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Applied and Environmental Microbiology, June 2003, p. 3110-3118, Vol. 69, No. 6
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.6.3110-3118.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Repeated Introduction of Genetically Modified Pseudomonas putida WCS358r without Intensified Effects on the Indigenous Microflora of Field-Grown Wheat

M. Viebahn,1* D. C. M. Glandorf,{dagger} T. W. M. Ouwens,1 E. Smit,2 P. Leeflang,2 K. Wernars,2 L. S. Thomashow,3 L. C. van Loon,1 and P. A. H. M. Bakker1

Faculty of Biology, Section of Phytopathology, Utrecht University, 3508 TB Utrecht,1 National Institute of Public Health and the Environment, Bilthoven, The Netherlands,2 U.S. Department of Agriculture, Washington State University, Pullman, Washington3

Received 6 November 2002/ Accepted 10 March 2003

To investigate the impact of genetically modified, antibiotic-producing rhizobacteria on the indigenous microbial community, Pseudomonas putida WCS358r and two transgenic derivatives were introduced as a seed coating into the rhizosphere of wheat in two consecutive years (1999 and 2000) in the same field plots. The two genetically modified microorganisms (GMMs), WCS358r::phz and WCS358r::phl, constitutively produced phenazine-1-carboxylic acid (PCA) and 2,4-diacetylphloroglucinol (DAPG), respectively. The level of introduced bacteria in all treatments decreased from 107 CFU per g of roots soon after sowing to less than 102 CFU per g after harvest 132 days after sowing. The phz and phl genes remained stable in the chromosome of WCS358r. The amount of PCA produced in the wheat rhizosphere by WCS358r::phz was about 40 ng/g of roots after the first application in 1999. The DAPG-producing GMMs caused a transient shift in the indigenous bacterial and fungal microflora in 1999, as determined by amplified ribosomal DNA restriction analysis. However, after the second application of the GMMs in 2000, no shifts in the bacterial or fungal microflora were detected. To evaluate the importance of the effects induced by the GMMs, these effects were compared with those induced by crop rotation by planting wheat in 1999 followed by potatoes in 2000. No effect of rotation on the microbial community structure was detected. In 2000 all bacteria had a positive effect on plant growth, supposedly due to suppression of deleterious microorganisms. Our research suggests that the natural variability of microbial communities can surpass the effects of GMMs.

* Corresponding author. Mailing address: Faculty of Biology, Section of Phytopathology, Utrecht University, P.O. Box 80084, 3508 TB Utrecht, The Netherlands. Phone: 31 30 2536860. Fax: 31 30 2518366. E-mail: M.Viebahn{at}bio.uu.nl.

{dagger} Present address: National Institute of Public Health and the Environment, Bilthoven, The Netherlands.

Applied and Environmental Microbiology, June 2003, p. 3110-3118, Vol. 69, No. 6
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.6.3110-3118.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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