Hyphal coil morphology and its relationship to thromboplerous hyphae in Rhizopogon roseolus.

While observing mycelial features of the ectomycorrhizal fungus Rhizopogon roseolus, we noted the formation of hyphal coils in laboratory cultures. The coiled hyphae initially formed at the hyphal tip in the presence or absence of the plant host (Pinus thunbergii), on both rich and poor modified Melin-Norkrans media. Hyphal coils formed from the hyphal tips toward the middle of the hyphae, with rope-like hyphal strands fused as extensive circular coils. Hyphal coils were generally oval and circular. Coils were composed of 1-5 layers of hyphae. Presence of the host had a significant impact on the diameter of the coil loops, which was consistently wider without the host than with the host present on both media. In addition, the terminal part of the mature coils was melanized and separated from the unmelanized coil by a septum. The melanized coils resembled thick condensed hyphae and were detached and scattered throughout the fungal colony. The observation of morphological characteristics suggested that mature coils detached into the mycelia were thromboplerous hyphae. This is the first report on hyphal coil morphogenesis and its potential relationship to the initial development of thromboplerous hyphae.

Basidium formation of Rhizopogon roseolus with Pinus thunbergii in agar medium.

The edible ectomycorrhizal (ECM) mushroom Rhizopogon roseolus usually develops basidium and basidiospores in the gleba of its basidiomata. Here, we report a novel production of basidia in laboratory cultures of the edible ECM mushroom. The basidium with sterigma was observed on the old mantle structure (> six months) of the ECM between R. roseolus and Pinus thunbergii in a modified Melin-Norkrans medium that was subjected to a temperature shock from 25 °C to 4 °C. The basidia were cylindrical to clavate, with prominent sterigmata and no basidiospores. The absence of basidiospores might indicate partial development of the basidium structure as a response to environmental stress and incomplete life cycle of R. roseolus. In addition, branched cystidia were evident in two or three clavate-to-ovoid cells. This study suggests the possibility of obtaining the primary mycelium of R. roseolus from pure cultures and may be an alternative genetic source for cultivation purposes. Further observations are required to induce basidiosporogenesis of R. roseolus basidia in an agar medium focusing on manipulation of temperature.

The impact of host plant (Pinus thunbergii) on the mycelial features of the ectomycorrhizal fungus Rhizopogon roseolus.

The impact of host plant on the mycelial features of mycorrhizal symbiont and its characteristics has been poorly investigated. This study aimed to compare and quantify (statistically tested) some of the mycelial features of an ectomycorrhiza (ECM)-forming fungus with and without the ECM host. The ECM-forming fungus, Rhizopogon roseolus, inoculated with or without Pinus thunbergii on both rich and poor nutrient media, was observed under laboratory conditions. On rich medium, fungi with the host had the highest colony diameter and consistently produced the highest hyphal length relative to fungi on other media. Thus, the host had a significant impact on the mycelium production of R. roseolus in both rich and poor media. Further, fungi without the host had a higher number of hyphal anastomoses per hyphal length on both poor and rich media than fungi with the host in the same medium. Anastomosis, which refers to the fusion of two parallel hyphae, was evident in all experiments. However, there was no significant impact of the host on the number of hyphal anastomoses. In addition, fungi without the host had more frequent hyphal branching on both rich and poor media than fungi with the host. The occurrence of a host only had a significant impact on the formation of the hyphal branch on poor medium. Further, a chlamydospore-like structure was identified, which had a higher diameter when formed with the host on both rich and poor media. The present data provide new insights into the host plant's impact on the mycelial features of ECM-forming fungi.

Cytological comparison of mycelial aggregates of Rhizopogon roseolus with and without the ectomycorrhiza host.

