title: Molecular approach for the rapid detection of Bacillus and Pseudomonas genera--dominant antagonistic groups--from diverse ecological niches using colony multiplex PCR.
abstract: Bacillus and Pseudomonas are the dominant groups of bacteria known for their antagonistic potential against many plant and animal pathogens. Presently, exploration of these genera with antagonistic property for disease management of aquaculture system is gaining more importance to overcome the use of antibiotics and related resistance issues. Rapid screening and identification of these genera from diverse bacterial populations by conventional methods is laborious, cost-intensive, and time-consuming. To overcome these limiting factors, in the present study, a colony multiplex PCR (cmPCR) method was developed and evaluated for the rapid detection of Bacillus and Pseudomonas. The technique amplifies the partial 16S rRNA gene of Bacillus and Pseudomonas with a product size of ~1,100 and ~375 bp, respectively, using single forward (BSF2) and two reverse primers (PAGSR and BK1R). Reliability of the cmPCR method was confirmed by screening 472 isolates obtained from ten different eco-stations, of which 133 isolates belonged to Bacillus and 32 to Pseudomonas. The cmPCR method also helped to identify six different Pseudomonas spp. and 14 different Bacillus spp. from environmental samples. Of the total 472 isolates studied, 46 showed antagonistic activity, among which 63 % were Bacillus and 17.4 % were Pseudomonas. Thus, the newly developed molecular approach provides a quick, sensitive, and potential screening tool to detect novel, antagonistically important Bacillus and Pseudomonas genera for their use in aquaculture. Further, it can also act as a taxonomic tool to understand the distribution of these genera from wide ecological niches and their exploitation for diverse biotechnological applications.
authors.affiliation: Marine Biotechnology Division, Central Marine Fisheries Research Institute (ICAR), Ernakulam North P.O, PB No: 1603, Kochi, 682018, Kerala, India.
authors.forename: Anusree V
authors.initials: AV
authors.lastname: Nair
authors.forename: M A
authors.initials: MA
authors.lastname: Pradeep
authors.forename: K K
authors.initials: KK
authors.lastname: Vijayan
mesh_terms: D000818:Animals; D001407:Bacillus / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D017753:Ecosystem; D057230:Limit of Detection; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D011549:Pseudomonas / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D015203:Reproducibility of Results
chemical_list: D012336:RNA, Ribosomal, 16S
references.citation: Appl Environ Microbiol. 1980 Jun;39(6):1212-22
references.pmid: 16345583
references.citation: J Gen Microbiol. 1984 Jul;130(7):1871-82
references.pmid: 6432953
references.citation: J Clin Microbiol. 2004 May;42(5):2074-9
references.pmid: 15131172
references.citation: World J Microbiol Biotechnol. 2012 Jul;28(7):2581-92
references.pmid: 22806164
references.citation: J Microbiol Methods. 2006 Jan;64(1):107-19
references.pmid: 15979744
references.citation: J Sci Food Agric. 2012 Oct;92(13):2652-6
references.pmid: 22555872
references.citation: Biotechnol Lett. 2011 Aug;33(8):1615-9
references.pmid: 21431847
references.citation: FEMS Microbiol Lett. 2005 Sep 1;250(1):55-62
references.pmid: 16039804
references.citation: Appl Microbiol Biotechnol. 2001 Sep;56(5-6):816-9
references.pmid: 11601635
references.citation: Biotechniques. 1992 Sep;13(3):350
references.pmid: 1389166
references.citation: Biotechniques. 1991 Jul;11(1):30-1
references.pmid: 1954013
references.citation: J Mol Biol. 1990 Oct 5;215(3):403-10
references.pmid: 2231712
references.citation: J Appl Microbiol. 2012 Apr;112(4):823-30
references.pmid: 22268759
references.citation: Microbiol Res. 2010 Sep 20;165(7):578-93
references.pmid: 20015629
references.citation: J Clin Microbiol. 2007 Sep;45(9):2761-4
references.pmid: 17626177
references.citation: Int J Syst Evol Microbiol. 2006 May;56(Pt 5):1031-1038
references.pmid: 16627650
references.citation: J Microbiol Methods. 2005 Apr;61(1):25-32
references.pmid: 15676193
references.citation: Curr Microbiol. 2007 Nov;55(5):409-12
references.pmid: 17680306
references.citation: J Appl Microbiol. 2001 Dec;91(6):1074-84
references.pmid: 11851816
references.citation: Int J Food Microbiol. 2009 Feb 28;129(3):282-7
references.pmid: 19157616
references.citation: Indian J Microbiol. 2010 Jun;50(2):229-32
references.pmid: 23100834
references.citation: J Clin Microbiol. 1999 Jun;37(6):1661-9
references.pmid: 10325304
references.citation: FEMS Microbiol Ecol. 2004 Jan 1;47(1):51-64
references.pmid: 19712346
references.citation: J Biotechnol. 1997 Jan 3;59(3):221-4
references.pmid: 9519482
references.citation: Dis Aquat Organ. 2011 Feb 22;93(3):199-206
references.pmid: 21516972
references.citation: Int J Food Microbiol. 2006 Mar 15;107(2):131-7
references.pmid: 16386323