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Applied and Environmental Microbiology, May 2000, p. 2071-2078, Vol. 66, No. 5
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Development of a Highly Sensitive Nested-PCR Procedure Using a
Single Closed Tube for Detection of Erwinia amylovora
in Asymptomatic Plant Material
Pablo
Llop,1
Anna
Bonaterra,2
Javier
Peñalver,1 and
María M.
López1,*
Instituto Valenciano de Investigaciones
Agrarias, 46113 Moncada (Valencia),1 and
Instituto de Tecnologia Agroalimentaria, Laboratorio de
Producción Vegetal, Universitat de Girona, 17071 Girona,2 Spain
Received 16 August 1999/Accepted 7 February 2000
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ABSTRACT |
A novel method, which involves a nested PCR in a single closed
tube, was developed for the sensitive detection of Erwinia amylovora in plant material. The external and internal primer pairs used had different annealing temperatures and directed the amplification of a specific DNA fragment from plasmid pEA29. The procedure involved two consecutive PCRs, the first of which was performed at a higher annealing temperature that allowed amplification only by the external primer pair. Using pure cultures of E. amylovora, the sensitivity of the nested PCR in one tube was
similar to that of a standard nested PCR in two tubes. The specificity
and sensitivity were greater than those of standard PCR procedures that
used a single primer pair. The presence of inhibitors in plant
material, very common in E. amylovora hosts, is overcome
with this system in combination with a simple DNA extraction protocol
because it eliminates many of the inhibitory compounds. In addition, it
needs a very small sample volume (1 µl of DNA extracted). With 83 samples of naturally infected material, this method achieved better
results than any other PCR technique: standard PCR detected 55% of
positive samples, two-tube nested PCR detected 71% of positive
samples, and nested PCR in a single closed tube detected 78% of
positive samples. When analyzing asymptomatic plant material, the
number of positive samples detected by the developed nested PCR was
also the highest, compared with the PCR protocols indicated previously (17, 20, and 25% of 251 samples analyzed, respectively). This method
is proposed for the detection of endophytic and epiphytic populations
of E. amylovora in epidemiological studies and for routine
use in quarantine surveys, due to its high sensitivity, specificity,
speed, and simplicity.
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INTRODUCTION |
Erwinia amylovora, the
causal agent of fire blight, is one of the most destructive
plant-pathogenic bacteria, affecting different rosaceous species of
economical importance (pear, apple, loquat, and several ornamental
species). This pathogen moves from one geographical area to another in
very diverse and effective ways (3, 7, 34, 40, 41, 44) and
in the last 20 years has undergone a rapid spread to many countries
around the world (35, 42; T. van der Zwet, Abstr.
8th Int. Workshop Fire Blight, p. 30, 1998). E. amylovora
can survive as an endophyte and an epiphyte (5, 8, 17), and
its systemic distribution in plants has been demonstrated (28,
36). This has prompted in the last years an increasing interest
for reliable and sensitive methods to analyze potentially infected but
symptomless plant material, because the inadvertent introduction of
infected plants to pathogen-free areas could result in the unstoppable
spread of E. amylovora (10). This in fact might
have been the reason for some of the outbreaks in certain Mediterranean
countries, which for many years have imported host plants from North
European countries where the disease is present.
The already available methods (2, 6, 9, 11, 13, 14, 15, 18, 21,
23, 26, 27, 31, 35) allow reliable detection of the pathogen with
a relatively good level of sensitivity in plant material with symptoms,
but all have some drawbacks. Isolation takes several days and needs
confirmation of the identity of the pathogen by other techniques
(6, 15, 21). Serological techniques are not sensitive
enough, except the enrichment-enzyme-linked immunosorbent assay
(ELISA) method (9), although it requires 3 days to complete
and the sensitivity could be affected by other bacteria present in the
sample. PCR inhibitors, which are very common in fire blight hosts,
present a serious drawback for conventional PCR techniques (13,
23, 27, 32). Furthermore, the actual population of epiphytic and
endophytic E. amylovora in symptomless plant material could
be well below the detection levels of these techniques. The
implementation of methodologies that overcome the above problems is
therefore necessary. In countries affected by fire blight, such methods
could help to improve the knowledge of the pathogen life cycle under
their specific ecological conditions. Additionally, the availability of
simple and sensitive protocols to analyze imported material and to
perform quarantine surveys is crucial in those countries that are still
free of the disease.
The rapidity and sensitivity of detection of this pathogen are
desirable characteristics that have been met by the use of a nested-PCR
procedure (27). However, the introduction of a second
amplification step, and the concomitant manipulation of the previously
amplified material, could lead to a significant increase of false
positives due to cross-contamination, making this approach too risky
for routine analysis. A realistic alternative to avoid the manipulation
of the PCR tubes between the first and second round of amplification is
the nested PCR in one tube (25, 27, 29, 30).
