phlF− mutant of Pseudomonas fluorescens J2 improved 2,4-DAPG biosynthesis and biocontrol efficacy against tomato bacterial wilt

Biological Control 78 (2014) 1–8

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phlF mutant of Pseudomonas fluorescens J2 improved 2,4-DAPG biosynthesis and biocontrol efficacy against tomato bacterial wilt Tian-Tian Zhou, Chun-Yu Li, Da Chen, Kai Wu, Qi-Rong Shen, Biao Shen ⇑ National Engineering Research Center for Organic-based Fertilizers, Jiangsu Key Lab and Engineering Center for Solid Organic Waste Utilization, Nanjing Agricultural University, Nanjing 210095, China

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 The phlF, negatively regulating

2,4-DAPG synthesis was deleted to obtain J2-phlF.   Mutant J2-phlF produced more 2,4-DAPG than wild strain J2.   Mutant J2-phlF colonization ability increased in rhizosphere soil.  Biocontrol efficacy against tomato bacterial wilt was improved by J2-phlF.

a r t i c l e

i n f o

Article history: Received 18 July 2013 Accepted 16 July 2014 Available online 25 July 2014 Keywords: P. fluorescens phlF mutant 2,4-Diacetylphloroglucinol Biocontrol Colonization

a b s t r a c t Pseudomonas fluorescens J2 can produce 2,4-diacetylphloroglucinol (2,4-DAPG) as the main antibiotic compound and effectively inhibits the wilt pathogens Ralstonia solanacearum and Fusarium oxysporum. The phlF which negatively regulates the 2,4-DAPG synthesis in strain J2 was disrupted by homologous recombination to construct a mutant strain J2-phlF. The mutant J2-phlF produced much more 2,4-DAPG and showed higher inhibitory effect on R. solanacearum than the wild type strain J2 in vitro. The mutant J2-phlF also showed more colonization of tomato roots and higher inhibition to R. solanacearum in soil than wild type strain J2. The biocontrol efficiency of mutant J2-phlF was higher against tomato bacterial wilt than wild type strain J2, but the differences were not significant. However, the application of both strains with organic fertilizer improved the colonization and biocontrol efficiency against tomato bacterial wilt and mutant strain J2-phlF showed higher biocontrol efficiency against tomato bacterial wilt than wild type strain J2. Both strains, J2 and J2-phlF, could also promote the growth of tomato plants. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction Pseudomonas fluorescens is widely used as a biocontrol agent in agricultural practices (Haas and Défago, 2005; Siddiqui and Shaukat, 2003; Weller et al., 2007; Quagliotto et al., 2009; Shruti and Naveen, 2012). The main mechanism that P. fluorescens strains use to repress the plant pathogens is the production of secondary metabolites, such as 2,4-diacetylphloroglucinol (2,4-DAPG) ⇑ Corresponding author. Fax: +86 25 84395212. E-mail address: [email protected] (B. Shen). http://dx.doi.org/10.1016/j.biocontrol.2014.07.006 1049-9644/Ó 2014 Elsevier Inc. All rights reserved.

(Bangera and Thomashow, 1999), hydrogen cyanide (Ramette et al., 2003), phenazine, pyoluterin and pyrrolnitrin (Mavrodi et al., 2001), and N-mercapto-4-formylcarbostyril (Walid et al., 2001). Among these secondary metabolites, 2,4-DAPG is considered to be the most important factor involved in the inhibitory activities against many phytopathogens(Keel et al., 1992; Bangera and Thomashow, 1999; Zhou et al., 2005). P. fluorescens strains producing 2,4-DAPG, such as P. fluorescens CHA0 (Keel et al., 1992), R62 (Saharan et al., 2011), Q8r1-96 and Q2-87V1 (Youn-Sig et al., 2012), TO7, SA3 and CA9 (Shruti and Naveen, 2012) were reported to protect crops from a number of plant

