Induced cytological aberrancies in basidiocarp morphology of Schizophyllum commune Fr.

Summary Compatible strains of Schizophyllum commune Fr. were grown on a minimal medium with adjustments in the CO₂ level and the carbon source. Acetate with glucose initiated reduced stipes and pilei of fruit-bodies, and changed lamellar cell structure. Sealed growth chambers increased CO₂ levels and inhibited basidiocarp formation while growth of dense hyphal aggregates in the dikaryotic mycelium was stimulated.     

Direct microscopic studies of clamp connection formation in growing hyphae of Schizophyllum commune

Summary Photomicroscopic studies of clamp connection formation were collated with microscopic measurements of apical extension and mitosis in rapidly growing dikaryotic hyphae of Schizophyllum commune. Intercalary clamp connection formation was described in sub-terminal regions of the dikaryon. Conventional (i.e., rearward) clamp initiation was compared to forward clamp connection formation. Primary branch emergence was observed from clamp connections in growing hyphae and contrasted to sub-basidial branching in the hymenium of dikaryotic fruit-bodies.     

Fruiting ofLyophyllum tylicolor in plate culture on Soytoneglucose agar and urea-treated soil extract agar

Lyophyllum tylicolor, which forms mycelial basidia (and basidiospores), produced fruit-bodies when cultivated at 20°C under continuous illumination of 400–700 lux on agar plates containing Bacto-Soytone and glucose or an extract from urea-treated soil. Under these conditions, mycelial basidia were also observed on the Soytone-glucose agar, but not on the soil extract agar. In darkness, fruit-bodies and mycelial basidia were not observed on either medium. In culture on the soil extract agar, fruit-body primordia were produced at the position of the margin of the colony when it was transferred from darkness to continuous light; stipes did not elongate under illumination of ca. 2000 lux; and mycelial basidia and basidiospores, but not fruit-bodies, developed when glucose concentration in the medium was as high as 1% (w/v).     

Bulleromyces genus novum (Tremellales), a teleomorph for Bullera alba,and the occurrence of mating in Bullera variabilis

Mating is observed in Bullera alba and B. variabilis, resulting in the formation of dikaryotic mycelium with clamps, haustorial branches, and lateral and terminal dikaryotic, clavate, lageniform or subglobose cells. These cells develop in B. alba into tremellaceous phragmobasidia. Karyogamy has been observed in young non-divided basidia. Germination of the phragmobasidia occurred by acropetal chains of yeast cells, ballistospores or hyphae. Septal pores are dolipores with parenthesomes made up of U-shaped vesicles (Tremellales type). For the teleomorph of B. alba a new genus, Bulleromyces, is proposed, with only one species, viz. Bulleromyces albus.     

Cultivation characteristics ofIsaria japonica

Cultivation characteristics of fruit-body (synnema) formation ofIsaria japonica were examined using liquid and solid media in order to produce fruit-bodies on a large scale. Mycelia grew well at 18–28°C on PDA medium with an initial pH of 7.0. The formation of fruit-bodies ofI. japonica was induced by lowering temperature to below 20°C in PD liquid medium. In sawdust-rice bran basal medium mixed with pupal powder prepared from silkworms (Bombyx mori), the fresh weight of fruit-bodies increased with increasing content of pupal powder. The highest yields of fruit-bodies were obtained in carbon-rich barley grain medium supplemented with pupal powder. The fruit-bodies grown under CO₂ concentrations of 1,000 μl/L had coral-like, many-branched synnemata with numerous conidiospores, whereas those formed under high concentrations (9,000 μl/L) of CO₂ had unbranched and longer synnemata. High concentrations of CO₂ remarkably inhibited conidiospore formation on synnemata. Continuous high-intensity illumination at 2.93 W·m⁻² inhibited the elongation of synnemata, and low-intensity illumination at 0.088 W·m⁻² slightly inhibited the branching of synnemata. Fruit-bodies were produced on the pupa metamorphosed from living larvae ofAgrotis fucosa placed on the surface of a culture ofI. japonica incubated in sawdust-rice bran medium.     

