Strain level diagnostics of a key forest pathogen from complex environmental DNA samples at scale

Austropuccinia psidii is an extreme broad host range pathogen known to cause myrtle rust disease on 528 species belonging to 76 genera in the Myrtaceae. Many of its hosts are environmentally and culturally important forest species in Australia. Currently, there is only a single strain of the pathogen present in Australia, named the pandemic biotype for its extensive global spread. Additional exotic strains are present in South America and other parts of the world. These strains represent high environmental biosecurity risks to Australia and are included in Australia’s National Priority Plant Pest list. Exotic strains of myrtle rust are indistinguishable from the pandemic strain with the naked eye and require molecular diagnostics to be differentiated.

Here we describe the development and application of a National Diagnostic Protocol for myrtle rust and its exotic strains. We illustrate how smart test and development cycles enabled us to simplify molecular biology processes. This allowed us to develop a multiplexed Nanopore sequence based diagnostic assay with fully automated bioinformatic reporting pipeline of the detected strains. Key to our innovation was the uplift of the original assay from spore and infected leaf samples to complex environmental DNA samples obtained via our state of the art spore-trapping network (www.sporetracker.com.au) in nine Australian Botanic Gardens. Our project demonstrates that it is possible to use sampling networks of complex environmental DNA (e.g. spore-trapping) to monitor pathogen distributions at the strain level and well beyond simple binary pathogen present-absence data. In future, these diagnostic approaches will enable monitoring of any phylogenetically or functionally important genetic locus of any airborne pathogen at scale.

This project highlights that cross-sectorial innovation between academia, government, and industry is key to unlocking step changes in biosecurity. The project was supported by the Australian Government Saving Native Species Program within the Department of Climate Change, Energy, the Environment, and Water (DEECCW).