Isolation and characterization of Pseudomonas

Microbiological Research 167 (2012) 388–394

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Isolation and characterization of Pseudomonas brassicacearum J12 as an antagonist against Ralstonia solanacearum and identification of its antimicrobial components Tiantian Zhou, Da Chen, Chunyu Li, Qian Sun, Lingzhi Li, Fang Liu, Qirong Shen, Biao Shen ∗ Jiangsu Provincial Key Lab for Organic Solid Waste Utilization, Nanjing Agricultural University, Nanjing 210095, China

a r t i c l e

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Article history: Received 25 November 2011 Received in revised form 6 January 2012 Accepted 8 January 2012 Keywords: Pseudomonas brassicacearum 2,4-DAPG Biocontrol phlACBD gene cluster Ralstonia solanacearum

a b s t r a c t A bacterial strain, J12, isolated from the rhizosphere soil of tomato plants strongly inhibited the growth of phytopathogenic bacteria Ralstonia solanacearum. Strain J12 was identified as Pseudomonas brassicacearum based on its 16S rRNA gene sequence. J12 could produce 2,4-diacetylphloroglucinol (2,4-DAPG), hydrogen cyanide (HCN), siderophore(s) and protease. The maximum growth and antagonistic activity were recorded at 30 ◦ C and pH 8. Glucose and tryptone were used as the most suitable carbon and nitrogen sources, respectively. Strain J12 significantly suppressed tomato bacteria wilt by 45.5% in the greenhouse experiment. The main antimicrobial compound of J12 was identified as 2,4-diacetylphloroglucinol (2,4DAPG) by HPLC–ESI-MS analysis. The gene cluster phlACBD, which is responsible for 2,4-DAPG production, was identified and expressed in the bacterial strain Escherichia coli DH5␣. © 2012 Elsevier GmbH. All rights reserved.

1. Introduction Ralstonia solanacearum is the causal agent of bacterial wilt, one of the most important bacterial diseases worldwide. Hundreds of different plant species, including many important agricultural crops such as potato, tomato, banana, and pepper plants, as well as trees, such as eucalyptus, are affected by this vascular pathogen (Hayward 1991). During infection, the pathogen bacteria become motile and travel throughout the vascular system of the plant. As the cell density increases, virulence genes are expressed and the cells become undergo proliferation and secrete exopolysaccharide and pectin-degrading enzymes, leading to the death of the plant (Clough et al. 1997; Saile et al. 1997). Bacterial wilt disease is widespread causing substantial crop losses in most areas of the world. Germicides are usually used as a solution to the problems of pathogen attack; however, their use results in serious environmental problems. Moreover, there is no effective chemicals to control bacterial wilt so far. Recently, there has been an increasing interest in using beneficial microorganisms as a solution to the overuse of potentially harmful pesticides (Fravel 1988; Weller 1988; Sakthivel and Gnanamanicham 1987; Xu and Gross 1986). Some rhizobacteria have been used extensively as biological agents to control many soil-born plant pathogens (Amico et al. 2005;

∗ Corresponding author. Tel.: +86 25 84395212; fax: +86 25 84395212. E-mail address: [email protected] (B. Shen). 0944-5013/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. doi:10.1016/j.micres.2012.01.003

Rajkumar et al. 2005). Many fluorescent Pseudomonas species from the soil environment could produce secondary metabolites to inhibite phytopathogenic bacteria, oomycetes and fungi (Morrissey et al. 2002; Raaijmakers et al. 2002; Haas and Defago 2005). Some of the fluorescent pseudomonads have currently received world-wide attention due to the production of a wide range of antifungal compounds i.e., fluorescent pigments (Stainier et al. 1966); siderophores (Neiland and Leong 1986); volatile compounds such as HCN (Defago and Haas 1990), antibiotics such as phenazine-1-carboxylic acid (Huang et al. 2009); pyoluteorin (Hu et al. 2005); phenazine-1-carboxamide, viscosinamide and tesin (Chin-A-Woeng et al. 2003); 2,4-diacetylphloroglucinol (DAPG) (Shanahan et al. 1992) and lytic enzymes. The phenolic metabolite 2,4-DAPG is an important component of the natural suppressiveness of certain agricultural soil to take all disease of wheat and black root of tobacco, and the active ingredient of many of the key biocontrol strains of Pseudomonas fluorescens (Picard and Bosco 2006; Rezzonico et al. 2007). There are numerous reports of the production of antibiotic compounds by Pseudomonas spp. Some of these antibiotics have been characterized chemically (Leisinger and Margraff 1979). Howell and Stipanovic (1979, 1980) provided evidence that different isolates of P. fluorescens were antagonistic to pathogens of cotton seedlings because of the production of the antibiotics pyrrolnitrin and pyoluteorin. Another antibiotic, produced by a Pseudomonas strain that was able to suppress take-all in wheat, was identified as a dimer of phenazine-1-carboxylate (Gurusiddaiah et al. 1986). Lam et al. (1987) reported a Pseudomonas isolate that produced an

