Structural and functional analysis of whole-soil microbial communities for risk and efficacy testing following microbial inoculation of wheat roots in diverse soils

Abstract The increasing use of genetically engineered or modified microorganisms (GEMs) has led to regulations regarding the safety of their use. Intended (target) effects and unintended (non-target) effects of GEMs must currently be evaluated prior to field testing or commercial use. We present soil and rhizosphere microbial community effects testing of two GEMs, Pseudomonas chlororaphis 3732RN-L11 and Pseudomonas fluorescens 2-79RN-L3, parental strains of these organisms and an uninoculated treatment using five diverse soils planted to wheat. An assay using BIOLOG ® GN plates measured microbial community functional responses on wheat roots with adhering soil. Overall differences using multivariate statistical methods were highest at inoculation, and these effects persisted while the inoculated organisms were detectible on selective media. Differentiation based on lacZY genes engineered to the chromosome of both GEMs was significant for the 3732 GEM in all five soils tested, but not for the 2-79 GEM in a single soil. Lactose utilization in uninoculated microbial communities varied around a low baseline value. Direct fatty acid extraction and analysis of soil from around wheat roots was also performed using a novel method. Fatty acid analysis differentiated the 3732 GEM from all other treatments, but did not distinguish the 3732 parent inoculated from uninoculated treatments. As with the BIOLOG assay, multivariate statistical differences from fatty acid analysis decreased between GEM inoculated and uninoculated populations as viable counts of the GEM declined. Neither assay showed measurable community-level effects when inoculated organisms declined below detection, though three of six soils with surviving GEM populations still had significant effects after 105 days.