IJERD – International Journal of Environmental and Rural Development (2012) 3-1

Research article
erd

Biocontrol of Phytopathogen by Pseudomonas fluorescens
R21, Isolated from Rice Rhizosphere in Thailand
PHRUEKSA LAWONGSA*
Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand
Email: phrueksa@gmail.com

NANTAKORN BOONKERD
Suranaree University of Technology, Nakhon Ratchasima, Thailand

FERGAL  O’GARA
University College Cork, National University of Ireland, Cork, Ireland

NEUNG TEAUMROONG
Suranaree University of Technology, Nakhon Ratchasima, Thailand
Received 14 December 2011

Accepted 18 April 2012(*: Corresponding Author)

Abstract Secondary metabolites produced by fluorescent Pseudomonas play key roles inthe suppression of various soilborne plant pathogens. However, the performance of thisbiocontrol agent varies depending on the environment and host plant species. In this study,
In vitro antagonistic activity against phytopathogens by Pseudomonas fluorescens R21,
which was isolated from rice rhizosphere in Thailand, was investigated in comparison to
Pseudomonas fluorescens F113, which was isolated from sugar beet rhizosphere in
Ireland. The result of in vitro antagonistic activity showed that Pythium spp. wassuppressed by strains R21 and F113. Then, strain R21 that has the ability to produce IAA
and to control plant pathogen was investigated for in vivo antagonistic activity and also
was screened for the production of secondary metabolites such as pyoluteorin, pyrrolnitrin,hydrogen cyanide and 2,4-diacetylphloroglucinol (DAPG). The result of in vivo
antagonistic activity confirmed that Pythium spp. was suppressed by strains R21 and F113.
In addition, the application of strain R21 to rice seeds significantly increased plant height,
shoots dry weight and roots dry weight of rice while the application of strain F113 to riceseeds showed no significant difference when compared to control.
Keywords Pseudomonas, rice, Pythium spp.

INTRODUCTION
Agriculture over the past few decades is heavily dependent on the application of chemical inputs.
However, many chemical pesticides are very toxic and thus result in contamination of environment.
Biological control is thus being considered as an alternative or a supplemental way of reducing theuse of chemicals in agriculture (Compant et al., 2005; Welbaum et al., 2004).
The introduction of P. fluorescens as a biocontrol agent offers a promising alternative tomanage soilborne plant pathogens. However, the production of an antimicrobial compound varies
among cultivars of the same species, and this has hampered the commercialization (Notz et al.,2001). The studies of the ability to produce antibiotic secondary metabolites and their plant growthpromoting potential are important not only for understanding their ecological roles in therhizosphere and their interaction with plants, but also for any biotechnological applications.
In this study, P. fluorescens R21, isolated from rice (Oryza sativa) rhizosphere in Thailand,that has the ability to produce indole-3-acetic acid (IAA) was selected (Lawongsa et al., 2008).
Strain R21 was investigated for in vitro and in vivo antagonistic activity of pathogenic fungi andalso screened for secondary metabolites production. These results will facilitate overcomingⒸISERD189

