Nanopore-Sequencing Metabarcoding for Identification of Phytopathogenic and Endophytic Fungi in Olive (Olea europaea) Twigs

Abstract

Metabarcoding approaches for the identification of plant disease pathogens and characterization of plant microbial populations constitute a rapidly evolving research field. Fungal plant diseases are of major phytopathological concern; thus, the development of metabarcoding approaches for the detection of phytopathogenic fungi is becoming increasingly imperative in the context of plant disease prognosis. We developed a multiplex metabarcoding method for the identification of fungal phytopathogens and endophytes in olive young shoots, using the MinION sequencing platform (Oxford Nanopore Technologies). Selected fungal-specific primers were used to amplify three different genomic DNA loci (ITS, beta-tubulin, and 28S LSU) originating from olive twigs. A multiplex metabarcoding approach was initially evaluated using healthy olive twigs, and further assessed with naturally infected olive twig samples. Bioinformatic analysis of basecalled reads was carried out using MinKNOW, BLAST+ and R programming, and results were also evaluated using the BugSeq cloud platform. Data analysis highlighted the approaches based on ITS and their combination with beta-tubulin as the most informative ones according to diversity estimations. Subsequent implementation of the method on symptomatic samples identified major olive pathogens and endophytes including genera such as Cladosporium, Didymosphaeria, Paraconiothyrium, Penicillium, Phoma, Verticillium, and others.

Implication of Nanopore Sequencing Technology for Pathogenic Diagnostics: A Mini-Review

“…However, this method was also effective in the pathogenic diagnostics of plants, animals, and environmental samples [26,[45][46][47]. For example, different phytopathogenic and endophytic fungi such as Cladosporium, Didymosphaeria, Paraconiothyrium, Penicillium, Phoma, and Verticillium species were detected in the olive tree using ONT sequencing for three DNA loci of ITS, betatubulin, and 28S LSU regions [48]. Additionally, a novel all-in-one diagnostic assay based on the nanopore sequencing method was developed for the quarantine of pathogens in plants that was completely done within 13 minutes [49].…”