Applied Microbiology and Biotechnology

, Volume 102, Issue 12, pp 5265–5278 | Cite as

Evaluation of MALDI-TOF mass spectrometry for the competitiveness analysis of selected indigenous cowpea (Vigna unguiculata L. Walp.) Bradyrhizobium strains from Kenya

  • Samuel Mathu Ndungu
  • Monika M. Messmer
  • Dominik Ziegler
  • Moses Thuita
  • Bernard Vanlauwe
  • Emmanuel Frossard
  • Cécile Thonar
Applied microbial and cell physiology


Cowpea N2 fixation and yield can be enhanced by selecting competitive and efficient indigenous rhizobia. Strains from contrasting agro-ecologies of Kilifi and Mbeere (Kenya) were screened. Two pot experiments were established consisting of 13 Bradyrhizobium strains; experiment 1 (11 Mbeere + CBA + BK1 from Burkina Faso), experiment 2 (12 Kilifi + CBA). Symbiotic effectiveness was assessed (shoot biomass, SPAD index and N uptake). Nodule occupancy of 13 simultaneously co-inoculated strains in each experiment was analyzed by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) to assess competitiveness. Strains varied in effectiveness and competitiveness. The four most efficient strains were further evaluated in a field trial in Mbeere during the 2014 short rains. Strains from bacteroids of cowpea nodules from pot and field experiments were accurately identified as Bradyrhizobium by MALDI-TOF based on the SARAMIS™ database. In the field, abundant indigenous populations 7.10 × 103 rhizobia g−1 soil, outcompeted introduced strains. As revealed by MALDI-TOF, indigenous strains clustered into six distinct groups (I, II, III, IV, V and VI), group III were most abundant occupying 80% of nodules analyzed. MALDI-TOF was rapid, affordable and reliable to identify Bradyrhizobium strains directly from nodule suspensions in competition pot assays and in the field with abundant indigenous strains thus, its suitability for future competition assays. Evaluating strain competitiveness and then symbiotic efficacy is proposed in bioprospecting for potential cowpea inoculant strains.


Bradyrhizobium Cowpea Symbiotic effectiveness Nodule occupancy Protein profile Bacteroid 



The authors thank Dr. Laurie Paule Schönholzer, Dr. Seher Bahar Aciksöz Özden, Monika Macsai, Carla Mosimann, Silvana Niedermann and Sämi Bickel for the technical support during experiments and Dr. Federica Tamburini for N analysis. Winnie Kimutai and Silas Kiragu are acknowledged for their support during field trials. Thanks to Dr. Abidine Traore for the cowpea nodules from which strain BK1 was isolated, and MEA for facilitating Biofix inoculant from which CBA (CB 1015) strain was isolated. Valuable input by the anonymous reviewers and the editor for improving this manuscript is also acknowledged.


This research was supported by funding from ETH Zurich Engineering for Development (E4D) scholarship through the Sawiris Foundation for Social development (Grant number 2-71060-13).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2018_9005_MOESM1_ESM.pdf (402 kb)
ESM 1 (PDF 402 kb)


