FEMS Microbiology Letters 236 (2004) 257–260

www.fems-microbiology.org

Development of PCR assay to identify Pseudomonas fluorescens

and its biotype
Mauro Scarpellini *, Laura Franzetti, Antoni€etta Galli

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Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Sez Microbiologia Agraria Alimentare Ecologica,
Via Celoria 2, 20133 Milano, Italy
Received 23 April 2004; received in revised form 26 May 2004; accepted 27 May 2004

First published online 9 June 2004

Abstract

The paper provides a simple protocol that uses the polymerase chain reaction to amplify a specific portion of the 16S gene, allowing
the recognition of Pseudomonas fluorescens from other group I Pseudomonas. The amplified DNA patterns of 16S rRNA and ITS1,
from the restriction fragment length polymorphisms VspI, HaeIII and TaqI digestion, produced band patterns that distinguished the
biotypes of Ps. fluorescens. In addition to distinguishing the biotypes C and 3 we used a phenotypical method for levan production.

2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Keywords: Pseudomonas fluorescens; 16S rRNA; Biotype; Primer species-specific

1. Introduction Ps. fluorescens is a heterogeneous species that can be

subdivided by various taxonomic criteria into several
The genus Pseudomonas is a group of ubiquitous biotypes [9].
Gram-motile rods characterised by elevated metabolic The aim of this work was: (i) to develop a rapid
versatility, utilising a wide range of organic compounds polymerase chain reaction (PCR)-based species-specific
and holding an important position in biosphere ecology; protocol to identify Ps. fluorescens and (ii) to distinguish
its members are saprophytic as well as pathogenic to the different biotypes of this species by combining mo-
plants, animals and humans [1–3]. The genus, first lecular techniques with traditional biochemical methods.
described time by Migula in 1894 [4], has undergone
repeated taxonomic revision, and the most recent DNA–
RNA hybridisation techniques have recognized 5 rRNA 2. Materials and methods
similarity groups; today, only group 1 is considered the
‘‘true’’ Pseudomonas or Pseudomonas ‘‘sensu stricto’’, 2.1. Microbial strains
while different designations have been proposed for the
members of other groups [5–8]. The strains used in this study are listed in Table 1.
Group 1 is the largest of the groups, and includes The strains were grown in Triptic Soy Broth (Difco-
both fluorescent and non fluorescent: the most impor- MD,USA) at 30 C for 48 h and were routinely
tant fluorescent species are Pseudomonas fluorescens, maintained at 4 C. For long-term maintenance, stock
Pseudomonas aeruginosa, Pseudomonas putida, Pseudo- solution cultures were stored in 20% (v/v) glycerol (v/v)
monas chlororaphis and plant pathogenic species (Pseu- on an appropriate liquid medium at )20 C.
domonas syringae and Pseudomonas chichorii).
2.2. DNA extraction

*
Corresponding author. Tel.: +39-2-50316734; fax: +39-2-50316694. Template DNA was prepared by boiling 200 ll of
E-mail address: mauro.scarpellini@unimi.it (M. Scarpellini). bacterial suspension in MilliQ (OD600 ¼ 0.6) in safe-lock

0378-1097/$22.00
2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.femsle.2004.05.043
258 M. Scarpellini et al. / FEMS Microbiology Letters 236 (2004) 257–260

Table 1 DNA 16S region amplification was performed using

Reference strains included in this study the primer set 16SF–16SR [11] (16SF 50 -AGA-
Strain Collection number GTTTGATCCTGGCTCAG-30 ; 16SR 50 -CTACGGC-
Pseudomonas aeruginosa DSMZ 50071a TACCTTGTTACGA-30 ) and the following thermal
Pseudomonas agarici DSMZ 11810a profile: 2 min at 94 C; 5 cycles consisting of 94 C for 45
Pseudomonas alcaligenes DSMZ 50342a s, 55 C for 1 min, 72 C for 2 min; 35 cycles consisting
Pseudomonas asplenii DSMZ 50254
Pseudomonas chichorii DSMZ 50259a
of 92 C for 45 s, 60 C for 45 s, 72 C for 2 min; final
Pseudomonas chlororaphis DSMZ 50083a extension of 72 C for 2 min; and final cooling at 4 C.
Pseudomonas fluorescens biotype A ATCC 17555 DNA 16S–23S intergenic spacer (ITS1) region am-
Pseudomonas fluorescens biotype B ATCC 17482 plification was performed using the primer set 16F945
Pseudomonas fluorescens biotype C ATCC 17559 and 23R458 [12] (16F945 50 -GGGCCCGCACA-
Pseudomonas fluorescens biovar 3 DSMZ 50124
Pseudomonas fluorescens bioytpe G ATCC 17518
AGCGGTGG-30 ; 23R458 50 -CTTTCCCTCACGG-

