Detection of root borne pathogen causing pea root rot by using minion sequencing

Abstract: Pea (Pisum sativum L.) is the most cultivated pulse in the temperate zone, economically important with high nutritional value (high protein content of 20-30%) and relatively low cost. Root rot is its predominant disease, it is referred to as root rot complex because it involves many pathogens. The most important pathogens of pea root rot are fungi and oomycetes. The aim of this project is to assess the possibility of using root samples with known disease severity index (DSI) to identify pea root rot causing pathogens using a MinION device from Oxford Nanopore technologies. DNA barcoding is the use of a standardized segment or region of the DNA to identify the organism to species level by comparison with a reference library. The ITS region is the barcode sequence marker of fungi. In this study, extracted DNA from pea plant roots of six naturally infected pea fields were sequenced. Two different primer pairs; ITS1Catta & ITS4ngsUni (fungi targeted) and ITS1OO & ITS4ngs (oomycota targeted) were used for the polymerase chain reaction (PCR) amplification. Taxonomic identification was done using Kraken2 bioinformatics tool and UNITE reference database. Organisms from fungal phyla Ascomycota, Basidiomycota and Oomycota were recovered from all the samples, but none of the pea rot root specific pathogens (Sclerotinia sclerotiorum, Pythium ultimum, Thielaviopsis basicola, Fusarium solani, Fusarium oxysporum, Ascochyta pinodella, Aphanomyces euteiches and Rhizoctonia solani) were identified. This project could not validate the use of nanopore sequencing using a MinIon device for the identification of specific pathogens causing pea root rots.