Chromatographic Detection of Antibacterial Antibiotics from Pseudomonas fluorescens (TNAU PF1) and Their Effect on Bacterial Wilt Pathogen Ralstonia solanacearum (KK2)

Main Article Content

Shiva Nagendran
Paranidharan Vaikuntavasan
Karthikeyan Muthusamy
Balachandar Dananjeyan
Balasubramani Venkatasamy


Bacterial wilt of tomato is a devastating disease incited by Ralstonia solanacearum (KK2) causing heavy yield loss in India. The antagonist Pseudomonas fluorescens (TNAU PF1) was characterized for antibacterial ability against the bacterial wilt pathogen. Thin-layer chromatography (TLC) identified six distinct antibiotics in crude metabolite of TNAU PF1 strain. The crude antibiotics and the antibiotics 2,4-diacetylphloroglucinol and Phenazine of TNAU PF1 showed best in vitro antibacterial activity against the bacterial wilt pathogen R. solanacearum. The ability of crude antibiotics in inhibiting Ralstonia colonies was higher than the individual antibiotics (2,4-diacetylphloroglucinol and Phenazine) under in vitro. Further, GC-MS analysis of the semi-purified crude extract of Pseudomonas fluorescens (TNAU PF1) identified six antibacterial compounds viz., Phloroglucinol dimethyl ether, Patulin, Hemipyocyanin, Phthalic acid, butyl 2-pentyl ester, Phenazine and 1-Phenazinecarboxylic acid that would be synergistically inhibited the colony multiplication of the pathogen. The study emphasized the antagonistic activity of the Pseudomonas fluorescens (TNAU PF1) for the management of bacterial wilt disease.

Bacterial wilt, biocontrol, Pseudomonas fluorescens, antibiotics, DAPG and phenazine, antibacterial.

Article Details

How to Cite
Nagendran, S., Vaikuntavasan, P., Muthusamy, K., Dananjeyan, B., & Venkatasamy, B. (2019). Chromatographic Detection of Antibacterial Antibiotics from Pseudomonas fluorescens (TNAU PF1) and Their Effect on Bacterial Wilt Pathogen Ralstonia solanacearum (KK2). International Research Journal of Pure and Applied Chemistry, 20(2), 1-10.
Original Research Article


De Curtis F, Lima G, Vitullo D, De Cicco V. Biocontrol of Rhizoctonia solani and Sclerotium rolfsii on tomato by delivering antagonistic bacteria through a drip irrigation system. Crop Prot. 2010;29:663-670.

Smith EF. A bacterial disease of the tomato, eggplant and Irish potato (Bacillus solanacearum n. sp.). US Dept. Agric. Div. Veg. Phys. Path; 1896.

Fegan M, Prior P. How complex is the Ralstonia solanacearum species complex, In Bacterial Wilt: The Disease and the Ralstonia solanacearum species complex (eds Allen C, Prior P, Hayward AC). American Phytopathol Society, St. Paul, MN. 2005;449-461.

Allen C, Prior P, Hayward AC. bacterial wilt disease and the Ralstonia solanacearum Species Complex, APS Press, St. Paul, MN, USA; 2005.

Singh D, Sinha S, Yadav DK, Sharma JP, Srivastava J. Characterization of biovar/ races of Ralstonia solanacearum the incitant of bacterial wilt in solanaceous crops. Ind Phytopathol. 2010;63:261-265.

Singh D, Yadav DK, Chaudhary G, Rana VS, Sharma RK. Potential of Bacillus amyloliquefaciens for biocontrol of bacterial wilt of tomato incited by Ralstonia solanacearum. J Plant Pathol Microbiol. 2016;7:327.

Roberts DP, Lohrke SM, Meyer SLF, Buyer JS, Bowers JH. Biocontrol agents applied individually and in combination for suppression of soil borne diseases of cucumber. Crop Prot. 2005;24:141-155.

Hafez EE, Hashem M, Balbaa MM, El-Saadani MA, Ahmed SA. Induction of new defensin genes in Tomato plants via pathogens-biocontrol agent interaction. J Plant Pathol Microb. 2013;4:167.

Lugtenberg BJJ, de Weger LA, Bennett JW. Microbial stimulation of plant growth and protection from disease. Curr Opin Microbiol. 1991;2:457-464.

