草菇减数分裂和担孢子形成细胞核行为

本文用苏木精染色和双苯并咪唑(Hoechst 33258)染色法,从草菇子实体“纽期”菌褶分化完开始,每3小时对同一个子实体连续切取菌褶进行染色观察。结果表明草菇子实体“纽期”菌褶形成时,约10％的担子发生了核配;在子实体发育过程中,尤其是子实体成熟期后,不断有少量新的双核担子产生,并发生核配,使草菇减数分裂的同步性不高;草菇从菌褶分化完成(此时已有10％担子发生核配)到子实体完全成熟,菌褶变成深粉红至褐色(此时约70％担子完成减数分裂)需要28—30小时;担子减数分裂的持续时间为18小时,其中细线期和偶线期5.9小时、粗线期6.2小时、双线期和终变期3.4小时、中期10.5小时、后期Ⅰ到四分体2小时;经过对粗线期、双线和终变期以及中期Ⅰ染色体条数的多次反复观察,认为草菇的染色体条数为11(n=11);减数分裂后,4个子核分别进入4个担孢子中,留下无核的担子;绝大部分担孢子是单核的,有约5％的担孢子是双核的。     

土红粉盖鹅膏菌减数分裂及核相变化的观察

用双苯并咪唑（Ｈｏｅｃｈｓｔ ３３２５８）染色法对不同大小的土红粉盖鹅膏菌子０实体切取菌褶进行观察。结果表明：土红盖鹅膏菌最初形成单倍性双核原担子,单倍性双核原担子发生核配,形成二倍性单核原担子,二倍性单核原担子细胞核发生染色体复制,经减数分裂过程形成４个染色体减半的单倍性子核,这４个子核分别进入形成的４个担孢子中,留下无核的空担子;在减数分裂过程中,出现明显的土星环式细胞;在担孢子中子核又发生有     

糙皮侧耳染色体计数和担孢子形成过程中细胞核行为的研究

糙皮侧耳(Pleurotus ostreatus)菌褶经有丝分裂阻断剂预处理、原生质体铺片和Gie-msa 染色并结合苏木精染色后,经过多次反复实验观察,证明糙皮侧耳的染色体条数为 9(n=9);糙皮侧耳从菌褶分化完成到子实体完全成熟的过程中,不断有少量新的双核担子产生,发生核配直到释放担孢子。其减数分裂同步性不高。减数分裂后,4个子核分别进入4个担孢子中,留下中空的担子。     

Modifications du rythme de fructification de Coprinus congregatus par le dépôt de carpophores mûrs exogènes à la surface des cultures

Changes in Fruiting Rhythm of Coprinus congregatus by Addition of Exogenous Mature Sporophores on the Surface of Cultures. Researches were performed over the regulating effect of mature sporophores on the fruiting rhythm of Coprinus congregatus Bull. ex Fr. Placing exogenous mature fruit-bodies on the vegetative mycelium before the accomplishment of the first flush gives an abundant and perfectly synchronized fructification for this flush. But the fruiting rhythm is then disturbed: compared to control cultures, less sporophores are produced in the following flushes, and, in most cases, the cultures are finally characterized by fewer flushes than the controls. The nature of the substance(s) hypothetically produced by mature sporophores, which may regulate the production of other fruit-bodies, and, correlatively, the fruiting rhythm, is as yet unknown.     

Karyological evidence for meiosis in the three different types of life cycles existing in Agaricus bisporus

In Agaricus bisporus all cytological studies performed until now concerned the pseudohomothallic and bisporic var. bisporus. In the past 12 y two tetrasporic varieties have been described, the heterothallic var. burnettii and the homothallic var. eurotetrasporus. Our aim was to compare the behavior of the nuclei in the vegetative and reproductive cells of the three varieties with light microscopy (Feulgen and DAPI staining) and transmission electron microscopy. Most of the vegetative cells contained 3-5 nuclei in the three varieties. Nuclear migrations through the septum were detected. In the basidia relative locations of nuclei and vacuoles, meiotic spindle alignments, relative content of nuclear DNA and synaptonemal complexes were measured or observed. From the observation of numerous asynchronous second division of meiosis within basidia of var. bisporus and var. burnettii a new hypothesis emerges to explain the nonrandom distribution of the four meiotic products in the two spores of the bisporic basidia. Karyogamy and meiosis similarly occurred in the three varieties. In the case of A. bisporus var. eurotetrasporus this implies that the reproductive mode is sexual and therefore homothallic in the strict sense. The three different types of life cycles are described.     

