MICROORGANISMS DELIBERATELY RELEASED INTO THE ENVIRONMENT AFTER RIGOROUS TESTING ARE UNLIKELY TO PROVE A DIRECT THREAT (I.E. AS PATHOGENS), AND PREDICTION OF SUCH RISKS IS ALMOST IMPOSSIBLE. HOWEVER, THE MAIN RISK IS THE PROSPECT OF CREATING POTENTIALLY HAZARDOUS NEW ORGANISMS FOLLOWING THE TRANSFER OF MOBILE GENES TO NATIVE BACTERIA. MANY BACTERIA CAN EXCHANGE GENETIC MATERIAL, INCLUDING PLASMIDS AND TRANSPOSONS; THEREFORE THE PERSISTENCE AND SPREAD OF BOTH THE ORGANISMS AND GENES ARE BOTH IMPORTANT AND WOULD BE AFFECTED BY A VARIETY OF SELECTIVE PRESSURES.
THE SPECIFIC AIM OF THE PROPOSED PROJECT IS TO MONITOR THE PERSISTENCE OF GENETICALLY MANIPULATED BACTERIA INTRODUCED INTO AGRICULTURAL SOILS AND TO SCREEN FOR THE SPREAD OF GENES CARRIED BY THESE MICROORGANISMS TO OTHER MEMBERS OF THE SOIL FLORA. THIS WILL PROVIDE A MODEL FOR ASSESSING THE RISKS INVOLVED IN INTRODUCING MORE DRASTICALLY ALTERED MICROORGANISMS INTO THE ENVIRONMENT. THE TEST ORGANISMS TO BE USED IN EACH PARTICIPATING COUNTRY WILL BE A RHIZOBIUM STRAIN GENETICALLY MODIFIED AT ROTHAMSED.
To investigate survival of introduced strains and their genes and to develop and assess monitoring methods, genetically marked derivatives of 2 common soil bacteria were used: Rhizobium which fixes atmospheric nitrogen in the root nodules of legumes (and has a long history) of safe and effective use as an agricultural inoculant) was used in both field and laboratory experiments; Enterobacter agglomerans which fixes nitrogen in association with cereal roots was studied in the laboratory. Strains were marked with genes conferring antibiotic resistance, either by selecting naturally occurring chromosomal mutations, or by insertion of transposon Tn5 to conjugative plasmids. In addition, native Rhizobium populations were screened for circumstantial evidence that genetic exchange occurs (over a long period) in the environment.
The Tn5 marker enabled extensive monitoring of the Rhizobium inoculant, which showed significant variation in survival in field soils of the collaborating countries. The host plant was not required for its establishment. Spread from the site of application over short distances indicated migration along extending roots; over moderate distances it was associated with standing surface water; and over greater areas by bulk soil movement. In the field, no Tn5 transfer to other Rhizobium (isolated from nodules) was found, although populations were below the level at which transfer could be detected in the laboratory. In culture, the inoculant could transfer Tn5 to Escherichia coli and E agglomerans, in addition to a range of Rhizobium species (in contrast to E agglomerans which transferred its plasmid only to closely related strains). Field populations of Rhizobium showed considerable evidence that genetic exchange between soil bacteria must occur in the long term.
THE AIM OF THIS PROJECT CONDUCTED AS AN INTEGRATED AND JOINT EFFORT BY THE PARTICIPATING LABORATORIES IS TO CONSTRUCT RHIZOBIUM STRAINS WITH SYM PLASMIDS CARRYING SELECTABLE, DETECTABLE MARKERS, AND ALSO CHROMOSOMAL MARKERS, TO RELEASE THEM IN THE DIFFERENT PARTICIPATING COUNTRIES IN CAREFULLY CHOSEN SITES, AND TO MONITOR PERSISTENCE AND EXCHANGE OF HOST AND MARKER GENES IN THE PRESENCE AND ABSENCE OF SELECTION BY PLANT HOSTS.
Funding SchemeCSC - Cost-sharing contracts