Curr Microbiol (2007) 55:247–253
DOI 10.1007/s00284-007-0120-3
Characterization of Pseudomonas corrugata Strain P94 Isolated
from Soil in Beijing as a Potential Biocontrol Agent
Yanbin Guo Æ Hui Zheng Æ Yiling Yang Æ
Huimin Wang
Received: 25 February 2007 / Accepted: 16 May 2007

Springer Science+Business Media, LLC 2007
Abstract In an attempt to obtain biologic control agents
for grey mildew of tomato, a total of 628 bacterial strains
were isolated from agricultural soil samples in Beijing,
China, and screened for in vitro antibiosis toward Botrytis
cinerea. Strain P94 exhibited the most obvious antagonistic
activity. It P94 had no pathogenicity and was identified as
Pseudomonas corrugata by the Biolog identification system
combined with 16S rDNA sequence analysis and biochem-
ical and physiologic characteristics. The specific products of
polymerase chain reaction with two pairs of specific primers
indicated that P94 belonged to P. corrugata genomic group
II. Strain P94 inhibited the growth of a number of phyto-
pathogenic fungal and bacterial species and showed inhibi-
tion activity to tomato grey mildew by tomato leaf testing
in vitro. Strain P94 showed a positive reaction for HCN,
protease, phosphatase, and indole acetic acid tests and a
negative reaction for siderophore-, chitinase-, and cellulase-
production tests. Therefore, the secondary metabolites pro-
ducing novel P. corrugata strain P94 exhibited an innate
potential of biocontrol activities in vitro.
Keywords Pseudomonas corrugata
Characterization

