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Biological control of rice bacterial blight by plant-associated bacteria producing 2,4-diacetylphloroglucinol

Palaniyandi Velusamy, J. Ebenezar Immanuel, Samuel S. Gnanamanickam, and Linda Thomashow

Abstract: Certain plant-associated strains of fluorescent Pseudomonas spp. are known to produce the antimicrobial antibiotic 2,4-diacetylphloroglucinol (DAPG). It has antibacterial, antifungal, antiviral, and antihelminthic properties and has played a significant role in the biological control of tobacco, wheat, and sugar beet diseases. It has never been reported from India and has not been implicated in the biological suppression of a major disease of the rice crop. Here, we report that a subpopulation of 27 strains of plant-associated Pseudomonas fluorescens screened in a batch of 278 strains of fluorescent pseudomonads produced DAPG. The DAPG production was detected by a PCR-based screening method that used primers Phl2a and Phl2b and amplified a 745-bp fragment characteristic of DAPG. HPLC, 1H NMR, and IR analyses provided further evidence for its production. We report also that this compound inhibited the growth of the devastating rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae in laboratory assays and suppressed rice bacterial blight up to 59%64% in net-house and field experiments. Tn5 mutants defective in DAPG production (Phl) of P. fluorescens PTB 9 were much less effective in their suppression of rice bacterial blight. Key words: biocontrol, 2,4-diacetylphloroglucinol, Pseudomonas fluorescens, rice, Xanthomonas oryzae pv. oryzae. Rsum : Certaines souches de Pseudomonas spp. fluorescentes associes aux plantes produisent un antibiotique antimicrobien, le 2,4-diactylphloroglucinol (DAPG). Il possde des proprits antibactriennes, antifongiques, antivirales et antihelminthiques, et a jou un rle important dans la lutte biologique contre les maladies du tabac, du bl et de la betterave sucre. On ne la jamais rapport en Inde et il na jamais t impliqu dans la lutte biologique contre les maladies principales touchant les rcoltes de riz. Nous rapportons ici quune sous-population de 27 souches de Pseudomonas fluorescens associes aux plantes, cribles partir dun lot de 278 souches de pseudomonades fluorescentes, a produit du DAPG. La production de DAPG a t dtecte par une mthode de criblage par PCR laide damorces Phl2a et Phl2b permettant damplifier un fragment caractristique de 745 pb. Des analyses en HPLC, 1H NMR et IR ont fourni des vidences supplmentaires de sa production. Nous rapportons aussi que ce compos a inhib la croissance de la rouille du riz Xanthomonas oryzae pv. oryzae, une bactrie pathogne dvastatrice, lors de tests de laboratoire, et quil a supprim la rouille bactrienne de 59 % 64 % lors de tests sur le terrain et en serres. Des mutants Tn5 de P. fluorescens PTB 9, dfectifs en production de DAPG (Phl), taient beaucoup moins efficace supprimer la rouille bactrienne du riz. Mots cls : lutte biologique, 2,4-diactylphloroglucinol, Pseudomonas fluorescens, riz, Xanthomonas oryzae pv. oryzae. [Traduit par la Rdaction] Velusamy et al. 65

Introduction
Annually, more than 40% of the worlds rice crop is lost owing to biotic stresses like insects, pests, pathogens, and weeds (Hossain 1996). Among several diseases caused by
Received 29 March 2005. Revision received 14 August 2005. Accepted 2 September 2005. Published on the NRC Research Press Web site at http://cjm.nrc.ca on 20 January 2006. P. Velusamy, J.E. Immanuel, and S.S. Gnanamanickam.1 Centre for Advanced Studies in Botany, University of Madras, Guindy Campus, Chennai 600 025, India. L. Thomashow. USDA-ARS, Department of Plant Pathology, Washington State University, Pullman, WA 99164-6430, USA.
1

Corresponding author (e-mail: gmanick@vsnl.com).

