Previous Article | Next Article 
Applied and Environmental Microbiology, June 2000, p. 2673-2677, Vol. 66, No. 6
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Detection of Pseudomonas savastanoi pv.
savastanoi in Olive Plants by Enrichment and PCR
Ramón
Penyalver,
Amparo
García,
Amparo
Ferrer,
Edson
Bertolini, and
María M.
López*
Departamento de Protección Vegetal y
Biotecnología, Instituto Valenciano de Investigaciones
Agrarias, 46113 Moncada, Valencia, Spain
Received 30 November 1999/Accepted 21 March 2000
 |
ABSTRACT |
The sequence of the gene iaaL of Pseudomonas
savastanoi EW2009 was used to design primers for PCR
amplification. The iaaL-derived primers directed the
amplification of a 454-bp fragment from genomic DNA isolated from 70 strains of P. savastanoi, whereas genomic DNA from 93 non-P. savastanoi isolates did not yield this amplified product. A previous bacterial enrichment in the semiselective liquid
medium PVF-1 improved the PCR sensitivity level, allowing detection of
10 to 100 CFU/ml of plant extract. P. savastanoi was
detected by the developed enrichment-PCR method in knots from different
varieties of inoculated and naturally infected olive trees. Moreover,
P. savastanoi was detected in symptomless stem tissues from
naturally infected olive plants. This enrichment-PCR method is more
sensitive and less cumbersome than the conventional isolation methods
for detection of P. savastanoi.
| |
TEXT |
Pseudomonas savastanoi
and its pathovars savastanoi, fraxini, and nerii incite a disease of
olive (Olea europaea L.), ash (Fraxinus excelsior
L.), other Oleaceae plants and oleander (Nerium oleander L.) that is characterized by tumorous outgrowths (4, 19). This development of knots is dependent on bacterial
production of the phytohormone indoleacetic acid (IAA) and cytokinins
(3, 7, 16, 18). P. savastanoi can conjugate IAA
with lysine to form 3-indoleacetyl-
-L-lysine
(IAA-lysine) (6). The two enzymes involved in IAA
biosynthesis are tryptophan monooxygenase, which converts tryptophan to
indoleacetamide, and indoleacetamide hydrolase, which catalyzes the
conversion of indoleacetamide to IAA (12). The enzyme
involved in the conversion of IAA to IAA-lysine is
(indole-3-acetyl)-L-lysine synthethase (5). The
genes for tryptophan monooxygenase (iaaM), indoleacetamide
hydrolase (iaaH), and IAA-lysine synthethase
(iaaL) reside on the 52-kb plasmid pIAA1 in the oleander
P. savastanoi strain EW2009, and they have been sequenced
(3, 5, 14, 20). Sequence analysis revealed that
iaaM and iaaH have significant similarity with
homologous genes of other plant-associated bacteria (13,
20). In contrast, to date, no nucleotide homologies have been
found with the iaaL gene.
Detection of P. savastanoi is currently based on bacterial
isolation followed by pathogenicity tests and biochemical or
serological techniques (2, 8, 17, 21). These conventional
methods are time-consuming and expensive, requiring bacterial
isolation. We used the published sequence of iaaL
(14) to design specific primers for amplification of this
gene. We report here the development of a new sensitive and specific
detection method for P. savastanoi based on amplification of
iaaL after a bacterial enrichment. The developed
enrichment-PCR assay can be applied to specifically detect low levels
of P. savastanoi in inoculated and naturally infected plants.
Specificity of the PCR assay.
Seventy strains of P. savastanoi isolated from olive, oleander, ash, and jasmine
(Jasminus officinalis L.) plants from different countries,
23 outgroup strains, and 70 saprophytic isolates from olive plants were
used to test for the specificity of the primers designed (Table
1). All strains were routinely grown in
King's medium B (9). The bacterial DNA preparation method
was based on the protocol described by Llop et al. (10). The
primers designed for iaaL amplification were as follows:
primer IAALF, 5'-GGCACCAGCGGCAACATCAA-3'; primer IAALR,
5'-CGCCCTCGGAACTGCCATAC-3'. PCR reactions were performed by
combining the following reagents in a reaction mix: 10× Taq buffer (GIBCO-BRL), 1.5 mM MgCl2, 5% formamide, 0.2 mM
concentrations of each deoxynucleoside triphosphate (Pharmacia LKB),
0.6 µM concentrations of each primer, and 1.5 U of Taq DNA
polymerase (GIBCO-BRL) per reaction. Then, 5 µl of the DNA extraction
from bacterial cultures (or plant samples) was added. Samples were
amplified through 1 cycle of 94°C (5 min), followed by 35 cycles of
94°C (30 s), 62°C (30 s), and 72°C (30 s) and then 1 cycle of
72°C for 5 min in a 9600 Perkin-Elmer thermocycler. Negative controls
with uninfected olive plants were included in every DNA extraction
series. Furthermore, to help detect carryover contamination, duplicate
samples and two sets of two negative controls each with sterile
purified water were routinely included in every reaction. One set was
loaded just after reaction mix preparation, and the other one was added just after loading the samples. Amplification was also conducted in a
separate laboratory. Amplified products (5 µl) were separated by
electrophoresis (100 V) on a 1.5% agarose gel or digested with the
restriction enzyme HaeIII (GIBCO-BRL) at 37°C for 2 h
and separated by electrophoresis (100 V) on a 2% agarose gel.
