A robust method for identification and in-planta detection of Verticillium dahliae in the infected olive trees, using real-time PCR and nested PCR

Verticillium wilt is a major disease of olive (Olea europaea) orchards worldwide. To control the disease, use of a reliable method for pre-planting screening of new planting stocks for infection by V. dahliae is essential. In this study, 20 symptomatic and asymptomatic olive trees, sampled from Zahak region of Sistan in Iran, were analyzed with real-time and nested PCR for in planta detection of V. dahliae. We could confirm the presence of V. dahliae in 12 out of 20 (60%) sampled trees using real-time PCR, whereas nested PCR produced amplicons for all tested symptomatic and asymptomatic samples (100% of tested trees), indicating that nested PCR was more efficient method than real-time PCR for in planta detection of V. dahliae. We used the designed nested PCR to determine also the pathotype of V. dahliae using Vdf2/Vdr2 primer set at the second round, which could produce the 297-bp amplicon for all tested samples infected with non-defoliating pathotype of V. dahliae. We showed that although the Verticillium wilt is spread in the olive cultivation areas in Zahak region of Sistan, nested PCR can be deployed for the timely detection and thereby prevention of the further spread of V. dahliae in the olive cultivation areas.