bacterial, fungal, and viral pathogens that devastate rice yields all over the world, bacterial blight (BB) (Xanthomonas oryzae pv. oryzae), blast (Magnaporthe grisea), sheath blight (Rhizoctonia solani), sheath rot (Sarocladium oryzae), and tungro virus are the most important. BB caused by X. oryzae pv. oryzae is one of the most important and oldest known diseases of rice. The disease was first observed by the farmers of Japan in 1884 (Tagami and Mizukami 1962). Crop losses of 10%20% in moderate conditions or severe losses of up to 50% in highly conducive conditions have been recorded in several Asian and Southeast Asian countries (Mew 1987; Ou 1985). Globally, its incidence has been reported from different parts of Asia, northern Australia, Africa, and the United States. In India, BB disease has been observed in most important rice-growing states like Andhra Pradesh, Bihar, Haryana, Kerala, Orissa,
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Punjab, and Uttar Pradesh. The disease occurred in an epidemic form during 1998 in the Palakkad district of Kerala (Venkatesan and Gnanamanickam 1999) and since then has been observed in severe proportions almost every year. BB disease reduces yields and yield stability. Some management tactics, such as chemicals, are harmful to the environment, while others, such as host plant resistance based on single genes, may not be durable in the field and might lead to frequent varietal breakdowns. Biological control, therefore, assumes special significance in being an ecology-conscious, cost-effective alternative strategy for BB management. This can also be used in integration with other strategies to afford greater levels of protection and sustain rice yields. Antagonistic bacteria are considered ideal biological control agents for obvious reasons, like rapid growth, easy handling, and aggressive colonization of the rhizosphere (Weller 1988). Bacterial antagonists have been evaluated with various degrees of success for the suppression of rice diseases of fungal origin (Vasudevan et al. 2002). However, there has been no detailed study on the use of antagonistic bacteria for suppression of BB except for a recent study with Bacillus spp. in our laboratory (Vasudevan 2002). The present study focuses on the use of Pseudomonas fluorescens strains from southern India that produce 2,4-diacetylphloroglucinol (DAPG) to suppress rice BB. The antagonistic fluorescent pseudomonads produce one or more metabolites, such as phenazine-1-carboxylic acid, DAPG, pyoluteorin, pyrrolnitrin (PRN), and oomycin A. Among these, DAPG is a polyketide antibiotic with a broad spectrum of antimicrobial properties and is produced by fluorescent Pseudomonas spp. of diverse geographic origin (Dowling and OGara 1994; Keel et al. 1996; Sharifi-Tehrani et al. 1998; Thomashow and Weller 1995; Raaijmakers et al. 1999; Notz et al. 2001). It has been implicated as the mechanism involved in the biological control of some of the most important crop diseases of wheat, tobacco, and sugar beet (Defago et al. 1990; de Souza and Raaijmakers 2003; de Souza et al. 2003; Garagulya et al. 1974; Keel et al. 1990, 1992; Kraus and Loper 1992; Levy et al. 1992; NowakThompson et al. 1994; Pidoplichko and Garagulya 1974; Vincent et al. 1991). Strains of P. fluorescens that produce DAPG also have had a key role in the natural biological control of take-all of wheat (caused by Gaeumannomyces graminis var. tritici) known as take-all decline (Raaijmakers et al. 1997, 1999; Raaijmakers and Weller 1998). Genes encoding biosynthesis and regulation of these metabolites involved in biocontrol have been cloned and in part sequenced (Fenton et al. 1992; Keel et al. 1996; Bangera and Thomashow 1996; Gardener et al. 2001). The biosynthetic locus of DAPG contains six genes, phlA, phlB, phlC, phlD, phlE, and phlF, in P. fluorescens Q2-87, and one of them, phlD, is essential for the synthesis of the DAPG precursor monoacetylphloroglucinol (Bangera and Thomashow 1999). In this study, we have used a PCR-based screening method to identify DAPG production by a subpopulation of P. fluorescens strains that are antagonistic to the bacterial leaf blight pathogen X. oryzae pv. oryzae and have tried to establish a role for DAPG in the biological suppression of rice BB. The two major questions we have addressed are

(i) Does DAPG production by P. fluorescens occur in the crop rhizosphere in India? and (ii) Does it play a measurable role in reducing the severity of rice BB?

