Comparative studies to assess bacterial communities on the clover phylloplane using MLST, DGGE and T-RFLP

Denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) were used to characterise the changes that occurred in Bacillus cereus group strains present in the phylloplane of clover Trifolium hybridum over 4 months. These strains had previously been analysed by multiple locus sequence typing (MLST). DGGE displayed many equally intense bands which indicated many equally abundant ribotypes. The bacterial community composition was variable and the leaves sampled as little as a week apart were found to have some temporal variability, indicating that diverse phylloplane bacterial communities follow sequential patterns from time to time. The B. cereus group community clearly clustered into early, mid and late branches, possibly due to multiple successional sequences occurring during growing seasons. The functionally and phylogenetically diverse microbial communities appeared to exhibit predictable successional patterns over shorter time scales. DGGE analysis with the molecular marker rpoB gave better resolution than 16S rRNA amplicons. There were no strong similarities between the dendrograms produced by DGGE, MLST and T-RFLP and the clustering produced by the automated T-RFLP method was variable even between the three restriction enzymes used. The DGGE–MLST method emerged as a superior method to T-RFLP–MLST for rapid typing of bacterial communities.