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J H Andrews

Publications and source records attributed to J H Andrews.

At least 19 recordsLinked to original sources
PubMed2000

Automated image analysis of live/dead staining of the fungus Aureobasidium pullulans on microscope slides and leaf surfaces.

An image analysis program and protocol for the identification and enumeration of live versus dead cells of the yeast-like fungus Aureobasidium pullulans was developed for both populations on microscope slides and leaf surfaces. Live cells took up CellTracker Blue, while nonviable cells stained with DEAD Red. Image analysis macro programs running under Optimas software were used to acquire images and to differentiate and enumerate viable from nonviable cells. The software was capable of discriminating green as a third parameter for identification and quantification of green fluorescent protein-expressing cells in a wild-type population.

Green Fluorescent Proteins↗
PubMed1999

Quantitative imaging and statistical analysis of fluorescence in situ hybridization (FISH) of Aureobasidium pullulans.

Image and multifactorial statistical analyses were used to evaluate the intensity of fluorescence signal from cells of three strains of A. pullulans and one strain of Rhodosporidium toruloides, as an outgroup, hybridized with either a universal or an A. pullulans 18S rRNA oligonucleotide probe in direct or indirect FISH reactions. In general, type of fixation (paraformaldehyde or methanol-acetic acid) had no apparent effect on cell integrity and minimal impact on fluorescence. Permeabilization by enzyme treatment for various times, though needed to admit high Mw detection reagents (avidin-FITC) in indirect FISH, tended to nonspecifically degrade cells and lower the signal. Digestion was unnecessary and undesirable for the directly labelled probes. Multilabelled (five fluorescein molecules) probes enhanced fluorescence about fourfold over unilabelled probes. Overall, direct FISH was preferable to indirect FISH and is recommended especially for studies of microbes on natural substrata.

Analysis of Variance↗
PubMed1999

Attachment of the yeast Rhodosporidium toruloides is mediated by adhesives localized at sites of bud cell development.

The basidiomycetous yeast Rhodosporidium toruloides (anamorph, Rhodotorula glutinis) is a common phylloplane epiphyte with biocontrol potential. To understand how R. toruloides adheres to plant surfaces, we obtained nonadherent fungal mutants after chemical mutagenesis with methane-sulfonic acid ethyl ester. Sixteen attachment-minus (Att-) mutants were identified by three methods: (i) screening capsule-minus colonies for loss of adhesive ability; (ii) enrichment for mutants unable to attach to polystyrene; and (iii) selection for reduced fluorescence of fluorescein isothiocyanate-concanavalin A (Con A)-stained cells by fluorescence-activated cell sorting. None of the 16 mutants attached to polystyrene or barley leaves. The lectin Con A eliminated adhesion in all of the wild-type isolates tested. Hapten competition assays indicated that Con A bound to mannose residues on the cell surface. Adhesion of wild-type R. toruloides was transient; nonadhesive cells subsequently became adhesive, with bud development. All Att- mutants and nonattaching wild-type cells lacked polar regions that stained intensely with fluorescein isothiocyanate-Con A and India ink. Lectin, enzyme, and chemical treatments showed that the polar regions consisted of alkali-soluble materials, including mannose residues. Tunicamycin treatment reduced wild-type adhesion, indicating that the mannose residues could be associated with glycoproteins. We concluded that compounds, including mannose residues, that are localized at sites of bud development mediate adhesion of R. toruloides to both polystyrene and barley leaf surfaces.

Journal Article↗
PubMed1998

Bacteria as modular organisms.

