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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 The genus Erwinia
comprises plant pathogens belonging to the family
Enterobacteriaceae. One group within the genus, the soft rot
erwinias, causes soft rot diseases of many plant species worldwide (28). The most important of the soft rot erwinias
commercially are E. chrysanthemi, E. carotovora subsp. carotovora, and E. carotovora subsp. atroseptica, which cause diseases of
potato and other commercially important crops. However, the other
subspecies, E. carotovora subsp.
betavasculorum, E. carotovora subsp.
odorifera, and E. carotovora subsp.
wasabiae, are also important pathogens (10, 11,
19). Since the host range of the soft rot erwinias has not been
elucidated fully, it is important to determine which of the pathogens
is responsible for a disease before disease control can be effective.
However, in current identification methods, which are based mainly on
biochemical and phenotypic characteristics, rapid and accurate
identification is not always possible. As a consequence, isolates may
be misidentified or, when results do not fit those expected, may be
labeled as "atypical."
Currently, the major soft rot erwinias are identified by their growth
and cavity formation on pectate-containing selective media, such as
crystal violet pectate (CVP) (3), at differential temperatures, i.e., E. carotovora subsp.
atroseptica grows at 27°C, E. carotovora
subsp. carotovora grows at 27 and 33.5°C, and
E. chrysanthemi grows at 27, 33.5, and 37°C. However,
these identifications often remain tentative, and growth at
differential temperatures is not always accurate, since some isolates
grow at temperatures outside their expected range (27).
Biochemical tests are currently the accepted standard for
identification and taxonomy of the soft rot erwinias (7, 8, 9,
23, 35), but on a routine level they are very time-consuming
(taking up to 14 days) and, when carried out by nonspecialist
laboratories, do not always provide a definitive identification.
A number of other methods have been used to identify the soft rot
erwinias although, as with biochemistry and growth on CVP, all have
limitations. The high serological heterogeneity and cross-reactivity within and between subspecies, respectively, have limited the use of
serology (7). Fatty acid profiling has been used to differentiate Erwinia species (24, 36) but has
been of only limited success in identifying individual subspecies
within E. carotovora (5, 29). The randomly
amplified polymorphic DNA (RAPD)-PCR method lacks reproducibility
(particularly between laboratories) and has not been tested on all
E. carotovora subspecies (21, 26, 32).
DNA-DNA hybridization is accurate but time-consuming and unsuitable for
routine use, especially when large numbers of strains are involved
(2, 10, 25, 34). PCR-restriction fragment length
polymorphism (PCR-RFLP) analysis of a pectate lyase gene has been used
but has been unsuccessful in identifying all E. carotovora subspecies (4, 15). Repetitive sequences and amplified fragment length polymorphisms have been used to fingerprint phytopathogens and is accurate, but due to the generation of 30 or more fragments for each isolate tested, both methods require
computer analysis for identification, which is not available to many
laboratories (20).
16S rRNA gene sequencing has been used to study the phylogenetic
relationships between Erwinia species (14, 18).
Although the taxonomy of the E. carotovora subspecies
has been examined by this method and does allow identification of
species and subspecies, sequencing for routine identification is
impractical at the present time. In addition, this method approaches
its limits of sensitivity below the species level (34).
The multicopy 16S-23S intergenic transcribed spacer (ITS), which
separates the rRNA genes, however, exhibits a greater sequence and
length variation that can be exploited in a simple PCR-RFLP-based test
and is suitable for differentiating below the species level (12,
13, 20, 22, 30). Jensen et al. (17) developed
universal primers and conditions for amplifying the ITS from all
prokaryotes, and these primers have been used subsequently to identify
both human (13, 22) and plant (12) pathogens.
In a limited number of cases this has been combined with RFLP analysis
of the ITS product (12, 30). This study describes the use
of ITS-PCR, in combination with ITS-RFLP, for the rapid and accurate
identification and differentiation of the soft rot erwinias.
Bacterial strains and media.
Bacterial strains used in this
study are listed in Tables 1 and
2. Reference strains are species type strains obtained
from the National Collection of Plant Pathogenic Bacteria (NCPPB; York, United Kingdom). In cases in which type strains were not available from
the NCPPB, strains identified by the NCPPB as belonging to certain
species and subspecies were used. In addition, a number of
uncharacterized and "atypical" strains were investigated. Bacterial strains were stored in freezing medium at
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
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
-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.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
80°C (1).
All cultures used in the study were maintained on nutrient agar at
18°C (Oxoid, Basingstoke, United Kingdom). When required,
Erwinia species and saprophytes were grown at 27°C, and
other enterobacteria were grown at 37°C in Luria-Bertani broth (LB)
for 18 h with shaking.