T. Zhou et al. / Microbiological Research 167 (2012) 388–394

unidentified antibiotic able to inhibit the fungus that causes Dutch elm disease. Pseudomonas is also a major group of rhizobacteria that aggressively colonize plant roots. It has been considered an important group for sustainable agriculture; thus, the screening of such bacteria with biofertilizing and biocontrol properties has been the subject of interest. In this paper, we describe the isolation and characterization of an antagonistic microorganism against phytopathogen R. solanacearum and present a preliminary characterization of the antagonistic mechanism. The optimal conditions for antagonistic activity and growth, the biocontrol activity against tomato bacterial wilt in the greenhouse and the characterizations of the antimicrobial compounds were investigated. 2. Methods 2.1. Isolation of antagonistic bacteria Antagonistic bacteria were isolated from the rhizospheric soil of tomato plants in Henan Province, China. The soil sample (5 g) was shaken in 100 ml sterilized water for 20 min. The soil suspension was then serially diluted and spread on King’s B agar (King et al. 1954). After incubating at 30 ◦ C for 2 days, single bacterial colonies were selected and streaked onto a new LB (Luria–Bertani) plate. The purified colonies were preserved in LB medium containing 10% glycerol at −80 ◦ C. The antagonistic activities of all the isolates against the pathogen R. solanacearum were screened by the dual inoculation technique (Bordoloi et al. 2001). Test plates were prepared with nutrient agar (NA) medium mixed with R. solanacearum (106 CFU/ml), and each of the bacterial candidates (107 CFU/ml) was spot inoculated as three replicates. Antagonistic activities were evaluated by measuring the widths of the inhibition zones after 2 days of incubation at 30 ◦ C. 2.2. Identification of the selected bacteria The primer pair used for amplification of the bacterial 16S rDNA was 27f and 1492r (Eden et al. 1991). The partial sequence of the 16S rRNA gene was analyzed using the NCBI BLAST tool for similarity comparison. Alignment of 16S rRNA genes from J12 and GenBank database was performed using the CLUSTAL W program. The unrooted tree was built by the Neighbor Joining method. 2.3. Production of antibacterial metabolites of strain J12 Production of siderophore by J12 was determined using the chrome azurol S (CAS) agar method (Alexander and Zuberer 1991). After incubation at 30 ◦ C for 3 days, siderophore production was assessed by a change in the color of the medium from blue to orange. The production of HCN was determined by spreading 1 ml of J12 broth culture (24 h old) on the King’s B plates and by incubating the plates with Whatman filter paper soaked with a solution containing 0.5% picric acid in 2% sodium carbonate placed in the lid plate (Bakker and Schippers 1987). The plates were sealed with the parafilm to avoid gas from escaping. A change in the color, from yellow to orange, of the filter paper was observed after 2 days. Protease production was determined on skim milk agar plates (Smibert and Krieg 1994). Protease activities were identified by a clear zone around the colony. To determine the phosphate solubilization ability of J12, a single colony was chosen and streaked onto the Pikovskaya’s agar medium (Pikovskaya 1948). After 3 days of incubation at 30 ◦ C, a clear zone around the colony was observed. In vitro biofilm formation assays were performed as described previously (Wei and Zhang 2006). Briefly, test strains were grown

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to saturation in LB media and then diluted 1:1000 in fresh LB media. The diluted culture (0.5 ml) was transferred to a polyvinyl chloride (PVC) plastic Eppendorf tube and incubated without shaking for 24 h at 30 ◦ C. The resulting biofilm was stained with 0.1% (w/v) crystal violet for 20 min, and then unattached cells and residual dye were removed. The dye was dissolved in 95% ethanol, and A570 nm of the dissolved dye was determined. 2.4. Optimum conditions for maximum growth and antagonistic activity of J12 The strain J12 was inoculated into nutrient broth and grown at various temperature ranges from 4 to 45 ◦ C for 24 h. Bacterial growth was monitored by measuring optical density (OD600 ) of the culture (Patton et al. 2006). To observe the pH effect on growth, the strain J12 was inoculated into nutrient broth with pH ranges of 3–10 and OD600 was recorded after 24 h of incubation at 30 ◦ C. Various carbon sources (glucose, lactose, sucrose, maltose, d-fructose and d-galactose) at a concentration of 2 g l−1 and nitrogen sources (beef extract, tryptone, yeast extract, urea, KNO3 , (NH4 )2 SO4 and NH4 NO3 ) at a concentration of 2 g l−1 were respectively added to modified mineral-based medium (MM) (Saddiqui and Shaukat 2004), along with a 1 ml trace salt stock solution (Soliveri et al., 1987). OD600 of the culture was measured after 2 days of incubation at 30 ◦ C. The influence of temperature, pH and carbon and nitrogen source for antagonistic activity were observed by the dual culture technique. The diameter of the inhibiting zone was recorded after 3 days incubation at 30 ◦ C. 2.5. Biocontrol activity against tomato bacteria wilt in greenhouse Pot experiment of biocontrol activity of J12 against tomato bacteria wilt was proceeded in greenhouse. The treatments were as following: control 1: not inoculated with any microbe; control 2: only inoculated with R. solanacearum; treatment: inoculated with R. solanacearum and J12. Tomato seeds were surface-sterilized in 2% NaClO for 3 min and 75% ethanol for 2 min and then rinsed 3 times in sterile water. The seeds were then germinated on 9cm plates covered with sterile wet filter paper at 25 ◦ C for 48 h. J12 cells were grown in LB medium. The bacteria were harvested by centrifuging at 5000 × g for 10 min and washing twice with phosphate-buffered saline (PBS) with a pH 7.0. The soil used in the experiment was inoculated with the J12 bacterial suspension to obtain final concentrations of 107 cells/g of natural soil. After inoculation, germinated seeds were transferred to the soil, irrigated with 1/2 hoagland medium and incubated in a growth chamber at 25 ◦ C under a 16-h light regimen. One week later 10 ml of R. solanacearum suspension (108 CFU/ml) was inoculated to the tomato seedlings. The percentage of disease incidence and severity were recorded on day 30 after sowing. Each treatment had 20 replicates. The calculation of control efficiency (CE) was according to the formula: CE (%) = (DIcontrol2 − DIx )/DIcontrol2 × 100% (DI: disease index, x: one of the treatments). Data were analyzed for significance using analysis of variance followed by Ducan’s leastsignificant-difference test (P = 0.05), using SPSS-software v. 13.0 (SPSS Inc., Chicago, IL, USA). All experiments were performed at least twice. 2.6. Characterization analysis of antibacterial compounds from J12 Antimicrobial compounds were extracted from a 4-day-old culture of strain J12. After the culture were centrifuged at

