European Journal of Plant Pathology

, Volume 153, Issue 1, pp 243–251 | Cite as

Re-evaluation of biosafety questions on genetically modified biocontrol bacteria

  • Debora C. M. GlandorfEmail author


Microorganisms have the potential to promote plant health and can be used to increase agricultural production that depends less on chemical control. The implementation of EU Directive 2009/128/EC, also called the Sustainable Use Directive, has led to a renewed interest in microbial biocontrol of plant diseases. Technological developments in biotechnology such as high throughput sequencing and genome editing using CRISPR/Cas open new possibilities for biocontrol applications of microorganisms. Some of these developments may involve the use of genetic modification to increase efficacy. This reopens biosafety questions posed for genetically modified microorganisms with respect to their environmental release. However, over the last decades quite some experience has been gained with genetically modified microorganisms, which could also be considered for the risk assessment of microorganisms obtained by recent techniques in biotechnology.

This paper describes experience gained from risk assessment studies with genetically modified microbial biocontrol agents under field conditions. The use of this experience in addressing current biosafety questions in biotechnology is discussed.


Biocontrol Biotechnology Genetically modified microorganisms Microbiome PGPR Pseudomonads 



I would like to thank Cecile van der Vlugt (RIVM), Jacqueline Scheepmaker (RIVM) and Peter Bakker (University Utrecht) for critical reading of the manuscript.

Compliance with ethical standards

Conflict of interest

The author declares that she has no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by the author.


