Does wheat genetically modified for disease resistance affect root-colonizing pseudomonads and arbuscular mycorrhizal fungi?

Abstract

This study aimed to evaluate the impact of genetically modified (GM) wheat with introduced pm3b mildew resistance transgene, on two types of root-colonizing microorganisms, namely pseudomonads and arbuscular mycorrhizal fungi (AMF). Our investigations were carried out in field trials over three field seasons and at two locations. Serial dilution in selective King's B medium and microscopy were used to assess the abundance of cultivable pseudomonads and AMF, respectively. We developed a denaturing gradient gel electrophoresis (DGGE) method to characterize the diversity of the pqqC gene, which is involved in Pseudomonas phosphate solubilization. A major result was that in the first field season Pseudomonas abundances and diversity on roots of GM pm3b lines, but also on non-GM sister lines were different from those of the parental lines and conventional wheat cultivars. This indicates a strong effect of the procedures by which these plants were created, as GM and sister lines were generated via tissue cultures and propagated in the greenhouse. Moreover, Pseudomonas population sizes and DGGE profiles varied considerably between individual GM lines with different genomic locations of the pm3b transgene. At individual time points, differences in Pseudomonas and AMF accumulation between GM and control lines were detected, but they were not consistent and much less pronounced than differences detected between young and old plants, different conventional wheat cultivars or at different locations and field seasons. Thus, we conclude that impacts of GM wheat on plant-beneficial root-colonizing microorganisms are minor and not of ecological importance. The cultivation-independent pqqC-DGGE approach proved to be a useful tool for monitoring the dynamics of Pseudomonas populations in a wheat field and even sensitive enough for detecting population responses to altered plant physiology.

Figure 1. Colonization of roots of transgenic and non-transgenic wheat by arbuscular mycorrhizal fungi (AMF) (A, C, E) and cultivable pseudomonads (B, D, F) in field trials performed in 2008, 2009 and 2010.

Roots were sampled at the tillering (B, D) and the milky ripe stage (A–F) at Reckenholz (A–F) and Pully (E, F). Relative colonization: root colonization by AMF was expressed for each wheat line/cultivar relative to the average colonization calculated for all wheat lines. Significant differences (P<0.05) are listed in Table 1. Pm3b = GM lines, Sb = non-GM sister lines. Bars indicate the standard error of the estimate. Significant differences (P<0.05) detected by pairwise comparisons A) Pm3b#1 vs Sb#1; B) tillering stage: Pm3b#1 vs Sb#1 and Pm3b#2; B) milky ripe stage: Pm3b#1 vs Sb#1 and Pm3b#2, Sb#1 vs Pm3b#2, Bobwhite vs Frisal and Toronit; D) milky ripe stage: Toronit vs Frisal; F) Reckenholz: Sb#2 vs Pm3b#2; F) Pully: Bobwhite vs Rubli.

Figure 2. Ordination biplots generated by correspondence analysis (CA) of pqqC-DGGE profiles from wheat root samples.

DGGE bands (letters a–n) incorporated in the CA are shown. The size and position of the blue bullets reflect the contribution of each band to the distribution pattern. Graph A shows plants grown in the Reckenholz field trial in 2008 (experiment 1) and sampled at the tillering (H1) and at the milky ripe stage (H2). Graph B additionally includes data from Reckenholz field trial 2009 obtained from plants harvested at the tillering stage (H1). GM and SIST = GM Bobwhite (pm3b) and non-GM Bobwhite sister lines; WT = conventional non-GM wheat cultivars Frisal (or Fris), Toronit (or Tor), Rubli (or Rub) and Bobwhite (or Bob).

Figure 3. Impact of plant production procedures on DGGE profiles.

Pseudomonas-specific pqqC DGGE was performed with DNA extracted from roots of wheat at the tillering stage grown in the Reckenholz field trial 2008. The four lanes derived from four replicate field plots per line/cultivar are shown. Arrows indicate the pqqC DGGE band J, which is enriched on root profiles of conventional wheat cultivars, and bands M and N which are enriched on root profiles of the Bobwhite GM and sister lines. These bands correspond to P. fluorescens subgroup 1e (band J) and P. fluorescens subgroup 1 g (band M and N), as defined by Meyer et al. . Pm3b#1 and Pm3b#2 are the GM lines with introduced pm3b transgene, Sb#1 and Sb#2 are their respective non-GM sister lines and Bobwhite is the parental line. Frisal, Toronit and Rubli are conventional wheat cultivars. Bobwhite GM and sister lines originate from T₀ GM plants regenerated from tissue cultures, sister lines lost the transgene in the T₁ generation due to segregation. Seeds of GM and sister lines used in the field trial were produced in the greenhouse, seeds of Bobwhite, Frisal, Toronit and Rubli were produced in the field. m, DGGE standard marker, consisting of pqqC fragments of Pseudomonas strains (from the upper to the lower band): Pseudomonas chlororaphis LMG1245, P. fluorescens Q2-87, P. fluorescens CHA0, P. fluorescens K94.37, P. corrugata LMG2172.

Figure 4. Impact of powdery mildew infection on population sizes of cultivable pseudomonads on roots of GM and non-GM wheat grown in field trials at Reckenholz 2008 (experiment 2) (A) and 2009 (B).

Roots were sampled at the milky ripe stage. PM infection = plants inoculated with powdery mildew. Fungicide = plants were treated with the fungicide Prosper. In 2009 data for the powdery mildew infected Toronit are missing. Bars indicate standard errors of the means. Mildew-infected plants differed significantly form fungicide treated plants in 2008, but not in 2009 (Table 1). Significant differences (P<0.05) detected by pairwise comparisons: A) PM infected: Pm3b#1 vs Sb#1, B) Fungicide: Toronit vs Frisal.