Metagenomic sequencing for detection and identification of the boxwood blight pathogen Calonectria pseudonaviculata.

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.

Metagenomic Sequencing for Identification of Xylella fastidiosa from Leaf Samples.

Xylella fastidiosa (Xf) is a globally distributed plant-pathogenic bacterium. The primary control strategy for Xf diseases is eradicating infected plants; therefore, timely and accurate detection is necessary to prevent crop losses and further pathogen dispersal. Conventional Xf diagnostics primarily relies on quantitative PCR (qPCR) assays. However, these methods do not consider new or emerging variants due to pathogen genetic recombination and sensitivity limitations. We developed and tested a metagenomics pipeline using in-house short-read sequencing as a complementary approach for affordable, fast, and highly accurate Xf detection. We used metagenomics to identify Xf to the strain level in single- and mixed-infected plant samples at concentrations as low as 1 pg of bacterial DNA per gram of tissue. We also tested naturally infected samples from various plant species originating from Europe and the United States. We identified Xf subspecies in samples previously considered inconclusive with real-time PCR (quantification cycle [Cq], >35). Overall, we showed the versatility of the pipeline by using different plant hosts and DNA extraction methods. Our pipeline provides taxonomic and functional information for Xf diagnostics without extensive knowledge of the disease. This pipeline demonstrates that metagenomics can be used for early detection of Xf and incorporated as a tool to inform disease management strategies. IMPORTANCE Destructive Xylella fastidiosa (Xf) outbreaks in Europe highlight this pathogen's capacity to expand its host range and geographical distribution. The current disease diagnostic approaches are limited by a multiple-step process, biases to known sequences, and detection limits. We developed a low-cost, user-friendly metagenomic sequencing tool for Xf detection. In less than 3 days, we were able to identify Xf subspecies and strains in field-collected samples. Overall, our pipeline is a diagnostics tool that could be easily extended to other plant-pathogen interactions and implemented for emerging plant threat surveillance.