Plasmids isolated from the sugar beet phyllosphere show little or no homology to molecular probes currently available for plasmid typing

From a representative sample of bacteria, isolated from mature sugar beet leaves (Beta vulgaris) grown at three separate locations in the UK, 79 (18%) were shown to contain plasmids ranging in size from 10 kb to 200 kb. A sensitive colony blot method was developed to facilitate the screening of both Gram-negative and Gram-positive isolates to determine the distribution of known plasmid incompatibility groups among plasmids isolated from the natural environment using the collection of inc/rep probes derived from basic replicons [rep FIA, FIIA, FIB, HI1, HI2,I1, B/O, L/M, N, P, Q, U, W and X, as described by Couturier et al. (1988) Microbiol Rev 52, 375-395]. After hybridization with each of the radiolabelled replicon probes, 54 of these 79 plasmid-containing natural isolates, which included Erwinia spp., Pseudomonas spp. and Gram-positive bacteria, failed to react. Reactivity was observed with 25 of the 29 Klebsiella and Erwinia isolates investigated. Of the plasmid-containing Enterobacteriaceae examined, 18 reacted with the repFIB probe, six with the repFIIA probe and one isolate, Erwinia salicis SBN169, hybridized to both. Southern hybridization demonstrated that the different isolates which shared homology with the repFIB probe contained a common 1 kb PstI fragment. By comparing the PstI restriction fragment patterns of total plasmid DNA, the Erwinia and Klebsiella isolates were divided into 10 distinct groups and the other non-reactive isolates divided into a further 24 groups. Plasmid type appeared to be restricted to the geographical location from which the host bacteria were isolated. This study illustrates that sugar beet phyllosphere bacteria support a diverse array of plasmids which cannot be readily classified at the molecular level into the recognized incompatibility groups commonly described for clinical isolates.