3.5.11
FIELD RELEASE OF GENETICALLY MODIFIED PSEUDOMONAS PUTIDA WCS358r::phz TO STUDY EFFECTS ON THE INDIGENOUS SOIL MICROFLORA


DCM GLANDORFl, P VERHEGGENl, T JANSENl, LS THOMASHOW2, E SMIT3, K WERNARS3, PAHM BAKKER1 and LC VAN LOON1

lDept. of Plant Ecology and Evolutionary Biology, Section of Plant Pathology, Utrecht University, P.O. Box 800.84, 3508 TB Utrecht, The Netherlands; 2USDA, Washington State University, Pullman WA, USA; 3National Institute of Public Health and the Environment, Bilthoven, The Netherlands

Background and objectives
One concern related to introduction of genetically modified biocontrol bacteria in the environment is their possible non-target effects on the saprophytic soil microflora. To address this concern experimentally, plant growth-promoting P. putida WCS358r was modified with the phz biosynthetic locus from strain P. fluorescens 2-79 [1], resulting in constitutive production of phenazine-l-carboxylic acid (PCA) by WCS358r. PCA production by WCS358r::phz resulted in growth inhibition of a number of pathogenic and saprophytic fungi in vitro. The WCS358r::phz derivates were introduced on wheat seed and compared to their parental strain regarding effects on activities, numbers and the composition of rhizosphere and soil microbial populations in a natural soil under field conditions.

Results and conclusions
During the growth season of wheat, population densities of WCS358r and its PCA-producing derivatives decreased at the same rate and populations had dropped below the detection limit at 130 days after sowing. Introduction of WCS358r and its PCA-producing derivatives did not significantly affect plant fresh weight, plant dry weight, plant length, soil respiration (SIR) or soil nitrifying potential. Fungal populations quantified on 2 % malt and Komada medium were significantly (p = 0.05) lower in time for the WCS358r::phz treated plants compared to the other treatments in the rhizosphere, but not in bulk soil. However, antifungal effects by WCS358r::phz in the field were minor and transient, and coincided with high population densities of the introduced bacteria. The transient effects on the fungal microflora by WCS358r::phz were further analyzed at the 18S RRNA level, using Amplified Ribasomal DNA Restriction Analysis (ARDRA).

References
1. Mavrodi DV, Ksenzenko VN, Chatuev BM, Thomashow LS, Boronin A, 1997. Molecular Biology 31, 62-68.