Isolation and characterization of compatible diploids of Schizophyllum commune.

Summary Common-AB diploids with several heterozygous biochemical markers were mated with appropriately marked haploid strains of S. commune in an effort to obtain compatible, common-A, and common-B diploid progeny with biochemical markers identical to those of the common-AB parent. The spores from these crosses were germinated on minimal medium. Five compatible diploids, but no common-A or common-B diploids, marked as desired, were isolated by this method. Two possessed some dikaryotic cells and two had many dikaryotic cells. One of the latter was shown to have peculiar behaviour associated with one of its B mating-type factors.     

Mating type of isolates derived from the spermogonial state ofPuccinia coronata var.coronata

Hyphal confrontation between two haploid cultures originating from single basidiospores was used to determine the mating type ofPuccinia coronata var.coronata. Pairs of 15 single-basidiospore cultures were placed approximately 1 mm apart on the medium in all possible combinations. Hyphae of the pairs of colonies came into contact with each other in all combinations approximately two weeks after confrontations. When the nuclear number of hyphal cells in a contact zone was investigated one month after confrontation, monokaryotic hyphae were observed in selfing combination. On the other hand, dikaryotic hyphae were observed in 90.5% of crossing combinations between different cultures. Two isolates are considered compatible if dikaryotic hyphae are present in the contact zone but incompatible if they are absent. The mating type of the fungus was found to be characterized by multiple-allelomorphic tetrapolar incompatibility controlled by the “A” and “B” incompatible factors. Contribution No. 116, Laboratories of Plant Pathology and Mycology, Institute of Agriculture and Forestry, University of Tsukuba     

Isolation of the <Emphasis Type="Italic">B</Emphasis> incompatibility factor mutants in <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis>

 B incompatibility factor mutants (Bmut) in Pleurotus ostreatus were recovered from common-B mating heterokaryons resulted from matings between wild-type monokaryons with different A but the same B factors (A1B2 and A2B2) after NTG mutagenesis. The mutant monokaryons such as A1B2mut and A2B2mut were observed to have regularly uninucleated hyphal cells and to be compatible with each other. Matings between A1B2mut and A2B2mut monokaryons produced stable heterokaryons (A1B2mut + A2B2mut) that had binucleated hyphal cells with true clamp connections and formed normal fruit-bodies. Mating tests using basidiospore progeny from each of these heterokaryons revealed the bipolar mating pattern. Genetic analysis suggested that the mutation of B factor in P. ostreatus might occur in the B incompatibility factor genes. Received: August 3, 2001 / Accepted: January 18, 2002     

Cytolytic Activity of Naegleria fowleri Cell-free Extract1

ABSTRACT. The cytotoxic activity of a cell-free extract of Naegleria fowleri amebae on B103 rat nerve cells in culture was investigated. The cell-free extract was prepared by subjecting lysed amebae to centrifugation at 100,000 g for 1 h, precipitation of the supernatant fluid with 30–60% saturated ammonium sulfate, and desalting by group exclusion chromatography utilizing Sephadex G-25. The supernatant fluid recovered from this procedure was termed the soluble fraction. The Naegleria cytotoxic activity present in the soluble fraction was assayed by ⁵¹Cr released from labeled B103 cells. The Naegleria soluble fraction, when added to nerve cells, elicited blebs on the B103 target cell surface within 5 min after exposure to the fraction. Later, holes were observed in the B103 cell plasma membrane. These alterations were never observed on untreated B103 cells. Phospholipase A, phospholipase C, and protease activities were associated with the desalted ammonium sulfate-precipitable cytotoxic activity of N. fowleri cell-free lysate. The cytotoxic activity was impaired by ethylenediamine-tetraacetate (EDTA), phospholipase A inhibitor (Rosenthal's reagent), heating at 50°C for 15 min, or incubation at pH 10 for 60 min. Repeated freeze-thawing and inhibitors of proteolytic enzymes had no effect on the cytotoxic activity. Small amounts of ethanol (5% v/v) enhanced cytotoxic activity of the fraction. Phospholipases A and C, as well as other as yet unidentified cytolytic factors may be responsible for producing ⁵¹Cr release from target cells by the soluble fraction of N. fowleri extracts.     

