Annual replication is essential in evaluating the response of the soil microbiome to the genetic modification of maize in different biogeographical regions

Autoři: Márton Szoboszlay aff001;  Astrid Näther aff001;  Ewen Mullins aff002;  Christoph C. Tebbe aff001
Působiště autorů: Thünen Institute of Biodiversity, Federal Research Institute for Rural Areas, Forestry and Fisheries, Braunschweig, Germany aff001;  Teagasc, Agriculture and Food Development Authority, Dept. Crop Science, Oak Park, Carlow, Ireland aff002
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article
doi: 10.1371/journal.pone.0222737


The importance of geographic location and annual variation on the detection of differences in the rhizomicrobiome caused by the genetic modification of maize (Bt-maize, event MON810) was evaluated at experimental field sites across Europe including Sweden, Denmark, Slovakia and Spain. DNA of the rhizomicrobiome was collected at the maize flowering stage in three consecutive years and analyzed for the abundance and diversity of PCR-amplified structural genes of Bacteria, Archaea and Fungi, and functional genes for bacterial nitrite reductases (nirS, nirK). The nirK genes were always more abundant than nirS. Maize MON810 did not significantly alter the abundance of any microbial genetic marker, except for sporadically detected differences at individual sites and years. In contrast, annual variation between sites was often significant and variable depending on the targeted markers. Distinct, site-specific microbial communities were detected but the sites in Denmark and Sweden were similar to each other. A significant effect of the genetic modification of the plant on the community structure in the rhizosphere was detected among the nirK denitrifiers at the Slovakian site in only one year. However, most nirK sequences with opposite response were from the same or related source organisms suggesting that the transient differences in community structure did not translate to the functional level. Our results show a lack of effect of the genetic modification of maize on the rhizosphere microbiome that would be stable and consistent over multiple years. This demonstrates the importance of considering annual variability in assessing environmental effects of genetically modified crops.

Klíčová slova:

Community structure – Fungal genetics – Maize – Polymerase chain reaction – Rhizosphere – Ribosomal RNA – Sequence databases – Slovakian people


1. ISAAA. Global status of commercialized biotech/GM crops in 2017: Biotech crop adoption surges as economic benefits accumulate in 22 years. 2017. ISAAA Brief No. 53. ISAAA, Ithaca, NY.

2. Koziel MG, Beland GL, Bowman C, Carozzi NB, Crenshaw R, Crossland L, et al. Field performance of elite transgenic maize plants expressing an insecticidal protein derived from Bacillus thuringiensis. Bio-Technol. 1993;11(2): 194–200. doi: 10.1038/nbt0293-194

3. European Commission Decision on 22 April 1998 concerning the placing on the market of genetically modified maize (Zea mays L line MON 810), pursuant to Council Directive 90/220/EEC (98/294/EC). 1998. p. 32–3.

4. EFSA. Guidance on the environmental risk assessment of genetically modified plants. EFSA J. 2010;8: 1879.

5. Schroter D, Cramer W, Leemans R, Prentice IC, Araujo MB, Arnell NW, et al. Ecosystem service supply and vulnerability to global change in Europe. Science. 2005;310(5752):1333–7. doi: 10.1126/science.1115233 16254151

6. Barrios E. Soil biota, ecosystem services and land productivity. Ecol Econ. 2007;64(2): 269–85. doi: 10.1016/j.ecolecon.2007.03.004

7. Raaijmakers JM, Paulitz TC, Steinberg C, Alabouvette C, Moenne-Loccoz Y. The rhizosphere: a playground and battlefield for soilborne pathogens and beneficial microorganisms. Plant Soil. 2009;321(1–2): 341–61. doi: 10.1007/s11104-008-9568-6

8. Dennis PG, Miller AJ, Hirsch PR. Are root exudates more important than other sources of rhizodeposits in structuring rhizosphere bacterial communities? FEMS Microbiol Ecol. 2010;72(3): 313–27. doi: 10.1111/j.1574-6941.2010.00860.x 20370828

