Experimental Mycology最新文献

Rivero, F., and Cerdá-Olmedo, E. 1994. Spore dormancy mutants of Phycomyces. Experimental Mycology 18, 221-229. The spores of the Zygomycete Phycomyces blakesleeanus are called dormant because few of them germinate when placed in a medium that sustains mycelial growth and development. Nearly all the spores germinate after activation, that is, exposure to heat or certain chemicals. We have looked for mutants whose spores would not need activation. Nine mutants formed authentic, but transient spores, which germinated spontaneously in the sporangium. Mutant mycelia had lower alcohol and aldehyde dehydrogenase activities and less glycogen than wild-type mycelia. The spontaneous germination and the metabolic alterations are attributed to the same recessive mutations. No differences were found between mutants and wild type in the cyclic AMP and fructose 2,6-bisphosphate concentrations in immature sporangia and the trehalase activity in the mycelia. In another mutant the spore primordia did not form spores, but remained viable for some time in the sporangium. The mutants were difficult to keep in the laboratory (except as lyophils); this stresses the importance of preventing spore germination in the sporangium.

Rodier, M.-H., El Moudni, B., Ghazali, M., Lacroix, C., and Jacquemin, J.-L. 1994. Electrophoretic detection of cytoplasmic serine proteinases (gelatinases) in Candida albicans. Experimental Mycology 18: 267-270. Proteolytic activities of Candida albicans were identified using sodium dodecyl sulfate-polyacrylamide gel electrophoresis with gels containing gelatin as proteinase substrate. Cytoplasmic extracts of five isolates were tested with this method. Two major proteolytic activities of M_r 50,000 and 60,000 were conserved between isolates whereas two other less intense activities of M_r 90,000 and 120,000 were only detected in three isolates. The two bands of proteolysis of M_r, 50,000 and 60,000 were revealed between pH 5.0 and pH 8.0, optimally active at pH 7.0, and their isoelectric point was 4.5. They were not influenced by the presence of 2-mercaptoethanol. Their sensitivity to phenylmethylsulfonyl fluoride and chymostatin allowed them to be characterized as serine proteinases. These two neutral proteinases were not secreted in culture supernatant.

Kerrigan, R. W., Imbernon, M., Callac, P., Billette, C., And Olivier, J.-M. 1994. The heterothallic life cycle of Agaricus bisporus var. burnettii and the inheritance of its tetrasporic trait. Experimental Mycology 18, 193-210. The new taxon Agaricus bisporus var. burnettii was recently proposed for a tetrasporic population of the species discovered in California. This tetrasporic variety is interfertile with the familiar bisporic variety of the species. In the present study we determined the heritability of parental basidial morphologies in 91 first-generation intervarietal hybrids descended from either of two genetically distinct strains of var. burnettii. Large samples of basidiospore offspring of one of these parental var. burnettii strains, and of one of the first-generation intervarietal hybrids, were evaluated with respect to their ploidy (n vs n + n). The life cycle of var. burnettii was deduced and the mating type alleles of the two var. burnettii parents stocks were determined. We found that: (1) The life cycle of this variety, of which more than 90% of the basidia were four-spored, was predominantly heterothallic. About 90% of the basidiospores were homokaryotic. (2) Its incompatibility system was unifactorial. Four different, previously unknown mating type alleles were found. (3) The tetrasporic trait was always transmitted to first-generation hybrids between bisporic and tetrasporic varieties. Of 91 intervarietal hybrids, 88 had a majority of four-spored basidia while 3 had predominantly three-spored basidia, and none had more than a small percentage of bisporic basidia. (4) The basidiospore offspring of these hybrids germinated normally, producing a high percentage of homokaryons. For one hybrid sporocarp this percentage was estimated at 77% by three methods (growth rate, mating competence, and multilocus allozyme genotype) which generally agreed with one another. These data shed new light on the genetics of A. bisporus and will be useful in related breeding work.

