Skip to main content

Studies of Phylogeny, Symbiotic Functioning and Ecological Traits of Indigenous Microsymbionts Nodulating Bambara Groundnut (Vigna subterranea L. Verdc) in Eswatini 

Abstract

Rhizobial microsymbionts of grain legumes are ubiquitous in soils and exhibit a wide range of diversity with respect to colony morphology, genetic variability, biochemical characteristics, and phylogenetic relationships. This study assessed the phylogenetic positions of rhizobial microsymbionts of Bambara groundnut from Eswatini exhibiting variations in morpho-physiology, adaptive characteristics, and N2-fixing efficiency. The isolates’ ERIC-PCR profiles revealed the presence of high genetic variation among them. These test isolates also exhibited differences in pH tolerance and IAA production. Multilocus sequence analysis based on the 16S rRNA, atpD, glnII, gyrB, and recA gene sequences of representative test isolates closely aligned them to the type strains of Bradyrhizobium arachidis, B. manausense, B. guangdongense, B. elkanii, and B. pachyrhizi. However, some isolates showed a high divergence from the known reference type strains, indicating that they may represent species yet to be properly characterized and described. Functional characterization in the glasshouse revealed that most of the isolates from the contrasting Agro-ecologies of Eswatini were efficient in N2 fixation, and therefore elicited greater stomatal conductance and photosynthetic rates in the homologous Bambara groundnut. Of the 75 isolates tested, 51% were more effective than the commercial Bradyrhizobium sp. strain CB756, with relative symbiotic effectiveness ranging from 138 to 308%. The findings of this study indicated that the analysis of housekeeping genes and functional traits of Bambara-nodulating microsymbionts can provide a clear view for understanding and predicting rhizobial community structure across environmental gradients.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

References

  1. 1.

    Mkandawire C (2007) Review of bambara groundnut (Vigna subterranea (L.) Verdc.) production in Sub Sahara Africa. Agric J 2:464–470

    Google Scholar 

  2. 2.

    Nkambule BS, Ossom EM, others (2010) Effects of jugo bean [Vigna subterranea (L.) Verdc.] plant population on physiological growth indices and yields under intercropping with sweetpotato [Ipomoea batatas (L.) Lam.]. Adv Environ Biol 4:201–215

    Google Scholar 

  3. 3.

    Karunaratne AS, Azam-Ali SN, Al-Shareef I et al (2010) Modelling the canopy development of bambara groundnut. Agric For Meteorol 150:1007–1015. https://doi.org/10.1016/j.agrformet.2010.03.006

    Article  Google Scholar 

  4. 4.

    Jørgensen ST, Liu F, Ouédraogo M, Ntundu WH, Sarrazin J, Christiansen JL (2010) Drought responses of two bambara groundnut (Vigna subterranea L. Verdc.) landraces collected from a dry and a humid area of Africa. J Agron Crop Sci 196:412–422. https://doi.org/10.1111/j.1439-037X.2010.00435.x

    Article  Google Scholar 

  5. 5.

    Mabhaudhi T, Chibarabada TP, Chimonyo VGP, Modi AT (2018) Modelling climate change impact: a case of bambara groundnut (Vigna subterranea). Phys Chem Earth 105:25–31. https://doi.org/10.1016/j.pce.2018.01.003

    Article  Google Scholar 

  6. 6.

    Musa M, Massawe F, Mayes S, Alshareef I, Singh A (2016) Nitrogen fixation and N-balance studies on bambara froundnut (Vigna subterranea L. Verdc) landraces grown on tropical acidic soils of Malaysia. Commun Soil Sci Plant Anal 47:533–542. https://doi.org/10.1080/00103624.2016.1141928

    CAS  Article  Google Scholar 

  7. 7.

    Cooper JE (2007) Early interactions between legumes and rhizobia: disclosing complexity in a molecular dialogue. J Appl Microbiol 103:1355–1365. https://doi.org/10.1111/j.1365-2672.2007.03366.x

    CAS  Article  PubMed  Google Scholar 

  8. 8.

