金针菇担孢子核相及遗传属性的研究

以3个不同的金针菇菌株为材料,研究了其担孢子的核相及遗传属性。荧光染色观察显示,担孢子核相以双核为主,双核孢子、单核孢子和无核孢子分别占80.2%、7.5%和12.3%。源于单孢分离物的菌丝为有隔膜、无锁状联合的多核菌丝。在交配试验中,源于不同菌株单孢分离物的菌丝原生质体的配对形成具锁状联合的菌落,而源于同一单孢分离物的菌丝原生质体的配对则形成无锁状联合的菌落,暗示担孢子中的两个核具有相同的交配型。RAPD分析显示,源于同一单孢分离物的菌丝原生质体为10个随机引物所扩增的图谱彼此完全相同,印证了担孢子中的双核是同质的。此外,观察表明,一个担子上着生有4个担孢子。因此,金针菇是一种具4个含同质双核担孢子的四极性蕈菌。     

Spore formation and karyological characterization of basidiosporogenesis,meiosis, post-meiotic and nuclear migration mitosis in Coprinus comatus

In Coprinus comatus, we sampled and observed different maturity stages of basidium to find the possible causes of binucleate basidiospores by microscopy using the HCI-Giemsa and DAPI staining methods. In basidium, following karyogamy, a single nucleolus was observed. We found evidence suggesting post-meiotic mitosis following nuclear migration in the spores. Post-meiotic mitosis occurred in the basidiospores, resulting in four binucleate basidiospores. This indicates that the basidiospore is a monokaryon, and that C. comatus shows the same pattern of basidiospore formation as Coprinopsis cinerea. This type of nuclear behavior was defined as pattern D, one of six distinct patterns (pattern A–F) of nuclear behavior during basidiosporogenesis have been described for basidiomycetes by Campos and Costa in 2010.     

95% of basidiospores fall within 1 m of the cap: a field-and modeling-based study

Plant establishment patterns suggest that ectomycorrhizal fungal (EMF) inoculant is not found ubiquitously. The role of animal vectors dispersing viable EMF spores is well documented. Here we investigate the role of wind in basidiospore dispersal for six EMF species, Inocybe lacera, Laccaria laccata, Lactarius rufus, Suillus brevipes, Suillus tomentosus and Thelephora americana. Basidiospores adhered to microscope slides placed on three 60 cm transects radiating from sporocarps. Morphological characteristics of species as well as average basidiospore volume were recorded. Number of basidiospores was quantified at specific distances to produce actual dispersal gradients. We found a negative exponential decay model using characteristics for each species fit the field data well. The 95% modeled downwind dispersal distance of basidiospores was calculated for each species. The 95% modeled downwind dispersal distance increased with increasing cap height and decreasing basidiospore volume for the species sampled, with 95% of basidiospores predicted to fall within 58 cm of the cap. Differences in anatomical characteristics of EMF species influence how far basidiospores are dispersed by wind. We discuss the role of wind dispersal leading to patterns of EMF establishment during primary succession.     

The number of nuclei in basidiospores of 63 species of ectomycorrhizal Homobasidiomycetes

The production of even a limited number of heterokaryotic spores would be advantageous for establishing new individuals after long distance dispersal. While Suillus and Laccaria species are known to produce binucleate, heterokaryotic spores, this condition is poorly studied for most ectomycorrhizal fungi. To begin addressing this matter the number of nuclei in basidiospores was recorded from 142 sporocarps in 63 species and 20 genera of ectomycorrhizal (EM) fungi. The mean proportion of binucleate basidiospores produced by sporocarps within a species ranged from 0.00 to 1.00, with most genera within a family showing similar patterns. Basidiospores from fungi in Amanita, Cortinariaceae and Laccaria were primarily binucleate but were likely still homokaryotic. Basidiospores from fungi in Boletaceae, Cantharellus, Rhizopogonaceae, Russulaceae, Thelephorales and Tricholoma were primarily uninucleate, but binucleate basidiospores were observed in many genera and in high levels in Boletus. Further research is needed to relate basidiospore nuclear number to reproductive potential in ectomycorrhizal species.     

