Impact of glyphosate application to transgenic Roundup Ready® soybean on horizontal gene transfer of the EPSPS gene to Bradyrhizobium japonicum and on the root-associated bacterial community

Abstract

In this study, two topics causing major public concern related to transgenic plants were investigated: The possibility of a horizontal gene transfer from plant to bacteria and the impact of transgenic plants after herbicide treatment on root associated bacteria. The transgenic plant chosen for this study was Roundup Ready® (RR) soybean, which is tolerant to the herbicide glyphosate and is the most commonly used genetically modified crop worldwide. Glyphosate, the active ingredient of Roundup Ready®, inhibits the EPSPS enzyme (5-enolpyruvylshikimate-3-phosphate synthase). EPSPS is an enzyme involved in the shikimic acid pathway leading to the aromatic amino acid biosynthesis and its inhibition leads to growth reduction of plants and microorganisms. RR crops are glyphosate tolerant due to the introduction of the CP4-EPSPS gene coding for a glyphosate insensitive EPSPS enzyme. The transgenic construct is under expression of a CaMV 35S promoter a nos transcriptional termination element from Agrobacterium tumefaciens. Horizontal gene transfer experiments with the EPSPS gene of the RR soybean were performed under controlled laboratory conditions and were targeted to the nitrogen fixing symbiont of soybean Bradyrhizobium japonicum. This bacterium comprises the requirements of a possible receptor for the glyphosate resistance trait, as it is sensitive to the herbicide and thus the acquirement of glyphosate resistance would signify a positive adaptation to glyphosate accumulated in the roots after herbicide application. Two key conditions for gene transfer from the CP4-EPSPS gene from the RR soybean to B. japonicum were evaluated in this study: The required specific conditions for B. japonicum to undergo natural transformation and the expression of the CP4-EPSPS gene in B. japonicum. For that purpose, the CP4-EPSPS gene was cloned into a B. japonicum chromosomal integration vector and was transferred by biparental mating into the B. japonicum genome. Subsequently, the expression of the CP4-EPSPS gene in B. japonicum was tested under increasing glyphosate selection pressure. Results of these experiments indicated that B. japonicum is not naturally transformable under any conditions known from the more than 40 so far reported naturally transformable bacteria. Furthermore, the CP4-EPSPS genetic construct, as contained in RR soybean, has been shown in this study to be not active in B. japonicum. Consequently, if there would be a gene transfer of the plant CP4-EPSPS to B. japonicum, this genetic construct does not confer glyphosate resistance to B. japonicum and does not constitute any adaptive advantage to the bacterium under glyphosate selection pressure. As the genetic trait of glyphosate resistance has been found in several bacteria, it would be more probable that the common mating exchange between bacterial groups could disperse the glyphosate resistance within an environment. Moreover, in the specific case of B. japonicum, a high spontaneous mutation rate for glyphosate resistance was observed, suggesting that B. japonicum can also adapt to the glyphosate selection pressure by mutation under natural conditions.
The impact of transgenic plants with their respective herbicide treatments on root associated bacteria was investigated in a greenhouse experiment. The composition and diversity of bacterial communities of RR soybean rhizospheres were analyzed and compared between glyphosate-treated and untreated plants. Samples from five harvests with two glyphosate applications were analysed by 16S rRNA gene T-RFLP analysis complemented with the evaluation of three clone libraries. Multivariate statistical analysis of the data was used to visualize changes in the microbial populations in response to glyphosate applications and in order to find groups of organisms responsible for the observed community shifts. A comparison of the rhizosphere communities revealed that a Burkholderia related group was significantly inhibited by glyphosate application, while the abundance of a group of Gemmatimonadetes related sequences increased significantly after the herbicide treatment. The significant increment of Gemmatimonadetes abundance after glyphosate application could indicate that these organisms are able to metabolize the herbicide. Shannon diversity indices were calculated based on the T-RFLP results with the aim to compare bacterial diversity in the rhizosphere of glyphosate-treated and non treated RR soybeans. Interestingly, the bacterial community associated to RR soybean roots after glyphosate application not only demonstrated effective resilience after the disturbance but in addition the bacterial diversity also increased in comparison to the untreated control samples. It is possible, that in an environment with organisms which are able to metabolize glyphosate, the key for enhancing diversity could be the succession of metabolites, which can be further utilized by a diverse range of bacteria.