    Badri DV, Weir TL, van der Lelie D, Vivanco JM (2009) Rhizosphere chemical dialogues: plant-microbe interactions. Curr Opin Biotechnol 20:642–650. https://doi.org/10.1016/j.copbio.2009.09.014

    CAS  Article  PubMed  Google Scholar 

  9. 9.

    Desbrosses GJ, Stougaard J (2011) Review root nodulation : a paradigm for how plant-microbe symbiosis influences host developmental pathways. CHOM 10:348–358. https://doi.org/10.1016/j.chom.2011.09.005

    CAS  Article  Google Scholar 

  10. 10.

    Mohale KC, Belane AK, Dakora FD (2014) Symbiotic N nutrition, C assimilation, and plant water use efficiency in Bambara groundnut (Vigna subterranea L. Verdc) grown in farmers’ fields in South Africa, measured using 15N and 13C natural abundance. Biol Fertil Soils 50:307–319

    CAS  Article  Google Scholar 

  11. 11.

    Grönemeyer JL, Kulkarni A, Berkelmann D, Hurek T, Reinhold-Hurek B (2014) Identification and characterization of rhizobia indigenous to the Okavango region in Sub-Saharan Africa. Appl Environ Microbiol 80:7244–7257. https://doi.org/10.1128/AEM.02417-14

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Puozaa DK, Jaiswal SK, Dakora FD (2017) African origin of Bradyrhizobium populations nodulating Bambara groundnut (Vigna subterranea L. Verdc) in Ghanaian and South African soils. PLoS One 12:e0184943. https://doi.org/10.1371/journal.pone.0184943

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Ibny FYI, Jaiswal SK, Mohammed M, Dakora FD (2019) Symbiotic effectiveness and ecologically adaptive traits of native rhizobial symbionts of Bambara groundnut (Vigna subterranea L. Verdc.) in Africa and their relationship with phylogeny. Sci Rep 9:1–17

    CAS  Article  Google Scholar 

  14. 14.

    Grönemeyer JL, Reinhold-Hurek B (2018) Diversity of bradyrhizobia in subsahara Africa: a rich resource. Front Microbiol 9:1–8. https://doi.org/10.3389/fmicb.2018.02194

    Article  Google Scholar 

  15. 15.

    Jaiswal SK, Dakora FD (2019) Widespread distribution of highly adapted Bradyrhizobium species nodulating diverse legumes in Africa. Front Microbiol 10:310

  16. 16.

    Mair P, Wilcox R (2019) Robust statistical methods in R using the WRS2 Package. Behav Res Methods 1–25

  17. 17.

    Mohammed M, Jaiswal S, Dakora F (2018) Distribution and correlation between phylogeny and functional traits of cowpea (Vigna unguiculata L. Walp.)-nodulating microsymbionts from Ghana and South Africa. Sci Rep 12:1–19. https://doi.org/10.1038/s41598-018-36324-0

    CAS  Article  Google Scholar 

  18. 18.

    Graham PH (2003) Chapter 2 Ecology of the root-nodule bacteria of legumes. 23–58

  19. 19.

    Somasegaran P, Hoben H (1994) Handbook for rhizobia methods in legume-Rhizobium technology. Springer-Verlag, New York

    Book  Google Scholar 

  20. 20.

    Mohammed M, Jaiswal SK, Dakora FD (2019) Insights into the phylogeny, nodule functioning and biogeographic distribution of microsymbionts nodulating the orphan Kersting’s groundnut [Macrotyloma geocarpum (Harms) Marechal & Baudet] in African soils. Appl Environ Microbiol 85(11)

  21. 21.

    Vincent JM (1970) A manual for the practical study of root-nodule bacteria. Blackwell Scientific, Oxford

    Google Scholar 

  22. 22.