  1. Ahemad, M., & Kibret, M. (2014). Mechanisms and applications of plant growth promoting rhizobacteria: Current perspective. Journal of King Saud University-Science, 1–20.Google Scholar
  2. Bakker, P. A. H. M., Glandorf, D. C. M., Viebahn, M., Ouwens, T. W. M., Smit, E., Leeflang, P., Wernars, K., Thomashow, L. S., Thomas-Oates, J. E., & van Loon, L. C. (2002). Effects of Pseudomonas putida modified to produce phenazine-1-carboxylic acid and 2,4-diacetylphloroglucinol on the microflora of field grown wheat. Antonie Van Leeuwenhoek, 81, 617–624.CrossRefGoogle Scholar
  3. Bakker, P. A. H. M., Berendsen, R. L., Doornbos, R. F., Wintermans, P. C. A., & Pieterse, C. M. J. (2013). The rhizosphere revisited: Root microbiomics. Frontiers in Plant Science, 4, 165.Google Scholar
  4. Berendsen, R. L., Pieterse, C. M. J., & Bakker, P. A. H. M. (2012). The rhizosphere microbiome and plant health. Trends in Plant Science, 17, 478–486.CrossRefGoogle Scholar
  5. Berendsen, R. L., van Verk, M. C., Stringlis, I. A., Zamioudis, C., Tommassen, J., Pieterse, C. M. J., & Bakker, P. A. H. M. (2015). Unearthing the genomes of plant-beneficial Pseudomonas model strains WCS358, WCS374 and WCS417. BMC Genomics, 16, 539.CrossRefGoogle Scholar
  6. Bortesi, L., & Fisher, R. (2015). The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnology Advances, 3, 41–53.CrossRefGoogle Scholar
  7. Chen, T., Hongdilokkul, N., Liu, Z., Thirunavukarasu, D., & Romesberg, F. E. (2016). The expanding world of DNA and RNA. Current Opinion in Chemical Biology, 34, 80–87.CrossRefGoogle Scholar
  8. Choi, H. Y., Ryder, M. H., Gillings, M. R., Stokes, H. W., Ophel-Keller, K. M., & Veal, D. A. (2003). Survival of a lacZY-marked strain of Pseudomonas corrugata following a field release. FEMS Microbial Ecology, 43, 367–374.CrossRefGoogle Scholar
  9. Compant, S., Duffy, B., Nowak, J., Clement, C., & Barka, E. (2005). Use of plant growth-promoting bacteria for biocontrol of plant diseases: Principles, mechanisms of action, and future prospects. Applied and Environmental Microbiology, 7, 4951–4959.CrossRefGoogle Scholar
  10. De Leij, F. A. A. M., Sutton, E. J., Whipps, J. M., Fenlon, J. S., & Lynch, J. M. (1995a). Field release of a genetically modified Pseudomonas fluorescens on wheat: Establishment, survival and dissemination. Nature Biotechnology, 13, 1488–1492.CrossRefGoogle Scholar
  11. De Leij, F. A. A. M., Sutton, E. J., Whipps, J. M., Fenlon, J. S., & Lynch, J. M. (1995b). Impact of field release of genetically modified Pseudomonas fluorescens on indigenous populations of wheat. Applied and Environmental Microbiology, 61, 3443–3453.Google Scholar
  12. De Leij, F. A. A. M., Thomas, C. E., Baily, M. J., Whipps, J. M., & Lynch, J. M. (1998). Effect of insertion site and metabolic load on the environmental fitness of a genetically modified Pseudomonas fluorescens isolate. Applied and Environmental Microbiology, 64, 2634–2638.Google Scholar
  13. De Lorenzo, V., Herrero, M., Jakubzik, U., & Timmis, K. N. (1990). Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing and chromosomal insertion of cloned DNA in gram-negative Eubacteria. Journal of Bacteriology, 172, 6568–6572.CrossRefGoogle Scholar
  14. De Lorenzo, V. (2009). Recombinant bacteria for environmental release: What went wrong and what we have learnt from it. Clinical Microbiology Infections, 15, 63–65.CrossRefGoogle Scholar
  15. De Lorenzo, V. (2010). Environmental biosafety in the age of synthetic biology: Do we really need a radical new approach? Bioessays, 32, 926–931.CrossRefGoogle Scholar
  16. EFSA (2011). Guidance on the risk assessment of genetically modified microorganism and their products intended for food and feed use. EFSA Journal, 9, 193–246.Google Scholar
  17. Faasse P.E. (2008). In splendid isolation: A history of the Willie Commelin Scholten Phytopathology Laboratory 1894–1992. History of science and scholarship in the Netherlands. Amsterdam: KNAW Press.Google Scholar
  18. Farrar, K., Bryant, D., & Cope-Selby, N. (2014). Understanding and engineering beneficial plant-microbe interactions: Plant growth promotion in energy crops. Plant Biotechnology Journal, 12, 1193–1206.CrossRefGoogle Scholar