Ultrastructure of developing basidiospores in <Emphasis Type="Italic">Rhizopogon roseolus</Emphasis> (= <Emphasis Type="Italic">R. rubescens</Emphasis>)

The ultrastructure of developing basidiospores in Rhizopogon roseolus is described. When viewed in the fruiting body chamber using scanning electron microscopy, basidiospores appear narrowly ellipsoid and have smooth walls. Eight basidiospores are usually produced on the apex of each sterigma on the basidium. Transmission electron micrographs showed that basidiospores formed by movement of cytoplasm (including the nuclei) via the sterigmata, and then each basidiospore eventually became separated from its sterigma by an electron-lucent septum. The sterigma and basidium subsequently collapsed, resulting in spore release. Freshly released spores retained the sterigmal appendage connected to the collapsed basidium. After spore release, the major ultrastructural changes in the spore concerned the lipid bodies and the spore wall. During maturation, lipid bodies formed and then expanded. Before release, the spore wall was homogeneous and electronlucent, but after release the spore wall comprised two distinct layers with electron-dense depositions at the inner wall, and the dense depositions formed an electron-dense third layer. The mature spore wall complex comprised at least four distinct layers: the outer electron-lucent thin double layers, the mottled electron-dense third layer, and the electron-lucent fourth layer in which electron-lucent granular substances were dispersed.     

长根小奥德蘑双孢菌株与四孢菌株核相变化的比较

用双苯并咪唑（Hoechst 33258）染色法分别对长根小奥德蘑Oudemansiella radicata双孢菌株和四孢菌株的菌丝、子实体、担孢子进行染色观察,结果表明：双孢长根小奥德蘑菌丝细胞多为单核,无锁状联合;原担子中单核进行一次有丝分裂形成两个横向或纵向排列的子核,这2个子核分别进入2个担孢子中,留下无核的空担子;成熟担孢子具有一个核。四孢长根小奥德蘑菌丝细胞大多数为双核,具有锁状联合;进入原担子中的两个单倍性细胞核先发生核配,形成一个二倍性的核,再经过减数分裂形成四个染色体减半的单倍性子核,     

食用外共生菌根真菌兰茂牛肝菌Lanmaoa asiatica纯培养条件下不同发育时期的核相

食用外共生菌根菌是一类可食用并与植物共生的大型真菌,其在纯培养条件下菌丝生长缓慢,不会扭结发育成原基,不能完成生活史。因此关于食用菌根菌纯培养条件下生活史的研究报道极少。本研究的兰茂牛肝菌Lanmaoa asiatica已能在纯培养条件下诱导出原基,这使生活史的研究得以完成。利用扫描电子显微镜、透射电子显微镜及荧光显微镜对兰茂牛肝菌子实体担子、担孢子、纯培养菌丝和原基的核相进行了观察。结果表明,子实体菌丝细胞为双核,担子经过减数分裂形成4个单核担孢子;担孢子萌发时长轴端长出芽状突起伸长为菌丝,原孢子中的细胞核不分裂,直接进入菌丝,形成单核初生菌丝;初生菌丝5d后变为双核菌丝;纯培养菌丝及原基细胞均为双核,其菌丝表面光滑,未观察到锁状联合。     

Seasonal and Diurnal Variation of Airborne Basidiomycete Spore Concentrations in Mexico City