In this study, we describe the development of a nested PCR in a single
closed tube which gives sensitivity levels equal to or higher than
those of previous detection methods and saves both time and reagents.
This method greatly reduces the cross-contamination risks and, due to
the low volume of sample used, is unaffected by the presence of PCR
inhibitors. The application of this method to several host plants
(apple, loquat, pear, quince, Cotoneaster spp.,
Crataegus spp., and Pyracantha spp.) and
different plant material (flowers, buds, shoots, stems, fruits, and
leaves) produced satisfactory results in all cases. Combined with an
efficient DNA extraction protocol previously developed in our
laboratory (22), this procedure could be used as a rapid and
sensitive technique for the routine detection of E. amylovora in plant material.
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MATERIALS AND METHODS |
Bacterial strains and sensitivity studies.
The E. amylovora strains employed in this study and their origins are
listed in Table 1. The
specificity tests were carried out with 71 E. amylovora
strains, 24 strains from other plant pathogenic species (one
Agrobacterium tumefaciens strain, one Agrobacterium
vitis strain, one Brenneria nigrifluens strain, one
Brenneria rubrifaciens strain, one Brenneria
quercina strain, six Pectobacterium carotovorum subsp.
carotovorum strains, one Pseudomonas corrugata strain, eight
Pseudomonas syringae strains, one Pseudomonas
savastanoi pv. savastanoi strain, one Ralstonia solanacearum strain, one Xylophilus ampelinus strain,
and one Xanthomonas vesicatoria strain) and 16 strains of
saprophytic bacteria isolated from fire blight hosts (5 identified as
Pantoea agglomerans strains and 11 identified as
Pseudomonas fluorescens strains). All the strains were grown
on King's medium B (18) at 25°C for 48 h, and a
suspension of each culture (ca. 108 CFU/ml) was prepared
for the PCRs in sterile ultrapure water.
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TABLE 1.
Summary of specificity assays with several E. amylovora strains using the nested procedure in one closed tube
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Serial dilutions ranging from 7 × 10
7 to 7 CFU/ml
were made from a concentrated suspension of
E. amylovora
strain PMV 6089
(mutant of strain CFBP 1430), and 5 µl from each was
used to compare
the sensitivity of the different PCRs (Table
2). Similar sensitivity
assays were
performed with bacterial suspensions added to pear,
apple, and
Pyracantha extracts obtained from comminuted shoots
of
greenhouse-grown plants in the buffer described by Gorris et
al.
(
9) (phosphate-buffered saline [pH 7.2] with 2%
polyvinylpyrrolidone
10, 1% mannitol, 10 mM ascorbic acid, and 10 mM
reduced glutathione).
The bacterial suspensions were mixed with the
plant extracts to
give a final concentration ranging from 5 × 10
5 to 5 CFU/ml. Bacterial counts were in all cases
confirmed by
plating 50 µl from each dilution in triplicate on
King's medium
B. With these samples, a simple DNA extraction protocol
was used
(
22). Briefly, 1 ml of sample was centrifuged at
10,000 ×
g for 10 min. The pellet was resuspended in
500 µl of extraction
buffer (200 mM Tris-HCl [pH 7.5], 250 mM NaCl,
25 mM EDTA, 0.5%
sodium dodecyl sulfate, 2% polyvinylpyrrolidone),
vortexed, and
left for 1 h at room temperature with continuous
shaking. After
centrifugation, 450 µl of the supernatant was taken,
mixed gently
with 450 µl of isopropanol, and left for 1 h at
room temperature.
The mixture was centrifuged, the supernatant was
discarded, and
the dried pellet was resuspended in 200 µl of sterile
water. Five
microliters of DNA extract was used for standard PCRs
(
2,
11,
23,
27) and for the first round of the two-tube
nested-PCR
assay (
27), while 1 µl was used for the nested
PCR in a single
closed tube. All the analyses were performed twice.
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TABLE 2.
Sensitivities of several sets of primers designed for the
detection of E. amylovora by various
PCR proceduresa
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Naturally infected samples.