TABLE 1.
Soft rot erwinia strains together with ITS-PCR and
ITS-RFLP patterns
TABLE 2.
Other soft rot and non-soft rot erwinia
strainsa
Biochemical and phenotypic tests.
Biochemical tests
acid
production from -methyl glucoside, palatinose, sorbitol, melibiose,
and lactose, the production of phosphatase and indole, reducing
substances from sucrose, the utilization of citrate, and growth in 5%
NaCl and on nutrient agar at 37°Cwere done as described previously
(8). Cavity formation on CVP medium at 27, 33.5, and
37°C was also assessed as described earlier (27).
Isolation of genomic DNA.
Bacterial genomic DNA (gDNA) was
extracted and purified using a DNA Mini Kit as described by the
manufacturer (Qiagen, Crawley, United Kingdom). Extracted DNA was
electrophoresed through a 1.2% agarose gel in Tris-borate-EDTA
(TBE) buffer and stained with ethidium bromide (0.5 µg
ml
1) as described previously (31).
DNA was stored at 20°C until required.
E. carotovora subsp. atroseptica-specific PCR. E. carotovora subsp. atroseptica gDNA was amplified using the E. carotovora subsp. atroseptica-specific primers ECA1f and ECA2r (6) obtained from MWG Biotech (Milton Keynes, United Kingdom) using a modified protocol described earlier (16).
PCR amplification and restriction digestion of the ITS. After the extraction of gDNA, the ITS was amplified using the primers G1 (5'-GAAGTCGTAACAAGG-3') and L1 (5'-CAAGGCATCCACCGT-3') as described by Jensen et al. (17). For each strain, 10 µl of the amplified product was digested with each of the restriction enzymes AluI, CfoI, HaeIII, HhaI, HpaII, MseI, MspI, MboI, RsaI, Sau3aI, TaqI, and ThaI, as described by the manufacturer (Life Technologies, Paisley, United Kingdom). Digested samples (10 µl) were electrophoresed through a 2% NuSieve GTG agarose gel (Flowgen, Ashby de la Zouch, United Kingdom) in TBE buffer for 1.5 h and stained as described above. Gel images were digitized and band sizes analyzed by Gel Compar software (Applied Maths, Kortrijk, Belgium). Fragment sizes were determined by comparison to a 1-kb Plus DNA molecular weight marker (Life Technologies, Paisley, United Kingdom).
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RESULTS AND DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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The main aim of this study was to develop a method, or to utilize an existing method, for the simple, rapid, and accurate identification and differentiation of the soft rot erwinias. This was accomplished by using a combination of ITS-PCR and ITS-RFLP.
After PCR amplification of the ITS (ITS-PCR) from all
Erwinia species, other plant and/or animal pathogens, and
plant and/or soil saprophytes, characteristic banding patterns were
generated on NuSieve agarose gels (Fig.
1) derived from multicopy rRNA operons (20). The majority of bands were clearly visible each time
that a PCR was carried out (primary bands). However, fainter bands did appear on occasion (secondary bands), but these proved less reliable for identification. Band sizes were thus recorded for primary
products only (Table 3). Bands from a
number of isolates were sequenced to reveal that each consisted of DNA
from the ITS region with deletions and/or insertions determining the
size of each band (data not shown). ITS-PCR generated unique patterns for all bacterial species tested (Fig. 1), and in most cases, these
patterns were similar for isolates within a species. Patterns generated
for human pathogens were similar but not identical to those described
by Jensen et al. (17), a result that might have been due
to the use of different strains (data not shown).
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Isolates of E. carotovora subspecies, E. chrysanthemi, and the closely related E. cacticida yielded unique banding patterns that clearly distinguished them from other Erwinia and non-Erwinia species tested (Fig. 1, lanes 1 to 8 and lanes 14 to 19). Primary products ranged from three to seven in number and from 440 to 1,170 bp in size. Within the soft rot erwinias, three PCR groups were distinguished based on differences in their banding patterns (groups I to III). Group I comprised E. carotovora subsp. atroseptica and subsp. betavasculorum and generated two patterns, each characteristic of a subspecies (Fig. 1, lanes 1 to 2). However, the difference between band sizes, i.e., a slight shift in the larger band, was deemed insufficient for a reliable identification. Group II comprised E. carotovora subsp. carotovora, subsp. odorifera, and subsp. wasabiae and E. cacticida and generated four different patterns (Fig. 1, lanes 3 to 8). Two of the patterns were present in both E. carotovora subsp. carotovora and E. carotovora subsp. odorifera, while the other two patterns were characteristic of E. carotovora subsp. wasabiae and E. cacticida, respectively. Again, small differences in band sizes were insufficient to distinguish reliably the members within group II. Groups I and II were clearly related based on the banding patterns generated. Group III comprised all E. chrysanthemi isolates and generated six different but related patterns (Fig. 1, lanes 14 to 19; Table 2), which differed from those of groups I and II (Fig. 1, lanes 1 to 8; Table 2). The variation within group III made identification difficult, especially since strains from other non-Erwinia species, e.g., Pantoea agglomerans (strain SCRI 459) and Enterobacter nimipressuralis (strain SCRI 491), gave similar patterns (Fig. 1, lanes 23 to 25).
