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Applied and Environmental Microbiology, September 2001, p. 4070-4076, Vol. 67, No. 9
Unit of Mycology, Bacteriology and
Nematology, Scottish Crop Research Institute, Invergowrie, Dundee
DD2 5DA, United Kingdom
Received 26 February 2001/Accepted 6 June 2001
Current identification methods for the soft rot erwinias are both
imprecise and time-consuming. We have used the 16S-23S rRNA intergenic
transcribed spacer (ITS) to aid in their identification. Analysis by
ITS-PCR and ITS-restriction fragment length polymorphism was found to
be a simple, precise, and rapid method compared to current molecular
and phenotypic techniques. The ITS was amplified from
Erwinia and other genera using universal PCR primers.
After PCR, the banding patterns generated allowed the soft rot erwinias to be differentiated from all other Erwinia and
non-Erwinia species and placed into one of three groups
(I to III). Group I comprised all Erwinia carotovora
subsp. atroseptica and subsp.
betavasculorum isolates. Group II comprised all
E. carotovora subsp. carotovora, subsp. odorifera, and subsp. wasabiae and
E. cacticida isolates, and group III comprised all
E. chrysanthemi isolates. To increase the
level of discrimination further, the ITS-PCR products were digested
with one of two restriction enzymes. Digestion with CfoI identified E. carotovora subsp.
atroseptica and subsp. betavasculorum (group I) and E. chrysanthemi (group III) isolates,
while digestion with RsaI identified E.
carotovora subsp. wasabiae, subsp.
carotovora, and subsp.
odorifera/carotovora and E. cacticida isolates
(group II). In the latter case, it was necessary to distinguish
E. carotovora subsp. odorifera and
subsp. carotovora using the
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.9.4070-4076.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Rapid Identification and Differentiation of the Soft Rot Erwinias
by 16S-23S Intergenic Transcribed Spacer-PCR and Restriction
Fragment Length Polymorphism Analyses
-methyl glucoside test.
Sixty suspected soft rot erwinia isolates from Australia were
identified as E. carotovora subsp.
atroseptica, E. chrysanthemi, E. carotovora subsp. carotovora, and
non-soft rot species. Ten "atypical" E.
carotovora subsp. atroseptica isolates were
identified as E. carotovora subsp.
atroseptica, subsp. carotovora, and
subsp. betavasculorum and non-soft rot species, and two
"atypical" E. carotovora subsp.
carotovora isolates were identified as E.
carotovora subsp. carotovora and subsp.
atroseptica.
*
Corresponding author. Mailing address: Unit of
Mycology, Bacteriology and Nematology, Scottish Crop Research
Institute, Invergowrie, Dundee DD2 5DA, United Kingdom. Phone:
44-1382-562731. Fax: 44-1382-562426. E-mail:
itoth{at}scri.sari.ac.uk.
Applied and Environmental Microbiology, September 2001, p. 4070-4076, Vol. 67, No. 9
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.9.4070-4076.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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