizoctonia-associated orchid Cypripedium debile, by using molecular barcoding of the mycobionts and stable isotope (C-13 and (15) N) analysis. Molecular barcoding of the mycobionts indicated that the green and albino individuals harbored Tulasnella spp., which formed a clade with the previously reported C. debile mycobionts. In addition, stable isotope analysis showed that both phenotypes were significantly enriched in C-13 but not in (15) N. Therefore, green and albino individuals were recognized as partial and full mycoheterotrophs, respectively. The green variants were estimated to obtain 42.5 +/- 8.2% of their C from fungal sources, using the C-13 enrichment factor of albino individuals as a mycoheterotrophic endpoint. The proportion of fungal-derived C in green C. debile was higher than that reported in other rhizoctonia-associated orchids. The high fungal dependence may facilitate the emergence of albino mutants. Our study provides the first evidence of partial mycoheterotrophy in the subfamily Cypripedioideae. Partial mycoheterotrophy may be more general than previously recognized in the family Orchidaceae.

We studied mycorrhizal symbioses of Cephalanthera falcata (Orchidaceae) in a population that included giant individuals, some of which were over 50 cm in height and had more than 10 shoots, as well as in two nearby habitats without giant individuals. For C. falcata adults in the habitat with giant individuals, a fungal lineage in the ectomycorrhiza-forming clade of Sebacinales was detected as the dominant mycobiont. Furthermore, higher C-13 enrichment, which is an indicator of mycoheterotrophy, was found in these C. falcata individuals compared to those in the two nearby habitats. These results indicate that the growth of giant C. falcata individuals may be a result of the increased ability of Sebacinales fungi to transfer carbon to the orchids. Furthermore, we attempted to discern the effect of mycobiont taxa on seedling development in the three habitats. Contrary to our expectations, Sebacinales fungi were not found to significantly impact seedling growth as this orchid was predominantly colonized by Thelephoraceae fungi at the seedling stage. These results suggest that seedling growth is mostly enhanced by symbiosis with Thelephoraceae fungi in this orchid, and that the growth of giant individuals due to colonization by Sebacinales fungi may occur subsequently.

Arbuscular mycorrhiza (AM) is a symbiotic interaction in terrestrial plants that is colonized by fungi in the Glomeromycotina. The morphological types of AM, including the Arum-type and Paris-type, are distinct, depending on the host plant species. A part of the regulatory pathways in Arum-type AM symbiosis has been revealed because most model plants form the Arum-type AM with a model AM fungus, Rhizophagus irregularis. Moreover, gibberellin (GA) is known to severely inhibit AM fungal colonization in Arum-type AM symbiosis. Recently, we showed that exogenous GA treatment significantly promoted AM fungal colonization in Paris-type AM symbiosis in Eustoma grandiflorum. In this study, we focused on the transcriptional changes in AM symbiosis-related genes in GA-treated E. grandiflorum. The expression levels of all examined E. grandiflorum genes were maintained or increased by GA treatment compared with those of the control treatment. Our new results suggest that signaling pathway(s) required for establishing AM symbiosis in E. grandiflorum may be distinct from the well-characterized pathway for that in model plants.

We investigated arbuscular mycorrhizal (AM) fungal communities in secondary forests and/or Chamaecyparis obtusa plantations at eight study sites in Japan's temperate region. In the secondary forests, AM plants of the families Lauraceae, Sapindaceae, Rutaceae, Araliaceae, Rosaceae, Magnoliaceae, Cornaceae, Piperaceae, and Anacardiaceae were found. The AM fungal communities were evaluated based on compositions of the AM fungal operational taxonomic units (OTUs), which were clustered at a 97% similarity threshold of the sequences of a partial small subunit of a nuclear ribosomal RNA gene obtained from the plant roots. The compositions of AM fungal OTUs were significantly correlated with the plant family compositions and were significantly differentiated among the study sites and between the study forests. Interestingly, only 19 OTUs remained after selecting for those that had more than 1.0% of the total reads, and these 19 OTUs accounted for 86.3% of the total rarefied reads that were classified into 121 OTUs. Furthermore, three dominant OTUs constituted 48.0% of the total reads, and the most dominant OTU was found at all study sites, except at one. These results indicate that AM fungal communities are primarily constituted by limited AM fungal taxa in the forest ecosystems with diverse plant taxa in Japan's temperate region. The results of basic local algorithm search tool (BLAST) searches against MaarjAM, a database of AM fungal sequences, also revealed that the AM fungi which were the three dominant OTUs are distributed in forest ecosystems on a worldwide scale.