IJERD – International Journal of Environmental and Rural Development (2012) 3-1

existing limitations in the understanding of plant-microbe interactions of strain R21. In this work, P.
fluorescens F113 isolated from sugar beet (Beta vulgaris) rhizosphere in Ireland was used as thereference strain to compare with strain R21.
MEHODOLOGY
Bacterial strains and culture conditions
Isolation of rice rhizosphere strain R21 was carried out by serial dilution. Nutrient agar andPseudomonas isolation agar (Sigma-Aldrich) were used. Strain R21 was identified on the basis of
growth characteristic, microscopy and biochemical tests. The strains of Pseudomonas fluorescens
were maintained on Luria-Bertani (LB) agar at 4°C and grown at 28°C overnight, shaking at 150
rpm.
In vitro biological control assay
An inhibition of phytopathogen by the Pseudomonas strains on Potato dextrose agar (PDA) plates
was performed as detailed in previous study (Lawongsa et al., 2008). Bacterial suspension ofstrains F113 and R21 was spotted 2 cm from the edge of the plate, and 0.1-0.3 cm square plug froma culture of Pythium spp. was placed at the center of the plate. The results were assessed after 3
days by measuring the distance between the edges of the bacterial colony and the fungal mycelium.
Rice and soil preparation for the assay of in vivo antagonistic activity
The cultivated rice (Oryza sativa cultivar Pathum Thani 1) was obtained from Pathum Thani Rice
Research Center, Pathum Thani, Thailand. The rice seeds were surface-sterilized with 70% ethanolfor 1 min and shaken in 10% (w/v) NaOCl solution for 30 min. Seeds were then washed three times
with sterilized distilled water by shaking (15 min each) (Prakamhang et al., 2009). Surface
sterilized seeds were gnotobiotically germinated on sterilized wet tissue paper. After 3 days, riceseedlings in each treatment were transferred into pots (cm in diameter, 60 cm high) containing
autoclaved soil. Three rice seeds were planted to a depth of 1 cm in each pot. Three replications foreach treatment were done. Rice pots were placed in growth chambers maintained at 25 °C (± 1 °C)
with 12 hours of light and 12 hours of darkness (Kato-Noguchi and Kanesawa, 2003).
Preparation of bacterial inocula and seed treatment for the assay of in vivo antagonistic
activity
Cells of antagonistic bacteria for use in the assay of in vivo antagonistic activity were grown inKing’s   Medium   B   broth   (KMB)   into a late exponential phase at 28°C with shaking at 150 rpm
overnight. Cells were then harvest by centrifugation (5000 rpm/min for 5 min), washed twice andresuspended in 0.5% sterile NaCl solution. The rice seeds were soaked with bacterial suspensionthat was adjusted to about 108 colony forming units (CFU/ml) for each experiment and appliedwith Pythium spp. (1 × 106 spores/ml) (Timms-Wilson et al., 2000). Control treatments were
inoculated in sterile distilled water and seedling with disease symptoms were recorded 4 weeks
after planting. Plants were arranged in a randomized completed block design with three replications.
Percentage of disease incidence was assessed 4 weeks after planting. Rice plants were sampled 4
weeks after planting to measure plant height, shoot and root dry weight.
Statistical analysis
Statistical analysis was performed using one way analysis of variance (ANOVA) followed by
Duncan's Multiple Range Test (DMRT) at 5 % level using the statistical package of the social
sciences (SPSS) version 17.0. The data presented are means standard deviation (SD) of three
replicates.
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Detection of pyoluteorin and pyrrolnitrin biosynthetic lociPrimers PltBf2 and PltBr were used for detection of the pyoluteorin biosynthetic locus of pltB.
Primers PrnCf and PrnCr were used for detection of the pyrrolnitrin biosynthetic locus of prnC.
PCR amplification was carried out in 25 l reaction mixture containing 1× Taq DNA polymerase
buffer, 200 M each of dATP, dTTP, dGTP, and dCTP, 20 pmol of each primer, 1.5 mM MgCl2,
and 0.06 units of GoTaq DNA polymerase (Promega). The PCR cycling program consisted ofinitial denaturation at 94°C for 2 min followed by 29 cycles of 94°C for 1 min, 58°C for 45 s, and72°C for 1 min. The amplification products were electrophoresed in 1% agarose gels in 1× TAE
buffer for 40 min at 100 Volt at room temperature, stained with ethidium bromide, andphotographed under UV light (Mavrodi et al., 2001).
Detection of hydrogen cyanide production
The production of hydrogen cyanide (HCN) was measured as described by Castric, 1975. Whatman
3MM paper was soaked in a chloroform solution containing copper (II) ethyl acetoacetate (5mg/ml) and 4,4'-methylene-bis-(N,N-dimethylaniline) (5 mg/ml), and subsequently dried andstored in the dark. A piece of paper was placed in the lid of a Petri dish in which bacteria had beenplaced on SA agar. The Petri dish was incubated overnight at 28 °C. Production of HCN by thebacteria was indicated by blue coloration of the paper (Castric, 1975).
Detection of genes involved with DAPG production by PCRStandard PCR mixtures and PCR conditions were employed: typically, an initial denaturation stepat 96°C for 3 min followed by 30 cycles of denaturation at 94°C for 30 s, then 30 s of primer
annealing with temperatures dependent on the primer pairs used, and an extension of 1 kb per min
at 72°C. DAPG genes were determined using phlD gene specific primers, Phl2a and Phl2b(Raaijmakers et al., 1997), and phlA gene specific primer, phlA-1f and PhlA-1r (Rezzonico et al.,2003).
RESULTS AND DISCUSSION
In vitro antagonistic activity
The antagonistic activity from both strains R21 and F113 was summarized in Table 1.
Table 1 Measurement of diameter of fungal inhibition ring (cm) of P. fluorescens R21
against Pythium spp.
PathogenDiameter of fungal inhibition ring (cm)R21F113
1.4 ± 0.05
1.4 ± 0.05

