A protocol for rapid identification of Brenneria nigrifluens among bacteria isolated from bark cankers in Persian walnut plants

A protocol based on API 20E, REP-PCR or Biolog analyses is described for rapid and reliable identification of Brenneria nigrifluens among the bacteria isolated from bark cankers in walnut plants. Bacteria that are soluble in 3 % KOH solution (Gram negative), oxidase negative and with oxidative (0) and fermentative (F) metabolism are subjected to APT 20E, REP-PCR or Biolog analyses. Isolates that generate a 7-digit code = 0005773 in the APT 20E system, a REP-PCR fingerprint or Biolog profile very similar (87-100%) to those of the B. nigrifluens reference strains are assigned to this bacterial species. Using the protocol, we identified 4 isolates of B. nigrifluens among the 28 gram negative isolates (14 with O/F and 14 with 0 metabolism) obtained from bark cankers in walnut plantations on 10 farms in Central Italy. Among the other ten O/F isolates, only 2, assigned to Pectobacterium chrysanthemi, were identified with reliability by Biolog analysis. This analysis also permits identification of isolates with 0 metabolism. A pathogenicity test performed on walnut plants revealed that only the 4 B. nigrifluens isolates provoked bark canker symptoms 3 months after the inoculation. We also found that one isolate of four and the type strain (LMG 2694(T)) of B. nigrifluens are urease negative, when determined in Dye's medium. Since our data are in contrast with those previously reported, this phenotypic character needs further verification for B. nigrifluens identification.