Skip to main content
SpringerLink
Log in

PCR assay for direct specific detection of Bradyrhizobium elite strain BR 3262 in root nodule extracts of soil-grown cowpea

  • Methods Paper
  • Published:
Plant and Soil Aims and scope Submit manuscript

Abstract

Aims

Successful inoculation of legume crops with rhizobia depends on dominating nodule occupancy with highly efficient strains. The aim of this study was to develop a rapid and reliable conventional PCR methodology to specifically detect an elite Bradyrhizobium strain in root nodule extracts from soil-grown cowpea plants.

Methods

The draft genome sequence of Bradyrhizobium pachyrhizi BR 3262 was compared to the closely related strain PAC 48T. BR 3262-specific regions were selected to design specific primer pairs, which were tested with respect to PCR amplification specificity and efficiency on extracted DNA, bacterial cells and root nodules from cowpea plants grown under gnotobiotic conditions and in soil.

Results

Eleven designed primer pairs were specific for BR 3262 amplification and two of them (pairs 2645 and 2736) were highly sensitive and selected for further analyses. Experiments with gnotobiotic and soil-grown plants showed that both primer pairs were suitable to reliably determine nodule occupancy and confirmed the competitiveness of strain BR 3262 in natural soil.

Conclusions

Primer pairs 2645 and 2736 are novel tools to accompany the fate of strain BR 3262 in inoculation experiments of cowpea in soil. This strategy should be applicable to other rhizobium/legume symbioses in the field.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  • Al-Hassan RM, Diao X (2007) Regional disparities in Ghana: policy options and public investment implications. International Food Policy Research Institute Washington, DC.

  • Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Baker G, Smith JJ, Cowan DA (2003) Review and re-analysis of domain-specific 16S primers. J Microbiol Methods 55:541–555

    Article  CAS  PubMed  Google Scholar 

  • Boddey RM, Fosu M, Atakora WK, Miranda CHB, Boddey LH, Guimaraes AP, Ahiabor BDK (2016) Cowpea (Vigna unguiculata) crops in Africa can respond to inoculation with rhizobium. Exp Agric:1–10. doi:10.1017/s0014479716000594

  • Bogino P, Banchio E, Bonfiglio C, Giordano W (2008) Competitiveness of a Bradyrhizobium sp. strain in soils containing indigenous rhizobia. Curr Microbiol 56:66–72

    Article  CAS  PubMed  Google Scholar 

  • Brockwell J, Bottomley PJ, Thies JE (1995) Manipulation of rhizobia microflora for improving legume productivity and soil fertility: a critical assessment. Plant Soil 174:143–180

  • Bushby HVA (1981) Quantitative estimation of rhizobia in non-sterile soil using antibiotics and fungicides. Soil Biol Biochem 13:237–239

    Article  Google Scholar 

  • Costa EM, Nóbrega RSA, Martins LDV, Amaral FHC, Moreira FDS (2011) Nodulação e produtividade de Vigna unguiculata (L.) Walp. por cepas de rizóbio em Bom Jesus, PI. Rev Ciênc Agron 42:1–7

    Article  Google Scholar 

  • Couillerot O, Poirier MA, Prigent-Combaret C, Mavingui P, Caballero-Mellado J, Moënne-Loccoz Y (2010) Assessment of SCAR markers to design real-time PCR primers for rhizosphere quantification of Azospirillum brasilense phytostimulatory inoculants of maize. J Appl Microbiol 109:528–538

    CAS  PubMed  Google Scholar 

  • de Alcantara MC, Xavier GR, Rumjanek NG, de Moura RM, dos Santos CJ (2014) Eficiencia simbiotica de progenitores de cultivares brasileiras de feij? o-caupi. Rev Ciênc Agron 45:1

    Article  Google Scholar 

  • de Almeida AL, de Alcântara RM, Nóbrega RS, Nóbrega JC, Leite LF, da Silva JA (2010) Produtividade do feijão-caupi cv BR 17 Gurguéia inoculado com bactérias diazotróficas simbióticas no Piauí. Braz J Agric Sci/Rev Bras Ciênc Agrár 5

  • de Freitas ADS, Silva AF, Sampaio EVDSB (2012) Yield and biological nitrogen fixation of cowpea varieties in the semi-arid region of Brazil. Biomass Bioenergy 45:109–114

