Optimization of a High-throughput Assay Enabling Early Detection of Anisogramma anomala in Hazelnuts

Anisogramma anomala (Peck) E. Müller is a fungal pathogen that causes eastern filbert blight (EFB) of hazelnuts (Corylus sp.).  Developing commercial-quality plants resistant to EFB is a major objective of hazelnut breeding programs in the United States.  However, the disease cycle of A. anomala makes screening for resistance difficult as symptoms (stromata and cankers) are only typically expressed sixteen to eighteen months after infection.  An assay was previously developed using real-time PCR (qPCR) to detect the fungus in young hazelnut seedlings.  However, it was based on a small sample size and was not optimized for screening large numbers of breeding progeny in an efficient and cost-effective manner.  Our goal was to develop a lower-cost qPCR method suitable for high-throughput sampling.  In this study, we examined several components of the assay.  We developed a second set of robust qPCR primers specific to hazelnuts to limit the occurrence of false negatives.  Further, we used the qPCR assay to track the movement and spread of A. anomalaover a period of eight weeks to determine the optimal location and timing of sampling to further minimize false positives and escapes.  Next, we examined methods to scale up to a high-throughput format in terms of DNA extractions and the qPCR assay.  As a final control, we compared the results of the qPCR assay with subsequent canker development in several large progenies of seedlings.  Based on these improvements, we can now rapidly sample high numbers of plants and determine the presence and quantity of the fungus within only a few weeks after inoculation, making the assay much more useful for applied breeding.