Experimental Mycology最新文献

Li, A., and Horgen, P. A. 1993. Evidence for cytosine methylation in ribosomal RNA genes and in a family of dispersed repetitive DNA elements in Agaricus bisporus and selected other Agaricus species. Experimental Mycology 17, 356-361. Evidence for the methylation of Agaricus repetitive DNA sequences was demonstrated using restriction enzyme analysis and DNA hybridization. Our results indicate that 5-methylcytosine (^5mC) was the only methylated base detected. The nucleotide was present as the internal cytosine of the CCGG recognition sequence in the ribosomal RNA genes and in a family of dispersed repetitive DNA elements of the commercial mushroom, Agaricus bisporus and selected other Agaricus species. Densitometry analysis revealed more methylation in the rDNA than in the dispersed repetitive elements. No methylation of cytosine residues was detected in mitochondrial ribosomal RNA genes nor in the inverted repeated sequences of the A. bisporus mitochondrial genome.

Corrêa, A., Jr., and Hoch, H. C. 1993. Microinjection of urediospore germlings of Uromyces appendiculatus. Experimental Mycology 17, 253-273. Urediospore germlings of Uromyces appendiculatus were microinjected with a variety of materials to assess the applicability of microinjection methodologies for studying fungal cell growth and development. Until now, hyphal cells have not been successfully microinjected without first being pretreated with osmotica to lower the turgor pressures of the cells. We describe the development of a hydraulicly actuated system and protocols for successfully microinjecting Uromyces cells on a routine basis. Synthetic fluorocarbon and silicone fluids, various fluorophore-conjugated dextrans, rhodamine-conjugated phalloidin, rhodamine 123, cyclic AMP, and cytochalasin E were all microinjected into living urediospore germlings that grew or responded in ways consistent with expected pharmacological effects of the injected material. Germlings that were microinjected with synthetic fluids maintained the ability to sense and respond to inductive topographies for the formation of appressoria. Materials were injected at volumes of ≤0.5 to ≤400 fl. The results of this investigation indicate that fungal cells can be successfully microinjected with a variety of materials and, importantly, continue to function as normal cells.

Kamada, T., Hirai, K., and Fujii, M. 1993. The role of the cytoskeleton in the pairing and positioning of the two nuclei in the apical cell of the dikaryon of the basidiomycete Coprinus cinereus. Experimental Mycology 17, 338-344. We examined the effects of tubulin mutations, the anti-microtubule agent benomyl, and the anti-microfilament agents cytochalasin B and E on the pairing and positioning of the two nuclei in the dikaryotic apical cell of the basidiomycete Coprinus cinereus. In the parental wild-type dikaryon, the two nuclei maintain an average separation of 19 μm and an average distance of about 76 μm from the growing hyphal apex; there was a weak tendency that the apex to nucleus distance decreases as cell length decreases. Examination of eight dikaryons homozygous for either one α- or one of seven β-tubulin mutations revealed that all the tubulin mutations disturbed the pairing of the two nuclei without changing the positioning of the leading nucleus. The anti-microtubule agent benomyl disturbed the nuclear pairing without changing the positioning of the leading nucleus. Neither cytochalasin B nor E affected either the pairing or the positioning. These results demonstrate that microtubules participate in the pairing of the two nuclei in the apical cell of the dikaryon and provide evidence against a direct involvement of either microtubules or microfilaments in the mechanism of their positioning relative to the hyphal apex.

Snetselaar, K. M. 1993. Microscopic observation of Ustilago maydis mating interactions. Experimental Mycology 17, 345-355. Ustilago maydis sporidia grown in liquid medium were concentrated by centrifugation, resuspended in sterile water, and incubated as drop cultures in petri dishes. Haploid and diploid strains with various combinations of mating-type alleles were incubated individually and in mixed cultures for up to 12 h, during which time sporidia were observed and compared with DIC and epifluorescence optics. When paired haploid strains carried unlike a alleles, sporidia formed mating hyphae and conjugated regardless of the b alleles they carded. If the strains also carded unlike b alleles, mating was followed by formation of rapidly growing dikaryotic hyphae. If sporidia with unlike a alleles and identical b alleles were incubated together, multiple fusions were common, and the few hyphae that formed grew slowly and were often multinucleate. When unlike a and disrupted b alleles were involved, more multiple fusions and fewer emergent hyphae resulted. When a diploid strain with compatible alleles at both loci was incubated alone, sporidia formed straight uninucleate hyphae without mating. A diploid strain that was isogenic except for a disrupted b1 locus mated indiscriminately, but straight, dikaryotic hyphae formed only when the mating partner carried an intact b1 allele. These results demonstrated that although the plate-mating assays in common use detect the vigorous filamentous growth associated with pathogenicity, they do not detect all kinds of filaments, nor do they assay mating per se. The correlation of mating cessation with the presence of compatible b alleles in a common cytoplasm implicates the active b product in negative regulation of mating-specific genes. The methods described here could be used to further define the roles of the a and b gene products in controlling mating behavior and subsequent development.