The majority of chlorophyllous orchids form mycorrhizal associations with so-called rhizoctonia fungi, a phylogenetically heterogeneous assemblage of predominantly saprotrophic fungi in Ceratobasidiaceae, Tulasnellaceae, and Serendipitaceae. It is still a matter of debate whether adult orchids mainly associated with rhizoctonia species are partially mycoheterotrophic. Here, we investigated the nutritional modes of green and albino variants of Goodyera velutina, an orchid species considered to be mainly associated with Ceratobasidium spp., by measuring their C-13 and N-15 abundances, and by molecular barcoding of their mycorrhizal fungi. Molecular analysis revealed that both green and albino variants of G. velutina harbored a similar range of mycobionts, mainly saprotrophic Ceratobasidium spp., Tulasnella spp., and ectomycorrhizal Russula spp. In addition, stable isotope analysis revealed that albino variants were significantly enriched in C-13 but not so greatly in N-15, suggesting that saprotrophic Ceratobasidium spp. and Tulasnella spp. are their main carbon source. However, in green variants, C-13 levels were depleted and those of N-15 were indistinguishable from the co-occurring autotrophic plants. Therefore, we concluded that the albino G. velutina variants are fully mycoheterotrophic plants whose C derives mainly from saprotrophic rhizoctonia, while the green G. velutina variants are mainly autotrophic plants, at least at our study site, in spite of their additional associations with ectomycorrhizal fungi. This is the first report demonstrating that adult nonphotosynthetic albino variants can obtain their nutrition mainly from nonectomycorrhizal rhizoctonia.

<p>千葉大学では平成26年度より文部科学省大学教育改革加速プログラム（AP）タイプIII（高大接続）「次世代才能スキップアップ」プログラムを千葉県内及び近隣都県高等学校と連係し実施してきた．この中で高校生を対象とした国際研究発表会を実施してきた．これは高校生が研究発表を海外の研究者・留学生に対して英語で行うものである．今回，平成28年度から平成30年度に参加した高校生の気づきと学びについて，アンケートの記述をテキストマイニング法により解析した．この結果，生徒は英語での会話に関して難しさを感じるとともに，研究発表は成功したと感じていることが示された．本プログラムは生徒に英語コミュニケーションの重要性に気付かせ，英語発表の自信につながると考えられる．</p>

Orchids produce minute seeds that contain limited or no endosperm, and they must form an association with symbiotic fungi to obtain nutrients during germination and subsequent seedling growth under natural conditions. Orchids need to select an appropriate fungus among diverse soil fungi at the germination stage. However, there is limited understanding of the process by which orchids recruit fungal associates and initiate the symbiotic interaction. This study aimed to better understand this process by focusing on the seed coat, the first point of fungal attachment. Bletilla striata seeds, some with the seed coat removed, were prepared and sown with symbiotic fungi or with pathogenic fungi. The seed coat-stripped seeds inoculated with the symbiotic fungi showed a lower germination rate than the intact seeds, and proliferated fungal hyphae were observed inside and around the stripped seeds. Inoculation with the pathogenic fungi increased the infection rate in the seed coat-stripped seeds. The pathogenic fungal hyphae were arrested at the suspensor side of the intact seeds, whereas the seed coat-stripped seeds were subjected to severe infestation. These results suggest that the seed coat restricts the invasion of fungal hyphae and protects the embryo against the attack of non-symbiotic fungi.

