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SUMMARY 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 (O) and fermentative (F) metabolism are subjected to API 20E, REP-PCR or Biolog analyses. Isolates that generate a 7-digit code = 0005773 in the API 20E system, a REP-PCR fingerprint or Biolog pro- file 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. nigriflu- ens among the 28 gram negative isolates (14 with O/F and 14 with O 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 relia- bility by Biolog analysis. This analysis also permits iden- tification of isolates with O 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 iso- late of four and the type strain (LMG 2694 T ) of B. nigri- fluens are urease negative, when determined in Dye's medium. Since our data are in contrast with those previ- ously reported, this phenotypic character needs further verification for B. nigrifluens identification.

A PROTOCOL FOR RAPID IDENTIFICATION OF BRENNERIA NIGRIFLUENS AMONG BACTERIA ISOLATED FROM BARK CANKERS IN PERSIAN WALNUT PLANTS