To improve the level of discrimination between the soft rot erwinias
further, ITS-PCR products were digested with either one or two of 12 four-base cutting restriction enzymes (ITS-RFLP). Of these enzymes, two
(CfoI and RsaI), used individually, produced restriction patterns that allowed identification and differentiation of
most species and subspecies within the soft rot groups (I to III) (Fig.
2). Double digests did not improve
the level of discrimination and were not used further. However, whether
CfoI or RsaI was used individually depended
on the ITS-PCR group (I to III), i.e., CfoI was used only
after identification of group I and III isolates, whereas
RsaI was used only for group II isolates. This prevented misidentification; e.g., the patterns generated for E. carotovora subsp. carotovora, subsp.
odorifera, and subsp. wasabiae (group II) using
CfoI were identical to that of E. carotovora
subsp. atroseptica (group I) (data not shown). After
identification of groups I and III using ITS-PCR, digestion with
CfoI clearly distinguished E. carotovora
subsp. atroseptica from E. carotovora subsp.
betavasculorum isolates (Fig. 2, lanes 1 to 2) and
distinguished the E. chrysanthemi (group III) isolates
from non-Erwinia species (Fig. 2, lanes 3 to 5).
E. carotovora subsp. atroseptica and subsp.
betavasculorum produced subspecies-specific patterns that
were identical for all isolates tested. E. chrysanthemi
isolates produced only three characteristic patterns (E. chrysanthemi1, E. chrysanthemi2, and E. chrysanthemi3) from the six ITS-PCR patterns
generated. RsaI digestion of group II products produced
characteristic patterns that could identify isolates of E. carotovora subsp. wasabiae, subsp.
carotovora, subsp. odorifera, and subsp.
carotovora and E. cacticida. However, it was not
always possible to differentiate E. carotovora subsp. carotovora and subsp. odorifera, since some
E. carotovora subsp. carotovora strains
appear to be too closely related to E. carotovora subsp. odorifera at the ITS DNA level (Fig. 2, lanes 6 to
13). This was evident by the generation of identical banding
patterns in many, but not all, cases using both ITS-PCR and
ITS-RFLP (Fig. 1 and 2) and has been shown previously by DNA-DNA
hybridization (10) and RFLP analysis (4, 15).
It was thus necessary to differentiate E. carotovora subsp. odorifera and subsp.
carotovora isolates using the -methyl glucoside test:
positive for E. carotovora subsp. odorifera
and negative for E. carotovora subsp.
carotovora (10), which could take a further 3 to 5 days. The E. carotovora subsp.
carotovora isolates tested produced five different
restriction patterns (E. carotovora subsp.
carotovora1,
carotovora2,
carotovora3,
carotovora4, and
carotovora5), while E. carotovora subsp. wasabiae and E. cacticida isolates produced their own characteristic patterns. All
E. carotovora subsp. odorifera isolates
produced a single pattern that was identical to E. carotovora subsp. carotovora2.
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Individual isolates within E. chrysanthemi and E. carotovora subsp. carotovora generated several different banding patterns with ITS-PCR and ITS-RFLP, respectively, showing a higher level of diversity within these than within other species or subspecies. This increased diversity has been shown previously at both the phenotypic and the genotypic levels (4, 7, 15, 21, 23, 26, 35). Although it is likely that new isolates could generate further patterns, a combination of ITS-PCR and ITS-RFLP would still allow the identification of these pathogens and their differentiation from other bacterial strains, including other soft rot erwinias.
