FATE OF GENETICALLY-MODIFIED RHIZOBIUM-LEGUMINOSARUM BIOVAR VICIAE DURING LONG-TERM STORAGE OF COMMERCIAL INOCULANTS

A study was carried out to assess the behaviour, in terms of strain su rvival and genetic stability, of genetically modified micro-organisms (GEMs) during their storage in commercial-type agricultural inoculants . Three genetically modified Rhizobium leguminosarum biovar viciae str ains were constructed, using a gene cassette containing an inducible l acZ gene from Escherichia coli and mercury resistance determinants fro m transposon Tn1831. In the first case the genes have been integrated into the chromosome, the second strain contains the inducible cassette on a plasmid, in the third case the cassette is carried by the same p lasmid, but the lacZ is constitutively expressed at high levels, due t o the removal of the regulatory structure (lac operator) between the g ene and its promoter. Three inoculum formulations, based on liquid, ve rmiculite and peat carriers, were prepared using the genetically modif ied strains, and were monitored during a period of up to 16 months. Re sults indicate a high stability of the chromosomally integrated marker s. The plasmid-borne modification also was very stable, though the pre sence of the plasmid affected the strain growth kinetics. In contrast, the strain containing the highly expressed lacZ showed dramatic marke r instability. Strain behaviour in stored inoculant packages reflected that observed in batch cultures; moreover, prolonged storage appeared to magnify differences found in in vitro cultures.