Detection and identification of decay fungi in spruce wood by restriction fragment length polymorphism analysis of amplified genes encoding rRNA.

We have developed a DNA-based assay to reliably detect brown rot and white rot fungi in wood at different stages of decay. DNA, isolated by a series of CTAB (cetyltrimethylammonium bromide) and organic extractions, was amplified by the PCR using published universal primers and basidiomycete-specific primers derived from ribosomal DNA sequences. We surveyed 14 species of wood-decaying basidiomycetes (brown-rot and white-rot fungi), as well as 25 species of wood-inhabiting ascomycetes (pathogens, endophytes, and saprophytes). DNA was isolated from pure cultures of these fungi and also from spruce wood blocks colonized by individual isolates of wood decay basidiomycetes or wood-inhabiting ascomycetes. The primer pair ITS1-F (specific for higher fungi) and ITS4 (universal primer) amplified the internal transcribed spacer region from both ascomycetes and basidiomycetes from both pure culture and wood, as expected. The primer pair ITS1-F (specific for higher fungi) and ITS4-B (specific for basidiomycetes) was shown to reliably detect the presence of wood decay basidiomycetes in both pure culture and wood; ascomycetes were not detected by this primer pair. We detected the presence of decay fungi in wood by PCR before measurable weight loss had occurred to the wood. Basidiomycetes were identified to the species level by restriction fragment length polymorphisms of the internal transcribed spacer region.

False-positive results obtained with the Alexon ProSpecT Cryptosporidium enzyme immunoassay.

Cryptosporidium is known to cause diarrhea in immunocompromised patients and is also associated with outbreaks of disease due to food-borne and waterborne parasites. Traditional procedures, involving iodine staining of wet mounts of stool sediments and trichrome staining, lack the sensitivity to detect Cryptosporidium. Special staining procedures, such as the modified acid-fast and safranin stains, are generally employed. Less labor-intensive antigen detection assays have simplified detection; however, careful attention to local epidemiology is important because false-positive tests occur. Here, we report two incidents involving 62 false-positive results obtained with the Alexon ProSpecT Cryptosporidium enzyme immunoassay, which were deemed false-positive based on negative results obtained from extensive microscopic examinations.

Use of fluorescent-antibody staining of cytocentrifuge-prepared smears in combination with cell culture for direct detection of respiratory viruses.

Over a 3-year period, 1,003 respiratory samples were collected and examined for selected respiratory viruses with cytocentrifuged prepared smears stained with fluorescently labeled antibodies (IFA) in conjunction with cell culture. IFA results were compared with results obtained by cell culture. Viruses were isolated or detected by direct means in 401 samples. Agreement between culture and IFA was 90%.

Environmental scanning electron microscopic observation of the hyphal sheath and mycofibrils in Postia placenta.

Environmental scanning electron microscopic observations of Postia placenta grown on a defined medium and on red spruce wood allowed for the examination of the hydrated sheath of P. placenta. In the wood environment, mature hyphae that were not adhering to the substrate were observed to have a mycofibrillar morphology whereas hyphal tips and branch points had a smooth sheath morphology. A mycofibrillar adhesive matrix was observed on the hyphae growing on glass slides in the defined medium. These morphologies for hyphal sheaths in P. placenta are similar to those previously described by investigators from other laboratories who have used traditional electron microscopic preparative protocols that include dehydration steps. The potential future usefulness of environmental scanning electron microscopic technology in the study of the fine details of extracellular matrices is briefly discussed.

Iron-binding compounds produced by wood-decaying basidiomycetes.

The chrome azurol-S universal siderophore assay and the rapid paper electrophoresis siderophore assay were used to screen 10 wood-decaying basidiomycete isolates for the formation of iron-chelating compounds. All 10 isolates were positive for chrome azurol-S reactivity on solid plating medium and in liquid cultures, and 9 of the 10 isolates produced fluorescent iron-binding compounds in the paper electrophoresis assay.

Monoclonal Antibodies to Glycoprotein Antigens of a Fungal Plant Pathogen, Phytophthora megasperma f. sp. glycinea.

Genetic studies of plants and their pathogens indicate that dominant alleles for resistance in hosts are complemented by corresponding dominant alleles for avirulence in pathogens. Products of these genes have not yet been identified. We have produced murine monoclonal antibodies (mAbs) to extracellular antigens of the fungal soybean pathogen Phytophthora megasperma f. sp. glycinea (Pmg, race 1) as part of a larger effort to identify antigenic determinants associated with particular avirulence genes. Thirty-six independent mAbs have been characterized by binding to Western blots of Pmg extracellular glycoproteins and by enzyme-linked immunosorbent assay with glycoproteins modified by treatment with periodate, alpha-mannosidase, and endo-beta-N-acetylglucosaminidase H. The mAbs are predominantly carbohydrate-specific and can be placed in six groups based on interactions with Pmg glycoproteins. Binding patterns of various mAbs to Western blots indicate that Pmg proteins may have single or multiple types of attached carbohydrate antigens. Races of Pmg with differing avirulence genes exhibit more characteristic differences by Western blot analysis than by protein staining of glycoprotein profiles. Several of the mAbs show much higher reaction levels to glycoproteins from race 1 than from two other races. All of the glycoprotein-specific mAbs cross-react with purified mycelial walls.

Exploratory numerical taxonomy based on biochemical and biophysical characters of the tymoviruses.

Three different exploratory analytical techniques, principal components analysis, reciprocal averaging and cluster analysis, were used to examine taxonomic relationships among the tymoviruses. The three techniques gave similar but not identical interpretations of the interrelationships within this group. Exploratory numerical analyses were useful in assessing relatedness, and they suggest that andean potato latent virus is misclassified as a strain of eggplant mosaic virus.

Determinants of perceived choice, number of options, and perceived time in making a selection.

An experiment was conducted to investigate the effects of number of choice options and perceived time taken to make a selection upon perceived choice. College students were asked to make a selection from a set of options which varied in numerosity; after making their selection, they were given information which varied their perception of time in making the selection. As predicted, perceived choice was found to be greater (1) the greater the number of options when a person perceived that he had taken a relatively short time; (2) if the selection involved a moderately large number of options than if it involved either a small or a very large number when a person perceived that he had taken a relatively long time; (3) the longer the time a person thought he had taken, except when the selection involved a very large number of options-in which case, perceived choice was greater the shorter the perceived time. The experiment also provided evidence about the relationship between perceived choice and perceived competence.