    Article  Google Scholar 

  • Deaker R, Roughley RJ, Kennedy IR (2004) Legume seed inoculation technology—a review. Soil Biol Biochem 36:1275–1288

    Article  CAS  Google Scholar 

  • Dowling D, Broughton W (1986) Competition for nodulation of legumes. Annu Rev Microbiol 40:131–157

    Article  CAS  PubMed  Google Scholar 

  • Fernandes P Jr, da Silva C, de Oliveira P, Rumjanek N, Martins L, Xavier G (2012) Performance of polymer compositions as carrier to cowpea rhizobial inoculant formulations: survival of rhizobia in pre-inoculated seeds and field efficiency. Afr J Biotechnol 11:2945–2951

    CAS  Google Scholar 

  • Ferreira DF (2008) SISVAR: um programa para análises e ensino de estatística. Revista symposium. Lavras

  • Ferreira LVM, Nóbrega RSA, Nóbrega JCA, de Aguiar FL, de Souza Moreira FM, Pacheco LP (2013) Biological nitrogen fixation in production of Vigna unguiculata (L.) Walp, family farming in Piauí, Brazil. J Agric Sci 5:153

    Google Scholar 

  • Giller KE (2001) Nitrogen fixation in tropical cropping systems. Cabi

  • Gillings M, Holley M (1997) Amplification of anonymous DNA fragments using pairs of long primers generates reproducible DNA fingerprints that are sensitive to genetic variation. Electrophoresis 18:1512–1518

    Article  CAS  PubMed  Google Scholar 

  • González-Andrés F, Ortiz JM (1998) Biodiversity of rhizobia nodulating Genista monspessulana and Genista linifolia in Spain. N Z J Agric Res 41:585–594

    Article  Google Scholar 

  • Harrison SP, Mytton LR, Skøt L, Dye M, Cresswell A (1992) Characterisation of rhizobium isolates by amplification of DNA polymorphisms using random primers. Can J Microbiol 38:1009–1015

    Article  CAS  PubMed  Google Scholar 

  • Josey D, Beynon J, Johnston A, Beringer J (1979) Strain identification in rhizobium using intrinsic antibiotic resistance. J Appl Bacteriol 46:343–350

    Article  Google Scholar 

  • Khalid A, Arshad M, Zahir Z (2004) Screening plant growth-promoting rhizobacteria for improving growth and yield of wheat. J Appl Microbiol 96:473–480

    Article  CAS  PubMed  Google Scholar 

  • Kremer RJ, Peterson HL (1982) Nodulation efficiency of legume inoculation as determined by intrinsic antibiotic resistance. Appl Environ Microbiol 43:636–642

    CAS  PubMed  PubMed Central  Google Scholar 

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

  • Lee I, Kim YO, Park S-C, Chun J (2016) OrthoANI: an improved algorithm and software for calculating average nucleotide identity. Int J Syst Evol Microbiol 66:1100–1103

    Article  CAS  Google Scholar 

  • Lupwayi N, Olsen P, Sande E, Keyser H, Collins M, Singleton P, Rice W (2000) Inoculant quality and its evaluation. Field Crop Res 65:259–270

    Article  Google Scholar 

  • Moawad M, Schmidt E (1987) Occurrence and nature of mixed infections in nodules of field-grown soybeans (Glycine max). Biol Fertil Soils 5:112–114

    Article  Google Scholar 

  • Mpepereki S, Wollum A II (1991) Diversity of indigenous Bradyrhizobium japonicum in North Carolina soils. Biol Fertil Soils 11:121–127

    Article  Google Scholar 

  • Mpepereki S, Javaheri F, Davis P, Giller K (2000) Soyabeans and sustainable agriculture: promiscuous soyabeans in southern Africa. Field Crop Res 65:137–149

    Article  Google Scholar 

  • Ndakidemi P, Dakora F, Nkonya E, Ringo D, Mansoor H (2006) Yield and economic benefits of common bean (Phaseolus vulgaris) and soybean (Glycine max) inoculation in northern Tanzania. Anim Prod Sci 46:571–577

    Article  Google Scholar 

  • Niemann S, Dammann-Kalinowski T, Nagel A, Pühler A, Selbitschka W (1999) Genetic basis of enterobacterial repetitive intergenic consensus (ERIC)-PCR fingerprint pattern in Sinorhizobium meliloti and identification of S. Meliloti employing PCR primers derived from an ERIC-PCR fragment. Arch Microbiol 172:22–30

