Molecular diagnosis of Pseudomonas syringae pv tomato.

Pseudomonas syringae pv. tomato, the causal agent of tomato bacterial speck, is an harmful pathogen for which early diagnosis in plant propagation material is mandatory (EEC directives 93/61 and 93/62). By analysis of the polymorphic amplicons generated with the primer OPA-19 from the DNA of ten isolates of P. syringae pv tomato collected from different geographical areas, a 250 bp fragment was identified and labelled with DIG-probe synthesis kit (Roche). In Southern blot analysis this fragment hybridised specifically to a fragment with similar size of the RAPD pattern generated by the type strain 1106 NCPPB of P. syringae pv tomato and used as control and by the other well-characterised strains ISFP 111, PD 170 and IPV BO 1611, IPV BO 2548 of P. syringae pv tomato kindly supplied by Dr. Marco Scortichini and Prof. Carlo Bazzi, respectively. No hybridisation signal was obtained with DNA extracted from pathovars actinidiae, populans, persicae, lacrymans, japonica, panici, pisi, aptata morsprunorum, syringae and from P. savastanoi pv savastanoi. The 250 bp fragment was ligated to the pGEM-T plasmid (Promega) and cloned in E. coli, strain DH5a. The nucleotide sequence was determined and deposited in EMBL database. When compared with other available sequences, this fragment was found 98% homologous to a region flanking a heat shock protein gene (section 16-21 of the complete genome) of the strain DC 3000 of P. syringae pv tomato (Acc. No AE016871). The use of this fragment as P. syringae pv tomato diagnostic tool is proposed for large scale testing.