Electrophoresis gels were stained with ethidium bromide.
A 454-bp product was obtained from all
P. savastanoi
strains, showing that
iaaL sequences were present in strains
from different
countries and isolated from four hosts (Table
1). Two
fragments
of 279 and 175 bp were generated from all amplified fragments
after
HaeIII digestion, a result in agreement with
previously
published sequence data (
14). Genomic DNA from
outgroup species
did not produce any discrete bands upon amplification.
Similarly,
we found no amplification of the 454-bp fragment with
genomic
DNA from isolates of olive microbiota (Table
1). Only the DNA
from one saprophytic isolate identified by biochemical tests as
Pseudomonas putida gave a smaller product of ca. 440 bp upon
amplification.
However, it was perfectly distinguishable from that of
P. savastanoi because after
HaeIII digestion we
did not find the corresponding
pattern of
P. savastanoi iaaL
(Table
1). Formamide (5%) was required
in the amplification cocktail
to eliminate nonspecific
bands.
Detection of P. savastanoi in plants.
Olive plant
samples (1 g of stem tissue from uninfected greenhouse-grown plants
comminuted in 50 ml of sterile distilled water) were subsequently
amended with known amounts of P. savastanoi 1628 from ca. 1 to 107 CFU/ml of the plant extract. Then, 0.5 ml of amended
plant samples from each bacterial concentration was amplified after
performing the DNA extraction described above or was added to 5 ml of
the nonselective King's medium B (9) and the semiselective
PVF-1 medium (17) for bacterial enrichments. Samples were
incubated for 3 days at 26°C, and bacterial DNA extracts using 500 µl of each sample were then subjected to PCR amplification as
described above. Three replicates of the experiment were performed. The detection limit was 102 to 103 CFU/ml of plant
extract (Fig. 1A). A previous bacterial
enrichment using King's medium B slightly improved the detection
level, allowing detection of ca. 102 CFU/ml of plant
extract. However, preenrichment in PVF-1 improved the detection level,
allowing detection of ca. 10 to 100 CFU/ml of plant extract (Fig. 1B).
View larger version (66K):
[in this window]
[in a new window]
|
FIG. 1.
Sensitivity of the PCR assay in detecting the
iaaL gene of P. savastanoi pv. savastanoi in
amended plant samples without enrichment (A) or with PVF-1 enrichment
(B). PCR products from amended plant samples were inoculated with
P. savastanoi 1628. Lanes 1, molecular marker (M); lanes 2, PCR-negative control; lanes 3, noninoculated plant material; lanes 4 to
11, PCR-amplified products obtained from amended plant samples
inoculated with increased concentrations of bacteria from 1 to
107 CFU/ml of plant extract.
|
|
We then evaluated the detection of
P. savastanoi in knots
from inoculated and naturally infected plants. The stems of 1-year-old
olive plants were wounded and subsequently inoculated with 10
µl of a
bacterial suspension (ca. 10
9 CFU/ml) of strain 1628 to
develop knots. They were analyzed 8
months after inoculation. In
addition, knots from olive, oleander,
and Spanish broom (
Retama
sphaerocarpa [Boiss] L.) from naturally
infected plants were
also collected. From inoculated and naturally
infected samples, 500 µl of comminuted knot tissues in sterile
water (1:50 [wt/vol]) were
subjected to the protocol described
above for amended plant samples,
and subsequently the amplified
product was digested with
HaeIII. Conventional bacterial isolations
were performed
from each sample before or after bacterial enrichments
by plating
serial dilutions on King's B (
9) and PVF-1 (
17)
media. The identity of the putative
P. savastanoi colonies
was
confirmed by biochemical tests (
21) and by the developed
PCR
assay. In all of the knot samples analyzed from inoculated plants,
we found the expected PCR product of 454 bp and also the corresponding
restriction profile (Table
2). We were
also able to isolate the
inoculated
P. savastanoi strain
(Table
2). We compared the detection
efficiency of the developed PCR
assay with that of the isolation
method in knot samples from naturally
infected plants, either
with a previous bacterial enrichment step or
not. When analyzing
37 knot samples,
P. savastanoi was
detected by PCR in 21 of the
37 samples, without preenrichment (Table
2). One of these amplified
products was double strand sequenced, and
the obtained sequence
was 100% similar to the previous
iaaL
reported sequence. A preenrichment
in King's medium B did not improve
the detection of
P. savastanoi by PCR in knot tissues from
naturally infected plants because
P. savastanoi was detected
in only 13 samples. However, a preenrichment
in PVF-1 medium improved
the detection of
P. savastanoi by PCR
to up to 29 of the 37 samples (Table
2).
