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Applied and Environmental Microbiology, November 2000, p. 4834-4841, Vol. 66, No. 11
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Application of Siderotyping for
Characterization of Pseudomonas tolaasii and
"Pseudomonas reactans" Isolates Associated with
Brown Blotch Disease of Cultivated Mushrooms
Patricia
Munsch,1
Valerie A.
Geoffroy,2
Tapani
Alatossava,1 and
Jean-Marie
Meyer2,*
Biotechnology Laboratory, REDEC of Kajaani, University of
Oulu, 88600 Sotkamo, Finland,1 and
Laboratoire de Microbiologie et de Génétique, CNRS UPRES-A
7010, Université Louis-Pasteur, 67000 Strasbourg,
France2
Received 23 March 2000/Accepted 2 August 2000
 |
ABSTRACT |
Pyoverdine isoelectric focusing analysis and pyoverdine-mediated
iron uptake were used as siderotyping methods to analyze a collection
of 57 northern and central European isolates of P. tolaasii
and "P. reactans." The bacteria, isolated from
cultivated Agaricus bisporus or Pleurotus
ostreatus mushroom sporophores presenting brown blotch disease
symptoms, were identified according to the white line test (W. C. Wong and T. F. Preece, J. Appl. Bacteriol. 47:401-407, 1979)
and their pathogenicity towards A. bisporus and were
grouped into siderovars according to the type of pyoverdine they
produced. Seventeen P. tolaasii isolates were recognized, which divided into two siderovars, with the first one
containing reference strains and isolates of various geographical origins while the second one contained Finnish isolates exclusively. The 40 "P. reactans" isolates divided into eight
siderovars. Pyoverdine isoelectric focusing profiles and cross-uptake
studies demonstrated an identity for some "P.
reactans" isolates, with reference strains belonging to the
P. fluorescens biovars II, III, or V. Thus, the easy
and rapid methods of siderotyping proved to be reliable by supporting
and strengthening previous taxonomical data. Moreover, two potentially
novel pyoverdines characterizing one P. tolaasii siderovar and one "P. reactans" siderovar were found.
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INTRODUCTION |
Pseudomonas tolaasii, the
causal agent of brown blotch disease on cultivated mushrooms, is
responsible of significant crop losses in mushroom growing houses. The
pathogen has been reported on Agaricus bisporus,
Agaricus bitorquis, Agaricus campestris, Pleurotus ostreatus, and Pleurotus eryngii
(2, 35). It has been classified in RNA-DNA homology group I
(31) and, more precisely, as closely related to biovar V of
Pseudomonas fluorescens (10, 17). However, it is
easily distinguished from P. fluorescens strains or
other fluorescent pseudomonads by two main features, which are (i) the
pathogenicity to mushrooms (13, 30) and (ii) the white line
reaction (37). The white line reaction is the result of a
specific interaction between two diffusible lipodepsipeptides, the
tolaasin toxin produced by P. tolaasii (3, 7, 28, 29, 33) and the so-called white line inducing principle
(26), which is produced by some pseudomonads associated with
mushrooms and referred to as "Pseudomonas reactans"
(37). "P. reactans" has been considered
a strictly saprophytic bacterial component in the microflora of
the cultivated mushrooms (32), and the white line inducing
principle has effectively been described as having no pathogenic
properties on mushroom tissues (34). However, some light
pathogenic symptoms have been recently observed for some of these
strains (36). Taxonomy studies on "P.
reactans" isolates already revealed a great heterogeneity as
observed from DNA-DNA hybridizations (17) and from
phenotypic characterizations which, moreover, demonstrated that some of
them belonged to P. fluorescens biovar III and that
some others belonged to P. fluorescens biovar V
(36). On the contrary, P. tolaasii strains
are considered phenotypically and genomically homogeneous (17,
36), although presenting phenotypic variations: old colonies
develop sectors which correspond to phenotypic variants described as
nonpathogenic and white line negative (8, 18).