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Pseudomonas fragi DSMZ 3456a TAC-30 ) and the following thermal profile: 5 min at 94
Pseudomonas mendocina DSMZ 50017a C; 30 cycles consisting of 94 C for 1 min, 55 C for 1
Pseudomonas pseudoalcaligenes DSMZ 50188a min, 72 C for 2 min; final extension of 72 C for 2 min.
Pseudomonas putida bv. A DSMZ 291a DNA 16S specific region for Ps. fluorescens amplifi-
Pseudomonas stutzeri DSMZ 5190a
Pseudomonas syringae DSMZ 10604a
cation was performed using the primer set 16SPSEfluF
Pseudomonas viridiflava DSMZ 11124a and 16SPSER (16SPSEfluF 50 -TGCATTCAAA-
ACTGACTG-30 ; 16SPSER 50 -AATCACACCGTG-
DSMZ refers to Deutsche Sammlung von Mikroorganismen,
Braunschweig, Germany; ATCC refers to American Type Culture GTAACCG-30 ).
Collection (Rockville, Maryland, USA). Following amplification 7 ll of product were analy-
a
Type strain. sed by electrophoresis at 100 V in (1% agarose gel, 0.2
lg of ethidium bromide ml1 ) in TAE buffer. The am-
plification was performed in a DNA thermal cycle
Eppendorf tubes for 10 min. The tubes were immedi- (Biometra Tgradient, Germany). The primer is reported
ately cooled on ice and centrifuged (20,000g
10 min, 5 in Fig. 1: primer forward is species specific, while the
C); the supernatants were subsequently kept on ice or reverse is family specific. The primers were developed
at )20 C. One microlitre of template DNA suspension and compared with partial regions 16S of Pseudomonas
was used for each reaction [10]. species belonging to group 1 (NCBI, National Center
for Biotechnology Information) by software DNAsis
2.3. PCR amplification 2.0.

All the PCR were performed in a volume of 50 ll 2.4. Restriction analysis of 16S rRNA
containing 50–100 ng of bacterial genomic DNA so-
lution, 5 ll of 10· PCR buffer, 200 lM of each dNTP, 2 Restriction digestion of each amplified 16S rRNA
mM of MgCl2 , 0.5 lM of each primer and 0.5 U of Taq was carried out for 16 h at 30 C in 25 ll reaction
Polymerase (Amersham-Pharmacia). mixture containing 15 ll of template DNA, 2.5 ll of 10·

Fig. 1. Nucleotide sequence comparison of Pseudomonas group I 16S rRNA region partial (482–521 bp; 1311–1351 bp).
 M. Scarpellini et al. / FEMS Microbiology Letters 236 (2004) 257–260 259

PCR buffer, and 18 U of one of the following restriction The differentiation of the various biotypes within Ps.
enzymes, HaeIII and VspI (Amersham Pharmacia Bio- fluorescens was performed by means of restriction
tech). Restriction digestion was then analysed by aga- analysis, and the RPFL patterns obtained are shown in
rose electrophoresis (3% w/v) containing 0.2 lg of Fig. 3.
ethidium bromide ml1 , TAE buffer [13]. The 16S rRNA region digested with VspI exhibited
two fragments (1150 and 450 bp) for the Ps. fluo-
2.5. Restriction analysis of spacer (ITS 1) rescens biotype C and biovar 3, while Ps. fluorescens
biotypes A, B and G had no VspI restriction sites
Restriction digestion of each amplified ITS1 was (Fig. 3).
carried out for 16 h at 65 C in 25 ll reaction mixture The Ps. fluorescens biotype B was discriminated from
containing 15 ll of template DNA, 2.5 ll of 10· PCR the other biotypes by the digestion of the same region
buffer, and 18 U of TaqI (Amersham Pharmacia Bio- with HaeIII: 2 fragments of 450 and 250 bp were ob-

