Annals of Microbiology

, Volume 68, Issue 5, pp 247–260 | Cite as

Diversity and symbiotic divergence of endophytic and non-endophytic rhizobia of Medicago sativa

Original Article
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Abstract

Knowledge of rhizobium diversity is helping to enable the utilization of rhizobial resources. To analyze the phenotypic and genetic diversity and the symbiotic divergence of rhizobia of Medicago sativa, 30 endophytic and non-endophytic isolates were collected from different parts of five alfalfa varieties in three geographic locations in Gansu, China. Numerical analyses based on 72 phenotypic properties and restriction fragment length polymorphism (RFLP) fingerprinting indicated the abundant phenotypic and genetic diversity of the tested strains. According to the phylogenetic analysis of 16S RNA, atpD, glnII, and recA gene sequences, Rhizobium and Ensifer were further classified into four different genotypes: Rhizobium radiobacter, Rhizobium sp., Rhizobium rosettiformans, and Ensifer meliloti. The differences in architecture and functioning of the rhizobial genomes and, to a lesser extent, environment diversification helped explain the diversity of tested strains. The tested strains exhibited similar symbiotic feature when inoculated onto M. sativa cvs. Gannong Nos. 3 and 9 and Qingshui plants for the clustering feature of their parameter values. An obvious symbiotic divergence of rhizobial strains was observed in M. sativa cvs. Longzhong and WL168HQ plants because of the scattered parameter values. Their symbiotic divergence differed according to alfalfa varieties, which indicated that the sensitivity of different alfalfa varieties to rhizobial strains may differ. Most of the tested strains exhibited plant growth-promoting traits including phosphate solubilization and production of indole-3-acetic acid (IAA) when colonizing plant tissues and soil.

Keywords

Medicago sativa Rhizobium Phenotypic diversity Genetic diversity Symbiotic divergence 

Notes

Acknowledgements

We thank the Key Laboratory of Grassland Ecosystem of Ministry of Education of Gansu Agricultural University for providing sampling sites and alfalfa plants. Thanks should go to Lin Xu for writing assistance.