Siderotyping (short for siderophore typing) has been recently proposed
as a rapid and efficient bacterial typing method for the
discrimination of fluorescent pseudomonad strains (24). It
is mainly based on the recognition of the different types of pyoverdine
(PVD), which is the typical fluorescent pigment and powerful
siderophore of the fluorescent Pseudomonas (11,
22). More than 30 structures of PVDs, differing mainly in their
peptide chain, have been so far described (4). These
differences in structure for PVD have a tremendous effect on the
biological activity of the siderophore since, as a general rule, a
fluorescent Pseudomonas recognizes exclusively the ferric
complex of its own PVD (19, 23). The combination of several
siderotyping methods, such as PVD isoelectric focusing (PVD-IEF) and
the PVD-mediated iron uptake method, was shown to be very effective for
the identification of well-defined P. aeruginosa strains into three different groups, or siderovars,
according to the type of PVD produced by the strains. The use of
the second method, PVD-mediated iron uptake, was particularly necessary
for analyzing strains having a defect in PVD synthesis but still
expressing the ferripyoverdine outer membrane receptor (24).
Siderotyping also proved to be an interesting way to
phenotypically discriminate the two recently proposed new
species, Pseudomonas brassicacearum and
Pseudomonas thivervalensis (1).
Moreover, IEF analysis allowed a rapid screening of numerous
fluorescent Pseudomonas and permitted a rapid detection of
structurally original PVDs. Thus, the method has proved to be an
interesting preceeding step before chemical characterization of new
PVDs (5, 16, 25).
The goal of the present study was to analyze through siderotyping a
collection of natural isolates belonging to the P. tolaasii species or to the taxonomically heterogeneous
"P. reactans" group for testing the discriminative
power and usefulness of the method in bacterial identification.
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MATERIALS AND METHODS |
Bacterial strains.
Most of the bacterial strains described
in the present study were isolated over a period of 18 months from
brown blotch disease-affected A. bisporus or
P. ostreatus mushrooms, kindly supplied by different northern and central European farms. Isolates are listed in Table 1 as well as their geographical origin.
Strains whose designations begin with PS were collected from
A. bisporus mushrooms; strains whose designations
begin with PL were collected from P. ostreatus mushrooms. One strain, PS22.2, was isolated from a wild
Agaricus sp. mushroom sporophore. Strain PS8.14V was
isolated as a phenotypic variant which spontaneously arose after
streaking the wild-type strain PS8.14 on an agar plate. Reference
strains were P. tolaasii LMG 2342T (also
known as NCPPB 2192T and ATCC 33618T),
P. tolaasii LMG 6641, and "P.
reactans" LMG 5329, obtained from the Belgian Collection, Ghent.
P. aeruginosa ATCC 15692, P. fluorescens ATCC 13525, and P. fluorescens ATCC
17400 were from the American Type Culture Collection, Manassas, Va.
Pseudomonas monteilii CFML 90.54 (12) and other
strains of the Collection de la Faculté de Médecine de
Lille (CFML), P. fluorescens 51W (25),
P. fluorescens 12 (15), and P. fluorescens 18.1 (6) were kindly provided by D. Izard
(Université de Lille 2, Lille, France), S. Shivaji (Center of
Cellular and Molecular Biology, Hyderabad, India), H. Budzikiewicz
(Universität zu Köln, Cologne, Germany), and M. Champomier-Verges (INRA, Jouy-en-Josas, France), respectively.
Isolation and growth conditions.
Blotched outer layer
tissues of affected mushrooms were recovered and suspended with
vigourous shaking in sterile water. Fifty microliters of an appropriate
serial dilution was spread on King's B agar medium (Difco
Laboratories), and one of each of the phenotypically different colonies
developing a fluorescent halo after 48 h of incubation at 25°C
was further purified by streaking it on the same medium. Routine growth
was in Luria-Bertani (LB) medium or King's B liquid medium. Strains
were preserved by mixing overnight LB culture with 50% glycerol (1:1,
vol/vol) and storage at 
80°C. Iron-poor liquid growth medium was
the Casamino Acid (CAA) medium, consisting of (per liter) 5 g of
low-iron Bacto Casamino Acid (Difco), 1.54 g of
K2HPO4 · 3H2O, and 0.25 g of MgSO4 · 7H2O, and was mainly used
for PVD-IEF analysis and PVD purification through the Amberlite XAD-4
(XAD) procedure as described previously (25). Another
iron-deficient medium used for PVD-mediated iron incorporation
experiments was the synthetic succinate medium (21). Media
were distributed in capped test tubes (180 by 18 mm; 7.5 ml) or 100-ml
flasks containing 40 ml of medium or in 1-liter Erlenmeyer flasks
containing 500 ml of medium. Light inoculations were done with sterile
wood sticks dipped in an overnight preculture in the same medium, and
the cultures were incubated on a rotary shaker (200 rpm) at 25°C.
White line and pathogenicity tests.