Leal-Morales, C. A., Bracker, C. E., and Bartnicki-Garcia, S. 1994. Distribution of chitin synthetase and various membrane marker enzymes in chitosomes and other organelles of the slime mutant of Neurospora crassa. Experimental Mycology 18, 168-179. The subcellular distribution of chitin synthetase was assessed in cell homogenates of the slime mutant of Neurospora crassa . The activities of several marker enzymes were monitored to establish the relationship between organelles containing chitin synthetase and other cytoplasmic organelles. Before cell breakage, the plasma membrane of the slime mutant was radiolabeled with traces of [³H]concanavalin A. Two major (I, II) and two minor (III, IV) bands of particles containing chitin synthetase were detected after sedimentation of crude cell homogenates on a discontinuous sucrose gradient [1-4 h at 81,500g (R_av)]. The main population (I) consisted of microvesicles (chitosomes) which sedimented more slowly than most other membranous organelles. The other major population (II) of chitin synthetase particles cosedimented with the plasma membrane markers, [³H]concanavalin A and a vanadate-sensitive ATPase. Despite differences in sedimentation velocity, the two major chitin synthetase populations (I and II) eventually equilibrated at the same buoyant density (1.12 g/ml). One of the two minor bands (III) of chitin synthetase was associated with plasma membrane surface marker. Band IV, the smallest fraction with chitin synthetase activity was not characterized. A soluble, dialyzable, partially thermostable factor found in the cytosol of N. crassa enhanced the chitin synthetase activity of chitosomes (4.6 to 4.8-fold); to a lesser extent (about 2-fold) it also enhanced the activity of the other chitin synthetase populations. This stimulatory factor found in the upper portion of the gradients affected the levels of chitin synthetase activity detected, particularly that of chirpspines in peak I. By a combination of centrifugation procedures, the subcellular populations of chitin synthetase were clearly separated from other organelles with similar [endoplasmic reticulum, d = 1.118, marker: phosphatidyl choline glyceride transferase] or different equilibrium densities [vacuole, d = 1.170 g/ml, marker: α-mannosidase; mitochondria, d = 1.175, marker: succinate dehydrogenase]. The absence of markers for plasma membrane, mitochondria, and vacuoles from the chitosome population and the partial but unequivocal separation of the endoplasmic reticulum marker from chitosomal chitin synthetase provide additional verification that these microvesicles are distinct subcellular structures and not fragments of other organelles. The findings support the existence of a unique secretory pathway based on chitosome microvesicles as the main conveyors of chitin synthetase to the cell surface.

Haindt, E., and Monzer, J. 1994. Elongation growth and gravitropic curvature in the Flammulina velutipes (Agaricales) fruiting body. Experimental Mycology 18, 150-158. 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 $\text{1}\text{8}$ 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.

Jones, L., and O'Shea, P. 1994. The electrostatic nature of the cell surface of Candida albicans: A role in adhesion. Experimental Mycology 18, 111-120. The yeast form of Candida albicans is subjected to particle electrophoresis in a variety of media, in order to determine whether the cell surface of the fungus conforms to a simple electrostatic system. It is found that C. albicans behaves essentially as a simple charged colloidal system. Similar measurements were performed with various glass surfaces in order to identify whether electrostatic interactions have any bearing on fungal adhesion. It was found that under all the circumstances studied, the fungi and glass were electronegative; the degree of adhesion was found to be affected by the magnitude of the coulombic repulsion. Significant adhesion still occurred, however, even when the coulombic repulsion was a maximum; this was taken to indicate that the fungal surface possesses other nonelectrostatic forces which are attractive. Both the electrostatic repulsive and the nonelectrostatic (presumably nonpolar) forces are considered to play a role in the adhesion of fungi to each other, to artificial surfaces such as glass, and presumably to other surfaces which occur in living systems.

Sherriff, C., Whelan, M. J., Arnold, G. M., Lafay, J.-F., Brygoo, Y., and Bailey, J. A. 1994. Ribosomal DNA sequence analysis reveals new species groupings in the genus Colletotrichum. Experimental Mycology 18, 121-138. The relatedness of a range of isolates of Colletotrichum species, selected to represent the major morphological forms of the genus, was studied by comparing their morphology with an analysis of an 886-bp region of their 28S rDNA sequences and ITS-2 regions. rDNA was amplified by PCR. An initial dendrogram for the full sequence was drawn using cluster analysis (UPGMA), with trees then constructed using the neighbor-joining method. The similarity matrices and the resulting dendrogram and trees indicate that the genus can be divided into two groups. One group, consisting of Colletotrichum lindemuthianum, Colletotrichum malvarum, Colletotrichum orbiculare, and Colletotrichum trifolii, was distinct from all the other species. Their rDNA was highly homologous and they had consistent morphological features, including their failure to produce septa during conidial germination, which readily distinguished them from Colletotrichum gloeosporioides and from all other isolates. It is concluded that isolates within this group may represent a single species, which should be referred to as C. orbiculare (Berk. and Mont. (v Arx)), in which distinct host-specific forms exist. Examination of the other group indicates that isolates presently included within C. gloeosporioides represent more than one species. The data also confirmed present day inaccurate identifications; e.g., the causal agent of cowpea anthracnose is not a form of C. lindemuthianum . The distinction between species with falcate conidia, i.e., Colletotrichum capsici, Colletotrichum graminicola, Colletotrichum caudatum, and Colletotrichum truncatum, was verified. The relationships revealed by analysis of a 886-base region between the isolates examined was also evident from selective analysis of domain 2 (206 sites) and ITS-2 (158 sites) sequences, suggesting that further analysis of either of these regions should rapidly extend the understanding of the taxonomy of this important plant pathogenic genus.