Materials and methods

Bacterial strains and media Rhizosphere samples of crops were collected from different locations in the states of Andhra Pradesh, Karnataka, Kerala, Maharashtra, and Tamil Nadu in India (listed in Table 1). These crops included rice, sorghum, sunn hemp, finger millet, black gram, and green gram. The rhizosphere samples were stored at 4 C. Pseudomonas spp. strains were isolated from the soil suspensions of these rhizosphere samples that were serially diluted and plated onto Kings B agar medium (King et al. 1954). The plates were incubated at 28 C for 2 days. Single colonies exhibiting bluish green fluorescence were picked under UV light ( = 365 nm) and further purified on the same medium. Such purified bacterial strains were stored in 50% glycerol at 4 C. These strains, which showed fluorescence on Kings B agar medium, were Gram-negative motile rods. They were characterized further by performing standard tests for oxidase and catalase production, gelatin liquefaction, arginine hydrolysis, and others for Pseudomonas spp., as outlined in Bergey (1982). The BB pathogen X. oryzae pv. oryzae strains were isolated from infected rice leaf and maintained on peptone sucrose agar (PSA) plates. Their pathogenicity to rice that led to the development of characteristic leaf blight lesions with wavy margins upon inoculation to leaves of IR24 rice confirmed their identity as BB pathogens. In vitro antibiosis Screening of the bacterial antagonists to X. oryzae pv. oryzae was carried out in laboratory dual-plate assays. The antagonists were selected based on their ability to produce a zone of growth inhibition around them when patched onto PSA plates spread-plated already with 100 L of suspension (106 CFU/mL) of X. oryzae pv. oryzae. The zone of inhibition produced by the bacterial strains was measured after incubation at 28 C for 24 days. These dual-plate assays led to the identification of efficient antagonists. PCR-based screening for DAPG Antagonistic fluorescent Pseudomonas were screened for DAPG production by a PCR-based method developed and described elsewhere (Raaijmakers et al. 1997). A strain of P. fluorescens CHAO (a gift from Dr. G. Defago, ETHZentrum, Zurich) known to produce DAPG was used as a positive control in all PCR-based selection of strains. To confirm the production of DAPG, intervening sequence specific primers Phl2a 20-mer (5-GAGGACGTCGAAGAC CACCA-3) and Phl2b 20-mer (5-ACCGCAGCATCGTGT ATGAG-3), which were developed from the phlD sequence of P. fluorescens Q2-87 (Raaijmakers et al. 1997), were used in the PCR analysis.
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Can. J. Microbiol. Vol. 52, 2006 Table 1. Plant-associated bacteria isolated from various rice-growing regions of India, their antibiosis towards Xanthomonas oryzae pv. oryzae, and detection of DAPG production by a PCR-based method. No. of bacterial strains obtained 36 22 19 15 10 39 12 15 35 20 21 16 17 22 11 13 21 13 12 17 10 31 12 10 14 12 13 12 10 11 15 11 17 11 12 13 10 15 12 637 No. of strains showing Xoo inhibition (% of bacterial strains in particular host)a 9 5 6 7 7 10 3 2 14 9 9 6 10 8 6 4 11 7 9 (25.0) (22.7) (31.6) (46.7) (70.0) (25.6) (25.0) (13.3) (40.0) (45.0) (42.9) (37.5) (58.8) (36.4) (54.5) (30.8) (52.4) (53.8) (75.0) No. of strains that produced DAPGb 2 1 1 1 1 1 1 1 2 2 3 1 1 1 2 1 2 1 1 1 27

Place of collection Karnataka Mandya Immavur Nanjangud Malavali Srirangapattinam Mysulipattinum Chamrajnagar Kanakapura Andhra Pradesh Nellur Vijayavada Sulur Cuddappah Nandyal Tada Kerala Pattambi Mannur Palghat Mancombu Cheramangalam Tamil Nadu Thiruvannamalli Vellore Thiruvercadu Gudiyatham Tiruchirappalli Madurai Sethuvali Virunchipuram Puducherry Valajabath Kavanur Theni Kovilpatti Rajapalayam Pudukkoittai Maharashtra Loanavola Khade Dapholi Miraj Karhad Total no. of strains

Code MAD IMV NJD BGR RDR MLP CJR KKP NEL VYA SLU CUD NAD TAD PTB MNR PAL MON CHE TVM VEL TVR GDY TRP MDR STV VGP PDY VLB KVR TNI KOV RJP PDU LVA KAD DPI KRA KAH

Host and (or) crop Rice Rice Rice Sunn hemp Finger millet Sunn hemp Sunn hemp Rice Rice Rice Rice Rice Rice Rice Rice Rice Rice Rice Rice Rice Finger millet Finger millet Rice Rice Rice Sorghum Sorghum Finger millet Rice Black gram Black gram Green gram Rice Rice Rice Black gram Rice Rice Rice

7 (41.2) 7 (70.0) 2 (6.5) 8 (66.7) 3 (30.0) 9 (64.3) 9 (75.0) 4 (30.8) 8 (66.7) 3 (30.0) 7 (63.6) 6 (40.0) 7 (63.6) 11 (64.7) 11(100.0) 4 8 9 8 5 278 (33.3) (61.5) (90.0) (53.3) (41.7)

Note: Xoo, Xanthomonas oryzae pv. oryzae; DAPG, 2,4-diacetylphloroglucinol. a Of 637 strains, 278 inhibited Xoo in laboratory dual-plate assays. b Production of DAPG was identified through a PCR-based screening procedure that amplified a 745-bp DNA fragment in 27 out of 278 tested strains.

