Comparison between real-time Scorpion PCR and traditional methods for the detection and quantification of Verticillium dahliae in soils and infected olive trees
A technique based on the real-time Scorpion polymerase chain reaction for the detection of Verticillium dahliae in soil and xylem of infected olive trees was developed. Two specific primer pairs (Ver2 -Ver3 and Vd7b-Vd10), previously designed by comparing sequences from the ribosomal DNA intergenic spacer of V. dahliae and related species, were used. A nested-PCR protocol was applied to detect the pathogen by using Ver2-Ver3 in the first step and Vd7B-Vd10 in the second step. Primers specifity and detection limit were assessed by using genomic DNA from V. dahliae isolates and other species of the same genera. Specific Scorpion-PCR amplification, using modified Vd7b primer (Vd7b-FAM), was successful in detecting V. dahliae from soil and infected olive wood. The reliability of the entire procedure was assessed using both artificially and naturally infested soils and woody materials.
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Compared to traditional methods the molecular approach proved to be rapid, sensitive, and enabled a large-scale analysis.