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5000 × g for 10 min, the supernatants were acidified to pH 2. After extraction with an equal volume of ethyl acetate, the organic layer was decanted and evaporated. Extracts were resuspended in 0.5 ml methanol. Samples were spotted on silica thin-layer chromatography (TLC) and developed by chloroform/acetone (9:1, v/v) and observed under UV light (254 nm). The distinct factions were recovered in ddH2 O and tested for antimicrobial activity against R. solanacearum. The fraction that exhibited strong antimicrobial activity in vitro was further analyzed by an HPLC system (Agilent 1200). The mobile phase was a 45% CH2 CN solution, containing 0.1% H3 PO4 , with a speed of 1.0 ml/min. The detection’s wavelength was fixed at 270 nm. The purified compound was subjected to liquid chromatography–electrospray ionization-mass spectrometry analysis (LC/ESI/MS) for molecular weight determination. The MS spectrum analysis was used for the main protonated compounds with an Agilent LC-MS 1100 instrument. The samples were ionized electrospray with a positive polarity. The electrospray ionization was operated at a spray voltage of 4.5 kV and a capillary temperature of 300 ◦ C. The samples were injected through an autosampler at a flow of 0.4 ml/min. Mobile phase A was water with 0.1% acetic acid, and mobile phase B was acetonitrile. The following elution condition was used: A:B = 60:40. Data acquisition was in the positive mode. The standard 2,4-DAPG chemical was purchased from Santa Cruz Biotechnology Inc.

2.7. Detection of the antibiotic genes in strain J12 and expression of the phlACBD gene cluster in Escherichia coli Detection of the genes responsible for the production of antibiotics was performed by PCR using gene-specific primers. The antibiotics of interest are 2,4-diacetylphloroglucinol (DAPG) (Mavrodi et al. 2001b), phenazine-1-carboxylic acid (PCA) (Raaijmakers et al. 1997), phenazine-1-carboxamide (PCN) (Mavrodi et al. 2001a), pyrrolnitrin (PRN) (Mavrodi et al. 2001b), pyoluteorin (PLT) (Mavrodi et al. 2001b) and hydrogen cyanide (HCN) (Ramette et al. 2003). PCR products were sequenced and compared with known genes in GenBank using BLAST N. To obtain the phlACBD gene cluster responsible for 2,4DAPG synthesis from strain J12, primers pF322 (5 -AAGGATCCAATGGAGCTCCGAG-3 ) and phl2a (5 -GAGGACGTCGAAGACCACCA-3 ) were designed based on the conserved amino acid sequences of the 2,4-DAPG gene cluster from P. fluorescens 2P24 (DQ083928) (Zhou 2005). The PCR product was cloned into the vector pMD19-T and transferred to E. coli DH5␣ for sequencing and BLAST analysis. The antibiosis activity of the E. coli DH5␣ containing the phlACBD recombination plasmid was tested on the NA plate. E. coli DH5␣ harboring pMD19-T only was used as a control. R. solanacearum was used as the target pathogen. The expression product was subjected to MS analysis.