Responses of Hebeloma radicosum fruit-bodies to light and gravity: negatively gravitropic and nonphototropic growth

Responses of the long-rooting agaric Hebeloma radicosum fruit-bodies to light and gravity were studied. In light from below or obliquely below, fruit-bodies grew straight downward with gills tilted and cap swollen and waved if they had emerged downward from the culture medium, or bent upward from the beginning if they had emerged obliquely downward. In light from above or obliquely above, they grew upward if they had emerged upward. Thus, they did not grow toward unilateral light from obliquely below or obliquely above, and hence their growth was nonphototropic and negatively gravitropic from the beginning of development. Even the straight downward growth seems to be latently negative-gravitropic. In the dark, fruit-bodies grew upward, forming pseudorhizas, but they remained immature; they matured only in the light. These characteristics may be related to the growth habits of the fungus colonizing deep in the ground, forming primordia there, and developing mature fruit-bodies on the ground. Received: March 26, 2001 / Accepted: July 12, 2001     

Comparison of Four Distinct Idiotypes of Monoclonal Antibodies against Hen’s Egg Ovomucoid

Two monoclonal antibodies (mAb 41B3 and mAb 51B3), directed against hen’s egg ovomucoid (OM) and different from those we previously reported (mAb 23E5 and mAb 32A8), were prepared and purified. A competitive radioimmunoassay using ¹²⁵I-labeled OM showed that both mAb 41B3 and mAb 51B3 could bind all three homologous domains (domains I (DI), II (DII) and III (Dili)) of OM and that they reacted most efficiently with Dill. To analyze the paratope specificities of these four mAbs, a sandwich assay and a competitive radioimmunoassay were done. Only the pair mAb 41B3 and mAb 51B3 could not simultaneously bind the OM or domains in the sandwich assay. Only mAb 41B3 inhibited the binding of mAb 51B3 to antigens and vice versa in the competitive radioimmunoassay. These results suggest that mAb 41B3 and mAb 51B3 recognized a closely related site distinct from the epitopes for mAb 23E5 or mAb 32A8. These mAbs may be useful for general studies on epitopes of protein antigens as well as for analyses of the antigenic determinants of OM.     

Cytopathic Action of Naegleria fowleri Amoebae on Rat Neuroblastoma Target Cells

The axenically cultured, weakly pathogenic Naegleria fowleri LEE and the highly pathogenic, mouse passaged N. fowleri LEEmp are cytopathic for B103 rat nerve cells in culture. Cytopathogenicity was measured by release of radiolabeled rubidium or radiolabeled chromium from B103 target cells. Cytopathogenicity was time-dependent for up to 18 h and dependent upon amoebae effector to nerve cell target ratios of less than 1:1. Release of⁵¹ Cr from B103 cells by either LEE or LEEmp amoebae was enhanced by addition of calcium or magnesium to medium free of these divalent cations but the ion-channel inhibitor, verapamil, or the ionophore A23187 and phorbol myristate acetate did not alter release of ⁵¹ Cr from B103 cells cocultured with the amoebae. Cycloheximide or actinomycin D impaired release of ⁵¹ Cr from B103 target cells injured by either LEE or LEEmp amoebae. Both strains of amoebae were fractionated by glass bead disruption and high speed centrifugation into membrane and soluble fractions. Each fraction was incubated with either ⁸⁶Rb or ⁵¹ Cr labeled nerve cells. The membrane fraction from LEEmp was more active than the soluble fraction in facilitating rubidium and chromium release. In contrast, the soluble fraction from LEE was more active than the membrane fraction in facilitating rubidium release from radiolabeled target cells. The sequential release of ⁸⁶Rb and ⁵¹ Cr from target cells rather than the simultaneous release of the two isotopes indicates that target cell death is due to the release of ions followed later by the release of large macromolecules. The results indicate that N. fowleri amoebae injure nerve cells by two alternate mechanisms, trogocytosis or contact-dependent lysis.     

