Real-time PCR assay to detect Verticillium albo-atrum and V. dahliae in hops: development and comparison with a standard PCR method

Abstract

Verticillium wilt, caused by Verticillium albo-atrum and V. dahliae, is a devastating disease that causes considerable economic crop losses in hop fields. The fungus can survive in soil for several years by producing resting structures, and due to the lack of effective fungicides, the disease is spreading. Thus, a fast and sensitive detection system is urgently needed. In this study we report a novel routine detection method for the identification of V. albo-atrum and V. dahliae on a molecular basis. The standard polymerase chain reaction (PCR) assay includes isolation of the fungus from affected tissue by following DNA extraction from fungal cultures and PCR identification by specific primers. In order to improve this detection assay, DNA was isolated directly from the hop bine using a commercially available DNA isolation kit. A multiplex real-time PCR assay for the simultaneous detection of V. albo-atrum and V. dahliae was established using a specific primer pairs/TaqMan probes combination. Ninety-six plants were collected from different culti-vars and locations in Germany and tested by standard and real-time PCR assay. The new detection system is more accurate, sensitive and time-saving than the standard PCR method and is suitable for routine use. The test provides a valuable tool for rapid and sensitive detection of V. albo-atrum and V. dahliae in plants and gives farmers and plant protection offices the chance to react accordingly and to evaluate measures for plant disease management against Verticillium.