Pseudomonas tremaeT (AJ492826)

63

Pseudomonas mandeliiT (AF058286)

51 17

Pseudomonas frederiksbergensisT (AJ249382) Pseudomonas liniT (AY035996)

15

Pseudomonas chlororaphisT (Z76673) Pseudomonas chlororaphisT (AY509898)

99

37

Pseudomonas chlororaphisT (DQ682655)

91

Pseudomonas corrugataT (D84012)

60

Pseudomonas kilonensisT (AJ292426) Pseudomonas thivervalensisT (AF100323)

35

50

Pseudomonas brassicacearumT (AF100321)

58 52

J12 (JN605747) Pseudomonas trivialisT (AJ492831)

50

Pseudomonas salomoniiT (AY091528) Pseudomonas veroniiT (AF064460)

31

Pseudomonas marginalisT (Z76663)

19

Pseudomonas rhodesiaeT (AF064459)

74

Pseudomonas grimontiiT (AF268029)

77 T

Pseudomonas migulae (AF074383) Pseudomonas brenneriT (AF268968) 69

Pseudomonas cedrinaT (AF064461)

0.001 Fig. 1. Phylogenetic tree of Pseudomonas species related to J12. The culture collection numbers of the strains are given after the species name. The bar indicates 0.1% estimated sequence divergence. Type strains are marked with T.


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Fig. 2. Production of antibacterial metabolites of strain J12. (a) Siderophore production on CAS assay. (b) Proteolytic activity on skim milk agar. (c) Phosphate solubilization on Pikovskaya’s agar medium. (d) Biofilm formation in a plastic Eppendorf tube.

3. Results

3.2. Production of antibacterial metabolites of strain J12

3.1. Isolation and identification of antagonistic bacteria strains

The appearance of a reddish-brown zone surrounding the J12 colony on CAS agar plates suggested the siderophore production (Fig. 2a). A remarkable change in color from yellow to reddishbrown suggested the HCN production of J12 (data not show). A clear zone on skim milk agar showed the strong protease activity of J12 (Fig. 2b). The clear zone surrounding the J12 colony on Pikovskaya’s agar medium indicated the phosphate solubilization activity (Fig. 2c). The trace of crystal violet in the PVC tube showed the biofilm formation of J12, suggesting its potential colonization ability on plant roots (Fig. 2d).

There were 9 isolates that showed effective antagonistic activity toward R. solanacearum. Blast analysis of the 16S rDNA sequences in the GenBank database revealed that they generally belong to Pseudomonas (three isolates), Brevibacillus (three isolates), Streptomyces (two isolates) and Bacillus (one isolate). Among them, the bacterial strain J12 showed the highest antagonist activity against pathogen R. solanacearum and also showed antagonism against the fungal pathogen Fusarium oxysporum. Therefore the J12 isolate was used for the further study. The colony of strain J12 was yellow pigmented, circular and smooth. The analysis of 16S rDNA sequences indicated that J12 (accession number: JN605747) shared a maximum 99% identity with Pseudomonas brassicacearum NFM421 (CP002585). Moreover, strain J12 clustered with a P. brassicacearum type strain (AF100321) in the phylogenetic tree (Fig. 1): clearly demonstrating that the strain was a member of P. brassicacearum of at the level of 16S rRNA gene sequence homology.

3.3. Effects of cultural conditions on biomass and inhibitory activity of strain J12 The tested pH range for strain J12 to grow and express inhibitory activities was between 4.0 and 10.0. The highest biomass (OD600 = 3.83) and maximum inhibitory zone (28.2 mm) were observed at pH 8.0. pH 7.0–10.0 was a suitable range for the strain to express higher bacterial biomass and

Fig. 3. Effect of culture conditions on growth and antagonistic activity of strain J12. (a) pH, (b) temperature, (c) carbon source, and (d) nitrogen source.

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Fig. 4. Mass spectrum analysis of antibiotic 2,4-DAPG from strain J12.

inhibitory activities, but it failed to grow under acidic conditions (Fig. 3a). Within the temperature range of 10–30 ◦ C, both the biomass and inhibition increased with the elevation in temperature. The temperature range for J12 to express higher biomass and stronger inhibitory activities was 20–30 ◦ C. The highest biomass (OD600 = 3.63) and maximum inhibition zone (24.7 mm) were observed at 30 ◦ C. It grew poorly when the temperature was lower than 10 ◦ C or higher than 37 ◦ C. Neither the growth nor the inhibitory activity was found at 45 ◦ C (Fig. 3b). Different carbon and nitrogen sources also significantly influenced on the growth and antagonistic activity of J12 (Fig. 3c and d). The effect of carbon and nitrogen sources on the antagonistic activity of J12 was identical to that on J12 growth in most cases. J12 expressed highest biomass (OD600 = 0.77) and antagonistic activity (25.3 mm) in the presence of glucose. The lowest biomass (OD600 = 0. 12) and antagonistic activity (13 mm) were observed in the presence of lactose (Fig. 3). Fig. 3d reveals that J12 could grow better in organic nitrogen sources than inorganic. Tryptone was the most suitable nitrogen source for growth and antagonistic activity of J12, followed by yeast extract, whereas the tested inorganic nitrogen sources gave comparatively lower levels of growth and antagonistic activity.