Economic Mass Cultivation of Paramecium tetraurelia on a 200-Liter Scale1

ABSTRACT. A new and inexpensive medium is described for axenic mass cultivation of Paramecium tetraurelia stock 51s and the double mutant pawn A/pawn B. Skim milk powder is the major carbon and nitrogen source in this medium. Growth characteristics (proliferation rate, final cell density, and cell size) are similar to those observed with other axenic culture media. Cultures were run in one-liter erlenmeyer flasks, a 20-liter, and a 250-liter airlift bioreactor. The yield of a large bioreactor is 750 g (wet wt.) Paramecium or 5 × 10⁹ cells. This easy, economical culture technique will greatly facilitate the use of Paramecium as a model organism for extensive biochemical studies.     

Agrobacterium tumefaciens-Mediated Transformation of the Vegetative Dikaryotic Mycelium of the Cultivated Mushroom Flammulina velutipes

Agrobacterium tumefaciens was used to transform the vegetative dikaryotic mycelium of Flammulina velutipes using a hygromycin B resistance gene as selectable marker. The gene coding for urogen III methyltransferase (cob) was introduced into F. velutipes dikaryotic cells. The resulting transformant cells generated a bright red fluorescence, indicating that cob is promising as a reporter gene in F. velutipes.     

The central role of septa in the basidiomycete Schizophyllum commune hyphal morphogenesis

The purpose of the present research was to observe in the filamentous basidiomycete Schizophyllum commune, the connection between the nuclear division and polymerization of the contractile actin ring with subsequent formation of septa in living hyphae. The filamentous actin was visualized using Lifeact-mCherry and the nuclei with EGFP tagged histone 2B (H2B). Time-lapse fluorescence microscopy confirmed that in monokaryotic and dikaryotic hyphae, the first signs of the contractile actin ring occur at the site of the nuclear division, in one to two minutes after division. At this stage, the telophase nuclei have moved tens of micrometers from the division site. The actin ring is replaced by the septum in six minutes. The apical cells treated with filamentous actin disrupting drug latrunculin A, had swollen tips but the cells were longer than in control samples due to the absence of the actin rings. The nuclear pairing and association with clamp cell development as well as the clamp cell fusion with the subapical cell was disrupted in latrunculin-treated dikaryotic hyphae, indicating that actin filaments are involved in these processes, also regulated by the A and B mating-type genes. This suggests that the actin cytoskeleton may indirectly be a target for mating-type genes.     

A New Method For Axenic Mass Cultivation of Paramecium Tetraurelia*

SYNOPSIS A method is described for the axenic mass cultivation of Paramecium tetraurelia strains 51s and 299s. the ciliate is grown in an enriched axenic medium developed by Soldo, Godoy & van Wagtendonk. Under continuous shaking on a rotary shaker, cultures were grown in one-liter Erlenmeyer flasks with 330 ml medium yield cell densities of 32,000 cell/ml and 20,000 cells/ml for strains 299s and 51s respectively. Doubling time is considerably shorter under these conditions than in the conventional static cultures. A 20-liter airlift bioreactor is described in detail which can be used successfully to otain up to 100 g wet weight of Paramecium in a single run; in this reactor the cell density reaches 38,000 cells/ml for strain 299s. and 23,000 cells/ml for 51s. This technic should facilitate the study of minor protein components of the ciliate.     

Antagonistic actions of Paucibacter aquatile B51 and its lasso peptide paucinodin toward cyanobacterial bloom-forming Microcystis aeruginosa PCC7806