9. Hargreaves SK, Williams RJ, Hofmockel KS. Environmental filtering of microbial communities in agricultural soil shifts with crop growth. PLOS ONE. 2015;10(7). doi: 10.1371/journal.pone.0134345 26226508

10. Matthews A, Pierce S, Hipperson H, Raymond B. Rhizobacterial community assembly patterns vary between crop species. Front Microbiol. 2019;10. doi: 10.3389/fmicb.2019.00581 31019492

11. Berendsen RL, Pieterse CMJ, Bakker P. The rhizosphere microbiome and plant health. Trends Plant Sci. 2012;17(8):478–86. doi: 10.1016/j.tplants.2012.04.001 22564542

12. Philippot L, Raaijmakers JM, Lemanceau P, van der Putten WH. Going back to the roots: the microbial ecology of the rhizosphere. Nature Rev Microbiol. 2013;11(11):789–99. doi: 10.1038/nrmicro3109 24056930

13. Li L, Li SM, Sun JH, Zhou LL, Bao XG, Zhang HG, et al. Diversity enhances agricultural productivity via rhizosphere phosphorus facilitation on phosphorus-deficient soils. P Natl Acad Sci USA. 2007;104(27): 11192–6. doi: 10.1073/pnas.0704591104 17592130

14. Hayat R, Ali S, Amara U, Khalid R, Ahmed I. Soil beneficial bacteria and their role in plant growth promotion: a review. Ann Microbiol. 2010;60(4): 579–98. doi: 10.1007/s13213-010-0117-1

15. Garcia-Alonso M, Raybould A. Protection goals in environmental risk assessment: a practical approach. Transgenic Res. 2014;23(6): 945–56. doi: 10.1007/s11248-013-9760-1 24154954

16. Lindahl BD, Nilsson RH, Tedersoo L, Abarenkov K, Carlsen T, Kjoller R, et al. Fungal community analysis by high-throughput sequencing of amplified markers—a user's guide. New Phytol. 2013;199(1): 288–99. doi: 10.1111/nph.12243 23534863

17. Vetrovsky T, Baldrian P. The variability of the 16S rRNA gene in bacterial genomes and its consequences for bacterial community analyses. PLOS ONE. 2013;8(2). doi: 10.1371/journal.pone.0057923 23460914

18. Cheeke TE, Schutte UM, Hemmerich CM, Cruzan MB, Rosenstiel TN, Bever JD. Spatial soil heterogeneity has a greater effect on symbiotic arbuscular mycorrhizal fungal communities and plant growth than genetic modification with Bacillus thuringiensis toxin genes. Mol Ecol. 2015;24(10):2580–93. doi: 10.1111/mec.13178 25827202

19. Philippot L, Kuffner M, Cheneby D, Depret G, Laguerre G, Martin-Laurent F. Genetic structure and activity of the nitrate-reducers community in the rhizosphere of different cultivars of maize. Plant Soil. 2006;287(1–2): 177–86. doi: 10.1007/s11104-006-9063-x

20. Oliveira AP, Pampulha ME, Bennett JP. A two-year field study with transgenic Bacillus thuringiensis maize: Effects on soil microorganisms. Sci Total Environ. 2008;405(1–3): 351–7. doi: 10.1016/j.scitotenv.2008.05.046 18656246

21. Barriuso J, Valverde JR, Mellado RP. Effect of Cry1Ab protein on rhizobacterial communities of Bt-maize over a four-year cultivation period. PLOS ONE. 2012;7(4). doi: 10.1371/journal.pone.0035481 22558158

22. Baumgarte S, Tebbe CC. Field studies on the environmental fate of the Cry1Ab Bt-toxin produced by transgenic maize (MON810) and its effect on bacterial communities in the maize rhizosphere. Mol Ecol. 2005;14(8): 2539–51. doi: 10.1111/j.1365-294X.2005.02592.x 15969733

23. Ondreickova K, Mihalik D, Ficek A, Hudcovicova M, Kraic J, Drahovska H. Impact of genetically modified maize on the genetic diversity of rhizosphere bacteria: A two-year study in Slovakia. Pol J Ecol. 2014;62(1):67–76. doi: 10.3161/104.062.0107