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diseases. The gene cluster phlACBD was found responsible for the biosynthesis of 2,4-DAPG in a number of Pseudomonad strains (Fenton et al., 1992; Bangera and Thomashow, 1999). The expression of phlACBD is negatively regulated by the repressor PhlF coded by gene phlF (Delany et al., 2000). PhlF was reported to bind specifically to the phlA-phlF intergenic region in strain F113 (Abbas et al., 2002; Delany et al., 2000). In P. fluorescens strain CHA0, and P. fluorescens Q2-87, PhlF was also found to function as a transcriptional repressor (Keel et al., 1992; Bangera and Thomashow, 1999). In this study, we used strain P. fluorescens J2, isolated from the soil with a history of tomato wilt disease, which could produce 2,4-DAPG and effectively suppressed the tomato bacterial wilt pathogen, Ralstonia solanacearum (Tiantian et al., 2012). We disrupted the phlF gene of P. fluorescens J2 to obtain a 2,4-DAPG over-producing mutant strain J2-phlF and compared its characteristics such as colonization in the rhizosphere and biocontrol abilities against tomato bacterial wilt with wild type strain J2. 2. Materials and methods 2.1. Bacterial strains, plasmids and growth conditions The bacterial strains and plasmids used in this study are listed in Table 1. P. fluorescens J2 (CGMCC No. 5104) was grown in Luria-Bertani (LB) or King’s B media (King et al., 1954) at 30 °C and R. solanacearum (ATCC No. 11696) was grown on nutrient agar (NA) or in nutrient broth (NB) medium. Fusarium oxysporum (ATCC16416) was grown on potato dextrose agar (PDA) medium. Escherichia coli DH5a was used for cloning hosts and was grown in LB broth at 37 °C. When required, the growth media were supplemented with 5-brome-4-chloro-3-indolyl-b-D-galactoside (X-Gal, 40 lg mL1), amplicillin (50 lg mL1), kanamycin (50 lg mL1), tetracycline (20 lg mL1), choramphenicol (20 lg mL1), or gentamycin (30 lg mL1). The X-Gal and antibiotics were purchased from Takara Biotechnology Co., Ltd., Dalian, PR China. 2.2. Extraction and analysis of antibacterial compounds from wild-type strain J2 Antimicrobial compounds were extracted from a 4-day-old cultures of wild-type strain J2. After the cultures were centrifuged

at 5000g for 10 min, the supernatants were acidified to pH 2 and extracted with an equal volume of ethyl acetate. The organic layer was evaporated and residues were suspended in 0.5 mL methanol. Samples were spotted on silica thin-layer chromatography (TLC), developed by chloroform/acetone (9:1 vol/vol) and observed under UV light (254 nm). The distinct fractions were recovered in ddH2O and tested for antimicrobial activity against R. solanacearum. The fraction that exhibited strong antibacterial activity was further analyzed by HPLC system (Agilent 1200). The mobile phase was a 45% CH2CN solution, containing 0.1% H3PO4, with a flow rate of 1.0 ml min1. The detection wavelength was fixed at 270 nm. The chemicals were purchased from Sigma-Aldrich Trading Co., Ltd., Shanghai, PR China. 2.3. Construction of a plasmid for homologous recombination A homologous recombination fragment containing the kanamycin resistance gene (kanr), upstream and downstream parts of phlF was constructed as follows. The 1.4 kb kanr fragment was amplified from the plasmid pET29a with the primer pair, kan-F/kan-R. A 1 kb fragment (F1), located upstream of phlF, was amplified by using primers G-speI (SpeI site introduced) and G-1044-R, and a 0.6 kb fragment (F2), located downstream of phlF, was amplified using primers A-1415-F and A-pstI (PstI site introduced) from the genomic DNA of strain J2 as the template. All primers used are listed in Table 1. Extensions of 25 to 40 nucleotides (nt) that were complementary to the 50 and 30 ends of the amplified kanr gene were added to the 50 end of the reverse primer of F1 fragment and forward primer of F2 fragment, respectively. All PCR products were purified using the AxyPrep DNA gel purification and extraction kit (Axygen, Shanghai, PR China). Primers syntheses were carried out by Invitrogen Biotechnology Co., Ltd., Shanghai, PR China. PCR kit was purchased from Takara Biotechnology Co., Ltd., Dalian, PR China. The kanr, F1 and F2 fragments obtained as above were fused by overlapping PCR (Yan et al., 2008) and a 3 kb fragment that was created carried kanamycin resistance gene (kanr), and upstream and downstream parts of phlF. The overlapping PCR steps are outlined below. Step A, 12 lL water, 5 lL PrimerSTAR buffer (5), 2 lL dNTP mix (2.5 mM each), 2 lL (20 ng) F1 fragment, 2 lL (20 ng) F2 fragment, 1.5 lL (15 ng) kanr fragment, and 0.5 lL PrimerSTAR HS DNA polymerase (5U ll1) were mixed in a PCR tube. The PCR

Table 1 Strains, plasmids and primers used in this study. Strains, plasmids, primers

Characteristics

Source

Strains P. fluorescens J2 P. fluorescens J2-phlF E. coli DH5a E. coli S17-1(k-pir) Ralstonia solanacearum Fusarium oxysporum

Wild type, 2,4-DAPG+, HCN+, Ampr,Tcr, Cmr phlF, 2,4-DAPG+, HCN+, Ampr,Kmr, Gms F- recAl endAl hsdRl7 deoR thi-l supE44 gyrA96 Thi pro hsdR recA;chromosomal RP4; tra+; Sm/SpR Bacterial pathogen caused tomato bacterial wilt Fungal pathogen caused Fusarium wilt

This study This study Takara Takara ATCC11696 ATCC16416

Plasmids pMD19-T pJQ200SK pRK2013 pJQ200SK-3k

Cloning vector, lacZ ori,Ampr Gene replacement vector; GmR oriT sacB traJ oriV Helper plasmid, Tra+, Mob+,ColE1, Kmr pJQ200SK with 3 kb PstI-SpeI fusion fragment carrying km gene insert into phlF, phlF

Takara Takara Lab stock This study

50 -GGACTAGTCGGCTTCTCACGCACTAT-30 50 -TCACGACACTTCTGCTGAAC-30 50 -ATCTATCGGTGGTGGGGTAACAAGGCAGCCGGAACCCCTATTTGTTTATT-30 50 -CGCAATACCCCGTTCAGCAGAAGTGTCGTGAGATACCTGTCCGCCTTTCTC-30 50 -GGCTGCCTTGTTACCCCACC-30

This study

Primers G-speI G-1044-R kan-R kan-F A-1415-F A-PstI

50 -AACTGCAGTCCAGATTCCGTTCTTTCA-30

The underlined sequences, ACTAGT and CTGCAG, indicate SpeI and PstI restriction sited, respectively.

