Extraction, Partial Purification and Detection of Antimicrobial Metabolites Produced by the Rhizobacterial Strain UPMP3 of Pseudomonas aeruginosa and UPMB3 of Burkholderia cepacia and their Antagonistic Activity against Ganoderma boninense In vitro
Waheeda Parvin1,2*, Md Mahbubur Rahman1,3 and Mui Yun Wong2,4
1Bangladesh Forest Research Institute, Chattogram, Bangladesh
2Department of Plant Protection, Faculty of Agriculture, Universiti Putra Malaysia, Serdang, Malaysia
3Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Serdang, Malaysia
4Institute of Plantation Studies, Universiti Putra Malaysia, Serdang, Malaysia
*Corresponding Author: Waheeda Parvin, Silviculture Genetics Division, Bangladesh Forest Research Institute, Chattogram, Bangladesh.
Received: July 31, 2021; Published: August 13, 2021
Antimicrobial metabolites are produced as secondary metabolites by microorganism as well as the plant growth promoting rhizobacteria. These compounds are widely distributed in nature, where they play an important role in regulating the microbial population of soil, water, sewage and compost. In the present investigation, some antimicrobial metabolites such as antibiotics, siderophores, and HCN were detected in vitro by TLC, CAS agar universal test plates and filter paper with alkaline picrate solution method respectively from the two rhizobacterial strains Pseudomonas aeruginosa UPMP3 and Burkholderia cepacia UPMB3. In vitro bioassay was carried out through antagonistic activity test against G. boninense based on the percentage inhibition of radial growth (PIRG). The strains showed antifungal activity against Ganoderma boninense that is responsible for the disease of basal stem rot in oil palm. The crude extracts obtained from ethyl acetate solvent extraction and analyzed by thin layer chromatography (TLC). Six different antibiotics were detected with different retention factors (Rf) on TLC plates. The Rf values were calculated as 0.88, 0.77, 0.63, 0.53, 0.47, 0.28 and 0.23 for 2-4 DAPG, pyoluteorin, phenazine, pyocyanin, phenazine-1-carboxamide (PCN) and pyrrolnitrin successively developed with different solvent systems. Among the different solvent systems ethyl acetate: chloroform was most effective in separating the active bands from the extracts. Siderophores were detected through colour change into blue to orange and HCN was in dark brown to red colour. The antagonistic activity of Pseudomonas aeruginosa UPMP3 and Burkholderia cepacia UPMB3 was evaluated. In case of bacterial antibiotics and volatile and non-volatile effects, the strain P. aeruginosa UPMP3 showed the highest 94.21% and 51.16% inhibitory on the mycelial growth of G. boninense than B. cepacia UPMB3 (21.27% and 8.89%) compared to control treatment after 7 days of incubation respectively. The findings of this study indicate that these two rhizo-bacterial strains are capable to control Ganoderma boninense through producing antimicrobial metabolites.
Keywords: Purification; Detection; Antifungal Metabolites; Pseudomonas aeruginosa UPMP3; Burkholderia cepacia UPMB3; Antagonistic Activity; Ganoderma boninense, In Vitro
- De Souza JT., et al. “Frequency, diversity, and activity of 2,4-diacetylphloroglucinol-producing fluorescent Pseudomonas in Dutch take all decline soils”. Phytopathology 93.1 (2003): 54-63.
- Raaijmakers JM., et al. “Antibiotic production by bacterial bio control agents”. Antonie Van Leeuwenhoek International Journal1-4 (2002): 537- 547.
- Weller DM and Cook RJ. “Suppression of take-all of wheat by seed treatment with fluorescent pseudomonads”. Phytopathology 3 (1983): 463-469.
- Parvin W., et al. “Detection of phenazines from UPMP3 strain of Pseudomonas aeruginosa and its antagonistic effects against Ganoderma boninense”. International Journal of Agriculture and Biology 3 (2016): 483-488.
- Ownley BH., et al. “Identification and manipulation of soil properties to improve the biological control performance of phenazine-producing Pseudomonas fluorescens”. Applied and Environmental Microbiology6 (2003): 3333-3343.
- Petterson M and Baath E. “Effects of the properties of the bacterial community on pH adaptation during recolonization of a humous soil”. Soil Biology and Biochemistry9 (2004): 1383-1388.
- Picard C., et al. “Frequency and biodiversity of 2, 4- diacetylphloroglucinol -producing bacteria isolated from the maize rhizosphere at different stages of plant growth”. Applied and Environmental Microbiology3 (2000): 948-955.
- Lodewyckx CJ., et al. “Endophytic bacteria and their potential applications”. Critical Reviews in Plant Sciences 6 (2002): 583-606.
