AimsTo develop real-time PCR assays for quantification of shoot infection levels of canker disease of stone fruits and nut crops caused by six fungal pathogen groups. Methods and ResultsThis study focused on six major canker-causing fungal pathogen groups: Phomopsis sp., Botryosphaeria dothidea, Lasiodiplodia sp., Cytospora sp., Neofusicoccum sp. and Diplodia sp., occurring in stone fruits and nut crops in California. DNA primers were designed to specifically target each of the six pathogen groups after the specificity tests using canker-causing and non-canker-causing pathogens and by using DNA sequences of other species from GenBank using blast. The quantitative real-time PCR (qPCR) systems were developed and used to quantify the infection levels of inoculated dried plum shoots. ConclusionsFor Neofusicoccum sp. and Phomopsis sp., which were used in inoculation of walnut shoots, the values of the molecular severity ranged from 560 to 694 during the 16days of latent infection period. The qPCR assays were more efficient, accurate and precise to quantify latent infections caused by canker-causing pathogens as compared to the traditional plating methods. Significance and Impact of the StudyThis study demonstrated the potential of using the developed qPCR systems for epidemiological studies on canker diseases of woody plants.