Biocontrol
Y. Guo
Y. Yang
H. Wang (&)
Department of Plant Pathology, China Agricultural University,
100094 Beijing, China
e-mail: wanghm69@cau.edu.cn
H. Zheng
Department of Food Sciences, Beijing University of Agriculture,
Beijing, China
Introduction
Pseudomonas corrugata was first described by Scarlett
et al. [29] as the causal agent of tomato pith necrosis in
Britain. The bacteria has subsequently been reported in
many other countries, including the United States, Canada,
Europe, South Africa, New Zealand, Israel [18], and China
[8]. In addition to water [29] and soil [31], P. corrugata
was also isolated from many parts of the world, such as
pepper [17], tomato [18], chrysanthemum [10], geranium
[21] (associated with induced pith necrosis), healthy alfalfa
roots [19], and grapevine stem [2], which suggested that
the bacterium could be considered ubiquitous.
P. corrugata has shown effective ability in suppressing
damping-off in tomato and maize [12, 24], but there have
been no reports on controlling tomato grey mildew caused
by Botrytis cinerea. In this article, we report the isolation
and characterization of a nonpathogenic P. corrugata strain
P94 that showed inhibition activity to grey mildew of to-
mato; we discuss its potential antagonistic activity against
phytopathogenic micro-organisms; and we describe the
spectrum, the biochemical characteristics, and some phe-
notypes related to biologic control.
Materials and Methods
Bacterial and Fungal Strains
The antagonistic bacterial strain P94 was isolated from
Beijing agricultural soil and identified in this study as
P. corrugata. P. corrugata strain ICMP 5819 was kindly
provided by Zhao Tingchang (Plant Protection Institute,
The Chinese Academy of Agricultural Sciences, Beijing,
China). B. cinerea, Ceratocystis fimbriata, Magnaporthe
123
248
grisea, Rhizoctonia cerealis, Phytophthora capsici, and
Alternaria solani were kindly provided by Liu Xili
(Department of Plant Pathology, China Agricultural Uni-
versity, Beijing, China). Monilinia laxa, Pythium aphan-
idermatum, Fusarium oxysporum f. sp. cucumerinum,
F. oxysporum f. sp. vasinfectum, and F. oxysporum f. sp.
lilii were kindly provided by Qiu Jiyan (Plant and Envi-
ronment Protection Institute, The Beijing Academy of
Agricultural and Forestal Sciences, Beijing, China). P.
syringae and Ralstonia solanacearum were kindly pro-
vided by Feng Jie (Plant Protection Institute, The Chinese
Academy of Agricultural Sciences, Beijing, China). Aci-
dovorax avenae was kindly provided by Dr. Zhang Liqun
(Department of Plant Pathology, China Agricultural Uni-
versity, Beijing, China). Agrobacterium rhizogenes K27
and A. vitis K308 were kindly provided by Kerr (Depart-
ment of Plant Pathology, Waite Agricultural Research
Institute, University of Adelaide, South Australia). Xan-
thomonas oryzae, X. campestris, and A. tumefaciens Cy4
were collected by our laboratory.
Bacterial Isolation and Screening
Soil samples were collected from the rhizospheres of dif-
ferent tomatoes infected with B. cinerea. After removing
approximately 5 cm of soil from the surface layer, the sam-
ples were placed in sterile plastic bags. The bacteria were
isolated from soil according to Mew et al. [22]. Each isolated
colony was transferred to a potato dextrose agar (PDA) plate
on which was placed a 5-mm mycelial plug of B. cinerea.
Bacteria were situated approximately 2.5 cm from the plug.
After incubation at 25C for 4 to 7 days, the most effective
strain that inhibited the fungal growth was selected.
Assessment of Antiphytopathogenic Activity In Vitro
The antagonistic ability of P. corrugata P94 strain was
checked against 11 phytopathogenic fungi and 9 phyto-
pathogenic bacteria. The inhibition activity of strain P94
against fungi was tested using the modified method of
Daayf et al. [9]. Two replicates of 5-ll aliquots of sus-
pension of P94 were spotted 180 apart and 2.5 cm from
the center of the PDA plate. A 5-mm mycelial plug of each
pathogenic fungus was transferred to the center of the
plate. A single plug of fungus placed on a PDA plate
without P94 inoculation served as control. The plates were
incubated at 25C, and duplicate diameters of fungal col-
onies were measured in a beeline of the bacteria spotted.
The inhibitory effect of strain P94 against fungus was
estimated based on the percent relative growth:
Relative growth ð%Þ ¼ ðT=CÞ  100;
123
Y. Guo et al.: Characterization of P. corrugata Strain P94
where T is the growth of the fungi when challenged with
the strain P94, and C is the growth of the control fungi. The
activity against phytopathogenic bacteria was examined
using the double-layer method of Stonier [32].
Test for Pathogenicity
Tomato and tobacco seedlings were grown in a greenhouse.
P. corrugata strain P94 was inoculated using 2-month-old
tomato and tobacco seedlings, 10 each of which were used
for bacterial inoculation; P. corrugata strain ICMP 5819
and sterile distilled water served as positive and a negative
controls, respectively. The plants were inoculated accord-
ing the method of Sutra et al. [33]. External and internal
symptoms were recorded 35 days after inoculation;
each plant was longitudinally cut, and pith necrosis was
observed.
Assays for Biologic Control Grey Mildew of Tomato
The inhibition activity of tomato grey mildew by P. cor-
rugata strain P94 was examined using the method of Weeds
et al. [36] with some modifications: Tomato leaves of the
same age were picked. The leaves were washed thrice with
sterile distilled water and placed in a large Petri dish (270
mm) with five layers of humid filter article on the bottom.
P94 was cultured for 48 hours at 28C in liquid potato dex-
trose (PD) medium, and the bacteria were diluted to optical
density (OD)600 of 0.6 (approximately 1.0 · 108 colony-
forming units/ml) in sterile buffered saline. Then the bacteria
suspension was sprayed onto the surface of tomato leaves.
