Effect of Genetically Modified Pseudomonas putida WCS358r on the Fungal Rhizosphere Microflora of Field-Grown Wheat

Abstract

We released genetically modified Pseudomonas putida WCS358r into the rhizospheres of wheat plants. The two genetically modified derivatives, genetically modified microorganism (GMM) 2 and GMM 8, carried the phz biosynthetic gene locus of strain P. fluorescens 2-79 and constitutively produced the antifungal compound phenazine-1-carboxylic acid (PCA). In the springs of 1997 and 1998 we sowed wheat seeds treated with either GMM 2, GMM 8, or WCS358r (approximately 10 7 CFU per seed), and measured the numbers, composition, and activities of the rhizosphere microbial populations. During both growing seasons, all three bacterial strains decreased from 10 7 CFU per g of rhizosphere sample to below the limit of detection (10 2 CFU per g) 1 month after harvest of the wheat plants. The phz genes were stably maintained, and PCA was detected in rhizosphere extracts of GMM-treated plants. In 1997, but not in 1998, fungal numbers in the rhizosphere, quantified on 2% malt extract agar (total filamentous fungi) and on Komada's medium (mainly Fusarium spp.), were transiently suppressed in GMM 8-treated plants. We also analyzed the effects of the GMMs on the rhizosphere fungi by using amplified ribosomal DNA restriction analysis. Introduction of any of the three bacterial strains transiently changed the composition of the rhizosphere fungal microflora. However, in both 1997 and 1998, GMM-induced effects were distinct from those of WCS358r and lasted for 40 days in 1997 and for 89 days after sowing in 1998, whereas effects induced by WCS358r were detectable for 12 (1997) or 40 (1998) days. None of the strains affected the metabolic activity of the soil microbial population (substrate-induced respiration), soil nitrification potential, cellulose decomposition, plant height, or plant yield. The results indicate that application of GMMs engineered to have improved antifungal activity can exert nontarget effects on the natural fungal microflora.

Dynamics of Fungal Communities in Bulk and Maize Rhizosphere Soil in the Tropics

“…Molecular fingerprinting techniques such as denaturing gradient gel electrophoresis (DGGE) analysis of ribosomal DNA (rDNA) fragments amplified from total community DNA have been mainly used to analyze the composition of bacterial communities (27). Only very few studies have used this approach to study fungal soil and rhizosphere communities (7,18,19,36,42). Since this technique can also be combined with cloning and sequencing, this allows us to analyze phylogenetic sequences of bands generated by community members (36).…”

Impact of root exudates and plant defense signaling on bacterial communities in the rhizosphere. A review

“…Whereas only a limited number of studies have investigated effects of plant defense on the non-pathogenic and beneficial microflora, most of these studies suggest that effects on the indigenous microflora are negligible. Development of methodologies to study shifts in the rhizosphere microflora have been stimulated by studies that have focused on the impact of introducing genetically modified bacteria that produce broad spectrum antibiotics (Glandorf et al 2001;Bakker et al 2002;Blouin-Bankhead et al 2004;Timms-Wilson et al 2004;Viebahn et al 2003Viebahn et al , 2006. Most of those studies used fingerprinting techniques of group specific PCR amplified 16S (bacteria) or 18S (fungi) rDNA, enabling detection of shifts but lacking power to identify the organisms affected.…”

Effects of Jasmonic Acid, Ethylene, and Salicylic Acid Signaling on the Rhizosphere Bacterial Community of Arabidopsis thaliana

“…After two weeks, germinated seedlings were transferred individually to 60-ml pots containing either a potting soil-sand mixture (12:5 vol/vol) (Pieterse et al 1996) or a clay soil (Glandorf et al 2001;Viebahn et al 2003). Both soils were either nonautoclaved or autoclaved (twice for 20 min at 121°C with a 24-h interval).…”