Indian Journal of Experimental Biology Vol. 52, September 2014, pp. 917-920
Production of biocontrol traits by banana field fluorescent Pseudomonads and comparison with chemical fungicide S S Shaikh, P R Patel, S S Patel, S D Nikam, T U Rane & R Z Sayyed* Department of Microbiology, PSGVP Mandal' Arts, Science & Commerce College, Shahada, Dist. Nandurbar, 425 409, India Received 17 May 2013; Revised 4 June 2014 Pseudomonas aeruginosa isolated from banana field rhizosphere produced different antifungal metabolites like bactriocin, hydrogen cyanide and siderophore. Bacteriocinogenic, siderophoregenic, and HCN rich broth of isolate inhibited the growth of phytopathogen like Aspergilus niger, Aspergilus flavus, Fusarium oxysporum and Alternaria alternata. The isolate exhibited more antifungal activity and comparatively low MIC vis-a-vis commonly used copper based systemic chemical fungicide;bil cop. Keywords: Antifungal, Bactriocin, HCN, PGPR, Pseudomonas, Siderophore
The rhizosphere zone has been defined as the volume of soil directly influenced by the presence of living plant roots or soil compartment influenced by the root1. It supports large and active microbial population of rhizobacteria or plant growth promoting rhizobacteria (PGPR) that exert the beneficial effects on the growth of the host plant2,3. PGPR are known to enhance plant growth directly by a variety of mechanisms: fixation of atmospheric nitrogen, solubilization of phosphorus, production of siderophores, and synthesis of phytohormones. Indirect mechanisms involve the bio-control of phyto pathogens, through the production of antibiotics, lytic enzymes, hydrogen cyanide, and siderophore4. A siderophore (Greek = iron carrier) is a low molecular weight (500-1000 Da), compound secreted by organisms to chelate the available iron and thereby restrict iron nutrition of respective phytopathogens. This helps in preventing growth and root colonization by phytopathogens. Siderophore producing PGPR have been seen as a sustainable means of controlling phytopathogens. They offer numerous advantages over chemical fungicide5,6. PGPR also provide iron nutrition to roots thereby promote the plant growth7. The present work focuses on detection and estimation and production and evaluation of biocontrol traits like siderophore, antibiotics and HCN ___________ * Corresponding author Telephone: (02565) 229576 Telefax: (02565) 229576 E-mail:
[email protected];
[email protected],
produced by Pseudomonas sp. and their comparison with copper based systemic chemical fungicide; bil cop. Fluorescent Pseudomonas was isolated from rhizosphere of banana field from Jalgoan, India. The fungal cultures like Aspergilus niger NCIM 1025, A. flavus NCIM 650, Fusarium. oxysporum NCIM 1008, were procured from NCIM Pune, and A. alternata IARI 715 was procured from Indian Agricultural Research Institute (IARI), New Delhi, India. The bacterial cultures like Pseudomonas sp, Staphylococcus aureus, Bacillus subtilis and Proteus vulgaris were obtained from culture depository of Microbiology laboratory of M. J. College, Jalgaon. Copper based systemic fungicide; bil cop containing copper oxychloride was procured from Bharat Insecticide, New Delhi. Phenotypic fingerprinting was done for utilization of 71 carbon sources and 23 chemical sensitivity assays on GEN III microplates and by BIOLOG system (BIOLOG Microstation ™ system), employing tetrazolium redox dye to colorimetrically indicate utilization of carbon sources. The colour development was red between 4, 6, 16 and 24 h of incubation, in Microstation Reader and BIOLOG Microlog version 5.1.1 software. Phenotypic fingerprinting profiles were compared and identification acknowledged when similarity index was ≥ 0.5 with the nearest entry. Genomic DNA was isolated from pure cultures using HiPurA™ Plant Genomic DNA Miniprep Purification Spin Kit (Hi-Media)8. Amplification of
