Environmental use of genetically-engineered microorganisms (GEMs) has raised concerns over potential ecological impact. Development of microcosm systems useful in preliminary testing for risk assessment will provide useful information for predicting potential structural, functional, and genetic effects of GEM release. This study was executed to develop techniques that may be useful in risk assessment and microbial ecology, to ascertain which parameters are useful in determining risk and to predict risk from releasing an engineered strain of Erwinia carotovora.

A terrestrial microcosm system for use in GEM risk assessment studies was developed for use in assessing alterations of microbial structure and function that may be caused by introducing the engineered strain of E. carotovora. This strain is being developed for use as a biological control agent for plant soft rot. Parameters that were monitored included survival and intraspecific competition of E. carotovora, structural effects upon both total bacterial populations and numbers of selected bacterial genera, effects upon activities of dehydrogenase and alkaline phosphatase, effects upon soil nutrients, and potential for gene transfer into or out of the engineered strain.

No significant difference was found in survival of the engineered strain as compared to its wildtype parent. Both strains survived for over two months in microcosms. The effects of both strains upon populations of total bacteria and selected bacterial genera were determined; while some effects upon community structure were observed, they were not significant.

The engineered strain was not found to be a superior competitor compared to its parent; three different doses of engineered and wildtype strains were used. ln addition, neither strain affected activities of dehydrogenase or alkaline phosphatase in soil. Likewise, no effects were observed upon the nutrients monitored.

However, transfer of the kanamycin resistance gene that had been inserted into the engineered E. carotovora strain may have occurred. Five species of indigenous bacteria displayed kanamycin resistance 15 days after being exposed to the engineered Erwinia. DNA from these strains was isolated, purified, and hybridization experiments executed to determine if any homology existed between these DNAs and the kanamycin resistance gene that had been inserted into E. carotovora. Using biotin-Iabeled probes and Iow-stringency washing conditions, homology was observed. However, before gene transfer can be proven, additional studies, including amplification and sequencing, may be required.

Although a simple microcosm design was employed, it yielded sufficient information to conclude that release of the engineered Erwinia carotovora will not affect any of the microbial measures of integrity that were studied in a manner different from that of the wildtype. Effects upon plant material and other higher taxa will be the focus of future studies.