Diagnosis of plant diseases using the Nanopore sequencing platform

Abstract

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 a restricted 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 tissues with symptoms from 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 samples with symptoms but unidentified pathogens were tested. Sequencing results were analysed with Nanopore data analysis tools. In all the inoculated plants, pathogens were identified in real time within 1–2 h of running the Nanopore sequencer and were classified to the species or genus 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.

Nanopore sequencing for the detection and the identification of Xylella fastidiosa subspecies and sequence types from naturally infected plant material

“…In the last fifteen years, NGS transformed genomic research with an inevitable impact also on diagnostics, taking us to a new scenario. These technologies have recently been applied for the diagnosis of plant pathogens (Bronzato Badial et al , 2018; Chalupowicz et al , 2019) and for the detection and identification of Xf (Bonants et al , 2019). Although several diagnostic methods are available for the detection of Xf (EPPO, 2016b), the identification of subspecies and ST are currently laborious and time-consuming.…”

“…The reason of the low sensitivity of the direct Nanopore sequencing should be addressed to the low quality of the DNA for the tested samples. Nanopore sequencing suffer of low throughput when the DNA used for the sequencing has contaminants and/or when reads are shorts (Chalupowicz et al , 2019). Further optimization of the DNA purification and size selection step may be necessary to maximize the performance of the Nanopore system.…”

“…A combination of whole transcriptome amplification and Nanopore sequencing device has been used to detect ‘ Candidatus Liberibacter asiaticus’ or plum pox virus in plants and insect vectors (Bronzato Badial et al , 2018). The reliability of Nanopore for the diagnosis of several plant pathogen was demonstrated (Chalupowicz et al , 2019) and a rapid MLST determination method was described for ST estimation of Klebsiella pneumoniae isolates by the ONT MinION device (Page and Keane, 2018).…”

High-Throughput Sequencing Application in the Diagnosis and Discovery of Plant-Infecting Viruses in Africa, A Decade Later

“…This unique property of unbiased and hypothesis-free testing of plant sample has been the principal reason behind integrating HTS as a method for simultaneous detection of multiple viruses regardless of their genome nature or structure [ 34 ]. Consequently, a number of HTS protocols targeting nucleic acid pools such as virion-associated nucleic acids, double-stranded RNAs, total RNAs, ribosomal-RNA-depleted RNAs, messenger RNAs, or small interfering RNAs have been developed concurrently with specific bioinformatics workflows for this purpose [ 35 , 36 , 37 , 38 , 39 ].…”

Strain-level identification of bacterial tomato pathogens directly from metagenomic sequences

“…Metagenomic sequencing with the MinION™ has already been used on several crops for identification of various pathogens (Chalupowicz et al 2019) using ONT’s software WIMP (Juul et al 2015) and on wheat to identify various fungal pathogens (Hu et al 2019) using the sequence alignment tool BLASTN (Camacho et al 2009) in combination with custom databases. The MinION™ has also been used for plant pathogen detection and identification starting from extracted RNA or DNA in combination with general or specific primers to increase the quantity of input for the MinION™ (Loit et al 2019; Badial et al 2018).…”