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。     

Sporophore production ofAgaricus bisporus in aseptic environments

Production of mature sporophores ofAgaricus bisporus was achieved for the first time in amended, autoclaved soil, gamma-sterilized soil, and soil-extract agar medium. The initiation of sporophores was triggered by metabolites of soil-inhabiting bacteria, particularly nodule forming isolates. Whether a single metabolite or several metabolites of these bacteria caused formation of sporophores could not be established; however, biotin alone when added to soil extract medium produced comparable results. The potentiality of different bacteria to induce sporophore formation varied considerably within species and isolates.Amino acids favored vegetative growth ofA. bisporus, but failed to induce formation of sporophores. Organic acids supported luxuriant growth and poor sporophore formation. Among several growth-promoting substances and vitamins, biotin induced abundant formation of mature sporophores.The authors are thankful to Dr. C. Corke, Department of Soil Microbiology, University of Guelph, Ontario, for providing some bacterial cultures used in this study.     

The effect of partial flower removal on the relation betwen root, shootand fruti growth in the indeterminate tomato

The root, vegetative shoot and fruit growth of November and January sown glasshouse tomato plants grown in flowing water culture was followed over 6–7 months. The relationship between vegetative and reproductive growth was examined after two-thirds of the flowers were removed from half the experimental plants. This resulted in larger plants which had fewer, larger fruits and eventually a fruit yield almost as large as the controls. In the control plants, fruit growth increased steadily until it reached 90% of the total incremental fresh weight of the plant 50–60 days after first anthesis. Leaf growth was markedly depressed at this stage and root growth ceased 4 wk after anthesis. Some root death was observed from anthesis onwards. When fruit growth subsequently diminished, vegetative growth recovered but to a lower rate than before fruiting commenced. Following partial flower removal, only 64% or less of the total increment of fresh weight went into the fruit. Although vegetative growth at this stage was thus greater than in the control plants, both shoot and root growth followed the same qualitative pattern with time. The ratio of vegetative shoot to root fresh weight remained essentially constant throughout the fruiting phase in plants of both sowings whether flowers were removed or not. This suggests that the fruit grew in competition with the vegetative organs as a whole, although, for a short period at early fruiting, root growth was more seriously affected. The pattern and amount of fruiting in this indeterminate plant was influenced by the size of the vegetative organs at fruiting, and by the effect of the existing developing fruit on further vegetative and reproductive growth.     

Isolation and characterization of a sporeless mutant in Pleurotus eryngii

A sporeless mutant dikaryon, completely defective in sporulation, was isolated from mycelial protoplasts of Pleurotus eryngii mutagenized by UV irradiation. Newly established dikaryons between one component monokaryon from the mutant, and 12 different wild type monokaryons from 3 other wild type dikaryons, all exhibited the sporeless phenotype, whereas those between the other monokaryon and the same wild type monokaryons all produced normal fruiting bodies. These results indicated that the sporeless mutation was induced in one of two nuclei of the mutant and was dominant. In the wild type basidia, the pattern of nuclear behavior during sporulation corresponded to the pattern C nuclear behavior as defined by Duncan and Galbraith. Cytological observation revealed that in the sporeless mutant meiosis was blocked at the meta-anaphase I in most basidia and hence basidiospores and sterigmata were not produced. Although fruiting bodies of the sporeless mutant showed a somewhat leaning growth, their gross morphology and its fruiting body productivity were comparable to that of the original wild type strain. Based on these results, it was considered that the sporeless mutant could serve as a potential material in breeding of sporeless P. eryngii commercial strains.     

Developmental regulator Le.CDC5 of the mushroom Lentinula edodes: analyses of its amount in each of the stages of fruiting-body formation and its distribution in parts of the fruiting bodies

Immunoblot analysis of Le.CDC5 (842 amino acid residues), the expressed product of the cDNA of Le.cdc5 gene that has been previously reported to be most actively transcribed in primordia and small immature fruiting bodies of the basidiomycete Lentinula edodes, showed that the primordia, immature fruiting bodies and mature fruiting bodies contain similar amounts of Le.CDC5 protein. This indicates that the Le.CDC5 protein molecules synthesized in the beginning and early stage of fruiting-body formation remains in mycelial tissues even after small immature fruiting bodies developed and matured. Immunohistochemical analysis showed that Le.CDC5 is present everywhere in the mycelial tissues of immature fruiting body, but prehymenophore, the border between pileus and stipe, and the bottom of stipe seem likely to contain larger amounts of Le.CDC5. Within the hymenophore of mature fruiting body, the hymenium (in/on which a large number of basidia and basidiospores are formed) contains the Le.CDC5 most exclusively.     