References

  1. Arnold AE (2007) Understanding the diversity of foliar endophytic fungi: progress, challenges, and frontiers. Fungal Biol Rev 21:51–66CrossRefGoogle Scholar
  2. Aserse AA, Räsänen LA, Aseffa F, Hailemariam A, Lindström K (2012) Phylogenetically diverse groups of Bradyrhizobium isolated from nodules of Crotalaria spp., Indigofera spp., Erythrina brucei and Glycine max growing in Ethiopia. Mol Phylogenet Evol 65: 595–609Google Scholar
  3. Aserse AA, Räsänen LA, Aseffa F, Hailemariam A, Lindström K (2013) Diversity of sporadic symbionts and nonsymbiotic endophytic bacteria isolated from nodules of woody, shrub, and food legumes in Ethiopia. Appl Microbiol Biot 97:10117CrossRefGoogle Scholar
  4. Bacon CW, Hinton DM (2006) Bacterial endophytes: the endophytic niche, its occupants, and its utility. In: Gnanamanickam SS (ed) Plant-associated Bacteria. Springer, Dordrecht, pp 155–194CrossRefGoogle Scholar
  5. Beltrangarcia MJ, Jr JFW, Prado FM, Prieto KR, Yamaguchi LF, Torres MS, Kato MJ, Medeiros MHG, Mascio PD (2014) Nitrogen acquisition in Agave tequilana from degradation of endophytic bacteria. Sci Rep 4:6938CrossRefGoogle Scholar
  6. Benabderrahim MA, Mansour H, Ali F (2009) Diversity of lucerne (Medicago sativa L.) populations in South Tunisia. Pakistan J Bot 41:2851–2861Google Scholar
  7. Beringer JE (1974) R factor transfer in Rhizobium leguminosarum. J Gen Microbiol 84:188–198PubMedGoogle Scholar
  8. Berrada H, Fikri-Benbrahim K (2014) Taxonomy of the rhizobia: current perspectives. British Microbiol Res J 4:616–639CrossRefGoogle Scholar
  9. Botha WJ, Jaftha JB, Bloem JF, Habig JH, Law IJ (2004) Effect of soil bradyrhizobia on the success of soybean inoculant strain CB 1809. Microbiol Res 159:219–231CrossRefPubMedGoogle Scholar
  10. Boukhatem ZF, Domergue O, Bekki A, Merabet C, Sekkour S, Bouazza F, Duponnois R, Lajudie PD, Galiana A (2012) Symbiotic characterization and diversity of rhizobia associated with native and introduced acacias in arid and semi-arid regions in Algeria. FEMS Microbiol Ecol 80:534–547CrossRefPubMedGoogle Scholar
  11. Cindy L, Jaco V, Fiona P, Edward RBM, Safieh T, Max M, Daniel van der L (2002) Endophytic bacteria and their potential applications. Crit Rev Plant Sci 21:583–606CrossRefGoogle Scholar
  12. Cooper JE (2007) Early interactions between legumes and rhizobia: disclosing complexity in a molecular dialogue. J Appl Microbiol 103:1355–1365CrossRefPubMedGoogle Scholar
  13. Deng ZS, Zhao LF, Kong ZY, Yang WQ, Lindström K, Wang ET, Wei GH (2011) Diversity of endophytic bacteria within nodules of the Sphaerophysa salsula in different regions of Loess plateau in China. FEMS Microbiol Ecol 76:463–475CrossRefPubMedGoogle Scholar
  14. Depret G, Laguerre G (2008) Plant phenology and genetic variability on root and nodule development strongly influence genetic structuring of Rhizobium leguminosarum biovar viciae populations nodulating pea. New Phytol 179:224–235CrossRefPubMedGoogle Scholar
  15. Doyle JJ (2011) Phylogenetic perspectives on the origins of nodulation. Mol Plant Microbe In 24:1289CrossRefGoogle Scholar
  16. Dudeja SS, Giri R, Saini R, Suneja-Madan P, Kothe E (2012) Interaction of endophytic microbes with legumes. J Basic Microbiol 52:248–260CrossRefPubMedGoogle Scholar
  17. Dudeja SS, Singh NP, Sharma P, Gupta SC, Chandra R, Dhar B, Bansal R, Brahmaprakash GP, Potdukhe SR, Gundappagol RC, Gaikawad BG, Nagaraj KS (2011) Biofertilizer technology and productivity of chickpea in India. In: Singh A, Parmar N, Kuhad RC (eds) Bioaugmentation, biostimulation and biocontrol, soil biology 28. Springer Verlag Berlin, Heidelberg, pp 43–63CrossRefGoogle Scholar
  18. Egamberdieva D, Kucharova Z (2009) Selection for root colonising bacteria stimulating wheat growth in saline soils. Biol Fertil Soils 45:563–571CrossRefGoogle Scholar
  19. Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791CrossRefPubMedGoogle Scholar
  20. Haag AF, Markus FF, Arnold MFF, Myka KK, Kercher B, Dall’angelo S (2013) Molecular insights into bacteroid development during Rhizobium -legume symbiosis. FEMS Microbiol Rev 37:364–383CrossRefPubMedGoogle Scholar