The white line test was
performed according to the method of Wong and Preece (37),
using the collection strains P. tolaasii LMG
2342T or P. tolaasii LMG 6641 and
"P. reactans" LMG 5329 as reference strains. On
King's B agar, bacterial patches of strains to be tested (3 µl of
1/10-diluted overnight LB cultures) were placed at a distance of 1 cm
from each other, on a line located between lines of P. tolaasii LMG 2342T (or P. tolaasii LMG
6641) and "P. reactans" LMG 5329 patches. The
appearance of the precipitate forming the white line in between patches
was examined after 72 h of incubation at 25°C. Pathogenicity tests were performed as a modification of the method described by
Olivier et al. (30): 20 µl of bacterial suspensions at
108 CFU per ml or 20 µl of sterile water as control was
deposited at the surface of A. bisporus mushroom
caps maintained at 16°C in a saturated-humidity chamber
(27). Brown discoloration around the inoculation spot within
72 h was considered a positive reaction. Healthy mushrooms for
this test were provided by Mykora Ltd., Kiukainen, Finland.
IEF analysis of PVDs.
The model 111 mini-IEF cell from
Bio-Rad was used. Casting of the gels (5% polyacrylamide containing
2% Bio-Lyte 3/10 ampholytes) and electric focusing were performed
according to the manufacturer's recommendations. One-microliter
samples of PVDs (aqueous XAD-purified solutions [6.5 mg/ml]), or of
culture supernatants (40-h CAA-grown culture supernatant concentrated
20-fold by lyophylization) were used. PVD bands in the gel were
visualized under UV light at 365 nm and photographed just after
focusing. Their respective isoelectric pH values (pI values) were
determined according to a calibration curve constructed by slicing the
electrophoresed gel into 0.5-cm bands, which were incubated in 2 ml of
10 mM KCl during 30 min before measuring the pH (Beckman pHmeter
equipped with a minielectrode). Repeated experiments on different gels
with different ampholine commercial samples demonstrated a standard
deviation of 0.1 for pI values above pH 6.0 or 0.2 for pI values below
pH 6.0. Therefore, the expected identity in PVD-IEF profiles was
controlled by performing a comigration of the concerned PVDs on the
same gel.
PVD-mediated iron uptake.
Bacterial cells from 40-h cultures
in succinate medium were harvested by centrifugation, washed once with
distilled water, and resuspended at an optical density at 600 nm of
0.33 in an incubation medium made of succinate medium with the nitrogen
source omitted. Label mix containing 59Fe-PVD complex
consisted of 5 µl of the commercial 59Fe3+
solution (iron chloride in 0.1 M HCl; specific activity, 110 to 925 MBq/mg of iron; Amersham) diluted first with 100 µl of water and then
mixed with 10 µl of a 6.5-mg/ml XAD-purified PVD solution. The final
volume of the label mix was adjusted after 30 min of incubation at room
temperature to 1 ml with incubation medium. Thirty five structurally
different PVDs, listed in the legend of Fig. 2, were used. Bacterial
suspension (1.8 ml) was mixed at time zero with 0.2 ml of label mix.
After 20 min of incubation with gentle shaking in a water bath at
25°C, 1 ml of each bacterial suspension was rapidly filtered through
a Whatman nitrocellulose filter (0.45-µm pore size), and the filters
were washed twice with 2 ml of fresh incubation medium. Each filter was
then wrapped in aluminium foil and radioactivity counts were determined
in a Gamma 4000 Beckman counter. The remaining 1-ml bacterial
suspension was directly counted to determine the total amount of
radioactivity present in the assay. Control assays without bacteria
were performed to verify the complete solubility of labeled iron
through PVD complexation. Uptake data expressed in the tables or
figures are average values of at least duplicate independent experiments.
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RESULTS AND DISCUSSION |
Raising of the collection of natural isolates of P. tolaasii and "P. reactans" through white
line and pathogenicity tests.
Numerous studies have ascertained
that the white line test together with a pathogenicity test
(37) is an accurate screening method for the isolation and
recognition of P. tolaasii and "P. reactans" strains (14, 17, 20, 34, 36-38). Thus,
these methods were used to isolate a collection of such bacteria from blotched A. bisporus or P. ostreatus mushroom sporophores provided by 14 farms located in
Finland, France, Germany, The Netherlands, and Sweden (Table 1).
