Electron Micrograph Study of the Asci and Ascospores of Metschnikowia Kamienski

Internal and surface structures of asci and ascospores were studied by transmission electron microscopy (TEM) and by scanning electron microscopy (SEM) to establish the character and number of ascospores within the ascus of Metschnikowia krissii. Enzyme digestion with snail gut enzymes and SEM examination suggested the presence of a single ascospore enclosed in a thick sheath of epiplasmic materials. Two closely associated ascospores without an epiplasmic sheath were clearly distinguishable from asci of M. bicuspidata var. chathamia when similarly treated. Ultramicrotomy and TEM established conclusively that M. krissii produced a single ascospore per ascus. Neither SEM nor TEM revealed any morphological detail of the ascospores of taxonomic significance.     

Iodine as an Aid in Identification of Asci and Ascospores of Schizosaccharomyces octosporus

Transverse paraffin sections of mature greenwood stems of rose (Rosa x hybrida) and flowering dogwood (Cornus florida L.) were stained with Bismarck brown followed by azure B or tolnidine blue O. The Bismarck brown was replaced by thiazin dye metachromasia in all structures except the cuticle which remained brown or yellow. The interface between the cuticle and exterior cell walls of the epidermis was delineated clearly.     

Dissection of Saccharomyces Cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae exists in either a haploid or diploid state. The ability to combine alleles from two haploids and the ability to introduce modifications to the genome requires the production and dissection of asci. Asci production from haploid cells begins with the mating of two yeast haploid strains with compatible mating types to produce a diploid strain. This can be accomplished in a number of ways either on solid medium or in liquid. It is advantageous to select for the diploids in medium that selectively promotes their growth compared to either of the haploid strains. The diploids are then allowed to sporulate on nutrient-poor medium to form asci, a bundle of four haploid daughter cells resulting from meiotic reproduction of the diploid. A mixture of vegetative cells and asci is then treated with the enzyme zymolyase to digest away the membrane sac surrounding the ascospores of the asci. Using micromanipulation with a microneedle under a dissection microscope one can pick up individual asci and separate and relocate the four ascopores. Dissected asci are grown for several days and tested for the markers or alleles of interest by replica plating onto appropriate selective media.     

Amylolytic activity of Byssochlamys fulva

Byssochlamys fulva was found to produce a glucoamylase (EC 3.2.1.3) that exhibited its maximal activity at 50°C and had a broad optimum pH range of 4.0–5.2. The K_m and V_max values of the crude enzyme for amylopectin were 0.15% and 17.9 mg glucose l^-1 min-^-1, respectively. The molecular weight of the enzyme as estimated by the gel-filtration method was 34 kDa.     

Optimization of production and reaction conditions of polygalacturonase from Byssochlamys fulva

In the present study, the optimization of production and reaction conditions of polygalacturonase produced by a fungus Byssochlamys fulva MTCC 505 was achieved. The production of polygalacturonase with a considerable activity of 1.28 IU/ml was found when the culture was shaken at 30°C for 5 days in 100 ml of medium containing (w/v) 10 g/l pectin, 2 g/l NaNO?, 1 g/l KH?PO?, 0.5 g/l KCl, 0.5 g/l MgSO?. 7H?O, 0.001 g/l FeSO?. 7H?O, 0.001 g/l CaCl?. The best carbon and nitrogen source for this enzyme were pectin (1%) and Ca(NO?)? (0.1%), respectively. The enzyme gave maximum activity at incubation time of 72 h, temperature of 30°C and pH 4.5. During the optimization of reaction conditions, the enzyme showed maximum activity in sodium citrate buffer (50 mM) of pH 5.5 at 50°C reaction temperature for 15 minutes of incubation. The enzyme showed greater affinity for polygalacturonic acid as substrate (0.5%). Km and Vmax values were 0.15 mg/ml and 4.58 μmol/ml/min. The effect of various phenolics, thiols, protein inhibitors and metal ions on the enzyme activity was investigated. The enzyme was quite stable at 4°C and 30°C. At 40°C the half life of the enzyme was 6 h and at 60°C it was 2 h.     

