A Rapid qPCR for the Detection of Verticillium nonalfalfae MLST2 - A Highly Pathogenic Fungus on Kiwifruit

A highly pathogenic fungus characterized as Verticillium nonalfalfae multilocus sequence type 2 (MLST2) is an emerging fungal pathogen causing Verticillium wilt on kiwifruit. Although V. nonalfalfae MLST2 has not been reported outside Chile, there is a risk that this pathogen could spread through the global movement of germplasms to other countries. Current diagnostic methods for this fungus rely on a laborious and time-consuming plating assay for morphological identification and DNA sequence analysis. In this study, we describe the development and validation of a novel quantitative polymerase chain reaction (qPCR) assay for rapid and specific detection of V. nonalfalfae MLST2 in plant tissues. The assay targets the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene and was shown to detect all tested isolates of V. nonalfalfae MLST2 with a detection limit of approximately 2 pg of pathogen genomic DNA. There was no cross-reaction with V. nonalfalfae MLST1, other Verticillium species, or non-target fungal species found on kiwifruit. This assay was duplexed with a plant internal control for simultaneous detection of the pathogen and cytochrome oxidase gene from the host plant. This new specific and sensitive qPCR assay is a valuable molecular diagnostic tool for rapid screening of imported plant material and would also be useful for testing samples collected from field surveillance activities to monitor the presence of V. nonalfalfae MLST2.