[要約] 千葉大学は文部科学省大学教育再生加速プログラムAP の委託を受け「次世代才能スキップアップ」プログラム実施している。このプログラムは理系に強い千葉大学の特長を生かす理系グローバル人材養成力強化の取り組みである。高校生が大学の学びを体感する体系的科学実験「基礎力養成講座」，さらにやる気と能力がある高校生に向けた課題研究支援「G- スキッパー」，グローバル力養成のための国際研究発表会，留学生派遣事業よりなる。本稿では平成29年度の基礎力養成講座4コース( 健康・医療，総合科学，テクノロジー，園芸学) を受講した高校生の学びを分析し，高大接続プログラムとしての本取組みの効果を考察したものである。高校生が各講座受講後に書いたレポートの自由記述をテキストマイニング法により解析し，高校生の学びを検討した。この結果，次世代スキップアッププログラムでの学びは高校生に学問への興味を喚起するものであり，また大学での学びを意識させるものであったことが示唆された。

© 2019, International Consortium of Landscape and Ecological Engineering. Arbuscular mycorrhizal fungi (AMF) are important components of the grassland ecosystems in terms of plant phosphorus uptake and accumulation of glomalin-related soil protein (GRSP). Though Mongolian grasslands are seriously degraded by livestock grazing, the effects of grazing on soil AMF and GRSP remain unclear. Here, we examined community composition and diversity of AMF as well as amount of GRSP at three different grazing intensities: lightly grazed (LG), moderately grazed (MG), and heavily grazed (HG) under two different types of grassland, mountain forest steppe at Hustai and desert steppe at Mandalgobi. The diversity and biomass of AMF-host and non-AMF plants strongly affected the overall AMF community composition and its diversity. When we separately analyzed the factors affecting soil AMF diversity at Hustai and Mandalgobi, decrease in the shoot biomass of Poaceae plants at Hustai and decreases in the species number and shoot biomass of AMF-host plants at Mandalgobi were significantly correlated with AMF diversity. GRSP decreased with increasing grazing intensity, which was significantly correlated with soil pH and total root biomass at Hustai. The decrease in plant biomass caused by grazing thus led to GRSP reduction. Our results showed that change in soil AMF community caused by livestock grazing were associated with change in the biomass and diversity of functional vegetation groups such as Poaeceae, AMF-host and non-AMF plants, indicating the importance to focus on such functional vegetation groups to evaluate the effect of grazing on AMF.

<p>ラン科植物は菌根共生への依存度が高く，さらに共生関係において宿主特異性が存在するため，パートナーとなりうる菌根菌が存在する環境でないと生育することができない。このため，やむを得ずラン科植物の移植を行う際には菌根菌が定着している環境に移植する必要がある。筆者らは鹿児島県に自生するオオバノトンボソウについて，シードパケット法を適用した種子発芽個体の解析に基づく，移植適地の判定に取り組んでおり，本稿ではオオバノトンボソウの種子発芽生態とともにその経過について報告する。</p>

Communities of arbuscular mycorrhizal (AM) fungi in Mongolian grassland were characterized under gradients of grazing intensity at three study sites of different aridity: mountain forest steppe at Hustai National Park (Hustai), and desert steppe at Mandalgovi and Bulgan. Grazing intensity was classified into three categories: lightly grazed (LG), moderately grazed (MG), and heavily grazed (HG). With regard to floristic composition, grazing decreased the shoot biomass of Poaceae species, especially Stipa spp. Distinctness of the AM fungal communities was observed among the three study sites, but most of the AM fungal operational taxonomic units (OTUs) that comprised over 1.0% of the total reads were ubiquitous. This result indicates that the AM fungal communities may be derived from similar AM fungal floras in correspondence with environmental factors. The composition of AM fungal communities differed significantly among the grazing intensities at all study sites. The relative abundance of the most dominant AM fungal OTU of the LG plots decreased with an increase in grazing intensity at all study sites. The mean proportions of the most dominant AM fungal OTUs also decreased with increased grazing intensity at Hustai. Dominance by a single AM fungal taxon may be a typical ecological feature of the AM fungal symbiosis, and grazing disturbs AM fungal community structure.

Achlorophylous and early developmental stages of chorolophylous orchids are highly dependent on carbon and other nutrients provided by mycorrhizal fungi, in a nutritional mode termed mycoheterotrophy. Previous findings have implied that some common properties at least partially underlie the mycorrhizal symbioses of mycoheterotrophic orchids and that of autotrophic arbuscular mycorrhizal (AM) plants; however, information about the molecular mechanisms of the relationship between orchids and their mycorrhizal fungi is limited. In this study, we characterized the molecular basis of an orchidmycorrhizal (OM) symbiosis by analyzing the transcriptome of Bletilla striata at an early developmental stage associated with the mycorrhizal fungus Tulasnella sp. The essential components required for the establishment of mutual symbioses with AM fungi or rhizobia in most terrestrial plants were identified from the B. striata gene set. A cross-species gene complementation analysis showed one of the component genes, calcium and calmodulin-dependent protein kinase gene CCaMK in B. striata, retains functional characteristics of that in AM plants. The expression analysis revealed the activation of homologs of AM-related genes during the OM symbiosis. Our results suggest that orchids possess, at least partly, the molecular mechanisms common to AM plants.