This This This This This

study study study study study

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program for step A was as follows: denaturing was conducted at 98 °C for 2 min, followed by 20 cycles of 98 °C for 10 s, annealing at 55 °C for 15 s and a final extension at 72 °C for 1.5 min. For step B, we used 32 lL water, 10 lL PrimerSTAR buffer, 4 lL dNTP mix (2.5 mM each), 1 lL forward primer of F1 fragment, 1 lL reverse primer of F2 fragment, 1 lL of unpurified PCR product from step A, and 1 lL PrimeSTAR HS DNA polymerase (5U ll1) in a PCR tube. The PCR program for step B was as follows: denaturing was conducted at 98 °C for 2 min, followed by 35 cycles of 98 °C for 10 s, annealing at 50 °C for 15 s, and extension at 72 °C for 3.5 min. The resulting PCR product was separated by electrophoresis on a 1% agarose gel. After digestion with SpeI and PstI, the fused 3 kb DNA fragment was cloned into pJQ200SK to create the homologous recombination plasmid pJQ200SK-3k, which contained the fragment carrying kanamycin resistance gene (kanr), and upstream and downstream parts of phlF. 2.4. Construction of the J2-phlF mutant The path of the construction of mutant J2-phlF is shown in Fig. 1a. Recombination plasmid pJQ200SK-3k was introduced into P. fluorescens J2 by triparental mating with helper strain E. coli DH5a containing plasmid pRK2013. Single exchange recombinant (KmrAmprGmr) in which plasmid pJQ200SK-3k was inserted into genomic DNA of J2 was selected on LB plates containing amplicillin (50 lg mL1), kanamycin (50 lg mL1) and gentamycin (30 lg mL1). The SacB gene in pJQ200SK-3k was used to screen double exchange recombinants by incubating single exchange recombinants in the LB agar containing 10% sucrose, amplicillin (50 lg mL1) and kanamycin (50 lg mL1) for 48 h because double exchange recombinants could grow in sucrose-containing media. Single recombinants did not grow out on this media. Those colonies grew out on media containing amplicillin (50 lg mL1) and kanamycin (50 lg mL1) but not containing gentamycin (30 lg mL1) were selected as double exchange recombinants J2-phlF (KmrAmprGms) because genr was lost in the double exchange recombinants. 2.5. Confirmation of phlF mutants Southern blots and PCR with the G-speI/A-pstI primers were used to confirm the double exchanged recombinants. Probes for hybridization were prepared from the 1.4 kb kanr fragment by PCR using kan-F/kan-R primers and labeled with digoxigenin

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following the manufacturer’s instructions (DIG DNA labeling kit, Roche Applied Science, Penzberg, Germany). Genomic DNA from P. fluorescens J2 and the phlF mutant were digested with restriction enzyme PstI. After electrophoresis on a 0.8% agrose gel, the DNA fragments were transferred to nylon membranes. Hybridization and immunological detection were conducted following the manufacturer’s instructions. 2.6. Quantification of 2,4-DAPG production For the extraction of 2,4-DAPG, strains were grown in flasks with 100 ml King’s B medium at 30 °C. The liquid cultures (40 ml) from flasks were taken after 12, 24 and 48 h, respectively and centrifuged at 5000g for 10 min. The supernatants were acidified to pH 2 with 1 M HCl and extracted with an equal volume of ethyl acetate. The organic layer was evaporated, residues were suspended in 0.5 mL methanol and analyzed by HPLC (Agilent 1200) using a C18reverse-phase column (4.6  150 mm) at 270 nm. The mobile phase was 45% CH2CN solution containing 0.1% H3PO4 with 1.0 ml min1 flow rate (Zhou et al., 2005). The standard 2,4-DAPG chemical was purchased from Santa Cruz Biotechnology, Inc. 2.7. Comparison of growth and biofilm formation between wild-type and mutant strains The growth comparison of wild-type J2 and mutant J2-phlF was conducted in LB medium at 30 °C. The absorbances of the cultures were determined at 600 nm with a spectrophotometer (Shimadzu UV-3600, Japan) every 2 h. Biofilm formation assays were performed as described previously (Wei and Zhang, 2006). Briefly, test strains were grown to the beginning of stationary phase in LB media and then diluted to 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 12, 24 and 48 h at 30 °C. The resulting biofilm were stained with 0.1% (w/v) crystal violet for 20 min and then unattached cells and residual dye were removed by pipette. The remaining dye in the biofilm was dissolved in 95% ethanol, and the A570nm of the dissolved dye was determined. 2.8. Antimicrobial activity of wild-type and mutant strains The cultures of wild type strain J2 and mutant strain J2-phlF at three different times (12, 24 and 48 h) were centrifuged at 5000g

