橘黄裸伞子实体个体发育研究

采用石蜡切片法对不同发育时期橘黄裸伞的原基或幼子实体进行个体发育研究。结果表明:橘黄裸伞原基的第一个形态分化是下部边缘菌丝平行排列进行垂直生长,形成菌柄;然后边缘菌丝迅速向外生长,外菌幕形成,菌盖原基生长,在菌盖原基下部观察到菌褶腔,菌褶腔上部连续排列着栅栏细胞;在菌褶形成过程中,囊状体在尖端上聚集,说明菌褶的生长点在与菌盖组织相连的基部而不是尖端;内菌幕由内菌幕原基、菌盖边缘平行向下生长的菌丝和菌柄上部边缘平行向外生长的菌丝共同发育形成,由于内菌幕与菌柄组织的同源性,菌环不易脱落。橘黄裸伞属于半被果型中的双菌幕发育型,发育顺序表明其为菌柄发育型。     

Gravitropism of the basidiomycete Flammulina velutipes: morphological and physiological aspects of the graviresponse

The fruiting body of the basidiomycete Flammulina velutipes shows a distinct negative gravitropic response. Maturing fruiting bodies in the rapid elongation phase become graviresponsive with basidiospore differentiation. Lateral gravistimulation by horizontal arrangement of the fruiting body results in unilateral growth regulation. Elongation in the upper Stipe side decreases to 40% during gravitropic reorientation of the fruiting body. Overshooting of the gravitropic response during reorientation is precisely regulated. The graviresponsiveness is concentrated to the apical area of the stipe, the transition zone between pileus and stipe, which features a prominent elongation capability. The small size and low vacuolization of the transition zone hyphae compared with differentiated basal stipe hyphae correspond with this physiological function on the light and electron microscopical levels. Curvature experiments using intact and explanted fruiting bodies demonstrated the graviperceptive role of the transition zone. The excision of various amounts of pilear tissue, even the disruption of the whole pileus, had no severe effect on gravitropic curvature, until the transition zone was damaged. Removal of the transition zone resulted in a dramatic loss of graviresponse, whereas the decrease of elongation was less drastic.     

Elongation growth and gravitropic curvature in the Flammulina velutipes (Agaricales) fruiting body

Differential elongation of stipe hyphae drives the gravitropic reorientation of Flammulina velutipes (Agaricales) fruiting bodies. The gravitropic curvature is strictly dependent on the presence of the transition zone between pileus and stipe. Elongation growth, providing the driving force for curvature, is also promoted by the pileus. Gravitropic curvature is successfully suppressed by clinostatic rotation, but the elongation rate is not affected. Explantation of fruiting body stipes lowers curvature and elongation rates corresponding to explant size reduction. In Flammulina, 25 mm length of transition zone explants is an efficient size for reproducible curvature and elongation during 48- to 72-h curvature tests. Submersion of specimens in aqueous medium causes cessation of the gravitropic curvature, but does not affect elongation. Thus the involvement of a diffusible factor in transmission of the curvature signal is probable. Splitting the fruiting body stipe in segments of 1/8 diameter does not suppress the gravitropic response, and the segments are individually reoriented to the vertical. It is concluded that the graviresponse of the Flammulina fruiting body is based on cellular perception of the gravistimulus and that a differential growth signal is transmitted in the stipe by a soluble factor that regulates hyphal elongation.     

Identification and expression analysis of a new glycoside hydrolase family 55 exo-β-1,3-glucanase-encoding gene in Volvariella volvacea suggests a role in fruiting body development

The edible straw mushroom Volvariella volvacea is an important crop in South East Asia and is predominantly harvested in the egg stage. Rapid stipe elongation and cap expansion result in a swift transition from the egg to elongation and maturation stage, which are subjected to fast senescence and deterioration. In other mushrooms, β-1,3-glucanases have been associated with degradation (softening) of the cell wall during stipe elongation and senescence. We present a new glycoside hydrolase family 55 (GH55) exo-β-1,3-glucanase gene, exg2, and highly conserved deduced EXG2 protein. The 3D model and presumed catalytic residues of V. volvacea EXG2 are identical to Lentinula edodes EXG2 and Phanerochaete chrysosporium Lam55A, supporting similar enzymatic functions. In addition to previous association to stipe elongation and senescence, our data clearly indicates a role for cap (pileus) expansion. Digital gene expression, quantitative PCR and isobaric tags for relative and absolute quantification analysis showed low exg2 and EXG2 levels in primordia, button, egg and elongation stages and significantly increased levels in the maturation stage. Subsequent relative quantitative PCR analysis designated expression of exg2 to the stipe in the elongation stage and to the pileus and stipe in the maturation stage. EXG2 cell wall softening activity, close correlation of exg2 expression with the principal expanding mushroom tissues and a strong conservation of expression patterns and protein sequences in other mushrooms, make V. volvacea exg2 an important candidate for future studies on mechanisms of fruiting body expansion and senescence causing commodity value loss.     