  1. Ampomah OY, Ofori-Ayeh E, Solheim B, Svenning MM (2008) Host range, symbiotic effectiveness and nodulation competitiveness of some indigenous cowpea bradyrhizobia isolates from the transitional savanna zone of Ghana. Afr J Biotechnol 7(8):988–996Google Scholar
  2. Asad S, Malik K, Hafeez F (1991) Competition between inoculated and indigenous Rhizobium/Bradyrhizobium spp. strains for nodulation of grain and fodder legumes in Pakistan. Biol Fertil Soils 12(2):107–111CrossRefGoogle Scholar
  3. Awonaike K, Kumarasinghe K, Danso S (1990) Nitrogen fixation and yield of cowpea (Vigna unguiculata) as influenced by cultivar and Bradyrhizobium strain. Field Crops Res 24(3–4):163–171CrossRefGoogle Scholar
  4. Batista L, Irisarri P, Rebuffo M, Jose Cuitino M, Sanjuan J, Monza J (2015) Nodulation competitiveness as a requisite for improved rhizobial inoculants of Trifolium pratense. Biol Fertil Soils 51(1):11–20. CrossRefGoogle Scholar
  5. Biswas S, Rolain J-M (2013) Use of MALDI-TOF mass spectrometry for identification of bacteria that are difficult to culture. J Microbiol Methods 92(1):14–24CrossRefPubMedGoogle Scholar
  6. Bottomley PJ (1992) Ecology of Bradyrhizobium and Rhizobium. In: Stacey G, Burris RH, Evans HJ (eds) Biological nitrogen fixation. Chapman and Hall, New York, pp 293–348Google Scholar
  7. Bouyoucos GJ (1962) Hydrometer method improved for making particle size analyses of soils. Agron J 54(5):464–465CrossRefGoogle Scholar
  8. Bremner J (1960) Determination of nitrogen in soil by the Kjeldahl method. J Agric Sci 55(1):11–33CrossRefGoogle Scholar
  9. Brockwell J (1963) Accuracy of a plant-infection technique for counting populations of Rhizobium trifolii. Appl Microbiol 11(5):377–383PubMedPubMedCentralGoogle Scholar
  10. Broughton WJ, Dilworth MJ (1970) Plant nutrient solutions. In: Somasegaran P, Hoben H (eds) Handbook for rhizobia: methods in legume-Rhizobium technology. Niftal Project. University of Hawaii, Honolulu, pp 245–249Google Scholar
  11. Checcucci A, Azzarello E, Bazzicalupo M, Galardini M, Lagomarsino A, Mancuso S, Marti L, Marzano MC, Mocali S, Squartini A (2016) Mixed nodule infection in Sinorhizobium melilotiMedicago sativa symbiosis suggest the presence of cheating behavior. Front Plant Sci 7:835. CrossRefPubMedPubMedCentralGoogle Scholar
  12. da Costa EM, de Almeida Ribeiro PR, de Lima W, Farias TP, de Souza Moreira FM (2017) Lima bean nodulates efficiently with Bradyrhizobium strains isolated from diverse legume species. Symbiosis 73(2):125–133. CrossRefGoogle Scholar
  13. Danso SKA, Owiredu JD (1988) Competitiveness of introduced and indigenous cowpea Bradyrhizobium strains for nodule formation on cowpeas Vigna unguiculata (L.) walp in 3 soils. Soil Biol Biochem 20(3):305–310. CrossRefGoogle Scholar
  14. de Almeida Ribeiro PR, dos Santos JV, da Costa EM, Lebbe L, Assis ES, Louzada MO, Guimarães AA, Willems A, de Souza Moreira FM (2015) Symbiotic efficiency and genetic diversity of soybean bradyrhizobia in Brazilian soils. Agric Ecosyst Environ 212:85–93CrossRefGoogle Scholar
  15. de Freitas ADS, Fernandes Silva A, Valadares de Sá Barretto Sampaio E (2012) Yield and biological nitrogen fixation of cowpea varieties in the semi-arid region of Brazil. Biomass Bioenergy 45:109–114. CrossRefGoogle Scholar
  16. Deaker R, Roughley RJ, Kennedy IR (2004) Legume seed inoculation technology—a review. Soil Biol Biochem 36(8):1275–1288CrossRefGoogle Scholar
  17. Ehlers J, Hall A (1997) Cowpea (Vigna unguiculata L. Walp.). Field Crops Res 53(1):187–204CrossRefGoogle Scholar
  18. FAOSTATS (2014) PUblisher. Accessed 28 Feb 2017
  19. Fening JO, Danso SKA (2002) Variation in symbiotic effectiveness of cowpea bradyrhizobia indigenous to Ghanaian soils. Appl Soil Ecol 21(1):23–29. CrossRefGoogle Scholar
  20. Ferreira E, Marques J (1992) Selection of Portuguese Rhizobium leguminosarum bv. trifolii strains for production of legume inoculants. Plant Soil 147(1):151–158CrossRefGoogle Scholar
