Optimization and production of salicylic acid by rhizobacterial strain Bacillus licheniformis MML2501

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1 ISPUB.COM The Internet Journal of Microbiology Volume 6 Number 1 Optimization and production of salicylic acid by rhizobacterial strain Bacillus licheniformis P Shanmugam, M Narayanasamy Citation P Shanmugam, M Narayanasamy. Optimization and production of salicylic acid by rhizobacterial strain Bacillus licheniformis. The Internet Journal of Microbiology Volume 6 Number 1. Abstract The Rhizobacterial isolate Bacillus licheniformis effectively inhibited the mycelial growth of fungal pathogens viz., Macrophomina phaseolina, Fusarium udum, Fusarium oxysporum, Bipolaris oryzae, Pyricularia oryzae, Alternaria alternata and Curvularia lunata. Bacillus licheniformis was tolerant to heavy metals such as zinc, cobalt and selenium. B. licheniformis was tolerant to fungicides such as propiconazole and tridemorph and showed moderate tolerance to tricyclazole and it was showed resistant to antibiotics such as ampicillin, streptomycin, bacitracin, cephalotitin, erythromycin and oxytetracycline. Bacillus licheniformis produced salicylic acid (SA), with a maximum yield of 18 µg/ml in optimized culture conditions such as ph 7.0, temperature 30 C, casaminoacids at a concentration of 0.4 % and at 200 rpm shaken condition. SA produced by Bacillus licheniformis was further confirmed by TLC and HPLC analyses, in which the Rf value and retention time of 0.61 and 5.24 min, respectively were matching with that of authentic SA. Therefore the present study suggests that the rhizobacterial strain Bacillus licheniformis has merits to be a beneficial bacteria for the crop protection through systemic resistance. INTRODUCTION Plants actively respond to a variety of chemical stimuli produced by soil- and plant-associated microbes. Such stimuli can either induce or condition plant host defenses through biochemical changes that enhance resistance against subsequent infection by a variety of pathogens. The important criteria for a good biocontrol strains is to show tolerance to heavy metals, fungicide and antibiotics (Brian, 2007) and thus the resistance and sensitive patterns of the bio control strain Bacillus licheniformis towards heavy metals, fungicides and antibiotics were tested. Bacterial strains isolated from the rhizosphere hold great promise as seed inoculants in innovative agriculture to induce resistance and reduce various plant diseases. Some biocontrol strains of Pseudomonas spp., Bacillus spp. and Trichoderma spp. are known to strongly induce plant host defenses (Harman, 2004; Haas and Defago, 2005). Nowadays most research and development efforts have focused towards the genus Bacillus (Brain et al., 2004). Induction of host resistance pathways, locally and systemically has been reported for some isolates of B. amyloliquefaciens, B. cereus, B. mycoides, B. pumilus, B. sphaericus and B. subtilis (Kloepper et al., 2004). Zhang et al. (2002) reported that plants treated with B. pumilus strain Se34 had greatly increased levels of salicylic acid, compared with that of non-treated plants or plants treated with two gram-negative bacteria. Salicylic acid (SA) is an essential signal elicitor for the induction of Induced systemic resistance (ISR) and the orchestration of the events that occur during the HR (Carl et al, 2005). With this context Indhiragandhi et al, (2008) reported that Acinetobacter sp., Pseudomonas sp.and Serratia sp. shows production of salicylic acid, which is important component in the induction of defense in plants. Thus in this study it will be worthwhile, to review the efficacy of Bacillus licheniformis as the producer of salicylic acid in optimized condition. MATERIALS AND METHODS ANTAGONISTIC BACTERIUM The isolate was obtained from the culture collection of the Biocontrol and Microbial Metabolites Lab (MML), Centre for Advanced Studies in Botany, University of Madras, Chennai. The isolate was originally isolated from the groundnut rhizosphere soil. 1 of 8