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Identification of antibacterial compounds produced by P. fluorescens Pseudomonas fluorescens CHAO and P. fluorescens strains identified to produce DAPG by the PCR-based method were grown on malt agar (15 g of malt extract, 17 g of agar, and 1 L of double-distilled water) at 28 C for 3 days. The agar mats were cut into small pieces and were extracted with 250 mL of 80% aqueous acetone, according to the procedure developed by Howell and Stipanovic (1979) with some modifications. The extract was filtered through Whatman No. 1 filter paper and condensed to one-third of the initial volume of acetone in vacuo at 45 C in a rotary evaporator (Buchler, Germany). The aqueous concentrates were acidified to pH 2 with 2 mol HCl/L and extracted three times in ethylacetate. The ethylacetate extracts were reduced to dryness in vacuo. The residue was dissolved in 3 mL of 65% methanol. For detection of PRN by thin-layer chromatography (TLC), bacterial strains were grown in 25 mL of a minimal medium containing 30 g glycerol/L, 3 g KH2PO4/L, 5 g NaCl/L, 0.5 g MgSO47H2O/L, and 0.61 g D-tryptophan/L under culture conditions that induced PRN production (Howell and Stipanovic 1979; Duffy and Defago 1999). TLC analyses of ethylacetate extracts of culture fluids were carried out according to the procedure described by de Souza and Raaijmakers (2003) by applying 50 L volumes to precoated silica gel plates (20 cm 20 cm, aluminium oxide 60 F254) (Merck, Germany) and separating with a chloroform acetone (9:1, v/v) solvent system. Values of Rf reported for PRN (0.80) and DAPG (0.54) were used to make tentative identification of the two major bands that were observed in P. fluorescens CHAO and 27 of the Indian strains of P. fluorescens evaluated. Purification Two millilitres of the crude extract was streaked on TLC plates coated with silica gel (Sigma Chemical Co., St. Louis, Missouri) and developed in tolueneacetone (4:1, v/v). These spots were visualized with UV light (short wavelength, 254 nm), and all active bands (corresponding to DAPG and PRN standards) were removed from the plates and eluted with 50 mL of acetone. The acetone eluate was taken to dryness in vacuo and taken up in hot hexane (1 mL) and the antibiotic was precipitated from it by slow cooling. Bioassays against X. oryzae pv. oryzae The purified compounds with tentative identification as DAPG and PRN were assayed for antibacterial activity to X. oryzae pv. oryzae in dual-plate assays by placing a 100 L aliquot of the concentrate in wells made in PSA plates spreadplated previously with an aqueous suspension of X. oryzae pv. oryzae (OD at 600 nm = 0.1 corresponding to 106 CFU/mL). The plates were incubated for 24 days at 28 C and observed for a zone of inhibition around the wells. DAPG analysis The purified compound was dissolved in 1 mL of 65% (v/v) methanol and approximately 50 L aliquots were chromatographed on TLC plate (silica gel F254) (Merck, Germany) with a DAPG standard using a tolueneacetone (4:1 v/v) solvent system. These spots were visualized with UV light (short wavelength, 254 nm) and Rf values were

compared with that of the DAPG standard. Production of DAPG was verified by using analytical high-performance liquid chromatography (HPLC) methods described by Keel et al. (1992). For analytical HPLC, 10 L of the purified sample was analyzed with a Waters Associates liquid chromatograph equipped with a reverse-phase column (4 mm 30 cm) packed with Nucleosil 120-5-C18. This was thermostatically controlled at 45 C. A solvent system consisting of acetonitrile water (1:1, v/v) was used to detect the compound. The samples were eluted with a three-step linear gradient of methanol, 18%23% (05 min), 23%53% (56 min), and 53%68% (515 min), in 0.43% (v/v) o-phosphoric acid (pH 2.8) with a flow rate of 1 mL/min. DAPG was detected with a UV diode array detector at 270 nm. The retention times for the peaks obtained in the crude samples of bacterial strains were compared with that of the DAPG standard. Quantitation was also done by a comparison of peak heights. The 1H NMR spectrum was recorded on a Bruker DPX 200 spectrometer using CDCl3 as the solvent. The chemical shifts were reported as parts per million with tetramethyl silane as the internal standard. The IR spectrum was recorded on a Perkin-Elmer 1600 series FT-IR spectrometer using a potassium bromide disc and the data expressed as per centimetre. Suppression of BB by DAPG-producing strains in nethouse and field experiments Selected DAPG-producing bacterial strains were evaluated for their ability to suppress the BB disease in a net-house experiment during JulyDecember 2002 and in a field experiment carried out during JulyDecember 2003. These experiments were conducted at the Regional Agriculture Research Station, Pattambi, Kerala, India. A highly BB-susceptible rice cultivar IR24 was used in these experiments. The DAPG producer strains were grown in LuriaBertani (LB) broth for 24 h at 28 C in a shaker (120 r/min), and rice seeds were soaked overnight in this bacterial suspension (prepared in 0.1% carboxymethylcellulose, 108 CFU/mL). After decanting the excess fluid, the treated seeds were sown in small pots. Seeds that were allowed to germinate in sterile water served as untreated control. Both bacteria-treated and untreated control seedlings were transplanted when they were 21 days old in net-house or field plots of 1 m 1 m size. At the time of transplanting, the seedlings were given a root dip in respective bacterial cell suspensions (containing 108 CFU/mL) or in sterile distilled water in the case of untreated control. When the seedlings were 35 and 45 days old, they received two additional foliar spray applications with respective bacteria at 108 CFU/mL of spray fluid suspension (prepared in 0.1% carboxymethylcellulose). In these experiments, each treated plot was covered with a plastic sheet on all four sides before spraying to avoid cross-contamination among different bacterial strains used as treatments. The plants were then clip-inoculated with X. oryzae pv. oryzae, and the development of BB lesions in bacterial treatments and in untreated controls was measured. Inoculation of X. oryzae pv. oryzae and scoring of BB disease Both the treated and untreated rice plants were then clipinoculated (Kauffmann et al. 1973) with a bacterial suspen 2006 NRC Canada