Hoffland E, Halilinen J, Van Pelt JA. Comparison of systemic resistance induced by avirulant and nonpathogenic Pseudomonas species. Phytopathol. 1996; 86:757-762.

Wei G, Kloepper JW, Tuzun S. Induced systemic resistance to cucumber diseases and increased plant growth by plant growth promoting rhizobacteria under field condition. Phytopathol. 1996;86:221-224.

Sullivan ODB, Gara OF. Traits of fluorescent Pseudomonas spp. involved in suppression of plant root pathogens. Microbiol Rev. 1992;56:662-676.

Prabhukarthikeyan SR, Raguchander T. Antifungal metabolites of Pseudomonas fluorescens against Pythium aphanidermatum. J of Pure and Appl Microbiol. 2016;10(1):579-584.

Leisinger T, Margraff R. Secondary metabolites of fluorescent Pseudomonas. Microbiol Rev. 1979;43:422-42.

Defago G, Haas D. Pseudomonads as antagonists of soil borne plant pathogens: Mode of action and genetic analysis. Soil Biochem. 1990;6:249-91.

Howell CR, Stipanovic RD. Suppression of Pythium ultimum-induced damping-off of cotton seedlings by Pseudomonas fluorescens and its antibiotic, pyoluteorin. Phytopathol. 1980;70:712-5.

Gurusiddaiah S, Weller DM, Sarkar A, Cook RJ. Characterization of an antibiotic produced by a strain of Pseudomonas fluorescens inhibitory to Gaeumannomyces graminis var. tritici and Pythium spp. Antimicrob Agents Chemother. 1986;29:488-99.

Sivapriya SL, Kumar K. Diversity of Azotobacter isolates from different rice soils of Tamil Nadu. Int J of Sci and Res. 2018;7(6):982-990.

King EO, Ward MK, Raney DE. Two simple media for the demonstration of pyocyanin and fluorescein. J Lab Clin Med. 1954;44:301-307.

Liu H, He Y, Jiang H, Peng H, Huang X, Zhang X, Thomashow LS, Xu Y. Characterization of a phenazine producing strain Pseudomonas chlororaphis GP72 with broad spectrum antifungal activity from green pepper rhizosphere. Curr Microbiol. 2007;54(4):302-306.

Chen CC, Chih-Cheng L, Hui-Ling H, Wen-Yu H, Han-Siong T, Tzu-Chieh W, Yin-Ching C, Ying-Chen L, Hung-Jen T. Antimicrobial activity of Lactobacillus species against Carbapenem-resistant Enterobacteriaceae. Front Microbiol. 2019; 10:789.

Dhingra OB, Sinclair JB. Basic Plant Pathology Methods. 2nd Edition, CRC Press, Boca Raton; 1995.

Kopka K. Current challenges and development in GC-MS based metabolite profiling technology. J of Biotech. 2006; 124(1):312-322.

De Oliveira AG, Murate LS, Spago, FR, Lopes LD, Beranger JPD, San Martin JAB. Evaluation of the antibiotic activity of extracellular compounds produced by the Pseudomonas strain against the Xanthomonas citri pv. Citri 306 strain. Biol Cont. 2011;56:125-131.

Lopes LP, de Oliveira AG, Beranger JPO, Gois CG, Vasconcellos FCS, San Martin JAB. Activity of extracellular compounds of Pseudomonas sp. against Xanthomonas axonopodis in vitro and bacterial leaf blight in eucalyptus. Trop Plant Pathol. 2012;37: 233-238.

Cardozo VF, de Oliveira, AG, Nishio EK, Perugini MRE, Andrade CGTJ, Silveira WD. Antibacterial activity of extracellular compounds produced by a Pseudomonas strain against methicillin-resistant Staphylococcus aureus (MRSA) strains. Ann Clin Microbiol Antimicrob. 2013;12:12.

Spago FR, Mauro CSI, de Oliveira AG, Beranger JPO, Cely MVT, Stanganelli MM. Pseudomonas aeruginosa produces secondary metabolites that have biological activity against plant pathogenic Xanthomonas species. Crop Prot. 2014;62: 46-54.

