Impact of Fluoride on Growth and its Accumulation in Plant Growth Promoting Rhizobacterial Species by IASIR

American International Journal of Research in Formal, Applied & Natural Sciences

Available online at http://www.iasir.net

ISSN (Print): 2328-3777, ISSN (Online): 2328-3785, ISSN (CD-ROM): 2328-3793 AIJRFANS is a refereed, indexed, peer-reviewed, multidisciplinary and open access journal published by International Association of Scientific Innovation and Research (IASIR), USA (An Association Unifying the Sciences, Engineering, and Applied Research)

Impact of Fluoride on Growth and its Accumulation in Plant Growth Promoting Rhizobacterial Species 1,2

Sheetal Trikha1 and R.S. Chundawat2 Department of Science, FASC, Mody University of Science and Technology (MUST) Lakshmangarh, Sikar, Rajasthan-332311 (India)

Abstract: In the present study the growth kinetics and plant growth promoting properties of two rhizobial strains P33 and P34 have studied under the presence of fluoride as a toxicant. The rhizobacterial strains were isolated from the root nodules and rhizosphere of chickpea plants(Cicer arietinum) grown in local agricultural fields of Fazilka district of Punjab and designated as Pseudomonas fluorescens strain Sufi-1 and Pseudomonas sp. strain Sufi-2 after 16S rDNA study. The impact of fluoride has examined on the growth at different intervals of time and pH range. The movement of fluoride from media to the bacterial cells was checked by ISE method. Data analysis clearly indicates the interference of fluoride on growth of the rhizobacteria by its absorption from the media but its toxicity effect is higher at neutral pH as compare to acidic and basic range. Keywords: Rhizobacteria, Soil microbes, Fluoride, pH, Growth kinetics, Plant Growth Promoting Rhizobacteria (PGPR), Ion Selective Electrode (ISE),

I. INTRODUCTION Fluorine is a naturally occurring and estimated to be the 13th most abundant element on the earth’s crust [14], whereas fluorides are defined as binary compounds or salts of fluorine and another element. They occur naturally in the earth’s crust in rocks, coal, clay, and soil. Fluorides come to the biosphere through volcanic eruptions, coal-fired power plants, aluminium smelters, phosphate fertilizer plants, glass, brick, tiles work and plastics factories [6], [17], [18]. Fluoride seems to be toxic for plants, animals as well as the soil microbes at applicable concentration and affect their various beneficial activities [2], [10], [11], [12], [19]. These PGPR’s show many beneficial activities like phosphate solubilisation, nitrogen fixation, plant growth hormone production, siderophore production etc. [4], [5], [7], [23]. The use of plant growth promoting rhizobacteria (PGPR) is progressively increasing in agriculture and offers an attractive way to supplement chemical fertilizers and pesticides. Therefore, in the present study attempts have been made to find the effect of fluoride as a toxicant on the isolated PGPR species by comparing their growth kinetics as compare to control and also to check its toxicity impact at different pH. II. MATERIAL AND METHODS Sampling and Isolation The bacterial strains P33 and P34 were isolated from the chick pea plant’s rhizosphere and root nodules respectively from fluoride endemic area in the Fazilka district of Punjab. The nodules were washed thoroughly with sterile distilled water and thereafter nodules were placed in to laminar air flow for further process. Nodules were surface-sterilized using 70% ethanol and 0.1% HgCl2, and repeatedly washed with sterile water. Sterile nodules were crushed in a sterilized tube with the help of sterilized glass rod in 1 ml sterilized distilled water and the resulting suspension was streaked on yeast extract mannitol (YEM) agar plates amended with Congo red and plates were incubated in an incubator at 28 ± 2°C for 24 to 36 hours [9]. After 24-36 hours, the translucent, glistening and elevated colonies were picked and purified by single colony streaking on the YEMA slants. Molecular characterization of the isolates The bacterial isolates have grown in YEM Broth on an incubator shaker (120 rpm) at 28 ± 2°C for 24 hours and used for genomic DNA extraction [21]. The approximately 1.5 kb rDNA fragment was amplified by Thermal Cycler using 16S rRNA universal primer. The PCR product was sequenced by Genetic Analyzer (ABI 3130 Genetic Analyzer) .The gene sequence of the isolates were deposited in the NCBI GenBank and assigned the accession number KP772219 and KP772220, designated as Pseudomonas fluorescens strain Sufi-1 and Pseudomonas sp. strain Sufi-2.