The body plan of modular organisms is based on an indeterminate structure composed of iterated units or modules arrayed at various levels of complexity (such as leaves, twigs, and branches). Examples of modular organisms include plants and many sessile benthic invertebrates. In contrast, the body of unitary organisms is a determinate structure consisting usually of a strictly defined number of parts (such as legs or wings) established only during embryogenesis. Mobile animals are examples. Unlike that of unitary creatures, the form of a modular organism derives from a characteristic pattern of branching or budding of modules, which may remain attached or become separated to live physiologically independent lives as parts of a clone. Modular organisms tend to be sessile or passively mobile and, as genetic individuals, have the capacity for exponential increase in size. They do not necessarily undergo systemic senescence, and do not segregate somatic from germ line cells. It is argued here that bacteria are essentially modular organisms where the bacterial cell, microcolony, and macrocolony are modules of different levels of complexity analogous to modules of macroorganisms. This interpretation provides a broad conceptual basis for understanding the natural history of bacteria, and may illuminate the evolutionary origins and developmental biology of modular creatures.

Bacteria↗
PubMed1997

Quantitative fluorescence in situ hybridization of Aureobasidium pullulans on microscope slides and leaf surfaces.

A 21-mer oligonucleotide probe designated Ap665, directed at the 18S rRNA of Aureobasidium pullulans and labelled with five molecules of fluorescein isothiocyanate, was applied by fluorescence in situ hybridization (FISH) to populations of the fungus on slides and apple leaves from growth chamber seedlings and orchard trees. In specificity tests that included Ap665 and a similarly labelled universal probe and the respective complementary probes as controls, the hybridization signal was strong for Ap665 reactions with 12 A. pullulans strains but at or below background level for 98 other fungi including 82 phylloplane isolates. Scanning confocal laser microscopy was used to confirm that the fluorescence originated from the cytoplasmic matrix and to overcome limitations imposed on conventional microscopy by leaf topography. Images were recorded with a cooled charge-coupled device video camera and digitized for storage and manipulation. Image analysis was used to verify semiquantitative fluorescence ratings and to demonstrate how the distribution of the fluorescence signal in specific interactions (e.g., Ap665 with A. pullulans cells) could be separated at a given probability level from nonspecific fluorescence (e.g., in interactions of Ap665 with Cryptococcus laurentii cells) of an overlapping population. Image analysis methods were used also to quantify epiphytic A. pullulans populations based on cell number or percent coverage of the leaf surface. Under some conditions, leaf autofluorescence and the release of fluorescent compounds by leaves during the processing for hybridization decreased the signal-to-noise ratio. These effects were reduced by the use of appropriate excitation filter sets and fixation conditions. We conclude that FISH can be used to detect and quantify A. pullulans cells in the phyllosphere.

Antibiosis↗
PubMed1997

Expression of green fluorescent protein in Aureobasidium pullulans and quantification of the fungus on leaf surfaces.

A red-shifted, mutated form of the jelly-fish green fluorescent protein (GFP) under control of a TEF promoter was expressed at high levels in the filamentous fungus Aureobasidium pullulans. In the three transformants studied, all morphotypes of the fungus, including pigmented chlamydospores, expressed GFP and fluoresced brightly. Confocal microscopy showed that the intra-cellular distribution of GFP was nonuniform. When applied to leaf surfaces, the transformants were readily visible and amenable to quantification by image analysis. Thus, GFP expression, together with quantitative image analysis, may provide a powerful method for ecological studies of plant-microbe relationships in nature.

Blotting, Southern↗
PubMed1996

Development of an oligonucleotide probe for Aureobasidium pullulans based on the small-subunit rRNA gene.

Aureobasidium pullulans, a cosmopolitan yeast-like fungus, colonizes leaf surfaces and has potential as a biocontrol agent of pathogens. To assess the feasibility of rRNA as a target for A. pullulans-specific oligonucleotide probes, we compared the nucleotide sequences of the small-subunit rRNA (18S) genes of 12 geographically diverse A. pullulans strains. Extreme sequence conservation was observed. The consensus A. pullulans sequence was compared with other fungal sequences to identify potential probes. A 21-mer probe which hybridized to the 12 A. pullulans strains but not to 98 other fungi, including 82 isolates from the phylloplane, was identified. A 17-mer highly specific for Cladosporium herbarum was also identified. These probes have potential in monitoring and quantifying fungi in leaf surface and other microbial communities.