Seasonal and diurnal changes in concentrations of airborne basidiomycete spores (basidiospores, rusts, smuts) were studied, using Burkard volumetric spore traps, in two areas of Mexico City with different degrees of urbanization and related to changes in climatic variables through 1991. Basidiomycete spores formed a large component of the total airborne fungal spore load in the atmosphere of Mexico City. They were the second most abundant spore type after Deuteromycotina (Hyphomycetes), forming 32% of the total fungal spores trapped in an urban-residential area and 28% in an urban-commercial area. The most abundant basidiomycete spores were basidiospores although smut-type spores were trapped on more days than basidiospores and rusts on fewer. Basidiospores occurred in concentrations up to 2,000 spores m-3 in the urban-residential area. Basidiospores showed a marked seasonal distribution, especially in the southern area, with their greatest abundance during the wet season. The correlation coefficients associated with regressions between basidiospore concentration and some environmental factors were increased when a lag period of 2 to 4 days was used between environmental measurements and the day of spore collection. Basidiospore concentrations exceeded the 75 percentile concentration (>400 spores m^-3) most often when rainfall was up to 6 mm and relative humidity was >70%. Basidiospores showed a diurnal periodicity with greatest concentrations in the early morning. The most common basidiospore type was Coprinus which formed 67% of basidiospores trapped in the southern area and 63% in the central area. Smut spores were trapped on 87% of days through the year while rust spores occurred in only 35%. Both rusts and smuts were present in only small concentrations.     

Electron microscope studies on Tomentella II. Hyphal anastomosis and reproductive organs

Light and electron microscope observations were made on the hyphal anastomosis, basidia and basidiospores ofTomentella bombycina andT. fuscoferruginosa. Three different types of anastomosis were observed and the presence of nuclei at the anastomosis area was seen in most cases. The probasidial cell appeared tetranucleate and nuclear migration to the sterigmata took place when basidiospore genesis was initiated. The basidiospores showed from 1 to 4 nuclei, but the nuclear identification on the electron microscope was difficult in mature basidiospores.T. fuscoferruginosa basidiospore cell wall appeared to be formed by one single layer, while theT. bombycina basidiospore wall showed three well-delimited layers with different electron density.

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Zusammenfassung Licht- und elektronmikroskopische Beobachtungen sind an der Hyphalanastomose, Basidia und Basidiosporen vonTomentella bombycina undT. fuscoferruginosa gemacht worden. Drei verschiedene Typen von Anastomose sind festgestellt worden und die Gegenwart der Kerne war in der Fläche der Anastomose in den meisten Fällen sichtbar. Die probasidiale Zelle erschien vierkernig und eine Kernwanderung zu den Sterigmata fand statt, wenn Basidiosporengenese eingeleitet war. Die Basidiosporen zeigten 1 bis 4 Sporen, aber die Identifikation der Kerne war durch Elektronmikroskopie in reifen Basidiosporen schwierig.T. fuscoferruginosa formed die Zellwand der Basidiospore durch eine einzige Schicht, während die Zellwand der Basidiospore vonT. bombycina drei gut begrenzte Schichten mit verschiedenen Elektrondichtigkeit zeigte.

     

担孢子介导传播对香菇双分体病毒LePV1群体形成的影响

【背景】LePV1为中国香菇种质资源中携带的主要病毒之一。在前期研究中,根据分子序列特征将LePV1带毒菌株群体分为两个分子类群(亚型I和亚型II),亚型I包含大部分带毒香菇菌株,而亚型II仅包含少数几个供试带毒香菇菌株,在地理上相距遥远的不同遗传背景香菇菌株中发现了同一LePV1分子类型。【目的】探究担孢子介导传播对香菇双分体病毒LePV1群体形成的影响。【方法】比较不同亚型菌株的有性担孢子后代带毒率差异,并采用单单杂交和单双杂交方式分析杂交对LePV1群体形成的影响等。【结果】亚型I中栽培菌株ZP51和野生菌株YS94的担孢子带毒率分别为70%和100%,亚型II中栽培菌株ZP28和野生菌株YS5的担孢子带毒率分别为45%和55%,暗示亚型I菌株担孢子传毒效率高于亚型II;当杂交配对的任一亲本携带LePV1时,无论是单单杂交还是单双杂交,获得的杂交子带毒率为100%。【结论】不同亚型担孢子病毒携带率的不同和杂交在香菇双分体病毒LePV1群体形成中可能发挥着重要作用;此外,LePV1不能在杂交不亲和的单核体之间传播,也不能在不亲和的单双杂交配对中进行传播;在杂交可亲和的单双杂交配对中,杂交成功的杂交子在与亲本双核体菌丝接触一段时间后,可以将病毒LePV1通过胞质传播传给亲本双核体菌丝体。该研究为明确香菇双分体病毒LePV1传播规律及LePV1群体形成机制提供了线索。     

