1a. Objectives (from AD-416):
Develop rapid molecular diagnostic tools for the boxwood blight fungal pathogen.
1b. Approach (from AD-416):
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.
3. Progress Report:
A real-time PCR detection assay for the boxwood blight fungus Calonectria pseudonaviculata was developed based on the nucleotide sequence of the beta tubulin marker. Detection of C. pseudonaviculata was duplexed with a second assay as an internal control, capable of detecting the DNA of the boxwood host plant. Testing was performed using a sample of (1) C. pseudonaviculata collected from the U.S., United Kingdom, Italy, and New Zealand; (2) sister Calonectria; and (3) uninfected boxwood and unrelated fungal endophytes. The assay was shown as highly sensitive and specific in these assays. However, application of the assay for testing soil led to late cycle false positives, minimizing usefulness. A second assay was developed using the nucleotide sequence of the histone H3 marker, also coupled with the internal control for boxwood DNA. The histone H3 assay was shown as highly sensitive and specific in these assays, including the soil-based analyses. A LAMP (loop mediated isothermal amplification) detection assay was developed based on the nucleotide sequence of the beta tubulin marker. Six sets of primer pairs were tested. A novel colorimetric detection method was developed simultaneously, for in-tube, visual detection of a positive reaction without the need for specialized equipment.