INDIAN J EXP BIOL, SEPTEMBER 2014
918
16S rRNA gene sequencing was performed using the primers fD1 (5′-AGAGTTTG ATCCTGGCTCAG-3′) and rP2 (3′-ACGGCTACCTTGTTACGACTT-5′). The nucleotide sequencing was done by using Big DyeR Terminator Cycle Sequencing and the sequences were analyzed with gapped BLASTn9 search algorithm. Phylogenetic analysis was performed by evolutionary distance and maximum likelihood with 1,000 bootstrap replicates. In order to screen siderophore production ability, fluorescent Pseudomonas was grown in sterile succinic acid medium (SAM)10 at 28±2 oC at 120 rpm for 24–48 h, followed by centrifugation at 10,000 rpm for 10 min. Siderophore in supernatant was detected by Chrome Azurol Sulphonate (CAS) assay6 and quantitatively estimated by CAS shuttle assay11. Optical density of sample (As) and reference (Ar) was measured at 630 nm to calculate the percent siderophore by using following formula; Siderophore (%) =
Ar − As ×100 Ar
Pseudomonas sp was grown in nutrient broth at 28±2 °C at 120 rpm for 24–48 h. The biomass was mixed with chloroform (v/v) and centrifuged at 5000 rpm for 20 min. The middle phase was collected and allowed to evaporate in water bath at 40 °C. The spectrum of antibacterial activity of the bacteriocinogenic preparation Pseudomonas sp was directed against pathogens like P. vulgaris, S. aureus, B. subtilis and beneficial rhizoflora like Ps. aeruginosa, A. vinelandii, R. meliotii, and B. japonicum by plate assay based on principle of diffusion12. Plates were incubated at 28 °C for 24-48 h and pattern of growth inhibition was determined with measurement of diameters zone of growth inhibition. HCN production and detection was carried out as per the method of Castric13 and was observed for colour change in HCN indicator paper from yellow to brownish.
In vitro phytopathogen suppression activity of siderophore and siderophoregenic culture preparations of Pseudomonas sp. was directed against A. niger NCIM 1025, A. flavus NCIM 650, F. oxysporum NCIM 1008, A. alternata IARI 71526. In vitro antifungal activity was based on the principle of diffusion. Inoculated plates after diffusion at 4 °C were incubated at 29±1 °C for 48 h and zone of growth was determined. In order to determine the MIC of the effective preparation, siderophore rich culture broth and cell free supernatant (20-100 µL) were individually added into PDA and NA previously seeded with fungal and bacterial pathogens respectively (one pathogen per plate). Inoculated plates after diffusion at 4 °C were incubated at 29±1 °C for 48 h and MIC was determined. For the purpose of studying the interaction of siderophore rich broth and bacteriocin rich broth of fluorescent Pseudomonas with useful soil rhizobia such as Ps. aeruginosa, A. vinelandii, R. meliotii, and B. japonicum, diffusion assay was performed as stated above, using nutrient agar for P. aeruginosa, Ashby’s mannitol agar for A. vinelandii and B. japonicum, and yeast extract mannitol agar for R. meliotii. The banana field isolate was Gram negative, motile, rod and fermented various sugars (Table 1). The DNAase, coagulase, catalase and oxidase negativity confirmed their non-pathogenic nature. It also showed a fluorescent pigment on King’s B medium; these characteristics very well resembled with the features of Pseudomonas sp. The comparison of the BLAST search of 16S rRNA gene sequences of isolate with the 16S rRNA gene sequences of the NCBI Gene Bank database showed 99.7% identity of the isolate to Pseudomonas aeruginosa. The phylogenetic tree based on 16S rRNA gene sequences of the isolate also formed a distinct group with Pseudomonas sp. hence the isolate was identified as Pseudomonas aeruginosa.