Influence of light on the morphological changes that take place during the development of the <Emphasis Type="Italic">Flammulina velutipes</Emphasis> fruit body

We show that fruit bodies of Flammulina velutipes can be induced in complete darkness after a sharp temperature reduction (23° to 16°C). However, the fruit bodies that form in complete darkness have a long stipe with an undeveloped pileus on the top (pinhead fruit bodies) and are thinner and whiter than the normal fruit bodies which are formed in the light. This finding suggests that F. velutipes fruit bodies cannot mature in complete darkness. However, when we irradiated the fruit bodies that had formed in complete darkness, a pileus developed immediately, and 4 days later the separation between the stipe and the pileus could be observed. Immediately after light exposure, the stipe also thickened and became increasingly pigmented. The stipe elongation was inhibited until 8 days after light exposure, although stipe elongation progressed very quickly thereafter. Basidospores were also visible in the gills 8 days after light exposure. We consider that the basidiospore development is involved in this rapid stipe elongation, which aids the effective dispersal of basidiospores.     

Stipe wall extension of Flammulina velutipes could be induced by an expansin-like protein from Helix aspersa

Expansin proteins extend plant cell walls by a hydrolysis-free process that disrupts hydrogen bonding between cell wall polysaccharides. However, it is unknown if this mechanism is operative in mushrooms. Herein we report that the native wall extension activity was located exclusively in the 10 mm apical region of 30 mm Flammulina velutipes stipes. The elongation growth was restricted also to the 9 mm apical region of the stipes where the elongation growth of the 1st millimetre was 40-fold greater than that of the 5th millimetre. Therefore, the wall extension activity represents elongation growth of the stipe. The low concentration of expansin-like protein in F. velutipes stipes prevented its isolation. However, we purified an expansin-like protein from snail stomach juice which reconstituted heat-inactivated stipe wall extension without hydrolytic activity. So the previous hypotheses that stipe wall extension was resulted from hydrolysis of wall polymers by enzymes or disruption of hydrogen bonding of wall polymers exclusively by turgor pressure are challenged. We suggest that stipe wall extension may be mediated by endogenous expansin-like proteins that facilitate cell wall polymer slippage by disrupting noncovalent bonding between glucan chains or chitin chains.     

黄鳞伞子实体发育

为了丰富大型真菌个体发育学研究数据,并依据个体发育学数据提出分类学建议,通过石蜡切片法对黄鳞伞子实体进行了发育学观察。结果显示:黄鳞伞子实体发育初期原基圆形至卵圆形,具外菌幕原基,内部菌丝弯曲致密,螺旋交织在一起呈束状,随着原基的发育,端向膨大生长,菌盖原基出现,与此同时原基基部在保持伸长生长外,直径也明显增大,菌柄原基由此形成。随后菌褶腔出现,栅栏状细胞的出现表明子实层发育的开始,发育过程中Y型菌褶清晰可见。黄鳞伞的发育属于菌盖菌柄型发育,发育类型是半被果双菌幕发育型。     

Protein expression during Flammulina velutipes fruiting body formation

Formation of the Flammulina velutipes fruiting body can be induced by lowering the ambient temperature (first treatment) in complete darkness. Fruiting bodies formed under these conditions elongate without pileus formation (pinhead fruiting body), suggesting that they cannot mature in complete darkness. However, after light treatment of the pinhead fruiting body (second treatment), a pileus develops immediately, and the stipe also thickens and becomes increasingly pigmented. The apical region swells as a result of cell division starting 2 days after light treatment, the pileus–stipe junction fracture and hymenium primordia form on day 4, and gills appear at day 6. Pf1 and Pf3 are specifically expressed after exposure to low temperature without light. The cell wall-associated protein [pileus-specific hydrophobin-like protein (PSH)] is specifically induced in the pileus, but not in the stipe, following the second light treatment to the pinhead fruiting body. These results suggest that Pf1 and Pf3 would be involved in fruiting body induction and that PSH would be involved in pileus formation. These phenomena will aid further histological and molecular biological investigations into the mechanisms behind fruiting body development in F. velutipes.     