Pythium spp.

The in vitro antagonism experiments with Pythium spp. (Figure 1) revealed that strain F113
showed the same level of antagonistic activity when compared to strain R21.
Assay of in vivo antagonistic activity
After two weeks, rice seedling inoculated with strains R21 and F113 showed damping-off
symptoms. An initial symptom was developed as lesions on sheaths of lower leaves near the water
line. After 4 weeks, the disease intensity in the treatment inoculated with Pythium spp. and strain
R21 was 58.3 % while the disease intensity in strain F113 was 66.7 %. However, there is no
significant difference in the disease intensity between applications of strain R21 and strain F113
against Pythium symptom (Fig. 2 and Table 2). These results indicated and confirmed the result ofⒸISERD191

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in vitro antagonistic activity that the colonization of roots by Pythium spp. was suppressed by bothstrains R21 and F113. Additionally, there is no significant difference in plant height, shoots dry
weight and roots dry weight of rice between applications of strain R21 and strain F113 against
Pythium spp. Interestingly, the application of strain R21 to rice seeds showed significantly
increased plant height, shoots dry weight and roots dry weight of rice when compared with control
while the application of strain F113 to rice seeds showed no significant difference when compared
to control.

Fig. 1 In vitro antagonistic activity against Pythium spp. by strain R21 and strain F113

Fig. 2 In vivo antagonistic activity of strain R21 and strain F113 against Pythium spp.
Table 2 Disease incidence, plant height, and dry weight of shoots and roots of riceinoculated with strain R21 grown in the presence of Pythium spp.
Sample% incidenceControl
R21
F113
Pythium spp.
R21+Pythium spp.
F1113+Pythium spp.
%CV

0c
0c
0c
100±0.00a
58.3±14.43b
66.7±14.43b
22.22

Plant height(cm)
21.70±1.57bc
28.77±2.81b
22.40±1.44b
9.40±3.65d
18.77±2.85bc
17.67±1.59c
12.46

Shoot dry mass
(mg)
32.47±2.50b
56.57±6.14b
33.00±7.05b
7.90±0.98d
23.57±5.95c
22.57±1.05c
15.94

Root dry mass
(mg)
11.44±0.30b
22.22±3.89b
10.15±3.21b
2.17±0.75c
5.56±0.70c
5.82±0.98c
20.36

Means in columns followed by different letters are significantly different at 5 % level according to the Duncan's Multiple
Range Test (DMRT)

Detection of secondary metabolites
Most biocontrol strains of Pseudomonas spp. with a proven effect in plant bioassays produce one orseveral antibiotic compounds. Pyoluteorin is composed of a resorcinol ring, derived throughⒸISERD192

