Archives of Microbiology

, Volume 201, Issue 9, pp 1151–1161 | Cite as

Diversity and nodulation effectiveness of rhizobia and mycorrhizal presence in climbing dry beans grown in Prespa lakes plain, Greece

  • Ioannis IpsilantisEmail author
  • Leonidas Lotos
  • Ioannis T. Tsialtas
Original Paper


The Prespa lakes plain is an isolated area where about 1000 ha are seeded to Phaseolus vulgaris L. and Phaseolus coccineus L. Nodulation, arbuscular mycorrhizal fungal (AMF) presence and the genetic diversity of rhizobia were evaluated by 16S-ITS-23S-RFLP patterns and by sequencing. The bean rhizobial population in the region was diverse, despite its geographic isolation. No biogeographic relationships were detected, apart from a Rhizobium tropici-related strain that originated from an acidic soil. No clear pattern was detected in clustering with bean species and all isolates formed nodules with both bean species. Most strains were related to Rhizobium leguminosarum and a number of isolates were falling outside the already characterized species of genus Rhizobium. Application of heavy fertilization has resulted in high soil N and P levels, which most likely reduced nodulation and AMF spore presence. However, considerable AMF root length colonization was found in most of the fields.


Arbuscular mycorrhizae Phaseolus vulgaris Phaseolus coccineus Symbiotic nitrogen fixation 



This work was supported by the Research Committee of Aristotle University of Thessaloniki (Grant number 89313). The authors would also like to thank Kyriaki Kosmanidou and the “Pelecanos” cooperative for their assistance in field sampling.

Supplementary material

203_2019_1679_MOESM1_ESM.docx (142 kb)
S 1. Agarose gel of the PCR products where multiple or single band may be observed before (left) and after HaeII digestion (right). Highlighted are the bands that were successfully sequenced. M: 100 base DNA ladder marker, 1: PYL2, 2: ORM3, 3: ORM1, 4: PYL6, 5: PYL7, 6: SLL3, 7: SLL7, 8: PYL6, 9: KAR6, 10: ORM4, 11: SLL8, 12: PYL5, 13: ERG2, 14: LAI3 (DOCX 142 kb)
203_2019_1679_MOESM2_ESM.docx (328 kb)
S 2. Agarose gel of the PCR products where multiple or single band may be observed before (up) and after HaeII digestion (down). Highlighted are the bands that were successfully sequenced. M: 100 base DNA ladder marker, 1: ORM2, 2: PYL3, 3: KAR1, 4: KAR7, 5: KAR9, 6: SLL1, 7: PYL2, 8: MIK3, 9: KAR2, 10: KAR8, 11: SLL6, 12: SLA2, 13: LAI2, 14: PYL4, 16: SLL12, 17: SLA5, 18: OPA1, 19: GRA4, 20: GRA5, 21: PYL1, 22: PYL9, 23: SLA1, 24: LAI5, 25: GRA2, 26: SLL4, 27: GRA3, 28: MIK 7, 29: MIK5, 30: JUM1, 31: MIK2, 32: LAI3, 33: GRA1, 34: LAI6, 35: LAI4, 36: OPA2, 37: MIK1 38: SLA4, 39: LAI8, 40: LAI7, 41: SLL5, 42: KAR4, 43: MIK4, 44: KAR5, 45: SLL13, 46: MIK6, 47: ERG1, 48: SLL2, 49: PYL10, 50: PYL11, 51: ORM5, 52: KAR10, 53: KAR11, 54: ORM6, 55: KAR12. Note that JUM1 is an isolate from Florina, an area close, but outside the Prespa lakes area (DOCX 328 kb)
203_2019_1679_MOESM3_ESM.docx (201 kb)
S 3. Agarose gel of the PCR products after MspI digestion. M: 100 base DNA ladder marker. 1: ERG1, 2: SLL11, 3: MIK6, 4: KAR3, 6: KAR6, 7: PYL5, 8: KAR1, 9: KAR4, 10: KAR7, 11: KAR9, 12: LAI6, 13: GRA1, 14: LAI7, 15: LAI1, 16: GRA1, 17: KAR5, 18: MIK4, 19: OPA1, 21: GRA2, 22: MIK5, 23: MIK2, 24: MIK7, 25: MIK3, 26: PYL2, 27: LAI3, 28: ERG2, 30: SLL3, 31: SLL7, 32: ORM1, 33: SLL4, 34: PYL4, 36: GRA2, 37: KAR2, 38: SLA1, 39: SLA1, 40: SLL1, 43: KAR3, 44: LAI2, 45: PYL6, 46: PYL7, 47: SLA2, 48: KAR8, 50: KAR9, 51: SLL7, 52: SLL12, 53: GRA3, 54: ERG1 (DOCX 200 kb)


