Effect of Azotobacter Chroococcum CL13 Inoculation on Growth and Curcumin Content of Turmeric (Curcuma Longa L.)
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Int.J.Curr.Microbiol.App.Sci (2014) 3(9) 275-283 ISSN: 2319-7706 Volume 3 Number 9 (2014) pp. 275-283 http://www.ijcmas.com Original Research Article Effect of Azotobacter chroococcum CL13 inoculation on growth and curcumin content of turmeric (Curcuma longa L.) Ajay Kumar, Ritu Singh, Deen Dayal Giri, Pawan Kumar Singh and Kapil D. Pandey* Centre of Advance study in Botany, Banaras Hindu University, Varanasi -221005, India *Corresponding author A B S T R A C T In the present study, Azotobacter chroococcum CL13 were isolated from the K e y w o r d s rhizospheric soil of turmeric and identified morphologically biochemically and through 16 S r RNA gene sequence analysis. The isolate significantly produced Turmeric, IAA, NH3 HCN and solubilized tricalcium phosphate during PGP trait analysis. Curcumin, The isolate used as inoculants in rhizome prior to sowing following standard Plant growth procedures. After inoculation plant growth parameters such as shoot height, shoot Promotion, fresh biomass, rhizome fresh biomass enhanced in A.chroococcum CL13 Bacterial inoculated turmeric than control (uninoculated). Curcumin is the most active and inoculation, important constituents of turmeric having various pharmacological activity. The Yields concentration of curcumin had been significantly enhanced by 6% in A. chroococcum CL13 inoculated turmeric. Introduction The turmeric, a rhizotomous herb of family chemo preventive agent in several cancer Zingibiraceae, is used as a spice, coloring (Rao et al., 1995). The rhizomes of turmeric agent and traditional medicine from the contain numbers of biochemical ingredients ancient time in South Asian and Middle like curcuminoids and sesquiterpenoids. For Eastern countries. Plant is used as a herbal the separation and quantification of medicine for the treatment of asthma curcuminoids a variety of methods have bronchial hyperactivity, rheumatism, been reported in the literature. He et al., diabetic wounds sinusitis, smallpox, skin (1988) reported online HPLC UV diode cancer, menstrual difficulties and abdominal array and electro spray mass spectrometer pain (Ammon and Wahl, 1990). The major methods to analyze curcuminoids in fresh curcuminoids, curcumin exhibited various turmeric extracts. Jayaprakasha et al., (2002) biological effects such as anti-inflammatory, reported the improved HPLC method for antioxidant, antimalerial, hypolipidemic the separation and quantification of activities. (Tonnesen, 1992; Reddy and curcumin demethoxycurcumin and Lokesh, 1992) and extensively studied as a bisdemethoxycurcumin in the turmeric. 275 Int.J.Curr.Microbiol.App.Sci (2014) 3(9) 275-283 Plant growth promoting rhizobacteria Materials and Methods (PGPR) is a group of free-living rhizosphere bacteria that enhances plant growth by Isolation and maintenance of bacterial performing activity of biofertilizers, isolates biopesticides or bio control agents. The most commonly found PGPR Pseudomonas Bacterial isolates were isolated from the fluorescence, Bacillus, Azotobacter, rhizospheric soil using standard Azospirillum and Klebsiella. PGPR facilitate microbiological techniques from healthy and the plant growth promotion directly by young turmeric (curcuma longa) growing in nutrient solubilization, nitrogen fixation and the Botanical Garden of Banaras Hindu by producing growth regulators and University, India. (200 18 N and 80° 36 E, antibiotics (Lucy et al., 2004), where as they elevation 80.71m). indirectly involved in production of hydrogen cyanide, siderophores, competitive Powdered (1g) rhizospheric soil was exclusion of pathogens and removal of suspended in 9.5 ml of sterilized distilled phototoxic substances produced by water and shaken on gravatory shaker for deleterious microorganisms. PGPR 1h. 0.1 ml of serially diluted soil suspension contribute to sustainable agriculture by inoculated on N free glucose medium diminishing the use of chemical pesticides, (Norris and Chapman, 1968) for the chemical fertilizer and also by protecting isolation of Azotobacter sp. Bacterial human health (Adesemoye and Kloepper isolates were selected and identified 2009). Recently PGPR have numerous according to Bergey s manual of systematic biotechnological applications in agriculture, bacteriology (Garrity, 2005). On the basis of horticulture, forestry and environmental morphological, biochemical screening protection (Zahir et al., 2004). Azotobacter isolates were selected and further identified is regarded as free living aerobic N2 fixer by 16s r RNA gene sequence analysis. present in soil. Besides nitrogen fixation (Kumar et al., 2006), the sequence was they also synthesize and secretes analyzed and queried with the BLAST considerable