JOURNAL of Soil Biology and Ecology

Tracking of inoculated bacterium Pantoea dispersa in the root zone soil of Ocimum tenuiflorum L. using molecular techniques

Author:E. JYOTHI, D. J. BAGYARAJ AND NARENDRA JAWALI

Abstract :

Beneficial soil bacteria promoting plant growth are usually referred to as plant growth promoting rhizobacteria (PGPR). Earlier studies brought out that inoculation of Ocimum tenuiflorum L. with the selected PGPR (Pantoea dispersa) plus arbuscular mycorrhizal fungus (AMF) (Funneliformis monosporus) significantly enhanced plant growth and yield of O. tenuiflorum under glasshouse condition and later in the field with 50% saving of NPK fertilizers. The objective of the present study was to track down the inoculated P. dispersa in the root zone soil of O. tenuiflorum in the field. Ninety days post-inoculation, the total DNA from root zone soil was isolated and subjected to partial 16s rDNA gene amplification of P. dispersa. The amplicons were cloned and DNA sequenced. The generated DNA sequences when subjected to BLASTn search; it identified the soil bacterium to be P. dispersa, the originally inoculated bacterium. A neighbour joining based phylogenetic tree also placed the bacterium in P. dispersa clade, thereby confirming the identity of the bacterium. Our results highlight the successful tracking of the inoculated PGPR (P. dispersa) in the root zone soil containing F. monosporus and other soil organisms.

Keywords: PGPR, Bacterial DNA, 16S rRNA, Phylogenetic analysis, BLASTn

References :

Altschul, S. F., Gish, W., Miller, W., Myers, E. W. and Lipman, D. J., 1990. Basic local alignment search tool. J. Mol. Biol., 215(3): 403–410.

Ayyaz, K., Zaheer, A., Rasul, G. and Mirza, M. S., 2016. Isolation and identification by 16S rRNA sequence analysis of plant growth-promoting Azospirilla from the rhizosphere of wheat. Br. J. Microbiol., 47:542-550.

Backer, R., Rokem, J. S., Ilangumaran, G., Lamont, J., Praslickova, D., Ricci, E., Subramanian, S. and Smith, D. L., 2018. Plant growth-promoting rhizobacteria: Context, mechanisms of action, and roadmap to commercialization of biostimulants for sustainable agriculture. Front. Plant Sci., 9: 1473.

Barghouthi, S. A., 2011. A universal method for the identification of bacteria based on general PCR primers. Ind. J. Microbiol., 51(4): 430–444.

Beneduzi, A., Ambrosini, A. and Passaglia, L. M. P., 2012. Plant growth-promoting rhizobacteria (PGPR): Their potential as antagonists and biocontrol agents. Gen. Mol. Biol., 35(4): 1044–1051.

Clarridge, J. E., 2004. Impact of 16S rRNA gene sequence analysis for identification of bacteria on clinical microbiology and infectious diseases. Clin. Microbiol. Rev., 17(4), 840–862.

De Leij, F. and Bainton, N., 2017. Tracking of specific microbes in the environment. In: Molecular Microbial Ecology Manual (Eds. G. A. Kowalchuk, F. J. de Bruijn, I. M. Head, A. D. Akkermans, & J. D. van Elsas), Springer, Dordrecht,  pp. 1185–1318.

Dojima, T. and Craker, L. E., 2016. Potential benefits of soil microorganisms on medicinal and aromatic plants. In: Medicinal and Aromatic Crops: Production, Phytochemistry and Utilization (Eds. V. D. Jeliazkov & C. L. Cantrell), American Chemical Society, Washington, DC, pp. 75–90.

Earanna, N., Farooqi, A. A., Bagyaraj, D. J. and Suresh, C. K., 2002. Influence of Glomus fasciculatum and plant growth promoting rhizomicroorganisms on growth and biomass of periwinkle. J. Soil Biol. Ecol., 22: 22–26.

Fatima, F., Pathak, N. and Verma, S. R., 2014. An improved method for soil DNA extraction to study the microbial assortment within rhizospheric region. Mol. Biol. Int., Article ID 518960. 

Fusco, V. and Quero, G. M., 2014. Culture-dependent and culture-independent nucleic-acid-based methods used in the microbial safety assessment of milk and dairy products. Compr. Rev. Food Sci. Food Saf., 13(4): 493–537.

