Beyond soil baiting: toward application of an eRNA diagnostic tool for Phytophthora

This project aims to advance environmental RNA (eRNA) sequencing techniques for the rapid identification of live, soil-borne plant pathogens, particularly Phytophthora species.

Labelled the ‘plant-destroyer’, Phytophthora are water moulds that can cause severe disease in a number of important plant species, including kauri, pine, and avocados, amongst others.

As RNA is found in living cells as the link between DNA and the building blocks of proteins, the idea is to extract eRNA directly from soil and use its presence to help diagnosticians determine which Phytophthora in the environment are currently active. RNA is less stable than DNA, degrading more rapidly, in days or weeks compared to years for DNA. Therefore, its detection can give a more biologically relevant snapshot of ‘viable’ populations in an environment.

The project aims to develop a Phytophthora testing procedure as an alternative to soil-baiting, the current ‘go-to’ method for surveillance of Phytophthora in soil. Soil-baiting, a time consuming method, relies on soil samples flooded with water to release zoospores which then infect leaves resting as bait at the surface. Infected baits are then plated on a medium selective for Phytophthora species for the results up to weeks later.

In a biosecurity setting, soil baiting is cumbersome for high risk site surveillance sampling. In addition, not all Phytophthora are culturable introducing the risk of missing some species with unknown biosecurity threat. Developing a repeatable and reliable eRNA protocol would significantly reduce the time from weeks to days, improve throughput capability, and theoretically provide a better representation of the Phytophthora community.

In this project, the team from Scion and Plant & Food Research will build on their previous B3 project ‘eRNA as a molecular diagnostic tool targeting viable Phytophthora spp. and other oomycetes in nurseries’. That work provided the proof-of-concept that high throughput sequencing (eRNA-HTS) could detect viable Phytophthora in soil samples.
The researchers aim to progress this method towards an operational diagnostic tool.

Protocols for extraction of RNA from soil will be optimised, using new methods to overcome the previous laborious approach not suited to rapid diagnostic processing. The high throughput sequencing aspect will also be improved by use of MinION (long-read DNA sequencing) to enable better taxonomic resolution for species-level identification of Phytophthora and other oomycetes and with the potential for portable or in-lab diagnostics. MinION is currently used for virus diagnostics at MPI-PHEL and has been shown to provide rapid turn-around times for diagnostic results.

Like the earlier proof-of-concept project, this will focus on detection of Phytohpthora in Pinus radiata nursery soils, but will extend to new environments.

Contact Project Leader Rebecca McDougal: [email protected].