  1. Abbaszadeh-dahaji P, Savaghebi GhR, Asadi-rahmani H, Rejali F, Farahbakhsh M, Moteshareh-zadeh D, Omidvari M, Lindstrom K (2012) Symbiotic effectiveness and plant growth promoting traits in some Rhizobium strains isolated from Phaseolus vulgaris L. Plant Growth Regul 68:361–370Google Scholar
  2. Adhikari D, Itoh K, Suyama L (2013) Genetic diversity of common bean (Phaseolus vulgaris L.) nodulating rhizobia in Nepal. Plant Soil 368:341–353Google Scholar
  3. Akter Z, Pageni BB, Lupwayi NZ, Balasubramanian PM (2014) Biological nitrogen fixation and nifH gene expression in dry beans (Phaseolus vulgaris L.). Can J Plant Sci 94:203–212Google Scholar
  4. Álvarez-Martínez ER, Valverde Á, Ramírez-Bahena MH, García-Fraile P, Tejedor C, Mateos PF, Santillana N, Zúñiga D, Peix A, Velázquez E (2009) The analysis of core and symbiotic genes of rhizobia nodulating Vicia from different continents reveals their common phylogenetic origin and suggests the distribution of Rhizobium leguminosarum strains together with Vicia seeds. Arch Microbiol 191:659–668PubMedGoogle Scholar
  5. Amarger N, Machere V, Laguerre G (1997) Rhizobium gallicum sp. nov. and Rhizobium giardinii sp. nov., from Phaseolus vulgaris nodules. Int J Syst Bacteriol 47:996–1006PubMedGoogle Scholar
  6. Andrade DS, Murphy PJ, Giller KE (2002) The diversity of Phaseolus-nodulating rhizobial populations is altered by liming of acid soils planted with Phaseolus vulgaris L. in Brazil. Appl Environ Micorbiol 68:4025–4034Google Scholar
  7. Anguilar OM, López MV, Donato M, Morón B, Soria-Diaz ME, Mateos C, Gil-Serrano A, Sousa C, Megías M (2006) Phylogeny and nodulation signal molecule of rhizobial populations able to nodulate common beans—other than the predominant species Rhizobium etli—present in soils from the northwest of Argentina. Soil Biol Biochem 38:573–586Google Scholar
  8. Aoki S, Kondo T, Prévost D, Nakata S, Kajita T, Ito M (2010) Genotypic and phenotypic diversity of rhizobia isolated from Lathyrus japonicus indigenous to Japan. Syst Appl Microbiol 33:383–397PubMedGoogle Scholar
  9. Aouani ME, Mhamdi R, Mars M, Elayeb M, Ghtir R (1997) Potential for inoculation of common bean by effective rhizobia in Tunisian soils. Agronomie 17:445–454Google Scholar
  10. Aserse AA, Räsänen LA, Assefa F, Hailemariam A, Lindström K (2012) Phylogeny and genetic diversity of native rhizobia nodulating common bean (Phaseolus vulgaris L.) in Ethiopia. Syst Appl Microbiol 35:120–131PubMedGoogle Scholar
  11. Baginsky C, Brito B, Scherson R, Pertuzé R, Seguelm O, Cañete A, Araneda C, Johnson WE (2015) Genetic diversity of Rhizobium from nodulating beans grown in a variety of Mediterranean climate soils of Chile. Arch Microbiol 197:419–429PubMedGoogle Scholar
  12. Bernal G, Graham PH (2001) Diversity in the rhizobia associated with Phaseolus vulgaris L. in Ecuador, and comparisons with Mexican bean rhizobia. Can J Microbiol 47:526–534PubMedGoogle Scholar