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sion of X. oryzae pv. oryzae culture (106 CFU/mL) on the 45th day after sowing. Development of BB lesions was scored 14 days after the inoculation. The lesion length and leaf lengths were measured, and the disease severity was determined in terms of the reduction in lesion length in the bacterial treatment as compared to that of the untreated control. The reduction in disease incidence in the treated plants was calculated, and the data were analyzed for statistical significance using the least significant difference (LSD) test (Gomez and Gomez 1976). The percent disease suppression afforded by the bacterial treatment from that of the untreated control was calculated with the following formula: % disease supression = mean lesion length in bacteria-treated rice leaves 100 mean lesion length in untreated control leaves Statistical analysis of data Data from the greenhouse and field experiments were subjected to analysis of variance and to LSD to make comparisons between the untreated control and each of the bacterial treatments. As every treatment had an equal number of replications, the following formulae were used for calculating the LSD value at 5% and at 1%: LSD0.05 = t 0.05 2 S 2 / r LSD0.01 = t 0.01 2 S 2 / r where t is the tabular value of t at either the 5% or the 1% level of significance for the error degrees of freedom from the analysis of variance table, S2 is the error mean square, and r is the number of replications. If the observed difference is larger than the computed LSD value, the two treatment (control and a treatment) means are significantly different at the level of significance, and if the difference between a pair of means is smaller than the computed LSD value, the treatment is not significantly different from the control. Generation of Phl mutants of DAPG-producing P. fluorescens PTB 9 To make a critical assessment of the role of DAPG in BB suppression, Phl mutants were generated by transposon mutagenesis. Transposons (in donor strains) were mobilized into DAPG-producing P. fluorescens PTB 9 by the method of Bangera and Thomashow (1999). The donor (S17-1(pir)/ pUTKm::phl TR#1) was placed onto an LB agar plate containing ampicillin (100 g/mL) and kanamycin (Km; 50 g/mL), and the recipient strain P. fluorescens PTB 9 was plated onto LB. Single colonies were picked and grown in liquid culture for 12 h with Km (100 g/mL) at 27 C. The donor Escherichia coli was diluted 1:100 in LB broth without antibiotics and the recipient was diluted 1:5 in LB broth. The recipient PTB 9 was grown for 3 h at 27 C. One millilitre of recipient and 1.5 mL of donor strains were transferred to an Eppendorf tube and spun down at 5000 r/min for 3 min (so that the pili are not disrupted). Cells were resuspended in 150 L of LB broth by gently pipetting up and down, and 25 L of the donor cells was spotted onto a filter followed by 25 L of the recipient. For a check, 25 L of each strain used was spotted onto filters in separate plates. Plates were

not turned over, as that would cause the suspension to run off of the filter squares. After incubation overnight, bacteria from the filter squares were resuspended in 1.0 mL of LB broth by vortexing and pipetting, if necessary. The cells were transferred to Eppendorf tubes and spun down. Most of the supernatant was aspirated, leaving about 100 L to resuspend the cells. The cells were resuspended in the 100 L of remaining media and were plated onto double selective media: the first medium contained antibiotic to kill the donor strain and the second medium to select for transformants of the recipient. If the recipient lacks a resistant marker, it may be possible to use minimal medium because the E. coli donor is an auxotroph. A total of 1125 transconjugants were screened in vitro by a dual-plate assay against the rice BB pathogen X. oryzae pv. oryzae to identify mutants defective in antibiotic activity. Five of the 50 strains that lacked the ability to inhibit X. oryzae pv. oryzae in the laboratory were selected and checked with PCR to confirm the insertion of the transposon. Greenhouse test to evaluate Phl mutants and the wildtype P. fluorescens PTB 9 for rice BB suppression in rice cultivar IR24 During JulyNovember 2004, a greenhouse experiment was carried out in our University greenhouse at Chennai to evaluate the biocontrol efficacy of the DAPG-producing wild-type strain P. fluorescens PTB 9 and its mutants that were defective in their antibiotic activity, PTB 9a, PTB 9b, PTB 9c, PTB 9d, and PTB 9e. Small batches of 5 g of rice seeds of a BB-susceptible cultivar (IR24) were seed-coated by bacterization (Gnanamanickam and Mew 1992) separately with cell suspensions of either PTB 9 or one of its Phl mutants at 108 CFU/mL in 0.1% carboxymethylcellulose. Seeds coated with plain 0.1% carboxymethylcellulose solution served as the untreated check. Plants raised from bacteriatreated and untreated seeds were clip-inoculated with X. oryzae pv. oryzae at 106 CFU/mL (by methods described earlier) when they were 45 days old. The lengths of BB lesions that developed were measured 14 days after the clipinoculation and reduction in disease severity was derived from reductions in lesion length.