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polyketide biosynthesis (Nowak-Thompson et al., 1997). It is an antibiotic that inhibits oomycete
fungi, including the plant pathogen Pythium ultimum, and suppresses plant diseases caused by thisfungus (Howell and Stipanovic, 1980). The pyoluteorin biosynthetic gene cluster pltLABCDEFG isrequired for pyoluteorin biosynthesis (Nowak-Thompson et al., 1999). In this experiment, strain
R21 was screened for the presence of pyoluteorin. P. fluorescens Pf-5 and CHA0 were used to be
the positive control. The PCR result showed no PCR product was amplified from strain R21 with
the PltBf2 and PltBr primers that amplified the predicted 773-bp fragment for detection of thepyoluteorin biosynthetic locus of pltB (Fig. 3). Pyrrolnitrin is a secondary metabolite derived fromtryptophan and has strong antifungal activity (Zhou et al., 1992). DNA region confers the ability to
produce pyrrolnitrin that contains four genes, prnABCD, each of which is required for pyrrolnitrin
production. Pyrrolnitrin has been described as an inhibitor of fungal respiratory chains (Tripathi
and Gottlieb, 1969) and synthetic analogues of pyrrolnitrin have been developed for use as
agricultural fungicides (Ligon et al., 2000). In this experiment, strain R21 was screened for thepresence of pyrrolnitrin. P. fluorescens Pf-5 and CHA0 were used to be the positive control. The
PCR result showed no PCR product was amplified from strain R21 with the PrnCf and PrnCr
primers that amplified the predicted 719-bp fragment of prnC (Fig. 3).

Fig. 3 PCR analysis of genes involved in pyoluteorin (pltB) and pyrrolnitrin (prnC)
production. Lane 1, 100 bp ladder (Invitrogen); lane 2, R21; lane 3, Pf-5;
lane 4, CHA0
HCN is a volatile compound which plays a role in biological control of some soilborne
diseases (Haas and Defago, 2005). The cyanide ion derived from HCN is a potent inhibitor of many
metalloenzymes, especially copper-containing cytochrome c oxidases (Blumer and Haas, 2000). In
this experiment, strain R21 was screened for the presence of HCN. P. fluorescens Pf-5 and F113
were used to be the positive control for HCN detection. Strain R21 showed a negative result ofhydrogen cyanide production (Fig. 4).

Fig. 4 Hydrogen cyanide production assay of strains Pf-5 (a), R21 (b) and F113 (c)DAPG is the best-known phloroglucinol compound in a family of related molecules that
includes MAPG and uncharacterized condensation products of DAPG and MAPG (Heeb et al.,2002). DAPG causes membrane damage to Pythium spp. and is particularly inhibitory to zoosporesof this oomycete (de Souza et al., 2003). In this experiment, strain R21 was screened for thepresence of DAPG. P. fluorescens Pf-5, CHA0, and F113 were used to be the positive control. The
result showed that the positive amplification PCR product was revealed by strain R21 usingⒸISERD193

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primers Phl2a and Phl2b that amplified the predicted 745-bp fragment of phlD (Fig. 5) and primers
phlA-1f and phlA-1r that amplified the predicted 418-bp fragment (Fig. 5).

Fig. 5 PCR analysis of genes involved in DAPG production (phlD and phlA).
Lane 1, 1 kb ladder (Promega); lane 2, R21; lane 3, Pf-5; lane 4,
CHA0; lane 5, F113
CONCLUSION
Among biocontrol agents, P. fluorescens producing the polyketide antibiotic DAPG are importantgroups of plant growth promoting rhizobacteria (PGPR) that suppress root and seedling diseases ona variety of crops. In this study, the results of in vitro and in vivo antagonistic activity showed that
Pythium spp. that caused damping-off and root rot was suppressed by strain R21 and F113.
Interestingly, the application of strain R21 to rice seeds showed a significantly increased of plantheight, shoots dry weight and roots dry weight of rice when compared with control while theapplication of strain F113 to rice seeds showed no significant difference when compared to control.
This information indicated that the ability to colonize rice roots is variable between rhizobacteria,
being these characteristics a reflection for their ability to compete for ecological niches in therhizosphere. Moreover, the exertion an appropriate biological control of soil borne fungi relies on
their ability to colonize roots efficiently; otherwise, their biocontrol character would be unusable.
Thus, introduction of biocontrol agent to agriculture requires appropriate and compatible PGPR for
the goal of making agriculture more sustainable. Moreover, an understanding of how biocontrol
bacteria regulate the inhibition of pathogens is important for predicting the optimum environmental
conditions of the bacteria to produce antagonistic compounds.
ACKNOWLEDGEMENT
This work was supported by a grant from Thailand Research Fund and Suranaree University ofTechnology.
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