  19. Glandorf, D. C. M., Brand, I., Bakker, P. A. H. M., & Schippers, B. (1992). Stability of rifampicin resistance as a marker for root colonization studies of Pseudomonas putida in the field. Plant and Soil, 146, 135–142.CrossRefGoogle Scholar
  20. Glandorf, D.C.M., Verheggen, P., Jansen, T., Jorritsma, J., Thomashow, L.S., Leeflang, P., Smit, E., Wernars, K., Laureijs, E., Thomas-Oates, J.E., Bakker, P.A.H.M., & van Loon, L.C. (2001). Effect of genetically modified Pseudomonas putida WCS358r on the fungal rhizosphere microflora of field-grown wheat. Applied and Environmental Microbiology, 67, 3371–3378.Google Scholar
  21. Glandorf, D. C. M. (2015). Categorization of field trials with GM plants in the Netherlands: Applicable to field trials with GM forest trees? IForest-Biogeosciences and Forestry, 8, 222–225.Google Scholar
  22. Glandorf, D.C.M. & Breyer, D. (2016). Field Trials with GM Trees: A Step-by-Step Approach. In: Biosafety of Transgenic Forest trees (pp. 141–154), Springer.Google Scholar
  23. Hogervorst, P.A.M., van den Akker. H.C.M., Glandorf, D.C.M., Klaassen, P., van der Vlugt, C.J.B. &Westra, J. (2018). Assessment of human health and environmental risks of new developments in modern biotechnology: Policy report. RIVM report 2018-0089,
  24. Jӓderlund, L., Hellman, M., Sundh, I., Baily, M. J., & Jansson, J. K. (2008). Use of a novel non-antibiotic triple marker gene cassette to monitor high survival of Pseudomonas fluorescens SBW25 on winter wheat in the field. FEMS Microbiology Ecology, 63, 156–168.CrossRefGoogle Scholar
  25. Kemal, R. A., Islamaiah, P. W. N., Sa’dah, M., & Lusiany, T. (2015). Synthetic biology for biocontrol: A mini-review. In Proceedings 6 th International Conference on Global Resource Conservation (pp. 92–94).Google Scholar
  26. Kloepper, J. W., Rodriguez-Kabana, R., Zehnder, G. W., Murphy, J. F., Sikora, E., & Fernandez, C. (1999). Plant root-bacterial interactions in biological control of soilborne diseases and potential extension to systemic and foliar diseases. Australasian Plant Pathology, 28, 21–26.CrossRefGoogle Scholar
  27. Lajoie, M.J., Rovner, A.J., Goodman, D.B., Aerni, H.R., Haimovich, A.D., Kuznetsov, G., Mercer, J.A., Wang, H.H., Carr, P.A., Mosberg, J.A., Rohland, N., Schultz, P.G., Jacobson, J.M., Rinehart, J., Church, G.M., &; Isaacs, F.J. (2013). Genomically recoded organisms expand biological functions. Science, 342, 357–360.Google Scholar
  28. Leeflang, P., Smit, E., Glandorf, D. C. M., Hannen, E. J., & Wernars, K. (2002). Effects of Pseudomonas putida WCS358r and its genetically modified phenazine producing derivative on the Fusarium population in a field experiment, as determined by 18S rDNA analysis. Soil Biology and Biochemistry, 34, 1021–1025.CrossRefGoogle Scholar
  29. Lenski R.E. (1991). Quantifying fitness and gene stability in microorganisms. In: L.R. Ginsburg (Ed.), Assessing ecological risks of biotechnology (pp.173–192), Butterworth-Heinemann, Boston, USA.Google Scholar
  30. Lilley, A. K., & Baily, M. J. (1997). The acquisition of indigenous plasmids by a genetically marked pseudomonad population colonising the sugar beet phytosphere is related to local environmental conditions. Applied and Environmental Microbiology, 63, 1577–1583.Google Scholar
  31. Lilley, A. K., Hails, R. S., Cory, J. S., & Baily, M. J. (1997). The dispersal and establishment of pseudomonad populations in the phyllosphere of sugar beet by phytophagous caterpillars. FEMS Microbiology Ecology, 24, 151–157.CrossRefGoogle Scholar
  32. Martinez-Garcia, E., Calles, B., Arevalo-Rodriquez, M., & de Lorenzo, V. (2011). PBMA1: An all-synthetic genetic tool for analysis and construction of complex bacterial phenotypes. BMC Microbiology, 11, 38.CrossRefGoogle Scholar
  33. Martinez-Viveros O., Jorquera M.A., Crowley D.E., Gajard G., & Mora, M.L. (2010). Mechanisms and practical considerations involved in plant growth promotion by rhizobacteria. Journal of Soil Science and Plant Nutrition, 10, 293–319.Google Scholar
  34. Mendes, R., Garbeva, P., & Raaijmakers, J. M. (2013). The rhizosphere microbiome: Significance of plant beneficial, plant pathogenic and human pathogenic microorganisms. FEMS Microbiology Reviews, 3, 634–663.CrossRefGoogle Scholar