Most cytology reports on ectomycorrhiza (ECM) have been less focused on the fungal structures, especially on the exploratory organ. This study aimed to evaluate the morphological response of explorative mycelia of ECM forming fungi with and without the occurrence of the ECM host. We assessed the mycelial aggregates of Rhizopogon roseolus which was inoculated with and without Pinus thunbergii under controlled laboratory conditions. The mycelial aggregates with the host produced strikingly complex hyphal strands relative to those without host. Light microscopy revealed that the cytology and plectology of both mycelial aggregates had the approximately similar architecture. The tubular hyphae diameter without the host was consistently larger than that with the host. This study confirmed that the septa diameter of the tubular hyphae conjunction of mycelial aggregates with the host were shorter than those without the host. In addition, partially and completely dissolved septa of the tubular hyphae were evident with and without host. We also described the thromboplerous hyphae, which have rarely been reported in vitro. These hyphae were produced in higher numbers near the fungal inoculum with and without the host. The current findings will add to the current knowledge regarding ECM fungi mycelial aggregate response to the host.

Detection of autophagy-related structures in fruiting bodies of edible mushroom, Pleurotus ostreatus.

Autophagy is involved in various fungal morphogenetic processes. However, there are limited reports regarding the role of autophagy in mushroom fruiting body formation. The purpose of this study was to reveal the autophagy-related structures in mushroom-forming fungi. The edible mushroom Pleurotus ostreatus was used in this study. Transmission electron microscopy revealed double-membrane bounded structures containing cytoplasmic components in the fruiting bodies of this fungus. Some of these double-membrane structures were observed to interact with the vacuoles. Additionally, curved flat cisternae of various lengths were detected in the cytoplasm. The shape, size and thickness of the limiting membrane of the double-membrane structures and the flat cisternae corresponded well with those of the autophagosomes and the isolation membranes, respectively. Regarding autophagosome formation, a membrane-bound specific zone was detected near the isolation membrane, which appeared to expand along the novel membrane. This is the first detailed report showing autophagy-related structures in P. ostreatus and provides a possible model for autophagosome formation in these filamentous fungi.

Effect of sodium chloride on basidiospore germination and vegetative mycelial growth of the ectomycorrhizal fungus Rhizopogon roseolus.

Rhizopogon roseolus is a basidiomycetous ectomycorrhizal fungus that inhabits mainly coastal areas. Understanding the response of this fungus to salinity at each stage of its life cycle will lead to elucidation of the strategies for its propagation. This study examined the effect of sodium chloride (NaCl) on basidiospore germination and mycelial growth of both homokaryotic and heterokaryotic strains of R. roseolus, on nutrient agar media with varying concentrations of NaCl (0, 50, 150, and 300 mM). Regardless of the presence of NaCl, R. roseolus basidiospores germinated and the germlings grew, forming compatible fusions. In addition, all multispore strains, including homokaryons and heterokaryons, grew under these NaCl conditions. Most of these strains had an effective concentration inhibiting mycelial growth by 50% value greater than 300 mM of NaCl. These results indicate that R. roseolus can germinate, grow, and mate in the presence of NaCl, allowing it to propagate in saline habitats.

Identification, characterization and expression of A-mating type genes in monokaryons and dikaryons of the edible mushroom Mycoleptodonoides aitchisonii (Bunaharitake).

Identifying the mating-type in Mycoleptodonoides aitchisonii is important for enhancing breeding and cultivation of this edible mushroom. To clarify the molecular mechanisms of the bipolar mating system in M. aitchisonii, the homeodomain protein gene 2 (Mahd2) was characterized. A genomic DNA fragment of Mahd2 in M. aitchisonii 50005-18 strain was 1,851 bp long and encoded a protein of 614 amino acids. Transcriptional analysis revealed that the expression of Mahd2 was higher in monokaryotic strains that produced clamp cells than in those that did not. The highest relative expression level of Mahd2 was observed in monokaryon TUFC 50005-4, which was capable of forming a true clamp. These results suggested that the formation of clamp cells is regulated by A-mating type homeodomain proteins, and the frequency of clamp cell formation might be promoted by high expression of the Mahd2 gene.

Nuclear behavior in Coprinus comatus from Japan and estimation of the lifecycle.

In order to elucidate the lifecycle of Coprinus comatus, we examined the number of nuclei in basidiospores, hyphal cells and oidia. Basidiospores isolated from the fruiting bodies of four Japanese strains were binucleate. In both primary and secondary mycelia, most of the cells were binucleate. In addition, oidia and oidiophores were observed for the first time in this mushroom and most of the oidia were binucleate. Based on these results, the lifecycle of C. comatus was inferred to be as follows. A homokaryotic binucleate basidiospore germinates and produces homokaryotic binucleate hyphae. After mating between compatible homokaryotic binucleate hyphae, a heterokaryotic binucleate secondary mycelium is produced. If environmental conditions are suitable for fruiting, homokaryotic binucleate basidiospores in the fruiting body are produced.