Viljoen, C. D., Wingfield, B. D., and Wingfield, M. J. 1993. Comparison of Seiridium isolates associated with cypress canker using sequence data. Experimental Mycology 17, 323-328. Three species of Seiridium have been associated with cypress canker. These include Seiridium cardinale, Seiridium unicorne, and Seiridium cupressi and are distinguished based on conidial appendage morphology. Some authorities believe that S. cupressi is conspecific with S. unicorne as these two species possess appendaged conidia while S. cardinale does not. Others are of the view that only one species of Seiridium with variable morphology is associated with cypress canker. In this study the variable first internal transcribed spacer region of the ribosomal RNA genes, from isolates of Seiridium associated with cypress canker, was sequenced and compared. Results suggest that species of Seiridium associated with cypress canker are closely related. Moreover sequence data support the view that S. cupressi and S. unicorne are synonyms of S. cardinale.

Li, S., Harris, C. P., and Leong, S. A. 1993. Comparison of fluorescence in situ hybridization and primed in situ labeling methods for detection of single-copy genes in the fungus Ustilago maydis. Experimental Mycology 17, 301-308. This report describes the use of fluorescence in situ hybridization for detection of single-copy genes in the fungus, Ustilago maydis . Either biotin- or digoxigenin-labeled DNA probes were hybridized to target nuclei of sporidia and subsequently rendered fluorescent by successive treatments with fluorescein (FlTC)-labeled avidin and biotinylated anti-avidin antibody or with anti-digoxigenin-FlTC or anti-digoxigenin-rhodamine. These results showed that the digoxigenin-based hybridization can be used successfully to specifically detect single-copy genes in nuclei of U. maydis with either 3.6- or 6.7-kb genomic DNA probes. By contrast, the biotin-based hybridization technique gave rise to nonspecific background. Primed in situ labeling with fluorescein-12-dUTP was also used successfully to specifically detect a 1.5-kb fragment of single-copy genomic DNA in nuclei of U. maydis sporidia. This technique has the advantages of lower staining background, shorter time of analysis, and a higher efficiency of hybridization of shorter DNA probes to target DNA, when compared to traditional in situ hybridization.

Terhune, B. T., and Hoch, A. C. 1993. Substrate hydrophobicity and adhesion of Uromyces urediospores and germlings. Experimental Mycology 17, 241-252. Adhesions of urediospores and urediospore germlings of Uromyces appendiculatus, the bean rust pathogen, to various substrata was evaluated with regard to surface wettability. A range of surface wettabilities, or conversely hydrophobicities, was obtained by coating glass or quartz substrates with various organosilanes. Adhesion of urediospores or germlings was evaluated after the spore or germling laden-silanized surfaces were washed. Both urediospores and germlings adhered most tenaciously to surfaces with wettability ratings less than 30. Such surfaces were polystyrene and glass treated with dimethyldichlorosilane, (tridecafluoro-1,1,2,2-tetrahydrooctyl)-1-trichorosilane, and diphenyldichlorosilane. The degree of germling contact to the various surfaces correlated closely with hydrophobicity and with the adhesion of germlings. Induction of appressoria on quartz substrates bearing inductive topographies (0.5-μm-deep grooves) was also closely associated with the degree of hydrophobicity.

Migheli, Q., Berio, T., and Gullino, M. L. 1993. Electrophoretic karyotypes of Fusarium spp. Experimental Mycology 17, 329-337. The electrophoretic karyotype of 17 antagonistic and pathogenic strains of Fusarium spp. has been established by using contour-clamped homogeneous electric field gel electrophoresis. Intact chromosomal DNA was prepared from fungal protoplasts with standard procedures. Up to 11 distinct chromosomal bands were resolved after 184 h of migration at 50 V. Polymorphic karyotypes were observed in different species of Fusarium, formae speciales of F. oxysporum , and races of F. oxysporum f.sp. dianthi. Using the Schizosaccharomyces pombe and Saccharomyces cerevisiae chromosomes as size standards, the size of the Fusarium genome was estimated to range from approximately 18.1 to 51.5 Mb. The suitability of electrophoretic karyotyping as a tool for strain characterization, as well as some applications in hybridization analysis of Fusarium spp., is discussed.

Francis, D. M., and Michelmore, R. W. 1993. Two classes of chromosome-sized molecules are present in Bremia lactucae. Experimental Mycology, 17, 284-300. The size and number of chromosomes from Bremia lactucae have been estimated using pulsed-field gel electrophoresis. The karyotype consists of a minimum of seven chromosomes between 3.0 and at least 8 Mb and a set of polymorphic molecules between 300 kb and 1.6 Mb. Genetic and hybridization analyses confirmed the distinction between the large chromosomes and the smaller polymorphic molecules. The polymorphic molecules are linear, nonribosomal, and located in the nucleus; they are related in sequence and do not segregate in a Mendelian fashion. The polymorphic molecules are therefore either B chromosomes or large linear plasmids. Chromosomes carrying two avirulence genes and several restriction fragment length polymorphism markers have been identified.

Bayles, C. J., Aist, J. R., and Berns, M. W. 1993. The mechanics of anaphase B in a basidiomycete as revealed by laser microbeam microsurgery. Experimental Mycology 17, 191-199. Cytoplasmic forces were found to be actively pulling on the spindle pole bodies during anaphase B in the dikaryotic, basidiomycete fungus, Helicobasidium mompa. When the spindle of one nucleus was severed with a laser microbeam at mid anaphase B, its two spindle pole bodies separated at a much faster rate than did those of the intact spindle in the other nucleus of the same cell. Since astral microtubule populations apparently reach their maximum during anaphase B in this fungus, we suggest that these microtubules may be involved in the cytoplasmic pulling forces. The spindle appears to act primarily as a governor, regulating the rate at which the spindle pole bodies are separated.