Development and evaluation of a real-time PCR seed lot screening method for Fusarium circinatum, causal agent of pitch canker disease

Fusarium circinatum is a serious pathogen of Pinus spp. worldwide, causing pitch canker disease. F.similar to circinatum can contaminate seeds both internally and externally and is readily disseminated via contaminated seed. Many countries require screening of pine seeds for F.similar to circinatum before they can be imported. The currently accepted screening method is based on culturing the pathogen on a semi-selective medium and identifying it using morphological traits. This method is time-consuming and does not allow for accurate identification of the pathogen to the species level. A bulk DNA extraction and real-time PCR procedure to screen seeds for the presence of F.similar to circinatum were developed in this study. The real-time PCR method resulted in the detection of F.similar to circinatum in 5 of 6 commercial seed lots tested and has a lower detection limit of 1 x 10-5 ng of F.similar to circinatum DNA per PCR. The culture-based method detected Fusarium spp. in four of six of the same seed lots. The real-time PCR method can be used to screen multiple seed lots in 2 days, whereas the culture-based method requires a minimum of 12 weeks. This new real-time PCR seed screening method allows for fast, sensitive and accurate screening and can be adapted to handle larger volumes of seeds.