Start Date: Jul 01, 2012
End Date: Jun 30, 2013
Comprehensively test and validate two types of molecular detection assays, using both boxwood tissue and soil. First, an isothermal recombinase polymerase assay (RPA), a method that can be used where thermal cycling equipment is unavailable, will be developed for detection of Calonectria pseudonaviculata (C. ps). Second, two independently developed real-time PCR assays will be tested and compared across 2 different cycling platform for specificity, sensitivity, and assay validation. Specificity of real-time PCR and RPA assays will be tested and validated on a comprehensive set of DNA controls (DNA from multiple cultured C. ps. isolates, DNA extracted from other Calonectria species, DNA from symptomatic and asymptomatic boxwood tissues, and DNA from other known fungal boxwood pathogens). Positive control DNA (synthesized long oligonucleotides corresponding to C. ps. DNA sequences) will be validated for application as a positive control as a substitute for biologically derived positive control DNA. It will be determined whether real-time PCR and RPA assays can detect C. ps. in soil using DNA extracted from artificially infested soil and lower limits of detection established. The sensitivity of this tool will be assessed with soil spiked with other Cylindrocladium species. Outcomes from these studies will allow growers to more effectively manage this threatening disease in the field, eliminate pathogen spread, and minimize the potential for economic losses through crop destruction.