Fig. 1. (a). The phlF disruption diagram by double crossover homologous recombination. (b). Southern blot analysis with the digoxigenin labeled 1.4 kb kanr fragment as probe. 1 and 2, J2-phlF; 3, J2; M, DNA Molecular Weight Marker III, DIG-labeled (Roche Applied Science). (c). PCR products confirming of J2 and mutants. 1 and 2, J2-phlF; 3, single homologous exchange recombinant; 4 and 5, wild-type J2; M, kDNA/HindIII marker.

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for 5 min, and the supernatants were filtered through a 0.22 lm membrane. Then 200 lL of cell-free supernatants were used for antibiosis assay against R. solanacearum and F. oxysporum by an agar diffusion assay (Tsui-Hsien et al., 2012). A 5 mm agar plug of fungus was placed in the middle of PDA plate. After the plates were incubated at 28 °C for 2 days, two Oxford cups were placed 2 cm apart from edge of fungal growth, and one Oxford cup was filled with 200 lL of cell-free supernatant of wild strain, and another one was filled with 200 lL of cell-free supernatant of phlF mutant. Inhibition zone was measured after 3 days incubation at 28 °C. For the antibiosis assay against R. solanacearum, 20 ml of NA agar media at 50 °C was mixed with the 1 ml of R. solanacearum over night culture and poured into sterile Petri dish, after the media solidified, two Oxford cups were placed on the plate and filled with 200 lL of cell-free supernatants of wild type strain J2 and mutant strain J2-phlF, respectively. After the plates incubated at 30 °C for 2 days, inhibition zones were measured. Each agar diffusion assay was performed in triplicate.

formula: CE(%) = (DIcontrol – DItreatment)/DIcontrol  100%. Data were analyzed for significance using analysis of variance followed by Duncan’s least-significant-difference test (P = 0.05), using SPSSsoftware v. 13.0 (SPSS Inc., Chicago, IL, USA). At days 7, 14, 21, and 28 after seedling transplantation, rhizosphere soils were collected from 3 randomly-selected plants in each treatment, and the population densities of strain J2 or J2-phlF in the rhizosphere soil were determined by plate count method. The soil was serially diluted with sterile water and soil solution was spread onto selection media plates. The J2-phlF numbers were counted on LB plate containing kanamycin (50 lg mL1) and amplicillin (50 lg mL1). J2 numbers were determined on LB agar containing amplicillin (50 lg mL1) and tetracycline (30 lg mL1). The numbers of R. solanacearum were counted on TTC medium plates (NA medium containing 0.005% 2,3,5-triphenyltetrazolium chloride) (Wei et al., 2011). The plates were incubated at 30 °C for 3 days and colony numbers were counted.

2.9. Biocontrol of tomato bacterial wilt by the wild-type strain J2 and mutant strain J2-phlF in greenhouse

3. Results

R. solanacearum, P. fluorescens strains J2 and J2-phlF were grown in King’s B medium, respectively at 30 °C for 24 h. After harvesting cells by centrifuging at 5000g for 10 min, the cells were washed with water and resuspended in water. The cell suspensions were used as liquid inoculants (final concentration 109 ml1). A pot experiment of biocontrol effect of strain J2 and J2-phlF on tomato bacterial wilt was conducted in a greenhouse. Each pot contained 3 kg of soil. R. solanacearum cell suspension (30 ml) was mixed evenly with soil to a final concentration of 107 cells g1 soil. The following treatments were designed: CK, chemical fertilizers were used; treatment 1, 30 g of organic fertilizer was used; treatment 2, 30 ml of strain J2 liquid inoculants and chemical fertilizers were used; treatment 3, 30 ml of strain J2 liquid inoculants and 30 g of organic fertilizer were used; treatment 4, 30 ml of strain J2-phlF-liquid inoculants and chemical fertilizers were used; treatment 5, 30 ml of strain J2-phlF liquid inoculants and 30 g of organic fertilizer were used. Bacterial cells and fertilizers were mixed thoroughly with soil. Each treatment had 30 pots as replicates and one seedling was transplanted into each pot. The organic fertilizer consisted of pig manure and rice straw compost and an amino acid organic fertilizer at a ratio of 6:4 (w/w). The organic fertilizer was supplied by Jiangsu Xintiandi Ltd (China). The organic fertilizer contained 35% organic matter, 2.0% nitrogen, 3.7% P2O5, and 1.1% K2O. The soil in CK, treatment 2 and treatment 4 was supplied with chemical fertilizers (urea, superphosphate and muriate of potash) containing nutrients (NPK) equivalent to that in 30 g of organic fertilizer treatments. Tomato (Lycopersicon esculentum L) seeds were surface-sterilized in 2% NaClO for 3 min followed by soaking in 75% ethanol for 2 min and then rinsed in sterile water 3 times. The seeds were then germinated on 9-cm plates covered with sterile wet filter paper at 25 °C for 48 h. The germinated seeds were moved into seedling trays in the greenhouse. At the 4-leaf stage, the seedlings were transplanted into pots. One seedling was transplanted into each pot. Wilt disease incidence (DI) and severity (expressed as disease index, di) were determined on day 28 after seedlings transplantaP tion. The DI was calculated as [ (numbers of diseased plants in this index  di)/(total number of plants investigated  the highest di)]  100%. Disease index were recorded based on five grades (0, 1, 2, 3, and 4) according to the method of Scherf et al. (2010), where 0, no leaves wilting; 1, 1–25% of leaves wilted; 2, 26–50% of leaves wilted; 3, 51–75% of leaves wilted, and 4, 76–100% of leaves wilted or dead. The control efficiency (CE) was calculated according to the

