A production strain of soybean nodule bacteria RZ300 Bradyrhizobium japonicum resistant to drying on the seed surface: cultural properties and genomic features

Pre-sowing treatment of cultivated legume seeds with nodule bacteria preparations is a standard agronomic practice. This is particularly important in soybean cultivation, as effective microsymbionts of soybeans are often absent from the soil. However, as many studies have shown, the efficacy of biopreparations depends largely on the survival of rhizobial cells on seeds during drying. In this study, we analyzed the viability of three production strains of Bradyrhizobium japonicum Kirchner (634b, 640 and RZ300) on soybean (Glycine max L.) seeds of various origins (varieties: EN Argenta, Bara and Prudence). The experiments evaluated several parameters: inoculant concentrations (10 and 100 %), drying temperatures (5, 15, and 25 °C), and protective polymer-carbohydrate formulations. The experiments revealed that the soybean variety had no noticeable effect on the viability of the studied rhizobial strains, while the strains themselves differed significantly in this regard. The RZ300 strain demonstrated the highest resistance to drying on soybean seeds. A comparative genomic analysis of this strain and the less resistant B. japonicum strain 634b revealed the presence of the opgC gene in the RZ300 strain (encodes the ОpgC protein involved in the biosynthesis of osmoregulated periplasmic glucans (OPGs)). This gene is absent in strain 634b and may potentially determine the increased resistance of nodule bacteria to drying on seeds. An evaluation of various protective formulations demonstrated that formulations based on 50 % sucrose provide the best protection, with rhizobia showing the highest resistance to drying at +5 °C. The results obtained in this study can be used both in the selection of effective inoculant strains and for providing technological support in the development of biological products. The genomic data support the development of genetic screening systems to identify promising strains and the potential introduction of the opgC gene into promising rhizobial strains to improve their manufacturability, i. e. to enable effective early seed inoculation.

1. Baker B.R., Ives C.M., Bray A., Caffrey M., Cochrane S.A. Undecaprenol kinase: function, mechanism and substrate specificity of a potential antibiotic target. Eur J Med Chem. 2021;210:113062. doi 10.1016/j.ejmech.2020.113062

2. Вashan Y., de-Bashan L., Prabhu S.R., Hernandez J. Advances in plant growth-promoting bacterial inoculant technology: formulations and practical perspectives (1998-2013). Plant Soil. 2014;378:1-33. doi 10.1007/s11104-013-1956-x

3. Bobkova Yu.A. Reaction of OAK Prudence soybeans to foliar fertilization with macro- and micro fertilizers in the conditions of the Orel region. Vestnik Agrarnoy Nauki = Bulletin of Agrarian Science. 2020;5(86):11-18. doi 10.17238/issn2587-666X.2020.5.11 (in Russian)

4. Bontemps-Gallo S., Lacroix J.M. New insights into the biological role of the osmoregulated periplasmic glucans in pathogenic and symbiotic bacteria. Environ Microbiol Rep. 2015;7(5):690-697. doi 10.1111/1758-2229.12325

5. Deaker R., Roughley R.J., Kennedy I.R. Desiccation tolerance of rhizobia when protected by synthetic polymers. Soil Biol Biochem. 2007; 39(2):573-580. doi 10.1016/j.soilbio.2006.09.005

6. Deriglazova G.M., Morozov A.N. Competitiveness of the domestic soybean variety EN Argenta compared with the Canadian breeding variety of OAK Prudence in the conditions of the Central Black Earth region. Zernobobovye i Krupyanye Kultury = Legumes Groat Crops. 2022;4(44):49-57. doi 10.24412/2309-348X-2022-4-49-57 (in Russian)

7. Diao J., Komura R., Sano T., Pantua H., Storek K.M., Inaba H., Ogawa H., … Yanagida H., Nishikawa J., Reid P.C., Cunningham C.N., Kapadia S.B. Inhibition of Escherichia coli lipoprotein diacylglyceryl transferase is insensitive to resistance caused by deletion of Braun’s lipoprotein. J Bacteriol. 2021;203(13):e00149-21. doi 10.1128/JB.00149-21

8. Doyle J.J., Doyle J.L. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull. 1987;19(1):11-15

9. Doyle J.J., Doyle J.L. Isolation of plant DNA from fresh tissue. Focus. 1990;12(1):13-15

10. Fred E.B. Some factors which influence the growth and longevity of the nodule bacteria. In: Fred E.B., Baldwin I.L., Mccoy E. (Eds) Root Nodule Bacteria and Leguminous Plants. Madison; Wisconsin: University of Wisconsin, 1932;104-117

11. Kincharova M.N., Matvienko E.V. The effectiveness of pre-sowing seed treatment in the fight against diseases of grain sorghum. Agrarnyy Vestnik Urala = Agrarian Bulletin of the Urals. 2021;9(212):2-10. doi 10.32417/1997-4868-2021-212-09-2-10 (in Russian)

12. Laktionov Yu.V., Kosulnikov Yu.V., Dudnikova D.V., Yahno V.V., Kojemyakov A.P. Pre-sowing protection of inoculated soybean Glycine max (L.) Merr. seeds by water-soluble polymer compositions and their solid-phase modification. Sel’skokhozyajstvennaya Biologiya = Agricultural Biology. 2019;54(5):1052-1059. doi 10.15389/agrobiology.2019.5.1052rus (in Russian)