Influences of environmental factors on fruiting body induction,development and maturation in mushroom-forming fungi

Mushroom-forming fungi (restricted to basidiomycetous fungi in this review) differentiate by sensing several environmental factors for fruiting body formation. For fruiting body induction, nutrient, temperature and light conditions are critical environmental factors. Higher nitrogen and carbon sources in the media will suppress fruiting body induction in many mushroom-forming fungi, with induction being triggered by lower nitrogen and carbon concentrations. Low temperature or temperature downshift is another critical influencing factor for fruiting body induction in many cultivated mushrooms, such as Flammulina velutipes, Lentinula edodes, and Volvariella volvacea. Fungal response toward starvation and cold involves the production of sexual spores as the next generation. Species like F. velutipes and Coprinopsis cinerea can form fruiting bodies in the dark; however, light accelerates fruiting body induction in some mushroom-forming fungi. Remarkably, fruiting bodies formed in the dark have tiny or no pileus on heads (called dark stipe, pinhead fruiting body, or etiolated stipe). Light is essential for pileus differentiation in many, but not all mushroom species; one exception is Agaricus bisporus. Mushrooms have positive phototropism and negative gravitropism for effective dispersal of spores. Carbon dioxide concentrations also affect fruiting body development; pileus differentiation is suppressed at a high concentration of carbon dioxide. Thus, the pileus differentiation system of mushrooms may allow the most effective diffusion of spores. Full expansion of the pileus is followed by pileus autolysis or senescence. In C. cinerea, pileus autolysis occurs during spore diffusion. Fruiting body senescence, browning of gill, and softening occur after harvesting in several mushroom species. Fruiting body induction, development, and maturation in mushroom-forming fungi are discussed in this review.     

Growth regulation inCoprinus radiatus

Some of the morphological and physiological parameters of stipe growth or elongation inCoprinus radiatus were investigated. During the development of the fruit body the number of cells in a row in the growing portion of the stipe doubled during the development of the button, and again during the phase of rapid stipe elongation. Also during the stage of rapid elongation the cells in the upper 2/3 of the stipe increased 6–8 fold in length. The existence of a growth regulator synthesized in the cap and exerting control over the stipe was demonstrated through decapitation experiments. The cap appears to be required for normal stipe development until the stipe reaches about 1/4 of its final length. Through decapitation and cap-stipe exchanges it was found that the cap produced growth regulator up to the time of autodigestion; however, the stipe responded to the regulator only during a brief period at the onset of elongation.     

Melanoderma boninense,a new species of Melanoderma (Polyporales,Agaricomycota) from the Bonin Islands in Japan

Melanoderma boninense is described and illustrated as a new species from the Bonin Islands, Japan, on the basis of morphological and phylogenetic investigations. This species is characterized by sessile basidiocarps with an ungulate pileus, a black crust on the pileus surface, pores 4–6/mm, a dimitic hyphal system comprising clamped generative hyphae and cyanophilous skeletal binding hyphae with or without a dextrinoid reaction, cylindrical basidiospores measuring 5.5–7.5 × 2–3 μm, and cystidioles on the sides of tubes and near the pore surface. Morphological examination of authentic specimens of other Melanoderma species revealed that the genus is variable in terms of the shape and size of cystidioles. A black crust on the pileus surface composed of palisade and highly agglutinated hyphae is a distinctive diagnostic morphological feature of Melanoderma that differentiates it from allied genera. Melanoderma boninense, which is currently known from a restricted area of the Bonin Islands, is potentially threatened by environmental reduction of the type locality due to the invasive tree Bischofia javanica.     