Superior antagonistic activity against axenic Microcystis aeruginosa PCC7806 was observed with Paucibacter sp. B51 isolated from cyanobacterial bloom samples among 43 tested freshwater bacterial species. Complete genome sequencing, analyzing average nucleotide identity and digital DNA–DNA hybridization, designated the B51 strain as Paucibacter aquatile. Electron and fluorescence microscopic image analyses revealed the presence of the B51 strain in the vicinity of M. aeruginosa cells, which might provoke direct inhibition of the photosynthetic activity of the PCC7806 cells, leading to perturbation of cellular metabolisms and consequent cell death. Our speculation was supported by the findings that growth failure of the PCC7806 cells led to low pH conditions with fewer chlorophylls and down-regulation of photosystem genes (e.g., psbD and psaB) during their 48-h co-culture condition. Interestingly, the concentrated ethyl acetate extracts obtained from B51-grown supernatant exhibited a growth-inhibitory effect on PCC7806. The physical separation of both strains by a filter system led to no inhibitory activity of the B51 cells, suggesting that contact-mediated anti-cyanobacterial compounds might also be responsible for hampering the growth of the PCC7806 cells. Bioinformatic tools identified 12 gene clusters that possibly produce secondary metabolites, including a class II lasso peptide in the B51 genome. Further chemical analysis demonstrated anti-cyanobacterial activity from fractionated samples having a rubrivinodin-like lasso peptide, named paucinodin. Taken together, both contact-mediated inhibition of photosynthesis and the lasso peptide secretion of the B51 strain are responsible for the anti-cyanobacterial activity of P. aquatile B51.     

Isolation and regeneration of protoplasts from gills ofAgaricus bisporus

Protoplasts were isolated from fruit-bodies ofAgaricus bisporus, and highest yields were derived from basidia. When gill fragments were treated with a combination of Novozyme 234, chitinase, and cellulase Onozuka R-10, and with 0.35m KCl as the osmotic stabilizer, high yields (3–4×10⁷ protoplasts/g fresh wt gills) were obtained within 1 h of incubation. About 20–30% of protoplasts regenerated in a solid MMNC medium. Investigation of specific activities of glucose-6-phosphate dehydrogenase and mannitol dehydrogenase indicated a highly active pentose phosphate pathway and a good capacity for mannitol synthesis in protoplasts, as well as in other cells of fruit-bodies of the species. The simple and efficient procedure provides a new approach for further investigation of the mushroom, and possibly of other basidiomycetes with hemiangiocarpus, by use of protoplasts.     

Morphological and cytological aspects of oidium formation in a basidiomycete,Pholiota nameko

Pholiota nameko produced abundant oidia on aerial hyphae from monokaryotic and dikaryotic test stocks, but oidia were rare on submerged hyphae. The oidia from the former stocks had a layer of hydrophobic protein between the cell wall and the inner cell membrane which was absent in the oidia from the latter. The only remarkable differences in the morphological features of the oidia from monokaryotic and dikaryotic mycelia was the slightly larger size of the latter. Observation of various test stocks on slide cultures revealed that about 80% of oidia were produced from the secondary branched hypha, and about 20% from the terminal hyphal, cell of the main hypha. In the former, the secondary hyphae were segmented to form several oidium cells; in the latter, a single or several oidia were formed at the terminal end of the main hypha. Most oidia from monokaryons and dikaryons had only one haploid nucleus, while the remainders were multinucleate. Among the stocks tested, most oidia had a DNA content with a haploid amount at the G1 phase of the cell cycle, but a few contained twice that amount corresponding to the G2 phase     

Responses of lignicolous-agaric fruit-bodies to light and gravity: A study to overview the fruit-body development in hymenomycetes

Our previous study revealed that, when exposed to light from below, fruit-bodies of humus-borne agarics grow straight downward both in the ‘Coprinus Type’ and ’Non-Coprinus Type’ species and that, in the latter, gills tilt by gravitropism, caps swell and wave, and stipes twist. The present study revealed that fruit-bodies of some lignicolous agarics also grew straight downward. Among themFlammulina velutipes andHypsizygus marmoreus fruit-bodies showed almost the same behaviour as the ‘Non-Coprinus Type’, butPleurotus ostreatus (assumedPolyporus Type) fruit-bodies did not show the gill tilting and cap waving. Rather, it redifferentiated to produce new gills or new fruit-bodies, possibly by gravimorphogenesis, or formed spiral stipes. Based on these results, graviresponses in hymenomycetes are overviewed.