Results
Laboratory assays to identify antagonists to X. oryzae pv. oryzae Two hundred and seventy-eight bacterial strains inhibited the growth of X. oryzae pv. oryzae in laboratory assays. Among the 637 strains screened, 44% were antagonists and their zone of pathogen inhibition ranged from 0.5 to 4.7 cm (Table 1). PCR-based screening method to detect DAPG production Following the PCR method using forward primer (Phl2a) and reverse primer (Phl2b), 27 strains of the 278 identified as antagonistic strains showed a characteristic 745-bp DNA fragment amplification. The PCR products of a DAPGproducing reference strain, P. fluorescens CHAO, also had this characteristic 745-bp fragment, while in the rest of the nonproducers, no PCR products were amplified (Fig. 1). The
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Velusamy et al. Fig. 1. PCR-based screening method for the production of 2,4diacetylphloroglucinol (DAPG) by plant-associated Pseudomonas fluorescens strains. Lanes 3 and 12, an amplified 745-bp fragment characteristic of the phlD gene, indicating DAPG production; lane 1, P. fluorescens CHAO, a known producer of DAPG; lane M, molecular mass marker (1-kb ladder).

61 Fig. 2. TLC detection of 2,4-diacetylphloroglucinol (DAPG) extracted from culture fluids of Pseudomonas fluorescens PTB 9. The antibiotic has an Rf value of 0.54 (right), similar to that of the DAPG standard (left) cochromatographed.

predicted 745-bp fragment of the same size was amplified from DNA of all 27 DAPG-producing Indian strains. On the basis of their biochemical profile, the DAPGproducing strains were identified as strains of P. fluorescens (data not shown). Metabolites of P. fluorescens and antibacterial activity In separate dual-plate assays, the growth of the rice BB pathogen X. oryzae pv. oryzae was inhibited in all 27 strains of P. fluorescens by metabolites that were identified by TLC analysis as PRN and DAPG. The antibacterial activity of PRN was not studied further. Relationship between DAPG production and BB suppression by P. fluorescens PTB 9 DAPG production and in vitro inhibition of pathogen Extracts of culture fluids (after 72 h growth) of P. fluorescens PTB 9, the most efficient strain and a producer of DAPG (identification made by the PCR method), yielded 40 g of the antibiotic in 1 mL of the culture. When this material was dissolved in 65% methanol and was assayed for antibiosis towards X. oryzae pv. oryzae in agar (PSA) well diffusion assays, 5075 g/mL inhibited the growth of the rice BB pathogen. The diameter of the inhibition zones ranged from 1.0 cm (for 50 g) to 1.752.00 cm (for 75 g). In control plates that had 65% methanol, there was no inhibition. DAPG analysis The DAPG extracted and purified from culture fluids of P. fluorescens PTB 9 was in the form of light orange crystals