3.1. Construction of mutant J2-phlF A mutant J2-phlF (KmrAmprGms) was constructed by double crossover homologous recombination as shown in Fig. 1a. Using the digoxigenin-labeled kanr gene as the probe, a southern hybridization signal appeared in J2-phlF but not in the wild type (Fig. 1b). A single 3 kb PCR product was obtained from J2-phlF and a single 2 kb fragment was obtained from the wild type J2 by the same primers of G-speI/A-pstI. In addition, there were two bands of a 2 and a 3 kb in the single exchange recombination strain (Fig. 1c). Southern hybridization and PCR analysis confirmed that the phlF gene was disrupted by kanr gene in mutant J2-phlF.

3.2. Production of 2,4-DAPG in wild-type strain J2 and mutant strain J2-phlF The production of 2,4-DAPG by the J2 and J2-phlF strains was analyzed by HPLC in a time-course experiment as shown in Fig. 2. In the first 12 h of growth, 2,4-DAPG was only detected in J2-phlF. After 48 h of growth 2,4-DAPG production was significantly higher in J2-phlF than in the wild type. As shown in Fig. 2, the mutant J2-phlF produced 3.14 lg mL1(OD600unit)1 of 2,4-DAPG while the wild-type strain J2 only produced 1.12 lg mL1 (OD600unit)1 after 48 h of growth. These results clearly demonstrated the negative role of the phlF gene on the production of 2,4-DAPG in wild type strain J2 and the disruption of

Fig. 2. 2,4-DAPG production of strains in different growth times. Note:Bars with the same letter are not statistically different among the treatments following Duncan’s test (P < 0.05).

T.-T. Zhou et al. / Biological Control 78 (2014) 1–8

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phlF gene led to an increased 2,4-DAPG production in mutant strain J2-phlF. 3.3. Growth and biofilm formation ability of wild-type strain J2 and mutant strain J2-phlF Under laboratory conditions, the growth patterns of the wildtype strain J2 and mutant strain J2-phlF were almost the same (Fig. 3). However, J2-phlF grew a little faster than the wild-type strain J2 at the early stage of growth. But in the late exponential phase their biomasses were almost similar. These results suggest that the disruption of the phlF gene did not affect cell growth in this medium under these conditions. The ability to produce a biofilm is an important function for bacteria to colonize and resist environmental stress. The data shows that the amount of biofilm produced by J2-phlF was a slightly higher than that produced by wild type J2, although there were no significant differences between them (Fig. 4). 3.4. Antimicrobial activity of wild-type strain J2 and mutant strain J2-phlF The inhibitory ability of mutant strain J2-phlF against R. solanacearum and F. oxysporum on the agar plates was significantly

Fig. 5. Inhibition activity of wild-type J2 and J2-phlF to R. solanacearum.

greater than wild-type stain J2 as shown in Table 2 and Fig. 5. This suggests that an increased production of the 2,4-DAPG antibiotic may be an important factor for the pathogen inhibition by these strains.

3.5. Biocontrol efficacies of wild-type strain J2 and mutant strain J2-phlF against tomato bacterial wilt and their colonization in soil

Fig. 3. Growth curves of wild-type J2 and mutant J2-phlF.

The wilt symptoms in tomato plants appeared first at the 12th and 15th day after seedling transplantation in treatment 1 and CK, respectively. The application of antagonistic strains delayed wilt development. The disease incidence at 28 days after seedling transplantation was 86.3% in CK. In treatment 1, the disease incidence was approximately 93.5%, higher than that of control, suggesting that organic fertilizer promoted the wilt development. The disease incidences in treatments 4 and 5 were 40.6% and 23.5%, respectively, lower than that in treatments 2 and 3 (46.8% and 32.3%), respectively, suggesting that the biocontrol effect of J2-phlF was better than that of J2. The results also showed that the application of antagonistic bacterial strain J2 or J2-phlF together with organic fertilizer could more effectively control tomato bacterial wilt than single use of bacterial strains J2 or J2-phlF. In treatment 3, the disease incidence was only 32.3%, much lower than that in treatment 2 (46.8%). In treatment 5, the disease incidence was only 23.5% that was also much lower than that in treatment 4 (40.6%) (Table 3).