To determine the effectiveness of ITS-PCR and ITS-RFLP in practice, 60 unidentified isolates from Australia (believed to be soft rot erwinias
due to their growth and cavity formation on CVP) were tested. After
ITS-PCR, all three soft rot groups were present within these isolates:
group I (13 isolates), group II (44 isolates), group III (one isolate),
and cavity-forming non-soft rot erwinias (2 isolates). After PCR,
ITS-RFLP identified all group I isolates as E. carotovora subsp. atroseptica (13 isolates), group II
isolates as E. carotovora subsp. carotovora
(26 isolates), and E. carotovora subsp.
carotovora/odorifera (18 isolates), and group III
isolates as E. chrysanthemi (1 isolate). These isolates were also extensively tested using phenotypic, biochemical (including -methyl glucoside) and E. carotovora subsp.
atroseptica-specific PCR-based tests, which confirmed the
above results and identified all 18 of the E. carotovora subsp. carotovora/odorifera
isolates as E. carotovora subsp. carotovora
(data not shown). This latter result was consistent with the use of the
-methyl glucoside test alone. ITS-PCR plus ITS-RFLP thus proved to
be considerably faster, more convenient, and more accurate than other
identification methods (CVP-differential temperature, biochemistry, and
E. carotovora subsp. atroseptica-specific
PCR) used in our laboratory and used in the present study to validate
the new method.
This success was further supported when 12 "atypical" isolates (10 "atypical" E. carotovora subsp. atroseptica and 2 "atypical" E. carotovora subsp. carotovora isolates) were tested. Of 10 "atypical" E. carotovora subsp. atroseptica isolates tested by ITS-PCR, 2 were placed in group I, 6 were placed in group II, and 2 gave non-soft rot erwinia patterns. ITS-RFLP identified the group I isolates as E. carotovora subsp. atroseptica (one isolate) and E. carotovora subsp. betavasculorum (one isolate) and the group II isolates as E. carotovora subsp. carotovora (six isolates). After ITS-PCR, the two "atypical" E. carotovora subsp. carotovora isolates were placed in groups I and II and were identified as E. carotovora subsp. atroseptica and E. carotovora subsp. carotovora by using ITS-RFLP. Again, the identification of the isolates was confirmed using phenotypic, biochemical, and E. carotovora subsp. atroseptica-specific PCR-based tests. Only 2 of the 12 strains tested were correctly identified at the time of isolation, although correct identifications, at that time, may have provided important information about the epidemiology of these organisms and their role in disease. In addition, using ITS-PCR/RFLP we were able to identify non-soft rot species, which were selected initially for their cavity formation on CVP and which originally led to misidentification of disease symptoms.
The Erwinia genus includes a diverse group of pathogens that
cause disease on a wide variety of plants (28). However,
visual disease symptoms are not always sufficient to make an
unequivocal identification of the pathogen involved. This is especially
true when any one of a number of soft rot erwinias can lead to the development of similar disease symptoms on a common host, e.g., E. carotovora subsp. atroseptica, subsp.
carotovora, and subsp. wasabiae and E. chrysanthemi all cause similar diseases on potato (11,
28). Thus, to ensure that a correct diagnosis is made and that
steps are taken toward reducing disease spread, identification systems
are required. While this can be achieved using detection systems that
identify an organism directly from plant extract, e.g., PCR-based
diagnostics (33), some soft rot erwinias, such as
E. carotovora subsp. carotovora, are widely
distributed in the environment and on plant surfaces, which can lead to
false-positive results. The direct detection of a soft rot erwinia in
plant extract is thus not necessarily an indication of its role in
disease, although it is a useful initial screen. Ultimately, the
isolation of the causative agent is needed, followed by a robust
identification method. ITS-PCR plus ITS-RFLP is such a method and
has proved to be the most simple, rapid, and with the exception of
biochemical testing, most accurate method currently available for the
identification of the soft rot erwinias. This is especially true when
large numbers of isolates are tested. ITS-PCR alone is a useful method
for the rapid identification of soft rot erwinia isolates (requiring
24 h, including DNA extraction) where an assumption has been made as to the species and/or subspecies present on a given host plant, e.g., E. carotovora subsp. atroseptica or
subsp. carotovora or E. chrysanthemi on
potato. However, it is recommended that ITS-PCR be used in conjunction
with ITS-RFLP in all cases so as to obtain an accurate identification
(requiring 48 h, including DNA extraction). When E. carotovora subsp. carotovora and subsp.
odorifera cannot be differentiated, it would still be
necessary to undertake an -methyl glucoside test, adding an
additional 3 to 5 days to the identification. However, this single
biochemical test is relatively simple to perform and requires minimal
labor time compared to performing multiple biochemical tests, which can
take up to 14 days. The method also appears to be suitable for the
identification of other Erwinia species, although more
testing is required to confirm this. Based on this information,
sequence analysis of the ITS may allow the phylogenetic relationships
between different soft rot and other Erwinia species to be determined.
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ACKNOWLEDGMENTS |
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This work was funded by the Scottish Executive Environment and Rural Affairs Department (SEERAD) and the British Potato Council.
We are grateful to Trevor Wicks and Barbara Morgan, The University of Adelaide, Adelaide, South Australia, Australia, for supplying isolates.
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FOOTNOTES |
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* 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.
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
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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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