Indian Journal of Experimental Biology Vol. 49, March 2011, pp. 229-233
Antibacterial activity of metabolite produced by Paenibacillus polymyxa strain HKA-15 against Xanthomonas campestris pv. phaseoli V Mageshwaran1*, Suresh Walia2, V Govindasamy3 & K Annapurna3 1
Chemical and Biochemical Processing Division, CIRCOT, Adenwala Road, Matunga, Mumbai 400 019, India 2 Division of Agricultural Chemicals, Indian Agricultural Research Institute, Pusa Campus, New Delhi 110 012, India 3 Division of Microbiology, Indian Agricultural Research Institute, Pusa Campus, New Delhi 110 012, India Received 13 May 2010; revised 28 October 2010
An antibacterial metabolite extracted from Paenibacillus polymyxa HKA-15 showed strong inhibition against Xanthomonas campestris pv. phaseoli strains CP-1-1 and M-5. Minimum inhibitory concentration (MIC) of crude extract against strains CP-1-1 and M-5 was found to be 1.7 mg/ml and 1.52 mg/ml, respectively. In UV-Vis range, the absorption peak of crude extract was maximum at 240 nm. The compound is resilience to wide range of temperature, pH, surfactants and organic solvents. The complete loss of activity was observed when crude metabolite was treated with pepsin (400 unit / ml). Characterization of crude metabolite suggested its hydrophobic and peptide nature. Inhibition of Xanthomonas campestris pv. phaseoli by peptide like metabolite produced by Paenibacillus polymyxa strain HKA-15 under in vitro conditions showed ecological and biotechnological potential of strain HKA-15 to control common blight disease in beans. Keywords: Antibacterial activity, Biocontrol, Paenibacillus polymyxa, Peptide antibiotic, Xanthomonas campestris pv. phaseoli
Indiscriminate use of chemicals for the control of plant diseases results in environmental degradation, food poisoning and mammalian toxicity. Researchers are focusing on alternative ways to manage plant diseases. Of the different management strategies, biocontrol of these pathogens by plant growth promoting bacteria (PGPB) offers the best alternative solution1. Bacillus spp and its related genera have been identified as potential biocontrol agent as they produce wide range of cyclic lipopeptides active against various microorganisms2. Pueyo et al.3 showed have that large group of lipopeptides produced by soil bacterium Bacillus megaterium are active against various plant pathogens. The culture filtrate of Bacillus amyloliquefaciens RC-2 show antimicrobial activity against Colletotrichum demantium, Rosellina necatrix, Pyricualria oryzae, Agrobacterium tumefaciens and Xanthomonas campestris pv. campestris4. Two active fractions viz., KB-8A and KB-8B are extracted by methanol from the culture filtrate of Bacillus polymyxa. Its MIC is 12.8 µg/ml for Fusarium oxysporum and Alternaria mali5. —————— * Correspondent author Telephone: 919769941511 E-mail:
[email protected],
Role of lipopeptides from Bacillus in antibiosis mechanism against Xanthomonas campestris pv. campestris has been studied by Monteiro et al.6. Under controlled conditions, treatment of soybean leaf surfaces with Bacillus subtilis 210 before 72 h of inoculation with pathogenic bacteria reduced the number of leisions by X. campestris7. Crude extract of antifungal metabolite produced by Paenibacillus lentimorbus strain WJ5 is thermostable and no loss of activity has been recorded when exposed to proteinase K, sodium dodecyl sulphate (1%), Tween-80 (1%) and glycerol (1%)8. Physico-chemical characterization of antimicrobial metabolite produced by P. peoriae strain NRRL BD-62 indicates that the compound retained the activity even after autoclaving (121° C for 10 min), treatment with organic solvents, hydrolytic enzymes and exposure to wide range of pH9. However, there is a need of characterization of antimicrobial metabolite active against Xanthomonas campestris pv. phaseoli causing common blight disease in French bean. Hence, this study was made with the objective to characterize the crude metabolite produced by Paenibacillus polymyxa HKA-15 having antibacterial activity against Xanthomonas campestris pv. phaseoli.