Expression of α-glucosidase during morphological differentiation in the basidiomycetous fungus Pholiota microspora.

The α-glucosidase gene from Pholiota microspora, designated PnGcs, was amplified and characterized. The open reading frame region of PnGcs, from ATG to the stop codon, is 2937 bp and encodes a protein of 979 amino acids with a signal peptide of 20 amino acids at the N-terminus. The predicted amino acid sequence of PnGcs indicated that it is a glycoside hydrolase family 31 protein. Quantitative reverse transcription PCR was used to investigate PnGcs expression in mycelia cultured in minimal medium containing various carbon sources, as well as in tissue during different stages of development of fruiting bodies. When P. microspora was grown in minimal medium supplemented with different carbon sources, PnGcs expression was highest when induced by maltose. During cultivation on sawdust medium, PnGcs expression increased dramatically at the fruiting body formation stage compared with the mycelial growth stage, which implied that PnGcs is closely associated with fruiting body development.

Maintenance of Chloroplast Structure and Function by Overexpression of the Rice MONOGALACTOSYLDIACYLGLYCEROL SYNTHASE Gene Leads to Enhanced Salt Tolerance in Tobacco.

In plants, the galactolipids monogalactosyldiacylglycerol (MGDG) and digalactodiacylglycerol (DGDG) are major constituents of photosynthetic membranes in chloroplasts. One of the key enzymes for the biosynthesis of these galactolipids is MGDG synthase (MGD). To investigate the role of MGD in the plant's response to salt stress, we cloned an MGD gene from rice (Oryza sativa) and generated tobacco (Nicotiana tabacum) plants overexpressing OsMGD. The MGD activity in OsMGD transgenic plants was confirmed to be higher than that in the wild-type tobacco cultivar SR1. Immunoblot analysis indicated that OsMGD was enriched in the outer envelope membrane of the tobacco chloroplast. Under salt stress, the transgenic plants exhibited rapid shoot growth and high photosynthetic rate as compared with the wild type. Transmission electron microscopy observation showed that the chloroplasts from salt-stressed transgenic plants had well-developed thylakoid membranes and properly stacked grana lamellae, whereas the chloroplasts from salt-stressed wild-type plants were fairly disorganized and had large membrane-free areas. Under salt stress, the transgenic plants also maintained higher chlorophyll levels. Lipid composition analysis showed that leaves of transgenic plants consistently contained significantly higher MGDG (including 18:3-16:3 and 18:3-18:3 species) and DGDG (including 18:3-16:3, 18:3-16:0, and 18:3-18:3 species) contents and higher DGDG-MGDG ratios than the wild type did under both control and salt stress conditions. These results show that overexpression of OsMGD improves salt tolerance in tobacco and that the galactolipids MGDG and DGDG play an important role in the regulation of chloroplast structure and function in the plant salt stress response.

Gymnopilins, a product of a hallucinogenic mushroom, inhibit the nicotinic acetylcholine receptor.

Gymnopilins are substances produced in fruiting bodies of the hallucinogenic mushroom, Gymnopilus junonius. Although, only a few biological effects of gymnopilins on animal tissues have been reported, it is believed that gymnopilins are a key factor of the G. junonius poisoning. In the present study, we found that gymnopilins inhibited ACh-evoked responses in neuronal cell line, PC12 cell, and determine the underlying mechanism. Gymnopilins were purified from wild fruiting bodies of G. junonius collected in Japan. Ca(2+)-imaging revealed that gymnopilins reduced the amplitude of ACh-evoked [Ca(2+)]i rises by about 50% and abolished the ACh responses remaining in the presence of atropine. Gymnopilins greatly reduced the amplitude of [Ca(2+)]i rises evoked by nicotinic ACh receptor agonists, 1,1-Dimethyl-4-phenylpiperazinium iodide (DMPP) and nicotine. In the whole-cell voltage clamp recording, gymnopilins inhibited the DMPP-evoked currents, but did not affect the voltage-gated Ca(2+) channel currents. These results indicate that gymnopilins directly act on nicotinic ACh receptors and inhibit their activity. This biological action of gymnopilins may be one of the causes of the G. junonius poisoning.

