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Title: INTACT SOIL-CORE MICROCOSMS FOR PRE-RELEASE TESTING OF MICROBES: EFFECTS OF INTRODUCED GEMS ON SOIL MICROBIAL COMMUNITY STRUCTURE AND FUNCTION

Author
item Gagliardi, Joel
item Buyer, Jeffrey
item ANGLE, JAY - UNIV. OF MD
item RUSSEK-COHEN, ESTELLE - UNIV. OF MD

Submitted to: Soil Biology and Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/22/2000
Publication Date: N/A
Citation: N/A

Interpretive Summary: Risk assessment requires determining if a genetically engineered microorganism (GEM) will adversely affect native species in the soil environment. Tests on native or non-target species were developed and assessed for use by risk assessors, utilizing intact soil-core microcosms originally used for survival testing of introduced GEMs. Assay A, specific for living organisms, detected the introduced GEMs by their differential ability to use various carbon sources for food. After the number of detectable GEMs declined in the soil, differences in carbon use patterns also decreased. Assay B analyzed cellular components, specifically the membrane fatty acids, of all organisms in the soil. Results were similar to Assay A. As long as there were detectable GEMs in soil, their effects could be distinguished from soils in which no inoculated GEM was present. When the number of GEMs became undetectable, the fatty acid component of cells in soil no longer showed differences when compared to soil that was not inoculated. These results are important for risk assessors who must determine if a GEM remains active in soil after it cannot be detected (in this case it did not), or whether its components persist after it has died out (in this case none were detected). These results also indicate that engineered traits were not transferred from the GEM to native species at detectable levels.

Technical 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 be evaluated prior to field testing or commercial use. Freshly obtained intact soil-core microcosms were inoculated with the GEMs Pseudomonas chlororaphis 3732RN-L11 and Pseudomonas fluorescens 2-79RN-L3, and their non-engineered parental strains 3732RN and 2-79RN. A functional assay utilizing BIOLOG(R) GN plates to measure rhizosphere microflora response to 95 different carbon sources was employed. The functional assay is applicable for soil or rhizosphere samples. It employs a mathematical correction for inoculum density and rhizosphere sample size, and a single plate incubation time and reading that all serve to normalize the assay. The functional assay detected changes that persisted while the inoculated organisms survived. Differentiation based on the lacZ gene incorporated into the chromosome of both GEMs was significant for 3732RN-L11 in five soils, but not for 2-79RN-L3 assayed in a single soil. Lactose utilization by the microbial communities in 3732RN-L11 inoculated soils declined steadily as levels of the introduced GEM declined. A structural assay utilizing direct transesterification and extraction of fatty acids from rhizosphere soil was employed for comparison to the functional assay. The structural assay differentiated GEM and parent inoculated soils from uninoculated soil and mirrored the effects seen with the functional assay. Differences between inoculated and uninoculated treatments decreased as relative numbers of the inoculated organisms declined.