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Molecular Detection of Apiosporina morbosa, Causal Agent of Black Knot in Prunus virginiana

A specific and sensitive polymerase chain reaction (PCR) assay was developed to detect Apiosporina morbosa, the causal agent of black knot disease on chokecherry, Prunus virginiana (including the cultivar ‘Shubert Select’). A pair of A. morbosa-specific forward and reverse primers (AMF and AMR) was designed from the internal transcribed spacer (ITS) regions of A. morbosa, preamplified by universal ITS primers ITS1 and ITS4, and compared with the ITS region sequences of Fusarium, Alternaria, Phoma, and Cladosporium species associated with black knots. The primers were tested for their specificity to A. morbosa detection in the PCR assays using DNA derived from 64 pure cultures, including 42 single-spore isolates of A. morbosa and 22 isolates of other fungi, as well as healthy and diseased plant branches collected from the field. A product of ~400 bp was amplified from DNA of all isolates belonging to A. morbosa. No product was amplified from DNA of other fungal species, confirming the specificity of the newly designed primers. Within plant tissues, the pathogen was detected at further distances from the edges of knots on thicker branches bearing larger knots compared with thinner branches bearing smaller knots. The PCR assay has shown high sensitivity, needing only 100 fg of the A. morbosa DNA for a reliable PCR amplification with the AMF and AMR primers.