Base Sequence↗
PubMed1993

Evidence for growth of Sporothrix schenckii on dead but not on living sphagnum moss.

When clinical isolates of Sporothrix schenckii were inoculated onto the apices of living or dead sphagnum moss plants maintained under growth chamber conditions, populations of the fungus, assessed by standard dilution plate methods, increased swiftly up to about 70-fold on moist, dead plants but did not increase on the live moss. Light and scanning electron microscopy revealed fungal growth and sporulation on and within dead plants, but no evidence of either on live plants. These data provide indirect support for the contention that S. schenckii does not grow on living sphagnum in bogs, but rather that sporotrichosis epidemics associated with sphagnum moss are likely to result from contamination of the dead plants at some point(s) in the chain of events during or after harvest. One practical implication of our results is that precautions should be taken to insure that sphagnum moss is stored dry and that it is not wetted any sooner than necessary before use. We also report here improvement of the Mycoses isolation medium by an increase in cycloheximide from 400 to 800 mg/l, chloramphenicol from 50 to 250 mg/l, and the addition of rifampicin at 20 mg/l.

Disease Reservoirs↗
PubMed1992

Enumeration and Characterization of Bacterial Colonists of a Submersed Aquatic Plant, Eurasian Watermilfoil (Myriophyllum spicatum L.).

A simple procedure for enumerating and grouping the bacterial colonists of Eurasian watermilfoil (Myriophyllum spicatum L.) is described. Colony characteristics of bacteria associated with M. spicatum were better defined and more stable on nutrient-poor, diluted nutrient broth agar than on high-nutrient media. Acinetobacter, Cytophaga, Flavobacterium, Moraxella, Pseudomonas and/or Alcaligenes, and Vibrio/Aeromonas spp., as well as two highly fastidious unidentified bacterial groups (gram-negative rods and gram-negative cocci), were associated with cultured watermilfoil during January, February, May, June, July, and August 1988. In Lake Wingra (Madison, Wis.), Micrococcus spp. and enterobacters were also associated with Eurasian watermilfoil during July, August, and October 1987.

Journal Article↗
PubMed1991

Genetic transformation of Aureobasidium pullulans.

Aureobasidium pullulans strain Y117 was transformed to hygromycin resistance using plasmid pDH33, which contains the bacterial hygromycin B phosphotransferase gene (hph) fused to promoter elements of the Aspergillus niger glucoamylase gene (glaA). Southern hybridizations of transformants revealed multiple, integrated copies of the vector. The glaA promoter was not induced by starch in A. pullulans as it is in A. niger; however, the transcriptional start points were the same in both species.

Aspergillus niger↗
PubMed1990

Spore Yield and Microcycle Conidiation of Colletotrichum gloeosporioides in Liquid Culture.

The effect of V8 juice concentration (5 to 40%, vol/vol), spore inoculum density (10 and 10 spores per ml), and liquid batch or fed-batch culture condition on mycelium and spore production by Colletotrichum gloeosporioides was evaluated. The amount of mycelium produced, the time required for initiation of sporulation following attainment of maximum mycelium, and the time for attainment of maximum spore concentration increased with increasing V8 juice concentration in batch culture. Cultures containing V8 juice at >10% achieved a similar spore density (apparent spore-carrying capacity) of about 0.8 mg of spores per ml (1 x 10 to 2 x 10 spores per ml) independent of inoculum density and V8 juice concentration. The relative spore yield decreased from a high of 64% of the total biomass for the low-inoculum 5% V8 culture, through 13% for the analogous 40% V8 culture, to a low of 2% for the high-inoculum 27% V8 culture. Fed-batch cultures were used to establish conditions of high spore density and low substrate availability but high substrate flux. The rate of addition of V8 juice was adjusted to approximate the rate of substrate utilization by the (increasing) biomass. The final spore concentration was about four times higher (3.0 mg of spores per ml) than the apparent spore-carrying capacity in batch culture. This high spore yield was obtained at the expense of greatly reduced mycelium, resulting in a high relative spore yield (62% of the total biomass). Microcycle conidiation occurred in the fed-batch but not batch systems. These data indicate that substrate-limited, fed-batch culture can be used to increase the amount and efficiency of spore production by C. gloeosporioides by maintaining microcycle conidiation conditions favoring allocation of nutrients to spore rather than mycelium production.