24. Cotta SR, Dias ACF, Marriel IE, Gomes EA, van Elsas JD, Seldin L. Temporal dynamics of microbial communities in the rhizosphere of two genetically modified (GM) maize hybrids in tropical agrosystems. Anton Leeuw Int J G. 2013;103(3): 589–601. doi: 10.1007/s10482-012-9843-7 23124960

25. Cheeke TE, Cruzan MB, Rosenstiel TN. Field evaluation of arbuscular mycorrhizal fungal colonization in Bacillus thuringiensis toxin-expressing (Bt) and non-Bt maize. Appl Environ Microbiol. 2013;79(13):4078–86. doi: 10.1128/AEM.00702-13 23624473

26. European Commission E. Regulation (EC) No 1107/2009 of the European Parliament and of the Council of 21 October 2009 concerning the placing of plant protection products on the market and repealing Concil Directives 79/117/EEC and 91/414/EEC. Official Journal of the European Union. 2009; L 309:1–50.

27. Kozich JJ, Westcott SL, Baxter NT, Highlander SK, Schloss PD. Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform. Appl Environ Microbiol. 2013;79(17): 5112–20. doi: 10.1128/AEM.01043-13 23793624

28. Klindworth A, Pruesse E, Schweer T, Peplies J, Quast C, Horn M, et al. Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Res. 2013;41(1): 11. doi: 10.1093/nar/gks808 22933715

29. Throbäck IN, Enwall K, Jarvis A, Hallin S. Reassessing PCR primers targeting nirS, nirK and nosZ genes for community surveys of denitrifying bacteria with DGGE. FEMS Microbiol Ecol. 2004(3);49: 401–417. doi: 10.1016/j.femsec.2004.04.011 19712290

30. Gardes M, Bruns TD. ITS primers with enhanced specificity for Basidiomycetes—Application to the identification of mycorrhizae and rusts. Mol Ecol. 1993;2(2): 113–8. doi: 10.1111/j.1365-294x.1993.tb00005.x 8180733

31. White TJ, Bruns T, Lee S, J. T. Amplifiation and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, eds. PCR Protocols: A guide to methods and applications. San Diego: Academic Press, Inc.; 1990. p. 315–22.

32. Callahan BJ, McMurdie PJ, Rosen MJ, Han AW, Johnson AJA, Holmes SP. DADA2: High-resolution sample inference from Illumina amplicon data. Nat Methods. 2016;13(7):581–+. doi: 10.1038/nmeth.3869 27214047

33. Pruesse E, Quast C, Knittel K, Fuchs BM, Ludwig WG, Peplies J, et al. SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nucleic Acids Res. 2007;35(21):7188–96. doi: 10.1093/nar/gkm864 17947321

34. Wang Q, Quensen JF, Fish JA, Lee TK, Sun YN, Tiedje JM, et al. Ecological patterns of nifH genes in four terrestrial climatic zones explored with targeted metagenomics using FrameBot, a new informatics tool. mBio. 2013;4(5):9. doi: 10.1128/mBio.00592-13 24045641

35. Fish JA, Chai BL, Wang Q, Sun YN, Brown CT, Tiedje JM, et al. FunGene: the functional gene pipeline and repository. Front Microbiol. 2013;4:14. doi: 10.3389/fmicb.2013.00014

36. Martin M. Cutadapt removes adapter sequences from high-throughput sequencing reads. 2011. 2011;17(1):3%J EMBnet.journal. Epub 2011-08-02. doi: 10.14806/ej.17.1.200

37. Koljalg U, Nilsson RH, Abarenkov K, Tedersoo L, Taylor AFS, Bahram M, et al. Towards a unified paradigm for sequence-based identification of fungi. Mol Ecol. 2013;22(21): 5271–7. doi: 10.1111/mec.12481 24112409

38. Oksanen J, Blanchet FG, Kindt R, Legendre P, Minchin PR, O'Hara RB, et al. vegan: Community Ecology Package. R package version 2.4–3. http://CRANR-projectorg/package = vegan. 2016.