- Hernandez ME., et al. “Phenazines and other redox-active antibiotics promote microbial mineral reduction”. Applied and Environmental Microbiology2 (2004): 921-928.
- Duijff BJ., et al. “Siderophore mediated competition for iron and induced resistance in the suppression of Fusarium wilt of carnation by Fluorescent Pseudomonas spp”. Netherlands Journal of Plant Pathology9 (1993): 277-289.
- Siddiqui IA., et al. “Role of cyanide production by Pseudomonas fluorescens CHAO in the suppression of root-knot nematode, Meloidogyne javanica in tomato”. World Journal of Microbiology and Biotechnology3 (2006): 641-650.
- Ahmad F., et al. “Screening of free-living rhizospheric bacteria for their multiple plant growth promoting activities”. Microbiological Research2 (2008): 173-181.
- Dowling DN and O’gara F. “Metabolites of Pseudomonas involved in the bio control of plant disease”. Trends in Biotechnology4 (1994): 133-141.
- Zaiton S., et al. “Isolation and Characterization of Microbial Endophytes from Oil Palm Roots: Implication as Biocontrol Agents against Ganoderma”. The Planter966 (2006): 587-597.
- Azadeh BF. and Sariah M. “Molecular characterization of Pseudomonas aeruginosa UPMP3 from oil palm rhizosphere”. American Journal of Applied Sciences11 (2009): 1915 -1919.
- Bernal G., et al. “Isolation and partial purification of a metabolite from a mutant strain of Bacillus with antibiotic activity against plant pathogenic agents”. Electronic Journal of Biotechnology 5.1 (2002): 01-09.
- Schwyn B and Neilands JB. “Universal Chemical Assay for the Detection and Determination of Siderophores”. Analytical Biochemistry1 (1987): 47-56.
- Reddy BP., et al. “Efficacy of antimicrobial metabolites of Pseudomonas fluorescens against rice fungal pathogens”. Current Trends in Biotechnology and Pharmacy1 (2008): 178-182.
- Montealegro JR., et al. “Selection of bio antagonistic bacteria to be used in biological control of Rhizoctonia solani in tomato”. Electronic Journal of Biotechnology2 (2003): 115-127.
- El-Sayed W., et al. “Isolation and Identification of Phenazine-1-Carboxylic acid from different Pesudomonas isolates and its Biological activity against Alternaria solani”. Research Journal of Agriculture and Biological Sciences6 (2008): 892-901.
- Rosales AM., et al. “Isolation and identification of antifungal metabolites produced by rice associated antagonistic Pseudomonas spp”. Phytopathology9 (1995): 1028-1032.
- Saosoong K., et al. “Isolation and Analysis of Antibacterial Substance Produced from aeruginosa TISTR 781”. Khon Kaen University Science Journal 37.2 (2009): 163-172.
- Thomas FC., et al. “Introduction of the phzH Gene of Pseudomonas chlororaphis PCL1391 Extends the Range of Biocontrol Ability of Phenazine-1-Carboxylic Acid-Producing Pseudomonas Strains”. Molecular Plant-Microbe Interactions 14.8 (2001): 1006-1015.
- Chang PC and Blackwood AC. “Simultaneous production of three phenazine pigments by Pseudomonas aeruginosa Mac 436”. Canadian Journal of Microbiology 5 (1969): 439-444.
- Hassanein WA., et al. “Characterization and antagonistic activates of Metabolite produced by aeruginosa Sha8”. Journal of Applied Sciences Research 5.4 (2009): 392-403.
- Keel C., et al. “Suppression of Root Diseases by Pseudomonas fluorescens CHA0: Importance of the Bacterial Secondary Metabolite 2,4-Diacetylphloroglucinol”. Molecular Plant-Microbe Interactions1 (1992): 4-13.
- Dikin A., et al. “Extraction of antimicrobial substances from antagonistic bacteria against Schizophyllum commune” In: Proceedings, 27th Symposium of the Malaysian Society for Microbiology, 24-27 November 2005. Grand Plaza Parkroyal, Penang, Malaysia. (2005).
- Hwang J., et al. “Pyrrolnitrin production by Burkholderia cepacia and biocontrol of rhizoctonia stem rot of poinsettia”. Biological Control1 (2002): 56-63.
- Lee CH., et al. “Cepacidine A, a novel antifungal antibiotic produced by Pseudomonas cepacia. I. Taxonomy, production, isolation and biological activity”. The Journal of Antibiotics12 (1994): 1402-1405.
