Development and Validation of Two Real‐Time
<scp>PCR</scp>
Assays for the Detection of ‘
Candidatus
<scp>Phytoplasma pruni</scp>
’ Strains Causing X‐Disease in Stone Fruits
ABSTRACT
X‐disease, caused by strains in the 16SrIII‐A subgroup of ‘
Candidatus
Phytoplasma pruni’, is a devastating disease of
Prunus
species (stone fruits). Multiple outbreaks of this disease have occurred across much of North America for more than a century, with the most recent one beginning around 2010 in the Pacific Northwest of the United States, causing severe damage to the stone fruit industry. Sensitive and specific detection of X‐disease is critical to prevent the propagation and spread of infected plant material; however, current PCR‐based detection workflows lack specificity to X‐disease and require one or more PCR assays followed by additional testing to confirm the species identity. In this study, two real‐time PCR assays, one targeting the
secY
gene and the other targeting the
tuf
gene, were developed for specific detection of X‐disease in stone fruits. The CPH‐TXD assay targeting the
tuf
gene provided high‐sensitivity target detection of as few as five target copies with significantly reduced cross‐reactivity compared to current methods. The CPH‐SXD assay targeting the
secY
gene detected a minimum of 30 target copies and showed no cross‐reaction with closely related strains. The repeatability and reproducibility of both assays were verified with acceptable results. Together, these assays provide efficient and high‐specificity detection methods to combat the growing threat of X‐disease in North America.