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tech) [12]. Restriction digestion was then analysed by tained (Fig. 3). The TaqI restriction profile of the ITS1
agarose electrophoresis (3% w/v) containing 0.2 lg of allowed the discrimination of the Ps. fluorescens biotype
ethidium bromide ml1 , TAE buffer. A, the only one that showed two characteristic bands of
280 and 180 bp. The Ps. fluorescens biotype G can be
2.6. Levan production recognised on comparing the RPFL profile patterns:
with TaqI it is can be differentiated from Ps. fluorescens
The method proposed by Lelliot et al. [14] was used. biotype A, with VspI from Ps. fluorescens biotypes 3 and
C, and with HaeIII from Ps. fluorescens biotype B.
Following this it was possible to discriminate the Ps.
3. Results and discussion fluorescens biotype C from Ps. fluorescens biovar 3 using
the levan test (Fig. 4): whereas Ps. fluorescens biotype C
The target regions for PCR primers 16SPSEfluF and is a levan producer, this test was negative for Ps. fluo-
16SPSER were identified at locations 482–521 bp and rescens biovar 3.
1311–1351 bp for all the investigated Ps. fluorescens The proposed PCR protocol (PSEfluF–PSER) offers
(Fig. 1). A single DNA fragment of 850 bp of 16S rRNA a rapid diagnostic tool to identify a Ps. fluorescens
was amplified only for Ps. fluorescens (Fig. 2). member of Pseudomonas ‘‘sensu strictu’’ (group 1).

Fig. 2. Agarose gel showing the species-specific amplification of the 850

bp fragment for Ps. fluorescens and related species obtained with the
primer 16SPSEflu F and 16SPSE R Lane 1 Marker Gene Ruler 100 bp
(MBI Fermentas), lane 2 Ps. fluorescens (ATCC 17555), lane 3 Ps. Fig. 3. Agarose gel showing the TaqI, restriction profile of PCR am-
fluorescens (ATCC 17482), lane 4 Ps. fluorescens (ATCC 17559), lane 5 plified ITS1 region of Ps. fluorescens biotype A (lane2), Ps. fluorescens
Ps. fluorescens (DSMZ 50124), lane 6 Ps. fluorescens (ATCC 17518), biotype B (lane 3), Ps. fluorescens biotype C (lane 4), Ps. fluorescens
lane 7 Ps. aeruginosa (50071), lane 8 Pseudomonas agarici (DSMZ biovar 3 (lane 5), Ps. fluorescens biotype G (lane 6); VspI, restriction
11810), lane 9 Pseudomonas alcaligenes (DSMZ 50342), lane 10 Pseu- profile of PCR amplified 16S rRNA region of Ps. fluorescens biotype A
domonas asplenii (DSMZ 50254), lane 11 Ps. chichorii (DSMZ 50259), (lane 8), Ps. fluorescens biotype B (lane 9), Ps. fluorescens biotype
lane 12 Ps. chlororaphis (DSMZ 50083), lane 13 Pseudomonas fragi C (lane 10), Ps. fluorescens biovar 3 (lane 11), Ps. fluorescens biotype G
(DSMZ 3456), lane 14 Pseudomonas mendocina (DSMZ 50017), lane (lane 12); HaeIII, restriction profile of PCR amplified 16S rRNA re-
15 Pseudomonas pseudoalcaligenes (DSMZ 50188), lane 16 Ps. putida gion of Ps. fluorescens biotype A (lane 14), Ps. fluorescens biotype B
(DSMZ 291), lane 17 Ps. syringae (DSMZ 10604), lane 18 Pseudo- (lane 15), Ps. fluorescens biotype C (lane 16), Ps. fluorescens biovar 3
monas stutzeri (DSMZ 51909), lane 19 Pseudomonas viridiflava (DSMZ (lane 17), Ps. fluorescens biotype G (lane 18), Marker Gene Ruler 100
11124), lane 20 Marker Gene Ruler 100 bp (MBI Fermentas). bp MBI Fermentas (lanes 1, 7 and 13).
260 M. Scarpellini et al. / FEMS Microbiology Letters 236 (2004) 257–260

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