Hirticrusta gen. nov. segregated from Neofomitella in Polyporaceae (Polyporales)

Taxonomic studies including morphological observations and phylogenetic analyses were conducted on Japanese “uragin-take”, an unidentified species from Amazonia, Brazil and their allies. Phylogenetic analyses using ITS, nrLSU and RPB2 regions revealed that “uragin-take”, Neofomitella polyzonata and the unidentified species formed a monophyletic clade separate from the clade including the other four Neofomitella spp. and that “uragin-take” is conspecific with N. polyzonata. Morphological investigations on authentic specimens revealed that Polyporus subradiatus is a prior name for N. polyzonata. We propose Hirticrusta gen. nov. typified by H. subradiata segregated from Neofomitella, and we erected H. amazonica sp. nov. for the unidentified species. Hirticrusta is characterized by annual to biennial and sessile basidiocarps, semicircular to dimidiate pileus, velutinous to tomentose hairs on pileus surface, buff to brown context with a crustose layer indicated by a dark brown line forming a longitudinal section below the superficial hairs, a trimitic hyphal system, crustose layer composed of parallel and densely arranged brown hyphae and cylindrical basidiospores. The new species, H. amazonica is distinguishable from other polypores by downy and long tomentum on the pileus surface (up to 20 mm thick), brown context with a dark brown layer below the tomentum and round pores (5–7/mm).     

Zur Entwicklung und funktionellen Anatomie des Phylloids vonLaminaria hyperborea

The persistent thallus of the phaeophyceanLaminaria hyperborea produces annually a new phylloid, formed by a meristematic zone between stipe and old phylloid. The growing frond is nourished with assimilates from the old one. Young and old phylloid are linked by a collar. Frond and stipe are of very similar anatomical structure. The frond is coated by a one-layer-meristoderm, which forms the external cortex. Using the position of slime ducts as border, the parenchymatous cortex can be subdivided into outer and inner cortex. Between inner cortex and medulla numerous transitions exist. The medulla itself is mainly composed of longitudinally arranged trumpet cells and hyphae. Frond enlargement is caused by the activity of both meristoderm and cortex. Not only cell divisions but also elongation in medulla and inner cortex contribute to growth of the frond. The frond diameter is decreased by this elongation process. Corresponding stretching occurs in the network of slime ducts. Swelling of longitudinal primary walls, accompanied by incorporation of alginate, facilitates the separation of cell strands, and across the medulla hyphae and crosslinks of both inner-cortex-layer cells form. Cells of the inner cortex continously differentiate to elements of the medulla. Trumpet cells within the outer part of the medulla are often branched and connected by hyphae to parenchymatous cells of the inner cortex. Toward the central part of the medulla, trumpet cells elongate and finally attain a length of 1000 µm; they form thick secondary longitudinal walls with ringshaped thickenings, reducing the lumen diameter. Crosswalls and whole cells are often plugged with callose and have, apparently, ceased to translocate.     

金针菇外切-β-1,3-葡聚糖酶家族基因鉴定及在不同发育时期的差异表达

金针菇具有很高的营养与保健价值,菌柄长短决定金针菇的产量与品质,而菌柄伸长的相关作用酶及分子机理尚不清楚。前期草菇中发现外切-β-1,3-葡聚糖酶基因（exg2）可能与菌柄伸长相关,但在金针菇中尚没有exg基因的相关报道。本研究首先在金针菇全基因组中鉴定到3个外切-β-1,3-葡聚糖酶家族基因（分别命名为：Ffexg1、Ffexg2、Ffexg3）,并进行了克隆验证。进一步采用定量PCR对3个基因在金针菇不同发育时期及组织部位的差异性表达进行了分析。结果显示：Ffexg1只在菌柄中高表达,Ffexg2与Ffexg3在菌柄中表达量先上升后下降,在菌盖中呈逐渐上升趋势。3个Ffexg基因均在菌柄发生伸长的部位表达量较高,且在菇体水平放置后菌柄弯曲程度较大的部位表达量较高。结果显示金针菇exg家族3个基因存在时空差异性表达,在菌柄中伸长较快的时期及部位伴随着Ffexg基因的高表达。结合其功能预测,Ffexg家族基因可能作用于细胞壁成分β-1,3-葡聚糖链,从而在金针菇菌柄及菌盖发育中起作用。     

金针菇信息素信号通路基因在子实体发育过程中的差异表达

【背景】子实体是食用菌的主要商品部位,也是真菌生殖生长的重要结构,其发育受到多种信号途径的调控。【目的】以金针菇(Flammulina filiformis)为材料,对转录组和基因组数据的信息素信号通路基因进行分析获得差异表达的基因,并对其在菌丝生长和子实体发育过程中的表达情况进行分析,以期为研究食用菌子实体发育提供参考。【方法】基于已有的金针菇基因组数据,注释了金针菇信息素信号通路。进一步通过转录组测序鉴定了该通路中参与金针菇子实体发育的关键基因,并对关键基因进行荧光定量PCR验证。【结果】cdc24和ste12基因在子实体发育不同时期的5个样品(原基、伸长期菌柄、伸长期菌盖、成熟期菌柄和成熟期菌盖)中的表达具有显著差异,使用荧光定量PCR技术进行验证与上述结果一致。【结论】cdc24和ste12这2个关键基因可能参与了金针菇子实体发育过程中的组织分化调控机制。     

