Mikrobiol. Z. 2018; 80(6):54-65. Ukrainian.
doi: https://doi.org/10.15407/microbiolj80.06.054

Survival of Bradyrhizobium japonicum Strains in Soil at their
Introduction into Soybean Agrocenosis

Krutylo D.V.

Institute of Agricultural Microbiology and Agro-Industrial Manufacture, NAAS of Ukraine
97 Shevchenko Str., Chernihіv, 14027, Ukraine

Objective. The objective of our work was to assess the ability of Bradyrhizobium japonicum strains with slow and intensive growth rate to survive in the soil and maintain their symbiotic activity for a long time. Methods. The survival of inoculum strains in the soil has been studied in the field experiments with soybean (Glycine max (L.) Merr.). The cowpea (Vigna unguiculata (L.) Walp.), mung bean (Vigna radiata (L.) Wilczek) and adzuki beans (Vigna radiata (L.) Wilczek) were used as trap plants for soybean rhizobia. The presence of studied strains in the nodules was determined in reaction of agglutination with specific antisera. Morphological and cultural properties of isolated rhizobia were studied by generally accepted methods. Sequencing of the 16S - 23S rDNA intergenic spacer of rhizobia was performed on ABI 3130 Genetic Analyzer. Results. The B. japonicum strains with different growth rates were introduced into soybean agrocenosis in the first year of experiment. When soybean was grown in monoculture, gradual replacement of slow-growing rhizobia (B. japonicum 46, B. japonicum М8 and B. japonicum 634b) from nodule populations by strain with intensive growth rate B. japonicum KB11 was observed. At the 4th year of the studies, these strains were absent in the soybean nodule, however they remained in the soil as saprophytic microorganisms. Their presence was detected using trap plants, which had symbiotic relationships with the studied microorganisms. The proportion of strains with slow growth rate in the nodules of different cowpea species ranged from 2.1 to 58.3 %. The presence of slow-growing strains in nodule populations of cowpea, mung bean and adzuki bean is proved by microbiological, serological (belonging to serogroups 46, M8, 634b) and molecular genetic methods. The rhizobia from mung bean nodules showed 99.4 - 99.8 % identity to B. japonicum strains (from GenBank) based on nucleotide sequences of ITS region. The highest level of homology (99.9 - 100.0 %) was noted with B. japonicum KC23 strain, which is a typical representative of the serogroup M8. Conclusion. The strains of B. japonicum with slow and intensive growth rates introduced in soybean agrocenosis persist in the soil for a long time. They occupy their ecological niche in the local rhizobial population, however they have different ex planta and in planta strategies. Within five years, the ratio of studied strains in the nodule populations has been significantly changed: from the presence of all strains in the nodules to complete dominance of B. japonicum KB11 strain with intensive growth rates and the transition of slow-growing rhizobia to saprophytic existence.

Keywords: Bradyrhizobium japonicum, isolates, trap-host, 16S-23S rDNA, soybean, cowpea, mung bean, adzuki bean.

Full text (PDF, in Ukrainian)