  21. Giller KE (2001) Nitrogen fixation in tropical cropping systems, 2nd edn. CAB International, WallingfordCrossRefGoogle Scholar
  22. Gopalakrishnan S, Sathya A, Vijayabharathi R, Varshney RK, Gowda CL, Krishnamurthy L (2015) Plant growth promoting rhizobia: challenges and opportunities. 3 Biotech 5(4):355–377CrossRefPubMedGoogle Scholar
  23. Guimarães AA, Duque Jaramillo PM, Abrahao Nobrega RS, Florentino LA, Silva KB, de Souza Moreira FM (2012) Genetic and symbiotic diversity of ditrogen-fixing bacteria isolated from agricultural soils in the western Amazon by using cowpea as the trap plant. Appl Environ Microbiol 78(18):6726–6733. CrossRefGoogle Scholar
  24. Howieson J, Dilworth M (2016) Working with rhizobia. Australian Centre for International Agricultural Research, CanberraGoogle Scholar
  25. Howieson J, Loi A, Carr S (1995) Biserrula pelecinus L.—a legume pasture species with potential for acid, duplex soils which is nodulated by unique root-nodule bacteria. Aust J Agric Res 46(5):997–1009CrossRefGoogle Scholar
  26. Hungria M, Franchini JC, Campo RJ, Crispino CC, Moraes JZ, Sibaldelli RN, Mendes IC, Arihara J (2006) Nitrogen nutrition of soybean in Brazil: contributions of biological N2 fixation and N fertilizer to grain yield. Can J Plant Sci 86(4):927–939CrossRefGoogle Scholar
  27. Jaetzold R, Schimdt H (1983) Farm management handbook: natural and farm management information, vol II/B. Ministry of agriculture, NairobiGoogle Scholar
  28. Jaetzold R, Schmidt H, Hornetz B, Shisanya C (2006) Ministry of agriculture farm management handbook of Kenya VOL. II-Part C Subpart C1. Ministry of agriculture, NairobiGoogle Scholar
  29. Ji ZJ, Yan H, Cui QG, Wang ET, Chen WF, Chen WX (2017) Competition between rhizobia under different environmental conditions affects the nodulation of a legume. Syst Appl Microbiol 40:114–119CrossRefPubMedGoogle Scholar
  30. Karanja D, Githunguri C, M'Ragwa L, Mulwa D, Mwiti S (2006) Variety, characteristics and production guidelines of traditional food crops. KARI Katumani Res Centre 5:9–14Google Scholar
  31. Kimiti JM, Odee DW (2010) Integrated soil fertility management enhances population and effectiveness of indigenous cowpea rhizobia in semi-arid eastern Kenya. Appl Soil Ecol 45(3):304–309CrossRefGoogle Scholar
  32. Krasova-Wade T, Diouf O, Ndoye I, Sall CE, Braconnier S, Neyra M (2006) Water-condition effects on rhizobia competition for cowpea nodule occupancy. Afr J Biotechnol 5(16):1457–1463Google Scholar
  33. Langyintuo A, Lowenberg-DeBoer J, Faye M, Lambert D, Ibro G, Moussa B, Kergna A, Kushwaha S, Musa S, Ntoukam G (2003) Cowpea supply and demand in West and Central Africa. Field Crops Res 82(2):215–231CrossRefGoogle Scholar
  34. Law IJ, Botha WF, Majaule UC, Phalane FL (2007) Symbiotic and genomic diversity of ‘cowpea’ bradyrhizobia from soils in Botswana and South Africa. Biol Fertil Soils 43(6):653–663. CrossRefGoogle Scholar
  35. Martins L, Xavier G, Rangel F, Ribeiro J, Neves M, Morgado L, Rumjanek N (2003) Contribution of biological nitrogen fixation to cowpea: a strategy for improving grain yield in the semi-arid region of Brazil. Biol Fertil Soils 38(6):333–339CrossRefGoogle Scholar
  36. Mathu S, Herrmann L, Pypers P, Matiru V, Mwirichia R, Lesueur D (2012) Potential of indigenous bradyrhizobia versus commercial inoculants to improve cowpea (Vigna unguiculata L. Walp.) and green gram (Vigna radiata L. Wilczek.) yields in Kenya. Soil Sci Plant Nutr 58(6):750–763. CrossRefGoogle Scholar
  37. McInnes A, Haq K (2007) Contributions of rhizobia to soil nitrogen fertility. In: Abbott LK, Murphy DV (eds) Soil biological fertility—a key to sustainable land use in agriculture. Springer, Dordrecht, pp 99–128Google Scholar