MADS-box转录因子编码基因Vvrin1在草菇不同发育时期的表达分析

草菇是我国土特产出口的主要种类之一,而采后易开伞问题限制了草菇的贮藏及运输。MADS-box转录因子对植物的成熟衰老和真菌的子实体发育起到重要的调控作用。目前尚未见草菇MADS-box转录因子的相关报道。本研究通过生物信息方法及分子生物学的手段对草菇的基因组、转录组数据进行分析,获得了草菇MADS-box转录因子基因Vvrin1。该基因全长1 392bp,含2个内含子,编码419个氨基酸残基。该转录因子含有一个MADS-box结构域,序列与双孢蘑菇Agaricus bisporus、斑玉蕈Hypsizygus marmoreus和灰盖鬼伞Coprinopsis cinerea的MADS-box转录因子相似性分别为71%、67%和61%。通过表达谱数据及荧光定量PCR分析表明Vvrin1基因在草菇子实体伸长期菌柄的表达量出现高峰,具有极显著性差异（P<0.01）,推测该转录因子参与调控草菇菌柄的伸长,菌盖的开伞。这些结果为草菇成熟衰老（特别是开伞）的调控研究提供数据支持。     

Mycena kentingensis,a new species of luminous mushroom in Taiwan,with reference to its culture method

Mycena kentingensis, a new luminous mushroom, was discovered in the tropical forest of Kenting National Park in Taiwan. The pileus of M. kentingensis is 3–8 mm in diameter and emits green light in the dark. The dorsal surface of the pileus is covered with short and white spines. The lamellae are nearly free. The stipe is puberulous with a white basal disc about 2 mm broad. Cheilocystidia are clavate to obpyriform, 16–40?×?4–8 μm, and have dense excrescences on the upper half or two-thirds of the cell. The pileus lacks cherocyte, and the stipe lacks caulocystidium. To further identify the species by the DNA barcode method, the internal transcribed spacer (ITS) DNA sequence of M. kentingensis was amplified by polymerase chain reaction, sequenced, and searched for in the GenBank. The hit with the maximum score was an uncultured fungus clone KFRI2121 (accession number HQ662846) with 88 % sequence identity. For those ITS sequences associated with Latin binominal names in the GenBank, the hit with the maximum score was Mycena stylobates with 87 % sequence identity. Phylogenetic analyses by Bayesian and maximum likelihood methods showed that M. kentingensis is close to M. stylobates and M. adscendens. To develop its culture method, mycelia of M. kentingensis were isolated. The optimal temperature for mycelial growth on potato-dextrose agar (PDA) medium was 24 °C. Commercial compost with the addition of 20 % rice bran and 50 % water was appropriate for fruit-body formation. The new species of Mycena kentingensis brings the total of luminous fungi in the world to 74 species.     

Two new species of <Emphasis Type="Italic">Coltricia</Emphasis> (Hymenochaetaceae,Basidiomycota) from southern China based on evidence from morphology and DNA sequence data

Two new species of Coltricia, C. austrosinensis and C. minima, are described from southern China on the basis of morphological characters and molecular evidence. Phylogenetic analysis based on the internal transcribed spacer (ITS) regions and nuclear large subunit (nLSU) ribosomal RNA gene regions indicated that the two new species were nested within the Coltricia clade in Hymenochaetales. Coltricia austrosinensis is characterized by centrally stipitate basidiocarps, lobed pileal margin, distinctly swollen stipe tip, cinnamon pore surface, large pores (1–3 per mm), and broadly ellipsoid basidiospores measuring 8–10?×?5.5–6.5 μm, with a distribution to date in subtropical China. Coltricia minima is characterized by tiny, centrally stipitate basidiocarps, entire pileal margin, uniform stipe, pileus bearing distinct concentric zones and pore surface dark greyish blue when fresh, small pores (3–4 per mm), narrow tramal hyphae (3.5–4 μm), and broadly ellipsoid to subglobose basidiospores measuring 6–7?×?4–5 μm, and occur in mixed tropical forests.