MONITORING THE FATE OF GENETICALLY-ENGINEERED BACTERIA SPRAYED ON THEPHYLLOPLANE OF BUSH BEANS AND GRASS

The fate of a Bacillus amyloliquefaciens with the recombinant plasmid pSB20 sprayed on the phyllosphere of grass, and of a Tn5 marked Pseudo monas syringae sprayed on the phyllosphere of bush beans was studied i n planted soil microcosms. B. amyloliquefaciens showed a decline from 1.5 x 10(8) to 3.1 X 10(2) cfu g(-1) on the phylloplane of grass in th e course of the experiment. B. amyloliquefaciens was easy to follow by selective cultivation due to the complete absence of bacterial backgr ound growth. Southern blot hybridization of HindIII digested genomic D NA showed plasmid restriction patterns identical with pSB20 indicating high plasmid stability. In total DNA extracts from phyllosphere bacte ria the recombinant plasmid was detectable by Southern blot hybridizat ion up to 6 X 10(4) cfu g(-1) (wet weight). Counts of hybridizing colo nies showed that P. syringae established on the phyllosphere of bush b eans at between 5 x 10(3) and 4 x 10(6) cfu g(-1) fresh weight. During senescence of the bean plants the strain was no longer detectable by selective cultivation and subsequent colony hybridization. In contrast , Tn5 marked DNA was detected after PCR amplification over the whole p eriod of the experiment.