From naturally infected plants,
we selected 83 samples that included material from parts of the plants
without symptoms as well as from organs showing fire blight symptoms
(Table 3). Also, 251 samples from
symptomless pear, apple, loquat, quince, Pyracantha sp.,
Cotoneaster sp., and Crataegus sp. plants were
obtained from different plots close to others with infected plants
where an outbreak was detected, to monitor for potential dissemination of the pathogen (Table 4). The samples,
consisting of flowers, buds, leaves, stems, and/or fruits, were
prepared according to the European Plant Protection Organization (EPPO)
methodology (6) for plants with symptoms using the buffer
described previously (9). For symptomless plants, the EPPO
method for plants with symptoms was also followed. In all samples,
isolation was performed according to the standard procedure
(6) on King's medium B (18) and on selective CCT
medium (15). Enrichment-ELISA-double-antibody sandwich
indirect (DASI) using specific monoclonal antibodies was assayed
(9) after an enrichment step in King's medium B and in CCT
medium (15, 18), and the DNA was extracted as described above. Greenhouse plant material previously determined to be free of
the bacterium was interspersed among the samples to monitor potential
cross-contamination during sample preparation. Additionally, up to five
negative controls were also placed among the sample tubes during PCR
analysis. All the PCR analyses, including the DNA extraction from each
sample, were repeated at least twice.
PCR design and comparison of amplifications.
We have
designed the nested PCR in a single closed tube considering primers
previously described because they have shown a good sensitivity and
specificity in this study and in our previous work. The criteria we
used for selecting the external and internal primer pairs were (i) the
external primer pair should amplify a fragment large enough to permit
the design of an appropriate internal couple, (ii) annealing
temperatures of the primer pairs should allow for the separation of
both PCRs only by this parameter, and (iii) high sensitivity of the
primers, to increase as much as possible the detection threshold of the
nested PCR in one tube. The standard PCRs were performed as described
by Bereswill et al. (2), using primers A and B; by McManus
and Jones (27), using primers AJ75-AJ76; by Maes et al.
(23), using primers EAF-EAR; and by Guilford et al.
(11), using primers EA71-EA72. After some sensitivity
assays, we choose as external primers those designed by McManus and
Jones (27), which were used at an annealing temperature of
72°C. We then designed as internal pair the primers PEANT1
(5'-TATCCCTAAAAACCTCAGTGC-3') and PEANT2
(5'-GCAACCTTGTGCCCTTTA-3'), which lie within 844 bases of
the fragment from the 29-kb plasmid pEA amplified by the external pair
(27). Since PEANT1 and PEANT2 produced amplification
products at 56°C but not at 72°C, it was thus possible to separate
the activity of the internal and the external primer pairs by modifying
the annealing temperature. PCRs were performed in a final volume of 50 µl with the following reagents: 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 3 mM MgCl2, 3% (vol/vol) formamide, a 200 µM concentration
of each deoxynucleoside triphosphate, 0.03 pmol each of external
primers AJ75 and AJ76 (27), 10 pmol each of internal primers
PEANT1 and PEANT2, and 3 U of Taq polymerase (Gibco BRL).
The reaction conditions were a denaturation step of 94°C for 4 min
followed by 25 cycles of 94°C for 30 s and 72°C for 1 min.
This first round of PCR was followed in the same thermocycler by a
second denaturation step of 94°C for 4 min and 40 cycles of 94°C
for 30 s, 56°C for 30 s, and 72°C for 45 s. The PCR
products were visualized after electrophoresis on 1.5% agarose gels.
Restriction fragment length polymorphisms and sequencing.
The restriction pattern of the amplification products obtained from the
bacterial suspensions was examined with DraI and
SmaI (Amersham/Pharmacia Biotech) to confirm their identity.
The fragments amplified from strains CFBP 1430 and PVM 6089 with the
primers designed by Bereswill et al. (2) were excised from
the gel, purified using the Concert Nucleic Acid purification kit
(Gibco BRL), and sequenced with the same primers. The resulting
sequences were then compared to the corresponding sequence obtained
from strain CA11 by McManus and Jones (27) (GenBank
accession no. U19245) using the program CLUSTAL W, version 1.5 (39).
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RESULTS |
Sensitivity and specificity tests.
We compared the sensitivity
of the nested PCR in a single closed-tube assay we developed with that
of a two-tube nested PCR and other PCR procedures that used a single
primer pair. The results of sensitivity assays performed with pure
E. amylovora cultures are shown in Table 2. The sensitivity
of the nested PCR in a single closed tube and the two-tube nested
assays was 7 × 10
1 CFU/ml, while in the best case
it was possible to detect only 7 × 10 cfu/ml with the standard
PCR procedures. When the assays were carried out with plant extracts
spiked with bacteria, the sensitivity levels of the nested procedures
were slightly reduced, to 5 CFU/ml, although they were still 100 to
1,000 times more sensitive than the standard one-round PCR assays. For
the sensitivity assays with plant material, we tested the effects of
different amounts of sample volume, looking for a balance between
minimum inhibitory effects of the extract on the PCR and the maximum
sensitivity (data not shown). For the standard PCRs, the volume was 5 µl, and for the two-tube nested PCR it was 5 µl in the first round and 2 µl in the second. For the nested PCR in a single closed tube,
only 1 µl of sample was necessary to obtain the strongest band signals.