    Morón B, Soria-Díaz ME, Ault J, Verroios G, Noreen S, Rodríguez-Navarro DN, Gil-Serrano A, Thomas-Oates J, Megías M, Sousa C (2005) Low pH changes the profile of nodulation factors produced by Rhizobium tropici CIAT899. Chem Biol 12:1029–1040. https://doi.org/10.1016/j.chembiol.2005.06.014

    CAS  Article  PubMed  Google Scholar 

  23. 23.

    Raddadi N, Cherif A, Boudabous A, Daffonchio D (2008) Screening of plant growth promoting traits of Bacillus thuringiensis. Ann Microbiol 58:47–52. https://doi.org/10.1007/BF03179444

    CAS  Article  Google Scholar 

  24. 24.

    Gordon SA, Weber RP (1951) Colorimetric estimation of inodoleacetic acid. Plant Physiol 26:192–195. https://doi.org/10.1016/0003-2697(76)90514-5

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  25. 25.

    De Bruijn FJ (1992) Use of epetitive (REP and ERIC) sequences and the PCR to fingerprint the genomes of Rhizobium meliloti isolates and other soil bacteria. Appl Environ Microbiol 58:2180–2187

    Article  Google Scholar 

  26. 26.

    Rohlf F (2009) NTSYSpc: numerical taxonomy system. ver. 2.21c

  27. 27.

    Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. 95–98

  28. 28.

    Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729. https://doi.org/10.1093/molbev/mst197

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111–120. https://doi.org/10.1007/BF01731581

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution (N Y) 39:783–791

    Google Scholar 

  31. 31.

    R Core Team (2019) R: a Language and Environment for Statistical Computing

  32. 32.

    Howieson JG, Dilworth MJ (2016) Working with rhizobia. Australian Centre for International Agricultural Research, Canberra

  33. 33.

    Gyogluu C, Jaiswal SK, Kyei-Boahen S, Dakora FD (2018) Identification and distribution of microsymbionts associated with soybean nodulation in Mozambican soils. Syst Appl Microbiol 41:506–515. https://doi.org/10.1016/j.syapm.2018.05.003

    Article  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Priefer UB, Aurag J, Boesten B, Bouhmouch I, Defez R, Filali-Maltouf A, Miklis M, Moawad H, Mouhsine B, Prell J, Schlüter A, Senatore B (2001) Characterisation of Phaseolus symbionts isolated from Mediterranean soils and analysis of genetic factors related to pH tolerance. J Biotechnol 91:223–236. https://doi.org/10.1016/S0168-1656(01)00329-7

    CAS  Article  PubMed  Google Scholar 

  35. 35.

    Ghosh PK, De TK, Maiti TK (2015) Production and metabolism of indole acetic acid in root nodules and symbiont ( Rhizobium undicola ) isolated from root nodule of aquatic medicinal legume Neptunia oleracea Lour. https://doi.org/10.1155/2015/575067

  36. 36.

    Duran D, Ormeno-Orrillo E, Rey L et al (2014) Bradyrhizobium paxllaeri sp. nov. and Bradyrhizobium icense sp. nov., nitrogen-fixing rhizobial symbionts of Lima bean (Phaseolus lunatus L.) in Peru. Int J Syst Evol Microbiol 64:2072–2078. https://doi.org/10.1099/ijs.0.060426-0

    Article  PubMed  Google Scholar 

  37. 37.

    Stackebrandt E, Frederiksen W, Garrity GM et al (2002) Taxonomic report of the ad hoc committee for the re-evaluation of the species definition in bacteriology:1043–1047. https://doi.org/10.1099/ijs.0.02360-0.02360

  38. 38.

    Naamala J, Jaiswal SK, Dakora FD (2016) Microsymbiont diversity and phylogeny of native bradyrhizobia associated with soybean (Glycine max L. Merr.) nodulation in South African soils. Syst Appl Microbiol 39:336–344. https://doi.org/10.1016/j.syapm.2016.05.009

    Article  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Chidebe IN, Jaiswal SK, Dakora FD (2018) Distribution and phylogeny of microsymbionts associated with cowpea (Vigna unguiculata) nodulation in three agroecological regions of Mozambique. Appl Environ Microbiol 84. https://doi.org/10.1128/AEM.01712-17

  40. 40.