Seventeen
P. tolaasii strains were isolated from eight
samples of blotched
A. bisporus sporophores
originating from Finland,
France, Germany, and The Netherlands. None
was detected on
P. ostreatus during the course of
this study. The isolates were retained
based on a positive white line
test response to "
P. reactans"
LMG 5329 (
37). Moreover, all
P. tolaasii
isolates gave a typical
brown blotch symptom when the pathogenicity
test (
27,
30)
was performed on healthy
A. bisporus sporophores with the exception
of strain PS8.14V.
This strain, isolated as a phenotypic variant
of
P. tolaasii PS8.14, did not react in the white line test and
was
nonpathogenic (results not shown), in accordance with the
literature
(
8,
18).
Forty "
P. reactans" isolates were selected after
they responded positively to
P. tolaasii LMG
2342
T in the white line test. They were isolated from
A. bisporus as
well as from
P. ostreatus mushrooms. Most of the isolates, when
subjected to
the pathogenicity test, gave negative responses.
A few of them,
however (for example, strain PS7.2), induced a
weak
discoloration when tested on
A. bisporus
sporophores (data
not
shown).
Characterization of P. tolaasii and
"P. reactans" PVDs by IEF.
Electrophoresis of
PVDs on ampholine-containing polyacrylamide gel (PVD-IEF) results in
the separation of the different molecular forms of PVD present in the
supernatant of an iron-starved fluorescent pseudomonad culture. Thus,
the method allows an easy discrimination between strains producing
structurally different PVDs (24). Most often, for a given
strain, these different molecular forms are succinic, succinamide,
malate, maleide, or 
-ketoglutarate forms (isoforms) of an otherwise
identical molecule (4). PVD isoforms appear on the
electrophoresed IEF gel exposed to UV light as fluorescent bands
with various intensities, depending on the respective concentrations
they reached in the culture supernatant during the bacterial growth.
Each band could be characterized by the pH value measured at the place
on the gel where the PVD isoform localizes (pI).
As shown in Fig
1, 10 different PVD-IEF
patterns were observed upon analyzing the culture supernatants of the
P. tolaasii and "
P. reactans"
strains grown under iron-deficient conditions
(CAA medium). Strains
developing an identical PVD-IEF profile
were grouped together to
form a so-called siderovar. Table
1 indicates the pI values, obtained
for all the PVD-producing natural
isolates and for the three
P. tolaasii and "
P. reactans" LMG
reference
strains, and their grouping into 10 siderovars.
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FIG. 1.
IEF profiles of PVDs synthesized by P. tolaasii and "P. reactans" strains
representatives of the 10 siderovars. Lane 1, P. tolaasii LMG 2342 (sv. 1); lane 2, P. tolaasii
PS3a (sv. 2); lane 3, "P. reactans" PL24.1(sv.1);
lane 4, "P. reactans" PS11.4 (sv. 2); lane 5, "P. reactans" PS7.2 (sv. 3); lane 6, "P. reactans" PS8.12 (sv. 4); lane 7, "P. reactans" PS3b (sv. 5); lane 8, "P. reactans" PL10.9 (sv. 6); lane 9, "P. reactans" PL4.14 (sv. 7); lane 10, "P. reactans" PS6.10 (sv. 8).
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P. tolaasii strains subdivided into two siderovars
(Table
1; Fig.
1).
P. tolaasii siderovar (sv.) 1 contains the reference
strains LMG 6641 and LMG 2342
T, as
well as six natural isolates, all isolated from
A. bisporus sporophores cultivated in three different countries
(Table
1).
The PVD-IEF profile of these strains, as illustrated in Fig.
1,
lane 1, for strain LMG 2342
T, consisted of three
well-separated bands which very likely should
correspond to the three
recognized isoforms (succinamide, succinate,
and
-ketoglutaric acid
forms) of the structurally known PVD of
P. tolaasii
NCPPB 2192
T (LMG 2342
T) (
9). The
same
P. tolaasii sv. 1 PVD-IEF pattern was also
observed for the Finnish strain PS22.2, isolated from a wild
Agaricus sp. mushroom sporophore, and for strain
PS8.14V, a nonpathogenic
variant which, thus, was confirmed as
belonging to the same siderovar
as the corresponding wild-type organism
PS8.14.
Another PVD-IEF pattern characterized the nine fluorescent
P. tolaasii strains representing
P. tolaasii sv. 2 (Table
1; Fig.
1). These strains originated
exclusively from
A. bisporus sporophores
harvested
from Finnish mushroom farms located in Kiukainen area.
