DNA-Barcoding Identification of Plant Pathogens for Disease Diagnostics

Abstract

The accurate identification of plant pathogens is a critical step to prevent their spread and attenuate their impact. Among the wide range of methods available, DNA-barcoding, i.e., the identification of an organism through the PCR amplification and sequencing of a single locus, remains one of the most straightforward and accurate plant-pathogen identification techniques that can be used in a generic molecular biology lab. This chapter provides a detailed protocol for the isolation of genomic DNA of fungal and oomycete pathogens from fresh field samples and the amplification and sequencing of the internal transcribed spacer (ITS) locus for DNA-barcoding purpose. Amendments to the protocol are provided to help in resolving issues related to the analysis of complicated samples and to the lack of species resolution that can be encountered with ITS barcodes.

Keywords: Bioinformatics; DNA-barcode; Fungal pathogen; ITS; Molecular identification; Oomycetes; Phytophthora; Pythium; Translation elongation factor 1-alpha 1 (EF-1α).