Table 1Preliminary identification of fluoroscent Pseudomonas sp. Characteristics/Result Gram Characteristics Motility Siderophore production HCN production Bacteriocin production
-ve = Negative, +ve = Positive
Utilization of carbohydrates - ve Motile + ve + ve + ve
Glucose Maltose Mannitol Lactose Xylose Arabinose
+ ve + ve - ve - ve + ve + ve
Enzyme production Catalase Oxidase Coagulase DNAse
- ve - ve - ve -ve
SHAIKH et al.: BIOCONTROL TRAITS BY BANANA FIELD FLUORESCENT PSEUDOMONADS
In shake flask studies change in colour of SAM from colourless to fluorescent green indicated siderophore production. Instant change in the colour of CAS reagent from blue to orange red due to the chelation of Fe2+ from HDTMA of CAS confirmed the presence of siderophore. The siderophore content of broth was 71% units. Similar results have been reported by Milagres et al14. In the preliminary shake flask studies bacteriocin production was noted as production of fluorescent green pigment in nutrient broth. Fluorescence under UV light and absorption spectra of extract within the 245-350 nm confirmed the presence of bacteriocin. Bacteriocinogenic preparation of fluorescent Pseudomonas aerugenosa inhibited growth of P. vulgaris, S. aureus and B. subtilis. The zone of growth inhibition of these pathogens was 28, 23 and 18 mm respectively while the growth of beneficial rhizobia was unaffected. Similar results have been recorded by Ivanova et al15. The modified King’s B agar plate inoculated with isolate, changed the color of HCN indicator paper
919
from initial yellow colour to brownish red. This may be due to the fact that HCN when reacts with picric acid solution amended on filter paper strip converts yellow colour of paper to brownish red13. Inhibition of all phytopathogens under test indicated the linear relation obtained with biomass of Ps. aeruginosa as antifungal activity. Based upon the degree of inhibition of mycelial growth; different phytopathogenic fungi showed varying sensitivity to the siderophore preparation of Ps. aeruginosa. A. flavus appeared as more sensitive phytopathogen as compared to the other fungal pathogens (Table 2a). Sayyed and Chincholkar6 have reported antifungal activity of siderophore producing A. feacalis. Saikia and Bezbruah16 have also reported antifungal activity of Azotobacter chrococcum RRL J203. Siderophore rich broth in less concentration 25 µL exerted more antifungal activity. This preparation was more potent inhibitor of fungal pathogens vis-a-vis chemical fungicide (Table 2b). More activity of broth indicated the role of other secondary metabolites in the growth inhibition of pathogenic fungi.
Table 2a Antifungal potential of Pseudomonas sp. vis-a`-vis chemical fungicide
Fungal sp. tested
Control (25 µL)
A. niger NCIM 1025 A. flavus NCIM 650 F. oxysporum NCIM 108 A. alternata IARI 715
– – – –
Diameter of zone of inhibition, (mm) Culture broth Culture supernatant (6 ×106 cells mL-1) (25 µL) (25 µL) 21.2 (0.66) 28.7 (0.76) 31.0 (0.95) 29.5 (0.29)
20.0 (0.71) 24.1 (0.81) 21.5 (0.07) 23.5 (0.07)
Bil cop (25 µg) 14.2 (0.03) 16.1 (0.05) 17.3 (0.01) 11.8 (0.03)
Each value is the average of three replicates. Numbers in parentheses are standard deviations. (—) No inhibition of fungal growth Table 2b MIC of siderophoregenic Pseudomonas sp. against common phytopathogenic fungi Antifungal preparations Culture Broth (µL)
Culture supernatant (µL)
Chemical fungicidebil cop (µg)
Amount 20 40 50 75 100 20 40 50 75 100 20 40 50 75 100
Diameter of zone of reduced growth (mm) A. niger A. flavus F oxysporum A. alternata 20.0 (0.71) 20.0 (0.71) 18.0 (0.60) 24.0 (0.12) 23.0 (0.74) 10.0 (0.74) 13.0 (0.67) 16.0 (0.71) 15.0 (0.91) 14.0 (0.71) 17.0 (0.37) 25.0 (0.12) 30.0 (0.39) 25.0 (0.52) 26.0 (0.71)