39. Gloor GB, Wu JR, Pawlowsky-Glahn V, Egozcue JJ. It's all relative: analyzing microbiome data as compositions. Ann Epidemiol. 2016;26(5):322–9. doi: 10.1016/j.annepidem.2016.03.003 WOS:000377641800004. 27143475

40. Fernandes AD, Reid JNS, Macklaim JM, McMurrough TA, Edgell DR, Gloor GB. Unifying the analysis of high-throughput sequencing datasets: characterizing RNA-seq, 16S rRNA gene sequencing and selective growth experiments by compositional data analysis. Microbiome. 2014;2:13. doi: 10.1186/2049-2618-2-13

41. Benjamini Y, Hochberg Y. Controlling the false discovery rate—A practical and powerful approach to multiple testing. J R Stat Soc Ser B-Methodol. 1995;57(1): 289–300.

42. Tan FX, Wang JW, Chen ZN, Feng YJ, Chi GL, Rehman SU. Assessment of the arbuscular mycorrhizal fungal community in roots and rhizosphere soils of Bt corn and their non-Bt isolines. Soil Biol Biochem. 2011;43(12): 2473–9. doi: 10.1016/j.soilbio.2011.08.014

43. Cheeke TE, Darby H, Rosenstiel TN, Bever JD, Cruzan MB. Effect of Bacillus thuringiensis (Bt) maize cultivation history on arbuscular mycorrhizal fungal colonization, spore abundance and diversity, and plant growth. Agr Ecosyst Environ. 2014;195:29–35. doi: 10.1016/j.agee.2014.05.019

44. Smalla K, Wieland G, Buchner A, Zock A, Parzy J, Kaiser S, et al. Bulk and rhizosphere soil bacterial communities studied by denaturing gradient gel electrophoresis: Plant-dependent enrichment and seasonal shifts revealed. Appl Environ Microbiol. 2001;67(10):4742–51. doi: 10.1128/AEM.67.10.4742-4751.2001 11571180

45. Schmalenberger A, Tebbe CC. Bacterial diversity in maize rhizospheres: conclusions on the use of genetic profiles based on PCR-amplified partial small subunit rRNA genes in ecological studies. Mol Ecol. 2003;12(1):251–61. doi: 10.1046/j.1365-294x.2003.01716.x 12492893

46. Dohrmann AB, Küting M, Jünemann S, Jaenicke S, Schlüter A, Tebbe CC. Importance of rare taxa for bacterial diversity in the rhizosphere of Bt- and conventional maize varieties. ISME J. 2013;7(1):37–49. doi: 10.1038/ismej.2012.77 22791236

47. Saxena D, Flores S, Stotzky G. Bt toxin is released in root exudates from 12 transgenic corn hybrids representing three transformation events. Soil Biol Biochem. 2002;34(1):133–7. doi: 10.1016/s0038-0717(01)00161-4

48. Coz I, Saxena D, Andow DA, Zwahlen C, Stotzky G. Microbial populations and enzyme activities in soil in situ under transgenic corn expressing Cry proteins from Bacillus thuringiensis. J Environ Qual. 2008;37(2): 647–62. doi: 10.2134/jeq2007.0352 18396552

49. Griffiths BS, Heckmann LH, Caul S, Thompson J, Scrimgeour C, Krogh PH. Varietal effects of eight paired lines of transgenic Bt maize and near-isogenic non-Bt maize on soil microbial and nematode community structure. Plant Biotechnol J. 2007;5(1):60–8. doi: 10.1111/j.1467-7652.2006.00215.x 17207257

50. Valldor P, Miethling-Graff R, Martens R, Tebbe CC. Fate of the insecticidal Cry1Ab protein of GM crops in two agricultural soils as revealed by 14C-tracer studies. Appl Microbiol Biotechnol. 2015;99(17):7333–41. doi: 10.1007/s00253-015-6655-5 25967657

51. Stotzky G. Persistence and biological activity in soil of the insecticidal proteins from Bacillus thuringiensis, especially from transgenic plants. Plant Soil. 2004;266(1–2):77–89.