3.6. Effects of wild-type strain J2, mutant strain J2-phlF and organic fertilizer on the growth of tomato plants

Fig. 4. Biofilm formation abilities of wild-type J2 and mutant J2-phlF.

Table 2 Inhibiting zone (mm) of J2 and J2-phlF culture against R.solanacearum and F. oxysporum. Strains

J2 J2-phlF

R. solanacearum

F. oxysporum

12 h

24 h

48 h

12 h

24 h

48 h

– 3.7 ± 0.3

4.7 ± 0.4b 8.8 ± 0.5a

6.9 ± 0.5b 11.2 ± 0.9a

– –

2.7 ± 0.5b 4.6 ± 0.3a

4.9 ± 0.3b 6.7 ± 0.3a

Values with different letter within the same column are significantly different at P < 0.05 according to Duncan’s test. Numbers followed by ‘‘ ± ’’ are standard errors (SE).

The application of both organic fertilizer and antagonistic strains (wild-type strain J2 and mutant strain J2-phlF) stimulated tomato growth as shown in Table 4. The height and biomass of tomato plants in treatments applied with wild-type strain J2 or mutant strain J2-phlF were much greater than those of plants in the control treatment indicating that both of wild-type strain J2 and mutant strain J2-phlF could promote tomato plant growth. But there was no significant difference of the promotion to tomato growth between wild-type strain J2 and mutant strain J2-phlF. The application of organic fertilizer together with wild-type strain J2 or mutant strain J2-phlF stimulated the tomato plant growth more effectively than the single use of organic fertilizer or wildtype strain J2 or mutant strain J2-phlF, but differences were nonsignificant.

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Table 3 Biocontrol efficacy of strain J2 and J2-phlF against tomato bacterial wilt. Treatments

Disease incidence (%)

Control efficacy (%)

CK 1 2 3 4 5

86.3a 93.5a 46.8b 32.3c 40.6b 23.5d

– 8.3 45.8 62.6 53.0 72.8

Note: CK, chemical fertilizer used; treatment 1, organic fertilizer used; treatment 2, strain J2 liquid inoculants used; treatment 3, both of strain J2 liquid inoculants and organic fertilizer used; treatment 4, strain J2-phlF liquid inoculants used; treatment 5, both of strain J2-phlF liquid inoculants and organic fertilizer used. Each treatment had 30 replicates. The organic fertilizer was used as 1% (w/w). The data in a column with a different letter differ significantly at Duncan’s significance level 0.05.

Table 4 Effects of antagonistic strain J2, J2-phlF and organic fertilizer on the growth of tomato plants (28 days). Treatments

Height (cm)

Dry weight (g)

CK 1 2 3 4 5

38.4 ± 2.1c 64.5 ± 3.1a 54.3 ± 4.2b 67.2 ± 4.8a 55.3 ± 3.5b 67.8 ± 2.7a

6.80 ± 0.2c 11.3 ± 0.3a 9.1 ± 0.2b 12.5 ± 0.1a 9.8 ± 0.2b 12.3 ± 0.3a

Note: Different lowercase letters in each column mean significant differences at a 0.05 level.

3.7. Population dynamics of R. solanacearum in soil Over all, during the first week of inoculation, the numbers of R. solanacearum decreased in all treatments (Fig. 6) and seven days later, the numbers of R. solanacearum in all treatments began to increase gradually. During the experiment period, the numbers of R. solanacearum in CK and treatment 1 were always higher than that in other treatments. The numbers of R. solanacearum were the highest in treatment 1, indicating that organic fertilizer

Fig. 7. Population dynamics of antagonistic bacterial strain J2 and J2-phlF in soil in different treatments. Note: Series 2,3,4,5 represent the same as shown in Fig.6.

promoted the growth of R. solanacearum in the soil, resulting in a higher disease incidence. The application of antagonistic wild-type strain J2 or mutant strain J2-phlF significantly reduced the population of R. solanacearum, and the application of mutant strain J2-phlF (treatment 4) more effectively inhibited the growth of R. solanacearum than the application of wild-type strain J2 (treatment 2) in soil. Combined application of antagonistic bacterial wild-type strain J2 (treatment 4) or mutant strain J2-phlF (treatment 5) with organic fertilizer could control the growth of R. solanacearum more effectively than the single use of wild-type strain J2 or mutant strain J2-phlF. The population of R. solanacearum was the lowest in treatment 5 than in all other treatments. 3.8. Population dynamics of the antagonistic wild-type strain J2 and mutant strain J2-phlF in soil The population dynamics of the antagonistic wild-type strain J2 and mutant strain J2-phlF in soil are shown in Fig. 7. The results showed that the numbers of wild-type strain J2 and mutant strain J2-phlF decreased gradually in all treatments during the whole experiment period after inoculation. But the mutant strain J2-phlF survived better than the wild-type strain J2 in soil. In treatments 3 and 5, the population of inoculated antagonistic strains decreased more slowly than in treatments 2 and 4, respectively, indicating that organic fertilizer aids the survival of inoculated antagonistic strains in soil. The higher numbers of strain J2 and J2-phlF in treatment 3 and 5 inhibited the growth of R. solanacearum and resulted in a lower soil count of R. solanacearum and a lower disease incidence, as shown in Fig. 6 and Table 3.

