Horizontal gene transfer to endogenous endophytic bacteria from poplar improves phytoremediation of toluene

Abstract

Poplar, a plant species frequently used for phytoremediation of groundwater contaminated with organic solvents, was inoculated with the endophyte Burkholderia cepacia VM1468. This strain, whose natural host is yellow lupine, contains the pTOM-Bu61 plasmid coding for constitutively expressed toluene degradation. Noninoculated plants or plants inoculated with the soil bacterium B. cepacia Bu61(pTOM-Bu61) were used as controls. Inoculation of poplar had a positive effect on plant growth in the presence of toluene and reduced the amount of toluene released via evapotranspiration. These effects were more dramatic for VM1468, the endophytic strain, than for Bu61. Remarkably, none of the strains became established at detectable levels in the endophytic community, but there was horizontal gene transfer of pTOM-Bu61 to different members of the endogenous endophytic community, both in the presence and in the absence of toluene. This work is the first report of in planta horizontal gene transfer among plant-associated endophytic bacteria and demonstrates that such transfer could be used to change natural endophytic microbial communities in order to improve the remediation of environmental insults.

FIG. 1.

Plant growth parameters for poplar cuttings calculated after 10 weeks of growth in the presence or absence of toluene (Tol) (0 or 500 mg liter⁻¹). Poplar cuttings inoculated with B. cepacia strain VM1468 of BU61 or noninoculated control plants were grown in conditions under which they were watered with half-strength Hoagland's solution to which 500 mg liter⁻¹ toluene was added, resulting in a step-wise wash-in of toluene. (A) Growth indexes were calculated as (Mt − M0)/M0 after 10 weeks of growth in the presence or absence of toluene (0 or 500 mg liter⁻¹), where M0 is the plant weight (in grams) before addition of toluene and Mt is the plant weight (in grams) 10 weeks after toluene addition. (B) Root and leaf weight were determined after 10 weeks of growth in the presence or absence of toluene (0 or 500 mg liter⁻¹). The data are the means of 10 replicates; standard deviations are indicated by error bars. The statistical significance of the results was confirmed at the 5% level using a two-way analysis of variance model, separately exploring treatment (bacterial inocula) and toluene doses.

FIG. 2.

Total amount of toluene (in micrograms) released from poplar into the upper cuvette compartment. Toluene was adsorbed with Chromosorb traps, and the toluene concentration was determined by GC-MS. The amount of evapotranspired toluene was calculated per square centimeter of leaf area. The data are the means of three replicates; standard deviations are indicated by error bars.