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Materials and Methods Microorganisms and culture conditions—Soybean bacterial endophyte Paenibacillus polymyxa HKA-15 was obtained from Division of Microbiology, IARI, New Delhi, India. Bacterial phytopathogen Xanthomonas campestris pv. phaseoli strains M-5 and CP-1-1 were obtained from Division of Plant Pathology, IARI, New Delhi. These bacterial strains were grown in nutrient broth at 30oC and preserved in 40% (v/v) glycerol at -20°C. Extraction of crude antibiotic—P. polymyxa strain HKA-15 grown in nutrient broth (absorbance = 0.5 at 560 nm) was inoculated in to 1L of nutrient broth. One hundred ml of culture was taken at different growth stages viz., 0, 2, 4, 8, 12, 24, 36, 48, 72 and 96 h for the extraction of metabolite. Absorbance was measured at 560 nm using spectrophotometer. About hundred ml of n-butanol was added to 100 ml of the culture and shaken well for 30 min and allowed to settle down. Organic phase was separated out and concentrated to dryness using vacuum evaporator. The dried substance redissolved in methanol and evaporated again. Finally, the crude extract was dissolved in 100 µl of methanol and subjected for bioassay against M-5 and CP-1-1. For small scale extraction of crude metabolite, 48 h old culture of P. polymyxa HKA-15 grown in 5L of nutrient broth at 30°C under shaking conditions (180 rpm) was added with equal volume of n-butanol. Organic phase was separated and concentrated to dryness using vacuum evaporator. From 5L of nutrient broth 137 mg of crude extract was obtained. It was dissolved in 10 ml of methanol to adjust the concentration to 13.7 mg/ml. The crude metabolite was subjected to bioassay against M-5 and CP-1-1. MIC, absorption spectrum and crude antibiotic partition—To find MIC, the crude extract was diluted from 1X to 10X. The least dilution showing inhibition of 5 mm was considered as MIC. Arbitary Units (AU) of antibacterial activity was calculated by the reciprocal of the highest dilution showing inhibition per ml of crude metabolite. To find the absorption spectrum, crude extract (100 µl) in methanol was taken, dried completely and suspended in 450 µl of sterile distilled water. The sample was scanned for their absorption spectrum at a wavelength range of ultra violet-visible rays by using Shimadzu-UV-160A spectrophotometer. The crude metabolite subjected to ultrafiltration in 10 KDa cut off membrane. Two distinct fractions (one bigger than 10KDa and other
smaller than equal to 10KDa) were obtained after this process were subjected to antibacterial activity. Effect of crude extract on growth of X. campestris pv. phaseoli cells—Overnight grown culture of X. campestris pv. phaseoli strains M-5 and CP-1-1 was mixed with crude extract at a final concentration of 1.5 mg/ml. To study the effect of crude extract on growth of X. campestris cells, absorbance at 560 nm and the number of viable cells (cfu/ml) were assessed at 2 h intervals from 0 to 8 h. Physical and chemical characterization —Effect of pH, enzymes, surfactants and organic solvents on crude extract was examined. To determine the effect of pH, sample (90 µl) mixed with 10 µl of 100 mM, HCl to give pH 2 (10 mM, HCl) and 90 µl of sample added with 10 µl of 1M, NaOH to give pH12 (100 mM, NaOH)). For enzymatic characterization, proteinase K (30 unit/ml) and pepsin (400 unit/ml) were added with 100 µl samples. The samples were boiled for 5 min to stop the activity of enzymes after incubation of 37oC for 3 h. Effect of glycerol (5%), triton X 100 (1%), and SDS (1%) on crude metabolite was tested. Methanol, ethanol, chloroform at the concentration of 10% (v/v) was added with crude extract and tested for bioactivity. The samples were incubated at 37°C for 3 h. To analyze the thermal stability, the samples was exposed to the autoclaving temperature (121°C for 15 min) and at -20°C for 3 h. The samples were tested for bioactivity against M-5 and CP-1-1. Control was maintained with no treatment and incubated at 37°C for 3 h. Results Extraction of crude antibiotic—Maximum absorbance reading at 560 nm was observed in 12 h of incubation. The antibacterial compounds were extracted using n-butanol. The antibacterial compounds production increased after 12 h of incubation and gradually decreased after 72 h of incubation. Bioassay of crude extract obtained from 48 h old culture showed higher inhibition zone of 5 and 6 mm against M-5 and CP-1-1, respectively (Figs 1 and 2). The small scale extraction of crude metabolite yielded 13.7 mg/ml. The crude antibiotic was showed inhibition against M-5 and CP-1-1. MIC, absorption spectrum and crude antibiotic partition—The least dilution showing the minimal inhibition (5 mm) was considered as MIC as has been described by Bernal et al.10. MIC of crude metabolite against CP-1-1 and M-5 were 8X and 9X which
