A rapid qPCR for the detection of Verticillium nonalfalfae MLST2 - a highly pathogenic fungus on kiwifruit

A highly pathogenic fungus characterized as multilocus sequence type 2 (MLST2) is an emerging fungal pathogen causing Verticillium wilt on kiwifruit. Although 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 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 MLST2 in plant tissues. The assay targets the glyceraldehyde-3-phosphate dehydrogenase ( ) gene and was shown to detect all tested isolates of MLST2 with a detection limit of approximately 2 pg of pathogen genomic DNA. There was no cross-reaction with MLST1, other 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 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 MLST2.