  1. Spaink HP, Kondorosi A, Hooykaas P. [The Rhizobiaceae: Molecular biology of model plant-associated bacteria]. Translated in rus. by I.A. Tikhonovich, N.A. Provorov. St.-Petersburg: Biont; 2002. Russian.
  2. Kots SYa, Morgun VV, Patyka VF et al. [Biological nitrogen fixation: legume-rhizobial symbiosis]. Kyiv: Logos; 2011; 2. Russian.
  3. Patyka VF, Krutylo DV, Kovalevska TM. [Effect of aboriginal populations of soybean nodule bacteria on symbiotic activity of introduced strain Bradyrhizobium japonicum 634b]. Mikrobiol Z. 2004; 66(3):14–21. Ukrainian.
  4. Provorov NA. [Evolution of plant-microbial symbioses: phylogenetic, population, genetic and selection aspects]. Thesis for the degree of doctor of biological sciences by speciality 03.00.15 – genetic. St.-Petersburg, 2009. Russian.
  5. Sullivan JT, Eardly BD, van Berkum P, Ronson CW. Four unnamed species of nonsymbiotic rhizobia isolated from the rhizosphere of Lotus corniculatus. Appl Environ Microbiol. 1996; 62:2818–2825.
  6. Sanginga N, Danso SKA, Mulongoy K, Ojeifo AA. Persistence and recovery of introduced Rhizobium ten years after inoculation on Leucaena leucocephala grown on an Alfìsol in southwestern Nigeria. Plant Soil. 1994; 159:199–204. https://doi.org/10.1007/BF00009281
  7. Krutylo DV, Nadkernychna OV, Kovalevska TM, Patyka VP. [Biological diversity of soybean nodule bacteria in soils of Ukraine]. Mikrobiol Z. 2008; 70(6):27–34. Ukrainian.
  8. Krutylo DV, Zotov VS. Genotypic analysis of nodule bacteria nodulating soybean in soils of Ukraine. Russian Journal of Genetics: Applied Research. 2015; 5(2):102–109. https://doi.org/10.1134/S2079059715020057
  9. Hardy RWF, Holsten RD, Jackson EK, Burns RC. The acetylene-ethylene assay for nitrogen fixation: laboratory and field evaluation. Plant Physiol. 1968; 43(8):1185–1207. https://doi.org/10.1104/pp.43.8.1185
  10. Silva FV, Simões-Araújo JL, Silva Júnior JP, Xavier GR, Rumjanek NG. Genetic diversity of Rhizobia isolates from Amazon soils using cowpea (Vigna unguiculata) as trap plant. Brazil J Microbiol. 2012; 43:682–691. https://doi.org/10.1590/S1517-83822012000200033
  11. Kebot E, Meyer B. [Experimental immunology]. Moscow: Medicina Publ.; 1968. Russian.
  12. Krutylo DV, Volkova IV. [Serological diversity of soybean nodule bacteria in Ukraine soils]. Agroecological journal. 2012; 4:66–71. Ukrainian.
  13. Bergey's Manual of Systematic Bacteriology. The Proteobacteria. New York: Springer SBM, 2005.
  14. [Methods of cultivation and long-term storage of nodule bacteria in the collections. Methodical recommendations]. Ed. by T.M. Kovalevska, S.F. Kozar, D.V. Krutylo, V.P. Gorban et al. Chernihiv: ICMAB NAAS; 2015. Ukrainian.
  15. Beringer JE. R1 transfer in Rhizobium leguminosarum. J. Gen. Microbiol. 1974; 84:188-198.
  16. Normand P, Ponsonnet C, Nesme X et al. ITS analysis of prokaryotes. Mol. Microbial Ecology Manual. 1996; 5(3–4):1–12.
  17. Ponsonnet C, Nesme X. Identification of Agrobacterium strains by PCR-RFLP analysis of pTi and chromosomal regions. Arch Microbiol. 1994; 161:300–309. https://doi.org/10.1007/BF00303584
  18. Dospekhov BA. [Field Experience Method]. Moscow: Agropromizdat; 1985. Russian.
  19. Tampakaki AP, Fotiadis CT, Ntatsi G, Savvas D. Phylogenetic multilocus sequence analysis of indigenous slow-growing rhizobia nodulating cowpea (Vigna unguiculata L.) in Greece. Syst Appl Microbiol. 2017; 40:179–189. https://doi.org/10.1016/j.syapm.2017.01.001
  20. Brunel B, Rome S, Ziani R, Cleyet-Marel JC. Comparison of nucleotide diversity and symbiotic properties of Rhizobium meliloti populations from annual species of Medicago. FEMS Microbiol Ecol. 1996; 19:71–82. https://doi.org/10.1111/j.1574-6941.1996.tb00200.x
  21. Vlassak K, Vanderleyden J, Franco A. Competition and persistence of Rhizobium tropici and Rhizobium etli in tropical soil during successive bean (Phaseolus vulgaris L.) cultures. Biol Fertil Soils. 1996; 21:61–68. https://doi.org/10.1007/BF00335994
  22. Bromfield E.S.P., Barran L.R., Wheatcroft R. Relative genetic structure of a population of Rhizobium meliloti isolated directly from soil and from nodules of alfalfa (Medicago sativa) and sweet clover (Melilotus alba). Mol. Ecol. 1995; 4:183–188.
  23. Krutylo DV. [Symbiotic relationship between Bradyrhizobium japonicum strains of different genetic groups and soybean plants]. Mikrobiol Z. 2017; 79(6):82–94. Ukrainian. https://doi.org/10.15407/microbiolj79.06.082
  24. Mpepereki S, Wollum AG, Makonese F. Diversity in symbiotic specificity of cowpea rhizobia indigenous to Zimbabwean soil. Plant Soil. 1996; 186:167–171. https://doi.org/10.1007/BF00035071