Lignocellulolytic enzymes profile during growth and fruiting of Pleurotus ostreatus on wheat straw and tree leaves

Cultivation of two commercial Pleurotus ostreatus (oyster mushroom) strains was performed in plastic bags. Tree leaves appeared to be an excellent growth substrate for the conversion into fruiting bodies with biological efficiency of 108-118%. The level of enzyme activity was strongly regulated during the life cycle of mushrooms. However, despite the quantitative variations, each strain had a similar pattern of enzyme accumulation in fermentation of both substrates. Laccase and MnP activities were high during substrate colonization and declined rapidly during fruiting body development. On the contrary, in substrate colonization P. ostreatus expressed comparatively low activity of hydrolases. When primordia appeared, the activity of these enzymes sharply increased. Both cellulase and xylanase activity peaked at the mature fruiting body stage. When mushrooms shifted to the vegetative growth, the activity of ligninolytic enzymes again gradually increased, whereas the activity of hydrolases decreased.     

Gibberellin-like Substances in the Sporophores of Agaricus bisporus (Lange) Imbach

Sporophores of cultivated Agaricus bisporus (Lange) Imbach,were shown to contain a gibberellin-like substance active inthe dwarf maize (d-5), -amylase and other bioassays. Ethyl-acetateextraction followed by paper, column, and thin-layer chromatographyrevealed the presence of one major active substance. Ficin hydrolysisof dried sporophore powder, after the complete removal of freesubstances, released more gibberellin-like substances, one ofwhich appeared identical to the free compound. The free substance was predominantly in the lamellae and residualpileus tissue. The major active substance released by ficinoccurred mostly in the lamellae but also in substantial equalamounts in both stipes and pilei. No activity was found in extractsof dikaryotic vegetative mycelium on malt agar. The level ofactivity in extracts from sporophores stored at – 20 °Cfell sharply after 7 d, and then remained constant over a periodof 6 weeks. The content of gibberellin-like substances in youngand old whole sporophores showed wide variation between experiments.In most cases young 2-d tissue had higher levels than old, 11-dtissue on a fresh-weight basis. Purified sporophore extractsand authentic gibberellins had no stimulating effect on growthof sporophores or of cultured vegetative mycelium. The inhibitorsof diterpene biosynthesis, CCC, and AMO-1618 induced a smallincrease in mycelial growth rate. Ethyl-acetate extraction ofhorse-straw compost prior to inoculation with Agaricus bisporusshowed the presence of gibberellin-like activity in significantamounts.     

Isolation and characterization of a sporeless mutant in <Emphasis Type="Italic">Pleurotus eryngii</Emphasis>

 A sporeless mutant dikaryon, completely defective in sporulation, was isolated from mycelial protoplasts of Pleurotus eryngii mutagenized by UV irradiation. Newly established dikaryons between one component monokaryon from the mutant, and 12 different wild type monokaryons from 3 other wild type dikaryons, all exhibited the sporeless phenotype, whereas those between the other monokaryon and the same wild type monokaryons all produced normal fruiting bodies. These results indicated that the sporeless mutation was induced in one of two nuclei of the mutant and was dominant. In the wild type basidia, the pattern of nuclear behavior during sporulation corresponded to the pattern C nuclear behavior as defined by Duncan and Galbraith. Cytological observation revealed that in the sporeless mutant meiosis was blocked at the meta-anaphase I in most basidia and hence basidiospores and sterigmata were not produced. Although fruiting bodies of the sporeless mutant showed a somewhat leaning growth, their gross morphology and its fruiting body productivity were comparable to that of the original wild type strain. Based on these results, it was considered that the sporeless mutant could serve as a potential material in breeding of sporeless P. eryngii commercial strains. Received: September 5, 2002 / Accepted: October 16, 2002 Acknowledgments We are grateful to Mrs. Motoe Masuda for her skillful technical assistance. Contribution no. 358 from the Tottori Mycological Institute Correspondence to:Y. Obatake     

Sensitivity of an Early Step in the Sporulation of Bacillus subtilis to Selective Inhibition by Ethidium Bromide

When a final concentration of 0.4 mug of ethidium bromide (EB) per ml, which is subinhibitory to vegetative growth, is added to sporulating cells of Bacillus subtilis Marburg during either stage 0 or the early part of stage 1, morphogenesis is blocked. If the given concentration of EB is added after the early part of stage 1, sporogenesis is unaffected. The synthesis of the serine protease and antibiotic, which are believed to be associated with sporulation events during the early part of stage 0, are not inhibited by EB. Enhanced binding of [(14)C]benzylpenicillin to sporulating cells during septation (stage 2) is a measure of the presence of terminal enzymes for germ cell wall peptidoglycan synthesis. EB does not interfere with the binding of penicillin to sporulating cells, but penicillin remains more permanently bound to EB-treated postlogarithmic cells than to untreated sporulating cells. The absence of an interval of increased penicillin binding activity during stage 2 by sporulating cells treated with EB indicates that EB blocks sporulation prior to the completion of the germ cell wall.     