P. savastanoi was isolated
by plating in
some samples without enrichment (eight samples);
however, the detection
efficiency of bacterial isolation was not
improved by any preenrichment
(Table
2).
To evaluate the detection of
P. savastanoi in asymptomatic
olive tissues, stem samples were collected from branches without
knots
taken from naturally infected plants. A total of 1 to 10
g of stem
tissues was processed, and 0.5 ml of the obtained samples
was analyzed
as described above. Uninfected greenhouse-grown plants
were used as
negative controls. When analyzing 38 asymptomatic
samples,
P. savastanoi was detected by the developed PCR and subsequent
restriction analysis in only 4 of the 38 samples (Table
3). One
of these amplified fragments was
double strand sequenced showing
that it corresponded to the
iaaL gene. A preenrichment using either
King's B
(
9) or PVF-1 (
17) medium greatly improved the
detection
of
P. savastanoi by PCR in symptomless stem
samples (Table
3).
The highest detection level was found with the
enrichment-PCR
method using PVF-1, which allowed the detection of
P. savastanoi in 23 of the 38 samples.
P. savastanoi was isolated by plating
in 10 samples without
preenrichment; however, the detection level
obtained by plating was not
improved by any preenrichment in these
asymptomatic samples (Table
3).
In this study an enrichment-PCR assay was developed for the detection
of
P. savastanoi in olive plants, and its efficiency
was
compared by isolation plating. We evaluated the specificity
of the PCR
assay by testing for amplification of the 454-bp DNA
in a collection of
P. savastanoi strains and other bacteria. Genomic
DNA from
all of the
P. savastanoi strains tested was amplified
with
the
iaaL primers, and subsequent restriction analysis of
the
amplified product yielded only the expected fragmentation
pattern. It
shows that this gene is present among the isolates
of
P. savastanoi from olive, oleander, ash, and jasmine hosts.
Genomic
DNA from all non-
P. savastanoi strains tested, including
outgroup species and different bacteria associated with olive
plants,
failed to produce the 454-bp PCR product upon amplification
under the
conditions described
above.
A bacterial enrichment prior to serological and molecular techniques
has been reported to improve the sensitivity of detection
of other
plant pathogenic bacteria (
11,
15). For
P. savastanoi detection in amended plant samples, knots, and
symptomless stem
tissues from naturally infected plants, a bacterial
enrichment
prior to PCR analysis in PVF-1 liquid medium (but not in
King's
medium B) greatly improved the detection level. This shows the
higher efficiency of selective enrichment versus bacterial enrichment
in common medium. With the developed enrichment-PCR assay it is
possible to detect living cells of
P. savastanoi at low
population
levels in plant material, thus avoiding the need to obtain
bacterial
cultures as required for the conventional detection
techniques.
In knot samples from inoculated plants the developed PCR assay was as
efficient as plating isolation because we recovered
the inoculated
P. savastanoi strain in all of the samples that
were also
positive by PCR. These samples came from fairly young
knots because the
analyses were done 8 months after the inoculations,
when the knot
tissues retain a high population of viable cells
as shown by the
isolation studies. In contrast, in knot samples
from naturally infected
plants the developed PCR assay was more
efficient than bacterial
isolation in detecting
P. savastanoi.
This finding could be
due to lower populations of viable bacteria
in old cracked knots. For
bacterial isolations in these samples,
an enrichment step, even using
the semiselective PVF-1 medium,
decreased the plating efficiency due to
the growth of bacteria
other than
P. savastanoi in the
plates. This finding suggests
that the enrichment allows the
multiplication of the
P. savastanoi living cells; however,
the rapid development of the colonies of
the microbiota from the
olive tissues on the isolation plates
(mainly
Pantoea
agglomerans and fluorescent pseudomonads) interfered
with the
growth of
P. savastanoi colonies but not with their
detection
in the enriched culture by PCR. Other authors (
1,
17) suggested
that fairly young olive knots should be used for
the isolation
of
P. savastanoi because the presence of a
number of other bacteria
and low viable populations of the pathogen in
the diseased tissues
from old, cracked knots often made isolations very
difficult.