[要約] ツインクルプログラムは日本とASEAN諸国での双方向型教員インターンシッププログラムとして平成24年から開始し，発展してきた。本報告では，プログラムの年度ごとの改変によりASEAN諸国の留学生の学びがどのように変化したかを，学生のレポートの記述をもとにテキストマイニングにより解析し，検討した。この結果，各年度とも「TWINCLE program」，「laboratory course work」，「culture」など，プログラムおよび活動内容を示す言葉がカテゴリとして抽出されるとともに，カテゴリ間の関係は年度を追ってシンプルになっていくことが示唆された。したがって，年度を追って活動内容が洗練されていったことが考えられた。一方で「laboratory course work」の実施方法などの課題も示され，今後の取組みに反映したい。

<p>Chiba University has carried out a student exchange program called "Twin college envoys (TWINCLE)" as part of Japan's re-inventory project with the support of MEXT since 2012. The TWINCLE program has 12 partner universities in 5 ASEAN countries—Indonesia, Thailand, Singapore, Cambodia, and Vietnam. During the 5 years of the program, 246 ASEAN students visited Japan. This study analyzes the effect of the program, focused on the influence on the careers of ASEAN students through the program activities, using students' comments in activity reports from 2013 to 2016 using text-mining methods and subsequently cluster analysis methods. The results suggest that ASEAN students were interested in each activity in the TWINCLE program and had good impressions after participating in the program.</p>

千葉大学では平成 24 年度より文部科学省大学世界展開力強化事業「ツインクル」プログラムをASEAN 諸国 12 大学と連係し実施してきた．今回，平成 25 年度平成 28 年度に参加した ASEAN 諸国の留学生の気づきと学びについて，アンケートの記述をテキストマイニングの手法により解析した．この結 果，カテゴリとしてプログラム内容に関する単語とともに culture を中心として科学や，学校に関するさまざまな Japan/Japanese に関する学びがあることを示す結果が得られた．さらに学生が日本での大学院での学びや日本での職業選択への希望が見出された．したがって本プログラム内容が，留学生の親日的 感情を高め，キャリア意識の形成への影響が示唆された．

Some green orchids obtain carbon from their mycorrhizal fungi, as well as from photosynthesis. These partially mycoheterotrophic orchids sometimes produce fully achlorophyllous, leaf-bearing (albino) variants. Comparing green and albino individuals of these orchids will help to uncover the molecular mechanisms associated with mycoheterotrophy. We compared green and albino Epipactis helleborine by molecular barcoding of mycorrhizal fungi, nutrient sources based on N-15 and C-13 abundances and gene expression in their mycorrhizae by RNA-seq and cDNA de novo assembly. Molecular identification of mycorrhizal fungi showed that green and albino E. helleborine harboured similar mycobionts, mainly Wilcoxina. Stable isotope analyses indicated that albino E. helleborine plants were fully mycoheterotrophic, whereas green individuals were partially mycoheterotrophic. Gene expression analyses showed that genes involved in antioxidant metabolism were upregulated in the albino variants, which indicates that these plants experience greater oxidative stress than the green variants, possibly due to a more frequent lysis of intracellular pelotons. It was also found that some genes involved in the transport of some metabolites, including carbon sources from plant to fungus, are higher in albino than in green variants. This result may indicate a bidirectional carbon flow even in the mycoheterotrophic symbiosis. The genes related to mycorrhizal symbiosis in autotrophic orchids and arbuscular mycorrhizal plants were also upregulated in the albino variants, indicating the existence of common molecular mechanisms among the different mycorrhizal types.