Twenty minutes later, 5-mm mycelial plugs of B. cinerea
were inverted onto the upper leaf surface when there were no
beads on the leaves. Leaves onto which sterile distilled water
was sprayed served as negative control. The plates were
closed to maintain high humidity and were incubated for 3
days at 20 to 25C under a diurnal cycle of daylight and
white fluorescent light. The area of disease blot was mea-
sured by staffs with small square grids (1 mm2/grid), and
grey mildew inhibition activity was calculated as follows:
Grey mildew inhibition ð%Þ ¼ ½ðC  TÞ=C  100;
where C is the average area of the disease blot of the
negative control group, and T is the average area of the
disease blot of tomato leaves treated with P94.
Identification of Strain P94: Biochemical and
Physiologic Characterization
The ability to use different carbon sources was tested using
the Biolog GN Microplate method (Biolog, Hayward,
CA).The morphologic, cultural, biochemical, and physio-
Y. Guo et al.: Characterization of P. corrugata Strain P94
logic characteristics of P94 were examined according to the
methods previously reported by Lopez et al. [18].
16S rDNA Gene Sequencing
Genomic DNA of P94 was extracted and purified according
to the method described by Sambrook et al. [28]. 16S
rDNA was amplified from P94 genomic DNA with the
primers 63F 5¢-CAGGCCTAACACATGCAAGTC-3¢ and
1494R 5¢-GGYTACCTTGTTACGACTT-3¢. PCR ampli-
fication was performed as follows: one cycle of 5 minutes
at 94C, followed by 30 cycles of 30 seconds each at 94C,
30 seconds at 56C, and 1 minute at 72C, followed by one
cycle of 5 minutes at 72C. PCR products were purified
using a gel extraction kit (Omega). The clean 16S rDNA
fragments were cloned into pMD18-T (TaKaRa) and se-
quenced with an ABI 3730 DNA sequencer at the Invi-
trogen Corporation in Beijing. DNA sequence homology
searches were performed using the online BLAST search
engine in GenBank (available at: http://www.ncbi.nlm.-
nih.gov).
Amplification of Specific PCR Products
P. corrugata was identified and distinguished into two
genomic groups (I or II) by using two pairs of specific
primers (PC 1/1 and PC 1/2 and PC 5/1 and PC 5/2) within
a single PCR reaction for amplification of two specific PCR
products (either 1100 or 600 bp) [6]. PCR was performed
as previously described by Catara et al. [6]. The strain was
distinguished by the presence of either of these two pos-
sible bands: 1100 (group I) or 600 bp (group II).
Transmission Electron Microscopy
For observation of stain P94 flagella by transmission
electron microscopy (TEM), cells were grown on a King’s
B (KB) plate and suspended in sterile buffered saline. A
small drop of the suspension was placed on a carbon-
coated copper grid, and the cells were negatively stained
with 0.5% phosphotungstic acid (pH 4.0) for observation
under the electron microscope (JEM-100CX; Jeol).
Detection of HCN and Siderophore Production
Production of HCN was observed according to the method
of Castric and Castric [5] with the following modification:
A single colony of strain P94 and non-HCN producing
P. fluorescens strain P12 (as a control) were inoculated into
an Eppendorf tube containing 0.5 ml of solid Luria-Bertani
medium, then a small piece of detective paper was hung
over the medium, and the tube was sealed with Parafilm
(Pechiney Plastic Packaging). The tube was incubated at
249
28C for 48 hours, and the result was investigated. The
change in color to blue of the HCN-sensitive paper indi-
cated the production of HCN. The amount of HCN released
by strain P94 was quantified according to the method of
Lambert et al. [16]. Siderophore production was deter-
mined by chrome azurol S (CAS) assay [30]. A yellow halo
around a colony after 4 to 5 days of incubation on blue agar
is indicative of siderophore excretion.
Determination of Hydrolytic Activity and
IAA Production
Protease production was determined using skim-milk agar
plates [15]. Chitinase activity was evaluated on chitin agar
(CA) plates [7]. Cellulase production was detected as de-
scribed by Teather and Wood [34]. Phosphatase activity
was detected using Pikovskaya’s agar plates [25], and
quantitative analysis of soluble phosphate was assessed
according to the method of King [13]. The production of
IAA was determined using the method of Bric et al. [3] in
nutrient broth with and without tryptophan (0.5 g/l).
Nucleotide Sequence Accession Number
The 16s rDNA sequence of 1462 bp has been deposited in
GenBank under accession number EF153018.
Results and Discussion
Six hundred twenty-eight bacterial strains were isolated
from agricultural soil samples collected from a greenhouse
in Beijing, China. Of all the strains tested, 11 bacterial
isolates (5 Pseudomonas sp., 3 Bacillus sp., 2 Rahnella sp.,
and 1 Achromobacter sp.) inhibiting B. cinerea growth were
selected, and the most potent one was strain P94 (data not
show). Strain P94 exhibited the most obvious antimicrobial
activity in vitro and showed significant growth-inhibitory
activity against a range of phytopathogenic fungi and phy-
topathogenic bacteria (Table 1), and the antifungal activity
was efficient compared with that of P. corrugata (inhibition
of Pythium ultimum) [11] and P. aeruginosa (inhibition of
B. cinerea and M. grisea) [15] previously reported, and the
antibacterial activity was as much as that of other antago-
nistic bacteria [35, 37]. Other isolates in this work differed
from P94 regarding their spectrum of antagonism to phy-
topathogenic fungi and were defective in antibacterial
activity (data not shown). However, strain P94 and the
P. corrugata strains described previously also showed dif-
ferences in spectrum-of-inhibition activity against phyto-
pathogenic fungi and bacteria. P. corrugata was isolated
from rhizosphere of maize (strains P. corrugata 1 and 7)
and tomato P. corrugata strains 1 and 3 as a potential
123
250 Y. Guo et al.: Characterization of P. corrugata Strain P94