  13. Bustos P, Santamaria RI, Pérez-Carrascal OM, Acosta JL, Lozano L, Juárez S, Martínez-Flores I, Martínez-Romero E, Cevallos MA, Romero D, Dávila G, Vinuesa P, Miranda F, Ormeρo E, González V (2017) Complete genome sequences of three Rhizobium gallicum symbionts associated with common bean (Phaseolus vulgaris). Genome Announc 11:e00030–17Google Scholar
  14. Caballero-Mellado J, Martínez-Romero E (1999) Soil fertilization limits the genetic diversity of Rhizobium in bean nodules. Symbiosis 26:111–121Google Scholar
  15. Cao Y, Wang ET, Zhao L, Chen WM, Wei GH (2014) Diversity and distribution of rhizobia nodulated with Phaseolus vulgaris in two ecoregions of China. Soil Biol Biochem 78:128–137Google Scholar
  16. Dall’Agnol RF, Ribeiro RA, Ormeño-Orrillo E, Rogel MA, Delamuta JRM, Andrade DS, Martínez-Romero E, Hungria M (2013) Rhizobium freirei sp. nov., a symbiont of Phaseolus vulgaris that is very effective at fixing nitrogen. Int J Syst Evol Microbiol 63:4167–4173PubMedGoogle Scholar
  17. Dar GH, Zargar MY, Beigh GM (1997) Biocontrol of Fusarium root rot in the common bean (Phaseolus vulgaris L.) by using symbiotic Glomus mosseae and Rhizobium leguminosarum. Microb Ecol 34:74–80Google Scholar
  18. Darriba D, Taboad GL, Doallo R, Posada D (2012) jModelTest 2: more models, new heuristics and parallel computing. Nat Methods 9:772PubMedPubMedCentralGoogle Scholar
  19. Elbanna K, Elbadry M, Gamal-Eldin H (2009) Genotypic and phenotypic characterization of rhizobia that nodulate snap bean (Phaseolus vulgaris L.) in Egyptian soils. Syst Appl Microbiol 32:522–530PubMedGoogle Scholar
  20. Embalomatis A, Papakosta DK, Katinakis P (1994) Evaluation of Rhizobium meliloti strains isolated from indigenous populations in northern Greece. J Agron Crop Sci 172:73–80Google Scholar
  21. García-Fraile P, Mulas-García D, Peix A, Rivas R, González- Andrés F, Velázquez E (2010) Phaseolus vulgaris is nodulated in northern Spain by Rhizobium leguminosarum strains harboring two nodC alleles present in American Rhizobium etli strains: biogeographical and evolutionary implications. Can J Microbiol 56:657–666PubMedGoogle Scholar
  22. Giller KE, Cadisch G (1995) Future benefits from biological nitrogen fixation—an ecological approach to agriculture. Plant Soil 174:255–277Google Scholar
  23. Graham PH, Ranalli P (1997) Common bean (Phaseolus vulgaris L.). Field Crops Res 53:131–146Google Scholar
  24. Graham PH, Rosas JC, Estevez de Jensen C, Peralta E, Tlusty B, Acosta-Gallegos J, Arraes Perreira PA (2003) Addressing edaphic constrains to bean production: the Bean/Cowpea CRSP project in perspective. Field Crops Res 82:179–192Google Scholar
  25. Gryndler H, Leština J, Moravec V, Přikryl Z, Lipavsky J (1989) Colonization of maize roots by VAM-fungi under conditions of long-term fertilization of varying intensity. Agric Ecosyst Environ 29:183–186Google Scholar
  26. Guindon S, Gascuel O (2003) A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52:696–704PubMedGoogle Scholar
  27. Hardarson G, Bliss FA, Cigales-Rivero MR, Henson RA, Kipe-Nolt JA, Longeri L, Manrique A, Pena-Cabriales JJ, Pereira PAA, Sanabria CA, Tsai SM (1993) Genotypic variation in biological nitrogen fixation by common bean. Plant Soil 152:59–70Google Scholar
  28. Haukka K, Lindström K, Young JPW (1996) Diversity of partial 16S rRNA sequences among and within strains of African rhizobia isolated from Acacia and Prosopis. Syst Appl Microbiol 19:352–359Google Scholar