52. Szoboszlay M, Dohrmann AB, Poeplau C, Don A, Tebbe CC. Impact of land-use change and soil organic carbon quality on microbial diversity in soils across Europe. FEMS Microbiol Ecol. 2017;93(12). doi: 10.1093/femsec/fix146 29087486

53. Peiffer JA, Spor A, Koren O, Jin Z, Tringe SG, Dangl JL, et al. Diversity and heritability of the maize rhizosphere microbiome under field conditions. Proc Nat Acad Sci USA. 2013;110(16): 6548–53. doi: 10.1073/pnas.1302837110 23576752

54. Yang Y, Wang N, Guo XY, Zhang Y, Ye BP. Comparative analysis of bacterial community structure in the rhizosphere of maize by high-throughput pyrosequencing. PLOS ONE. 2017;12(5). doi: 10.1371/journal.pone.0178425 28542542

55. Cheeke TE, Pace BA, Rosenstiel TN, Cruzan MB. The influence of fertilizer level and spore density on arbuscular mycorrhizal colonization of transgenic Bt 11 maize (Zea mays) in experimental microcosms. FEMS Microbiol Ecol. 2011;75(2): 304–12. doi: 10.1111/j.1574-6941.2010.01013.x 21198682

56. Porras-Alfaro A, Herrera J, Natvig DO, Sinsabaugh RL. Effect of long-term nitrogen fertilization on mycorrhizal fungi associated with a dominant grass in a semiarid grassland. Plant Soil. 2007;296(1–2): 65–75. doi: 10.1007/s11104-007-9290-9

57. Hudson CM, Kirton E, Hutchinson MI, Redfern JL, Simmons B, Ackerman E, et al. Lignin-modifying processes in the rhizosphere of arid land grasses. Environ Microbiol. 2015;17(12): 4965–78. doi: 10.1111/1462-2920.13020 26279186

58. Mounier E, Hallet S, Cheneby D, Benizri E, Gruet Y, Nguyen C, et al. Influence of maize mucilage on the diversity and activity of the denitrifying community. Environ Microbiol. 2004;6(3): 301–12. doi: 10.1111/j.1462-2920.2004.00571.x 14871213

59. Sun RB, Guo XS, Wang DZ, Chu HY. Effects of long-term application of chemical and organic fertilizers on the abundance of microbial communities involved in the nitrogen cycle. Appl Soil Ecol. 2015; 95:171–8. doi: 10.1016/j.apsoil.2015.06.010

60. Kandeler E, Deiglmayr K, Tscherko D, Bru D, Philippot L. Abundance of narG, nirS, nirK, and nosZ genes of denitrifying bacteria during primary successions of a glacier foreland. Appl Environ Microbiol. 2006;72(9):5957–62. doi: 10.1128/AEM.00439-06 16957216

61. Azziz G, Monza J, Etchebehere C, Irisarri P. nirS- and nirK-type denitrifier communities are differentially affected by soil type, rice cultivar and water management. Euro J Soil Biol. 2017; 78:20–8. doi: 10.1016/j.ejsobi.2016.11.003

62. Dandie CE, Wertz S, Leclair CL, Goyer C, Burton DL, Patten CL, et al. Abundance, diversity and functional gene expression of denitrifier communities in adjacent riparian and agricultural zones. FEMS Microbiol Ecol. 2011;77(1): 69–82. doi: 10.1111/j.1574-6941.2011.01084.x 21385191

63. Jones CM, Spor A, Brennan FP, Breuil MC, Bru D, Lemanceau P, et al. Recently identified microbial guild mediates soil N2O sink capacity. Nat Clim Change. 2014;4(9): 801–5. doi: 10.1038/nclimate2301

64. Hallin S, Philippot L, Loffler FE, Sanford RA, Jones CM. Genomics and Ecology of Novel N2O-Reducing Microorganisms. Trends Microbiol. 2018;26(1):43–55. doi: 10.1016/j.tim.2017.07.003 28803698

65. Hannula SE, de Boer W, van Veen JA. Do genetic modifications in crops affect soil fungi? A review. Biol Fert Soils. 2014;50(3):433–46. doi: 10.1007/s00374-014-0895-x

Článek vyšel v časopise


2019 Číslo 12