Long-read DNA sequencing using MinION (Oxford Nanopore) is routinely used at the Ministry for Primary Industries’ Plant Health and Environment Laboratory (PHEL) for plant viral diagnostics. The challenge is to apply this to the much larger genomes of bacterial and fungal pathogens and, further to that, expand the repertoire of diagnostic markers able to detect and distinguish multiple pathogens simultaneously in living plants.
In this project, the researchers will firstly use the MinION for metagenomics, sequencing genetic material across the microbial community within the sample of a plant. This aspect builds on a previous B3 project that discovered diagnostic markers to distinguish between pathogenic and non-pathogenic strains of pseudomonad bacteria, irrespective of their species or strain name.
Pseudomonas can infect a wide variety of fruits, vegetables, and ornamental plants and cause symptoms such as dead buds, spots or blackening of the plant, shoot-tip die-back and inhibition of seed production. A simple PCR test derived from that research is now used routinely by MPI.
In this subsequent project, focus will move to native plants. Microorganisms infecting taonga plants growing overseas remain a largely untapped source of biosecurity knowledge that could provide Māori, MPI, and the Department of Conservation with important information on the potential susceptibility of taonga species in New Zealand.
Material repatriated from sentinel plants growing in Californian botanic gardens will be used, including from karaka, pōhutukawa and kauri shown previously to be infected with the high-risk bacterial pathogen Xylella fastidiosa. Read about a related B3 project using the sentinel plant network.
MinION data from this material will be screened for other key bacterial species that may have also infected those plant samples. For example, the bacterial genera Pantoea and Xanthomonas and the fungi Fusarium and Alternaria which have been identified by MPI-PHEL in border interceptions to be taxa of high diagnostic uncertainty.
The second aspect of the research aims to use MinION to assess RNA-seq as a method to screen the microbial community in a plant for markers of infection, as opposed to the metagenomic DNA markers of pathogen presence. The focus will be on Pantoea in onion as a model system. These bacteria are present in a wide variety of plants and are increasingly intercepted at the border, but it is difficult to distinguish whether strains are dangerous to plants or not.
Pantoea-host infection models that span pathogenic and endophytic interactions will be established, then RNA sequences will be generated from them using MinION to look for differentially, co-expressed genes in pathogens that are shared across hosts as potential biomarkers indicative of plant disease.
The outcome of this project will be a diagnostic package that incorporates developments in nanopore sequencing, biological risk information, and mātauranga Māori from taonga plants. It will underpin aspects of diagnostics that are reliant on genomic understanding of pathogen complexes and the pathogenicity that they display on New Zealand hosts. It will provide the proof of principle for a concurrent identification and classification system for high-risk pathogens detected by new sequencing technology to overcome the limitations of single-locus diagnostic targets or multi-locus assay customization for individual pathogen systems.
Contact Project Leader Sandra Visnovsky: Sandra.Visnovsky@plantandfood.co.nz