Diagnosis of plant diseases using Nanopore sequencing platform

Reliable detection and identification of plant pathogens are essential for disease control strategies. Diagnostic methods commonly used to detect plant pathogens have limitations such as requirement of prior knowledge of the genome sequence, low sensitivity and are limited in ability to detect several pathogens simultaneously. The development of advanced DNA sequencing technologies has enabled determination of total nucleic acid content in biological samples. The possibility of using the single molecule sequencing platform of Oxford Nanopore, as a general method for diagnosis of plant diseases was examined. It was tested by sequencing DNA or RNA isolated from symptomatic tissues of plants of several families inoculated with known pathogens (e.g., bacteria, viruses, fungi, phytoplasma). Additionally, samples of groups of 200 seeds containing one infected seed of each of two or three pathogens, as well as symptomatic samples with unidentified pathogens were tested. Sequencing results were analyzed with Nanopore data‐analysis tools. In all the inoculated plants the pathogens were identified in real time within one to two hours of running the Nanopore sequencer, and were classified to the species or pathovar level. DNA sequencing or direct RNA sequencing of samples with unidentified disease agents were validated by conventional diagnostic procedures (e.g., PCR, ELISA, Koch test), which supported the results obtained by Nanopore sequencing. The advantages of this technology include: long read lengths, fast run times, portability, low cost and the possibility of use in every laboratory. This study indicates that adoption of the Nanopore platform will be greatly advantageous for routine laboratory diagnosis.