Real-Time and Conventional PCR Tools for Detection and Discrimination of Calonectria pseudonaviculata and C....

Calonectria pseudonaviculata and C. henricotiae are the causal agents of boxwood blight, a devastating disease of boxwood that has caused significant economic impact on the nursery and landscape industries in the U.S. and in Europe. The two species are genetically distinct and are found in different geographic areas but are difficult to distinguish based on morphology and pathogenicity. Fast, accurate, and inexpensive methods to detect and differentiate these species is critical in stopping the spread of the disease. We designed primer pairs based on available sequences of four conserved regions-calmodulin, histone H3, internal transcribed spacer, and β-tubulin-and tested their ability to differentiate the two Calonectria species. Here we report three primer pairs derived from sequence differences in the histone H3 region that can be used to specifically detect C. pseudonaviculata, C. henricotiae, or both species. Specificity of these primers was tested against nine isolates of C. pseudonaviculata, three isolates of C. henricotiae, 13 other Calonectria species, and five isolates from related genera using conventional and real-time PCR. These are the first primers available that can be used with either a multiplexed conventional PCR or SYBR-based real-time PCR to specifically detect and differentiate the two fungal species.