3.5. TLC and HPLC analysis of antimicrobial compounds Four main fractions were exhibited on the TLC plate. The fraction having Rf value 0.70 showed strong antibacterial activity on plate (data not show). The biologically active fraction was isolated for further characterization. HPLC analysis showed that the retention time of the antibacterial compound was 5.9 min, which was the same with that of standard 2,4-diacetylphloroglucinol (2,4-DAPG). HPLC quantification suggested that 2,4-DAPG production of J12 was approximately 40 ␮g ml−1 (OD600 unit−1 ). 3.6. HPLC–ESI-MS Identification of antibiotic 2,4-DAPG The mass spectrum of the antibiotic compound was characterized by three intense peaks with m/e values of 210, 192, and 174 (Fig. 4), which was similar to that of 2,4-DAPG reported by Raaijmakers et al. (1999). The highest mass peak at 210 is the molecular weight of 2,4-DAPG. The peak at m/e 192 is most likely due to a neutral fragment of 2,4-DAPG loss of ·H2 O (M(+) −18 at m/e 210 = 192), whereas the peak at m/e 174 corresponds to loss of 2·H2 O (M(+) −36 at m/e 210 = 174) from the parent molecular ion. In addition to these three major peaks, the mass spectrum of 2,4-DAPG also is also characterized by three minor peaks with m/e values of 150, 118, and 87.

3.4. Biocontrol activity against tomato bacteria wilt in greenhouse Pot experiment showed that wilt disease incidence was 47% when soil inoculated with P. brassicacearum J12, whereas that was 86.2% in non-inoculated control. Biocontrol efficiency was 45.5% when treated with J12 (Table 1). The result indicated that J12 could effectively suppress tomato bacteria wilt caused by R. solanacearum.

Table 1 Control efficacy of J12 strain against tomato bacterial wilt caused by R. solanacearum. Treatments

Disease index (%)

Control effect (%)

Control 1 Control 2 Treatment

0±0c 86.2 ± 9 a 47 ± 6 b

45.5

Control 1 (not inoculated with any microbe), Control 2 (only inoculated with R. solanacearum), Treatment (inoculated with R. solanacearum and J12). Values with the different letter within the same column are significantly different at P < 0.05 according to Duncan’s test. Numbers followed by the “±” are standard errors.

Fig. 5. PCR detection of antibiotic genes in P. brassicacearum strain J12. 1: phlD fragment (745 bp); 2: hcnBC fragment (587 bp); 3: DL2000 DNA Marker.


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Fig. 6. Antibiosis activity against R. solanacearum. (a) E. coli DH5␣ containing pMD19-T plasmid only. (b) E. coli DH5␣ harboring pMD19-T plasmid with a 4.3 kb phlACBD gene fragment. (c) HPLC/ESI/MS analysis of expression product of phlACBD gene cluster in E. coli strain.

3.7. Detection of antibiotic genes by PCR and expression of the phlACBD gene cluster in E. coli Strain J12 showed the presence of gene fragments for 2,4-DAPG and hydrogen cyanide synthesis (Fig. 5). The phlD fragment (745 bp) showed high homology with that of P. brassicacearum NFM421 (CP002585) (99% identity) and P. fluorescens F113 (DQ083928) (95% identity), the hcnBC fragment (587 bp) showed 99% identity with that of P. brassicacearum NFM421(CP002585). J12 did not show the presence of PRN, PLT, PCA and PCN genes when tested in PCR using the gene-specific primer. A 4.3kb gene fragment of phlACBD gene cluster was amplified from J12 genome and submitted to GenBank (accession number: JN561597). A database search has revealed that the 4.3 kb nucleotide sequence showed high homology with some known 2,4-DAPG biosynthesis sequences, such as those of P. brassicacearum NFM421 (CP002585) (99% identity) and P. fluorescens F113 (DQ083928) (96% identity). The phlACBD gene cluster was cloned into pMD19-T and expressed in E. coli DH5␣. The expression product effectively inhibited R. solanacearum on plates (Fig. 6b). Mass analysis of the expression product showed a peak of 211 [M+H+ ] (Fig. 6c), which indicated the molecular weight of 2,4-DAPG. The result demonstrated that the phlACBD gene cluster was responsible for antibiotic 2,4-DAPG production. 4. Discussion In this study, P. brassicacearum J12 was isolated and found to produce 2,4-DAPG, HCN, siderophores, and protease that contribute to pathogen growth inhibition, suggesting its potential in biocontrol of plant disease. The main active compound against R. solanacearum was isolated from J12 and further identified as 2,4-DAPG. This compound is toxic to fungi and bacteria, exhibits herbicidal activity resembling that of 2,4-dichlorophenoxyacetate (2,4-D) (Katar’yan and Torgashova 1976), and has anthelmintic (Bowden et al. 1965) and antiviral properties (Tada et al. 1990). Katar’yan and Torgashova (1976) reported on some herbicidal effects of DAPG, and Reddi and Borovkov (1970) studied its activity against a range of bacteria and fungi. Surprisingly these authors found that DAPG was very active against gram-positive bacteria but less active against fungi and many gram-negative bacteria. However, our study showed that 2,4-DAPG strongly inhibited R. solanacearum, a gram-negative bacterial pathogen. The broad spectrum activity of DAPG suggested that it targets one or more essential cellular processes. An assessment of the physiological effects of DAPG on Pythium ultimum, however, found that DAPG can inhibit zoospore swimming and hyphal growth, and can cause