The specificity of the procedure developed in this work was tested
using pure cultures of 40 strains from 14 species of phytopathogenic
and saprophytic bacteria. No unspecific banding was observed with
any
of the bacteria analyzed (data not shown), while all of the
71
E. amylovora strains examined produced a single amplification
band
(Table
1). With only three of these strains the amplified
fragment was
447 bp long, as predicted from the sequence obtained
from strain CA11
(P. McManus and A. Jones, accession no.
U19245)
from which the primers
were designed, while 63 strains produced
391-bp fragments and bands of
intermediate size were amplified
from 5 strains (Table
1; Fig.
1). Nonetheless, digestion of the
amplicons with
DraI or
SmaI in all cases produced
two fragments
whose sizes were as predicted or slightly smaller,
supporting
the identity of the amplified fragment. To investigate the
reasons
for the discrepancies in the size of the amplicons, we obtained
the nucleotide sequence of the fragments amplified from strains
CFBP
1430 and PMV 6089 (391 bp). Both sequences were identical
and showed a
deletion of 56 nucleotides with respect to the sequence
from strain
CA11, comprising seven 8-bp tandem repeats (GAATTACA)
(Fig.
2).
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FIG. 1.
Diversity of fragments obtained after amplification
following the nested-PCR method in a single closed tube. The sizes
vary, including the expected 447 bp (lanes 4 and 7), 391 bp (lanes 1, 2, 3, and 9), and intermediate values (lanes 5, 6, 8, 10, and 11).
Numbered lanes contain samples from the NCPPB collection (strain number
and country of origin are given in parentheses): lane 1, 2292 (United
States); lane 2, 2293 (United States); lane 3, 2950 (United States);
lane 4, 311 (Canada); lane 5, 683 (United Kingdom); lane 6, 1734 (Egypt); lane 7, 1819 (United States); lane 8, 2080 (New Zealand); lane
9, 2791 (United States); lane 10, 3159 (The Netherlands); lane 11, 3548 (Turkey); lane M, marker (100-bp DNA ladder; Gibco BRL).
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FIG. 2.
Location and extent of the deletion in the amplified
fragments. The fragments amplified by the one-round PCR using primers
designed by Bereswell et al. (2) from strains CFBP 1430 and
PMV 6089 that gave a band of 391 bp by the nested PCR in a single
closed tube were sequenced and compared to the corresponding sequence
of strain CA11 (447 bp) using the program CLUSTAL W. For simplicity,
only the sequence surrounding the 56-bp deletion present in strains
CFBP 1430 and PMV 6089 is shown, since the rest was identical for the
three strains. The 8-bp repeats are indicated by arrows.
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Detection in naturally infected plant material.
To test its
suitability for routine analyses, the nested PCR in a single closed
tube was compared to standard one-round and two-tube nested procedures
(2, 23, 27) using naturally infected plant material. The
primers proposed by Guilford et al. (11) were not included
in this comparison due to their low sensitivity in bacterial cultures
and spiked plant material (Table 2).
We first tested our method with material from plants naturally infected
with
E. amylovora. All of the PCR procedures tested
detected
E. amylovora in samples both with and without symptoms,
although the nested procedure in a single closed tube allowed
the
detection of the pathogen in the largest number of samples:
65 positives out of 83 samples versus 46 positives in the best
case with
standard one-round PCRs and 59 for the nested PCR in
two tubes (Table
3). A further advantage of the nested PCR in
a single closed tube is
its greater specificity and thus a higher
reliability for diagnosis,
since no spurious bands were observed
in any of the samples analyzed in
this work. In contrast, using
the standard PCR procedures we commonly
observed the appearance
of several unspecific amplification bands that
hampered the interpretation
of the results, as shown in Fig.
3. The presence of the pathogen
in the
positive samples was confirmed by its isolation in culture
medium
and/or by enrichment-ELISA. The controls employed to monitor
the
reliability of the sample preparation and the PCRs were all
negative.
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FIG. 3.
Specificity of the nested PCR in a single closed tube
compared to that of other PCR methods. Samples were taken from
naturally infected plants and analyzed by one-round PCR using the
primers described by Bereswill et al. (2) (A), the primers
described by McManus and Jones (27) (B), the nested PCR
developed in this work (C). Note that the first two pairs of primers
produce unspecific amplifications. Samples: 1, Pyrus
communis 1892-b.1; 2, Pyracantha sp. 1952-b.5; 3, Pyracantha sp. 1952-b.9; 4, Pyracantha sp.
