Detection of the nondefoliating pathotype of Verticillium dahliae in infected olive plants by nested PCR

An increasing incidence and distribution of verticillium wilt has occurred in the last few years in newly established olive orchards in southern Spain . This spread of the disease may result from use of Verticillium dahliae-in fected planting material. The early in planta detection of the pathogen wou ld aid the implementation of certification schemes for pathogen-free planti ng material. In this work, a nested polymerase chain reaction (PCR) method was developed for the in planta detection of the nondefoliating (ND) V. dah liae pathotype, aimed especially at nursery-produced olive plants. For this purpose, specific primers were designed from the sequence of a 1958-bp ran dom amplified polymorphic DNA (RAPD) marker of ND V. dahliae, and a procedu re for the extraction of PCR-quality total genomic DNA from infected root a nd stem tissues of young olive plants was tested and further optimized. Nes ted PCR assays detected ND V. dahliae in 4- to 14-month-old artificially in fected plants of three olive cultivars. The ND-specific PCR product was not amplified from total genomic DNA extracted from olive plants infected with the defoliating V. dahliae pathotype. Detection of the ND pathotype was ef fective from the very earliest moments following artificial inoculation of olive plants with a V. dahliae conidial suspension. Also, detection was ach ieved in inoculated, though symptomless, olive plants as well as in plants that were symptomatic but became symptomless by 217 days after inoculation.