disorganization of hyphal tips including alteration of the plasma membrane, vacuolisation and cell content disintegration (De Souza et al. 2003). It has been suggested that the ability of DAPG to inhibit a wide range of organisms including fungi and protists may indicate a general target such as membrane integrity or electron transport (Jousset et al. 2006). Gleeson et al. established that inhibition of fungal growth is caused by impairment of mitochondrial function (Gleeson et al. 2010). A number of Pseudomonas strains have been shown to produce 2,4-DAPG (Broadbent et al. 1976; Garagulya et al. 1974; Keel et al., 1990), but little was known about 2,4-DAPG produced by P. brassicacearum. This study demonstrates that, P. brassicacearum J12 could produce 2,4-DAPG confirmed by HPLC and MS. In strain J12, the 4.3 kb phlACBD gene fragment responsible for the production of 2,4-DAPG was identified and expressed in E. coli. The action of HCN in root pathogen control remains unclear. It is possible that HCN production in the rhizosphere could have beneficial or deleterious effects on different plants (O’Sullivan and O’Gara 1992). The antagonistic activities are presumably due to the production of siderophores (Kloepper et al. 1980; Leong 1986). Siderophores are iron-chelating compounds produced by many microorganisms growing under iron-limiting conditions. It has been hypothesized that siderophores function as biostatic compounds by drastically reducing the amount of ferric ions available to certain rhizosphere microflora. However, siderophores may contribute to the control of plant diseases only when soil conditions are favorable to their production (Thomashow and Weller 1995). The ability to solubilize phosphate promoted the growth of plants, and biofilm formation may be helpful during root colonization and protects plants from invasion by soil-born pathogens. Several abiotic factors such as oxygen, temperature, specific carbon and nitrogen sources, and microelements have been identified or proposed to influence in situ antibiotic production by fluorescent Pseudomonas spp. (Duffy and Défago 1997; Howie and Suslow 1991; Shanahan et al. 1992). Our data also showed that temperature, pH and carbon and nitrogen sources affected the growth and antagonistic activity of P. brassicacearum J12. In conclusion, P. brassicacearum J12 produces 2,4-DAPG, HCN, siderophores and proteases, and effectively suppresses tomato bacteria wilt. Thus, J12 is a potential biocontrol strain.

Acknowledgements This research was financially supported by the projects of the China Agriculture Ministry (201103004), the National Nature Science Foundation of China (40871126) and the Chinese Ministry of Science and Technology (2010AA10Z401).