  29. Havlin JJ, Tisdale SL, Nelson WL, Beaton JD (2014) Chapter 4: Nitrogen. In: Havlin JJ, Tisdale SL, Nelson WL, Beaton JD (eds) Soil fertility and fertilizers, 8th edn. Pearson, Prentice Hall, Upper Saddle River, pp 117–184Google Scholar
  30. Hayman DS (1986) Mycorrhizae of nitrogen-fixing legumes. MIRCEN J 2:121–145Google Scholar
  31. Hayman DS, Barea JM, Azcon R (1976) Vesicular-arbuscular mycorrhiza in southern Spain: its distribution in crops growing in soil of different fertility. Phytopathol Mediterr 15:1–6Google Scholar
  32. Herrera-Cervera JA, Caballero-Mellado J, Laguerre G, Tichy HV, Requena N, Amarger N, Martínez-Romero E, Olivares J, Sanjuan J (1999) At least five rhizobial species nodulate Phaseolus vulgaris in a Spanish soil. FEMS Microbiol Ecol 30:87–97Google Scholar
  33. Hou BC, Wang ET, Li Y, Jia RZ, Chen WF, Man CX, Sui XH, Chen WX (2009) Rhizobial resource associated with epidemic legumes in Tibet. Microb Ecol 57:69–81PubMedGoogle Scholar
  34. Huang YY, Cho ST, Lo WS, Wang YC, Lai EML, Kuo CH (2015) Complete genome sequence of Agrobacterium tumefaciens Ach5. Genome Announc 3:e00570–15PubMedPubMedCentralGoogle Scholar
  35. Junier P, Alfaro M, Guevara R, Witzel KP, Carú M (2014) Genetic diversity of Rhizobium present in nodules of Phaseolus vulgaris L. cultivated in two soils of the central region in Chile. Appl Soil Ecol 80:60–66Google Scholar
  36. Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol 30:772–780PubMedPubMedCentralGoogle Scholar
  37. Kosmas C, Moustakas N, Tsatiris B, Danalatos N (1990) Evaluation of soil resources of the Prespa region, Greece. EEC Project B6617-25-89. Agricultural University of Athens, AthensGoogle Scholar
  38. Kwon SW, Park JY, Kim JS, Kang JW, Cho YH, Lim CK, Parker MA, Lee GB (2005) Phylogenetic analysis of the genera Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium on the basis of 16S rRNA gene and internally transcribed spacer region sequences. Int J Syst Evol Microbiol 55:263–270PubMedGoogle Scholar
  39. Laguerre G, Mavingui P, Allard MR, Charnay MP, Louvrier P, Mazurier SI, Rigottier-Gois L, Amarger N (1996) Typing of rhizobia by PCR DNA fingerprinting and PCR-restriction fragment length polymorphism analysis of chromosomal and symbiotic gene regions: application to Rhizobium leguminosarum and its different biovars. Appl Environ Microbiol 62:2029–2036PubMedPubMedCentralGoogle Scholar
  40. López-López A, Rogel-Hernández MA, Barois I, Ceballos AIO, Martínez J, Ormeño-Orrillo E, Martínez-Romero E (2012) Rhizobium grahamii sp. nov., from nodules of Dalea leporina, Leucaena leucocephala and Clitoria ternatea, and Rhizobium mesoamericanum sp. nov., from nodules of Phaseolus vulgaris, siratro, cowpea and Mimosa pudica. Int J Syst Evol Microbiol 62:2264–2271PubMedGoogle Scholar
  41. Mavromatis AG, Arvanitoyannis S, Korkovelos AE, Giakountis A, Chatzitheodorou VA, Goulas CK (2010) Genetic diversity among common bean (Phaseolus vulgaris L.) Greek landraces and commercial cultivars: nutritional components, RAPD and morphological markers. Span J Agric Res 8:986–994Google Scholar
  42. Mhamdi R, Jebara M, Aouani ME, Ghrir R, Mars M (1999) Genotypic diversity and symbiotic effectiveness of rhizobia isolated from roots nodules of Phaseolus vulgaris L., grown in Tunisian soils. Biol Fertil Soil 28:313–320Google Scholar