Gamalero, E., Berta, G. and Glick, G. R., 2009. The use of microorganisms to facilitate the growth of plants in saline soils. In: Microbial Strategies for Crop Improvement (Eds. M. S. Khan, A. Zaidi, & J. Musarrat), Springer‐Verlag, Berlin, Heidelberg, pp. 1–22.

Grayston, S. J., Wang, S., Campbell, C. D. and Edward, A. C., 1998. Selective influence of plant species on microbial diversity in the rhizosphere. Soil Biol. Biochem., 30: 369–378.

Holben, W. C., Jansson, J. K., Cheim, B. K. and Tiedje, J. M., 1988. DNA probe methods for the detection of specific microorganisms in the soil bacterial community. Appl. Environ. Microbiol., 54: 703–711.

Horwarth, W. R., Elliot, L. F. and Lynch, J. M., 1998. Influence of soil quality on the function of inhibitory rhizobacteria. Lett. Appl. Microbiol. 26: 87–92.

Jyothi, E. and Bagyaraj, D. J., 2016. Effect of plant growth promoting bacteria on growth, nutrient uptake and yield of Ocimum sanctum. J. Soil Biol. Ecol., 36, 44–50.

Jyothi, E. and Bagyaraj, D. J., 2017. Inoculation with microbial consortia enhances the growth, nutrition and oil concentration of Ocimum sanctum. Medicinal Plants: Int. J. Phytomed. Rel. Ind., 9: 237–241.

Jyothi, E., Bagyaraj, D. J. and Prakasa Rao, E. V. S., 2018. Microbial consortia developed for Ocimum tenuiflorum reduces application of chemical fertilizers by 50% under field conditions. Medicinal Plants: Int. J. Phytomed. Rel. Ind., 10: 89–95.

Kostylev, M., Otwell, A. E., Richardson, R. E. and Suzuki, Y., 2015. Cloning should be simple: Escherichia coli DH5α-mediated assembly of multiple DNA fragments with short end homologies. PLOS ONE, 10, e0137466. 

Kumar, S., Stecher, G. and Tamura, K., 2016. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol., 33(7): 1870–1874.

Pickup, R. W., 1991. Development of molecular methods for the detection of specific bacteria in the environment. J. Gen. Microbiol., 137: 1009–1019.

Ruppel, S., Rühlmann, J. and Merbach, H., 2006. Quantification and localization of bacteria in plant tissues using quantitative real-time PCR and online emission fingerprinting. Plant Soil, 286: 21–35.

Santoro, M. V., Cappellari, L., Giordano, W. and Banchio, E., 2015. Systemic induction of secondary metabolite biosynthesis in medicinal aromatic plants mediated by rhizobacteria. In: Plant Growth Promoting Rhizobacteria (PGPR) and Medicinal Plants Vol 42.  (Eds. D. Egamberdieva, S. Smriti, & A. Varma), Springer, Cham, pp. 7–13). 

Sorensen, J., 1997. The rhizosphere as a habitat for soil microorganisms. In: Modern Soil Microbiology (Eds. J. D. van Elsas, J. D. Trevors, & E. M. H. Wellington), Marcel Dekker Inc., New York, pp. 21–45.

Triveni, K., Singh, A. K., Kumar, R., Gupta, V. and Tripathi, K., 2013. Ocimum sanctum Linn: A review on phytopharmacology and therapeutic potential of Tulsi. Int. J. Pharma. Phytopharmacol. Res., 3(2): 148–151.

Van Elsas, J. D., Trevors, J. T. and Starodub, M., 1998. Bacterial conjugation between Pseudomonads in the rhizosphere of wheat. FEMS Microbiol. Lett., 53: 299–306.

Vinod, V., Kumar, A. and Zachariah, T. A., 2014. Isolation, characterization and identification of pericarp-degrading bacteria for the production of off-odour-free white pepper from fresh berries of Piper nigrum L. J. Appl. Microbiol., 116(4), 890–902.

Xu, X., Passey, T., Wei, F., Saville, R. and Harrison, R. J., 2015. Amplicon-based metagenomics identified candidate organisms in soils that caused yield decline in strawberry. Hort. Res., 2:15022. 

Yeates, C., Gillings, M. R., Davison, A. D., Altavilla, N. and Veal, D. A., 1998. Methods for microbial DNA extraction from soil for PCR amplification. Biol. Proced. Online, 1: 40–47.