with a melting point of 165167 C (Campbell and Coppinger 1951). The purified compound was cochromatographed with the DAPG standard. TLC showed an identical Rf value of 0.54 for the DAPG extracted from efficient P. fluorescens PTB 9 and for the DAPG standard (Fig. 2). The presence of DAPG in the condensed extract of P. fluorescens PTB 9 was also confirmed by HPLC. HPLC analysis of the purified compound of strain PTB 9 showed a major peak at a retention time of 7.25 min that corresponded to the peak obtained for the DAPG standard at a retention time of 7.12 min. Peak areas were converted to micrograms of the antibiotic from a calibration curve prepared by the injection of the DAPG standard. Antibiotic (4.5 ng) was detected in 10 L of the extract injected. The 1H NMR spectrum of DAPG exhibited (i) a six-proton singlet at 2.84 owing to the methyl protons of the two acetyl methyl groups, (ii) a one-proton singlet at 5.99 owing to the aromatic proton, and also (iii) a broad singlet at 3.56 owing to the three hydroxyl protons. The Fourier transform IR spectrum of the DAPG sample showed a broad band at 3304/cm owing to the phenolic hydroxyl groups and a sharp broad band at 1620/cm owing to the acetyl carbonyl groups (Silverstein et al. 1981). The purified DAPG extracted from P. fluorescens PTB 9 had Rf and retention time values that matched those of the DAPG standard. Its spectral (1H NMR, IR) profiles plus its melting point of 165167 C confirmed the production of DAPG. Evaluation of DAPG producers for biological suppression of rice BB in net-house and field plots In the net-house experiments, all 27 strains of P. fluorescens suppressed BB and the disease reductions ranged from 8.2% to 58.8% (Table 2). Nine strains (five rice strains and four nonrice strains) reduced BB incidence by more than 50%, and among them, the most efficient strains (rice strain PTB 9
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Can. J. Microbiol. Vol. 52, 2006 Table 2. Evaluation of Pseudomonas fluorescens strains producing DAPG in the biological suppression of BB in rice cultivar IR24. Net-house experiment Diameter of inhibition of Xoo in dual-plate assay (cm) 3.6 4.1 2.8 3.0 4.4 4.2 1.2 3.5 2.8 2.1 2.3 1.7 2.3 2.1 4.1 1.8 3.5 1.4 3.8 1.7 2.5 3.6 3.0 2.4 1.8 2.9 2.7 0 Mean BB lesion length (cm)a 9.04** 8.66** 13.25** 15.20** 8.64** 16.83** 17.54* 16.07** 17.39* 14.52** 10.15** 13.90** 16.87** 14.53** 8.99** 9.82** 9.35** 13.08** 8.63** 13.91** 9.24** 15.50** 12.85** 18.80 ns 16.52** 19.25 ns 17.41* 20.96 3.0 4.0 % BB suppression 56.87 56.68 36.78 27.48 58.78 19.70 16.32 23.33 17.03 30.73 51.57 33.68 53.15 30.68 57.11 19.51 55.39 37.60 58.83 33.64 55.92 26.05 38.69 10.31 21.18 8.16 16.94 0 Field experiment Mean BB lesion length (cm)a 10.15** 9.53** 14.72** 12.62** 7.83** 13.85** 16.85** 11.07** 18.17* 12.90** 12.05** 14.42** 11.38** 16.90** 10.04** 14.09** 11.19** 11.87** 9.50** 20.03 ns 13.42** 15.82** 19.89 ns 21.92 ns 20.13 ns 19.43 ns 21.58 ns 22.03 3.4 4.5 % BB suppression 53.93 56.74 33.18 42.71 64.46 37.13 23.51 50.75 17.52 41.44 45.30 34.54 48.34 23.29 54.43 36.04 51.21 46.12 51.88 9.08 39.08 23.19 9.71 0.50 8.62 11.80 2.04 0

Strain KAD 7 IMV 14 IMV 2 BGR 19 PTB 9 MON 1 TVM 8 VEL 17 VEL 10 GDY 4 GDY 7 TRP 5 TRP 18 MDR 9 MDR 7 STR 7 VGP 13 MDR 16 PDY 7 VLB 7 KVR 5 TNI 13 KOV 8 RJP 31 KOV 3 PDU 1 PDU 9 Untreated check LSD0.05 LSD0.01

Note: Experiments were performed either in a net house or in the field at the Regional Agriculture Research Station, Pattambi, Kerala, India. Xoo, Xanthomonas oryzae pv. oryzae; BB, bacterial blight; DAPG, 2,4-diacetylphloroglucinol; LSD, least significant difference. a Each value is a mean of 40 observations. Reduction in lesion length: **, significant at the 1% level; *, significant at the 5% level; ns, not significant.

and finger millet strain PDY 7) reduced BB by 58.8% (Tables 1 and 2). When these 27 DAPG-producing P. fluorescens strains were evaluated for BB suppression in a field experiment planted with cultivar IR24 rice, the mean leaf BB lesion length in bacteria-treated plants ranged from 7.8 to 21.9 cm, while in the untreated plants, it was 22.0 cm (Table 2). The untreated plants showed severe BB disease symptoms with long and spreading blight lesions, while the plants treated with some of the DAPG producer strains were relatively healthy and had shorter lesions of less than 3 cm. Of the 27 strains, seven strains, IMV 14, PTB 9, MDR 7, KAD 7, VEL 17, VGP 13, and PDY 7, showed more than 50% (56.74%, 64.46%, 54.43%, 53.9%, 50.8%, 51.2%, and 51.9%, respec-

tively) BB suppression compared with untreated plants (Fig. 3). Among the seven efficient strains, three (IMV 14, PTB 9, and MDR 7) were rice-associated bacterial strains and the other four (KAD 7, VEL 17, VGP 13, and PDY 7) were non-rice-associated bacterial strains. Maximum BB suppression (64.46%) was afforded by strain PTB 9, a riceassociated strain. Loss of DAPG production results in loss of biocontrol efficiency In PCR analysis, five Phl mutants (PTB 9a, PTB 9b, PTB 9c, PTB 9d, and PTB 9e) of P. fluorescens PTB 9 generated through transpositional mutagenesis (Tn5-Km) defective in DAPG production showed transposon insertion. Their heat 2006 NRC Canada

Velusamy et al. Fig. 3. Biological suppression of bacterial blight (BB) lesion length in rice cultivar IR24 owing to treatments with Pseudomonas fluorescens strains. The leaves detached from field plots raised with rice plants that were treated with six strains show reductions in BB lesion length. Strain PTB 9 afforded a maximum of 64.5% reduction. A leaf from the untreated control plot showing a longer spreading BB lesion of more than 22 cm is on the far left.