Combined molecular and morphological data for improving phylogenetic hypothesis in Dacrymycetes.

We analyzed the DNA sequences of four gene regions, 28S and 18S rDNA, the ITS region and rpb2, to obtain a high resolution phylogenetic tree of Dacrymycetes. In addition, we comparatively studied micro- and macromorphological characteristics of representative species. The traditional generic classification based on morphological characteristics was not reflected by our molecular phylogenies. Ancestral state reconstructions indicated that the morphology of basidia and clamp connections are evolutionarily stable. In contrast, basidiocarps and basidiospore septation patterns appear variable. Dacrymyces unisporus shares the dolipores with non-perforate parenthesomes typical of other dacrymycetous taxa but is a unique species having predominantly non-bifurcate basidia and subglobose to ovoid basidiospores with transverse and longitudinal septa. In molecular phylogenies this species is a member of Dacrymycetes but always occupies a sister position in relation to the rest of the Dacrymycetes. Based on our results we propose a new genus, Unilacryma, for D. unisporus. For proper accommodation of this taxon, we introduce the family Unilacrymaceae and the order Unilacrymales.

Multiple effects of gymnopilin on circulatory system of the rat.

Gymnopilin is one of the substances produced by the hallucinogenic mushroom, Gymnopilus junonius. In this study, we examined effects of gymnopilins purified from wild fruiting bodies of G. junonius on contractile activity of aorta preparations and blood pressure in rats. Gymnopilins at lower concentrations than 5 mg/mL did not evoke contraction of helical strips of the thoracic aorta. In contrast, gymnopilins (5 mg/mL) applied to the aorta strips pre-contracted by norepinephrine (100 nM) caused relaxation. This relaxing action did not depend on the activity of the endothelium cells. The relaxing effect of 5-mg/mL gymnopilins was observed in aorta strips contracted by angiotensin II (10 nM) and the high K+ solution (60 mM). Moreover, the adenylyl cyclase inhibitor, SQ-22536, significantly inhibited the relaxing effect of gymnopilins at 1 mg/mL on the norepinephrine-contracted strips. These results suggested that gymnopilins acted directly on smooth muscle cells of the aorta and activated the cAMP-dependent cascade to cause the vasodilation. Paradoxically, gymnopilins injection into the jugular vein transiently increased blood pressure without affecting the heart rate. This result suggests that gymnopilins increase cardiac output and/or tension of the artery through the excitation of the vasomotor nerve that overcame the direct relaxing effect on the vascular smooth muscle.

Gymnopilin--a substance produced by the hallucinogenic mushroom, Gymnopilus junonius--mobilizes intracellular Ca(2+) in dorsal root ganglion cells.

Gymnopilus junonius is a widely spread mushroom in Japan and well known as a hallucinogenic mushroom. Gymnopilin was purified from the fruiting body of G. junonius and was reported to act on the spinal cord and depolarize motoneurons. This is the only evidence that gymnopilin has a biological effect on animals and no mechanism of the action has been determined at all. In this study, we examined effects of gymnopilin on intracellular Ca(2+) concentrations ([Ca(2+)](i)) of cultured cells isolated from the dorsal root ganglion (DRG) of the rat. The cell culture consisted of neurons and non-neuronal cells. Gymnopilin increased [Ca(2+)](i) in both the types of cells. The gymnopilinevoked [Ca(2+)](i) rise in the non-neuronal cells was inhibited by cyclopiazonic acid and U-73122, inhibitors of Ca(2+)-ATPase of the intracellular Ca(2+) store and phospholipase C, respectively, but not by removal of extracellular Ca(2+). These results indicate that gymnopilin activated phospholipase C and mobilize Ca(2+) from the intracellular Ca(2+) store in non-neuronal cells from the DRG. This is the first report to show that gymnopilin directly acts on cells isolated from the mammalian nervous system.

