Diagnostic tools for the identification of Brenneria nigrifluens, the causal agent of persian walnut bark canker

After the first report in 1998 in North Italy (Veneto and Piemonte), bark canker of persian walnut (Juglans regia L.), caused by Brenneria nigrifluens, has later been described in other italian regions (Lazio and Campania). In 2002, we found the symptoms of the disease in walnut plantations for timber production located in 13 farms of Central Italy (Umbria and Marche). All of the 44 isolates obtained from diseased plants were Gram-negative and had oxidative and fermentative metabolism. Twenty of them were submitted to API 20E system (bioMérieux) for the identification because they were oxidase negative and therefore belonged to the Enterobacteriaceae. Four isolates (1 from Umbria and 3 from Marche) gave the 7-digit code0005773, which is identical to those observed for the type strain LMG 2694T and the two reference strains LMG 5107 and LMG 5953 of B. nigrifluens. When trunks of young walnut plants were inoculated with these 4 isolates, they caused typical symptoms of the disease 3 months after the inoculation. A number of isolates associated with bark cankers and not pathogenic on walnut plants were identified as Erwinia rhapontici by API 20E system. Rep-PCR performed with the primers REP showed that the pathogenic isoaltes had high similarity (89%) with the type and reference strains of B. nigrifluens. At present, we intend to develop a diagnostic molecular assay exploiting a 905 bp fragment amplified during REP-PCR, which seems to be characteristic of B. nigrifluens.