2 MAINTENANCE OF CULTURES Fungal pathogens were maintained on potato dextrose agar (PDA) and the isolate was maintained on nutrient agar (NA). The bacterium was also preserved at -20 C as glycerol (15%) stocks. 16S RDNA SEQUENCING The Bacillus sp. was identified based on the 16S rdna sequence homology ANTAGONISTIC ACTIVITY OF AGAINST PHYTOPATHOGENS The isolate Bacillus licheniformis was screened for antagonistic activity against the following phytopathogens: Fusarium oxysporum, Fusarium udum, Rhizoctonia solani, Macrophomina phaseolina, Pyricularia oryzae, Alternaria alternata and Curvularia lunata on PDA by dual culture technique. The mycelial discs measuring 9 mm were cut out from 4 days old culture of fungal phytopathogens pre-cultured on PDA plate and inoculated at the center of a fresh petriplate containing PDA. The isolate was streaked in the periphery of each petriplate and incubated at room temperature. The growth of fungal phytopathogens was observed periodically and the growth inhibition was measured after 6 days of incubation. ANTIBIOTIC SENSITIVITY/RESISTANCE PATTERN FOR The Mueller-Hinton agar (Hi Media) was prepared as per the manufacturer instructions, poured into petriplates and allowed to solidify. B. licheniformis was inoculated evenly on the surface of the medium by swabbing the culture and kept for 2-3 min for the agar surface to dry. Then a disc containing 14 antibiotics (Hi-media) with specific concentration was placed on the surface of the culture with the help of a sterile forceps and slightly pressed so that the disc adhered to the surface of the agar. Plates with out antibiotic discs served as control. The plates were incubated at room temperature for 4 days and the zone of inhibition around each antibiotic disc was observed, measured to determine the resistance and sensitivity of B. licheniformis. HEAVY METAL TOLERANCE SPECTRUM FOR The tolerance of B. licheniformis to various heavy metals was studied as follow. A loop full of overnight grown culture was streaked on Nutrient agar plate amended with various concentrations (2 10 mm) of heavy metals 2 of 8 viz., cobalt, zinc, magnesium, iron, copper, mercury, silver, cadmium, arsenic, lead and selenium. The plates were incubated at room temperature for 24 h and observed for the growth of B. licheniformis. The MSM plates without heavy metals served as controls. FUNGICIDE TOLERANCE SPECTRUM OF The tolerance of B. licheniformis to various commercial fungicides was studied as follow. A loop full of (24hr) grown culture was streaked on Nutrient agar plate amended with various concentrations ( ppm) of commercial fungicides viz., propiconazole, tridemorph, carbendazim, mancozeb, hexaconazole and tricyclazole. The plates were incubated at room temperature for 48 h and observed for the growth of B. licheniformis. The Nutrient agar plates without fungicide served as controls. DETECTION OF SALICYLIC ACID (SA) PRODUCTION BY (VISCA., 1993) The efficiency of B. licheniformis to produce SA was tested in casamino acids medium with different concentrations of casamino acids ( %) at different ph (3-10), temperature (20-55 C) and in static and shaken conditions. The broth cultures were incubated for 4 days and cells were separated by centrifugation at 8000 rpm for 10 min. All the harvesting procedures were carried out in dim light with samples maintained in covered ice baths. QUANTIFICATION OF SA The SA was extracted from the acidified culture supernatant. The filtrate was adjusted to ph 2 with 1M HCl and extracted twice with double volume of ethyl acetate. To 1 ml of the extraction, 2 ml of 2 M FeCl3 and 1 ml of distilled water were added. The SA reacts with 2 M FeCl3 to form a purple iron + SA complex in the aqueous phase with a maximum absorbance at 527 nm. The difference in absorbance was recorded at a Beckman DU40 spectrophotometer. The SA concentration in extraction was determined using a calibration curve of standard SA. TLC ANALYSIS OF SA The extracted SA was evaporated to dryness in a flash evaporator, solubilized in a minimal volume of methanol and spotted on pre coated silica gel plates. Then the plates were developed in a solvent system consisting of chloroform:acetic acid:ethanol at the ratio of 95:5:2.5 (v/v). The plates were viewed in blue fluorescence emission under UV light (256 nm) immediately after removal from the