  21. Hoagland DR, Arnon DS (1950) The water culture method for growing plants without soil. CA, BerkeleyGoogle Scholar
  22. Huo PH (2014) Antimicrobial-resistant rhizobia screening and effect verification of undesired microbe control in the prepared rhizobia inoculant. Gansu Agricultural University, DissertationGoogle Scholar
  23. ICSP Subcommittee on the Taxonomy of Rhizobium and Agrobacterium: Diversity, phylogeny and systematics. 2013 Rhizobial Taxonomy Up-to-date Submitted by Vinuesa 2013–01-20 20, 23Google Scholar
  24. Kalita M, Malek W (2010) Genista tinctoria microsymbionts from Poland are new members of Bradyrhizobium japonicum bv. Genistearum. Syst Appl Microbiol 33:252–259CrossRefPubMedGoogle Scholar
  25. Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111–120CrossRefPubMedGoogle Scholar
  26. Kuklinsky-Sobral K, Araujo WL, Mendonca C, Geran LC, Piskala A, Azevedo JL (2004) Isolation and characterization of soybean-associated bacteria and their potential for plant growth promotion. Environ Microbiol 6:1244–1251CrossRefPubMedGoogle Scholar
  27. Larsen MV, Cosentino S, Rasmussen S, Friis C, Hasman H, Marvig RL, Jelsbak L, Sicheritz-ponten T, Ussery DW, Aarestrup FM, Lund O (2012) Multilocus sequence typing of total-genome-sequenced bacteria. J Clin Microbiol 50:1355–1361CrossRefPubMedPubMedCentralGoogle Scholar
  28. Leite J, Fischer D, Rouws FM, Fernandesjúnior PI, Hofmann A, Kublik S, Schloter M, Xavier GR, Radi V (2017) Cowpea nodules harbor non-rhizobial bacterial communities that are shaped by soil type rather than plant genotype. Front Plant Sci 7:2064CrossRefPubMedPubMedCentralGoogle Scholar
  29. Li JF, Zhang SQ, Shi SL, Huo PH (2009) Position and quantity of endogensis rhizobia in alfalfa plant. Chinese J Eco-Agriculture 17:1200–1205CrossRefGoogle Scholar
  30. Li JF, Zhang SQ, Shi SL, Huo PH (2011) Mutational approach for N2-fixing and P-solubilizing mutant strains of Klebsiella pneumoniae, RSN19 by microwave mutagenesis. World J Microb Biot 27:1481CrossRefGoogle Scholar
  31. Li JH, Wang ET, Chen WF, Chen WX (2008) Genetic diversity and potential for promotion of plant growth detected in nodule endophytic bacteria of soybean grown in Heilongjiang province of China. Soil Biol Biochem 40:238–246CrossRefGoogle Scholar
  32. Li JF, Shi SL, Zhang SQ (2010) Effects of the pH value of an acid environment on early growth and physiology of Medicago sativa W525. Acta Prataculturae Sinica 19:47–54Google Scholar
  33. Martínez-Romero E (2009) Coevolution in rhizobium-legume symbiosis? DNA Cell Biol 28:361CrossRefPubMedGoogle Scholar
  34. Miao YY, Shi SL, Zhang JG, Mohamad OA (2017) Migration, colonization and seedling growth of rhizobia with marine treatment in alfalfa (Medicago sativa L.). Acta Agr Scand B-S P 1–13Google Scholar
  35. Mierzwa B, Wdowiak-Wro′bel S, Malek W (2010) Robinia pseudoacacia in Poland and Japan is nodulated by Mesorhizobium amorphae strains Antonie Van Leeuwenhoek 97: 351–361Google Scholar
  36. Miliute I, Buzaite O, Baniulis D, Stanys V (2015) Bacterial endophytes in agricultural crops and their role in stress tolerance: a review. Zemdirbyste 102:465–478CrossRefGoogle Scholar
  37. Minoru M, Doris ZD (2015) Phenotypic and molecular differences among rhizobia that nodulate Phaseolus lunatus in the Supe valley in Peru. Ann Microbiol 65:1803–1808CrossRefGoogle Scholar
  38. Muresu R, Polone E, Sulas L, Baldan B, Tondello A, Delogu G, Cappuccinelli P, Alberghini S, Benhizia Y, Benhizia H, Benguedouar A, Mori B, Calamassi R, Dazzo FB, Squartini A (2008) Coexistence of predominantly nonculturable rhizobia with diverse, endophytic bacterial taxa within nodules of wild legumes. FEMS Microbiol Ecol 63:383CrossRefPubMedGoogle Scholar
  39. Nandwani R, Dudeja SS (2009) Molecular diversity of a native Mesorhizobial population of nodulating chickpea (Cicer arietinum L.) in Indian soils. J Basic Microbiol 49:463–470CrossRefPubMedGoogle Scholar
  40. Nei M, Li WH (1979) Mathematical model for studying genetic variations in terms of restriction endonucleases. P Natl Acad Sci USA 76:52–69CrossRefGoogle Scholar