Table 1 Antagonistic activity of P. corrugata P94 to the indicator

phytopathogenic fungi and phytopathogenic bacteria
Relative growth (%)
4d 6d

Indicator phytopathogenic fungi

B. cinerea 65 ± 3.2a 40 ± 1.7
C. fimbriata 51 ± 3.1 32 ± 2.4
M. laxa 53 ± 2.4 35 ± 1.1
M. grisea 64 ± 0.9 57 ± 1.0
P. aphanidermatum 77 ± 2.6 57 ± 2.1
P. capsici 68 ± 1.2 60 ± 2.2
A. solani –b –
R. cerealis – –
F. oxysporum f. sp. – –
cucumerinum
F. oxysporum f. sp. – –
vasinfectum
F. oxysporum f. sp. lilii – –
Inhibition zone diameter
(mm)
Fig. 1 Tomato stem split longitudinally to show symptoms of pith
Indicator phytopathogenic bacteria necrosis. Lane 1 = tomato plants inoculated with P. corrugata ICMP
P. corrugata ICMP 5819 23 ± 0.34a 5819. Lane 2 = tomato plants inoculated with P. corrugata P94. Lane
P. syringae 25 ± 0.58 3 = tomato plants inoculated with water as control.
A. avenae 28 ± 0.68
R. solanacearum 22 ± 0.29
X. oryzae –
X. campestris –
A. tumefaciens –
A. rhizogenes –
A. vitis –
a
SE of 15 independent experiments
b
–, Non growth-inhibitory activity

biologic control agent [12, 23]. Strains P. corrugata 1 and 7

inhibited the growth of Alternaria alternata, Fusarium
moniliforme, and Rhizoctonia [23], but P94 did not exhibit
inhibitory activity against the phytopathogenic fungi men-
tioned above. The antibacterial activity of strains P. cor-
rugata 1 and 7, as well as tomato strains 1 and 3, was not
mentioned in the previous study [12, 23]. Strain P94 may
produce bacteriocin because it was only active against
closely related classical Pseudomona and was inactive
against Xanthomonas sp. and Agrobacterium sp. A common
trait of bacteriocins is their narrow spectrum of activity, and
bacteriocins are only toxic to bacteria closely related to the
producing strain [14]. Previously, many Pseudomonas sp.
strains had been reported to show growth inhibition in vitro
of pathogenic bacteria by bacteriocin [35].
In the pathogenicity test, plants inoculated with P. Fig. 2 Inhibition activity to tomato grey mildew. Lane 1 = tomato
plants inoculated with B. cinerea treated with distilled water. Lane
corrugata strain ICMP 5819 produced typical symptoms of 2 = tomato plants inoculated with B. cinerea treated with P94. Lane
necrosis and pith hollowing, but they showed no difference 3 = no B. cinerea as control.

123
Y. Guo et al.: Characterization of P. corrugata Strain P94 251

Table 2 Biochemical and physiologic characteristics of P. corrugata Table 2 continued

strain 94 and strain NCPPB 2445 reported by Lopez et al. (1994)
Test Strain 94 2445 NCPPB
Test Strain 94 2445 NCPPB
Putrescine + +
Gram reaction –a – Threonine + +
Fluorescence on KB – – Histamine + NT
Levan – –
a
+, positive reaction; –, negative reaction; w, weak reaction; NT,
Oxidase + +
not tested
Arginine dihydrolase + – b
Tested by Biolog identification system
Starch hydrolysis – –
Gelatin hydrolysis + NT
Pyovendin – NT when inoculated with strain P94 (Fig. 1). Strain P94
Aesculin utilization – – showed the ability to protect tomato against B. cinerea in
Pectate liquefaction – – tomato leaf testing in vitro (Fig. 2), and the biologic con-
Nitrate reduction + + trol effect of P94 was 55.3% by testing 90 tomato leaves
Lecithinase + + (30 leaves treated with P94, 30 leaves as negative control,
Lipase – + and 30 leaves treated with water only as blank control).
Poly-b-hydroxybutyrate + + This result was in accordance with recent studies showing
Anaerobic growth – – that P. fluorescens (strains PTA-268 and PTA-CT2) sup-
Catalase + NT pressed grapevine grey mildew caused by B. cinerea (45%
Urase + NT to 70%) [20]. We had not previously found the reports
H2S production – NT about P. corrugata biocontrol of B. cinerea. P. corrugata
Hypersensitivity reaction – NT was isolated from rhizosphere of maize, which had shown
Carbohydrates and polyalcohols effective ability in suppressing damping-off [11, 12, 24],
D-glucoseb + + and further work should be done to determine if strain P94
D-fructoseb + + could also suppress damping-off.
D-galactose b
+ + Strain P94 was identified as P. corrugata using the
D-arabinoseb – – Biolog identification system, and that was confirmed by
L-rhamnoseb – – 16S rDNA sequence analysis and biochemical and physi-
Sucroseb + +
ologic characteristics. This procedure in identification was
Cellobioseb – –
more accurate than the traditional method. The 16S rRNA
Maltoseb – –
gene sequence of this strain exhibited 99.7% similarity to
that of P. corrucata. The results of biochemical and
Trehaloseb +w +
physiologic testing is listed in Table 2. Strain P94 cells
Inositolb – NT
were Gram negative, rod shaped (0.7 · 1.8 lm), and motile
Arabitolb – NT
by ‡2 polar flagellum. The bacteria was aerobic and pro-
Erythritolb – NT
b duced a diffusible nonfluorescent, yellowish-green pig-
Sorbitol – –
ment. This strain showed positive reactions for oxidase,
Adonitolb – –
gelatin hydrolysis, poly-beta-hydroxybutyrate (PHB)
Mannitolb +w +
accumulation, and nitrate reduction, but it was negative for
D-Xylose – –
starch hydrolysis and levan production, which corresponds
D-Ribose + +
with P. corrugata reports in the literature [18, 33]. The
Organic salts
biochemical and physiologic characteristics of strain P94
Malate + +
produced similar results to those of P. corrucata strain
Malonate + + NCPPB 2445, with the exception of arginine dihydrolase
Gluconate + + and lipase. Arginine dihydrolase activity was positive for
Benzoate – – strain P94 and negative for strain NCPPB 2445. Lipase
Fumarate + + activity was negative for strain P94 and positive for
Oxalate – – NCPPB 2445. However, Sutra et al. [33] found that not all
Succinate + + P. corrugata strains were negative for arginine dihydrolase.
meso-Tartrate + NT The size of specific PCR product with the mixed primers
Nitrogenous compounds was 600 bp, indicating that strain P94 belonged to the P.
Homoserine + + corrugata genomic group II (Fig. 3), and further support-

