Simultaneous detection of Candidatus Phytoplasma prunorum and Plum Pox Virus in Prunus spp. by crude RNA-DNA extraction and RT-qPCR.

Many efforts have been made to develop a rapid and sensitive method for phytoplasma and virus detection. Taking our cue from previous works, different rapid sample preparation methods have been tested and applied to "Candidatus Phytoplasma prunorum" detection by RT-qPCR. A duplex RT-qPCR has been optimized using the crude sap as template to simultaneously amplify a fragment of 16S rRNA of the pathogen and 18S rRNA of the host plant. The specific plant 18S rRNA internal control allows comparison and relative quantification of samples. A comparison between DNA and RNA contribution to qPCR detection is provided, showing higher sensitivity for the latter. The method presented here has been validated on more than a hundred samples of apricot, plum and peach trees in field and in nursery since 2010. Finally, a triplex RT-qPCR assay has been optimized to simultaneously detect "Candidatus Phytoplasma prunorum" and Plum Pox Virus in Prunus, as these pathogens may occur in the same plant species, are both detrimental and localized in the phloem. Therefore sampling can be done from the same tissue and RT-qPCR can be performed on the same crude sap, without the need to extract nucleic acids. The described triplex test could decrease costs, time and labour of stone fruit trees screening in areas susceptible to both pathogens.