  41. Pavlo A, Leonid O, Iryna Z, Natalia K, Maria PA (2011) Endophytic bacteria enhancing growth and disease resistance of potato (Solanum tuberosum L.) Biol Control 56:43–49CrossRefGoogle Scholar
  42. Qi CM (2004) The study on biodiversity and phylogensis of rhizobia isolated from Trifolium and Medicago M.D. candidates. Dissertation, Sichuang Agricultural UniversityGoogle Scholar
  43. Qi J (2006) Screening endogenous rhizobia from alfalfa seeds and their promoting alfalfa seedlings growth property. Dissertation, Gansu Agricultural UniversityGoogle Scholar
  44. Rai R, Dash PK, Mohapatra T, Singh A (2012) Phenotypic and molecular characterization of indigenous rhizobia nodulating chickpea in India. Indian J Exp Biol 50:340–350PubMedGoogle Scholar
  45. Rasul A, Amalraj ELD, Kumar GP, Grover M, Venkateswarlu B (2012) Characterization of rhizobial isolates nodulating Millettia pinnata, in India. FEMS Microbiol Lett 336:148CrossRefPubMedGoogle Scholar
  46. Rivas R, Martens M, De Lajudie P, Willems A (2009) Multilocus sequence analysis of the genus Bradyrhizobium. Syst Appl Microbiol 32:101–110CrossRefPubMedGoogle Scholar
  47. Rohlf FJ (1993) NTSYS-pc: numerical taxonomy and multivariate analysis system. Version 2.0. Exeter Software, New YorkGoogle Scholar
  48. Rouhrazi K, Khodakaramian G (2015) Phenotypic and genotypic diversity of root-nodulating bacteria isolated from chickpea (Cicer arietinum, L.) in Iran. Ann Microbiol 65:2219–2227CrossRefGoogle Scholar
  49. Sachs JL, Kembel SW, Lau AH, Simms EL (2009) In situ phylogenetic structure and diversity of wild Bradyrhizobium communities. Appl Environ Microbiol 75:4727–4735CrossRefPubMedPubMedCentralGoogle Scholar
  50. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425PubMedGoogle Scholar
  51. Samir BR, Mustapha T, Mohamedelarbi A, Philippede L, Ridha M (2009) The diversity of rhizobia nodulating chickpea (Cicer arietinum) under water deficiency as a source of more efficient inoculants. Soil Biol Biochem 41:2568–2572CrossRefGoogle Scholar
  52. Sánchez AC, Gutiérrez RT, Santana RC, Urrutia AR, Fauvart M, Michiels J, Vanderleyden J (2014) Effects of co-inoculation of native Rhizobium, and Pseudomonas, strains on growth parameters and yield of two contrasting Phaseolus vulgaris L. genotypes under Cuban soil conditions. Eur J Soil Biol 62:105–112CrossRefGoogle Scholar
  53. Senthilkumar M, Anandham R, Madhaiyan M, Venkateswaran V, Sa T (2011) Endophytic bacteria: perspectives and applications in agricultural crop production. In: Maheshwari (ed) Bacteria in agrobiology crop ecosystems. Springer publishing, New York, pp 61–96Google Scholar
  54. Shamseldin A, Moawad H, Abd El-Rahim WM, Sadowsky MJ (2013) Near-full length sequencing of 16S rRNA and RFLP indicates that Rhizobium etli is the dominant species nodulating Egyptian winter Berseem clover (Trifolium alexandrinum L.). Syst Appl Microbiol. http:// doi: 10.1016 / j. syapmGoogle Scholar
  55. Shetta ND, Alshahrani TS (2016) The symbiotic efficiency of legume tree rhizobia for host range legumes in Central Saudi Arabia. Int J Agri Biol 18:851–857CrossRefGoogle Scholar
  56. Shi SL (2005) The analysis for factors that affect the ability of growth promotion of alfalfa rhizobia in cold and drought regions and screening of high efficient strains. Gansu Agricultural University, DissertationGoogle Scholar
  57. Silva C, Kan FL, Martinez-Romero E (2007) Population genetic structure of Sinorhizobium meliloti and S. medicae isolated from nodules of Medicago spp.in Mexico. FEMS Microbiol Ecol 60:477–489CrossRefPubMedGoogle Scholar
  58. Sneath PA, Sokal R (1973) Principles of numerical taxonomy. Freeman and Co., San FranciscoGoogle Scholar
  59. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28: 2731–2739.  https://doi.org/10.1093/molbev/msr121
  60. Tang Z, An H, Deng L, Wang Y, Zhu G, Shangguan Z (2016) Effect of desertification on productivity in a desert steppe. Sci Rep 6:27839CrossRefPubMedPubMedCentralGoogle Scholar
  61. Tanuja BSC, Mishra PK (2013) Ascending migration of endophytic Bacillus thuringiensis, and assessment of benefits to different legumes of D.W. Himalayas. Eur J. Soil Biol 56: 56–64Google Scholar
