A Barcoded ITS Primer-Based Nanopore Sequencing Protocol for Detection of Alternaria Species and Other Fungal Pathogens in Diverse Plant Hosts

<i>Alternaria</i> is a genus that contains several important plant pathogens affecting nearly 400 plant species worldwide, including economically important crops such as grapes, citrus, and ornamental plants. Rapid, scalable, and efficient methods of pathogen detection are crucial for managing plant diseases and ensuring agricultural productivity. Current amplicon sequencing protocols for <i>Alternaria</i> detection often require the enzymatic barcoding of amplicons, increasing hands-on time, cost, and contamination risk. We present a proof-of-concept study using custom barcoded primers, combining universal primers targeting ITS1 and ITS2 regions (600 bp) coupled with Oxford Nanopore Technologies (ONT) barcode sequences. Sequencing was performed on infected grapevine, mandarin orange, thuja, and maple tree samples. In total, we analyzed 38 samples using qPCR; 8 tested positive for <i>Alternaria</i>, which were sequenced using a newly developed protocol. As a result, we could identify <i>Alternaria</i> in every positive sample, and besides the pathogen of interest, we could identify the associated mycobiome. This protocol reduces hands-on time and cost, making a significant advancement over current sequencing methods. Future work will focus on optimizing our approach for high-throughput sequencing of up to 96 samples and determining the method's applicability for large-scale mycobiome analysis.