Another
P. tolaasii isolate of identical origin, PS7.5, was not
typeable by the IEF method because of its inability to produce
PVD
under all three tested iron-starved growth conditions (CAA
medium,
succinate medium, or liquid King's
medium).
The 40 "
P. reactans" strains presented eight
different PVD-IEF profiles, most of which were well differentiated from
the PVD-IEF
profiles of the two
P. tolaasii siderovars
(Fig.
1). Thus, the
"
P. reactans" strains were
grouped into eight siderovars ("
P. reactans"
sv. 1 to "
P. reactans" sv. 8) (Table
1).
Fourteen strains,
isolated from
A. bisporus (six
isolates) or
P. ostreatus (eight
isolates) originating
from nine different geographical areas (Table
1), formed a major
siderovar ("
P. reactans" sv. 2) (Table
1).
"
P. reactans" sv. 1 (two strains) and
"
P. reactans" sv. 8 (six
strains) were each
characterized by a similar although distinguishable
PVD-IEF pattern
(Table
1), with the band at pI 9.2 systematically
more pronounced for
the strains belonging to the "
P. reactans"
sv. 8 (Fig.
1, lanes 3 and 10). The six isolates of "
P.
reactans"
sv. 7 were characterized by a very pronounced PVD band
at pI 5.4,
which allowed us to easily distinguish them from the
"
P. reactans"
sv. 2 strains (Fig
1, lanes 9 and 4, respectively). The PVD-IEF
patterns of the strains belonging to
"
P. reactans" sv. 3 and "
P. reactans" sv. 4 were very similar (Fig.
1, lanes 5 and 6) and
required a comigration of the respective PVDs to ascertain their
classification. Strain PL10.9 showed a PVD-IEF pattern very close
to
the PVD-IEF pattern of
P. tolaasii sv. 2 strains (Fig.
1, lanes
8 and 2, respectively) but unique among "
P.
reactans" strains.
Thus, it was recognized as the unique
representative of "
P. reactans"
sv.
6.
Discrimination of P. tolaasii and "P.
reactans" siderovars by PVD-mediated iron uptake.
In order
to ascertain the classification reached by PVD-IEF, all the strains
were analyzed for their capacity to incorporate iron under the form of
a PVD-iron complex. The PVD produced by one representative of each
siderovar was purified by the XAD filtration procedure (4)
and tested for its capacity to mediate 59Fe iron uptake in
each of the strains belonging to the corresponding siderovar. Table
2 reports that the PVD of strain LMG
2342T (P. tolaasii sv. 1) efficiently
mediated iron incorporation in all the strains belonging to
P. tolaasii sv. 1. Conversely, the PVD of strain PS3a
(P. tolaasii sv. 2) was well recognized by the strains
belonging to P. tolaasii sv. 2. The PVD of
P. tolaasii PS3a was also recognized by strain PS7.5 (a
PVD-deficient isolate) (Table 2). Accordingly, this strain could then
be classified in siderovar 2. Table 2 indicates also that the
P. tolaasii PVDs should be considered siderovar
specific since none of the strains was able to use both compounds
efficiently as an iron transporter.
Intrasiderovar efficiency was also conclusive for the PVDs produced by
the "
P. reactans" strains previously grouped into
eight
siderovars (Table
1). Each strain belonging to a defined
siderovar,
according to its IEF profile, was able to use the
corresponding
siderovar-specific PVD, at an efficiency identical or
very close
to that of the PVD producer strain. Parts of these data are
shown
in Table
3 for the two strains
belonging to "
P. reactans" sv.
1 and for the six
strains of "
P. reactans" sv. 7. Unlike
P. tolaasii,
some cross-recognitions between different
"
P. reactans" siderovars
and their respective PVDs
were observed, but the highest iron
incorporation was always reached in
the homologous system. This
was particularly evident for the four
siderovars "
P. reactans"
sv. 2 to 5 (Table
4). As an example, strain PS20.11 of
"
P. reactans"
sv. 4 was able to use the PVD of
strain PL5.13 ("
P. reactans"
sv. 2) at half
efficiency compared to its own PVD. The reciprocity
of
cross-recognition was effective for this couple of strains
but was not
a general rule, as illustrated in Table
4 for strains
PL5.13 and PS3b
or strains PL10.9 and PL4.14.
