Phylogenetic diversity and cross-inoculation of indigenous isolated Bradyrhizobium from nodules of peanut in Liaoning province of China

Document Type: Original article


1 College of Land and Environment, Shenyang Agricultural University, Shenyang, China

2 College of Agronomy, Shenyang Agricultural University, Shenyang, China

3 Department of Biotechnology, University of Sargodha, Sargodha Pakistan

4 College of Bioscience and Biotechnology, Shenyang Agricultural University, Shenyang, China


Arachis hypogaea. L is a legume of economic importance, which is nodulated by Bradyrhizobium, a slow-growing bacteria. However there is no well characterization of this rhizobia in many areas of China. In the present study, cross-inoculation experiments were performed in cowpea and soybean. The isolated bacteria strains were characterized physiologyically, biochemically and identified through 16S rDNA sequence analysis showing that it belongs to Bradyrhizobium japonicum. The genetic diversity of the seventeen isolated strains were assessed through PCR-RFLP of 16S rDNA and 16S-23S rDNA IGS region. Cross inoculation test indicated that isolates could nodulate cowpea but not soybean. The cluster analysis based on physiological and biochemical characteristics showed the lower correlation between isolates and sites. The isolates were grouped into four clusters based on 16S rDNA gene sequence analysis. Thirteen polymorphisms were variable across all observations in 16S rDNA RFLP and six different IGS types from isolates. The results implies that there was some association between geographical factor and phylogenetic diversity of indigenous Bradyrhizobium isolates.


1. Cleveland CC, Townsend AR, Schimel DS, Fisher H, Howarth RW, Hedin LO, Perakin SS, Latty EF, Von Fischer JC, Elseroad A, Wasson MF. Global patterns of terrestrial biological nitrogen (N2) fixation in natural ecosystems. Global Biogeochem 1999;13:623-645.

2. Sprent J, Legume N. A Global Perspective, Wiley Blackwell,Oxford, UK 2009;183.

3. Dresler-Nurmi A, Fewer DP, Räsänen LA, Lindström K. The diversity and evolution of Rhizobia. Microbiol Monogr 2009;8:3-41.

4. Mahmood A, Athar M. Cross inoculation studies: Response of Vigna mungoto inoculation with rhizobia from tree legumes growing under arid Environment. Int J Environ Sci Tech 2008;5:135-139.

5. Osei YA, Kerstin H. Genetic diversity of root nodule bacteria nodulating Lotus corniculatus and Anthyllis vulneraria in Sweden. Syst Appl Microbiol 2011;34:267-275.

6. Zhang YF, Wang ET, Tian CF, Wang FQ, Han LL, Chen WF, Chen WX. Radyrhizobium elkanii, Bradyrhizobium yuanmingense and Bradyrhizobium japonicum are the main rhizobia associated with Vigna unguiculata and Vigna radiata in the subtropical region of China. FEMS Microbiol Lett 2008;285:146-154.

7. Taurian T, Aguilar OM, Fabra A. Characterization of nodulating peanut rhizobia isolated from a native soil population in Córdoba, Argentina. Symbiosis 2002;3:59-72.

8. Vincent M. A manual for the practical study of root nodule bacteria. In: BP handbook, Blackwell Scientific Publications, Oxford and Edinburgh 1970;15.

9. Papa SS, Takeo Y, Yuichi S. Phylogenetic diversity of indigenous cowpea Bradyrhizobia from soils in Japan based on sequence analysis of the 16S-23S rRNA internal transcribed spacer (ITS) region. Syst Appl Microbiol 2011;34:285-292.

10. Boulila F, Depret G, Boulila A,  Belhadi D, Benallaoua S, Laguerre G. Retama species growing in different ecological-climatic areas of northeastern Algeria have a narrow range of rhizobia that form a novel phylogenetic clade within the Bradyrhizobium genus. Syst Appl Microbiol 2009;32:245-255.

11. Vanina M, Fernando I, Maria LT. Phenotypic and phylogenetic characterization of native peanut Bradyrhizobium isolates obtained from Córdoba, Argentina. Syst Appl Microbiol 2011;34:446-452.

12. Mahaveer PS, Khushboo S, Sushil KS. Biochemical characterization and metabolic diversity of soybean rhizobia isolated from Malwa region of Central India. Plant Soil Environ 2010; 56:375-383.

13. Parveen G, Ansari DL, Rao N. Soybean Rhizobia in Indian Soils: Populations, Host Specificity and Competitiveness. Proc Natl Acad Sci, India, Sect B Biol Sci 2014;84:457-464.

14. Mark DS, Allan RJ. Physiological and symbiotic characteristics of fast-growing Rhizobium japonicum. Plant Soil 1984;77:3-14.

15. Hameed S, Yasmin S, Malik KA, Zafar Y, Hafeez FY. Rhizobium, Bradyrhizobium and Agrobacterium strains isolated from cultivated legumes. Biol Fertil Soils 2004;39:179-185.

