Development and enhancement of plant pathogens diagnostic protocols based on Nanopore sequencing

In recent times, we have witnessed a concerning rise in the accidental introduction of harmful organisms into both floriculture and agricultural production. This trend poses significant threats, particularly when it comes to quarantine pathogens, many of which are notoriously difficult to diagnose. The presence of quarantine phytopathogens in floricultural plants not only jeopardizes crop health but also inflicts severe damage on the floriculture and broader agricultural sectors. Phytopathogen detection requires rapid and sensitive assays to reach effective management of crop diseases. The main limitation of current diagnostic testing is the inability to combine broad and sensitive pathogen detection with the identification of key strains, pathovars, and subspecies. This thesis work has its base in the GARDING project (Bando 2018 d.d.s. n. 4403 del 28/03/2018), sponsored by the Lombardy region and born from the collaboration between the University of Milan, the University of Verona and VIVAI NORD Snc for the protection of the territory from the invasion of potentially harmful pathogen species. The project aims to build barcoding protocols for the identification of phytopathogens directly at customs, thus eliminating the need for sample transfers to external laboratories to perform rapidly multiple pathogens tests with less time required compared to present methods. One promising solution is implementing Nanopore Third Generation Sequencing technology for pathogen detection. This technology is portable, and it allows the detection of microorganisms even at low concentrations. It validates results from existing molecular methods and extends diagnostic capabilities beyond laboratory settings. The results presented in this thesis show the development of an innovative diagnostic method based on Nanopore third-generation sequencing technology capable of identifying several quarantine pathogens in simulated infected plants, even discerning among pathogens subspecies. These outcomes prove the potential of this technology to facilitate the advancement of next-generation diagnostic assays for efficiently monitoring other plant pathogens.