On the contrary, a high specificity of recognition characterized the
strains and the PVDs synthesized by isolates belonging
to
"
P. reactans" sv. 1 and "
P.
reactans" sv. 8, for which only
the homologous system was
efficient in iron incorporation. Even
though the PVD of strain PL10.9
("
P. reactans" sv. 6) was highly
specific to its
producing strain, the strain was also able to
use, at a lower
efficiency, however, the PVDs of "
P. reactans"
sv.
4, 5, and 7 (Table
4). Moreover, strain PL10.9 was unable
to use the
PVD of the
P. tolaasii sv. 2 strains, and conversely,
no uptake was detected for strain PS3a (
P. tolaasii sv.
2) when
tested with PVD(PL10.9) (data not shown). Although they have
similar
IEF patterns (Fig.
1, lanes 2 and 8), these two PVDs should,
therefore,
be different in
structure.
Altogether, the data reached by PVD-mediated iron uptake studies well
confirmed the siderovar grouping first reached by PVD-IEF.
IEF of PVDs
appears, thus, to be the method of choice for siderotyping
fluorescent
pseudomonads, especially because of its rapidity,
allowing also the
simultaneous analysis of as many as 15 strains
in one run. However, the
uptake method was necessary for the siderotyping
of strain PS7.5, which
was able to incorporate iron through PVD
but presented a defect in PVD
biosynthesis and, therefore, was
nontypeable by IEF. Furthermore, the
method is required for discriminating
strains having similar IEF
patterns and, therefore, should be
used to assess the grouping of
strains as reached by PVD-IEF.
Attempts for taxonomical recognition of "P.
reactans" strains through siderotyping.
Siderotyping, by
discriminating eight siderovars among the "P.
reactans" strains, already supported the taxonomic diversity recognized for such bacteria by previous investigations (17, 36) which, furthermore, identified some "P.
reactans" strains as being closely related to P. fluorescens biovar III and P. fluorescens biovar V
(36). Therefore, we tested one strain of each
"P. reactans" siderovar for 59Fe iron
incorporation mediated by a collection of PVDs of different bacterial
origin. The 35 compounds tested, referred to as the heterologous PVDs,
have been obtained from strains representative of a wide variety of
fluorescent Pseudomonas species
among them, strains
belonging to P. fluorescens biovar III or biovar Vand were characterized each by a particular peptidic structure (most structures of these 35 PVDs have been already published [see
references 4 and 23 for a
compilation], while some others are presently under investigation).
As shown in Fig.
2, each "
P.
reactans" siderovar type strain had a specific pattern of
heterologous PVD recognition. Strain
PS6.10, the representative of
"
P. reactans" sv. 8, presented the
most restricted
pattern since none of the 35 heterologous PVDs
was recognized. Strain
PL24.1 ("
P. reactans" sv. 1) and strain
PL10.9
("
P. reactans" sv. 6) each well recognized a single
heterologous
PVD, respectively, the PVD of
P. fluorescens ATCC 17400 (Fig.
2 [PVD 13]) and the PVD of
P. fluorescens 51W (Fig.
2 [PVD 5]).
These two PVDs
were not recognized by any of the other "
P.
reactans"
siderovars. The representatives of siderovars 2 to 5 and siderovar
7 were able to recognize two or more of the PVDs produced
by the
following
Pseudomonas strains (Fig.
2)
P. fluorescens ATCC 13525
(PVD 16),
P. aeruginosa
ATCC 15692 (PVD 17),
P. fluorescens 18.1
(PVD 18),
P. fluorescens 12 (PVD 19),
P. rhodesiae CFML 92.104
(PVD 26),
P. veronii CFML
92.124 (PVD 27), and
Pseudomonas sp.
CFML 90.33 (PVD 28). In
some cases, the efficiencies of iron uptake
mediated by these
heterologous PVDs were close, if not identical,
to the one reached by
the homologous PVD, suggesting a structural
identity in between those
of the PVDs of concern, i.e., the PVDs
of
P. fluorescens ATCC 17400 and "
P. reactans"
PL24.1 (sv. 1),
P. fluorescens 18.1 and
"
P. reactans" PS11.4 (sv. 2),
P. fluorescens 51W and "
P. reactans" PL10.9 (sv.
6), or
P. fluorescens 12 and
"
P.
reactans" PL4.14 (sv. 7) (Fig.
2). Therefore, we analyzed
the
capacities of some of the reference strains to incorporate
the
corresponding "
P. reactans" PVDs. IEF patterns of
the PVDs
of concern were compared as well. As shown in Table
3,
P. fluorescens ATCC 17400 recognized the PVD of
"
P. reactans" PL24.1 (sv. 1)
as well as its own
PVD.
