Long amplicon nanopore sequencing of Botrytis cinerea and other fungal species present in infected grapevine leaf samples

Botrytis cinerea is a well-known plant pathogen responsible for grey mould disease infecting more than 500 plant species. It is listed as the second most important plant pathogen scientifically and economically. Its impact is particularly severe in grapes since it affects both the yield of grape berries and the quality of wines. While various methods for detecting B. cinerea have been investigated, the application of Oxford Nanopore Technology (ONT) for complete ribosomal operon sequencing, which has proven effective in human and animal fungal research and diagnostics, has not yet been explored in grapevine (Vitis vinifera) disease research. In this study, we sequenced complete ribosomal operons (∼5.5 kb amplicons), which encompass the 18S, ITS1, 5.8S, ITS2, and 28S regions, from both pure cultures of B. cinerea and infected grapevine leaf samples. Minimap2, a sequence alignment tool integrated into the EPI2ME software, served as a taxonomy classifier, utilizing the custom reference database FRODO. The results demonstrate that B. cinerea was detectable when this pathogen was not the dominant fungal species in leaf samples. Additionally, the method facilitates host DNA-free sequencing and might have a good potential to distinguish other pathogenic and non-pathogenic fungal species hosted within grapevine's infected leaves, such as Alternaria alternata, Saccharomyces cerevisiae, Saccharomyces boulardii, Mucor racemosus, and Ascochyta rabie. The sequences were uploaded to the NCBI database. Long amplicon sequencing method has the capacity to be broadened to other susceptible crops and pathogens, as a valuable tool for early grey rot detection and mycobiome research. Future large-scale studies are needed to overcome challenges, such as comprehensive reference databases for complete fungal ribosomal operons for grape mycobiome studies.