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 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.

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Plant growth promoting rhizobacteria Materials and Methods (PGPR) is a group of free-living rhizosphere 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, 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.

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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

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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 inoculated turmeric with the growth. round, mucoid, smooth colonies. Increase in morphological parameters, shoot Morphological and biochemical height, shoots biomass and rhizome biomass characteristics of isolates have been was significant (p<0.05) with the growth presented in (table.1). One of the pure strain periods (Table 2). CL13 on 16s r RNA gene sequence analysis Peak identification identified as Azotobcter chroococcum species, which show 100% similarity with The HPLC chromatogram of control and A. the strain A. chroococcum GD5. The gene chroococcum CL13 inoculated turmeric at sequence were deposited in GeneBank under the wavelength of 425 nm three peaks were the accession number [KJ001770] observed at the different retention time (RT). The peak at the retention time of Plant growth promoting (PGP) traits of 8.50±0.55 (average of three replicates) was bacterial isolates identified as curcumin. The purity of the compounds was checked by the standard The PGP traits for the isolates A. compounds of curcumin (Rt. 8.90). In both chroococcum CL13 revealed that they the standard and sample peak, Peak purity produced IAA in the presence of different was greater than 0.9899. The UV-spectral concentrations (50 to 500 µg/ml) of matching was found between 0.9899 tryptophan, they did not produce IAA in 0.9788. absence of tryptophan. The content of IAA was produced by A. chroococcum CL13 Analysis of curcumin content in the sample (1.33 µg/ml) at 50 µg/ml of tryptophan and of control and A. chroococcum CL13 10.97 µg/ml of IAA at 500 µg/ml IAA. turmeric were performed using HPLC-PDA Phosphate solubilization (zone of clearance detector. The variation in amount of 11 mm), production of siderophore and curcumin was measured by comparing the NH3were detected positive during plant area covered by the curcumin in the growth promoting properties. chromatogram of respective sample with the standard curve of curcumin (Fig.1). The Growth and yield of PGPR treated standard curve were made by injecting turmeric different concentration 0.5 to 7 mg/ml of curcumin, which show linear relation The various morphological parameters like between 1 to 7 mg/ml, The correlation number of leaves shoot height, shoot and Coefficient (R2) of curcumin was 0.9980 rhizome fresh biomass were measured in and linear regression equation for the curve control and A. chroococcum CL13 was 215e+004x-2.34e+004 achieved.

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Table.1 Morphology and biochemical characterization of isolates

Biochemical Azotobacter isolate characters Gram stain, cell shape Gram ve, Rod Colony morphology Watery, mucilaginous white and Pigmentation with exuberant margin Growth of N2 free + medium Polysaccharides - production + citrate test + Carbohydrate utilization Glucose + Lactose - Sucrose + Mannitol + Hydrolysis + Starch + Lipid + Biotin

Table.2 Various growth and yield parameters of Azotobacter chroococcum CL13 inoculated turmeric

Month Shoot Height(cm) Shoot Biomass(g) Rhizome Biomass(g) r r r m m m e e e u u u t t t l l l c c c c c c o o o 3 3 3 c c c r r r a a a t 1 t 1 t 1 o o o b b b c c c n n n L L L o o o o o o o o o t t t C C C o o o o o o C C C r r r z z z h h h A A A c c c

June 0 0 0 0 7.5 7.5 July 20±0.55a 22±0.55b 13±0.92k 14±0.71l 8±0.63ab 8±0.66bc August 33±0.63c 37±0.72d 20±0.66 m 25±0.58 n 10±0.62cd 10±0.58ef September 55 ±0.46e 59±0.61f 36±0.61 o 39±0.57 p 33±0.61fg 38±0.31gh October 69±0.66 g 73±0.66h 46±0.60 q 51±0.55 r 63±0.39ij 67±0.57kl November 78±0.81i 81±0.68 j 57±0.60 s 61±0.58 t 135±0.70mn 142±0.60op

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Fig.1 HPLC chromatogram of control and A. chroococcum CL13inoculated turmeric at 425nm

0.10 U A 0.05

0.00 0.00 5.00 10.00 15.00 Minutes

Fig.1a HPLC Chromatogram of standard curcumin at 425 nm

1.20

1.00

0.80 U

A 0.60

0.40

0.20

0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 Minutes

Fig1.b HPLC chromatogram of control turmeric (uninoculated) at 425 nm

2.50

2.00

1.50 AU 1.00

0.50

0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 Minutes

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Fig1.c HPLC Chromatogram of Azotobacter chroococcum CL13 inoculated turmeric at 425 nm

