Scortichini 2005 consequently further assessment to

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Scortichini 2005). Consequently, further assessment to verify the genetic relationship of this strain with representative strains of E. amylovora was underta- ken. In the present study, after the screening of the gen- etic variability of 93 E. amylovora strains obtained from different host plants belonging to 12 different genera and inferred using rep-PCR, it was observed that the strain from Amelanchier sp. and three strains isolated from Rubus spp. had a different genomic fingerprint. The objectives of this study were to: (i) establish the genetic relationship between the strain isolated from Amelanchier sp. (Maloideae) and the other strains from Maloideae and Rosoideae using amplified ribosomal DNA restriction analysis (ARDRA) and 16S rDNA gene sequen- cing; (ii) assess the collection using restriction fragment length polymorphism (RFLP) analysis for checking the variability in length of the Pst I fragment of pEA29 plas- mid and to verify the reliability of strain grouping with this method; (iii) further analyse the occurrence and the number of SSR units in E. amylovora from different host plants and to check the number of SSR units in 32 strains obtained from two pear orchards showing for the first time symptoms of fire blight. Materials and methods Bacterial strains and growth medium Erwinia amylovora strains used in this study are listed in Table 1. The strains marked with Istituto Sperimentale per la Frutticoltura (ISF) were isolated for this study from diseased pear specimens, all the others were obtained from international or national culture collec- tion (Table 1). Pear twigs or fruits showing symptoms of fire blight were used for isolation. Fragments of tissue at the margin of lesion were crushed in sterile mortars containing 5 ml of sterile saline (SS) (0 Æ 85% of NaCl in distilled water). Ten-fold serial dilutions in tubes were performed. Subsequently, aliquots of 0 Æ 1 ml were spread on to Petri dishes containing nutrient sucrose agar (NSA) [28 Æ 0 g of nutrient agar (Oxoid, Basingstoke, UK) supplemented with 50 Æ 0 g of sucrose, per litre]. The plates were incubated at 25–27 Ŷ C for 3 days. With the levaniform, whitish colonies suspected to belong to E. amylovora , confirmatory tests (i.e. tobacco hypersensi- tivity, absence of fluorescent pigments on the medium B of King et al. (1954) (KB), induction of necrosis and oozing in immature pear fruits, SDS-PAGE of whole-cell protein extracts and comparison with E. amylovora type- strain NCPPB 683 and other representative strains pre- viously isolated in Italy was carried out. The isolates inducing the hypersensitivity reaction in tobacco leaves, negative for the fluorescent pigments production on KB, inciting necrosis and oozing on immature pear fruits and showing the same protein profile as E. amylovora NCPPB 683 and the other representative Italian strains, were retained to belong to E. amylovora . For this study, all the 93 strains used were routinely grown on NSA, at 25–27 Ŷ C.
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  • Spring '08
  • Devartanian
  • DNA, DNA sequencing, Restriction enzyme, Greece Greece Greece Greece Greece Greece Greece Greece Hungary Hungary Hungary Hungary Hungary Hungary Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy USA USA Italy Italy Italy Italy, E. amylovora

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