Journal Article↗
PubMed1989

Colonization of a Submersed Aquatic Plant, Eurasian Water Milfoil (Myriophyllum spicatum), by Fungi under Controlled Conditions.

A laboratory assay to assess colonization of a submersed aquatic plant, Eurasian water milfoil (Myriophyllum spicatum), by fungi was developed and used to evaluate the colonization potential of Colletotrichum gloeosporioides, Acremonium curvulum, Cladosporium herbarum, Aureobasidium pullulans, a Paecilomyces sp., and an unidentified sterile, septate fungus. Stem segments of plants were first immersed in suspensions of fungal propagules for 24 h and then washed to remove all but the tightly attached component of the population. Inoculation was followed by two growth cycles of 3 days each. At the start of each cycle, washed plants were transferred to a mineral salts medium to provide an opportunity for the attached fungal populations to grow. After each growth period, plants were again washed, and fungal populations in the medium (nonattached), loosely attached and tightly attached to the plant, and within the plant (endophytic) were assayed by dilution plating. The fungi differed in the extent to which they attached to water milfoil and in their ability to grow in association with it. There were relatively few significant differences among the tightly attached fungal populations after 24 h, but growth of the better colonizers led to a greater number of significant differences after 4 and 7 days. In addition, the better colonizers showed sustained regrowth of loosely and nonattached fungal propagules in the face of intermittent removal by washing. A milfoil pathogen, C. gloeosporioides, was the only endophytic colonizer; it was also among the best epiphytic colonizers but was not demonstrably better than A. curvulum, a fungus commonly found as an epiphyte on watermilfoil. The yeastlike hyphomycete Aureobasidium pullulans was the only fungus that consistently failed to establish an increasing population on the plant.

Journal Article↗
PubMed1988

Sources of variability in the measurement of fungal spore yields.

VARIABILITY IN THE PRODUCTION OF FUNGAL SPORES AND IN THE MEASUREMENT OF SPORE YIELDS WAS INVESTIGATED IN FOUR SPECIES OF FUNGI: Colletotrichum gloeosporioides, Colletotrichum coccodes, Colletotrichum phomoides, and Acremonium strictum. When the fungi were grown on solid medium in microplates and spore yields were measured by counting the subsamples with a hemacytometer, the variability among hemacytometer squares was always the largest source of variation, accounting for 51 to 91% of the total variation. Variability among replicate cultures and results of repeat experiments were generally also significant. The effect of square-to-square variability on the precision of spore yield measurement was minimized by counting a moderate number (ca. 30) of squares per culture. Culture-to-culture variability limited the practical precision of spore production measurements to a 95% confidence interval of approximately the mean +/- 25%. We provide guidelines for determining the number of replicate cultures required to attain this or other degrees of precision. Particle counter-derived spore counts and counts based on spore weights were much less variable than were hemacytometer counts, but they did not improve spore production estimates very much because of culture-to-culture variability. Results obtained by both of these methods differed from those obtained with a hemacytometer; particle counter measurements required a correction for spore pairs, while the relationship between spore weights and spore counts changed as the cultures aged.

Journal Article↗
PubMed1987

Microplate assay for colletotrichum spore production.