  62. Thompson JD, Higgins DG, Gibson TJ (1994) Clustal W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucl Acids Res 22:4673–4680CrossRefPubMedPubMedCentralGoogle Scholar
  63. Torres TG, Rogel MA, Ormeñoorrillo E, Althabegoiti MJ, Nilsson JF, Niehaus K, et al. (2016) Rhizobium favelukesii sp. nov. isolated from the root nodules of alfalfa (Medicago sativa L.). Int J Syst Evol Microbiol 66: 4451–4457Google Scholar
  64. Vinuesa P, Silva C, Lorite MJ, Izaguirremayoral ML, Bedmar EJ, Martínez-Romero E (2005) Molecular systematics of rhizobia based on maximum likelihood and Bayesian phylogenies inferred from rrs, atpD, recA and nifH sequences, and their use in the classification of Sesbania microsymbionts from Venezuelan wetlands. Syst Appl Microbiol 28:702CrossRefPubMedGoogle Scholar
  65. Wand ET, Berkum PV, Beyene D, Sui XH, Dorado O, Chen WX, Martinez-Romero E (1998) Rhizobium huautlense sp. nov., a symbiont of Sesbania herbacea that has a close phylogenetic relationship with Rhizobium galegae. Int J Syst Bacteriology 48:687–699CrossRefGoogle Scholar
  66. Wang L, Ma F, Qu Y, Sun D, Li A, Guo J, Yu B (2011) Characterization of a compound bioflocculant produced by mixed culture of Rhizobium radiobacter, F2 and Bacillus sphaeicus, F6. World J Microb Biot 27:2559–2565CrossRefGoogle Scholar
  67. Weir BS (2012) The current taxonomy of rhizobia. New Zealand Rhizobia Webstie Google Scholar
  68. Weisburg WG, Barns SM, Pelletier DA, Lane DJ (1991) 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173:697–703CrossRefPubMedPubMedCentralGoogle Scholar
  69. Wielbo J (2012) Rhizobial communities in symbiosis with legumes: genetic diversity, competition and interactions with host plants. Cent Eur J Biol 7:363–372Google Scholar
  70. Wielbo J, Marek-Kozaczuk M, Mazur A, Kubik-Komar A, Skorupska A (2010) Genetic and metabolic divergence within a Rhizobium leguminosarum bv. Trifolii population recovered from clover nodules. Appl Environ Microbiol 76:4593–4600CrossRefPubMedPubMedCentralGoogle Scholar
  71. Wielbo J, Marek-Kozaczuk M, Mazur A, Kubik-Komar A, Skorupska A (2011) The structure and metabolic diversity of population of pea microsymbionts isolated from root nodules. British Microbiology Research Journal 1:55–69CrossRefGoogle Scholar
  72. Xu L, Zhang Y, Wang L, Chen W, Wei G (2014) Diversity of endophytic bacteria associated with nodules of two indigenous legumes at different altitudes of the Qilian mountains in China. Syst Appl Microbiol 37:457–465CrossRefPubMedGoogle Scholar
  73. Yao T (2002) Characteristics and biofertilizer of plant growth promoting rhizobacteria isolated from oat and wheat in Northwest China. Gansu Agricultural University, DissertationGoogle Scholar
  74. Yoon SH, Ha SM, Kwon S, Lim J, Kim Y, Seo H, Chun J (2017) Introducing EzBioCloud: a taxonomically united database of 16s rRNA gene sequences and whole-genome assemblies. Int J Syst Evol Microbiol 67:1613CrossRefPubMedPubMedCentralGoogle Scholar
  75. Zaied KA, Kosba ZA, Nassef MA, EI-saied AI (2009) Induction of rhizobium inoculants harboring salicylic acid gene. Australian J Basic Appl Sci 1386–1411Google Scholar
  76. Zhang XF, Shi SL, Nan LL, Chen JS, Man YR (2009) Phenotype diversities of alfalfa rhizobium strains collected in different ecological regions in Gansu Province. J Gansu Agric Univ 3:106–111Google Scholar
  77. Zhang SQ (2012a) Migration of rhizobia inside alfalfa plants and influencing factors. Gansu Agricultural University, DissertationGoogle Scholar
  78. Zhang YM (2012b) Ecological characteristics and comparative genomics of soybean rhizobia with biogeographic feature. China Agricultural University, DissertationGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature and the University of Milan 2018

Authors and Affiliations

  1. 1.College of Grassland ScienceGansu Agricultural UniversityLanzhouChina
  2. 2.Key Laboratory of Grassland Ecosystem of Ministry of EducationLanzhouChina
  3. 3.College of Agriculture and BiotechnologyHexi UniversityZhangyeChina

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