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References Amico ED, Cavalca L, Andreoni V. Analysis of rhizobacterial communities in perennial Graminaceae from polluted water meadow soil, and screening of metal resistant, potentially plant growth-promoting bacteria. FEMS Microbiol Ecol 2005;52:153–62. Alexander DB, Zuberer DA. Use of chrome azurol S reagents to evaluate siderophore production by rhizosphere bacteria. Biol Fertil Soils 1991;12:39–45. Bakker AW, Schippers B. Microbial cyanide production in therhizosphere in relation to potato yield reduction and Pseudomonas spp. mediated plant growth stimulation. Soil Biol Biochem 1987;19:451–7. Bordoloi G, Kumari B, Guha A, Bordoloi MJ, Roy MK, Bora TC. Isolation and structural elucidation of a new antifungal and antibacterial antibiotic produced by Streptomyces sp. 201. Biotechnol Biochem 2001;65:1856–8. Broadbent D, Mabelis RP, Spenser H. C-Acetyphloroglucinols from Pseudomonas fluorescens. Phytochemistry 1976;15:1785. Bowden K, Broadbent JL, Ross WJ. Some simple anthelmintics. Br J Pharmacol 1965;24:714–24. Chin-A-Woeng TFC, Bloemberg GV, Lugtenberg BJJ. Phenazines and their role in biocontrol by Pseudomonas bacteria. New Phytol 2003;157:503–23. Clough S, Flavier A, Schell M, Denny T. Differential expression of virulence genes and motility in Ralstonia (Pseudomonas) solanacearum during exponential growth. Appl Environ Microbiol 1997;63:844–50. Defago G, Haas D. Pseudomonads as antagonists of soil borne plant pathogens: mode of action and genetic analysis. Soil Biochem 1990;6:249–91. De Souza JT, Arnould C, Deulvot C, Lemanceau P, Gianinazzi-Pearson V, Raaijmakers JM. Effect of 2, 4-diacetylphloroglucinol on pythium: cellular responses and variation in sensitivity among propagules and species. Phytopathology 2003;93:966–75. Duffy BK, Défago G. Zinc improves biocontrol of Fusarium crown and root rot of tomato by Pseudomonas fluorescens and represses the production of pathogen metabolites inhibitory to bacterial antibiotic biosynthesis. Phytopathology 1997;87:1250–7. Eden PA, Schmidt TM, Blakemore RP, Pace NR. Phylogenetic analysis of Aquaspirillum magnetotacticum using polymerase chain reaction-amplified 16S rRNA specific DNA. Int J Syst Bacteriol 1991;41:324–5. Fravel DR. Role of antibiosis in the biocontrol of plant diseases. Annu Rev Phytopathol 1988;26:75–91. Garagulya AD, Kiprianova EA, Boiko OI. Antibiotic effect of bacteria from the genus Pseudomonas on phytopathogenic fungi. Microbiol Zh (Kiev) 1974;36: 197–202. Gleeson O, O’Gara F, Morrissey JP. The Pseudomonas fluorescens secondary metabolite 2,4 diacetylphloroglucinol impairs mitochondrial function in Saccharomyces cerevisiae. Anton Van Leeuw 2010;97:261–327, 3. Gurusiddaiah S, Weller DM, Sarkar A, Cook RJ. Characterization of an antibiotic produced by a strain of Pseudomonas fluorescens inhibitory to Gaeumannomyces graminis var. tritici and Pythium spp. Antimicrob Agents Chemother 1986;29:488–99. Haas D, Defago G. Biological control of soil-borne pathogens by fluorescent pseudomonads. Nat Rev Microbiol 2005;3:307–19. Hayward HC. Biology and epidemiology of bacterial wilt caused by Pseudomonas solanacearum. Annu Rev Phytopathol 1991;29:65–87. Howie WJ, Suslow TV. Role of antibiotic biosynthesis in the inhibition of Pythium ultimum in the cotton spermosphere and rhizosphere by Pseudomonas fluorescens. Mol Plant Microbe Interact 1991;4:393–9. Howell CR, Stipanovic RD. Control of Rhizoctonia solani on cotton seedlings with Pseudomonas fluorescens and with an antibiotic produced by the bacterium. Phytopathology 1979;69:480–2. Howell CR, Stipanovic RD. Suppression of Pythium ultimum-induced damping-off of cotton seedlings by Pseudomonas fluorescens and its antibiotic, pyoluteorin. Phytopathology 1980;70:712–5. Huang J, Xu Y, Zhang H, Li Y, Huang X, Ren B, et al. Temperature-dependent expression of phzM and its regulatory genes lasI and ptsP in rhizosphere isolate Pseudomonas sp. strain M185. Appl Environ Microbiol 2009;75:6568–80. Hu HB, Xu YQ, Cheng F, Zhang XH, Hur B. Isolation and characterization of a new Pseudomonas strain produced both phenazine 1-carboxylic acid and pyoluteorin. J Microbiol Biotechnol 2005;15:86–90. Jousset A, Lara E, Wall LG, Valverde C. Secondary metabolites help biocontrol strain Pseudomonas fluorescens CHA0 to escape protozoan grazing. Appl Environ Microbiol 2006;72:7083–90. Raaijmakers JM, Bonsall Robert F, Weller DM. Effect of Population Density of Pseudomonas fluorescens on Production of 2,4-Diacetylphloroglucinol in the Rhizosphere of Wheat. Appl Environ Microbiol 1999;89:470–5. Raaijmakers JM, Vlami M, de Souza JT. Antibiotic production by bacterial biocontrol agents. Anton Van Leeuw 2002;81:537–47. Katar’yan BT, Torgashova GG. Spectrum and activity of the herbicidal effect of 2,4diacetylphloroglucinol. Dokl Akad Nauk Arm SSR 1976;63:109–12. Keel C, Wirthner P, Oberhansli T, Voisard C, Burger U, Haas D, et al. Pseodomonas as antagonists of plant pathogens in the rhizosphere, role of the antibiotic 2,4-diacetylphloroglucinols in the suppression of black root rot of tobacco. Symbiosis 1990;9:327–41. King EO, Ward MK, Raney DE. Two simple media for demonstration of pyocyanin and fluorescein. J Lab Clin Med 1954;44:301–7.

