Abstract
Pathogen detection and identification are key elements in outbreak control of human, animal, and plant diseases. Since many fungal plant pathogens cause similar symptoms, are difficult to distinguish morphologically, and grow slowly in culture, culture-independent, sequence-based diagnostic methods are desirable. Whole genome metagenomic sequencing has emerged as a promising technique because it can potentially detect any pathogen without culturing and without the need for pathogen-specific probes. However, efficient DNA extraction protocols, computational tools, and sequence databases are required. Here we applied metagenomic sequencing with the Oxford Nanopore Technologies MinION to the detection of the fungus Calonectria pseudonaviculata, the causal agent of boxwood (Buxus spp.) blight disease. Two DNA extraction protocols, several DNA purification kits, and various computational tools were tested. All DNA extraction methods and purification kits provided sufficient quantity and quality of DNA. Several bioinformatics tools for taxonomic identification were found suitable to assign sequencing reads to the pathogen with an extremely low false positive rate. Over 9% of total reads were identified as C. pseudonaviculata in a severely diseased sample and identification at strain-level resolution was approached as the number of sequencing reads was increased. We discuss how metagenomic sequencing could be implemented in routine plant disease diagnostics.
Highlights
The sooner a disease outbreak is detected and the causative agent is identified, the faster the outbreak can be controlled by implementing testing, quarantine, and isolation
While we focused on Calonectria pseudonaviculata (Cps) and boxwood, the developed approach should be adaptable to most fungal pathogens of most plants and contribute to the improvement of plant disease diagnostics for outbreak control in general
The rationale was that sonication can be expected to maximize the DNA of microorganisms that are separated from the host plant and should minimize contaminating plant DNA, whereas homogenization in liquid nitrogen efficiently frees DNA from all cells and can be expected to increase fungal DNA yield while increasing plant DNA contamination
Summary
The sooner a disease outbreak is detected and the causative agent is identified, the faster the outbreak can be controlled by implementing testing, quarantine, and isolation. Other molecular methods are based on Loop-mediated isothermal amplification (LAMP) and have been shown to exhibit high specificity for pure cultures These assays can discriminate between the target pathogen and closely related species that may be present in the rhizosphere with no falsepositive results. Next-generation sequencing (NGS) using Illumina technology has been used to identify Cps as the pathogen causing Sarcococca blight This method was able to identify Calonectria at the species-rank, but only after DNA was obtained from pure fungal cultures[10]. Whole genome metagenomic sequencing is a promising new approach for pathogen detection and identification for disease diagnosis[11,12] This culture-independent method consists in sequencing all DNA or RNA present in a sample, for example from a symptomatic host, and has been shown to provide accurate diagnosis. This portable sequencer has been used for metagenomic sequencing in medical research to successfully detect and sequence pathogens like Ebolavirus[16] and SARS-CoV-217
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