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Applied and Environmental Microbiology, February 2001, p. 598-607, Vol. 67, No. 2
Department of Plant Quality, Bulb Research
Centre, 2160 AB Lisse,1 and Department
of Molecular Microbiology, IMBW/BCA Faculty of Biology, Vrije
Universiteit, 1081 HV Amsterdam,2 The
Netherlands
Received 7 July 2000/Accepted 16 November 2000
A sensitive and specific detection method was developed for
Xanthomonas hyacinthi; this method was based on
amplification of a subsequence of the type IV fimbrial-subunit gene
fimA from strain S148. The fimA gene was
amplified by PCR with degenerate DNA primers designed by using the
N-terminal and C-terminal amino acid sequences of trypsin fragments of
FimA. The nucleotide sequence of fimA was determined and
compared with the nucleotide sequences coding for the fimbrial subunits
in other type IV fimbria-producing bacteria, such as Xanthomonas
campestris pv. vesicatoria, Neisseria gonorrhoeae,
and Moraxella bovis. In a PCR internal primers JAAN and
JARA, designed by using the nucleotide sequences of the variable central and C-terminal region of fimA, amplified a 226-bp
DNA fragment in all X. hyacinthi isolates. This PCR was
shown to be pathovar specific, as assessed by testing 71 Xanthomonas pathovars and bacterial isolates belonging to
other genera, such as Erwinia and Pseudomonas.
Southern hybridization experiments performed with the labelled 226-bp
DNA amplicon as a probe suggested that there is only one structural
type IV fimbrial-gene cluster in X. hyacinthi. Only two
Xanthomonas translucens pathovars cross-reacted weakly in
PCR. Primers amplifying a subsequence of the fimA gene of
X. campestris pv. vesicatoria (T. Ojanen-Reuhs, N. Kalkkinen, B. Westerlund-Wikström, J. van Doorn, K. Haahtela,
E.-L. Nurmiaho-Lassila, K. Wengelink, U. Bonas, and T. K. Korhonen, J. Bacteriol. 179: 1280-1290, 1997) were shown to be
pathovar specific, indicating that the fimbrial-subunit sequences are
more generally applicable in xanthomonads for detection purposes. Under
laboratory conditions, approximately 1,000 CFU of X. hyacinthi per ml could be detected. In inoculated leaves of
hyacinths the threshold was 5,000 CFU/ml. The results indicated that
infected hyacinths with early symptoms could be successfully screened
for X. hyacinthi with PCR.
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.2.598-607.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Analysis of the Type IV Fimbrial-Subunit Gene
fimA of Xanthomonas hyacinthi: Application in
PCR-Mediated Detection of Yellow Disease in Hyacinths
*
Corresponding author. Mailing address: Department of
Plant Quality, Bulb Research Centre, P. O. Box 85, 2160 AB Lisse,
The Netherlands. Phone: 31(0)252462173. Fax: 31(0)252417762. E-mail: Joop.van.Doorn{at}lbo.agro.nl.
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