Kloepper JW, Leong J, Teintze M, Schroth NM. Enhanced plant growth by siderophores produced by plant growth promoting rhizobacteria. Nature (London) 1980;286:885–6. Lam BS, Strobel GA, Harrison LA, Lam ST. Transposon mutagenesis and tagging of fluorescent Pseudomonas: antimycotic production is necessary for control of Dutch elm disease. Proc Nat Acad Sci (USA) 1987;84:6447–51. Leisinger T, Margraff R. Secondary metabolites of fluorescent pseudomonas. Microbiol Rev 1979;43:422–42. Leong J. Siderophores: their biochemistry and possible role in biocontrol of plant pathogens. Annu Rev Phytopathol 1986;24:187–209. Mavrodi DV, Bonsall RF, Delaney SM, Soule MJ, Phillips G, Thomashow LS. Functional analysis of genes for biosynthesis of pyocyanin and phenazine-1-carboxamide from Pseudomonas aeruginosa PAO1. J Bacteriol 2001a;183:6454–65. Mavrodi OV, Gardener BBM, Mavrodi DV, Bonsall RF, Weller DM, Thomashow LS. Genetic diversity of phlD from 2,4-diacetylphloroglucinol-producing fluorescent Pseudomonas spp. Phytopathology 2001b;91:35–43. Morrissey JP, Walsh UF, O’Donnell A, Moenne-Loccoz Y, O’Gara F. Exploitation of genetically modified inoculants for industrial ecology applications. Anton Van Leeuw 2002;81:599–606. Neiland JB, Leong SA. Siderophores in relation to plant disease. Annu Rev Plant Physiol 1986;37:187–208. O’Sullivan DJ, O’Gara F. Traits of Fluorescent Pseudomonas spp. involved in suppression of plant pathogens. Microbiol Rev 1992;56:662–72. Patton T, Barrett J, Brennan J, Moran N. Use of aspectrophotometric bioassay for determination of microbial sensitivity to manuka honey. J Microbiol Methods 2006;64:84–95. Picard C, Bosco M. Heterozygosis drives maize hybrids to select elite 2,4diacethylphloroglucinol-producing Pseudomonas strains among resident soil populations. FEMS Microbiol Ecol 2006;58:193–204. Pikovskaya RI. Mobilization of phosphorous in soil in connection with vital activity of some microbial species. Mikrobiologiya 1948;17:363–70. Raaijmakers J, Weller DM, Thomashow LS. Frequency of antibiotic producing Pseudomonas spp. in natural environments. Appl Environ Microbiol 1997;63:881–7. Rajkumar M, Lee WH, Lee KJ. Screening of bacterial antagonists for biological control of Phytophthora blight of pepper. J Basic Microbiol 2005;45:55–63. Ramette A, Frapolli M, Defago G, Monenne Y. Phylogeny of HCN synthase-encoding hcnBC genes in biocontrol fluorescent pseudomonas and its relationship with host plant species and HCN synthesis ability. Mol Plant Microbe Interact 2003;16:525–35. Reddi TK, Borovkov AV. Antibiotic properties of 2,4-diacetylphloroglucinol produced by Pseudomonas fluorescens strain 26-0. Antibiotiki (Moscow) 1970;15:19–21. Rezzonico F, Zala M, Keel C, Duffy B, Moenne-Loccoz Y, Defago G. Is the ability of biocontrol fluorescent pseudomonads to produce the antifungal metabolite 2,4diacetylphloroglucinol really synonymous with higher plant protection? New Phytol 2007;173:861–72. Saddiqui IA, Shaukat SS. Liquid culture carbon, nitrogen and inorganic phosphate source regulate nematicidal activity by fluorescent pseudomonads in vitro. Lett Appl Microbiol 2004;38:185–90. Saile E, McGarvey J, Schell M, Denny T. Role of extracellular polysaccharide and endoglucanase in root invasion and colonization of tomato plants by Ralstonia solanacearum. Phytopathology 1997;87:1264–71. Sakthivel N, Gnanamanicham SS. Evaluation of Pseudomonas fluorescens for suppression of sheath rot disease and for enhancement of grain yields in rice (Oryza sativa L.). Appl Environ Microbiol 1987;53:2056–9. Shanahan P, O’Sullivan DJ, Simpson P, Glennon JD, O’Gara F. Isolation of 2,4diacetylphloroglucinol from a fluorescent pseudomonad and investigation of physiological parameters influencing its production. Appl Environ Microbiol 1992;58:353–8. Smibert RM, Krieg NR. Phenotypic characterization. In: Gerhardt P, Murray RGE, Wood WA, Krieg NR, editors. Methods for general and molecular bacteriology. Washington, DC: American Society of Microbiology; 1994. p. 607–54. Soliveri J, Arias ME, Laborda E. PA5 and Pa7 pantane and heptane macrolide antibiotics produced by a new isolate of Streptoverticillium from Spanish soil. Appl Microbiol Biotechnol 1987;25:366–71. Stainier RY, Palleroni NJ, Doudoroff M. The aerobic Pseudomonads: a taxonomic study. J Gen Microbiol 1966;41:159–271. Tada M, Takakuwa T, Nagai M, Antiviral Takao Y. antimicrobial activity of 2,4-diacetylphloroglucinols, 2-acylcyclohexane-1, 3-diones and 2carboxymidocyclohexane-1,3-diones. Agric Biol Chem 1990;54:3061–3. Thomashow LS, Weller DM. Current concepts in the use of introduced bacteria for biological disease control: mechanisms and antifungal metabolites. In: Stacey G, Keen N, editors. Plant microbe interactions. New York: Chapman and Hall; 1995. p. 187–235. Wei HL, Zhang LQ. Quorum-sensing system influences root colonization and biological control ability in Pseudomonas fluorescens 2P24. Anton Van Leeuw 2006;89:267–80. Weller DM. Biological control of soilborne plant pathogens in the rhizosphere with bacteria. Annu Rev Phytopathol 1988;26:379–407. Xu GW, Gross DC. Selection of fluorescent pseudomonads antagonistic to Erwinia cartovora and suppressive of potato seed piece decay. Phytopathology 1986;76:414–22. Zhou HY. The genetic regulation of biosynthesis 2,4-diacetylphloroglucinol in Pseudomonas fluorescence 2P24, PhD thesis. China Agricultural University; 2005.