MacKichan, J. K., Tuininga, A. R., and Kerwin, J. L. 1994. Preliminary characterization of phospholipase A₂ in Lagenidium giganteum. Experimental Mycology 18, 180-192. Phospholipase A₂ (PLA₂) hydrolyses the fatty acyl ester bond at the sn-2 position in glycerophospholipids. To better understand its regulatory roles, factors affecting PLA₂ activity in Lagenidium giganteum were investigated: divalent ions; chelators: inhibitors; pH; and substrate concentration. PLA₂ activity of L. giganteum whole cell homogenates was determined using 1-stearoyl-2-[1-¹⁴C]arachidonoyl phosphatidylcholine as substrate. The divalent cations Ca²⁺, Mg²⁺, and Mn²⁺ all enhanced PLA² activity, while Co²⁺, Fe²⁺, and Zn²⁺ were either slightly inhibitory or without effect. High concentrations of EGTA enhanced activity, low concentrations of the chelators were slightly inhibitory, while high concentrations of EDTA had little effect. EGTA, which has a higher affinity for Ca²⁺ and Mn²⁺ than Mg²⁺, reduced hydrolysis less than a comparable concentration of EDTA. Two pH optima were found, at both acid (ca. 5.5) and alkaline (ca. 11.5) levels. Four classical inhibitors, nordihydroguaiaretic acid, ellagic acid, gossypol, and 4-bromophenacylbromide, reduced PLA₂ activity by about 80% at 5 mM concentration, 50% with 1 mM inhibitor, and had no effect at 100 μM. The relatively high levels of these compounds needed to inhibit PLA₂ hydrolysis may have been due to the presence of a cocktail of enzymes, some of which were not susceptible to inhibition. All inhibitors at 1 mM concentration in live cell cultures effectively shut down oosporogenesis, without adverse effects to the mycelia. PLA₂ activity was highest in the late oospore stage of the life cycle, although the enzymes were probably not metabolically active in these stationary cultures. Cultures grown on cholesterol-supplemented defined media had significantly higher levels of PLA₂ activity relative to cultures grown on sterol-free media. The enzyme was found to be associated primarily with microsomal membranes, but there was significant activity in cytosolic fractions. Separation of cell homogenates by column chromatography revealed that there were at least nine enzymes capable of cleaving fatty acids in the sn -2 position of phospholipids.

Iranzo, M., Marcilla, A., Elorza, M. V., Mormeneo, S., and Sentandreu, R. 1994. Preparation of anti-protein and anti-mannan antisera against fungal cell wall by affinity chromatography. Experimental Mycology 18, 159-167. A novel and easy chromatographic method has been developed for the isolation of anti-protein and anti-mannan antisera from a population of polyclonal antibodies obtained against Candida albicans and Yarrowia lipolytica cell wall mannoproteins. The technique is based on the immobilization of mannan (to be used as immunoadsorbent) by Affi-Prep H_z resin after the oxidation of neighboring hydroxyl groups of the polysaccharide with sodium periodate. For Y. lipolytica polyclonal antiserum, a single chromatographic step using the homologous mannan was sufficient to obtain an antiprotein antibody preparation free of antimannan antibodies. For C. albicans, three chromatographic processes using homologous and heterologous mannan were needed to obtain a satisfactory antiprotein antiserum. The potential application of the anti-protein antiserum obtained has been demonstrated by indirect immunofluorescence assays of whole cells and electrophoretic analysis of wall proteins in C. albicans and Saccharomyces cerevisiae .