Deep Sequencing for Trunk Disease Diagnostics

The aim of this multi-year project is to develop rapid and cost-effective diagnostic methods for detection, identification and quantification of trunk pathogens in asymptomatic and symptomatic grape wood. In the 2015 -2016 grant cycle, we have tested different NGS-based methods for the detection of trunk pathogen in symptomatic infected field material and compared the results with traditional diagnostic approaches. All methods we tested were in agreement with traditional diagnostic methods. NGS-based methods also detected simultaneously multiple pathogen species with no need of tedious and hands-on sample culturing and colony purification. Additionally, unlike traditional qualitative diagnostics, deep sequencing also allowed to determine the relative abundance of the different species. Among all methods tested, ITS deep sequencing (ITS-seq) remains the most cost effective until library prep costs for DNA and RNA sequencing do not decline significantly. For this reason, ITS-seq will be the subject of protocol optimization of the second year of the project. Both sensitivity and specificity of the method need to be improved for diagnostics purposes. The results of this first phase of the project demonstrate that unlike traditional approaches NGS-based detection delivers rapid simultaneous identification and quantification of multiple species in infected tissue with no need of culturing and isolation.