1952-b.11; 5, Pyrus communis 1961-d.1; 6, Pyrus
communis 1961-e.2; 7, Pyrus communis 1961-e.3; 8, Pyrus communis 1961-f; 9, Pyrus communis
1961-g.3; 10, Malus domestica 1899-h. All the samples were
positive except number 10. Sample number 6 gave a faint band. C+,
positive control; M, marker (100 bp; New England Biolabs). The negative
control is not shown in this figure.
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Secondly, we tested the validity of our method for routine detection by
assaying 251 samples from symptomless plants from
areas where fire
blight outbreaks were reported. The nested PCR
in a single closed tube
allowed the detection of the pathogen
in 62 samples, the standard
one-round PCRs allowed detection in
42 samples, and the two-tube nested
PCR allowed detection in 51
samples (Table
4). The samples that were
negative for
E. amylovora with the single closed-tube method
were also negative by the other
PCR analysis. Three months after the
analysis, six of the plants
in which the pathogen was detected by the
nested PCR in a single
closed tube, but not by the other PCR
procedures, developed typical
fire blight symptoms and the pathogen
could be recovered from
affected tissues. Fifty-three out of 62 of the
positive samples
obtained by the proposed method could be confirmed by
other methods
like isolation, enrichment-ELISA, and other PCR systems
(Table
5), so only nine samples remained
with no confirmation.
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DISCUSSION |
The importance of controlling the spread of the fire blight is
well known in the United States, the European Community, and other
countries (17, 42; van der Zwet, Abstr. 8th Int.
Workshop Fire Blight). Recent outbreaks in several countries
(10, 17; P. Battilani, L. Mazzoli, and U. Mazzuchi,
Abstr. 8th Int. Workshop Fire Blight, p. 17, 1998) show how difficult
the control of this disease is and how fast it spreads, even when
different measures of control are taken (4, 12, 16, 20, 24,
38, 43; Battilani et al., Abstr. 8th Int. Workshop Fire
Blight). In addition, no symptoms are observed in winter in deciduous
species, and the surveys, made mainly by visual detection of typical
lesions, are useless. Apparent healthy plants can carry latent
infections (5, 8, 23, 43), and from these E. amylovora could be distributed from nurseries to other parts of
the country or other countries, where it will only take favorable
conditions for symptoms to develop. As pointed out by other authors, in
spite of being a very useful and sensitive technique, PCR is still
seriously limited due to inhibition by different compounds (13,
23, 27, 32). In fact, our experience in diagnosing fire blight
has shown the importance of this problem, sometimes detecting fewer
positive samples by the standard PCR technique than by plating or
enrichment-ELISA (data not shown). The nested PCR in a single closed
tube developed in this work solves the main drawbacks of this
technique, since we overcome the problem of false-negative results by
reducing the volume of sample used, thus avoiding plant inhibitors, and by minimizing sample manipulations, which drastically reduces the
possibility of cross-contamination. The comparison of the two nested
systems with symptomless samples shows the inhibitory effect of the
plant material on the PCR. The slightly larger amount of sample volume
employed in the two-tube nested procedure (5 µl instead of 1 µl)
seems enough to affect the first round of PCR, and thus, the whole
nested reaction. The results obtained in the sensitivity assays are
concordant with what we expected from the nested technology
(27), the two nested systems being 100 to 1,000 times more
sensitive than the standard PCR systems. The highest sensitivity of the
nested PCR in a singleclosed tube, compared to the other PCR systems,
was observed with asymptomatic material, with 62 positive samples
versus 51 (two-tube nested PCR) and 42 (standard PCRs) (Table 4). In
this assay, some of the samples that were positive by the nested PCR in
a single closed tube were confirmed by other techniques, such as
isolation, enrichment-ELISA, or other types of PCR (Table 5).
Furthermore, 3 months later typical fire blight symptoms appeared in
some of the analyzed plants. The bacterium could then be isolated,
corroborating the presence of latent infections of E. amylovora, as demonstrated by other authors (5, 8, 23,
43). The use of this highly sensitive method allows a more rapid
detection of the pathogen in asymptomatic plants, since it overcomes
the need to wait for the results of time-consuming techniques and for
the appearance of typical symptoms. Moreover, we do not know much about
the survival of the bacterium or after applying different control
treatments (4, 16, 17, 24, 43; Battilani et al.,
Abstr. 8th Int. Workshop Fire Blight). Thus, this system could be
useful for monitoring the effectiveness of some of these methods.