P. fluorescens 51W and "
P.
reactans" PL10.9
(sv. 6) both reacted similarly. On the
contrary,
P. monteilii CFML 90.54, whose PVD was also
efficient in mediating iron uptake
in strain PL10.9 (sv. 6) but was
less efficient than PVD(51W)
(Fig.
2, PVDs 5 and 24), was unable to
efficiently use PVD(51W)
or PVD(PL10.9) as illustrated in Table
3.
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FIG. 2.
Heterologous PVD-mediated 59Fe incorporation
by "P. reactans" strains belonging to the eight
siderovars. Ordinate values correspond to 59Fe
radioactivity expressed in counts per minute as measured after 20 min
of incubation (see Materials and Methods). Abscissa numbers 1 to 36 correspond to the structurally different PVDs tested, originating from
the following bacterial strains (PVD numbers are given in parentheses):
Pseudomonas strain E8 (1), P. syringae ATCC
19310 (2), P. fluorescens 9AW (3), P. putida ATCC 12633 (4), P. fluorescens 51W (5),
P. aeruginosa Pa6 (6), P. fluorescens
CCM 2798 (7), P. fluorescens CHA0 (8), P. tolaasii NCPPB 2192 (9), P. aeruginosa ATCC 27853 (10), P. fluorescens ii (11), P. fluorescens SB8.3 (12), P. fluorescens ATCC 17400 (13), P. fluorescens 1.3 (14), Pseudomonas
strain 267 (15), P. fluorescens ATCC 13525 (16),
P. aeruginosa ATCC 15692 (17), P. fluorescens strain 18.1 (18), P. fluorescens 12 (19), P. fluorescens CFBP 2392 (20),
Pseudomonas strain L1 (21), Pseudomonas sp.
strain ATCC 15915 (22), P. putida WCS358 (23),
P. monteilii CFML 90-54 (24), "P.
mosselii" CFML 90-77 (25), P. rhodesiae CFML
92-104 (26), P. veronii CFML 92-124 (27),
Pseudomonas sp. strain CFML 90-33 (28),
Pseudomonas sp. strain CFML 90-51 (29),
Pseudomonas sp. strain CFML 90-52 (30),
Pseudomonas sp. strain CFML 95-307 (31),
Pseudomonas sp. strain 2908 (32), Pseudomonas sp.
strain A214 (33), P. fluorescens PL7 (34),
P. fluorescens PL8 (35), and the PVD synthesized by the
strain under investigation (36). The counts per minute were corrected
for the blank values obtained in assays without bacteria.
|
|
The comparison of the IEF patterns of the considered pyoverdines
was in full agreement with a structural identity for PVD(PL24.1)
and PVD(17400) (Fig.
3, lanes 1 and 2),
PVD(PL10.9) and PVD(51W)
(Fig.
3, lanes 3 and 4), PVD(PL4.14)
and PVD(Pfl12) (Fig.
3, lanes
6 and 7), or PVD(PS7.2) and
PVD(13525) (Fig.
3, lanes 14 and 15).
Figure
3 also indicates that
P. monteilii CFML 90.54 is producing
a PVD with a
particular IEF pattern (Fig.
3, lane 5) and which,
therefore, and in
agreement with the incorporation data, should
be different in structure
from the PVD of strain PL10.9 (Fig.
3, lane 3). It could also be
deduced from the comparison of the
IEF patterns that strains PS3b (sv.
5), PS11.4 (sv. 2), and PS8.12
(sv. 4) are producing specific PVDs,
structurally different from
the heterologous PVDs capable of mediating
iron uptake in these
strains, i.e., the PVDs of
P. fluorescens 18.1 (Fig.
2, PVD 18;
Fig.
3, lane 8),
P. aeruginosa ATCC 15692 (Fig.
2, PVD 17; Fig.
3, lane 9),
P. rhodesiae CFML 92.104 (Fig.
2, PVD 26; Fig.
3,
lane
12),
P. veronii CFML 92.124 (Fig.
2, PVD 27; Fig.
3,
lane
13), and
Pseudomonas sp. CFML 90.33 (Fig.
2, PVD 28;
two bands
with pI values of 4.2 and 3.8 [data not shown]).
View larger version (112K):
[in this window]
[in a new window]
|
FIG. 3.