Allergenic Implications of Airborne Leptosphaeria Ascospores

In order to investigate potential acroallergens of the Auckland region, a quantitative and qualitative study of the air spora was conducted at three different sites over the 12-month period, September 1979 to August 1980. Burkard volumetric spore traps were operated simultaneously at each of the three sites viz. Mt. Eden, Henderson and Waitakeres, located on a 20-km axis extending westward from Auckland City and encompassing residential, horticultural, agricultural, commercial and forested contexts. In the individual category of ascomycetous fungi, Leptosphaeria ascospores were recorded as an important component of air spora of the two non-forested sites. Ascospores concentration displayed a seasonal peak in late summer (February) and early autumn (March) and a diel periodicity with a distinct nocturnal maxima at all sites, confirming Leptosphaeria to be a component of the “rising air” or “damp-air spora”. The maximum concentration exceeded 4430 m^?3 of air around midnight (24/25 March, early autumn). A comparison of the results from the three sites showed that 61% of the total Leptosphaeria ascospores were trapped at Henderson (a satellite town in a rural setting) 29% at Mt. Eden (commercial/residential area) and 10% at Waitakeres (mainly forest site). Our data for Leptosphaeria ascospores combined with a high regional incidence of respiratory allergic diseases particularly bronchial asthma in late summer and autumn, indicate that a thorough investigation of the role of Leptosphaeria as a potential aeroallergen is warranted.     

Enzymatic synthesis of esters in organic medium with lipase from Byssochlamys fulva

Enzymatic esterification of sugars and fatty acids in tertiary butyl alcohol with lipase from Byssochlamys fulva NTG 9 was studied. Of different fatty acids examined, linoleic acid yielded the highest percentage of esterification of sugar (65.5%). Fructose gave a much higher percentage of esterification of fatty acid (71.3%) than glucose (47.8%), lactose (0%), maltose (67%) and sucrose (36.6%).     

Heat resistance of Byssochlamys ascospores.

Ascospores from 25 strains of Byssochlamys were studied for their ability to resist heat treatment in a standard defined medium. Seven of these were able to survive heating at 90 degrees C for 25 min or longer, when initial numbers were frequently near 10(6)/ml. Ascospores from five resistant strains suspended in the medium at pH 5.0 were usually more resistant than those at pH 3.6. Rapid heat inactivation occurred for one strain at pH 6.6. Nonlogarithmic heat death rate was observed in all strains tested.     

Heat resistance of Byssochlamys ascospores.

Ascospores from 25 strains of Byssochlamys were studied for their ability to resist heat treatment in a standard defined medium. Seven of these were able to survive heating at 90 degrees C for 25 min or longer, when initial numbers were frequently near 10(6)/ml. Ascospores from five resistant strains suspended in the medium at pH 5.0 were usually more resistant than those at pH 3.6. Rapid heat inactivation occurred for one strain at pH 6.6. Nonlogarithmic heat death rate was observed in all strains tested.     

Morphogenesis of Ascospores in Saccharomyces cerevisiae

Ultrastructural changes associated with ascospore formation in Saccharomyces cerevisiae were investigated by using freeze-etching and thin-sectioning techniques. The first nuclear division (meiosis I) is indicated by the appearance of spindle fibers within the nucleus. The nucleus subsequently elongates and eventually assumes a barbell shape; the second nuclear division (meiosis II) occurs before nuclear separation. The spindle fibers involved in meiosis II appear to be oriented perpendicular to those observed in meiosis I. A discrete bilaminar structure (forespore wall) progressively delineates each ascospore nucleus and encloses cytoplasmic material including mitochondria and endoplasmic reticulum. The forespores then elongate, close off, and become separated from the ascus cytoplasm by membranes. The ascospores assume a spherical shape as spore coat material is laid down; the latter stages of ascospore formation are characterized by thickening of the ascospore wall and disintegration of the ascus cytoplasm. No structures which could be identified as chromosomes were observed.     

Scanning Electron Microscopy of Ascospores of Schwanniomyces

Ascospores of the four recognized species of Schwanniomyces were examined by scanning electron microscopy. Spores of S. alluvius, S. castellii, and S. occidentalis, which were essentially identical, had abundant, long protuberances and wide, thin equatorial rings. The two known strains of S. persoonii differed from the other species as well as from each other. One strain had spores with a wide ring but only a few short protuberances; spores from the second strain were covered with craterlike depressions and had a narrow ring. Also examined were spores of Schwanniomyces hominis (=Saccharomyces rosei) which lacked a ring and were covered with short irregularly shaped protuberances, a finding consistent with the morphology of spores from other strains of S. rosei.     