MAGESHWARAN et al.: ANTIBACTERIAL ACTIVITY OF PAENIBACILLUS POLYMYXA HKA-15
corresponds to the concentration of 1.71 mg/ml and 1.52 mg/ml, respectively. AU of crude metabolite against Xanthomonas campestris pv. M-5 and CP-1-1 were 300 and 266, respectively (Fig. 3). Absorption spectrum of n-butanol extracted crude metabolite fell in the range of 200-400 nm wavelengths. The absorption peak was maximum at 240 nm in UV range (Fig. 4). Crude metabolite was separated into filtrate (F) higher than 10 kDa and retentate (R) less than 10 KDa based on ultrafiltration in a 10 KDa microcon cut off membrane. Retentate showed higher inhibition against CP-1-1 than filtrate. Whereas, both retentate and filtrate showed inhibition against M-5. Effect of crude extract on growth of Xanthomonas campestris pv. phaseoli cells—A addition of crude extract (1.5 mg/ml) at final concentration to cell suspension of X. campestris pv. phaseoli caused a large decrease in the number of viable cells in both the strains M-5 and CP-1-1 after 4 h of contact. However, the contact of crude extract with cell
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suspension up to 4 h did not result in much change in cell density. There was a slight decrease in absorbance after 4 h of incubation of both crude extract and cell suspension (Fig. 5). Physical and chemical characterization —No loss of activity was observed when crude metabolite exposed to temperatures ranging from -20°C and 121°C for 15 min (Table 1). The antibiotic retained full activity when they were subjected to pH 2 and pH 12 at 37°C at overnight incubation. Enzymatic treatment with proteinase K showed no loss of activity against CP-1-1 and M-5, while treatment with
Fig. 3—Minimum inhibitory concentration (MIC).[♦Zone of Inhibition against M-5, ■Zone of Inhibition against CP-1-1 ]
Fig. 1—Antibacterial activity of crude extract from Paenibacillus polymyxa strain HKA-15 against X.campestris pv.phaeoli strains at various time intervals. [■Absorbance at 560 nm, Zone of Inhibition against M-5, Zone of Inhibition against CP-1-1 ]
Fig. 4—Absorption spectrum of crude antibiotic
Fig. 2—Inhibition of growth of Xanthomonas campestris pv. phaseoli CP-1-1 by crude extract at different incubation period (A) 0 to 24 h; and (B) 24 to 96 h
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pepsin resulted in complete loss of activity. Treatment with organic solvents such as acetone, ethanol, methanol and chloroform showed that no loss of activity and they were soluble in these solvents. Antibacterial compound treated with glycerol (5%) resulted in half a reduction of activity. Treatment with triton X 100 (1%) showed no loss of activity against M-5 and some loss of activity against CP-1-1. No loss of activity was observed when antibacterial compound was treated with 1% SDS. Discussion In the present study, the crude antibacterial metabolite from Paenibacillus polymyxa HKA-15 was isolated and characterized. P. polymyxa HKA-15 produced maximum antibacterial compound at 48 h of incubation (late stationary phase) in nutrient broth under shaking conditions at 30°C. Experiments
Fig. 5—Effect of crude antibiotic on growth of Xanthomonas campestris pv. phaseoli [■log cfu/ml of M-5, ♦OD of M-5 ▲log cfu/l CP-1-1 ■OD of CP-1-1 ] Table 1―Physical and chemical characterization of crude extract Treatement to crude extract
Zone of Inhibition (mm) X. c pv. M-5 8
X. c pv. CP-1-1 Control (None added) 8 incubated for 3 h at 37oC Exposed to 8 8 -20oC. 121oC 8 8 pH 2 8 8 pH 12 8 8 Proteinase K (0.5 mg/ml) 8 8 Pepsin nil nil (0.5 mg/ml) Glycerol (5%) 4 4 Triton X-100 (1%) 8 5 SDS (1%) 8 8 Methanol 8 8 Chloroform 8 8 Ethanol 8 8 X.c pv. M-5 = Xanthomonas campestris pv. phaseoli M-5 X.c pv. CP-1-1 = Xanthomonas campestris pv. phaseoli CP-1-1