Byrne JM, Dianese AC, Jia P, Campbell HL, Cuppels DA, Louws FJ, Miller SA, Jones JB, Wilson M. Biological control of bacterial spot of tomato under field conditions at several locations in North America. Biol Cont. 2005;32:408-418.

Hussein, ZR, Atia SS. Antimicrobial effect of pyocyanin extracted from Pseudomonas aeruginosa. Eur J Exp Bio. 2016;6(6):1-4.

Kavitha K, Mathiyazhagan S, Sendhilvel S, Nakkeeran S, Chandrasekar G, Dilantha WGF. Broad spectrum action of phenazine against active and dormant structures of fungal pathogens and root knot nematode. Arch. of Phytopathol and Pl Prot. 2005; 38(1):69-76.

Karen DB, David AS, Patricia JS. Pyrrolnitrin production by biological control agent Pseudomonas cepacia B37w in culture and in colonized wounds of potatoes. Appl and Envi Microbial. 1994; 2031-2039.

Rosales AM, Thomashow L, Cook RJ, Mew TW. Isolation and identification of antifungal metabolites produced by rice associated antagonistic Pseudomonas spp. Phytopathol. 1995;85(9):1029-1032.

Baron SS, Rowe JJ. Antibiotic action of pyocyanin. Antimicrob Agents Chemother. 1981;20:814-820.

Rhitu R, Srinivasamurthy R, Prasanta KD, Payal G. Isolation, characterization and evaluation of the biocontrol potential of Pseudomonas protegens RS-9 against Ralstonia solanacearum in tomato. Int J of Exp Biol. 2017;55:595-603.

Yoshihisa H, Zenji S, Fukushi H, Katsuhiro K, Haruhisa S, Takahito S. Production of antibiotics by Pseudomonas cepacia as an agent for biological control of soil borne plant pathogens. Soil Biol aind Biochem. 1989;21(5):723-728.

Thomashow LS, Weller DM, Bonsall RF, Pierson LS. Production of antibiotic phenazine 1- carboxylic acid by fluorescent pseudomonad species in the rhizosphere of wheat. Appl Environ Microbial. 1990;56: 908-912.

Tiantian Z, Da C, Chunyu L, Qian S, Lingzhi Li, Fang L, Qirong S, Biao S. Isolation and characterization of Pseudomonas brassicacearum J12 as an antagonist against Ralstonia solanacearum and identification of its antimicrobial components. Microbiol Res. 2012;167: 388-394.

Pierson LS, Pierson EA. Metabolism and function of phenazines in bacteria: impacts on the behavior of bacteria in the environment and biotechnological processes. Appl Microbiol Biotec. 2010;86: 1659-1670.

Khatiwora E, Adsul VB, Kulkarni M, Deshpande NR, Kashalkar RV. Antibacterial activity of Dibutyl Phthalate: A secondary metabolite isolated from Ipomoea carnea stem. J Pharm Res. 2012;5(1):150-152.

Khan N, Shagufta R, Shahid AK, Sher BK. A new antibacterial dibenzofuran-type phloroglucinol from Myrtus communis Linn. Nat Prod Res. 2019;1-6.

Mittal N, Haben HT, Andrew MH, Silvia TC, John LS. Synthesis and antibiotic activity of novel acylated phloroglucinol compounds against methicillin-resistant Staphylococcus aureus. The J of Antibiot. 2019;1-7.

Mbundi L, Hector G, Mohammad RK, Jonathan LB, Sara L, Rosa B. Advances in the analysis of challenging food contaminants: nanoparticles, bisphenols, mycotoxins, and brominated flame retardants. Adv in Mol Toxicol. 2014;8: 35-70.

Thuraya AM, Maha AMA. Antimicrobial activity of pyocyanin for inhibition of Pseudomonas aeruginosa urinary tract pathogens. Asian J of Med and Health. 2016;4(4):1-9.

Srinivasan GV, Sharanappa P, Leela NK, Sadashiva CT, Vijayan KK. Chemical composition and anti¬microbial activity of the essential oil of Leea indica (Burm. f.) Merr flowers. Nat Prod Rad. 2009;8(5): 488-493.

Mezaache AS, Guechi A, Zerroug MM, Nicklin J, Strange RN. Antimicrobial activity of Pseudomonas secondary metabolites. Pharma Com. 2013;3(3):39-44.