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source for dna digestion - Journal of Applied Microbiology...

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ORIGINAL ARTICLE Characterization of Erwinia amylovora strains from different host plants using repetitive-sequences PCR analysis, and restriction fragment length polymorphism and short-sequence DNA repeats of plasmid pEA29 D. Barionovi 1 , S. Giorgi 1 , A.R. Stoeger 2 , W. Ruppitsch 2 and M. Scortichini 1 1 C.R.A.-Istituto Sperimentale per la Frutticoltura, Ciampino Aeroporto, Rome, Italy 2 Austrian Agency for Health and Food Safety, Spargelfeldstrasse 191, Vienna, Austria Introduction Erwinia amylovora (Burrill) Winslow et al. causes one of the most severe diseases affecting the Rosaceae family worldwide. Plant species, especially those belonging to Maloideae and Rosoideae subfamilies, can be infected by the bacterium. Plant quarantine measures may slow but often fail to prevent the eventual introduction into new areas. Assessing the genetic variability of pathogens is a fundamental prerequisite to develop effective diagnostic Keywords ±re blight, genetic diversity, Maloideae, rep- PCR, Rosaceae, Rosoideae. Correspondence Marco Scortichini, C.R.A.-Istituto Sperimentale per la Frutticoltura, Via di Fioranello, 52, I-00040 Ciampino aeroporto (Roma), Italy. E-mail: mscortichini@yahoo.it 2005/0555: received 20 May 2005, revised 26 August 2005 and accepted 20 October 2005 doi:10.1111/j.1365-2672.2006.02813.x Abstract Aims: The three main aims of the study were the assessment of the genetic relationship between a deviating Erwinia amylovora strain isolated from Amel- anchier sp. (Maloideae) grown in Canada and other strains from Maloideae and Rosoideae, the investigation of the variability of the Pst I fragment of the pEA29 plasmid using restriction fragment length polymorphism (RFLP) analy- sis and the determination of the number of short-sequence DNA repeats (SSR) by DNA sequence analysis in representative strains. Methods and Results: Ninety-three strains obtained from 12 plant genera and different geographical locations were examined by repetitive-sequences PCR using Enterobacterial Repetitive Intergenic Consensus, BOX and Repetitive Extragenic Palindromic primer sets. Upon the unweighted pair group method with arithmetic mean analysis, a deviating strain from Amelanchier sp. was ana- lysed using ampli±ed ribosomal DNA restriction analysis (ARDRA) analysis and the sequencing of the 16S rDNA gene. This strain showed 99% similarity to other E. amylovora strains in the 16S gene and the same banding pattern with ARDRA. The RFLP analysis of pEA29 plasmid using Msp I and Sau 3A restriction enzymes showed a higher variability than that previously observed and no clear-cut grouping of the strains was possible. The number of SSR units reiterated two to 12 times. The strains obtained from pear orchards showing for the ±rst time symptoms of ±re blight had a low number of SSR units. Conclusions: The strains from Maloideae exhibit a wider genetic variability than previously thought. The RFLP analysis of a fragment of the pEA29 plas- mid would not seem a reliable method for typing E. amylovora strains. A low number of SSR units was observed with ±rst epidemics of ±re blight.
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source for dna digestion - Journal of Applied Microbiology...

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