4. Discussion

Fig. 6. Population dynamics of R. solanacearum in soil in different treatments. Note: CK, chemical fertilizer used; treatment 1, organic fertilizer used; treatment 2, strain J2 liquid inoculants used; treatment 3, both of strain J2 liquid inoculants and organic fertilizer used; treatment 4, strain J2-phlF liquid inoculants used; treatment 5, both of strain J2-phlF liquid inoculants and organic fertilizer used.

Fluorescent Pseudomonas species are widely utilized as effective biocontrol agents against plant root disease caused by soil-borne pathogens (Weller et al., 2007; Quagliotto et al., 2009; Couillerot et al., 2009; Shruti and Naveen, 2012). Production of phenolic compound, 2,4-DAPG by fluorescent Pseudomonas species was considered as an important factor to inhibit the soil-borne pathogens (Weller et al., 2007; Shruti and Naveen, 2012; Almario et al., 2013). Increase of 2,4-DAPG production by molecular biological manipulation in fluorescent Pseudomonas species will be potential strategy for the better biocontrol of plant diseases.

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In our previous study, P. fluorescens J2 showed antagonistic activity against pathogens R. solanacearum and F. oxysporum and produced 2,4-DAPG, hydrogen cyanide (HCN), siderophore(s) and protease. But the production of 2,4-DAPG was the main antimicrobial compound (Tiantian et al., 2012). In order to improve the production of 2,4-DAPG in wild-type strain J2, phlF gene which negatively regulates the biosynthesis of 2,4-DAPG, was disrupted by homologous recombination and mutant J2-phlF was obtained. The results showed that mutant strain J2-phlF produced more 2,4DAPG than the wild-type strain J2. Our results were not in agreement with some previous reports like Delany et al. (2000) who reported that the phlF deletion mutant of P. fluorescens F113 (F113-phlF) increased the production of 2,4-DAPG as compared to the wild-type strain only in the early growth phase, but at the later growth stages, 2,4-DAPG productions were similar in both mutant and wild-type F113. However, similar results were reported by Schnider-Keel et al. (2000) that the production of 2,4-DAPG increased four times in phlF mutant strain of P. fluorescens CHA0 than wild-type strain, but the growth of phlF mutant strain was slower than the wild type strain. In this study, the growth of the mutant strain J2-phlF was a little faster than the wild-type strain J2, but differences were not significant and 2,4DAPG synthesis in mutant strain J2-phlF was 2.8 times higher than in wild-type strain J2. So different phlF mutants may show different behaviors in growth and production of 2,4-DAPG. Antibiotics production of biological control bacteria has been shown to be a key determinant in the pathogen inhibition. However, antibiotic production in the soil will depend on a variety of ecological parameters (Duffy and Defago, 1999; Haas and Keel, 2003; Okubara and Bonsall, 2008). Although the mutant strain J2-phlF produced more 2,4-DAPG and showed stronger antagonistic activity against R. solanacearum than the wild-type strain J2 on the plates, the application of mutant strain J2-phlF alone did not show significantly improved protection against tomato bacterial wilt caused by R. solanacearum in pot experiment than the wildtype strain J2. The reason might by that the mutant strain J2-phlF did not produce more 2,4-DAPG in rhizosphere as in culture. How to educe the mutant’s potential to produce more 2,4-DAPG in the rhizosphere is a challenge faced by microbiologists. Okubara and Bonsall (2008) found that the accumulation of Pseudomonasderived 2,4-DAPG on wheat seedling roots is influenced by host cultivar. So, screening of suitable plants for mutant strain J2-phlF is also needed to be considered in future. Some research reports showed that the combined application of organic fertilizers and antagonistic microorganisms had much higher control efficacy than the use of antagonistic microorganisms alone (Trillas et al., 2006; Sanjay et al., 2008; Wei et al., 2011). These researchers believed that the organic fertilizers could supply nutrients for antagonistic microorganisms and aid their survival after the inoculation in soils. In this study, the application of antagonistic wild-type strain J2 or mutant strain J2-phlF combined with organic fertilizer also showed much higher biocontrol efficiency of tomato bacterial wilt than single use of wild-type strain J2 or mutant strain J2-phlF. The nutrient status of soil improved by the organic fertilizer might induced the capacity of strains J2 or J2-phlF to produce more 2,4-DAPG in soil, which helped the antagonists to fight better with the pathogen strain. Selection of more suitable organic fertilizer for strain J2-phlF to produce more 2,4-DAPG is a prospective way to effectively control soil-borne plant diseases. Biological control efficacy of soil-borne plant diseases using bacterial agents is largely based on their efficient colonization in plant rhizosphere during the proper period (Lugtenberg and Dekkers, 1999). It has been reported that the biosynthesis of phenazine antibiotics contributes to the long-term survival of Pseudomonas strains in soil habitats (Mazzola et al., 1992). In this study,

