PMID- 9251214
OWN - NLM
STAT- MEDLINE
DCOM- 19970918
LR  - 20210526
IS  - 0099-2240 (Print)
IS  - 1098-5336 (Electronic)
IS  - 0099-2240 (Linking)
VI  - 63
IP  - 8
DP  - 1997 Aug
TI  - Quantitative fluorescence in situ hybridization of Aureobasidium pullulans on 
      microscope slides and leaf surfaces.
PG  - 3261-7
AB  - 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.
FAU - Li, S
AU  - Li S
AD  - Department of Plant Pathology, University of Wisconsin, Madison 53706, USA.
FAU - Spear, R N
AU  - Spear RN
FAU - Andrews, J H
AU  - Andrews JH
LA  - eng
PT  - Journal Article
PT  - Research Support, U.S. Gov't, Non-P.H.S.
PL  - United States
TA  - Appl Environ Microbiol
JT  - Applied and environmental microbiology
JID - 7605801
RN  - 0 (Oligonucleotide Probes)
RN  - 0 (RNA, Ribosomal, 18S)
SB  - IM
MH  - Antibiosis
MH  - Colony Count, Microbial
MH  - Cytoplasm/microbiology
MH  - Fungi/genetics/isolation & purification
MH  - Image Processing, Computer-Assisted
MH  - In Situ Hybridization, Fluorescence/*methods
MH  - Microscopy, Confocal
MH  - Mitosporic Fungi/genetics/*isolation & purification
MH  - Oligonucleotide Probes/*genetics
MH  - Plant Leaves/*microbiology
MH  - RNA, Ribosomal, 18S/*genetics
MH  - Sensitivity and Specificity
MH  - Trees/microbiology
PMC - PMC168625
EDAT- 1997/08/01 00:00
MHDA- 1997/08/01 00:01
PMCR- 1997/08/01
CRDT- 1997/08/01 00:00
PHST- 1997/08/01 00:00 [pubmed]
PHST- 1997/08/01 00:01 [medline]
PHST- 1997/08/01 00:00 [entrez]
PHST- 1997/08/01 00:00 [pmc-release]
AID - 10.1128/aem.63.8.3261-3267.1997 [doi]
PST - ppublish
SO  - Appl Environ Microbiol. 1997 Aug;63(8):3261-7. doi: 
      10.1128/aem.63.8.3261-3267.1997.