Our work agrees with these findings and extends it to
oleander
and broom
knots.
The semiselective medium PVF-1 was more effective than King's medium B
for
P. savastanoi enrichment and subsequent PCR detection,
probably due to the relative low growth rate of
P. savastanoi compared to that of some other saprophytic bacteria
present in
plant samples. This finding also suggests that the
enrichment
effect was not primarily due to the dilution effect of the
potential
PCR inhibitors present in plant material, as previously
suggested
(
15).
With the developed enrichment-PCR method we detected
P. savastanoi in many samples in which we were not able to isolate
the
bacterium. We were then concerned about the possibility of
false-positives
with the PCR technique. To help detect
cross-contamination among
samples and PCR reagents, multiple controls
were used in all steps
of the protocol as described in Materials and
Methods. Negative
controls from every experiment were consistently
negative. On
the other hand, the designed primers were highly specific,
and
confirmation that the PCR product is from amplification of the
target DNA was always made by restriction analysis and by DNA
sequencing in two samples. Besides the conventional isolation
methods,
no other serological or molecular techniques are available
for further
confirmation of
P. savastanoi detection directly in
plant
samples.
In conclusion, this study indicates that PCR amplification of
P. savastanoi with the
iaaL-derived primers and the
developed
preenrichment assay is a sensitive and specific method for
the
detection of this phytopathogenic bacterium in plant material.
This
enrichment-PCR method has advantages over all presently used
detection
methods for
P. savastanoi in time, sensitivity, and
cost.
This assay may be useful for the early detection of low
levels of
P. savastanoi in olive plants during plant material
propagation for certification purposes or epidemiological
studies.
| |
ACKNOWLEDGMENTS |
We thank P. G. Psallidas and N. Iacobellis for kindly
providing bacterial strains and J. Piquer for technical support with plant management. We are grateful to A. Olmos for technical advice.
R. Penyalver is a recipient of a contract from the Spanish MEC
(Programa de Incorporación de Doctores a Grupos de
Investigación en España). This work was supported by grant
OLI96-2179 from the CICYT of Spain.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Departamento de
Protección Vegetal y Biotecnología, Instituto Valenciano
de Investigaciones Agrarias, Apartado Oficial, 46113 Moncada, Valencia,
Spain. Phone: 34-96-1391000. Fax: 34-96-1390240. E-mail:
mlopez{at}master.ivia.es.
| |
REFERENCES |
| 1.
|
Azad, H. R., and D. A. Cooksey.
1995.
A semiselective medium for detecting epiphytic and systemic populations of Pseudomonas savastanoi from oleander.
Phytopathology
85:740-745.
|
| 2.
|
Casano, F. J.,
S. Y. Hung, and J. M. Wells.
1987.
Differentiation of some pathovars of Pseudomonas syringae with monoclonal antibodies.
Eur. Mediterr. Plant Prot. Organ. Bull.
17:173-176.
|
| 3.
|
Comai, L., and T. Kosuge.
1980.
Involvement of plasmid deoxyribonucleic acid in indoleacetic acid synthesis in Pseudomonas savastanoi.
J. Bacteriol.
143:950-957[Abstract/Free Full Text].
|
| 4.
|
Gardan, L.,
C. Bollet,
M. Abu Ghorrah,
F. Grimont,
P. A. D. Grimont, and P. A. D. Grimont.
1992.
DNA relatedness among the pathovar strains of Pseudomonas syringae subsp. savastanoi Janse (1982) and proposal of Pseudomonas savastanoi sp. nov..
Int. J. Syst. Bacteriol.
42:606-612[Abstract/Free Full Text].
|
| 5.
|
Glass, N. L., and T. Kosuge.
1986.
Cloning of the gene for indoleacetic acid-lysine synthetase from Pseudomonas syringae subsp. savastanoi.
J. Bacteriol.
166:598-603[Abstract/Free Full Text].
|
| 6.
|
Hutzinger, O., and T. Kosuge.
1968.
3-Indoleacetic-L-lysine, a new conjugate of 3-indoleacetic acid produced by Pseudomonas savastanoi, p. 183-194.