Histochemistry and storage of organic compounds during basidiosporogenesis in the ectomycorrhizal fungus <Emphasis Type="Italic">Pisolithus microcarpus</Emphasis>

Knowledge on the distribution and storage of different organic compounds during basidiosporogenesis in P. microcarpus is paramount to a better understanding of basidiospore recalcitrance to germination. The objective of this work was to detect the presence and distribution of phenolics, reducing sugars, starch, glycogen, total polysaccharides, RNA, and proteins during P. microcarpus basidiosporogenesis. Starch and reducing sugars were not detected in the fungal basidiocarps, while other polysaccharides predominated in the extracellular matrix at the base of the basidiocarp containing unconsolidated peridioles. Phenolics were also detected in this region. Glycogen was present inside the hyphae, basidia, and basidiospores and constitutes an important storage compound in the fungal basidiocarps. In mature basidiospores, RNA accumulation occurred at discrete locations in the cytoplasmatic periphery, while polysaccharides and proteins were shown to predominate in the cell wall. The presence of glycogen, RNA, and proteins inside the basidiospores strongly indicates provision for future germination and suggests that other factors may also influence basidiospore recalcitrance.     

Isolation of a homothallic mutant in <Emphasis Type="Italic">Lentinula edodes</Emphasis>

To obtain a homothallic mutant in Lentinula edodes, basidiospores derived from the common Bmut dikaryon (A1B1mut × A2B1mut) were treated with UV irradiation. Of a total of approximately 5000 monosporous cultures recovered, a single basidiospore isolate was found to produce the hyphae bearing clamp connections without mating. This mutant strain could form fruit bodies, and all its single basidiospore isolates developed into colonies with clamp connections. Such homothallic behaviors were transmitted from the mutant strain to the next generation. During the germination and following hyphal elongation in a single basidiospore of mutant strain, clamp connections were clearly detected in multicellular hyphae, which contained two nuclei in each cell. Their clamp connections were morphologically variable, viz., pseudo, abnormal, and true clamps. Amplified fragment length polymorphism (AFLP) profiles among the basidiospore isolates of mutant strain were identical, indicating that the mutant strain produced isogenic basidiospore progeny. Contribution no. 385 from the Tottori Mycological Institute     

Cytological studies of <Emphasis Type="Italic">Agaricus brasiliensis</Emphasis>

Agaricus brasiliensis is a medicinal mushroom native to Brazil. It was first identified as Agaricus blazei and its scientific name continues to be debated. We examined the cytology of different Brazilian commercial strains of A. brasiliensis and the nuclear behavior of strain CS1 during basidiospore development using fluorescent microscopy. All strains have multinucleate hyphae and no significant differences in nuclei numbers were observed between them. Basidia from A. brasiliensis strain CS1 are typically tetrasporics and produce binucleate basidiospores, demonstrating that a postmeiotic mitosis occurs during basidiospore development. This result suggests that A. brasiliensis is primarily a heterothallic species.     