Aryl Sulfatase in Ascospores of Neurospora crassa

Neurospora crassa ascospores normally do not contain aryl sulfatase even when formed under conditions of sulfur limitation. However, when one of the parental strains is the nonrepressible mutant scon(c), the resulting (mixed) ascospores contain significant levels of aryl sulfatase even when formed under conditions of sulfur abundance.     

Activity and Heat Stability of Trehalase from the Mycelium and Ascospores of Neurospora

Trehalases from the ascospores of Neurospora tetrasperma and the mycelium of N. crassa were compared. Enzymes from both sources have identical electrophoretic mobilities, K(m)'s, responses to pH, immunological reactions, and activities in low-molarity buffers. Because both enzymes are so similar, conclusions about the properties of the ascospore enzyme may, be made by studying mycelial trehalase. Mycelial trehalase is most active and stable in low-molarity buffers. The enzyme exists in at least three species; the smallest has a molecular weight between 105,000 and 125,000 and is predominant in low-molarity buffers at 37 C. The stability of trehalase to heating at 65 C can be increased by increasing enzyme concentration and by the addition of polyols. Ascospores contain large amounts of trehalose, which protects trehalase from heat inactivation at 65 C. The importance of this phenomenon in vivo and its relationship to the localization of trehalase in ascospores is discussed.     

Simple Method for the Separation of Ascospores

A simple method for the separation of ascospores is described. To isolate single spores from adhesive ascospores and the mycelium, the suspension was sucked through a combination of sintered-glass plates with different pore sizes.     

The ultra-structure of spores of Byssochlamys fulva

The ultra-structures of conidio- and ascospores of the ascomyceteByssochlamys fulva have been investigated in the electron microscope by means of ultrathin sections embedded in Vestopal and in Epon 812. The structure of conidiospores is completely different from that of ascospores. The most striking difference is the formation of extremely thick intermediate space between the cellwall and the cytoplasmic membrane of the ascospores. Within the intermediate space another cell-layer could be detected which might represent an additional protection for the cytoplasm against external influences.

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Zusammenfassung Die Ultrastrukturen der Konidio- und Askosporen der AskomyzetenByssochlamys fulva sind im Elektronmikroskop mittels ultradünner Schnitte, eingebettet in Vestopal und in Epon 812, untersucht worden. Die Struktur der Konidiosporen ist von denen der Askosporen völlig verschieden. Der auffallendste Unterschied ist die Bildung eines äußerst dicken Zwischenraumes zwischen der Zellwand und der zytoplasmatischen Membrane der Askosporen. Innerhalb des Zwischenraumes war eine andere Zellenschicht sichtbar, die wohl einen zusätzlichen Schutz des Zytoplasmas gegen äußere Einwirkung darstellen mag.

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This work was supported by grant FG-Austria-102 from the United States Department of Agriculture.     

Immunodetection of Venturia inaequalis Ascospores with Phage Antibodies

We describe the bacterial expression of single chain variable fragment (scFv) antibodies that bind specifically to the ascospores of Venturia inaequalis (Cooke). A scFv phage display library was prepared from the expressed V‐gene repertoire of a mouse immunized with whole V. inaequalis ascospores. Affinity selection was then carried out using intact, non‐germinated ascospores. The binding of selected phage antibodies was monitored by enzyme‐linked immunosorbent assay, flow cytometry and fluorescence microscopy. Several scFv antibodies were found to bind specifically to V. inaequalis ascospores. No cross‐reactivity was detected with spores from other phytopathogenic fungi, such as Plectosphaerella cucumerina, Cladosporium spp. and Alternaria brassicicola. Moreover, the scFvs did not bind to V. inaequalis conidiospores or mycelia. This is the first report describing the immunodetection of V. inaequalis ascospores by phage‐derived scFv antibody fragments. The degree of specificity of the antibodies is sufficiently high to allow rapid detection of ascospores within environmental probes such as those from particle samplers.