The amplification of plasmid sequences for detection of a given
pathogen could produce misleading results if (i) plasmidless cells
remain virulent or (ii) the plasmid is transferred to other bacterial
species (1). Nevertheless, virulent E. amylovora strains without the plasmid have not been found in nature (1, 31), and the transfer of the plasmid to other species or to other
genera has not been reported. On the other hand, the advantages of
using primers designed to amplify pEA29 sequences are a higher sensitivity and specificity.
The size of the amplified bands was variable in samples from plant
material as well as from the E. amylovora collection strains analyzed. This can be explained by the variability in the number of
8-bp repeats in the amplified sequence, which has already been described (33). This was confirmed by the comparison of the sequence from strain CA11 with those of strains CFBP 1430 and PMV 6089, which were used as positive controls. Previous works (1, 19, 27,
33) have reported the amplification of different sized fragments,
rather than the 900 bp reported by Bereswill et al. (2),
from several strains from the United States and New Zealand
(33) and from Europe (19). Nevertheless, these small variations in the size of the amplicons do not compromise the
validity of the one-tube nested system for detection. The use of two
consecutive and specific amplification reactions greatly reduces the
possibility of obtaining false positives, while making it possible to
further confirm the identity of the amplified fragment by restriction analysis.
The combination of the nested PCR in a single closed tube with a simple
and effective DNA extraction protocol that involves little handling and
does not employ toxic compounds such as phenol or chloroform
(22) has led to very high levels of sensitivity. The large
number of species of naturally infected plant material tested and their
different origins show that the method developed here can be of
universal use for fire blight detection and epidemiological applications. The probability of contamination by amplicons under the
system presented here is as low as that with standard PCRs, although
the sensitivity is at least as good as that of the two-tube nested PCR,
thus allowing the implementation of the one-tube nested approach for
routine detection. As far as we know, this is the first development of
such a methodology for the detection of a bacterial plant pathogen, and
the features it presents could be applied to other plant-pathogenic bacteria.
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ACKNOWLEDGMENTS |
We thank J. Laurent, J. P. Paulin, D. Stead, N. Alivizatos,
D. Hayes, D. Berra, M. Borruel, and J. Murillo for kindly providing some of the E. amylovora strains employed and J. Cubero and
B. Lastra for critical reading of the manuscript. We give special thanks to J. Murillo for extensive revision of our English and useful comments.
We are grateful to the Subdirección General de Sanidad Vegetal,
MAPA, Madrid, Spain, CICYT project AGF98 0402CO302, and SMT project
4-CT98 2252 for funding.
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FOOTNOTES |
*
Corresponding author. Mailing address: Departamento de
Protección Vegetal y Biotecnología, Instituto Valenciano
de Investigaciones Agrarias, Apartado Oficial, Carretera
Moncada-Náquera km 4,5, 46113 Moncada, Valencia, Spain. Phone:
34-96-1391000. Fax: 34-96-1390240. E-mail: mlopez{at}ivia.es.
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REFERENCES |
| 1.
|
Bereswill, S.,
P. Bugert,
I. Bruchmuller, and K. Geider.
1995.
Identification of the fire blight pathogen, Erwinia amylovora, by PCR assays with chromosomal DNA.
Appl. Environ. Microbiol.
61:2636-2642[Abstract].
|
| 2.
|
Bereswill, S.,
A. Pahl,
P. Bellemann,
F. Berger,
W. Zeller, and K. Geider.
1992.
Sensitive and species-specific detection of Erwinia amylovora by PCR analysis.
Appl. Environ. Microbiol.
58:3522-3526[Abstract/Free Full Text].
|
| 3.
|
Clark, G. G.,
K. D. Hickey, and J. W. Travis.
1991.
Fireblight management: evaluation of the role of aphids in transmission of bacteria and development of a computerized management system for growers.
Penn. Fruit News
71:43-44.
|
| 4.
|
Covey, R. P., and W. R. Fischer.
1990.
Timely cutting of fire blight infections reduces losses.
Acta Hortic.
273:351-353.
|
| 5.
|
Crepel, C.,
J. Geenen,
M. Maes, and W. G. Bonn.
1996.
The latent survival of Erwinia amylovora in hibernating shoots.
Acta Hortic.
411:21-25.
|
| 6.
|
European Plant Protection Organization.
1992.
Quarantine procedure no. 40. Erwinia amylovora. Sampling and test methods.
Bull. EPPO
22:225-231.
|
| 7.
|
Ficke, W.,
F. Ehrig,
M. Nachtigall,
K. Naumann,
K. Richter,
H. J. Schaefer, and R. Zielke.
1990.