26.0 (0.71) 21.0 (0.07) 31.0 (0.61) 24.5 (0.54) 24.5 (0.31) 23.0 (0.74) 20.0 (0.71) 25.0 (0.12) 15.0 (0.37) 21.0 (0.07) 21.0 (0.07) 18.0 (0.60) 28.0 (0.57) 31.0 (0.61) 29.0 (0.60)
25.0 (0.12) 27.0 (0.64) 21.0 (0.07) 36.0 (0.71) 22.0 (0.56) 21.5 (0.07) 18.0 (0.60) 20.0 (0.07) 25.0 (0.12) 15.0 (0.91) 12.0 (0.61) 13.0 (0.67) 20.0 (0.71) 20.0 (0.71) 23.0 (0.74)
Each value is the average of three replicates. Numbers in parentheses indicate standard deviations
20.0 (0.70) 18.0 (0.60) 21.0 (0.07) 21.0 (0.07) 20.0 (0.70) 14.0 (0.71) 15.0 (0.37) 16.0 (0.71) 23.0 (0.74) 21.0 (0.07) 20.0 (0.70) 24.0 (0.12) 26.0 (0.71) 28.0 (0.57) 27.0 (0.39)
920
INDIAN J EXP BIOL, SEPTEMBER 2014
Plate assay revealed that none of the preparations of Pseudomonas aeroginosa inhibit the growth of useful soil rhizobia. Sayyed and Chincholkar6 have reported that A. fecalis has no inhibitory effect on useful soil rhizobia. Pseudomonas aeroginosa produced antifungal metabolites like siderophore, bacteriocin and HCN. Siderophore and HCN inhibite fungal phytopathogens while bacteriocin exerts toxic effect on bacteria. These metabolites did not inhibit the growth of any of the beneficial rhizobia. RZS is thankful to UGC New Delhi for financial support in the form of major research project. Reference 1 Atlas RM & Batrha R, Interaction between microorganism and plants, Microbial ecology: Fundamentals and applications, (Benjamin/Cummings, Menlo Park, CA.) 1998, 99. 2 Juanda JIH, Screening of soil bacteria for plant growth promoting activities in vitro, J Agri Sci, 4 (2005) 27. 3 Ahmad F, Ahmad I & Khan MS, Screening of free-living rhizospheric bacteria for their multiple growth promoting activities, Microbiol Res, 163 (2008) 173. 4 Sayyed RZ, Naphade BS & Chincholklar SB, Siderophore producing A. feacalis promoted the growth of Safed musali and Ashwagandha, J Med Aromat Plants, 29 (2007) 1. 5 Nehl DB, Allen SJ & Brown JF, Deleterious rhizosphere bacteria: an integrating prospective, Appl Soil Ecol, 5 (1996) 1. 6 Sayyed RZ & Chincholkar SB, Siderophore producing A. feacalis more biocontrol potential vis-a-vis chemical fungicide, Curr Microbiol, 58 (2009) 47–51
7 Sayyed RZ, Patel DC & Patel PR, Plant growth promoting potential of P solubilizing Pseudomonas sp. occuring in acidic soil of Jalgaon, Asian J Microbiol Biotechnol Environ Sci, 4 (2007) 925. 8 Sambrook J, Fritschm EF & Maniatis T, Molecular cloning: A laboratory manual. (Cold Spring Harbor Laboratory, New York) 1989. 9 http://www.ncbi.nlm.nih.gov 10 Meyer JM & Abdallah MA, The fluorescent pigments of Fluorescent pseudomonas: biosynthesis, purification and physicochemical properties, J Gen Microbiol, 107 (1978) 319. 11 Payne SM, Detection, isolation and characterization of siderophores, Methods in enzymology, Vol. 235, edited by Clark VL and Bovil PM (Academic Press, New York) 1994, 329. 12 Schillingger U & Lucke FK, Antibacterial activity of Lactobacillus sake isolated from meat, J Appl Bacteriol, 70 (1989) 473. 13 Castric PA, Hydrogen cyanide, a secondary metabolite of Psuedomonas aeruginosa. Can J Microbiol, 21 (1975) 613. 14 Milagres AMF, Machuca A & Napoleao D, Detection of siderophore production from several fungi and bacteria by a modification of chrome Azurol S (CAS) agar plate assay. J Microbiol Methods, 37 (1999) 1. 15 Ivanova I, Kabadjova P, Pantev A, Danova S & Dousset X, Detection, purification and partial characterization of a novel bacteriocin substance produced by lactoccous lactis subsp. lactis b14 isolated from boza-bulgarian traditional cereal beverage. Biocatalysis: Fundamentals and applications, Proceedings of the International Conference biocatalysis-2000: fundamentals & applications Moscow, Russia, 2000. 47. 16 Saikia N & Bezbruah B, Iron dependent plant pathogen inhibition through Azotobacter RRL J203 isolated from iron rich acid soil, Indian J Exp Biol, 35 (1995) 571.