The concentration of curcumin in control enhanced in A. chroococcum CL13 (4.52±0.78 g) increased by 6% (4.79±0.45 inoculation compared to control. This may g) in A. chroococcum CL13 inoculated be due to the production of IAA, NH3 and turmeric (amount are the average of three phosphate solubilization by A. chroococcum replicates). CL13 which was observed during PGP trait analysis in this study. The plant growth In the intensive agricultural system to obtain promotion due to bacterial IAA production the maximum amount of product, huge during root colonization is reported by other amount of chemicals as fertilizers and workers also. The IAA producing bacteria pesticides are being applied for nutrient survive on root and proliferate by utilizing supplement and pest control. On the other nutrients exuded by them and efficiently hand continuous uses of chemicals adversely colonize the entire root system (Bloemberg affect soil fertility, soil ecology, and and Lugtenberg, 2001). Reports of environment as well as produce harmful Rodríguez and Fraga (1999) stated that P. effect on human health (Ayala and Rao, solubilizing bacteria are capable to increase 2002). Hence, environment friendly the aviability of phosphorous in soil, which sustainable agriculture is preferred to obtain is beneficial for the plant growth. A. the desirable yields. chroococcum CL13 is known as N2 fixer in plant rhizosphere/soil and its inoculation The present study has shown that the growth increased the shoot height, shoot fresh of shoot and yield of rhizome biomass biomass, rhizome fresh biomass.

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Planta Med., 57: 1 7. Curcuminoids is an important component of Ayala, S., Rao, E.V.S. 2002. Perspective of turmeric. Amongst curcuminoids, curcumin soil fertility management with a focus is an important constituent. In this present on fertilizer use for crop productivity. study, A. chroococcum CL13 inoculation Curr. Sci., 82: 797 807. enhanced the concentration of curcumin. Bloemberg, G.V., Lugtenberg, B.J.J. (2001). The exact mechanism through which A. Molecular basis of plant growth chroococcum CL13 modulate the synthesis promotion and biocontrol by of curcumin had been not studied in this rhizobacteria. Curr. Opin. Plant Biol., experiment but its chemotactic behavior 4: 343 350. were studied by workers in cotton and wheat Brick, J.M., Bostock, R.M., Silverstone, seedlings. The results showed that this S.E. (1991). Rapid In situ assay for bacterium had strong chemotactic indole acetic acid production by movements towards few amino acids like bacteria immobilized on nitrocellulose (Glutamic acid, Arginine, Threonine) and membrane. Appl. Environ. Microbiol., organic acids like (citric acid, succinic acid, 57: 535 538. Maleic acid and Malonic acid) (Kumar et Cappuccino, J.C., Sherman, N. (1992). In: al., 2007). The rhizome of turmeric contains Microbiology: A Laboratory Manual, number of phenolic compounds like New York, pp. 125 179. curcuminoids and sesquiterpenoids. There Garrity, G. (2005). The , Part exudates likely to attract A. chroococcum B the gamma proteobacteria. In: CL13 more suitably and effectively Brenner, D.J., Krieg, N.R., Staley, J.T. colonized roots resulting enhanced the (Eds), Bergey s Manual of Systematic production of curcumin. Hence positive Bacteriology, 2nd Edn,. Springer, New response of A. chroococcum CL13 towards York, NY, 2: 323 379. curcumin production was obtained. The A. He, X.G., Lin, L.Z., Lian, L.Z., chroococcum CL13 during PGP traits Lindernmaier, M. (1988). Liquid analysis produced IAA, solubilize P and chromatography-electrospray mass produced NH3, its inoculation might enhance spectrometric analysis of curcuminoids the morphological yields and curcumin and sesquiterpenoids in turmeric concentration. (Curcuma longa). J. Chromatogr. A., 818: 127 132. Acknowledgement Jayaprakasha, G.K., Rao, J.M., Sakariah, K.K. (2002). Improved HPLC methods Authors are thankful to University Grant for the determination of curcumin, Commission (UGC), New Delhi, India for demethoxycurcumin, and financial assistance bisdemethoxy curcumin. J. Agric. Food Chem., 50: 3668 3672. References Kumar, B., Kumar, M.S., Annapurna, K. and Maheshwari, D.K. (2006). Genetic Adesemoye, A.O., Kloepper, J.W. (2009). diversity of plant growth-promoting Plant microbes interactions in rhizobia isolated from a medicinal enhanced fertilizer-use efficiency. legume, Mucuna pruriens Linn. Curr. Appl. Microbiol. Biotechnol., 85: 1 Sci., 9(11): 1524 1529 12. Kumar, R., Bhatia, R., Kukreja, K., Behl, Ammon, H.P.T., Wahl, M.A. (1990). R.K., Dudeja, S.S., Narula, N. (2007). Pharmacology of Curcuma longa.

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