amount of phytohormones to enhance the plants growth and pathogenic Plant growth promoting (PGP) traits of diseases tolerance (Van Loon, 2007). bacterial isolates In recently past Azotobacter broadly used as Azotobacter isolates were characterized for a soil or plant inoculants in agronomic field plant growth promoting properties including trails for the diseases management and indole acetic acid production (IAA) (Brick growth enhancement (Amein et al. 2008; et al., 1991), phosphate solubilization (Laslo Maheshwari et al., 2012). In the present et al., 2012), HCN production (Lorck, study, we evaluate the influence of PGPR 1948), siderophore production (Schwyn and strains Azotobacter inoculation on the Neilands, 1987) NH3 production morphological parameters as well as on the (Cappuccino and Sherman 1992) as per concentration of nutritional and medicinally standard protocols. important curcumin of turmeric. 276 Int.J.Curr.Microbiol.App.Sci (2014) 3(9) 275-283 Rhizome bacterization The curcumin stock solution was prepared in methanol at a different concentration Young growing rhizotomus buds of turmeric between 0.5 to 7mg/ml collected from Botanical garden of Banaras Hindu University were bacteriazed by Chemical and reference compounds method of Weller and Cook (1983). Rhizotomus buds (7.5g) were surface All the chemicals and solvents used were of sterilized with 1% HgCl2 for 30 second and analytical grade (E. Merck, Mumbai, India). then washed with distilled water for 5 6 Standard sample of curcumin obtained from times, cell biomass of A. chroococcum CL13 Sigma Aldrich, (Bangalore, India) and were harvested through centrifugation before use, all solvents were filtered through (10,000g, 10 min) at 4°C from 72 h old 0.2 µm millipore membrane filter. culture. Equipment and chromatic condition The pellets resuspended in sterile distilled water (108 cfu ml-1) and viable bacterial The HPLC analysis was performed on a number was measured. The rhizomes were system consisting of Hewlett-Packard then coated with 20 ml bacterial inoculums quaternary HP1090 Series (Hewlett-Packard (108cfu ml-1) using 1% carboxymethyl palo Atto CA, USA) with multi wave length cellulose (CMC) slurry as an adhesive. The Photodiode-Array detector set between 200 rhizome coated with 1% slurry without nm to 500 nm and managed by computer bacterial strain served as control. The system HP 9000 workstation. The bacterial coated rhizome of turmeric sown in quantification of compounds was performed the experimental pots for (6 month) by using Luna RP-C18 prepacked column completion of life cycle under controlled (150nm × 3m) with a particle size of 5 mm. natural condition. Acetonitrile and 2% acetic acid 60:40 (v/v) used as mobile phase with the flow rate of Plant materials for curcumin 0.5 ml/min, the injected volume was 20 µl. quantification Calibration and linearity The linearity For the quantification of curcumin in the range of standards was determined by bacterial inoculated turmeric, young and analyzing series of standard curcumin. Test mature rhizomes of control (uninoculated) solution ranging (0.5 7.0) mg/ml of and A. chroococcum CL13 inoculated curcumin was prepared and injected three turmeric were collected from the times for linearity test. The linear regression experimental pot. The rhizomes of turmeric curve was obtained by plotting the peak area (1.0 g each) were treated with hexane (50 count of curcumin at (y axis) separately ml) by using a soxhlet extractor (30 min) for against the concentration (x axis) of each extraction. The hexane was removed injection separately. Linearity was found in through rotary evaporator, extracted samples the concentration range between 1 7mg were dissolved in 50 ml of methanol for 2 h. with high reproducibility and accuracy. Before using, the extracted samples were Regression analysis of experimental data filtered through 0.2 µm millipore filter. showed linear relationship. The limit of detection (LOD) and limit of quantification Preparation of stock solution (LOQ) were determined during HPLC by injecting series of standard solutions until 277 Int.J.Curr.Microbiol.App.Sci (2014) 3(9) 275-283 the signal to noise ratio (S/N) ratio for each inoculated turmeric up to 6 month (from compound was 3 for LOD and 10 for LOQ. June to November 2011). All these morphological parameters increased Results and Discussion significantly (Multiple Tukey HSD, SPSS.16). The difference in leaves number Isolation of bacterial isolates (same in both) were not significant between control and A. chroococcum CL13 Total 13 bacterial isolates were grown on N inoculated turmeric. Shoot height, shoot free agar specific plate, which were same in fresh biomass and rhizome fresh biomass the morphology and biochemical tests. The increased significantly in A. chroococcum isolates were Gram-negative, rod shaped, CL13