Research Article | Volume 11, Issue 1, January, 2023

Occurrence of two endophytic associative nitrogen-fixing Caulobacter spp., from three non-nodulating endemic legumes based on nifH gene analysis

N. Thamizhseran Ganesh V. Shendye   

Open Access   

Published:  Nov 22, 2022

DOI: 10.7324/JABB.2023.110123

Legumes are cosmopolitan plants, and nodulating legumes are well known for their symbiotic nitrogen-fixing ability by rhizobia-legume interaction. Biological nitrogen fixation in non-nodulating endemic legumes by associative or endophytic symbiotic bacteria requires much needed attention. Three non-nodulating legumes, namely, Humboldtia brunonis Wall., Kunstleria keralensis C.N. Mohanan and N.C. Nair, and Bauhinia phoenicea Wight and Arn., endemic to the Western Ghats regions of Karnataka state, were studied. Employed techniques of selective culture media to understand diazotrophic diversity inside the roots of these plants. The isolates that can grow in a nitrogen-free semisolid agar medium have been considered positive for nitrogen-fixing ability. nifH gene is taken as the marker gene to ascertain the nitrogen-fixing ability of the bacteria. The qualified bacterium in the previous steps is identified using 16s RNA sequencing and the Sanger sequence method. The results obtained showed B. phoenicea Wight and Arn. and H. brunonis Wall. as Caulobacter segnis, and in K. keralensis C.N. Mohanan & N.C. Nair, it is Caulobacter crescentus. The presence of the nifH gene is demonstrated through molecular methods. This work adds to the diverse works of Caulobacter as a successful plant growth-promoting endophyte even in the nitrogen-deficient, slopy soils of the Western Ghats.

Keyword:     nifH gene analysis Nitrogen fixation Nitrogen-free medium Non-nodulating endemic legume Plant growth-promoting endophyte


Thamizhseran N, Shendye GV. Occurrence of two endophytic associative nitrogen-fixing Caulobacter spp., from three non-nodulating endemic legumes based on nifH gene analysis. J App Biol Biotech. 2023;11(1):171-175.

Copyright: Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license.

HTML Full Text

1. Mitter EK, Tosi M, Obregón D, Dunfield KE, Germida JJ. Rethinking crop nutrition in times of modern microbiology: Innovative biofertilizer technologies. Front Sustain Food Syst 2021;5:606815.

2. Zhang K, Wu Y, Hang H. Differential contributions of NO3-/NH4+ to nitrogen use in response to a variable inorganic nitrogen supply in plantlets of two Brassicaceae species in vitro. Plant Methods 2019;15:86.

3. Mokhele B, Zhan X, Yang G, Zhang X. Review: Nitrogen assimilation in crop plants and its affecting factors. Can J Plant Sci 2012;92:399-405.

4. Bruijn F. Biological nitrogen fixation book summary. Adv Microbiol 2016;6:407-11.

5. Gaiero JR, McCall CA, Thompson KA, Day NJ, Best AS, Dunfield KE. Inside the root microbiome: Bacterial root endophytes and plant growth promotion. Am J Bot 2013;100:1738-50.

6. Nayar T, Beegam AR, Sibi M. Flowering Plants of the Western Ghats, India. Vol. 1. Thiruvananthapuram, India: Jawaharlal Nehru Tropical Botanic Garden and Research Institute; 2014.

7. Tiwari S, Moghe S, Gurnule WB, Bhagat DS, Gunjal A. Habitat-specific microbial community associated with the biodiversity hotspot. In: Gunjal A, Shinde S, editors. Microbial Diversity in Hotspots. Ch. 2. Cambridge: Academic Press; 2022. p. 25-43.

8. Baldani JI, Reis VM, Videira SS, Boddey LH, Baldani VL. The art of isolating nitrogen-fixing bacteria from non-leguminous plants using N-free semisolid media: A practical guide for microbiologists. Plant Soil 2014;384:413-31.