Background: In nature, orchid plants depend completely on symbiotic fungi for their nutrition at the germination and the subsequent seedling (protocorm) stages. However, only limited quantitative methods for evaluating the orchid-fungus interactions at the protocorm stage are currently available, which greatly constrains our understanding of the symbiosis. Here, we aimed to improve and integrate quantitative evaluations of the growth and fungal colonization in the protocorms of a terrestrial orchid, Blettila striata, growing on a plate medium.Results: We achieved both symbiotic and asymbiotic germinations for the terrestrial orchid B. striata. The protocorms produced by the two germination methods grew almost synchronously for the first three weeks. At week four, however, the length was significantly lower in the symbiotic protocorms. Interestingly, the dry weight of symbiotic protocorms did not significantly change during the growth period, which implies that there was only limited transfer of carbon compounds from the fungus to the protocorms in this relationship. Next, to evaluate the orchid-fungus interactions, we developed an ink-staining method to observe the hyphal coils in protocorms without preparing thin sections. Crushing the protocorm under the coverglass enables us to observe all hyphal coils in the protocorms with high resolution. For this observation, we established a criterion to categorize the stages of hyphal coils, depending on development and degradation. By counting the symbiotic cells within each stage, it was possible to quantitatively evaluate the orchid-fungus symbiosis.Conclusions: We describe a method for quantitative evaluation of orchid-fungus symbiosis by integrating the measurements of plant growth and fungal colonization. The current study revealed that although fungal colonization was observed in the symbiotic protocorms, the weight of the protocorm did not significantly increase, which is probably due to the incompatibility of the fungus in this symbiosis. These results suggest that fungal colonization and nutrition transfer can be differentially regulated in the symbiosis. The evaluation methods developed in this study can be used to study various quantitative aspects of the orchid-fungus symbiosis.

<p>The TWINCLE program is a teacher internship program which aims to enhance mutual understanding of foreign cultures and to develop students' ability to succeed in overseas internships. In this study, reports written by students who participated in the program were analyzed by a text mining method to evaluate the changes in their ideas about the development and practice of the teaching materials. The results indicate that the students' attitudes towards science classes changed from 'simple lecture oriented classes' to 'problem-solving learning oriented classes'. Therefore, participating in the TWINCLE program is an effective way to change the students' image of the science class.</p>

<p>平成26 年度に科学教育を主体とする交換留学プログラムツインクルに参加したASEAN 諸国の留学生の気づきと学びについて、アンケートの記述をテキストマイニングの手法により解析した。この結果、カテゴリとしてプログラム内容に関する単語とともにgood, learn という感想を示す単語、さらに culture, Japanese も同様に抽出され、学びと日本とに対する良い感情が表現されていた。また今後の進 路への影響に関する回答からは学生が日本と関連付けて、仕事を得ようとする姿勢が見られた。したがって本プログラム内容が、留学生にとって前向きに将来を考えるものとして機能している可能性が示唆された。</p>

Petrosavia sakuraii (Petrosaviaceae) is a rare, mycoheterotrophic plant species that has a specific symbiotic interaction with a narrow clade of arbuscular mycorrhizal (AM) fungi. In the present study, we tested the hypothesis that the distribution and abundance of mycobionts in two P. sakuraii habitats, Nagiso and Sengenyama (central Honshu, Japan), determine the distribution pattern of this rare plant. Nagiso is a thriving habitat with hundreds of P. sakuraii individuals per 100 m(2), whereas Sengenyama is a sparsely populated habitat with fewer than 10 individuals per 100 m(2). AM fungal communities associated with tree roots were compared at 20-cm distances from P. sakuraii shoots between the two habitats by molecular identification of AM fungal partial sequences of the small subunit ribosomal RNA gene. The percentage of AM fungal sequences showing over 99 % identity with those of the dominant P. sakuraii mycobionts was high (54.9 %) in Nagiso, but low (13.2 %) in Sengenyama. Accordingly, the abundance of P. sakuraii seems to reflect the proportion of potential mycobionts. It is likely that P. sakuraii mycobionts are not rare in Japanese warm temperate forests since 11.2 % of AM fungal sequences previously obtained from a deciduous broad-leaved forest devoid of P. sakuraii in Mizuho, central Honshu, Japan, were > 99 % identical to those of the dominant P. sakuraii mycobionts. Thus, results suggest that the abundant mycobionts may be required for sufficient propagation of P. sakuraii, and this quantitative trait of AM fungal communities required for P. sakuraii may explain the rarity of this plant.