    Article  CAS  PubMed  Google Scholar 

  • Norris D, Mannetje L (1964) The symbiotic specialization of African Trifolium spp. in relation to their taxonomy and their agronomic use. East Afr Agric For J 29:214–235

    Google Scholar 

  • Overbeek R, Olson R, Pusch GD, Olsen GJ, Davis JJ, Disz T, Edwards RA, Gerdes S, Parrello B, Shukla M (2014) The SEED and the rapid annotation of microbial genomes using subsystems technology (RAST). Nucleic Acids Res 42:D206–D214

    Article  CAS  PubMed  Google Scholar 

  • Rogozin IB, Makarova KS, Natale DA, Spiridonov AN, Tatusov RL, Wolf YI, Yin J, Koonin EV (2002) Congruent evolution of different classes of non-coding DNA in prokaryotic genomes. Nucleic Acids Res 30:4264–4271

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Rutherford K, Parkhill J, Crook J, Horsnell T, Rice P, Rajandream M-A, Barrell B (2000) Artemis: sequence visualization and annotation. Bioinformatics 16:944–945

    Article  CAS  PubMed  Google Scholar 

  • Sanginga N, Dashiell K, Okogun J, Thottappilly G (1997) Nitrogen fixation and N contribution by promiscuous nodulating soybeans in the southern Guinea savanna of Nigeria. Plant Soil 195:257–266

    Article  CAS  Google Scholar 

  • Simões-Araújo JL, Leite J, Rouws LFM, Passos SR, Xavier GR, Rumjanek NG, Zilli JÉ (2016) Draft genome sequence of Bradyrhizobium sp. strain BR 3262, an effective microsymbiont recommended for cowpea inoculation in Brazil. Braz J Microbiol 47:783–784

  • Somasegaran P, Hoben HJ (2012) Handbook for rhizobia: methods in legume-rhizobium technology. Springer Science & Business Media, New York, pp 450

  • Stets MI, Alqueres SMC, Souza EM, de Oliveira PF, Schmid M, Hartmann A, Cruz LM (2015) Quantification of Azospirillum brasilense FP2 bacteria in wheat roots by strain-specific quantitative PCR. Appl Environ Microbiol 81:6700–6709

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tamura K (1992) Estimation of the number of nucleotide substitutions when there are strong transition-transversion and G+ C-content biases. Mol Biol Evol 9:678–687

    CAS  PubMed  Google Scholar 

  • Thies JE, Wijkstra G, Ronson C (1999) What does strain persistence really mean? In: Martínez E, Hernández G (eds) Highlights of nitrogen fixation research. Springer, New York, pp. 85–90

  • Thies J, Holmes E, Vachot A (2001) Application of molecular techniques to studies in rhizobium ecology: a review. Anim Prod Sci 41:299–319

    Article  CAS  Google Scholar 

  • Thuita M, Pypers P, Herrmann L, Okalebo RJ, Othieno C, Muema E, Lesueur D (2012) Commercial rhizobial inoculants significantly enhance growth and nitrogen fixation of a promiscuous soybean variety in Kenyan soils. Biol Fertil Soils 48:87–96

    Article  Google Scholar 

  • Turco RF, Moorman TB, Bezdicek DF (1986) Effectiveness and competitiveness of spontaneous antibiotic-resistant mutants of rhizobium leguminosarum and rhizobium japonicum. Soil Biol Biochem 18:259–262

    Article  Google Scholar 

  • Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M, Rozen SG (2012) Primer3—new capabilities and interfaces. Nucleic Acids Res 40:e115–e115

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • van Berkum P, Fuhrmann JJ (2000) Evolutionary relationships among the soybean bradyrhizobia reconstructed from 16S rRNA gene and internally transcribed spacer region sequence divergence. Int J Syst Evol Microbiol 50:2165–2172

    Article  PubMed  Google Scholar 

  • van Berkum P, Elia P, Song Q, Eardly BD (2012) Development and application of a multilocus sequence analysis method for the identification of genotypes within genus Bradyrhizobium and for establishing nodule occupancy of soybean (Glycine max L. Merr). Mol Plant-Microbe Interact 25:321–330

  • Versalovic J, Koeuth T, Lupski R (1991) Distribution of repetitive DNA sequences in eubacteria and application to finerpriting of bacterial enomes. Nucleic Acids Res 19:6823–6831