9. Gaby JC, Buckley DH. A comprehensive evaluation of PCR primers to amplify the nifH gene of nitrogenase. PLoS One 2012;7:e42149.

10. Rösch C, Bothe H. Diversity of total, nitrogen-fixing and denitrifying bacteria in an acid forest soil. Eur J Soil Sci 2009;60:883-94.

11. Dhabak MR, Nandikar MD. Lectotypification of Humboldtia brunonis (Fabaceae) and notes on its petal numbers. Ann Bot Fenn 2021;58:285-7.

12. Singh RK, Diwakar PG, Sudhakar JV. Bauhinia phoenicea B. Heyne ex Wight and Arn., the less known endemic and threatened liana of the southern Western Ghats, requires immediate conservation. J Threat Taxa 2015;7:7676-82.

13. Sreeja K, Unni PN. Floristic diversity of Vallikkaattu Kaavu, a sacred grove of Kozhikode, Kerala, India. J Ecol Nat Environ 2016;8:175-83.

14. MSSBG RET Plant List 2021. Puthurvayal: MSSBG; 2022.

15. Yamaji K, Watanabe Y, Masuya H, Shigeto A, Yui H, Haruma T. Root fungal endophytes enhance heavy-metal stress tolerance of Clethra barbinervis growing naturally at mining sites via growth enhancement, promotion of nutrient uptake and decrease of heavy-metal concentration. PLoS One 2016;11:e01609089.

16. Mefteh F, Bouket AC, Daoud A, Luptakova L, Alenezi FN, Gharsallah N, et al. Metagenomic insights and genomic analysis of phosphogypsum and its associated plant endophytic microbiomes reveals valuable actors for waste bioremediation. Microorganisms 2019;7:382.

17. López AC, Alippi AM. Feasibility of using RFLP of PCR-amplified 16S rRNA gene(s) for rapid differentiation of isolates of aerobic spore-forming bacteria from honey. J Microbiol Methods 2019;165:105690.

18. Frank JA, Reich CI, Sharma S, Weisbaum JS, Wilson BA, Olsen GJ. Critical evaluation of two primers commonly used for amplification of bacterial 16S rRNA genes. Appl Environ Microbiol 2008;74:2461-70.

19. Gaby JC, Buckley DH. A comprehensive evaluation of PCR primers to amplify the nifH gene of nitrogenase. PLoS One 2012;7:e42149

20. Ando S, Goto M, Hayashi H, Yoneyama T, Meunchang S, Thongra- Ar P, et al. Detection of nifH sequences in sugarcane (Saccharum officinarum L.) and pineapple (Ananas comosus [L.] Merr.). Soil Sci Plant Nutr 2010;51:303-8.

21. Quiagen. QIAamp DNA Mini Kit. Genomic DNA; 2019.

22. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990;215:403-10.

23. Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 2018;35:1547-9.

24. Gaby JC, Buckley DH. A comprehensive aligned nifH gene database: A multipurpose tool for studies of nitrogen-fixing bacteria. Database (Oxford) 2014;2014:bau001.

25. Kaga H, Mano H, Tanaka F, Watanabe A, Kaneko S, Morisaki H. Rice seeds as sources of endophytic bacteria. Microbes Environ 2009;24:154-62.

26. Singha KM, Singh B, Pandey P. Host specific endophytic microbiome diversity and associated functions in three varieties of scented black rice are dependent on growth stage. Sci Rep 2021;11:12259.

27. Mehnaz S. Plant growth-promoting bacteria associated with sugarcane. Bacteria in agrobiology: Crop ecosystems. Berlin, Heidelberg: Springer; 2011. p. 165-87.

28. Berrios L, Ely B. Plant growth enhancement is not a conserved feature in the Caulobacter genus. Plant Soil 2020;449:81-95.

Article Metrics

2 Absract views 14 PDF Downloads 16 Total views

Related Search

By author names

Citiaion Alert By Google Scholar