Sensitive and specific detection of Pseudomonas avellanae using primers based on 16S rRNA gene sequences

A rapid polymerase chain reaction (PCR)-based procedure was developed for the detection of Pseudomonas avellanae, the causal agent of hazelnut (Corylus avellana) decline in northern Greece and central Italy. The partial sequence of the 16S rRNA gene of P. avellanae strain PD 2390, isolated in central Italy, was compared with the sequence coding for the same gene of P. syringae pv. syringae type-strain LMG 1247(t1). Primers PAV 1 and PAV 22 were chosen, and after the PCR, an amplification product of 762 base pairs was specifically produced only by 40 strains of P. avellanae isolated from northern Greece and central Italy. No other bacterial species among those tested showed an amplification product under optimized PCR conditions. The adding of 4% BLOTTO (10% skim milk powder and 0.2% NaN3) in the PCR mixture proved essential in order to avoid interference of hazelnut extracts during the amplification. The procedure proved more effective than repetitive PCR with ERIC primer sets in diagnosing apparently healthy hazelnut trees as infected. This technique could be of great help for screening the hazelnut propagative material as well as for monitoring the wild C. avellana trees growing in the woods near the infected hazelnut orchards.