conducted earlier to study the antibacterial metabolite production from different bacterial systems have shown maximum metabolite production at poststationary phase8-11. These results are in agreement with our study. The crude extract was subjected to study MIC, UV-VIS range and molecular weight partition. MIC of crude antibiotic against CP-1-1 was 8X and MIC of crude extract against M-5 was 9X. Similarly Bernal et al.10 have reported that the antibacterial metabolite produced by Bacillus sp. having MIC of 11X against Erwinia caratovora. AU of antibacterial activity of metabolite was found 300 against M-5 and 266 against CP-1-1. The results were in agreement with Wu et al.12 who have reported AU of subpeptin JM4-A and subpeptin JM4-B from Bacillus subtilis to be 1280. The cell suspension of X. campestris pv. phaseoli strain M-5 and CP-1-1 showed a drastic reduction in number of viable cells after 4 h of contact with crude extract (final concentration of 1.5 mg/ml). Similar results have been obtained when cerein 8A at final concentration of 400 AU/ml caused a significant decrease of number of viable cells in Listeria monocytogenes and Bacillus cereus with in 75 min and 60 min of contacts, respectively13. Absorption maximum of crude metabolite extracted with n-butanol in UV-Vis range suggested the peptide nature of antibacterial metabolite. Kim et al.2 and Bernal et al.10 have reported that metabolite from Bacillus strains shows major peaks at 231, 259 and 212 nm corresponding to polyene, lactone and iturin like peptides. Ultrafiltration of crude extract showed that active compound against M-5 and CP-1-1 have molecular wt. more than and less than 10 kDa, respectively. Weid et al.9 have suggested that two fractions obtained after ultrafiltration in centricon system of culture supernatant of P. peoriae. The fraction less than 10 kDa shows inhibitory activity against Micrococcus sp. and Ag. tumefaciens. An antibacterial metabolite with the molecular weight of 30 kDa from Pseudomonas sp. strain 4B shows inhibition to spoilage bacteria14. The present result suggested involvement of more than one compound in crude extract responsible for antibacterial activity. To conclude, antibacterial metabolite from P. polymyxa strain HKA-15 revealed the hydrophobic and peptide nature and their resilient to wide range of physical and chemical stresses. The strong inhibition of test organism X. campestris pv. phaseoli by peptide antibiotic produced by P. polymyxa strain HKA-15 under in vitro conditions suggested the ecological and
MAGESHWARAN et al.: ANTIBACTERIAL ACTIVITY OF PAENIBACILLUS POLYMYXA HKA-15
biotechnological potential of strain HKA-15 to control common blight disease in beans. Acknowledgement The author (VM) thanks to Jawaharlal Nehru Memorial Fund, New Delhi and Indian Agricultural Research Institute, New Delhi for financial assistance for this study. References 1 Compant S, Duffy B, Nowak J, Clement C & Barka E A, Use of pant growth-promoting bacteria for biocontrol of pant disease: Principles, mechanisms of action and future prospects, Appl Environ Microbiol, 71 (2005) 4951. 2 Kim H S, Park J, Choi S W, Choi K H, Lee G P, Ban S J, Lee C H & Kim C S, Isolation and characterization of Bacillus strains for biological control, J Microbiol, 41 (2003) 196. 3 Pueyo M T, Bloch C J, C-Ribeiro A M & Masico P, Lipopeptides produced by a soil Bacillus megaterium, Microb Ecol, 57 (2009). 4 Yoshida S, Hiradate S, Tsukamot T, Hatakeda K & Shirata A, Antimicrobial activity of culture filtrate of Bacillus amyloliquefaciens RC-2 isolated from mulberry leaves, Biol control, 91 (2001) 181. 5 Hyun J W, Kim Y H, Lee Y S & Park W M, Isolation and evaluation of protective effect against Fusarium wilt of sesame plants of antibiotic substance from Bacillus polymyxa KB-8, Plant Pathol J, 15 (1999) 152. 6 Monteiro L, Mariano R, Lima D R & S-Maior A M, Antagonism of Bacillus spp. against Xanthomonas campestris pv. campestris, Braz Arch Biol Technol, 48 (2005) 23.
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