13. Lamptey S., Ahiabor B.D.K., Yeboah S., Asamoah C. Response of soybean (Glycine max) to rhizobial inoculation and phosphorus application. J Exp Biol Agric Sci. 2014;2(1):72-77

14. Magomedov R.D., Tsekhmeistruk N.G., Shelyakin V.A., Ryabukha S.S., Didovich S.V. The influence of various strains Rhizobium japonicum (Kircher) Buchanan on soybean yield. Maslichnyye Kul’tury = Oil Crops. 2011;2(148-149):159-162 (in Russian)

15. Pancholi V., Boël G., Jin H. Streptococcus pyogenes Ser/Thr kinaseregulated cell wall hydrolase is a cell division plane-recognizing and chain-forming virulence factor. J Biol Chem. 2010;285(40):30861- 30874. doi 10.1074/jbc.M110.153825

16. Parakhin N.V., Lysenko N.N., Petrova S.N., Kuzmicheva Yu.V., Ryzhov I.A. Evaluation of herbicide system efficacy in agrocenosis of different soybean varieties depending on tillage method. Zemledeliye. 2017;2:39-43 (in Russian)

17. Regar M.K., Meena R.H., Jat G., Mundra S.L. Effect of different rhizobial strains on growth and yield of soybean [Glycine max (L.) Merrill]. Int J Curr Microbiol App Sci. 2017;6(11):3653-3659. doi 10.20546/ijcmas.2017.611.427

18. Reina-Bueno M., Argandoña M., Nieto J.J., Hidalgo-García A., Iglesias-Guerra F., Delgado M.J., Vargas C. Role of trehalose in heat and desiccation tolerance in the soil bacterium Rhizobium etli. BMC Microbiol. 2012;12(1):207. doi 10.1186/1471-2180-12-207

19. Roth M., Goodall E.C.A., Pullela K., Jaquet V., François P., Henderson I.R., Krause K.-H. Transposon-directed insertion-site sequencing reveals glycolysis gene gpmA as part of the H2O2 defense mechanisms in Escherichia coli. Antioxidants (Basel). 2022;11(10):2053. doi 10.3390/antiox11102053

20. Rudra P., Hurst-Hess K.R., Cotten K.L., Partida-Miranda A., Ghosh P. Mycobacterial HflX is a ribosome splitting factor that mediates antibiotic resistance. Proc Natl Acad Sci USA. 2020;117(1):629-634. doi 10.1073/pnas.1906748117

21. Skorupska A., Janczarek M., Marczak M., Mazur A., Król J. Rhizobial exopolysaccharides: genetic control and symbiotic functions. Microb Cell Fact. 2006;5(1):7. doi 10.1186/1475-2859-5-7

22. Smith R.S. Legume inoculant formulation and application. Can J Microbiol. 1992;38(6):485-492. doi 10.1139/m92-080

23. Soria M.A., Pagliero F.E., Correa O.S., Kerber N.L., Garcia A.F. Tolerance of Bradyrhizobium japonicum E109 to osmotic stress and the stability of liquid inoculants depend on growth phase. World J Microbiol Biotechnol. 2006;22(1):1235-1241. doi 10.1007/s11274-006-9166-9

24. Vasilchikov A.G., Gurev G.P. Adaptation of soybean varieties with different growing seasons to the soil and climatic conditions of the Oryol region. Zernobobovye i Krupyanye Kultury = Legumes Groat Crops. 2018;4(28):49-53. doi 10.24411/2309-348X-2018-00001 (in Russian)

25. Vavilov P.P., Posypanov G.S. Legumes and the Issue of Vegetable Protein. Moscow, 1983 (in Russian)

26. Vincent J.M., Thompson J.A., Donovan K.O. Death of root nodule bacteria on drying. Aust J Agric Res. 1961;13(2):258-270. doi 10.1071/AR9620258

27. Volobueva O.G., Trukhachev S., Belopukhov C., Seregina I. Comparative study of symbiotic activity of legumes when using Risotorphin and Epin-extra. Braz J Biol. 2023;83(3):e264218. doi 10.1590/1519-6984.264218

28. Vriezen J.A., de Bruijn F.J., Nüsslein K. Desiccation responses and survival of Sinorhizobium meliloti USDA 1021 in relation to growth phase, temperature, chloride and sulfate availability. Lett Appl Microbiol. 2006;42(2):172-178. doi 10.1111/j.1472-765X.2005.01808.x

29. Vriezen J., Bruijn F., Nüsslein K. Responses of rhizobia to desiccation in relation to osmotic stress, oxygen, and temperature. Appl Environ Microbiol. 2007;73(11):3451-3459. doi 10.1128/AEM.02991-06

30. Wu L., Fan Z., Guo L., Li Y., Chen Z., Qu L. Over-expression of the bacterial nhaA gene in rice enhances salt and drought tolerance. Plant Sci. 2005;168(2):297-302. doi 10.1016/j.plantsci.2004.05.033

31. Yadav J., Verma J.P., Rajak V.K., Tiwari K.N. Selection of effective indigenous Rhizobium strain for seed inoculationof chickpea (Cicer aritenium L.) production. Bacteriol J. 2011;1(1):24-30. doi 10.3923/bj.2011.24.30

32. Zhang Y., Ku Y.S., Cheung T.Y., Cheng S.S., Xin D., Gombeau K., Cai Y., Lam H.M., Chan T.F. Challenges to rhizobial adaptability in a changing climate: genetic engineering solutions for stress tolerance. Microbiol Res. 2024;288:127886. doi 10.1016/j.micres.2024.127886