Exploring the diversity and distribution of Sclerotinia sclerotiorum in oilseed rape through molecular techniques

Abstract: Sclerotinia sclerotiorum, a fungus with a broad host range, causes plant diseases, while its closely related counterpart, Sclerotinia subarctica, has a more limited host range and prefers colder environments. Sclerotinia stem rot is a disease caused by the fungus, leading to economic losses for farmers. In this study, the aim was to utilize PCR and Sanger sequencing to identify and differentiate various isolates of S. sclerotiorum and S. subarctica obtained from sclerotia collected in eight fields, while also employing nanopore sequencing to examine oilseed rape leaves from three distinct fields for the detection of fungal pathogens. To confirm the identification of S. sclerotiorum or S. subarctica isolates, the ITS regions of ribosomal DNA from the leaves were amplified using the primer pairs ITS1Catta and ITS4ngsUni for targeted amplification, and ITS2AF and ITS2AR for amplification of the rRNA ITS region from the sclerotia. Based on the Sanger sequencing results from sclerotia samples, the study determined that S. sclerotiorum was the identified fungi in all of the samples. Nanopore sequencing was performed on the PCR amplified fungal ITS region from leaves, and the resulting data was analyzed using Kraken2 and UNITE databases. The analysis using Kraken2 and UNITE databases revealed successful identification of fungal sequences, with S. sclerotiorum not detected but other plant-infecting fungi identified. Ascomycota and Saccharomycodes ludwigii were predominant using Kraken2, while Streptophyta and Brassica napus were abundant using UNITE. Molecular-based methods like fungal ITS sequencing are essential for accurate identification of plant-infecting fungi.