  38. Mehlich A, Pinkerton A, Robertson W, Kempton R (1962) Mass analysis methods for soil fertility evaluation. Ministry of agriculture, NairobiGoogle Scholar
  39. Mehta A, Silva LP (2015) MALDI-TOF MS profiling approach: how much can we get from it? Front Plant Sci 6(184).
  40. Müller P, Pflüger V, Wittwer M, Ziegler D, Chandre F, Simard F, Lengeler C (2013) Identification of cryptic Anopheles mosquito species by molecular protein profiling. PLoS One 8(2):e57486. CrossRefPubMedPubMedCentralGoogle Scholar
  41. Ndiso J, Chemining’wa G, Olubayo F, Saha H (2015) Participatory selection of cowpea varieties in Kilifi county of Kenya. IJPSS 4(2):1–10 IJPSS.21843CrossRefGoogle Scholar
  42. Ndungu SM, Messmer MM, Ziegler D, Gamper HA, Mészáros É, Thuita M, Vanlauwe B, Frossard E, Thonar C (2018) Cowpea (Vigna unguiculata L. Walp) hosts several widespread bradyrhizobial root nodule symbionts across contrasting agro-ecological production areas in Kenya. Agric Ecosyst Environ.
  43. Novák K (2011) Determination of symbiotic nodule occupancy in the model Vicia tetrasperma using a fluorescence scanner. Ann Bot 107(4):709–715CrossRefPubMedPubMedCentralGoogle Scholar
  44. O’Hara G (2001) Nutritional constraints on root nodule bacteria affecting symbiotic nitrogen fixation: a review. Anim Prod Sci 41(3):417–433CrossRefGoogle Scholar
  45. Olsen SR (1954) Estimation of available phosphorus in soils by extraction with sodium bicarbonate. United States department of agriculture, WashingtonGoogle Scholar
  46. Pérez-Giménez J, Quelas JI, Lodeiro AR (2011) Competition for nodulation. In: El-Shemy HA (ed) Soybean physiology and biochemistry. InTech, Rijeka, pp 139–166Google Scholar
  47. Pistorio M, Balagué L, Del Papa M, Pich-Otero A, Lodeiro A, Hozbor D, Lagares A (2002) Construction of a Sinorhizobium meliloti strain carrying a stable and non-transmissible chromosomal single copy of the green fluorescent protein GFP-P64L/S65T. FEMS Microbiol Lett 214(2):165–170CrossRefPubMedGoogle Scholar
  48. Pule-Meulenberg F, Belane AK, Krasova-Wade T, Dakora FD (2010) Symbiotic functioning and bradyrhizobial biodiversity of cowpea (Vigna unguiculata L. Walp.) in Africa. BMC Microbiol 10:89. CrossRefPubMedPubMedCentralGoogle Scholar
  49. Rengel Z (2002) Breeding for better symbiosis. Plant Soil 245(1):147–162CrossRefGoogle Scholar
  50. Rufini M, Da Silva MAP, Ferreira PAA, de Souza Cassetari A, Soares BL, De Andrade MJB, de Souza Moreira FM (2014) Symbiotic efficiency and identification of rhizobia that nodulate cowpea in a Rhodic Eutrudox. Biol Fertil Soils 50(1):115–122CrossRefGoogle Scholar
  51. Sadowsky MJ, Tully RE, Cregan PB, Keyser HH (1987) Genetic diversity in Bradyrhizobium japonicum serogroup-123 and its relation to genotype-specific nodulation of soybean. Appl Environ Microbiol 53(11):2624–2630PubMedPubMedCentralGoogle Scholar
  52. Singh B, Ajeigbe H, Tarawali SA, Fernandez-Rivera S, Abubakar M (2003) Improving the production and utilization of cowpea as food and fodder. Field Crops Res 84(1):169–177CrossRefGoogle Scholar
  53. Slattery J, Coventry D, Slattery W (2001) Rhizobial ecology as affected by the soil environment. Anim Prod Sci 41(3):289–298CrossRefGoogle Scholar
  54. Somasegaran P, Hoben HJ (1994) Handbook for rhizobia: methods in legume-Rhizobium technology. Springer-Verlag, NewYorkCrossRefGoogle Scholar
  55. Spriggs AC, Dakora FD (2009) Assessing the suitability of antibiotic resistance markers and the indirect ELISA technique for studying the competitive ability of selected Cyclopia Vent. rhizobia under glasshouse and field conditions in South Africa. BMC Microbiol 9:142CrossRefPubMedPubMedCentralGoogle Scholar
  56. Thies JE, Singleton PW, Bohlool BB (1991) Influence of the size of indigenous rhizobial populations on establishment and symbiotic performance of introduced rhizobia on field-grown legumes. Appl Environ Microbiol 57(1):19–28PubMedPubMedCentralGoogle Scholar