Karyological characterization of meiosis, post-meiotic mitosis and nuclear migration in the ectomycorrhizal fungus Rhizopogon roseolus (= R. rubescens).

Karyological characteristics during basidiosporogenesis of Rhizopogon roseolus, a member of the hypogeous Agaricomycetes, were studied by light and transmission electron microscopy. More than 1000 tissue fragments of young basidiomata were stained with HCl-Giemsa and observed by a light microscopy to evaluate nuclear behavior. Basidium morphology in the hymenium was observed by transmission electron microscopy. Meiosis and post-meiotic mitosis took place in the center of the basidium. Sterigmata appeared when the first meiotic division occurred, and the center of the basidium became constricted when the second meiotic division occurred. Asynchronous nuclear migration from the basidium into the basidiospores occurred after post-meiotic mitosis, producing eight uninucleate basidiospores. The nucleus migrated patchily into basidiospores. The pattern of post-meiotic mitosis of R. roseolus, in which post-meiotic mitosis took place in the center of the basidium, is reported for the first time.

Abscisic acid substantially inhibits senescence of cucumber plants (Cucumis sativus) grown under low nitrogen conditions.

Low nitrogen (N) availability such as that found in both dry land and tropical regions limits plant growth and development. The relationship between the level of abscisic acid (ABA) in a plant and its growth under low-N conditions was investigated. The level of ABA in cucumber (Cucumis sativus) plants under low-N conditions was significantly higher at 10 and 20 d after transplantation compared with that under sufficient-N conditions. Chlorophyll was preserved in the aerial parts of cucumber plants grown under low-N conditions in the presence of ABA, while there was no significant difference between control plants and ABA-applied plants under sufficient-N conditions. ABA suppressed the reduction of chlorophyll biosynthesis under low-N conditions but not under sufficient-N conditions. On the other hand, ABA decreased the expression of the chlorophyll degradation gene in older cucumber plants grown under both conditions. In addition, transcript and protein levels of a gene encoding a chlorophyll a/b binding protein were positively correlated with ABA concentration under low-N conditions. The chloroplasts in control plants were round, and the stack of thylakoid membranes was reduced compared with that of plants treated with ABA 10(-5) M. These results strongly suggest that ABA is accumulated in cucumber plants grown under low-N conditions and that accumulated ABA promotes chlorophyll biosynthesis and inhibits its degradation in those plants.

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.

A-mating-type gene expression can drive clamp formation in the bipolar mushroom Pholiota microspora (Pholiota nameko).

In the bipolar basidiomycete Pholiota microspora, a pair of homeodomain protein genes located at the A-mating-type locus regulates mating compatibility. In the present study, we used a DNA-mediated transformation system in P. microspora to investigate the homeodomain proteins that control the clamp formation. When a single homeodomain protein gene (A3-hox1 or A3-hox2) from the A3 monokaryon strain was transformed into the A4 monokaryon strain, the transformants produced many pseudoclamps but very few clamps. When two homeodomain protein genes (A3-hox1 and A3-hox2) were transformed either separately or together into the A4 monokaryon, the ratio of clamps to the clamplike cells in the transformants was significantly increased to ca. 50%. We therefore concluded that the gene dosage of homeodomain protein genes is important for clamp formation. When the sip promoter was connected to the coding region of A3-hox1 and A3-hox2 and the fused fragments were introduced into NGW19-6 (A4), the transformants achieved more than 85% clamp formation and exhibited two nuclei per cell, similar to the dikaryon (NGW12-163 x NGW19-6). The results of real-time reverse transcription-PCR confirmed that sip promoter activity is greater than that of the native promoter of homeodomain protein genes in P. microspora. Thus, we concluded that nearly 100% clamp formation requires high expression levels of homeodomain protein genes and that altered expression of the A-mating-type genes alone is sufficient to drive true clamp formation.

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.

Rapid species identification of cooked poisonous mushrooms by using real-time PCR.

Species-specific identification of the major cooked and fresh poisonous mushrooms in Japan was performed using a real-time PCR system. Specific fluorescence signals were detected, and no nonspecific signals were detected. Therefore, we succeeded in developing a species-specific test for the identification of poisonous mushrooms within 1.5 h.