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more colonization of mutant strain J2-phlF than wild-type J2 in the rhizosphere soil was observed. This might be due to the fact that the mutant strain J2-phlF produced more 2,4-DAPG, which inhibited the other indigenous microorganisms. But the numbers of wild-type strain J2 and mutant strain J2-phlF decreased gradually during the experiment period after inoculation. How to improve the colonization of antagonistic bacteria in soil is very important for the biocontrol of soil-born pathogens. In this research, the application of organic fertilizer improved the soil colonization of antagonistic bacterial strains J2 and J2-phlF. If applied with organic fertilizer, strains J2 and J2-phlF maintained much more numbers. The higher soil count of strain J2 and J2-phlF inhibited the growth of R. solanacearum better which resulted in a lower disease incidence. In conclusion, disruption of the phlF gene in wild-type strain J2 significantly enhanced the antibiotic 2,4-DAPG production and mutant strain J2-phlF showed higher antagonistic activity against R. solanacearum. The mutant strain J2-phlF also colonized the rhizosphere better and showed higher biocontrol activity against tomato bacterial wilt than wild-type strain J2. Organic fertilizer could improve the colonization of antagonistic bacterial strain J2 and J2-phlF and protection against tomato bacterial wilt. Acknowledgments 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). References Abbas, A., Morrissey, J.P., Marquez, P.C., Sheehan, M.M., Delany, I.R., O’Gara, F., 2002. Characterization of interactions between the transcriptional repressor PhlF and its binding site at the phlA promoter in Pseudomonas fluorescens F113. J. Bacteriol. 184 (11), 3008–3016. Almario, J., Moënne-Loccoz, Y., Muller, D., 2013. Monitoring of the relation between 2,4-diacetylphloroglucinol-producing Pseudomonas and Thielaviopsis basicola populations by real-time PCR in tobacco black root-rot suppressive and conducive soils. Soil Biol. Biochem. 57, 144–155. Bangera, M.G., Thomashow, L.S., 1999. Identification and characterization of a gene cluster for synthesis of the polyketide antibiotic 2, 4-diacetylphloroglucinol from Pseudomonas fluorescens Q2–87. J. Bacteriol. 181 (10), 3155–3163. Couillerot, O., Prigent-Combaret, C., Caballero-Mellado, J., Moënne-Loccoz, Y., 2009. Pseudomonas fluorescens and closely-related fluorescent pseudomonads as biocontrol agents of soil-borne phytopathogens. Lett. Appl. Microbiol. 48 (5), 505–512. Delany, I., Sheehan, M.M., Fenton, A., Bardin, S., Aarons, S., O’Gara, F., 2000. Regulation of production of the antifungal metabolite 2,4-diacetylphloroglucinol in Pseudomonas fluorescens F113: genetic analysis of PhlF as a transcriptional repressor. Microbiology 146 (pt2), 537–543. Duffy, B.K., Defago, G., 1999. Environmental factors modulating antibiotic and siderophore biosynthesis by Pseudomonas fluorescens biocontrol strains. Appl. Environ. Microbiol. 65 (6), 2429–2438. Fenton, A.M., Stephens, P.M., Crowley, J., O’Callaghan, M., O’Gara, F., 1992. Exploitation of gene(s) involved in 2,4-diacetylphloroglucinol biosynthesis to confer a new biocontrol capability to a Pseudomonas strain. Appl. Environ. Microbiol. 58 (12), 3873–3878. Haas, D., Défago, G., 2005. Biological control of soil-borne pathogens by fluorescent pseudomonas. Nat. Rev. Microbiol. 3 (4), 307–319. Haas, D., Keel, C., 2003. Regulation of antibiotic production in root-colonizing Pseudomonas spp and relevance for biological control of plant disease. Annu. Rev. Phytopathol. 41, 117–153. Keel, C., Schnider, U., Maurhofer, M., Voisard, C., Laville, J., Burger, U., Wirthner, P., Haas, D., Défago, G., 1992. Suppression of root disease by Pseudomonas fluorescens CHA0: importance of the bacterial secondary metabolite 2,4diacetylphloroglucinol. Mol. Plant Microbe Interact. 5 (1), 4–13. King, E.O., Ward, M.K., Raney, D.E., 1954. Two simple media for the demonstration of pyocyanin and fluorescein. J. Lab. Clin. Med. 44, 301–307. Lugtenberg, B.J.J., Dekkers, L.C., 1999. What makes Pseudomonas bacteria rhizosphere competent. Environ. Microbiol. 1 (1), 9–13. Mavrodi, D.V., Bonsall, R.F., Delaney, S.M., Soule, M.J., Phillips, G., Thomashow, L.S., 2001. Functional analysis of genes for biosynthesis of pyocyanin and phenazine1-carboxamide from Pseudomonas aeruginosa PAO1. J. Bacteriol. 183 (21), 6454– 6465.

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