63

wild-type strain P. fluorescens PTB 9 had shorter lesions of 35 cm (Table 3). The wild-type strain afforded a BB reduction of 59.52%, while the mutants (PTB 9a, PTB 9b, PTB 9c, PTB 9d, and PTB 9e) defective in DAPG production afforded 19.65%, 17.11%, 23.77%, 20.81%, and 18.11% BB reductions, respectively.

Discussion
This perhaps is the first systematic research effort in India to investigate the possible use of Pseudomonas sp. strains as DAPG producers and to highlight a role for DAPG in the suppression of BB. Therefore, this is an important landmark study on the biological control of BB in both India and the tropics. There has been no previous report from India on the production of DAPG by plant-associated bacteria or on its suppression of rice BB. DAPG production has been a very well-known mechanism in the biological control of some of the major fungal pathogens of the temperate regions and has assumed much importance as the factor that has contributed to the take-all decline in wheat (Raaijmakers et al. 1997, 1999; Raaijmakers and Weller 1998). Further, its antibacterial activity against soft-rot Erwinia spp. was also previously known. Yet, in the present study, DAPG production has been implicated as an antibacterial compound involved in the suppression of one of the most important and devastating bacterial crop diseases of the tropics, the BB of rice. Results presented in Table 2 show BB suppression by DAPG-producing strains, and among these, a superior rice-associated strain of P. fluorescens PTB 9 was the most effective. It suppressed BB by 58.8% in the nethouse and by 64.5% in the field consistently in greenhouse, net-house, and field experiments carried out in two locations over a period of 2 years (20022004). The generation and use of Phl mutants further clarifies the role of DAPG in BB suppression (Table 3). The Phl mutants have shown substantial loss of their ability to protect the rice plants against X. oryzae pv. oryzae. PRN produced by the same DAPG producer strains is also antibacterial towards X. oryzae pv. oryzae; its role in BB suppression remains to be studied. Until PRN production is also carefully studied in greenhouse and field experiments, its share of possible BB suppression does remain uncertain. Therefore, what we have presented is a careful analysis that confirms the biological activity of DAPG, extracted and purified from a superior strain of P. fluorescens PTB 9, in the inhibition of the growth of the BB pathogen X. oryzae pv. oryzae and that establishes a causal relationship between DAPG production and suppression of BB severities in rice through net-house and field experiments. The results from the greenhouse experiment (Table 3) obtained with Phl mutants of P. fluorescens PTB 9 do lend further support to the importance of DAPG production in BB suppression.

lyzed bacterial DNA did not show the amplification of the characteristic 745-bp fragment, whereas the DNA of the wild-type strain PTB 9 had the fragment. In the greenhouse assay, rice plants raised from seeds treated with Phl mutants, PTB 9a, PTB 9b, PTB 9c, PTB 9d, and PTB 9e, had BB lesions of 1525 cm in length, while the plants raised from seeds that were treated with the

Acknowledgements
We thank Dr. G. Defago for providing the P. fluorescens CHAO positive strain. We are grateful to Dr. P.V. Balachandran, Associate Director, Regional Agricultural Research Station, Pattambi, Kerala, India, for providing the field space. We also thank the Department of Biotechnology, Government of
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64

Can. J. Microbiol. Vol. 52, 2006 Table 3. Evaluation of Pseudomonas fluorescens PTB 9 and its Phl mutants for suppression of BB in rice cultivar IR24. Difference in lesion length from control (cm)b 11.27** 3.72 3.24 4.50 3.94 3.43 0 5.8 6.6 ns ns ns ns ns

Strain Wild-type P. fluorescens PTB 9 Phl mutant PTB 9a PTB 9b PTB 9c PTB 9d PTB 9e Check LSD0.05 LSD0.01

Mean BB lesion length (cm)a 7.66** 15.21 15.69 14.43 14.99 15.50 18.93 5.8 6.6 ns ns ns ns ns

% BB suppression 59.52 19.65 17.11 23.77 20.81 18.11 0 5.8 6.6

Note: Greenhouse experiments were performed in Chennai, southern India, JulyNovember 2004. BB, bacterial blight. a Mean of three replications. Reduction in lesion length: **, significant at 1% by the LSD method of analysis; ns, not significant. b Mean of normalized lesion lengths (mean lesion length in untreated control/check deducted from mean lesion length in bacteria-treated plants). Reduction in lesion length: **, significant at 1% by the LSD method of analysis; ns, not significant.

India, for the financial support to carry out this research. We are thankful to the Director, CAS in Botany, University of Madras, for his help and encouragement.

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