  35. Moe-Behrens, G. H. G., Davis, R., & Haynes, K. A. (2013). Preparing synthetic biology for the world. Frontiers in Microbiology, 4, 5.1–5.10.CrossRefGoogle Scholar
  36. Moënne-Locoz, Y., Powell, J., Higgins, P., McCarthy, J., & O’Gara, F. (1998). An investigation of the impact of biocontrol Pseudomonas fluorescens F113 on the growth of sugar beet and the performance of subsequent clover-Rhizobium symbiosis. Applied Soil Ecology, 7, 225–237.CrossRefGoogle Scholar
  37. Moënne-Locoz, Y., Tichy, H.-V., O’Donnel, A., Simon, R., & O’Gara, F. (2001). Impact of 2,4-Diacetylphloroglucinol-producing biocontrol strain Pseudomonas fluorescens F113 on intraspecific diversity of resident culturable pseudomonads associated with the roots of field-grown sugar beet seedlings. Applied and Environmental Microbiology, 67, 3418–43425.CrossRefGoogle Scholar
  38. Nakashima, N., & Miyazaki, M. (2014). Bacterial cellular engineering by genome editing and gene silencing. International Journal of Molecular Science, 15, 2773–2793.CrossRefGoogle Scholar
  39. Nekrassov, V., Wang, C., Win, J., Lanz, C., Weigel, D., & Kamoun, S. (2017). Rapid generation of a transgene-free powdery mildew resistant tomato by genome deletion. Nature Scientific reports, 7, 482.CrossRefGoogle Scholar
  40. Oburger, E., & Schmidt, H. (2016). New methods to unravel rhizosphere processes. Trends in Plant Science, 21, 243–255.CrossRefGoogle Scholar
  41. OECD (1986). Recombinant DNA considerations. Paris: OECD.Google Scholar
  42. Prosser, J. I., Bohannan, B. J. M., Curtis, T. P., Ellis, R. J., Firestone, M. K., Freckleton, R. P., Green, J. L., Green, J. E., Killham, K., Lennon, J. J., Osborn, A. M., Solan, M., van der Gast, C. J., & Young, J. P. W. (2007). The role of ecological theory in microbial ecology. Nature Reviews Microbiology, 5, 384–392.CrossRefGoogle Scholar
  43. Raaijmakers, J.M. (2015). The Minimal Rhizosphere Microbiome. In: Lugtenberg, B. (ed.), Principles of Plant-Microbe Interactions (pp. 411–417), Springer. Google Scholar
  44. Rovner, A. J., Haimovich, A. D., Katz, S. R., Li, Z., Grome, M. W., Gassaway, B. M., Amiram, M., Patel, J. R., Gallagher, R. R., Rinehart, J., Farren, J., & Isaacs, F. J. (2015). Recoded organisms engineered to depend on synthetic amino acids. Nature, 518, 89–93.CrossRefGoogle Scholar
  45. Ryan, P. R., Dessaux, Y., Thomashow, L. S., & Weller, D. M. (2009). Rhizosphere engineering and management for sustainable agriculture. Plant and Soil, 32, 363–383.CrossRefGoogle Scholar
  46. Scheepmaker, J. W. A., Hogervorst, P. A. M., & Glandorf, D. C. M. (2016). Future introductions of genetically modified microbial biocontrol agents in the EU: Are current EU legislation and risk assessment fit for purpose? Accessed 11 Aug 2016
  47. Scherwinski, K., Grosch, R., & Berg, G. (2008). Effects of bacterial antagonists on lettuce: Active biocontrol of Rhizoctonia solani and negligible, short term effects on non-target microorganism. FEMS Microbiology Ecology, 64, 106–116.CrossRefGoogle Scholar
  48. Schippers, B., & Roosje, G. S. (1997). Hundred years of history and the future of the foundation ‘Willie Commelin Scholten Phytopathological laboratory. European Journal of Plant Pathology, 10, 667–671.CrossRefGoogle Scholar
  49. Schmidt, M., & de Lorenzo, V. (2012). Synthetic constructs in/for the environment: Managing the interplay between natural and engineered biology. FEBS Letters, 586, 2199–2206.CrossRefGoogle Scholar
  50. Schmidt, M., & de Lorenzo, V. (2016). Synthetic bugs on the loose: Containment options for deeply engineered (micro)organisms. Current Opinions in Biotechnology, 38, 90–96.CrossRefGoogle Scholar
  51. Sigler, W. V., Nakatsu, C. H., Reicher, Z. J., & Turco, R. F. (2001). Fate of biological control agent Pseudomonas aureofaciens TX-1 after application on turfgrass. Applied and Environmental Microbiology, 67, 3542–3548.CrossRefGoogle Scholar
