Evaluation of Polymerase Chain Reaction (PCR)-Based Methods for Rapid, Accurate Detection and Monitoring of Verticillium dahliae in Woody Hosts by Real-Time PCR.

Abstract

Verticillium wilt, caused by Verticillium dahliae, is one of the most economically important diseases of woody hosts such as ash (Fraxinus spp.), sugar maple (Acer saccharum), and redbud (Cercis canadensis). The causal agent has a broad host range, including not only woody hosts but also important vegetable and field crops, and it is distributed worldwide. Diagnosis of V. dahliae in infected woody hosts is often based on the occurrence of vascular discoloration and time-consuming isolation. However, not all woody hosts exhibit vascular discoloration, and not all vascular discoloration symptoms are due to infection by V. dahliae. In this study, real-time polymerase chain reaction (PCR)-based assays were evaluated and employed for rapid and accurate detection of V. dahliae in different woody hosts. High-quality DNA was extracted in large quantities from presumptively infected woody hosts by collecting drill-press shavings from sample tissue, bead beating, and extracting using a cetyltrimethylammonium bromide method. Six published primer sets were evaluated against genomic DNA of V. dahliae as well as selected negative controls, and two sets (VertBt-F/VertBt-R and VDS1/VDS2) showed promise for further evaluation using DNA extracts from field samples. The VertBt primers amplified a species-specific 115-bp fragment of the expected size, while the VDS primers amplified the expected specific 540-bp fragment. However, the VertBt primer set exhibited higher sensitivity in detection of V. dahliae even in asymptomatic trees. The PCR-based methods developed here could be used as rapid tools for pathogen detection and monitoring, thus informing plant pathogen management decisions.