Possibilities for detecting the causal agent of fireblight (Erwinia amylovora (Burrill) Winslow et al.) in the air space of orchards.
Zentbl. Mikrobiol.
145:121-133.
|
| 8.
|
Ge, Q.,
T. van der Zwet, and W. G. Bonn.
1996.
Persistence and recovery of endophytic Erwinia amylovora in apparently healthy apple tissues.
Acta Hortic.
411:29-33.
|
| 9.
|
Gorris, M. T.,
M. Cambra,
P. Llop,
M. M. López,
P. Lecomte,
R. Chartier,
J. P. Paulin, and W. G. Bonn.
1996.
A sensitive and specific detection of Erwinia amylovora based on the ELISA-DASI enrichment method with monoclonal antibodies.
Acta Hortic.
411:41-46.
|
| 10.
|
Griffo, R.,
A. Tartaglia,
F. Capriolo, and F. Adamo.
1998.
Fire blight in Campania.
Inform. Agrar.
54:73-75.
|
| 11.
|
Guilford, P. J.,
R. K. Taylor,
R. G. Clark,
C. N. Hale,
R. L. S. Forster, and W. G. Bonn.
1996.
PCR-based techniques for the detection of Erwinia amylovora.
Acta Hortic.
411:53-56.
|
| 12.
|
Hasler, T.,
J. Vogelsanger,
B. Schoch, and W. G. Bonn.
1996.
Disinfection of fire blight contaminated tools.
Acta Hortic.
411:369-371.
|
| 13.
|
Henson, J. M., and R. French.
1993.
The polymerase chain reaction and plant disease diagnosis.
Annu. Rev. Phytopathol.
31:81-109[CrossRef][Medline].
|
| 14.
|
Hutschemakers, J.,
M. Verhoyen, and H. Bazin.
1987.
Production of rat monoclonal antibodies specific to Erwinia amylovora.
Acta Hortic.
217:71-76.
|
| 15.
|
Ishimaru, C., and E. J. Klos.
1984.
New medium for detecting Erwinia amylovora and its use in epidemiological studies.
Phytopathology
74:1342-1345.
|
| 16.
|
Keck, M.,
R. Chartier,
W. Zislavsky,
P. Lecomte, and J. P. Paulin.
1995.
Heat treatment of plant propagation material for the control of fire blight.
Plant Pathol.
44:124-129.
|
| 17.
|
Keck, M.,
H. Reich,
R. Chartier,
J. P. Paulin, and W. G. Bonn.
1996.
First record of fire blight (Erwinia amylovora) in Austria. Preliminary experiments on the survival on fruit boxes.
Acta Hortic.
411:9-11.
|
| 18.
|
King, E. O.,
M. Ward, and D. E. Raney.
1954.
Two simple media for the demonstration of pyocyanin and fluorescein.
J. Lab. Clin. Med.
44:301-307[Medline].
|
| 19.
|
Lecomte, P.,
C. Manceau,
J. P. Paulin, and M. Keck.
1997.
Identification by PCR analysis on plasmid pEA29 of isolates of Erwinia amylovora responsible for an outbreak in Central Europe.
Eur. J. Plant Pathol.
103:91-98.
|
| 20.
|
Lecomte, P., and J. P. Paulin.
1992.
Chemical control of bacterial blight: balance sheet and prospects after ten years of experimentation in France.
Fruit Belge
60:204-216.
|
| 21.
|
Lelliot, R. A., and D. E. Stead.
1987.
Methods for the diagnosis of bacterial diseases of plants.
Methods Plant Pathol.
2:1-216.
|
| 22.
|
Llop, P.,
P. Caruso,
J. Cubero,
C. Morente, and M. M. López.
1999.
A simple extraction procedure for efficient routine detection of pathogenic bacteria in plant material by polymerase chain reaction.
J. Microbiol. Methods
37:23-31[CrossRef][Medline].
|
| 23.
|
Maes, M.,
P. Garbeva, and C. Crepel.
1996.
Identification and sensitive endophytic detection of the fire blight pathogen Erwinia amylovora with 23S ribosomal DNA sequences and the polymerase chain reaction.
Plant Pathol.
45:1139-1149[CrossRef].
|
| 24.
|
Manceau, C.,
L. Gardan, and J. P. Paulin.
1987.
Use of antibiotics to control fire blight in France: environmental hazards and established legislation.
Acta Hortic.
217:195-202.
|
| 25.
|
Mathis, A.,
R. Weber,
H. Kuster, and R. Speich.
1996.
Reliable one-tube nested PCR for detection and SSCP-typing of Pneumocystis carinii.