Page 24

Sheetal Trikha et al., American International Journal of Research in Formal, Applied & Natural Sciences, 13(1), December, 2015February, 2016, pp. 24-27

Effect of fluoride on Growth Kinetics and its accumulation MIC Determination: The minimal inhibitory concentration (MIC) of Fluoride was determined by the standard broth agar dilution method. Viability was tested by CFU on YEMA media plates [2]. So the 15 ppm concentration of fluoride has been selected for the experiments. Growth Kinetics: The Parent strains were fully grown in YEM broth media at 30oC for 30 hours at neutral pH range. Suspensions used for inoculation were diluted to 0.08 A620 nm (1x 108 cell /ml) with fresh sterile media [22]. Bacterial growth was monitored at different time intervals of 3, 6, 9, 24, 27 and 30 hours using Spectrophotometer at 620 nm for control as well as toxicant simultaneously under same growth conditions and the % age change in growth due to fluoride has been determined. Estimation of Fluoride Absorption: Ion Selective Electrode (ISE) has used for the fluoride estimation in the media. Before fluoride estimation we centrifuge 30 hours old culture. After centrifugation we took supernatant as well as pellet and digest them with triple acid (H2SO4:HNO3:HClO4 = 1:1:0.5) solution. A total-ionic strength adjustment buffer (TISAB) is used to adjust the pH [13]. Effect of Different pH Ranges on Fluoride Toxicity: In addition to the investigation of the impact of fluoride, the influence of the initial pH value was examined. The effect of pH was evaluated on the both strains at three different pH ranges. These were pH 4 for acidic range, pH 7 as neutral and pH 9 in basic range. The microbes were allowed to grow all these pH ranges for 30 hours under same growth conditions for both control as well as toxicant as mentioned above for growth kinetics study. After 30 hours % age change in growth between control and toxicant were compared in cultures grown at the 4, 7 and 9 pH respectively. III. RESULTS Fluctuation in growth at different pH: Growth data was collected from both isolated species P33 and P34 in presence of fluoride at 3, 6, 9, 12, 24, 27 and 30 hours incubations. Control was considered as 100% and any fluctuation due to fluoride presence were shown in graphs (Figure 1 and 2) respectively at pH=7. Both the isolates showed significant decrease in growth at each interval of time due to the presence of fluoride. Based on the optical density measurement, there was very less growth when the pH of the media was reduced to 4 in comparison with the reference nutrient broth medium with a pH of 7. There was intermediate growth observed when the initial pH of the medium was increased to 9 (Figure 3). Graphical data shows the % age change in the growth at acidic, neutral and basic pH for both rhizobacterial species after the 30 hours incubation time. Alteration of fluoride concentration: The fluoride movement was observed by assessing media and bacterial cell pellet separately after 30 hours incubation. 15 ppm was considered as 100% for media and any fluctuation in concentration after 30 hours is shown in graph. The results clearly indicate that fluoride was absorbed by the bacterial cell so there was significant drop in media fluoride concentration (Figure 4). Simultaneously, significant fluoride absorption by bacterial cells was observed (Figure 5).

Figure 1: Growth kinetics of P33 rhizobacterial species for control and in the presence of toxicant (pH=7).

Figure 2: Growth kinetics of P34 rhizobacterial species for control and in the presence of toxicant (pH=7).

Page 25

Sheetal Trikha et al., American International Journal of Research in Formal, Applied & Natural Sciences, 13(1), December, 2015February, 2016, pp. 24-27

Figure 3: % age change in growth of P33 and P34 rhizobacterial species at acidic pH=4, neutral pH=7 and basic pH= 9) due to fluoride.

Figure (4-5) represent the decrease of fluoride concentration from media and its increase in bacterial cell pellets for P33 and P34 species respectively after 30 hours incubation.

IV. DISCUSSION In the present study, significant response has observed for both isolates with respect to the growth and fluoride accumulation. In soil, fluoride enters bacterial cell mainly in the form of HF [15]. Fluoride ones enter in cell cause acidic inhibition of glycolytic enzymes like enolase may cause lipid per-oxidation and disturbs various metabolic pathways [8]. This is the reason due to which there is a decrease in growth in presence of fluoride [20]. The permeability of fluoride to the bacterial cell is most at neutral pH therefore the % age change in growth is highest at the pH 7. At pH 4 the change is least because this acidic environment is not optimum for the growth of microbes. So the decrease in growth is mainly due to the acidic environment in both control as well as toxicant. Rest the change which appeared may be due to fluoride toxicity [16]. At pH 9 lesser % age change in growth appeared in contrast to neutral broth because at this basic environment the fluoride may bind with the other salts present in the media so less fluoride is available in free form to enter the microbial cell [1]. So the fluoride shows its lesser toxicity as compare to the neutral pH. Bacterial cell fight against toxicant, by its glutathione pool and try to maintain its internal homeostasis, but when stress is much higher it leads to the decrease in colony forming units (CFU), causes cell death and as a consequence overall productivity decreases [20]. REFERENCES [1] [2] [3] [4] [5]

A. Monballiu, N. Cardon, M. T. Nguyen, C. Cornelly, B. Meesschaert and Y. Chiang, " Tolerance of chemoorganotrophic bioleaching microorganisms to heavy metal and alkaline stresses," Bioinorganic chemistry and application, 2015. A. Ram, P. Verma and B. R. Gadi, "Effect of fluoride and salicylic acid on seedling growth and biochemical parameters of watermelon (Citrullus lanatus)," Fluoride,vol. 47 , 2014, pp. 49-55. A. R. Shakoori, K. S. Zaidi, "Cadmium resistant Enterobacter clacae and Klebsiella sp isolated from industrial effluents and their possible role in cadmium detoxification," World Journal of Microbiology and Biotechnology, vol.15, 1998, pp. 249-254. C. R. Howell, R. C. Beier and R. D. Stipanovic," Production of Ammonia by Enterobacte cloacae and its possible role in the biological control of Phythium pre-emergance damping- off by the bacterium," Phytopathology, vol. 78, 1998, pp. 1075-1078. D. P. Verma, M. G. Fortin, J. Stanley, V. P. Mauro, S. Purohit and N. Morrison," Nodulins and nodulin genes of Glycine max," Plant Molecular Biology , vol. 7, 1986, pp. 51-61.