IEF profiles of PVDs for some "P.
reactans" isolates and some other pseudomonads having similar
uptake efficiencies. PVDs originated from the indicated strains: lane
1, "P. reactans" PL24.1 (sv. 1); lane 2, P. fluorescens ATCC 17400; lane 3, "P.
reactans" PL10.9 (sv. 6); lane 4, P. fluorescens
51W; lane 5, P. monteilii CFML 90.54; lane 6, "P. reactans" PL4.14 (sv. 7); lane 7, P. fluorescens 12; lane 8, P. fluorescens 18.1; lane
9, P. aeruginosa ATCC 15692; lane 10, "P.
reactans" PS11.4 (sv. 2); lane 11, "P.
reactans" PS3b (sv. 5); lane 12, P. rhodesiae
CFML 92.104; lane 13, P. veronii CFML 92.124; lane 14, "P. reactans" PS7.2 (sv. 3); lane 15, P. fluorescens ATCC 13525.
|
|
Thus, siderotyping identified four of the eight "
P.
reactans" PVDs as being identical in structure to
well-characterized PVDs,
some of them being produced by taxonomically
well-defined strains.
Therefore, a correlation is suggested between the
"
P. reactans"
sv. 1 strains and
P. fluorescens biovar III (to which belongs
P. fluorescens ATCC 17400) and between strain PL10.9 of
"
P. reactans"
sv. 6 and
P. fluorescens biovar V (to which belongs
P. fluorescens 51W). Some uncertainty remains for the
"
P. reactans" sv. 3 strains,
which could be related
to
P. fluorescens ATCC 13525, the type
strain of the
P. fluorescens biovar I, but also to the
P. chlororaphis species since
P. chlororaphis ATCC
9446 produces the same PVD
as
P. fluorescens ATCC 13525 (
4).
P. fluorescens 12 is a natural
isolate
whose precise taxonomic assignment among the
P. fluorescens biovars remains unknown. However, it could be
tentatively linked
to the
P. marginalis group belonging
to
P. fluorescens biovar
II, since
P. marginalis CFBP 4044 has been shown by siderotyping
and structural
identification to produce the same PVD as
P. fluorescens 12 (R. Fuchs, J. M. Meyer, and H. Budzikiewicz,
unpublished
results).
Indeed, these correlations between "
P. reactans"
strains and more- or less-defined fluorescent
Pseudomonas
strains need to
be confirmed by phenotypic and genomic taxonomy
studies. It should
be mentioned, however, that siderotyping has already
been successfully
used for a rapid and convenient identification of
P. aeruginosa isolates (
24). It has also
already proved to be a reliable method
for the discrimination of
numerous
Pseudomonas species recently
described, e.g.,
P. brassicacearum,
P. thivervalensis,
P. monteilii,
P. rhodesiae,
P. veronii,
P. jessenii, and
P. mandelii (
1;
V. Coulanges, L. Gardan, D. Izard, P. Lemanceau, and J. M. Meyer,
Abstr. 5°
Congr. Soc. Fr. Microbiol. 1998, abstr. 47, 1998). Moreover,
the fact
that some of the correlations suggested in the present
work well
corresponded to previous taxonomic assignments (
36)
supports
siderotyping as a powerful method for taxonomy studies
within
fluorescent
Pseudomonas strains. The usefulness of the
method is well demonstrated in the present study by the rapid
identification of strain PS8.4V. Because of the loss of the white
line
and pathogenicity phenotypic features, PS8.14V would not
be so easily
identifiable, unless a rather time-consuming study
based on classical
taxonomic methods were undertaken. Thanks to
siderotyping,
PS8.14V was recognized as a
P. tolaasii isolate
within a few hours following growth
completion.
| |
ACKNOWLEDGMENTS |
We deeply acknowledge N. J. Palleroni for critical reading
of the manuscript. P. M. is grateful to S. Lakovaara for
providing excellent research facilities and to J.-C. Hubert for
training provided in his laboratory. M.-M. Kytöviita is
acknowledged for the gift of the wild Agaricus sp.
sporophore. MYKORA Ltd. (Finland) is thanked for kindly providing the
mushrooms used in the pathogenicity tests.
P.M. is grateful to the Runar Bäckström Foundation for
financial support.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratoire de
Microbiologie et de Génétique, 28 rue Goethe, 67083 Strasbourg Cedex, France. Phone (33) 3 88 24 41 50. Fax: (33) 3 88 35 84 84. E-mail: meyer{at}gem.u-strasbg.fr.
| |
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Top
Abstract
Introduction