    Menna P, Hungria M (2011) Phylogeny of nodulation and nitrogen-fixation genes in Bradyrhizobium: supporting evidence for the theory of monophyletic origin, and spread and maintenance by both horizontal and vertical transfer. IJSEM. 61:3052–3067. https://doi.org/10.1099/ijs.0.028803-0

    CAS  Article  PubMed  Google Scholar 

  41. 41.

    Dabo M, Jaiswal SK, Dakora FD (2019) Phylogenetic evidence of allopatric speciation of bradyrhizobia nodulating cowpea (Vigna unguiculata L. walp) in South African and Mozambican soils. FEMS Microbiol Ecol 95:fiz067

    CAS  Article  Google Scholar 

  42. 42.

    Ardley JK, Reeve WG, O’hara GW et al (2013) Nodule morphology, symbiotic specificity and association with unusual rhizobia are distinguishing features of the genus Listia within the southern African crotalarioid clade Lotononis sl. Ann Bot 112:1–15

    CAS  Article  Google Scholar 

  43. 43.

    Thrall PH, Laine AL, Broadhurst LM, Bagnall DJ, Brockwell J (2011) Symbiotic effectiveness of rhizobial mutualists varies in interactions with native Australian legume genera. PLoS One 6:1–11. https://doi.org/10.1371/journal.pone.0023545

    CAS  Article  Google Scholar 

  44. 44.

    Etesami H, Alikhani HA, Akbari AA, others (2009) Evaluation of plant growth hormones production (IAA) ability by Iranian soils rhizobial strains and effects of superior strains application on wheat growth indexes. World Appl Sci J 6:1576–1584

    CAS  Google Scholar 

  45. 45.

    Etesami H, Alikhani HA, Hosseini HM (2015) Indole-3-acetic acid (IAA) production trait, a useful screening to select endophytic and rhizosphere competent bacteria for rice growth promoting agents. MethodsX 2:72–78. https://doi.org/10.1016/j.mex.2015.02.008

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We are grateful to the South African Research Chair in Agrochemurgy and Plant Symbioses, the National Research Foundation, and the Tshwane University of Technology for financial support to FDD’s research, and for a SARCHI Chair master bursary to STD. We also acknowledge Malkerns Research Station, Department of Grain Legumes in Eswatini for providing us seeds and land for the experiment.

Data Accessibility

The nucleotide sequences of all test genes were submitted in the NCBI Genbank database under accession numbers MT522377 - MT522397 (16S rRNA); MT514533 - MT514555 (atpD); MT514556 - MT514581 (glnII); MT514582 - MT514607 (gyrB); MT514634 - MT514658 (recA); and MT514608 - MT514633 (nifH).

Author information

Affiliations

Authors

Contributions

S.K.J and F.D.D conceived the ideas and developed the research questions. S.T.D. executed the research. M.M. recorded gas exchange data. S.K.J and S.T.D. analyzed the data and led the writing of the manuscript. All authors gave their final approval for publication.

Corresponding authors

Correspondence to Sanjay K. Jaiswal or Felix D. Dakora.

Ethics declarations

Conflict of Interest

The authors declare that they have no conflict of interest.

Supplementary Information

ESM 1

(DOCX 356 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Dlamini, S.T., Jaiswal, S.K., Mohammed, M. et al. Studies of Phylogeny, Symbiotic Functioning and Ecological Traits of Indigenous Microsymbionts Nodulating Bambara Groundnut (Vigna subterranea L. Verdc) in Eswatini . Microb Ecol 82, 688–703 (2021). https://doi.org/10.1007/s00248-021-01684-0

Download citation

Keywords

  • Genotypes
  • Agro-ecosystem
  • Housekeeping and symbiotic genes
  • Horizontal gene transfer
  • PCA
  • Correlation