Multiphasic approach for the identification of the different classification levels of Pseudomonas savastanoi pv.phaseolicola

The relationships between strains of Pseudomonas savastanoi pv. phaseolicol a (P. sav. phaseolicola), P. syringae pv. tabaci (P. syr. tabaci) and P. sy r. syringae which all cause disease on bean; the related species P. sav. gl ycinea and P. syr. actinidiae, and reference bacteria, were evaluated by st udying the phenotypic and genetic diversity of a collection of 62 strains. All the P. sav. phaseolicola strains tested produced characteristic waterso aked lesions on bean pods. Other pathovars produced varying combinations of symptoms including necrotic lesions, with or without watersoaked centres a nd sunken tissue collapse of the lesion (P. syr. tabaci) and necrotic lesio ns with or without sunken collapse (P. syr. syringae). At the genomospecies level, all the strains of P. sav. phaseolicola, P. sav. glycinea and P. sy r. tabaci, belonging to genomospecies 2, could be separated from P. syr. sy ringae strains (genomospecies 1) and P. syr. actinidiae strains (unknown ge nomospecies) by BOX-PCR and DNA/DNA hybridisation. To distinguish P. sav. p haseolicola, within genomospecies 2, from P. sav. glycinea and P. syr. taba ci, it was necessary to perform nutritional characterisations (myo-inositol negative and p-hydroxy benzoate positive for P. sav. phaseolicola strains) , PCR with specific primers designed from the tox region (positive for all of the P. sav. phaseolicola strains) and serotyping, as 71% of the P. sav. phaseolicola strains reacted as O-serogroup PHA1. Important intrapathovar v ariation was seen by genomic fingerprinting with REP and ERIC primers, as w ell as with RAPD primers (AE7 and AE10) and esterase profilings. While RAPD fingerprinting detected variability correlated with two race-associated ev olutionary lines, REP, ERIC and esterase profiles revealed intrapathovar va riation linked to some host origins, that separated the kudzu isolates, and the mungbean isolates, from the other P. sav. phaseolicola strains.