  43. Miller RM, Reinhardt DR, Jastrow JD (1995) External hyphal production of vesicular-arbuscular mycorrhizal fungi in pasture and tallgrass prairie communities. Oecologia 103:17–23PubMedGoogle Scholar
  44. Mnasri B, Mrabet M, Laguerre G, Aouani ME, Mhamdi R (2007) Salt-tolerant rhizobia isolated from a Tunisian oasis that are highly effective for symbiotic N2-fixation with Phaseolus vulgaris constitute a novel biovar (bv. mediterranense) of Sinorhizobium meliloti. Arch Microbiol 187:79–85PubMedGoogle Scholar
  45. Mnasri B, Saïdi S, Chihaou SA, Mhamdi R (2012) Sinorhizobium americanum symbiovar mediterranense is a predominant symbiont that nodulates and fixes nitrogen with common bean (Phaseolus vulgaris L.) in a northern Tunisian field. Syst Appl Microbiol 35:263–269PubMedGoogle Scholar
  46. Mnasri B, Liu TY, Saidi S, Chen WF, Chen WX, Zhang XX, Mhamdi R (2014) Rhizobium azibense sp. nov., a nitrogen fixing bacterium isolated from root nodules of Phaseolus vulgaris. Int J Syst Evol Microbiol 64:1501–1506PubMedGoogle Scholar
  47. Nelson M, Guhlin J, Epstein B, Tiffin P, Sadowsky MJ (2018) The complete replicons of 16 Ensifer meliloti strains offer insights into intra-and inter-replicon gene transfer, transposon-associated loci, and repeat elements. Microb Genom 4:e000174PubMedCentralGoogle Scholar
  48. Oliveira JP, Galli-Terasawa LV, Enke CG, Cordeiro VK, Armstrong LCT, Hungria M (2011) Genetic diversity of rhizobia in a Brazilian oxisol nodulating Mesoamerican and Andean genotypes of common bean (Phaseolus vulgaris L.). World J Microbiol Biotechnol 27:643–650Google Scholar
  49. Pavel BA, Vasile CI (2012) PyElph—a software tool for gel images analysis and phylogenetices. BMC Bioinform 13:9Google Scholar
  50. Pérez-Ramírez NO, Rogel MA, Wang E, Castellanos JZ, Martínez-Romero E (1998) Seeds of Phaseolus vulgaris bean carry Rhizobium etli. FEMS Microbiol Ecol 26:289–296Google Scholar
  51. Prévost D, Antoun H (2007) Root nodule bacteria and symbiotic nitrogen fixation, Chapter 31. In: Carter MR, Gregorich EG (eds) Soil sampling and methods of analysis. CRC Press, Boca Raton, pp 379–397Google Scholar
  52. Rahmani ΗΑ, Räsänen LA, Afshari M, Lindström K (2011) Genetic diversity and symbiotic effectiveness of rhizobia isolated from root nodules of Phaseolus vulgaris L. grown in soils of Iran. Appl Soil Ecol 48:287–293Google Scholar
  53. Redecker D, von Berswordt-Wallrabe P, Beck DP, Werner D (1997) Influence of inoculation with arbuscular mycorrhizal fungi on stable isotopes of nitrogen in Phaseolus vulgaris. Biol Fert Soils 24:344–346Google Scholar
  54. Rodriguez-Navarro DN, Buendia AM, Camacho M, Lucas MM, Santamaria C (2000) Characterization of Rhizobium spp. bean isolates from south west Spain. Soil Biol Biochem 32:1601–1613Google Scholar
  55. Santamaría RI, Bustos P, Pérez-Carrascal OM, Miranda-Sánchez F, Vinuesa P, Martínez-Flores I, Juárez S, Lozano L, Martínez-Romero E, Cevallos MA, Romero D, Dávila G, Ormeño-Orrillo E, González V (2017) Complete genome sequences of eight Rhizobium symbionts associated with common bean (Phaseolus vulgaris). Genome Announc 5:e00645–17PubMedPubMedCentralGoogle Scholar
  56. Sessitch A, Ramirez-Saad H, Hardarson G, Akkermans ADL, DeVos WM (1997) Classification of Austrian rhizobia and the Mexican isolate FL27 obtained from Phaseolus vulgaris L. as Rhizobium gallicum. Int J Syst Bacteriol 47:1097–1101Google Scholar