16. Weisburg WG, Barns SM, Pelletier DA, Lane DJ. 16S ribosomal DNA mplification for phylogenetic study. J Bacteriol 1991;173:697-703.

17. Laguerre G, Allard MR, Revoy F, Amarger N. Rapid identification of rhizobia by restriction fragment length polymorphism analysis of PCR amplified 16S rRNA genes. Appl Environ Microbiol 1994;60:56-63.

18. Tamura K, Dudley J, Nei M, Kumar S. Molecular Evolutionary Genetics nalysis (MEGA) software version 4.0. Mol Biol Evol 2007; 24:1596-1599.

19. Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 1987;44:406-425.

20. Chang YL, Wang JY, Wang ET, Liu HC, Sui XH, Chen WX. Bradyrhizobium lablabi sp. nov., isolated from effective nodules of Lablab purpureus and Arachis hypogaea. Int J Syst Evol Micr 2011;61:2496-2502.

21. Gillette WK, Elkan GH. Bradyrhizobium (Arachis) sp. strain NC92 contains two nod genes involved in the repression of nodA and a nolA gene required for the efficient nodulation of host plants. J Bacteriol 1996;178:2757-2766.

22. Li J, Fan H, Li L, Cao F, Xin J. Cross-Nodulation test for Bradyrhizobial strains and their phylogeny analysis based on 16S and 23S rDNA PCR-RFLP. Chin J Appl Environ Biol 2003;9:59-62.

23. Kumar D, Shivay YS, Dhar S, Kumar C, Parsad R. Rhizospheric flora and the influence of agronomic practices on thema review. Proc Natl Acad Sci India Sect B Biol Sci 2013;83:1-14.

24. Ridha M, Gisele L, Mohamed EA, Mars M, Amarger N. Different species and symbiotic genotypes of field rhizobia can nodulate Phaseolus vulgaris in Tunisian soils. FEMS Microbiol Ecol 2002;41:77-84.

25. Laguerre G, Mavingui P, Allard MR,  Charnay MP,  Louvrier P,  Mazurier SI,  Rigottier-Gois L,  Amarger N. Typing of rhizobia by PCR DNA fingerprinting and PCR-restriction fragment length polymorphism analysis of chromosomal and symbiotic gene regions: application to Rhizobium leguminosarum and its different biovars. Appl Environ Microbiol 1996;62:2029-2036.

26. Cem TG, Ibrahim O, Islam G. Genetic diversity of Vicia faba L. and Pisum sativum L.nodulating rhizobia in the central Black Sea region of Turkey. Ann Microbiol 2013;16:638-643.

27. Yang JK, Xie FL, Zou J, Zhou Q, Zhou JC. Polyphasic characteristics of Bradyrhizobia isolated from nodules of peanut (Arachis hypogaea) in China. Soil Biol Biochem 2005;37: 141-153.

28. Fiorela N, Pablo B, Natalia N. Genotypic analysis of isolated peanut- nodulating rhizobial strains reveals differences among populations obtained from soils with different cropping histories. Appl Soil Ecol 2012;53:74-82.

29. Adhikari D, Kaneto M, Itoh K, Suyama K, Pokharel BB, Gaihre YK. Genetic diversity of soybean-nodulating rhizobia in Nepal in relation to climate and soil properties. Plant Soil 2012;357:131-145.

30. Dinesh A, Kazuhito I, Kousuke S. Genetic diversity of common bean (Phaseolus vulgaris L.) nodulating rhizobia in Nepal. Plant Soil 2013;368:341-353.

31. El-Akhal M, Rincon A, Arenal F, Lucas M, El Mourabit N, Barrijal S. Pueyo J. Genetic diversity and symbiotic efficiency of rhizobial isolates obtained from nodules of Arachis hypogaeain northwestern Morocco. Soil Biol Biochem 2008;40:2911-2914.

32. El-Akhal M, Rincon A, Mourabit NE, Pueyo JJ. Barrijal S. Phenotypic and genotypic characterizations of rhizobia isolated from root nodules of peanut (Arachis hypogaea L.) grown in Moroccan soils. J Basic Microbiol 2009;49:415-425.

33. Taurian T, Ibanez F, Fabra A, Aguilar O. Genetic diversity of rhizobia nodulating Arachis hypogaea L. in central Argentinean soils. Plant Soil 2006; 282:41-52.

34. Mellal H, Yacine B, Boukaous L, Khouni S, Benguedouar A, Castellano-Hinojosa A, Bedmar EJ. Phylogenetic diversity of Bradyrhizobium strains isolated from root nodules of Lupinus angustifolius grown wild in the North East of Algeria. Systematic Appl Microbiol 2019;

35. Salmi A, Boulila F, Bourebaba Y, Roux C, Belhadi D, Lajudie P. Phylogenetic diversity of Bradyrhizobium strains nodulating Calicotome spinosa in the Northeast of Algeria. System Appl Microbiol 2018;41:452-459.