UDC 576.8
CHARACTERIZATION OF THE FUNGICIDAL ACTIVITY OF Calothrix elenkinii USING CHEMICAL METHODS AND MICROSCOPY
© 2012 C. Natarajan*, R. Prasanna*, V. Gupta*, P. Dureja**, and L. Nain*
*Division of Microbiology, Indian Agricultural Research Institute (IARI), New Delhi - 110012, India **Division of Agricultural Chemicals, Indian Agricultural Research Institute (IARI), New Delhi — 110012, India
e-mail: radhapr@gmail.com Received May 24.2011
An investigation was directed towards biochemical characterization of cyanobacterium Calothrix elenkinii and analysis of the chemical nature and mode of action of its fungicidal metabolite(s) against oomycete Pyth-ium debaryanum. Biochemical characterization of the culture in terms of carbohydrate utilization revealed the facultative nature of C. elenkinii. Unique antibiotic markers were also found for this strain. 16S rDNA sequencing of the strain revealed 98% similarity with Calothrix sp. PCC7101. The fungicidal activity was tested by disc diffusion assay of different fractions of the culture filtrate. A minimum inhibitory concentration of 10 pl was recorded for ethyl acetate fraction of the 7-weeks old culture filtrates. HPLC, followed by NMR spectral analysis demonstrated the presence of a substituted benzoic acid in the ethyl acetate fraction. Microscopic examination revealed distinct granulation, followed by disintegration of the hyphae of Pythium sp., indicating the presence of an active metabolite in the culture filtrates of Calothrix sp. The fungicidal activity of C. elenkinii can be attributed to the presence of 3-acetyl-2-hydroxy-6-methoxy-4-methyl benzoic acid. This is the first report of a benzoic acid derivative having fungicidal activity in cyanobacteria.
Cyanobacteria comprise the only coherent group of prokaryotes which proliferate both in eutrophic and oligotrophic type of marine and freshwater habitats. Besides these habitats, they are widespread in tropical/temperate rice fields, in which they play a significant role in the maintenance of soil fertility [1]. They have been identified as one of the most promising group of organisms, which produce a wide range of biologically active and novel secondary metabolites such as cyclic peptides, alkaloids and lipopolysaccharides [2—4]. The production of secondary metabolites gives a competitive advantage to these microorganisms for survival in diverse environmental conditions.
Research on biological control of plant pathogens has increased considerably over the past years due to public concern about the use of hazardous chemical pesticides. A number of cyanobacteria belonging to genera Anabaena, Nostoc, Microcystis exhibit algicid-al, antibacterial and antifungal properties [3, 5]. The antifungal cyclic peptides — laxaphycin A and B are known to be produced by Anabaena laxa [6]. Calothrix fusca was found to produce calophycin, a cyclic de-capeptide compound of 1248 Da molecular weight, exhibiting fungicidal activity [7].
In this study, we used Calothrix elenkinii strain which had shown fungicidal activity against Rhizocto-nia bataticola, Pythium aphanidermatum and Pythium debaryanum [8, 9]. The aim of the study is to investigate the mode of action and chemical nature of the fungicidal metabolite(s) of C. elenkinii.
MATERIALS AND METHODS
Strains and growth conditions. Calothrix elenkinii strain (CCC 124) was originally isolated from the rice fields of Indian Agricultural Research Institute (New Delhi, India) and tested for fungicidal activity against phytopathogenic fungus Pythium debaryanum ITCC 95 taken from Indian Type Culture Collection, Division of Plant Pathology, Indian Agricultural Research Institute. The cyanobacterial strain was axenized by standard procedures employing a set of antibiotics [10]. Cultivation of C. elenkinii was done in nitrogen free BG-11 medium [11] at 28 ± 2°C under light: dark cycle (16 : 8 h) and intensity of white light 5W m-2. The fungal culture was grown [8, 9], and maintained in potato dextrose agar at 28°C in BOD incubator (Col-to, India)
Characterization of C. elenkinii. The antibiotic sensitivity profile of C. elenkinii was generated by taking 5 ml of homogenized suspension of the axenic culture, mixing well with 0.8% top agar by vortexing and plated on the solidified bottom agar (1.2%) on Petri dish with diameter (9 mm). The agar plates were allowed to solidify and incubated under optimal conditions of light and temperature. After 5 days, 5 mm discs containing different antibiotics (HiMedia Laboratories, India) were placed on the cyanobacterial lawn and zones were recorded up to 10 days of incubation.
In order to characterize the strain, in terms of carbohydrate utilization pattern, 50 ^l of the cell suspension of the axenized late log phase (21 d) culture of C. elenkinii was inoculated in each well of HiCarbohy-
drate Kit (HiMedia Laboratories, India). Different sets were incubated in 16 : 8 light: dark cycle and continuous dark conditions (24 h) and colour changes were recorded according to manufacturer's instructions.