123
252
Fig. 3 PCR amplification with a mixture of primers (PC1/1, PC1/2,
PC5/1, and PC5/2). Lanes 1 = marker. Lane 2 = P. fluorescens P12 as
negative control. Lane 3 = P. corrugata P94 belonged to P. corrugata
genomic group II. Lane 4 = P. corrugata ICMP 5819 belonged to
P. corrugata genomic group I
ing evidence for this result came from biochemical tests.
Genomic groups I and II corresponded, respectively, to the
subphenas 1a and 1b of Sutra et al. [33], and the genetic
groups were not correlated with differences in pathoge-
nicity or virulence of the strains [6]. Use of meso-tartrate,
isobyturate, and histamine was the distinguishable char-
acteristic of P. corrugata subphenas 1a and 1b [33]. Strain
P94 fell into P. corrugata subphena 1b because it could
assimilate meso-tartrate and histamine (Table 2).
It has been documented (1) that HCN and cell wall–
degrading enzymes–such as cellulases, proteases, and
chitinases–could be involved in antagonism toward phyto-
pathogens and contribute to biocontrol of plant diseases [1,
4] and (2) that production of IAA and phosphatase by plant-
beneficial bacteria enhance the development of the host plant
root system and thus help in the growth of plants [1, 27].
Strain P94 showed a positive reaction for production of
HCN, protease, phosphatase, and IAA and a negative reac-
tion for production siderophore, chitinase, and cellulase.
These characteristics of P. corrugata have not been
described previously. Quantitative dating confirmed the
production of 21.8 and 7.1 lg/ml HCN in the presence and
123
Y. Guo et al.: Characterization of P. corrugata Strain P94
absence of glycine, respectively. However, the amount of
HCN released by P94 was greater than that achieved with P.
aeruginose PAO1 or Pseudomonas sp. NJ-101, whatever the
glycine condition [1, 26]. Strain P94 produced 15.97 lg/ml
and 6.97 lg/ml IAA in nutrient broth with and without
tryptophan, respectively. Quantitative analysis revealed the
release of 82.4 lg/ml soluble phosphate from tricalcium
phosphate in the medium. The phosphate-solubilizing
activity of P. corrugata P94 was stronger than that of
Pseudomonas sp. NJ-101 [1]. However, IAA production was
low compared with that reported in the earlier study [1, 15].
In this study, for the first time, we reported P. corrugata
P94 as a new nonpathogenic strain of tomato rhizosphere
soil origin with the production of HCN, IAA, phosphatase,
and protease and without the production of siderophore,
chitinase, and cellulase. P. corrugata P94 showed the
ability to control tomato grey mildew caused by B. cinerea,
inhibit phytopathogenic fungi and bacteria, and produce
plant growth–promoting substances, such as IAA and
phosphatase, that would enhance the potential use of P94 as
an effective biocontrol agent.
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