DOI QR코드

DOI QR Code

Nodulation Experiment by Cross-Inoculation of Nitrogen-Fixing Bacteria Isolated from Root Nodules of Several Leguminous Plants

  • Ahyeon Cho (Department of Agricultural Chemistry, Jeonbuk National University) ;
  • Alpana Joshi (Department of Bioenvironmental Chemistry, Jeonbuk National University) ;
  • Hor-Gil Hur (School of Earth Sciences and Environmental Engineering, Gwangju Institute of Science and Technology) ;
  • Ji-Hoon Lee (Department of Agricultural Chemistry, Jeonbuk National University)
  • Received : 2023.10.19
  • Accepted : 2023.12.21
  • Published : 2024.03.28

Abstract

Root-nodule nitrogen-fixing bacteria are known for being specific to particular legumes. This study isolated the endophytic root-nodule bacteria from the nodules of legumes and examined them to determine whether they could be used to promote the formation of nodules in other legumes. Forty-six isolates were collected from five leguminous plants and screened for housekeeping (16S rRNA), nitrogen fixation (nifH), and nodulation (nodC) genes. Based on the 16S rRNA gene sequencing and phylogenetic analysis, the bacterial isolates WC15, WC16, WC24, and GM5 were identified as Rhizobium, Sphingomonas, Methylobacterium, and Bradyrhizobium, respectively. The four isolates were found to have the nifH gene, and the study confirmed that one isolate (GM5) had both the nifH and nodC genes. The Salkowski method was used to measure the isolated bacteria for their capacity to produce phytohormone indole acetic acid (IAA). Additional experiments were performed to examine the effect of the isolated bacteria on root morphology and nodulation. Among the four tested isolates, both WC24 and GM5 induced nodulation in Glycine max. The gene expression studies revealed that GM5 had a higher expression of the nifH gene. The existence and expression of the nitrogen-fixing genes implied that the tested strain had the ability to fix the atmospheric nitrogen. These findings demonstrated that a nitrogen-fixing bacterium, Methylobacterium (WC24), isolated from a Trifolium repens, induced the formation of root nodules in non-host leguminous plants (Glycine max). This suggested the potential application of these rhizobia as biofertilizer. Further studies are required to verify the N2-fixing efficiency of the isolates.

Keywords

Acknowledgement

This study was supported by the Cooperative Research Program for Agricultural Science and Technology Development [Project No. PJ015716032023 and RS-2021-RD009903] of the Rural Development Administration, Republic of Korea.