J. Eukaryot. Microbiol.
43:7S[Medline].
|
| 26.
|
McLaughlin, R. J.,
T. A. Chen, and J. M. Wells.
1989.
Monoclonal antibodies against Erwinia amylovora: characterization and evaluation of a mixture for detection by enzyme-linked immunosorbent assay.
Phytopathology
79:610-613.
|
| 27.
|
McManus, P. S., and A. L. Jones.
1995.
Detection of Erwinia amylovora by nested PCR and PCR-dot-blot and reverse blot hybridizations.
Phytopathology
85:618-623[CrossRef].
|
| 28.
|
Momol, M.,
J. Norelli,
D. Piccioni,
E. A. Momol,
H. L. Gustafson,
J. N. Cummins, and H. S. Aldwinckle.
1998.
Internal movement of Erwinia amylovora through symptomless apple scion tissues into the rootstock.
Plant Dis.
82:646-650.
|
| 29.
|
Niepold, F., and B. Schober.
1997.
Application of the one-tube PCR technique in combination with a fast DNA extraction procedure for detecting Phytophtora infestans in infected potato tubers.
Microb. Res.
152:345-351.
|
| 30.
|
Orou, A.,
B. Fechner,
H. J. Menzel, and G. Utermann.
1995.
Automatic separation of two PCRs in one tube by annealing temperature.
Trends Genet.
11:127-128[Medline].
|
| 31.
| Paulin, J. P. In J. Vanneste (ed.), Fire
blight: the disease and its causative agent, Erwinia
amylovora, part II. The pathogen, in press.
|
| 32.
|
Pulawska, J.,
M. Maes,
T. Deckers, and P. Sobiczewski.
1997.
The influence of pesticide contamination on detection of epiphytic Erwinia amylovora using PCR, p. 959-962.
In
Proceedings of the 49th International Symposium on Crop Protection, vol. 62. , part IV.
|
| 33.
|
Schnabel, E. L., and A. L. Jones.
1998.
Instability of a pEA29 marker in Erwinia amylovora previously used for strain classification.
Plant Dis.
82:1334-1336[CrossRef].
|
| 34.
|
Seidel, M.,
E. Steffen,
D. Seidel, and A. Walter.
1994.
Survival of Erwinia amylovora (Burrill) Winslow et al. on bird feet.
Arch. Phytopathol. Plant Protection
29:25-27.
|
| 35.
|
Sobiczewski, P.,
T. Deckers, and J. Pu awska.
1997.
Fire blight (Erwinia amylovora): some aspects of epidemiology and control.
Research Institute of Pomology and Floriculture, Skierniewice, Poland.
|
| 36.
|
Steiner, P. W., and P. Suleman.
1993.
The systemic nature of fire blight: implications for control.
Penn. Fruit News
73:16-22.
|
| 37.
|
Thomson, S. V.,
S. C. Gouk, and A. J. Popay.
1992.
The effect of rain on the development of Erwinia amylovora and E. herbicola populations on apple flowers, p. 301-303.
In
Proceedings of the Forty-Fifth New Zealand Plant Protection ConferenceWellington, New Zealand.
|
| 38.
|
Thomson, S. V., and W. G. Bonn.
1996.
Solarization of pear and apple trees to eradicate bacteria in fire blight cankers.
Acta Hortic.
411:337-339.
|
| 39.
|
Thompson, J. D.,
D. G. Higgins, and T. J. Gibson.
1994.
CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties, and weight matrix choice.
Nucleic Acids Res.
22:4673-4680[Abstract/Free Full Text].
|
| 40.
|
de Wael, L.,
M. de Greef, and O. van Laere.
1990.
The honeybee as a possible vector of Erwinia amylovora (Burr.) Winslow et al.
Acta Hortic.
273:107-113.
|
| 41.
|
van der Zwet, T.
1994.
The various means of dissemination of the fire blight bacterium Erwinia amylovora.
Bull. EPPO
24:209-214.
|
| 42.
|
van der Zwet, T.
1996.
Present worldwide distribution of fire blight.
Acta Hortic.
411:7-8.
|
| 43.
|
van der Zwet, T., and S. V. Beer.
1995.
Fire blight: its nature, prevention and control, p. 1-91.
In
A practical guide to integrated disease management. U.S. Department of Agriculture Agriculture Information Bulletin 631, U.S. Department of Agriculture, Washington, D.C.
|
| 44.
|
van der Zwet, T., and R. L. Bell.
1993.
Survival of Erwinia amylovora on apple and pear pollen.
Acta Hortic.
338:111.
|
Applied and Environmental Microbiology, May 2000, p. 2071-2078, Vol. 66, No. 5
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Abstract
Introduction