  57. Thrall PH, Laine A-L, Broadhurst LM, Bagnall DJ, Brockwell J (2011) Symbiotic effectiveness of rhizobial mutualists varies in interactions with native Australian legume genera. PLoS One 6(8):e23545CrossRefPubMedPubMedCentralGoogle Scholar
  58. Uhlik O, Strejcek M, Junkova P, Sanda M, Hroudova M, Vlcek C, Mackova M, Macek T (2011) Matrix-assisted laser desorption ionization (MALDI)-time of flight mass spectrometry-and MALDI biotyper-based identification of cultured biphenyl-metabolizing bacteria from contaminated horseradish rhizosphere soil. Appl Environ Microbiol 77(19):6858–6866CrossRefPubMedPubMedCentralGoogle Scholar
  59. Ulzen J, Abaidoo RC, Mensah NE, Masso C, AbdelGadir AH (2016) Bradyrhizobium inoculants enhance grain yields of soybean and cowpea in northern Ghana. Front Plant Sci 7:1770CrossRefPubMedPubMedCentralGoogle Scholar
  60. Van Berkum P (1990) Evidence for a third uptake hydrogenase phenotype among the soybean bradyrhizobia. Appl Environ Microbiol 56(12):3835–3841PubMedPubMedCentralGoogle Scholar
  61. Vincent JM (1970) A manual for the practical study of the root-nodule bacteria. Blackwell Scientific Publications, OxfordGoogle Scholar
  62. Wade TK, Le Quere A, Laguerre G, N'Zoue A, Ndione J-A, dorego F, Sadio O, Ndoye I, Neyra M (2014) Eco-geographical diversity of cowpea bradyrhizobia in Senegal is marked by dominance of two genetic types. Syst Appl Microbiol 37(2):129–139. CrossRefPubMedGoogle Scholar
  63. Walkley A, Black IA (1934) An examination of the Degtjareff method for determining soil organic matter, and a proposed modification of the chromic acid titration method. Soil Sci 37(1):29–38. CrossRefGoogle Scholar
  64. Wittwer M, Heim J, Schar M, Dewarrat G, Schurch N (2011) Tapping the potential of intact cell mass spectrometry with a combined data analytical approach applied to Yersinia spp.: detection, differentiation and identification of Y. pestis. Syst Appl Microbiol 34(1):12–19. CrossRefPubMedGoogle Scholar
  65. Wongphatcharachai M, Wang P, Staley C, Chun CL, Ferguson JA, Moncada KM, Sheaffer CC, Sadowsky MJ (2015) Site-specific distribution and competitive ability of indigenous bean-nodulating rhizobia isolated from organic fields in Minnesota. J Biotechnol 214:158–168CrossRefPubMedGoogle Scholar
  66. Xu KW, Penttinen P, Chen YX, Chen Q, Zhang X (2013) Symbiotic efficiency and phylogeny of the rhizobia isolated from Leucaena leucocephala in arid–hot river valley area in Panxi, Sichuan, China. Appl Microbiol Biotechnol 97(2):783–793CrossRefPubMedGoogle Scholar
  67. Zahran HH (1999) Rhizobium-legume symbiosis and nitrogen fixation under severe conditions and in an arid climate. Microbiol Mol Biol Rev 63(4):968–989PubMedPubMedCentralGoogle Scholar
  68. Zhang JJ, Yu T, Lou K, Mao PH, Wang ET, Chen WF, Chen WX (2014) Genotypic alteration and competitive nodulation of Mesorhizobium muleiense against exotic chickpea rhizobia in alkaline soils. Syst Appl Microbiol 37(7):520–524CrossRefPubMedGoogle Scholar
  69. Ziegler D, Mariotti A, Pflueger V, Saad M, Vogel G, Tonolla M, Perret X (2012) In Situ identification of plant-invasive bacteria with MALDI-TOF mass spectrometry. PLoS One 7(5):e31789. CrossRefGoogle Scholar
  70. Ziegler D, Pothier JF, Ardley J, Fossou RK, Pflüger V, De Meyer S, Vogel G, Tonolla M, Howieson J, Reeve W, Perret X (2015) Ribosomal protein biomarkers provide root nodule bacterial identification by MALDI-TOF MS. Appl Microbiol Biotechnol 99(13):5547–5562CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Institute of Agricultural SciencesETH Zurich, Plant Nutrition groupLindauSwitzerland
  2. 2.Research Institute of Organic Agriculture (FiBL)FrickSwitzerland
  3. 3.International Institute of Tropical Agriculture (IITA)NairobiKenya
  4. 4.Mabritec AGRiehenSwitzerland
  5. 5.AgroBioChem Department, Gembloux Agro-Bio TechUniversity of LiègeGemblouxBelgium

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