- Sajeed SA and Vidhale NN. “Antagonistic Activity of Siderophore Producing Pseudomonas aeruginosa against Aspergillus and Candida albicans”. Research Journal of Pharmaceutical, Biological and Chemical Sciences 3.4 (2012): 719-726.
- Katy Dı´az P., et al. “Production of phytohormones, siderophores and population fluctuation of two root-promoting rhizobacteria in Eucalyptus globulus cuttings”. World Journal of Microbiology and Biotechnology 5 (2012): 2003-2014.
- Martha P., et al. “Siderophore Producing Pseudomonas as Pathogenic Rhizoctonia solani and Botrytis cinerea Antagonists”. Universitas Scientiarum1 (2005): 65-74.
- Hu Q-P., et al. “Isolation and identification of a potential bio control agent Bacillus subtilis QM3 from Qinghai yak dung in China”. World Journal of Microbiology and Biotechnology11 (2008): 2451-2458.
- Castric PA. “Glycine Metabolism by Pseudomonas aeruginosa: Hydrogen Cyanide Biosynthesis”. The Journal of Bacteriology2 (1977): 826-831.
- Rakh RR., et al. “Biological Control of Sclerotium rolfsii, Causing Stem Rot of Groundnut by Pseudomonas cf. monteilii 9”. Recent Research in Science and Technology3 (2011): 26-34.
- Bergsma-Vlami M., et al. “Assessment of Genotypic Diversity of Antibiotic-Producing Pseudomonas Species in the Rhizosphere by Denaturing Gradient Gel Electrophoresis”. Applied and Environmental Microbiology2 (2005): 993-1003.
- Stockwell VO., et al. “Antibiosis contributes to biological control of fire blight by Pantoea agglomerans strain Eh252 in orchards”. Phytopathology11 (2002): 1202-1209.
- Heungens K and Parke JL. “Post infection biological control of oomycete pathogens of pea by Burkholderia cepacia AMMDR1”. Phytopathology4 (2001): 383-391.
- Singh A., et al. “Biocontrol of collar rot disease of betelvine (Piper betle) caused by Sclerotium rolfsii by using rhizosphere-competent Pseudomonas fluorescens NBRI-N6 and P. fluorescens NBRI-N”. Current Microbiology 47.2 (2003): 153-158.
- De La Fuente L., et al. “Pseudomonas fluorescens UP61 isolated from birdsfoot trefoil rhizosphere produces multiple antibiotics and exerts a broad spectrum of biocontrol activity”. European Journal of Plant Pathology7 (2004): 671-681.
- Manwar AV., et al. “Siderophore production by a marine Pseudomonas aeruginosa and its antagonistic action against phytopathogenic fungi”. Applied Biochemistry and Biotechnology 1-3 (2004): 243-252.
- Dikin A., et al. “Mode of action of antimicrobial substances from Burkholderia multivorans and Microbacterium testaceum against Schizophyllum commune Fr”. International Journal of Agriculture and Biology2 (2007): 311-314.
- Ramette A., et al. “Prevalence of fluorescent pseudomonads producing antifungal phloroglucinols and/or hydrogen cyanide in soils naturally suppressive or conducive to tobacco root rot”. FEMS Microbiology Ecology1 (2003): 35-43.
- Voisard C., et al. “Cyanide production by Pseudomonas fluorescens helps suppress black root rot of tobacco under gnotobiotic conditions”. The EMBO Journal2 (1989): 351-358.
- Archibold DD., et al. “Identifying natural volatile compounds that control gray mold (Botrytis cinerea) during postharvest storage of strawberry, blackberry and grape”. Journal of Agricultural and Food Chemistry10 (1997): 4032-4037.
- Andersen RA., et al. “Structure-antifungal activity relationships among C6 and C9 aliphatic aldehydes, ketones and alcohols”. Journal of Agricultural and Food Chemistry7 (1994): 1563-1568.
- Fernando WGD and Linderman RG. “Inhibition of Phytophthora vignae and root rot of cowpea by soil bacteria”. Biological Agriculture and Horticulture1 (1994): 1-14.
- Rahman MA., et al. “Screening of antagonistic bacteria for biocontrol activities on Colletotrichum gloeosporioides in papaya”. Asian Journal of Plant Sciences 1 (2007): 12-20.
- Diby P., et al. “Antagonistic mechanisms of fluorescent Pseudomonas against Phytophthora capsici Leonian in black pepper (Piper nigrum)”. Journal of Spices and Aromatic Crops 14.2 (2005): 94-101.
- Watanabe T., et al. “Potential of antago-nistic isolate obtained from Lentinus lepideus basidiospores as a biocontrol agent”. Mycoscience 1 (2000): 79-82.