High genetic diversity in a population of the ectomycorrhizal basidiomycete laccaria amethystina in a 150-year-old beech forest

The genetic structure of a population of the ectomycorrhizal basidiomycete Laccaria amethystina (Bolt. ex Hooker) Murr. was assessed in a closed 150-year-old beech (Fagus sylvatica L.) forest in the Vosges Mountains in northeastern France. During the autumn of 1994 and 1997, sporophores were collected from three 100-m2 sampling plots located along a 120-m transect crossing the beech stand. The genetic variation of 676 sporophores was initially estimated using heteroduplex analysis of the ribosomal DNA intergenic spacer (IGS1). Ten unique IGS1 heteroduplex/homoduplex patterns were identified, although three types represented most of the sporophores analysed. Each group of IGS1 type was then analysed using random amplified microsatellite analysis (RAMS). RAMS resolved 388 different genotypes amongst the 634 sporophores analysed from the three plots during the autumn of 1994 and 1997. Density as high as 130 genets per 100 m2 was observed during the autumn of 1994. The largest clone covered approximately 1 m2, but most genets covered a few cm2 and produced only one to three sporophores. Only eight genotypes identified in 1994 were found in 1997. Although L. amethystina has the capacity for vegetative persistence, the present study indicates that its populations maintain a genetic structure more consistent with a high frequency of sexual reproduction. This suggests that beech trees could be recolonized by new genotypes each year. Alternatively, this spatial distribution may also arise from erratic fruiting of underground persistent genets. These features (i.e. numerous genets of small size), typical of ruderal species, contrast with studies carried out on other ectomycorrhizal basidiomycetes occurring in mature closed forests.     

Dielectric characterization of forespores isolated from Bacillus megaterium ATCC 19213

Isolated stage III forespores of Bacillus megaterium ATCC 19213 in aqueous suspensions were nearly as dehydrated as mature spores, as indicated by low dextran-impermeable volumes of ca. 3.0 ml per g (dry weight) of cells compared with values of ca. 2.6 for mature spores and 7.3 for vegetative cells. The forespores lacked dipicolinate, had only minimal levels of calcium, magnesium, manganese, potassium, and sodium, and were more heat sensitive than vegetative cells. The effective homogeneous conductivities and dielectric constants measured over a frequency range of 1 to 200 MHz indicated that the inherent conductivities of the forespores were unusually low, in keeping with their low mineral contents, but that the forespores could be invaded by environmental ions which could penetrate dielectrically effective membranes. Overall, our findings support the view that the dehydration of a forespore during stage III of sporogenesis may be the result of ion movements out of the forespore into the sporangium.     

Cultivation of mouse oocytes in vitro: the ability to resume meiosis

It is well established that fully grown oocytes from sexually mature mice are capable of resuming meiosis when cultured in vitro. However it has not been established at what stage during the growth the oocyte acquires the ability to mature. The present study was undertaken to determine the precise stage of growth at which the ability to resume meiosis is acquired in ICR strain mice. Oocytes of various sizes were isolated from the ovary by mechanical dissection. This method yielded about 30% growing and fully-grown oocytes from the ovary. Cumulus-free oocytes dissociated from the ovaries of 5-week-old mice were cultured in vitro in a chemically defined medium for 3 hr. None of the 61- to 65-microns-diameter oocytes resumed meiosis; however 81.8% of the 66- to 70-microns-diameter oocytes were able to resume meiosis. Fifty- to 60-microns-diameter oocytes recovered by enzymatic dispersal of the basement membrane of isolated growing follicles were unable to resume meiosis. Follicle-enclosed 50- to 60-microns-diameter oocytes did not resume meiosis even when cultured in a medium containing Ca-ionophore A 23187 or 8-bromoadenosine 3', 5'-cyclic monophosphate which are known to induce resumption of meiosis of oocytes in Graafian follicles. These results indicate that the ability to resume meiosis is acquired dramatically at a specific stage of oocyte growth.