The use of 16S and 16S-23S rDNA to easily detect and differentiate common Gram-negative orchard epiphytes

The identification of Gram-negative pathogenic and non-pathogenic bacteria commonly isolated from an orchard phylloplane may result in a time consumin g and tedious process for the plant pathologist. The paper provides a simpl e "one-step" protocol that uses the polymerase chain reaction (PCR) to ampl ify intergenic spacer regions between 16S and 23S genes and a portion of 16 S gene in the prokaryotic rRNA genetic loci. Amplified 16S rDNA, and restri ction fragment length polymorphisms (RFLP) following EcoRI digestion produc ed band patterns that readily distinguished between the plant pathogen Erwi nia amylovora (causal agent of fire blight in pear and apple) and the orcha rd epiphyte Pantoea agglomerans (formerly E. herbicola). The amplified DNA patterns of 16S-23S spacer regions may be used to differentiate E. amylovor a at the intraspecies level. Isolates of E. amylovora obtained from raspber ries exhibited two major fragments while those obtained from apples showed three distinct amplified DNA bands. In addition, the size of the 16S-23S sp acer region differs between Pseudomonas syringae and Pseudomonas fluorescen s. The RFLP pattern generated by HaeIII digestion may be used to provide a rapid and accurate identification of these two common orchard epiphytes. (C ) 2001 Published by Elsevier Science B.V.