<p>教員インターンシッププログラムであるツイン型学生派遣プログラム(ツインクル)は，日本の学 ［キーワード］科学実験授業，ASEAN 諸国，教員インターンシップ，文理融合，テキストマイニング 生が文理融合チームを組み，ASEAN 諸国の高等学校において現地の大学生と協働して科学実験授業を実施するものである。本研究では，ASEAN 諸国での科学教育活動前後での学生の学びと成長について，学生の自由記述アンケートをテキストマイニングにより分析した。特に本報告では参加前後での授業に対する学生の意識変化について解析をした。この結果，学生が派遣前には授業を子ども中心の視点で考えていたが，派遣によるASEAN 諸国の高校での授業体験を経て，それぞれの授業の目的や目的に基づくあり方に変容することが示唆された。</p>

我々は,これまで中・高校生を対象に理系人材育成支援に取り組んできた。今回,この活動のノウハウを活かし,アセアンを含む東アジアにおける早期才能支援教育プログラムの開発を実施した。具体的な取り組みとして①中国版生命科学系講座における学習教材の開発,②タイの理系大学生対象の生命科学系実験プログラムの実施,③アセアン展開型科学教育プログラムの開発・実施を行った。この結果,東アジアにおける早期理系人材才能育成の一助となる学習プログラムの開発に成功した。本研究は科学研究費補助金基盤研究B「「ラボon theデスク」に基づく東アジア普及型早期才能支援プログラムの開発」(課題番号24300265),文部科学省公募事業平成24年度「大学の世界展開力強化事業」,科学研究費補助金基盤研究C「ASEANと日本をつなぐ科学教育実験教材開発」(課題番号26350226)の一部により実施いたしました。

<p>千葉大学はスーパーグローバル大学として学生のグローバル能力強化のための教育を推進している。ツイ ン型学生派遣プログラム(ツインクル)はASEANの学校において，日本の学生が文理融合チームを組み現地の学生と協働して科学実験授業を実施する，教員インターンシッププログラムである。本研究では，ASEANでの科学教育活動を通して，学生にどのような学びと成長があったかについて，学生の自由記述アンケートをテキストマイニングにより分析した。平成25年度後期と平成26年度のアンケートを分析した結果，学生がいくつもの困難を乗り越え，ASEANで教員体験をし，グローバル人材としての資質を身につけたことが示唆された。</p>

Petrosaviaceae is a monocotyledonous plant family that comprises two genera: the autotrophic Japonolirion and the mycoheterotrophic Petrosavia. Accordingly, this plant family provides an excellent system to examine specificity differences in mycobionts between autotrophic and closely related mycoheterotrophic plant species. We investigated mycobionts of Japonolirion osense, the sole species of the monotypic genus, from all known habitats of this species by molecular identification and detected 22 arbuscular mycorrhizal (AM) fungal phylotypes in Archaesporales, Diversisporales, and Glomerales. In contrast, only one AM fungal phylotype in Glomerales was predominantly detected from the mycoheterotrophic Petrosavia sakuraii in a previous study. The high mycobiont diversity in J. osense and in an outgroup plant, Miscanthus sinensis (Poaceae), indicates that fungal specificity increased during the evolution of mycohetrotrophy in Petrosaviaceae. Furthermore, some AM fungal sequences of J. osense showed >99 % sequence similarity to the dominant fungal phylotype of P. sakuraii, and one of them was nested within a clade of P. sakuraii mycobionts. These results indicate that fungal partners are not necessarily shifted, but rather selected for in the course of the evolution of mycoheterotrophy. We also confirmed the Paris-type mycorrhiza in J. osense.