3 developing chamber. The SA was detected by observing a UV reflected band with an Rf value corresponding to that of the standard SA. Figure 1 Table 1: Antagonistic activity of against plant pathogens HPLC ANALYSIS OF SA The HPLC system used was having C18 column (Gemini) with a mobile phase, 2.3 g monobasic ammonium phosphate in 850 ml of water, 150 ml acetonitrile, 1 g sodium pentane sulphonate salt with ph adjustment 2.5 using phosphoric acid. The configuration of system is Shimadzu prominence. The flow rate is 1 µl and the injecting volume is 20 µl. For each sample, the peak heights corresponding to the retention times of SA were measured on chromatographs from detector, and the F/FC ratio was also calculated. The ratios from each of three replications of each culture filtrate extraction were compared to an SA standard. RESULTS IDENTIFICATION OF THE BACTERIA According to the 16S rdna sequencing analysis the Bacillus sp. was identified as Bacillus licheniformis and designated as. The 16S rdna sequence of B. licheniformis generated with 1549 bases was deposited in the GenBank with the accession number of EU and the culture was deposited in IMTECH and the deposition number is ANTAGONISTIC ACTIVITY OF AGAINST FUNGAL PLANT PATHOGENS Screening of against a wide range of plant pathogens revealed that it effectively inhibited the mycelial growth of Macrophomina phaseolina, Fusarium oxysporum, Fusarium udum, Bipolaris oryzae, Pyricularia oryzae, Alternaria alternata and Curvularia lunata. The maximum zone of inhibition (ZOI) was observed against Bipolaris oryzae (2.2 cm) followed by Macrophomina. phaseolina (2.0 cm). However In other pathogens, the ZOI ranged from 1.0 cm to 1.9 cm (Table 1). 3 of 8 Values are mean of three replicates. In a column, values followed by the same letter are not significantly different at P = ANTIBIOTIC SENSITIVITY/RESISTANCE PATTERN OF B. licheniformis exhibited resistance to ampicillin, streptomycin, bacitracin, cephalotitin, erythromycin and oxytetracycline. It showed sensitivity to remaining 14 antibiotics (Table 2).

4 Figure 2 Table 2: Antibiotic sensitivity/resistance pattern of tolerance to tricyclazole and slight tolerance to carbendazim, hexaconazole and mancozeb (Table 4). Figure 4 Table 4: Fungicide tolerance spectrum of DETECTION OF SALICYLIC ACID PRODUCTION BY The optimal conditions for the SA production by B. licheniformis were standardized. STATIC/SHAKEN CONDITIONS HEAVY METAL TOLERANCE SPECTRUM OF B. licheniformis exhibited good tolerance to zinc, cobalt and selenium and moderate tolerance to manganese, cadmium and silver. It showed slight tolerance to lead and sensitivity to iron, mercury and arsenic (Table 3). Among the static and shaken conditions, culturing B. licheniformis at 100 rpm was found to be optimum for the production of SA (14 µg/ml) on the 2 nd day of incubation (Fig. 1). Figure 5 Figure 3 Table 3: Heavy metal tolerance spectrum of Among different ph tested, the ph 7.0 favoured the maximum SA production of 16 g/ml as against 7-12 g/ml in rests of the ph (Fig. 2). FUNGICIDE TOLERANCE SPECTRUM OF B. licheniformis exhibited good tolerance to propiconazole and tridemorph. It exhibited moderate 4 of 8

5 Figure 6 Figure 8 TEMPERATURE Among different temperature tested, B. licheniformis produced maximum SA of 17 g/ml at 30 C. The SA production in rest of the temperature ranged between 5 g/ml and 15 g/ml. It has been observed that in both the lower and higher temperature (20, 25, 45 C and 50 C), there was no SA production (Fig. 3). Figure 7 DETERMINATION OF SA PRODUCTION BY THIN LAYER CHROMATOGRAPHY The SA produced by the B. licheniformis was confirmed by the blue bands that appeared on pre-coated silica gel viewed under UV illumination. The Rf value of SA (0.61) produced by B. licheniformis was matched with the Rf value (0.61) of the authentic SA (Fig. 5). Figure 9 CONCENTRATION OF CASAMINO ACIDS Different concentrations of casamino acids as a substrate ranging from % was tested for the production of SA, in which 0.4% was found to be optimum for the production of SA with 18 µg/ml on the 2 nd day of incubation (Fig. 4). DETERMINATION OF SA PRODUCTION BY HPLC The extracted SA from the culture filtrate of B. licheniformis showed similar peak to that of the authentic SA peak in HPLC analysis. The retention time of the extracted SA is 5.24 min (Fig. 6) as that of the authentic SA (Fig. 7). 5 of 8

