Oomycete plant pathogens threaten global food security, agriculture, and environmental health. Molecular identification of oomycetes typically relies on short high-copy number DNA markers (~500-bp or less), such as ribosomal DNA or mitochondrial cytochrome oxidase subunits, but these markers may lack species- or subspecies-level resolution due to factors like insufficient nucleotide variation or inconsistent amplification. We developed a primer set that amplifies ~92.5% of the cox1-spacer-cox2 region and report a long-read amplicon approach using Oxford Nanopore Technologies to sequence 2.0-2.3 kbp amplicons. Oomycete specificity was tested with positive amplification from 24 oomycete genera and 88 oomycete species obtained from axenic cultures and plant tissue. Tested fungi did not yield amplicons, and environmental sequences were limited to oomycetes, confirming marker specificity. Phylogenetic analysis showed taxonomic resolution consistent with multi-locus and genome-scale datasets. Environmental testing showed direct oomycete species identification from three different systems. Turfgrass samples identified novel taxa of a new Pythium species and a new genus of oomycetes basal to Albugo. Sequencing of 52 diseased corn and sugarcane leaves differentiated Peronosclerospora philippinensis from P. sacchari, resolving a previously difficult taxonomic distinction. Metabarcoding of Florida citrus orchards roots and soils identified seven oomycete species including two major citrus pathogens (Phytophthora palmivora and P. nicotianae). This cox1-spacer-cox2 protocol enables high resolution oomycete species identification directly from environmental samples and is comparable with existing cox1 and cox2 molecular datasets. Overall, the COX1-2 primer set and sequencing approach provides enhanced identification of oomycetes across agricultural and horticultural systems.
Groben et al. (Sun,) studied this question.