Abstract
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.
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Claims 4
Claim · model-written summary
The probe signal was strong for 12 target strains and near background for 98 other fungi.
- Taxon
- apple
Malus domesticae1 - Interactor
- Aureobasidium pullulans
Aureobasidium pullulanst1other - Outcome
- hybridization signale0
- Value
- 12 A. pullulans strainse0
- Comparator
- 98 other fungie0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“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”
e1“apple leaves from growth chamber seedlings and orchard trees”
t1“Aureobasidium pullulans”
Claim · model-written summary
Image analysis quantified epiphytic fungus by cell number or leaf surface coverage.
- Taxon
- appleMalus domesticae1
- Interactor
- A. pullulansAureobasidium pullulanse0other
- Outcome
- epiphytic A. pullulans populationse0
- Value
- cell number or percent coveragee0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Image analysis methods were used also to quantify epiphytic A. pullulans populations based on cell number or percent coverage of the leaf surface”
e1“apple leaves from growth chamber seedlings and orchard trees”
Claim · model-written summary
Leaf autofluorescence and released compounds reduced signal to noise under some conditions.
- Taxon
- appleMalus domesticae1
- Outcome
- signal-to-noise ratioe0
- Qualifier
- Under some conditionse0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Under some conditions, leaf autofluorescence and the release of fluorescent compounds by leaves during the processing for hybridization decreased the signal-to-noise ratio”
e1“apple leaves from growth chamber seedlings and orchard trees”
Claim · model-written summary
Appropriate excitation filters and fixation reduced these effects.
- Taxon
- appleMalus domesticae1
- Outcome
- These effects were reducede0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“These effects were reduced by the use of appropriate excitation filter sets and fixation conditions”
e1“apple leaves from growth chamber seedlings and orchard trees”