6 Figure 10 DISCUSSION In the present study, emphasis was laid on a rhizobacterial Bacillus strain as it is resistance to adverse conditions and can withstand in soil for long period to give sustainable crop protection against pathogens. The spore forming nature of Bacillus and its sustainable crop protection in agriculture is well documented (Leclere et al., 2005). With this background, the antagonistic potential of the isolate was tested in dual plate in which the bacteria effectively inhibited both soil-borne as well as foliar pathogens and showed good activity against Macrophomina phaseolina, Bipolaris oryzae, Pyricularia oryzae and considerable activity against Curvularia lunata, Fusarium oxysporum, and Alternaria alternata in dual plate culture. Many rhizosphere Bacillus strain such as Bacillus subtilis D1/2, CBE4, PRBS1, Bacillus amyloliquefaciens FZB42 (Chan et al., 2003; Kavitha et al., 2005, Fernando et al., 2005; Koumoutsi et al., 2004) showed antagonistic activity against different fungal phytopathogens. The isolate was further characterized for its antibiotic resistance and sensitive pattern, heavy metal and fungicide tolerance. Resistance to different antibiotics, heavy metal and fungicides attribute better survivability of a strain in the natural environment, where the use of heavy metal containing fungicides are in agricultural field practice. The results of experiments to determine the tolerance spectrum revealed that the strain B. lichenformis is resistant to Ampillicin, Streptomycin, Bacitracin, Cephalotitin, Erythromycin and Oxytetracycline antibiotics. Similarly, it showed good tolerance to the heavy metals, Zinc, Cobalt and Selenium and the fungicides, Propiconazole and Tridemorph. The above findings show that the possibility of using B. lichenformis in a xenobiotic environment is high owing to its tolerance to antibiotics, heavy metals and fungicides. It was reported that the B. subtilis relatively tolerant to the killing and lytic effects of a cell wall antibiotic (Jolliffe et al., 1982). Yilmaz (2003) reported that the Bacillus circulans is very much 6 of 8 tolerant to many heavy metals and antibiotics. Picket and Dean (1979) reported that a strain of B. subtilis was tolerant to cadmium and zinc. Bacillus-based biocontrol agents BAC J and BAC B were tolerant to 10 ppm of propiconazole and in field plots, BAC J alone or in combination with the fungicides propiconazole, benzimidazole, azoxystrobin and tetraconazole were used for fungal diseases (Jacobsen, 2000). Apart from the production of secondary metabolites, induced systemic resistance in plants by rhizobacteria may also be attributed to the diseases suppression. ISR mediation through salicylic acid is already well established (Van loon et al., 1998). Enhancement of induced disease resistance by salicylic acid dependent pathways against bacterial pathogen was carried out in Arabidopsis thaliana (Van Wees et al 2000). In order to determine the ability of the B. licheniformis for the production of salicylic acid, experiments were conducted under in vitro and in vivo conditions. Under optimal ph, temperature, concentration of substrate and shaken conditions, B. licheniformis showed maximum production of 18 µg/ml of SA, which is important component in the induction of plant mediated defense enzymes. Similarly salicylic acid has been found synthesized in the culture supernatants of Pseudomonas aeruginosa and Pseudomonas cepacia (Viska et al., 1993). Indiragandhi et al (2008) also reported that Serratia sp. PSGB13, Acinetobacter sp. PRGB16 and Pseudomonas sp. PRGB06 produces extra cellular salicylic acid with the concentration of 10.0 ± 0.7, 7.2 ± 0.6 and 6.8 ± 0.4 g/ml respectively. Meyer and Hofte (1997) stated that some plant growth promoting bacteria (PGPB) do trigger a salicylic acid dependent signaling pathway by producing small amount of salicylic acid in rhizosphere. The present study indicated that the rhizobacterial strain Bacillus sp. as a high salicylic acid producing organism, has the merits to be explored for its ISR mediated defense induction in agricultural crops and can also be used in the xenobiotic environment. References 1. Brain, B. Gardener, M. and Driks, A. (2004). Overview of the nature and application of biocontrol microbes: Bacillus sp. Phytopathology. 94, Brian. Denton (2007). Advances in Phytoremediation of Heavy Metals Using Plant Growth Promoting Bacteria and Fungi. MMG 445 Basic Biotechnology. 3, Carl N. Mayers, Kian-Chung Lee, Catherine A. Moore, Sek-Man Wong and John P. Carr. (2005). Salicylic AcidInduced Resistance to Cucumber mosaic virus in Squash and Arabidopsis thaliana: Contrasting Mechanisms of Induction and Antiviral Action. 18(5), Chan, YK. McCormick, WA. and Seifert, KA. (2003). Characterization of an antifungal soil bacterium and its antagonistic activities against Fusarium species. Can. J.

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8 Author Information Prashanth Shanmugam, Ph.D. Bio control and Microbial Metabolite Lab, Centre for Advanced Studies in Botany, University of Madras Mathivanan Narayanasamy, Ph.D. Bio control and Microbial Metabolite Lab, Centre for Advanced Studies in Botany, University of Madras 8 of 8