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Versalovic J, Schneider M, De Bruijn FJ, Lupski JR (1994) Genomic fingerprinting of bacteria using repetitive sequence-based polymerase chain reaction. Methods Mol Cell Biol 5:25–40

    CAS  Google Scholar 

  • Vincent JM (1970) A manual for the practical study of the root-nodule bacteria. Blackwell Scientific, pp. 164

  • Woomer P, Bennett J, Yost R (1990) Overcoming the inflexibility of most-probable-number procedures. Agron J 82:349–353

    Article  Google Scholar 

  • Yokoyama T, Ando S, Tsuchiya K (1999) Serological properties and intrinsic antibiotic resistance of soybean bradyrhizobia isolated from Thailand. Soil Sci Plant Nutr 45:505–515

    Article  Google Scholar 

  • Zilli J, Ferreira E, Neves M, Rumjanek N (1999) Efficiency of fast-growing rhizobia capable of nodulating cowpea. An Acad Bras Cienc 71:553–560

    Google Scholar 

  • Zilli J, Marson LC, Marson BF, Rumjanek NG, Xavier GR (2009) Contribuição de estirpes de rizóbio para o desenvolvimento e produtividade de grãos de feijão-caupi em Roraima. Acta Amazon 39:749–758

    Article  Google Scholar 

Download references

Acknowledgements

We are grateful for the financial assistance from Alliance for Green Revolution in Africa (AGRA) through the Soil Health Project (Grant Number 2013 SHP025) and Africa-Brazil Agricultural Innovation Marketplace project (ID 1705). We also express our gratitude to Embrapa Agrobiologia for the technical and infrastructural assistance.

Author information

Authors and Affiliations

  1. Embrapa Agrobiologia, Rodovia BR 465 km 07, 23891-000, Seropédica, Rio de Janeiro, Brazil

    Ophelia Osei, Jean L. Simões Araújo, Jerri E. Zilli, Robert M. Boddey & Luc F. M. Rouws

  2. Department of Crop and Soil Sciences, Faculty of Agriculture, Kwame Nkrumah University of Science and Technology, PMB, Kumasi, Ghana

    Ophelia Osei

  3. CSIR, Savannah Agricultural Research Institute, P.O.Box 52, Tamale, Ghana

    Benjamin D. K. Ahiabor

  4. Department of Theoretical and Applied Biology, Kwame Nkrumah University of Science and Technology, PMB, Kumasi, Ghana

    Robert C. Abaidoo

  5. International Institute of Tropical Agriculture, PMB, 5320, Ibadan, Nigeria

    Robert C. Abaidoo

Authors
  1. Ophelia Osei
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jean L. Simões Araújo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jerri E. Zilli
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Robert M. Boddey
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Benjamin D. K. Ahiabor
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Robert C. Abaidoo
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Luc F. M. Rouws
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Luc F. M. Rouws.

Additional information

Responsible Editor: Euan K. James.

Electronic supplementary material

Fig. S1

Molecular phylogenetic analysis of strain B. pachyrhizi strains BR 3262 and PAC48T (in bold) and other Bradyrhizobium strains based on partial recA gene sequences. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura 3-parameter model (Tamura 1992). Bootstrap values are shown when the represented relationships were observed in at least 50% of 500 pseudoreplicates. The scale bar represents 0.02 nucleotides substitutions per site. There was a total of 375 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 (Kumar et al. 2016). Genbank accession numbers are indicated between parentheses. (DOCX 392 kb)

Fig. S2

Graphical representation of the genomic context of the BR 3262-specific target sequences for PCR amplification. Graphical representations of primer target sites were generated using the Artemis software (Rutherford et al. 2000) after re-annotation using rhe RAST (Overbeek et al. 2014). Amplicons are represented by yellow boxes identified by ‘Amp’ followed by the primer pair identification number as provided in Table 1. Blue arrows represent annotated protein functions. (PPTX 345 kb)

Table S1

General characteristics of (draft) genome sequences used in this study (DOCX 12 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Osei, O., Simões Araújo, J.L., Zilli, J.E. et al. PCR assay for direct specific detection of Bradyrhizobium elite strain BR 3262 in root nodule extracts of soil-grown cowpea. Plant Soil 417, 535–548 (2017). https://doi.org/10.1007/s11104-017-3271-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11104-017-3271-4

Keywords

Search

Navigation