The amount of protein in pellet and filtrate of different age culture was determined spectrophotometri-cally with bovine serum albumin as standard [12].
16S rDNA analysis. DNA isolation was done using Plant Ultra Clean DNA isolation kit (MoBio, USA) and quantified by Alpha Imager Gel Documentation system 1220 (Alpha Innotech Corporation, USA). PCR amplification and agarose gel electrophoresis were performed according to standard procedures [13]. DNA ladders were purchased from MBI Fermentas (Burlington, Canada) and all PCR related chemicals were obtained from Bangalore Genei (India). PCR amplification of the 16S rDNA was performed by the same set of primers as used earlier [14— 16]. PCR amplification was done using 10 ng genomic DNA. PCR conditions consisted of initial denatur-ation at 94°C for 5 min, 30 cycles at 95°C (2 min), 42°C (30 s), and 72°C (2 min), plus one additional cycle with a final 10 min chain elongation. The single bands of desired amplicons were excised from the gels, purified using gel extraction kit (Qiagen, USA) and sequenced directly using the same set of primers as used for amplification. The CAP program was used for sequence assembly [17].
In silico analysis of16S rDNA sequences of C. elen-kiniiwas done by NCBI BLASTN program [18]. Multiple sequence analysis was made with available 16S rDNA sequences of different Calothrix species from the NCBI data base, using CLUSTALW software. The phylogenetic tree in the form of dendrogram was generated using the neighbor-joining method and maximum-parsimony algorithms as implemented in the program MEGA package version 4.1 [19].
Disc diffusion assay for antifungal activity. The cell pellet and filtrate of culture of different ages (1 to 7 weeks) were separated by centrifugation at 6.000 g for 10 min. The cells in pellet were broken down by sonication (at output 2 s pulse, 50% duty cycles, output control setting 8 for 2 min using Labsonic L.B. Braun Sonicator Biotech International, Germany) and used to evaluate fungicidal activity by disc diffusion assay [3]. The inhibition zone formed was evaluated as positive for antifungal activity and its diameter (in mm) was measured. The culture filtrates were also tested by the same procedure. The culture filtrates were then partitioned using ethyl acetate as solvent and both the phases were used for disc diffusion assay to determine the nature of solubility of the toxic compound.
Microscopy. Microscopic examination was also undertaken in order to investigate the mode of anti-fungal action of sample using phase contrast light microscope attached with Canon Power Shot S50 Digital Camera and Canon utilities Remote Capture Version
2.7.2.16 software (Japan). The lactophenol cotton blue stained hyphal specimens were observed at 40X magnification.
Minimum Inhibitory concentration (MIC). MIC
was determined in microtitre plates using 50 ^l obtained from 1 ml of filtrate generated using 10 mg dry biomass of organism. The samples included culture filtrates and the solvent and aqueous phases of 1 ml culture filtrate extracted with equal volume of ethyl acetate. Different dilutions of the filtrates and extracts were tested keeping the final volume as 50 p,l. Pythium debaryanum grown in PDA plates was removed and suspended in sterile water (100 mg of fungal hy-phae/ml sterile water) before use as inoculum. Microtitre plates were prepared with 200 ^l of PDA per well and inoculated with 50 ^l of homogenized fungal inoculum. After 1 day of growth at 28°C, 50 ^l of different age culture filtrate(s) of C. elenkinii were inoculated along with controls of ethyl acetate (50 ^l), nystatin (100 U/50 ^l) and sterile water (50 ^l). Inhibition was observed visually after 2 days of incubation and scored for calculation of MIC and IC50.
Chemical characterization of fungicidal compound.
The cell free filtrates (from 1—7 weeks old cultures of C. elenkinii) were analyzed using preparative thin layer chromatography (TLC). For partial purification of fungicidal compound, ethyl acetate fraction of cell free filtrate (from 6- and 7-weeks old culture) was condensed using Rotary vacuum film evaporator (Perfit, India) kept at 500 mm Hg at 45°C. The extract was dissolved in acetone and separated by TLC (Silica gel 60, Merck, USA) using different solvent systems containing benzene-acetone (45 : 5) and n-hexane-ben-zene (1 : 1). The different fractions obtained were elut-ed using acetone (90%), tested for fungicidal activity against Pythium sp. and purified.
Samples were also analyzed by HPLC (Varian Prostar, UK) with an RP18 reverse phase column (4.5 cm length) as stationary phase and methanol-water (80 : 20 v/v) as mobile phase maintained at a flow rate of 1 ml min-1 with the detector wavelength set at 270 nm.
The spectra were generated using 1H-NMR and 13C-NMR, in the Division of Agricultural Chemicals, IARI, (New Delhi, India). The proton nuclear magnetic resonance sp
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