Page 26

Sheetal Trikha et al., American International Journal of Research in Formal, Applied & Natural Sciences, 13(1), December, 2015February, 2016, pp. 24-27 [6] F.V. Zohoori, P. J. Moynihan, N. Omid, L. Abuhaloob and A. Maguire, "Impact of water fluoride concentration on the fluoride content of infant foods and drinks requiring preparation with liquids before feeding," Community Dental Oral Epidemiology, vol.40, 2012, pp. 432-440. [7] H. Rodriguez and R. Fraga, " Phosphate solubilising bacteria and their role in plant growth promotion," Biotechnology Advances, vol.17, 1999, pp. 319-339. [8] H.S. Spets, L. Seppa, A. Korhonen and P. Alakujala, " Accumulation of strontium and fluoride in approximal dental plaqueand changes in plaque microflora after rinsing with chlorohexidine-fluoride-strontium solution," Oral diseases, vol. 4, 1998, pp. 114-119. [9] J. M. Vincent, A manual for the practical study of the root-nodule bacteria, International Biological Programme handbook no. 15, Blackwell Scientific Publications, Oxford, 1970. [10] K. C. Pal, N. K. Mondal, S. Chatterjee, T. S. Ghosh and J. K. Datta, "Characterization of fluoride-tolerant halophilic Bacillus flexus NM25 (HQ875778) isolated from fluoride-affected soil in Birbhum district, West Bengal, India," Environment Monitering Assessment, 2014; vol.186, pp. 699-709. [11] M. Baunthiyal and S. Ranghar, "Physiological and biochemical responses of plants under fluoride stress: An overview," Fluoride, vol. 47, 2014, pp. 287-293. [12] N. K. Mondal, K. C. Pal, M. Dey, S. Ghosh, C. Das and J. K. Datta, " Seasonal variation of soil enzymes in areas of fluoride stress in Birbhum district, West Bengal, India," Journal of Taibah University of Science, vol.9, 2015, pp. 133-142. [13] NIOSH Manual of Analytical Methods, 4th Edn., Cincinnati, 1994. [14] P. K. Rakshit, "Studies on estimation of fluoride and fluoridation of drinking water," Dissertation , IISc Bangalore, 2004. [15] R. E. Marquis, S. A. Clock and M. M. Meira," Fluoride and organic weak acids as modulators of microbial physiology," FEMS Microbiology Reviews, 2002. [16] R. Sharma, M. Tsuchiya, Z. Skobe, B. A. Tannous and J.D. Bartlett," The acid test of fluoride: how pH modulates toxicity," PloS One, Vol.5, 2010. [17] S. J. Cronin, V. Manoharan, M. J. Hedley and P. Loganathan," Fluoride: A review of its fate, bioavailability, and risks of fluorosis in grazed-pasture systems in New Zealand," New Zealand Journal of Agricultural Research, vol. 43, 2000, pp. 295-321. [18] S. Jagtap , M. K. Yenkie, N. Labhsetwar and S. Rayalu, "Fluoride in Drinking Water and Defluoridation of Water," Chemical Review, vol.112, 2012, pp. 2454-2466. [19] S. L. Choubsia," Status of fluorosis in animals, "Proceedings in National Academy of Sciences, India," Section B Biological Sciences, vol.82 (3), 2012, pp. 331-339, DOI 10.1007/s 40011-012-0026-0. [20] S. Trikha and R. S. Chundawat," Identification of suitable rhizobacterial species for fluoride contaminated areas: A comparative growth kinetics study," International journal of Science and Research, vol.4, 2015, pp.1349-1352. [21] T. T. Kuo, Y. S. Chao, Y. H. Lin, B. Y. Lin, L. F. Liu and T. Y. Feng, "Integration of the DNA of filamentous bacteriophage Cflt into the chromosomal DNA of its host," Journal of Virology, vol. 61(1), Jan 1987, pp. 60â&#x20AC;&#x201C;65. [22] T.V. Bhuvaneshwari , B.G. Turgeon ,B.D. Wolfgang, " Early events in the infection of soyabean (Glycine max L.Merr) by Rhizobium japonicum," Plant Physiology (66) pp. 1027-1031, 1980. [23] W. Frankenberger and M. Arshad, Phytohormones in soils: Microbial production and function, New York, Marcel Dekker, 1995.

ACKNOWLEDGEMENT Authors are thankful to the Mody University of Science and Technology, Lakshmangarh, Sikar (Rajasthan) for providing the necessary laboratory facilities and fellowship for the above research work.

Page 27