References

  1. Zhu YG, Peng J, Chen C, Xiong C, Li S, Ge A, et al. 2023. Harnessing biological nitrogen fixation in plant leaves. Trends Plant Sci. 28: 1391-1405.  https://doi.org/10.1016/j.tplants.2023.05.009
  2. Kneip C, Lockhart P, Voss C, Maier UG. 2007. Nitrogen fixation in eukaryotes-new models for symbiosis. BMC Evol. Biol. 7: 55. 
  3. Soumare A, Diedhiou AG, Thuita M, Hafidi M, Ouhdouch Y, Gopalakrishnan S, et al. 2020. Exploiting biological nitrogen fixation: a route towards a sustainable agriculture. Plants 9: 1011. 
  4. Masson-Boivin C, Sachs JL. 2018. Symbiotic nitrogen fixation by rhizobia-the roots of a success story. Curr. Opin. Plant. Biol. 44: 7-15.  https://doi.org/10.1016/j.pbi.2017.12.001
  5. Ferguson BJ, Mens C, Hastwell AH, Zhang M, Su H, Jones CH, et al. 2019. Legume nodulation: the host controls the party. Plant. Cell. Environ. 42: 41-51.  https://doi.org/10.1111/pce.13348
  6. Wang D, Yang S, Tang F, Zhu, H. 2012. Symbiosis specificity in the legume-rhizobial mutualism. Cell. Microbiol. 14: 334-342.  https://doi.org/10.1111/j.1462-5822.2011.01736.x
  7. Liu CW, Murray JD. 2016. The role of flavonoids in nodulation host-range specificity: an update. Plants (Basel) 5: 33. 
  8. Bosse MA, Silva MBD, Oliveira NGRM, Araujo MA, Rodrigues C, Azevedo JP, et al. 2021. Physiological impact of flavonoids on nodulation and ureide metabolism in legume plants. Plant Physiol. Biochem. 166: 512-521.  https://doi.org/10.1016/j.plaphy.2021.06.007
  9. Peters NK. 1997. Nodulation: finding the lost common denominator. Curr. Biol. 7: R223-R226.  https://doi.org/10.1016/S0960-9822(06)00106-0
  10. Angel R, Nepel M, Panholzl C, Schmidt H, Herbold CW, Eichorst SA, et al. 2018. Evaluation of primers targeting the diazotroph functional gene and development of NifMAP - A bioinformatics pipeline for analyzing nifH amplicon data. Front. Microbiol. 9: 703. 
  11. Hoy JA, Hargrove MS. 2008. The structure and function of plant hemoglobins. Plant. Physiol. Biochem. 46: 371-379.  https://doi.org/10.1016/j.plaphy.2007.12.016
  12. Kosmachevskaya OV, Nasybullina EI, Shumaev KB, Topunov AF. 2021. Expressed soybean leghemoglobin: effect on Escherichia coli at oxidative and nitrosative stress. Molecules (Basel) 26: 7207. 
  13. Madhaiyan M, Poonguzhali S, Senthilkumar M, Sundaram S, Sa T. 2009. Nodulation and plant-growth promotion by methylotrophic bacteria isolated from tropical legumes. Microbiol. Res. 164: 114-120.  https://doi.org/10.1016/j.micres.2006.08.009
  14. Yang J, Lan L, Jin Y, Yu N, Wang D, Wang E. 2022. Mechanisms underlying legume-rhizobium symbioses. J. Integr. Plant Biol. 64: 244-267.  https://doi.org/10.1111/jipb.13207
  15. Dinkins RD, Hancock JA, Bickhart DM, Sullivan ML, Zhu H. 2022. Expression and variation of the genes involved in rhizobium nodulation in red clover. Plants (Basel). 11: 2888. 
  16. Maluk M, Giles M, Wardell GE, Akramin AT, Ferrando-Molina F, Murdoch A, et al. 2023. Biological nitrogen fixation by soybean (Glycine max [L.] Merr.), a novel, high protein crop in Scotland, requires inoculation with non-native bradyrhizobia. Front. Agron. 5: 1196873. 
  17. Kumar S, Diksha, Sindhu SS, Kumar, R. 2021. Biofertilizers: an ecofriendly technology for nutrient recycling and environmental sustainability. Curr. Resear. Microb. Sci. 3: 100094. 
  18. Teale WD, Paponov IA, Palme K. 2006. Auxin in action: signaling, transport and the control of plant growth and development. Nat. Rev. Mol. Cell Biol. 7: 847-859.  https://doi.org/10.1038/nrm2020
  19. Bal HB, Das S, Dangar TK, Adhya TK. 2013. ACC deaminase and IAA producing growth promoting bacteria from the rhizosphere soil of tropical rice plants. J. Basic Microbiol. 53: 972-984.  https://doi.org/10.1002/jobm.201200445
  20. Lebrazi S, Fadil M, Chraibi M, Fikri-Benbrahim K. 2020. Screening and optimization of indole-3-acetic acid production by Rhizobium sp. strain using response surface methodology. J. Genet. Eng. Biotechnol. 18: 21. 
  21. Jaiswal SK, Mohammed M, Ibny FYI, Dakora FD. 2021. Rhizobia as a source of plant growth-promoting molecules: potential applications and possible operational mechanisms. Front. Sustain. Food Syst. 4: 619676. 
  22. Kai S, Matsuo Y, Nakagawa S, Kryukov K, Matsukawa S, Tanaka H, et al. 2019. Rapid bacterial identification by direct PCR amplification of 16S rRNA genes using the MinIONTM nanopore sequencer. FEBS Open Bio 9: 548-557.  https://doi.org/10.1002/2211-5463.12590
  23. Galkiewicz JP, Kellogg CA. 2008. Cross-kingdom amplification using bacteria-specific primers: complications for studies of coral microbial ecology. Appl. Environ. Microbiol. 74: 7828-7831.  https://doi.org/10.1128/AEM.01303-08
  24. Wang Y, Chen Y, Xue Q, Xiang Q, Zhao K, Yu X, et al. 2021. The abundance of the nifH gene became higher and the nifH-containing diazotrophic bacterial communities changed during primary succession in the Hailuogou Glacier chronosequence, China Front. Microbiol. 12: 672656. 
  25. Sarita S, Sharma PK, Priefer UB, Prell J. 2005. Direct amplification of rhizobial nodC sequences from soil total DNA and comparison to nodC diversity of root nodule isolates. FEMS Microbial. Ecol. 54: 1-11.  https://doi.org/10.1016/j.femsec.2005.02.015