We attempted to introduce ectomycorrhizal (ECM) fungi onto seedlings of Dipterocarpus alatus (Dipterocarpaceae) by soil inocula collected from tree stands of D. alatus. Top soils collected from beneath the trees of D. alatus in a dry evergreen forest and a 15-year-old plantation were inoculated onto germinating seeds of D. alatus. After 7 months of seedling cultivation, ECM fungal communities in the seedlings were investigated based on the sequences of ITS rDNA. The ECM fungi detected were divided into 19 phylotypes by molecular analysis. Most of the phylotypes were identified as ECM fungal taxa, i.e., Clavulina, Laccaria, Lactarius, Tomentella, Pyronemataceae, and Tricholomataceae. Accordingly, we can confirm that soil inoculation is a simple method to induce ECM formation with diverse fungi in pot cultured seedlings, which would be useful for introducing diverse ECM fungi to dipterocarp plantations.

Communities of arbuscular mycorrhizal (AM) fungi were investigated in Stipa krylovii, Leymus chinensis (Poaceae), Allium bidentatum (Liliaceae), and Astragalus brevifolius (Fabaceae) in the Mongolian steppe to examine the effect of plant species on the communities in this study. The AM fungal communities were examined by molecular analysis based on the partial sequences of a small subunit of the ribosomal RNA gene. The sequences obtained were divided into 23 phylotypes by the sequence similarity >98%. Many of the AM fungal phylotypes included AM fungi previously detected in high-altitude regions in the Tibet and Loes plateaus, which suggested that these AM fungi may have wide distribution with stressful conditions of aridity and coldness. Among the 23 phylotypes, 12 phylotypes were found in all four plants, and 87.4% of the all obtained sequences were affiliated into these 12 types. For the distribution of the AM fungal phylotypes, overlapping of the phylotypes among the four plant species were significantly higher than that simulated by random chance. These results suggested that AM fungal communities were less diversified among the examined plant species. (C) 2013 The Mycological Society of Japan. Published by Elsevier B. V. All rights reserved.

We investigated communities of arbuscular mycorrhizal fungi (AMF) in the fine roots of Pyrus pyrifolia var. culta, and Plantago asiatica to consider the relationship between orchard trees and herbaceous plants in AMF symbioses. The AMF communities were analyzed on the basis of the partial fungal DNA sequences of the nuclear small subunit ribosomal RNA gene (SSU rDNA), which were amplified using the AMF-specific primers AML1 and AML2. Phylogenetic analysis showed that the obtained AMF sequences were divided into 23 phylotypes. Among them, 12 phylotypes included AMF from both host plants, and most of the obtained sequences (689/811) were affiliated to them. Canonical correspondence analysis showed that the host plant species did not have a significant effect on the distribution of AMF phylotypes, whereas the effects of sampling site, soil total C, soil total N and soil-available P were significant. It was also found that the mean observed overlaps of AMF phylotypes between the paired host plants in the same soil cores (27.1% of phylotypes shared) were significantly higher than the mean 1,000 simulated overlaps (14.2%). Furthermore, the same AMF sequences (100% sequence identity) were detected from both host plants in 8/12 soil cores having both roots. Accordingly, we concluded that Py. pyrifolia and Pl. asiatica examined shared some AMF communities, which suggested that understory herbaceous plants may function as AMF inoculum sources for orchard trees.

We examined arbuscular mycorrhizal (AM) fungi colonizing the roots of Stipa krylovii, a grass species dominating the grasslands of the steppe zone in Hustai and Uvurkhangai in Mongolia. The AM fungal communities of the collected S. krylovii roots were examined by molecular analysis based on the partial sequences of a small subunit of ribosomal RNA gene as well as AM fungal colonization rates. Almost all AM fungi detected were in Glomus-group A, and were divided into 10 phylotypes. Among them, one phylotype forming a clade with G. intraradices and G. irregulare was the most dominant. Furthermore, it was also found that most of the phylotypes include AM fungi previously detected in high altitude regions in the Eurasian Continent. Significant correlations were found among soil total N, total plant biomass and AM fungal colonization ratio, which suggested that higher plant biomass may be required for the proliferation of AM fungi in the environment. Meanwhile, redundancy analysis on AM fungal distribution and environmental variables suggested that the effect of plant biomass and most soil chemical properties on the AM fungal communities were not significant. (C) 2012 The Mycological Society of Japan. Published by Elsevier B. V. All rights reserved.

