Detection Of Soil Borne Pathogens Causing Pea Root Rot Using Minion

Abstract: Peas are small spherical seedpod of the pod fruit Pisum sativum. It is of great nutritional value containing vitamins, carbohydrates, proteins, and several minerals. A variety of diseases affects peas through a number of pathogens including insects, viruses, bacteria and fungi. Fungi and fungi like organism (oomycetes) are eukaryotic organisms known to be detrimental to plants. They are inconspicuous due to their small sizes and cryptic lifestyle in soil and are often difficult to detect. DNA barcoding uses sections of a gene to identify unknown species or parts of an organism. The official DNA barcoding marker for specie level identification of fungi, the nuclear ribosomal internal transcribed spacer ITS, has shown to contain less intraspecific variations than interspecific variation making it suitable for most fungal studies. In this study, DNA extracted from six naturally infected pea fields’ samples were amplified using two different primer pairs. ITS1Catta & ITS4ngsUni targeted fungi phylum; Ascomycetes, Zygomycetes and Basidiomycota while ITS1OO & ITS4ngs targeted phylum Oomycota. MinION sequencing was used to read the amplicons. Further taxonomic identification was done using the Kraken2 bioinformatics tool. Several reference databases were explored like UNITE, which contains fungal ITS, NCBI which covers plants, bacteria and fungi. The primer pair ITS1OO & ITS4ngs recovered 4,195 oomycetes reads while the primer pair ITS1Catta & ITS4ngsUni recovered 1983 ascomycetes and 370 basidiomycetes. MinION provides a fast and efficient alternative to current methods of identifying pea root rot causing fungi and oomycetes.