  52. Sutherland, W., Bardsley, S., Clout, M., Depledge, M. H., Dicks, L. V., Fellman, L., Fleishman, E., Gibbons, D. W., Keim, B., Lickorish, F., Margerison, C., Monk, K. A., Norris, K., Peck, L. S., Prior, S. V., Scharlemann, J. P. W., Spalding, M. D., & Watkinson, A. R. (2013). A horizon scan of global conservation issues for 2013. Trends in Ecology & Evolution, 28, 16–22.CrossRefGoogle Scholar
  53. Tebbe C.C. (2015). Risk assessment considerations of genetically modified micro-organisms for releases. In: Biosafety and the environmental used of micro-organisms, Conference proceedings OECD.Google Scholar
  54. Van Elsas, J. D., Turner, S., & Bailey, M. J. (2003). Horizontal gene transfer in the phytosphere. New Phytologist, 157, 525–537.CrossRefGoogle Scholar
  55. Van Overbeek, L., van Veen, J. A., & van Elsas, J. D. (1997). Induced reporter gene activity, enhanced stress resistance, and competitive ability of a genetically modified Pseudomonas fluorescens strain released into a field plot planted with wheat. Applied and Environmental Microbiology, 6, 1965–1973.Google Scholar
  56. Viebahn, M., Glandorf, D. C. M., Ouwens, T. W. M., Smit, E., Wernars, K., Leeflang, P., Thomashow, L. S., van Loon, L. C., & Bakker, P. A. H. M. (2003). Repeated introduction of genetically modified Pseudomonas putida WCS358r without intensified effects on the indigenous microflora of field-grown wheat. Applied and Environmental Microbiology, 69, 3110–3118.CrossRefGoogle Scholar
  57. Viebahn, M., Doornbos, R., Wernars, K., van Loon, L. C., Smit, E., & Bakker, P. A. H. M. (2005). Ascomycete communities in the rhizosphere of field-grown wheat are not affected by introductions of genetically modified Pseudomonas putida WCS358r. Applied and Environmental Microbiology, 7, 1775–1785.Google Scholar
  58. Viebahn, M., Wernars, K. Smit, E., van Loon L.C., de Santis T.Z., Andersen G.L. &. Bakker, P.A.H.M (2006). Microbial diversity in wheat rhizosphere as affected by genetically modified Pseudomonas putida WCS358r. In: J.M. Raaijmakers and R.A. Sikora (eds.) Multitrophoic interactions in Soil and Integrated Control, IOBC/WPRS Bull 29: 167–172.Google Scholar
  59. Viebahn, M., Smit, E., Glandorf, D. C. M., Wernars, K., & Bakker, P. A. H. M. (2009). Effects of genetically modified bacteria on ecosystems and their potential benefits for bioremediation and biological control of plant diseases: A review. Sustainable Agricultural Reviews, 2, 45–69.Google Scholar
  60. Wang, Y., Cheng, X., Shan, Q., Zhang, Y., Liu, J., Gao, C., & Qiu, J. (2014). Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nature Biotechnology, 32, 947–952.CrossRefGoogle Scholar
  61. Walsh, U., Morrissey, J. P., & O'Gara, F. (2001). Pseudomonas for biocontrol of phytopathogens: From functional genomics to commercial exploitation. Current Opinions in Biotechnology, 12, 289–295.CrossRefGoogle Scholar
  62. Weller, D. M. (1988). Biological control of soil borne plant pathogens in het rhizosphere with bacteria. Annual Review of Phytopathology, 26, 379–407.CrossRefGoogle Scholar
  63. Weller, D. M., Raaijmakers, J. M., Gardener, B. B., & Thomashow, L. S. (2002). Microbial populations responsible for specific soil suppressiveness to plant pathogens. Annual Review of Phytopathology, 40, 309–348.CrossRefGoogle Scholar
  64. Weller, D.M. & Thomashow, L.S. (2015). Phytosanitation and the development of transgenic biocontrol agents. In: Biosafety and the environmental used of micro-organisms, Conference proceedings OECD.Google Scholar
  65. Winding, A., Binnerup, S. J., & Pritchard, H. (2004). Non-target effects of bacterial biocontrol agents suppressing root pathogenic fungi. FEMS Microbiology Ecology, 47, 129–141.CrossRefGoogle Scholar
  66. Zhang, Y., Ptacin, J. L., Fischer, E. C., Aerni, H. R., Caffaro, C. E., San Jose, K., Feldman, A. W., Turner, C. R., & Romesberg, F. E. (2017). A semi-synthetic organism that stores and retrieves increased genetic information. Nature, 551, 644–647.CrossRefGoogle Scholar

Copyright information

© Koninklijke Nederlandse Planteziektenkundige Vereniging 2018

Authors and Affiliations

  1. 1.Department of Gene Technology and Biological Safety, Centre for Safety of Substances and ProductsNational Institute of Public Health and the Environment (RIVMBilthovenThe Netherlands

Personalised recommendations