Recovery of Sugars from Ionic Liquid Biomass Liquor by Solvent Extraction

The dissolution of biomass into ionic liquids (ILs) has been shown to be a promising alternative biomass pretreatment technology, facilitating faster breakdown of cellulose through the disruption of lignin and the decrystallization of cellulose. Both biological and chemical catalysis have been employed to enhance the conversion of IL-treated biomass polysaccharides into monomeric sugars. However, biomass-dissolving ILs, sugar monomers, and smaller carbohydrate oligomers are all soluble in water. This reduces the overall sugar content in the recovered solid biomass and complicates the recovery and recycle of the IL. Near-complete recovery of the IL and the holocellulose is essential for an IL-based pretreatment technology to be economically feasible. To address this, a solvent extraction technique, based on the chemical affinity of boronates such as phenylboronic acid and naphthalene-2-boronic acid for sugars, was applied to the extraction of glucose, xylose, and cellobiose from aqueous mixtures of 1-ethyl-3-methylimidazolium acetate. It was shown that boronate complexes could extract up to 90% of mono- and disaccharides from aqueous IL solutions, 100% IL systems, and hydrolysates of corn stover containing IL. The use of boronate complexes shows significant potential as a way to recover sugars at several stages in ionic liquid biomass pretreatment processes, delivering a concentrated solution of fermentable sugars, minimizing toxic byproducts, and facilitating ionic liquid cleanup and recycle.     

Cytology of teliospore germination and basidiospore formation inUromyces appendiculatus var.appendiculatus

Summary The cytology of teliospore germination and basidiospore formation inUromyces appendiculatus var.appendiculatus was characterized with light and fluoroscence microscopy. Meiosis of the diploid nucleus occurred in the metabasidium. The four haploid daughter nuclei migrated into the basidiospore initials where they underwent a post meiotic mitosis. Each basidiospore was delimited from the meatabasidium by a septum at the apex of the sterigma. Seventy-five percent of mature basidiospores were binucleate, 24.5% uninucleate, and 0.5% trinucleate. Mature, released basidiospores measured 16×9 m, were smooth-surfaced, and reniform to ovate-elliptical in shape.This study represents portion of a dissertation submitted by the senior author to the Faculty of Biology of the University of Constance in March, 1983, in partial fulfillment of the requirements for the degree of Doctor of Natural Sciences (Dr. rer. nat.).     

Nuclear Degeneration during Ballistospore Formation of Cronartium asclepiadeum (Uredinales)*

When basidia of Cronartium asclepiadeum (Uredinales) develop basidiospores, nuclei migrate from the basidial cells into the basidiospores. A mitotic nuclear division yields two nuclei in the basidiospore. One of these nuclei degenerates if the basidiospore develops a secondary ballistospore. Several stages in the degeneration of the nuclei can be recognized: (1) Condensation of chromatin, (2) separation of the nucleus into a portion containing the chromatin and a portion containing the nucleoplasm by an invagination of the nuclear envelope, (3) reduction of nuclear volume, (4) enveloping and (5) twisting of nuclear membranes around the degenerating nucleus, (6) homogenization of the chromatin with reduction of the nuclear envelope, and reduction of the enclosing membrane complex from a multilamellar structure to a single membrane layer, and (7) invagination or splitting off of the spiralled membranes. The nuclear behavior during early developmental stages of secondary spore formation is similar to that of budding basidiomycetous yeasts. Basidiomycetes producing ballistospores may possibly have arisen from those whose reproducing phase is yeast-like.     

Tilletia vankyi, a new species of reticulate-spored bunt fungus with non-conjugating basidiospores infecting species of Festuca and Lolium

A bunt fungus, exhibiting a spore germination pattern unique to known reticulate-spored species of Tilletia was found infecting plants in seed production fields of Festuca rubra ssp. rubra (red fescue) and F. rubra ssp. fallax (Chewing's fescue) in Oregon, and in seed lots of Lolium perenne (perennial ryegrass) from Australia and Germany. Teliospores germinated to form 20–40 uninucleate, non-conjugating basidiospores, and colonies derived from single basidiospores produced teliospores in culture. In inoculation studies using single basidiospore colonies, perennial ryegrass and L. perenne ssp. multiflorum (Italian or annual ryegrass) were infected. A phylogenetic analysis, based on ITS region rDNA, eukaryotic translation elongation factor 1 alpha, and the second largest subunit of RNA polymerase II demonstrated that the fescue and ryegrass bunts are conspecific, and distinct from known species of Tilletia.     