In
F. Wightman, and G. Setterfield (ed.), Biochemistry and physiology of plant growth substances. The Runge Press, Ltd., Ottawa, Ontario, Canada.
|
| 7.
|
Iacobellis, N. S.,
A. Sisto,
G. Surico,
A. Evidente, and E. Di Maio.
1994.
Pathogenicity of Pseudomonas syringae pv. savastanoi mutants defective in phytohormone production.
J. Phytopathol.
140:238-248.
|
| 8.
|
Janse, J. D.
1991.
Pathovars discrimination within Pseudomonas syringae subsp. savastanoi using whole-cell fatty acids and pathogenicity as criteria.
Syst. Appl. Microbiol.
13:79-84.
|
| 9.
|
King, E. O.,
M. K. Ward, and D. E. Raney.
1954.
Two simple media for the demonstration of pyocanin and fluorescin.
J. Lab. Clin. Med.
44:301-307[Medline].
|
| 10.
|
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].
|
| 11.
|
López, M. M.,
M. T. Gorris,
P. Llop,
J. Cubero,
B. Vicedo, and M. Cambra.
1997.
Selective enrichment improves isolation, serological and molecular detection of plant pathogenic bacteria, p. 117-121.
In
H. W. Dehne, et al. (ed.), Diagnosis and identification of plant pathogens. Kluwer Academic Publishers, Dordrecht, The Netherlands.
|
| 12.
|
Maggie, A. R.,
E. E. Wilson, and T. Kosuge.
1963.
Indoleacetamide as an intermediate in the synthesis of indoleacetic acid in Pseudomonas savastanoi.
Science
141:1281-1282[Abstract/Free Full Text].
|
| 13.
|
Mazzola, M., and F. F. White.
1994.
A mutation in the indole-3-acetic acid biosynthesis pathway of Pseudomonas syringae pv. syringae affects growth in Phaseolus vulgaris and syringomycin production.
J. Bacteriol.
176:1374-1382[Abstract/Free Full Text].
|
| 14.
|
Roberto, F. F.,
H. Klee,
F. White,
R. Nordeen, and T. Kosuge.
1990.
Expression and fine structure of the gene encoding N -(indole-3-acetyl)-L-lysine synthetase from Pseudomonas savastanoi.
Proc. Natl. Acad. Sci. USA
87:5797-5801[Abstract/Free Full Text].
|
| 15.
|
Schaad, N. W.,
S. S. Cheong,
S. Tamaki,
E. Hatziloukas, and N. J. Panopoulos.
1995.
A combined biological and enzymatic amplification (BIO-PCR) technique to detect Pseudomonas syringae pv. phaseolicola in bean seed extracts.
Phytopathology
85:243-248.
|
| 16.
|
Smidt, M., and T. Kosuge.
1978.
The role of indole-3-acetic acid accumulation by alpha-methyl tryptophan-resistant mutants of Pseudomonas savastanoi in gall formation in oleander.
Physiol. Plant Pathol.
13:203-214.
|
| 17.
|
Surico, G., and P. Lavermicocca.
1989.
A semiselective medium for the isolation of Pseudomonas syringae pv. savastanoi.
Phytopathology
79:185-190.
|
| 18.
|
Surico, G.,
N. S. Iacobellis, and S. Sisto.
1985.
Studies on the role of indole-3-acetic acid and cytokinins in the formation of knots on olive and oleander plants by Pseudomonas syringae pv. savastanoi.
Physiol. Plant Pathol.
26:309-320.
|
| 19.
|
Wilson, E. E.
1965.
Pathological histogenesis in oleander tumors induced by Pseudomonas savastanoi.
Phytopathology
55:1244-1249.
|
| 20.
|
Yamada, T.,
C. J. Palm,
B. Brooks, and T. Kosuge.
1985.
Nucleotide sequences in the Pseudomonas savastanoi indolacetic acid genes show homology with Agrobacterium tumefaciens T-DNA.
Proc. Natl. Acad. Sci. USA
82:6522-6526[Abstract/Free Full Text].
|
| 21.
|
Young, J. M., and C. M. Triggs.
1994.
Evaluation of determinative tests for pathovars of Pseudomonas syringae van Hall 1902.
J. Appl. Bacteriol.
77:195-207[Medline].
|
Applied and Environmental Microbiology, June 2000, p. 2673-2677, Vol. 66, No. 6
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Perez-Martinez, I., Zhao, Y., Murillo, J., Sundin, G. W., Ramos, C.
(2008). Global Genomic Analysis of Pseudomonas savastanoi pv. savastanoi Plasmids. J. Bacteriol.
190: 625-635
[Abstract]
[Full Text]
Top
Abstract
Text