A simple microplate method was devised to assay spore production by Colletotrichum gloeosporioides by growing the fungus on 1 ml of solid media in the wells of tissue culture plates. Growth and sporulation on microplates were compared at days 4 and 8 with growth and sporulation in 100-ml liquid batch cultures that involved 11 common media. Spore production per unit volume of medium was the same for solid and liquid forms of the media. Qualitative assessment of mycelial growth measured on microplates agreed with that of growth measured in liquid cultures. The microplate assay indicated that V8 juice was the best medium and that an organic content of about 6 mg/ml was optimal for high sporulation and low mycelium production. The assay provides a convenient, rapid, and inexpensive means of screening media for the production of fungal conidia in large numbers, to be used, for example, in biological control programs.

Journal Article↗
PubMed1987

Microcycle Conidiation and Spore-Carrying Capacity of Colletotrichum gloeosporioides on Solid Media.

The effect of spore inoculum density, medium concentration, and temperature on slime-spot formation, spore yield, and mycelium production by Colletotrichum gloeosporioides on agar media were studied with a simple microplate assay. A steady-state spore yield (spore-carrying capacity) independent of inoculum density was reached only on media that supported good fungal growth and sporulation. The spore-carrying capacity was reached earlier, the denser the inoculum. On standard mycological media a high inoculum density (2.5 x 10 spores per ml) resulted in a slimy mass of conidia forming a slime spot, a phenomenon associated with greatly reduced mycelium formation and indicative of microcycle conidiation. In contrast, for a similar inoculum density, enhanced mycelial growth preceded sporulation and overrode slime-spot formation on highly concentrated media; a very low medium concentration resulted in much less mycelium, but spore production was also decreased. Exposure to suboptimal growth temperatures of 36 to 48 degrees C for up to 8 days did not induce microcycle conidiation from inocula that did not form a slime spot at 28 degrees C.

Journal Article↗
PubMed1979

The effects of a pesticide program on microbial populations from apple leaf litter.

The leaf litter microbial community was quantitatively and qualitatively changed when a standard pesticide schedule that comprised an insecticide, a bactericide, and a fungicide was applied to McIntosh apple trees in the summer. Effects were observed for two winters by four indirect assays and three direct methods. Populations were altered qualitatively both years, but the most striking difference was the quantitative impact from year to year. Bacteria, filamentous fungi, and yeasts from treated leaves were reduced 10- to 10 000-fold between November 1976 and April 1977 and did not recover until snow cover had melted in March. Reductions in 1977-1978 were negligible. The marked seasonal difference is attributed to meteorological influences. Fluorescent pseudomonads were among the bacteria depressed by chemicals. Of the 49 genera of fungi and yeasts isolated, Coniothyrium sp., Penicillium spp., Arthrobotrys spp., and Nodulisporium sp. were appreciably reduced, whereas Typhula spp., Pleurophomella sp., Sporobolomyces spp., and Rhodotorula spp. were substantially enhanced by the spray program.

Azinphosmethyl↗
PubMed1978

The effects of a pesticide program on non-target epiphytic microbial populations of apple leaves.

The epiphytic microbial community was quantitatively and qualitatively altered when a standard pesticide schedule that comprised applications of an insecticide, a bactericide, and a fungicide was applied to McIntosh apple trees. Effects on non-target organisms were observed for two seasons by three indirect methods and three direct methods: plating of leaf washings, imprinting of leaves onto five different media, spore fall patterns, light microscopy, scanning electron microscopy, and isolation of propagules from leaves incubated in humidity chambers. Magnitude of reduction of bacteria, filamentous fungi, yeasts, and actinomycetes varied annually and between categories of microflora. Populations from treated leaves were reduced 10- to 1000-fold in 1976 and up to 50-fold in 1977. Qualitatively, fluorescent pseudomonads and lactic acid-type bacteria were among those depressed by pesticide. Fungal populations on treated leaves were less diverse than on control leaves. Aureobasidium was only slightly affected and incidence of Sporobolomyces was substantially higher on treated leaves than on controls. The results suggest that numbers of antagonists to foliar pathogens of apple which may occur as part of the natural epiphytic microbial community may be reduced by current pesticide programs and hence have possible implications for the development of biological approaches to integrated control strategies.

Bacteria↗