Basidiospore formation in monokaryotic fruiting bodies of a mutant strain of Coprinus macrorhizus

The process of basidiospore formation in a mutant strain Fis^c of Coprinus macrorhizus, a heterothallic species of Basidiomycete, which forms monokaryotic fruiting bodies was examined. A single nucleus in a young basidium divided mitotically and two daughter nuclei were fused subsequently. The fused nucleus then divided meiotically forming four basidiospores on a basidium. The typical chromosome behaviours in the first meiotic prophase were observed. Synaptonemal complexes were observed in a basidium at the first meiotic prophase. A continuous illumination of fruiting bodies was effective to arrest meiosis in monokaryotic fruiting bodies at the particular stage of meiotic division.     

桦纤孔菌有性生殖相关特征及交配型位点分析

对桦纤孔菌菌株MDJCBS88的显微形态、菌丝及担孢子核相进行了观察。采用棉籽壳培养基对担孢子萌发形成的菌株进行栽培试验,筛选出不形成子实体或子实体发育不完整的菌株,将这些菌株在平板上进行了亲和试验,分析桦纤孔菌的有性生殖方式;并基于基因组序列进行交配型基因克隆验证,分析桦纤孔菌的交配型位点结构。显微观察发现,桦纤孔菌菌丝没有锁状联合结构,菌丝细胞无核到多核;子实层担孢子可含0-4个不等的细胞核,不同时期弹射的担孢子含有的细胞核数量不同。桦纤孔菌担孢子萌发率极低,能萌发的担孢子多为早期弹射的担孢子;培养基也影响担孢子的萌发率,与PDA培养基和CYM培养基相比,桦木屑培养基最适合桦纤孔菌担孢子萌发,萌发率为4.55%。从担孢子萌发的96个菌株中获得了2个不结实菌株和9个结实不产孢菌株,占11.5%,这些菌株间亲和试验出现不同的表现特征,包括形成产孢子实体,产生菌丝纽结,相互融合和相互拮抗等现象,认为桦纤孔菌的有性生殖以次级同宗结合为主,并受交配型基因控制。交配型位点克隆测序后分析发现,桦纤孔菌交配型A位点共14 034 bp,含有一个MIP基因和两组HD1和HD2基因;交配型B位点包含3个疑似信息素受体基因和1个信息素前体编码基因。     

Development of Novel Ionic Liquid-Based Microemulsion Formulation for Dermal Delivery of 5-Fluorouracil

The present study was aimed at synthesizing an imidazole-based ionic liquid 1-butyl-3-methylimidazolium bromide (BMIMBr) and subsequent development of a novel ionic liquid-in-oil (IL/o) microemulsion (ME) system for dermal delivery of a poorly permeating drug 5-fluorouracil (5-FU). A significant enhancement in the solubility of 5-FU was observed in BMIMBr. IL/o MEs of 5-FU were prepared using isopropyl myristate, Tween 80/Span 20, and BMIMBr. Results of ex vivo skin permeation studies through mice skin indicated that the selected IL/o ME exhibited 4-fold enhancement in percent drug permeation as compared to aqueous solution, 2.3-fold as compared to hydrophilic ointment, and 1.6-fold greater permeation than water in oil (w/o) ME. The results of in vivo studies against dimethylbenz(a)anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mice skin carcinogenesis demonstrated that the IL/o ME could effectively treat skin cancer in 4 weeks. In addition, the side effects such as erythema and irritation associated with the conventional formulations were not observed. Histopathological studies showed that the use of IL/o ME caused no anatomic and pathological changes in the skin structure of mice. These studies suggest that the use of IL-based ME system can efficiently enhance the solubility and permeability of 5-FU and hence its therapeutic efficacy.