  26. Tamura K, Nei M, Kumar S. 2004. Prospects for inferring very large phylogenies by using the neighbor-joining method. Proc. Natl. Acad. Sci. USA 101: 11030-11035.  https://doi.org/10.1073/pnas.0404206101
  27. Ngwenya ZD, Mohammed M, Jaiswal SK, Dakora FD. 2022. Phylogenetic relationships among Bradyrhizobium species nodulating groundnut (Arachis hypogea L.), jack bean (Canavalia ensiformis L.) and soybean (Glycine max Merr.) in Eswatini. Sci. Rep. 12: 10629. 
  28. Godeke J, Binnenkade L, Thormann KM. 2012. Transcriptome analysis of early surface-associated growth of Shewanella oneidensis MR-1. PLoS One 7: e42160. 
  29. Rahman A, Sitepu IR, Tang SY, Hashidoko Y. 2010. Salkowski's reagent test as a primary screening index for functionalities of rhizobacteria isolated from wild dipterocarp saplings growing naturally on medium-strongly acidic tropical peat soil. Biosci. Biotechnol. Biochem. 74: 2202-2208.  https://doi.org/10.1271/bbb.100360
  30. Zhang W, Dun S, Ping Y, Wang Q, Tana S, Tana A, et al. 2022. Differentially expressed long noncoding RNAs and mRNAs in PC12 cells under lysophosphatidylcholine stimulation. Sci. Rep. 12: 19333. 
  31. Menna P, Hungria M, Barcellos FG, Bangel EV, Hess PN, Martinez-Romero E. 2006. Molecular phylogeny based on the 16S rRNA gene of elite rhizobial strains used in Brazilian commercial inoculants. Syst. Appl. Microbiol. 29: 315-332.  https://doi.org/10.1016/j.syapm.2005.12.002
  32. Yarza P, Yilmaz P, Pruesse E, Glockner FO, Ludwig W, Schleifer KH, et al. 2014. Uniting the classification of cultured and uncultured bacteria and archaea using 16S rRNA gene sequences. Nat. Rev. Microbiol. 12: 635-645.  https://doi.org/10.1038/nrmicro3330
  33. Zhou L, Li H, Zhang Y, Han S, Xu H. 2014. Development of genus-specific primers for better understanding the diversity and population structure of Sphingomonas in soils. J. Basic Microbiol. 54: 880-888.  https://doi.org/10.1002/jobm.201200679
  34. Johnson JS, Spakowicz DJ, Hong BY, Petersen LM, Demkowicz P, Chen L, et al. 2019. Evaluation of 16S rRNA gene sequencing for species and strain-level microbiome analysis. Nat. Commun. 10: 5029. 
  35. Siqueira AF, Ormeno-Orrillo E, Souza RC, Rodrigues EP, Almeida LG, Barcellos FG, et al. 2014. Comparative genomics of Bradyrhizobium japonicum CPAC 15 and Bradyrhizobium diazoefficiens CPAC 7: elite model strains for understanding symbiotic performance with soybean. BMC Genom. 15: 420. 
  36. Bender FR, Nagamatsu ST, Delamuta JRM, Ribeiro RA, Nogueira MA, Hungria M. 2022. Genetic variation in symbiotic islands of natural variant strains of soybean Bradyrhizobium japonicum and Bradyrhizobium diazoefficiens differing in competitiveness and in the efficiency of nitrogen fixation. Microb. Genom. 8: 000795. 
  37. Moulin L, Bena G, Boivin-Masson C, Stkepkowski T. 2004. Phylogenetic analyses of symbiotic nodulation genes support vertical and lateral gene co-transfer within the Bradyrhizobium genus. Mol. Phylogenet. Evol. 30: 720-732.  https://doi.org/10.1016/S1055-7903(03)00255-0
  38. Mpai T, Jaiswal SK, Cupido CN, Dakora FD. 2021. Ecological adaptation and phylogenetic analysis of microsymbionts nodulating Polhillia, Wiborgia and Wiborgiella species in the Cape fynbos, South Africa. Sci. Rep. 11: 23614. 
  39. Lu YL, Chen WF, Wang ET, Guan SH, Yan XR, Chen WX. 2009. Genetic diversity and biogeography of rhizobia associated with Caragana species in three ecological regions of China. Syst. Appl. Microbiol. 32: 351-361.  https://doi.org/10.1016/j.syapm.2008.10.004
  40. Shokri D, Emtiazi G. 2010. Indole-3-acetic acid (IAA) production in symbiotic and non-symbiotic nitrogen-fixing bacteria and its optimization by Taguchi design. Curr. Microbiol. 61: 217-225.  https://doi.org/10.1007/s00284-010-9600-y
  41. Datta C, Basu PS. 2000. Indole acetic acid production by a Rhizobium species from root nodules of a leguminous shrub. Microbiol. Res. 155: 123-127.  https://doi.org/10.1016/S0944-5013(00)80047-6
  42. Sridevi M, Mallaiah KV. 2007. Production of indole-3-acetic acid by Rhizobium isolates from sesbania species. Afr. J. Microbiol. Res. 1: 125-128. 
  43. 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. Microbial. 39: 336-344.  https://doi.org/10.1016/j.syapm.2016.05.009
  44. Minakata C, Wasai-Hara S, Fujioka S, Sano S, Matsumura A. 2023. Unique rhizobial communities dominated by Bradyrhizobium liaoningense and Bradyrhizobium ottawaense were found in vegetable soybean nodules in Osaka prefecture, Japan. Microbes Environ. 38: ME22081. 
  45. Favero VO, de Carvalho RH, Leite ABC, Santos DMT, Freitas KM, Zilli JE, et al. 2022. Cross-inoculation of elite commercial Bradyrhizobium strains from Cowpea and soybean in mung bean and comparison with mung bean isolates. J. Soil. Sci. Plant. Nutr. 22: 4356-4364.  https://doi.org/10.1007/s42729-022-01034-0
  46. Singh RK, Singh P, Li HB, Song QQ, Guo DJ, Solanki MK, et al. 2020. Diversity of nitrogen-fixing rhizobacteria associated with sugarcane: a comprehensive study of plant-microbe interactions for growth enhancement in Saccharum spp. BMC Plant Biol. 20: 220.