Mixotrophy, obtaining carbon by mycoheterotrophy and photosynthesis, has been suggested in Cephalanthera species (Orchidaceae) by analyses on stable isotopes of carbon. In this study, we examined the growth of Cephalanthera falcata in pot cultured tripartite symbioses with Thelephoraceae fungi and Quercus serrata. Mycorrhizal fungi were isolated from roots of C. falcata in natural habitats. Two fungal isolates identified as Thelephoraceae were cultured and inoculated to fine roots of non-mycorrhizal seedlings of Q. serrata (Fagaceae). After the ectomycorrhizal formation, non-mycorrhizal seedlings of C. falcata were co-planted. The pots with tripartite symbioses were cultured in greenhouse for 30 months, and growth of C. falcata seedlings was examined. Fresh weight of C. falcata seedlings was significantly increased by the tripartite symbioses even in those with no shoot, thus providing further evidence for the mycoheterotrophic nature of this orchid. The achievement of seedling culture in tripartite symbioses would be valuable for conserving many forest orchids and for conducting experiments to understand their physiology and ecology.

We examined the colonization rate and communities of arbuscular mycorrhizal fungi (AMF) in the roots of Pyrus pyrifolia var. culta (Japanese pear) in orchards to investigate the effect of phosphorus (P) fertilization on AMF. Soil cores containing the roots of Japanese pear were collected from 13 orchards in Tottori Prefecture, Japan. Soil-available P in the examined orchards was 75.7 to 1,200 mg kg(-1), showing the extreme accumulation of soil P in many orchards. The AMF colonization rate was negatively correlated with soil-available P (P <0.01). AMF communities were examined on the basis of the partial fungal DNA sequences of the nuclear small-subunit ribosomal RNA gene (SSU rDNA) amplified by AMF-specific primers AML1 and AML2. The obtained AMF sequences were divided into 14 phylotypes, and the number of phylotypes (species richness) was also negatively correlated with soil-available P (P <0.05). It was also suggested that some AM fungi may be adapted to high soil-available P conditions. Redundancy analysis showed the significant effects of soil pH, available P in soil, and P content in leaves of P. pyrifolia var. culta trees on AMF distribution. These results suggested that the accumulation of soil-available P affected AMF communities in the roots of Japanese pear in the orchard environment.

The community structure of arbuscular mycorrhizal (AM) fungi associated with Ixeris repens was studied in coastal vegetation near the Tottori sand dunes in Japan. I. repens produces roots from a subterranean stem growing near the soil surface which provides an opportunity to examine the effects of an environmental gradient related to distance from the sea on AM fungal communities at a regular soil depth. Based on partial sequences of the nuclear large subunit ribosomal RNA gene, AM fungi in root samples were divided into 17 phylotypes. Among these, five AM fungal phylotypes in Glomus and Diversispora were dominant near the seaward forefront of the vegetation. Redundancy analysis of the AM fungal community showed significant relationships between the distribution of phylotypes and environmental variables such as distance from the sea, water-soluble sodium in soil, and some coexisting plant species. These results suggest that environmental gradients in the coastal vegetation can be determinants of the AM fungal community.

Lecanorchis is a nonphotosynthetic plant genus in Vanilloideae, Orchidaceae. Because of the distribution of many Lecanorchis taxa in various climate conditions, we hypothesized that mycorrhizal diversity and specificity are different among the different taxa of Lecanorchis. In the present study, identities of mycorrhizal fungi were examined for 90 individuals of 10 Lecanorchis taxa at 26 sites from Niigata to Okinawa Prefectures in Japan. Phylogenetic analyses of Lecanorchis taxa based on the internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene (rDNA) divided the examined Lecanorchis taxa into three groups, groups A, B, and C. ITS rDNA sequences suggested that fungi associating with Lecanorchis were ectomycorrhiza-forming fungi in Lactarius, Russula, Atheliaceae, and Sebacina, with Lactarius and Russula dominant. Our results suggested some degree of mycorrhizal specialization among Lecanorchis taxa. Interestingly, the Lecanorchis group C had some specific relationships with Lactarius, whereas less specificity was found in the relationships with Russula. However, observed specificity results may be biased by geographic opportunity, and we suggest further research to assess whether Lecanorchis species are limited to the associations we observed.

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.similar to minor roots. We tested for a 13C and 15N 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.similar to densiflora seedlings. Stable isotope analyses indicated mixotrophic nutrition of P.similar to 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.

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 mu m. 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 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.

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.