  57. Smith SE, Read DJ (1997) Mycorrhizal symbiosis. Academic Press, San DiegoGoogle Scholar
  58. Somasegaran P, Hoben HJ (1985) Methods in legume-rhizobium technology. University of Hawaii NifTAL Project and MIRCENGoogle Scholar
  59. Sylvia DM (1994) Vesicular-arbuscular mycorrhizal (VAM) fungi. In: Weaver RW, Angle JS, Bottomley PJ, Bezdicek D, Smith S, Tabatabai A, Wollum AG (eds) Methods of soil analysis, Part 2. Microbiological and biochemical properties, vol 5. Soil Science Society of America, Madison, pp 351–378Google Scholar
  60. Tamimi SM (2002) Genetic diversity and symbiotic effectiveness of rhizobia isolated from root nodules of common bean (Phaseolus vulgaris L.) grown in the soils of the Jordan valley. Appl Soil Ecol 19:183–190Google Scholar
  61. Tertivanidis K, Koutita O, Papadopoulos II, Tokatlidis IS, Tamoutsidis EG, Pappa-Michailidou V, Koutsika-Sotiriou M (2008) Genetic diversity in bean populations based on random amplified polymorphic DNA markers. Biotechnology 7:1–9Google Scholar
  62. Thies JE, Bohlool BB, Singleton PW (1992) Environmental effects on competition for nodule occupancy between introduced and indigenous rhizobia and among introduced strains. Can J Microbiol 38:493–500Google Scholar
  63. Valverde A, Ingua JM, Peix A, Cervantes E, Velásquez E (2006) Rhizobium lusitanum sp. nov. a bacterium that nodulates Phaseolus vulgaris. Int J Syst Evol Microbiol 56:2631–2637PubMedGoogle Scholar
  64. Van Cauwenberghe J, Verstraete B, Lemaire B, Lievens B, Michiels J, Honnay O (2014) Population structure of root nodulating Rhizobium leguminosarum in Vicia cracca populations at local to regional geographic scales. Syst Appl Microbiol 37:613–621PubMedGoogle Scholar
  65. Wang L, Cao Y, Wang ET, Qiao YJ, Jiao S, Liu ZS, Zhao L, Wei GH (2016) Biodiversity and biogeography of rhizobia associated with common bean (Phaseolus vulgaris L.) in Shaanxi province. Syst Appl Microbiol 39:211–219PubMedGoogle Scholar
  66. Wei GH, Zhang ZX, Chen C, Chen WM, Ju WT (2008) Phenotypic and genetic diversity of rhizobia isolated from nodules of the legume genera Astragalus, Lespedeza and Hedysarum in northwestern China. Microbiol Res 163:651–662PubMedGoogle Scholar
  67. Yan H, Ji ZJ, Jiao YS, Wang ET, Chen WF, Guo BL, Chen WX (2016) Genetic diversity and distribution of rhizobia associated with the medicinal legumes Astragalus spp. and Hedysarum polybotrys in agricultural soils. Syst Appl Microbiol 39:141–149PubMedGoogle Scholar
  68. Zurdo-Piñeiro JL, García-Fraile P, Rivas R, Peix A, León-Barrios M, Willems A, Mateos PF, Martínez-Molina E, Velázquez E, van Berkum P (2009) Rhizobia from Lanzarote, the Canary Islands, that nodulate Phaseolus vulgaris have characteristics in common with Sinorhizobium meliloti isolates from mainland Spain. Appl Environ Microbiol 75:2354–2359PubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Laboratory of Soil Science, Faculty of AgricultureAristotle University of ThessalonikiThessalonikiGreece
  2. 2.Laboratory of Plant Pathology, Faculty of AgricultureAristotle University of ThessalonikiThessalonikiGreece
  3. 3.Laboratory of Agronomy, Faculty of AgricultureAristotle University of ThessalonikiThessalonikiGreece

Personalised recommendations