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Ongoing project

Improving preparedness of the Australian horticultural sector to the threat potentially posed by Xylella fastidiosa (a severe biosecurity risk) (MT17006)

Key research provider: The Victorian Department of Jobs, Precincts and Regions

What’s it all about?

This multi-industry investment will review and allow Australia to adopt world’s best practice methods for detecting and identifying strains of the Xylella fastidiosa bacteria, should it come to our shores. As well as developing state-of-the-art diagnostic tools, technologies and protocols to screen plant material entering the country and to support active surveillance programs, it will provide associated training to technical staff in diagnostic laboratories.

The project’s work will ultimately allow for quick and effective detection of what is considered to be the number one plant biosecurity threat to Australia and New Zealand, to facilitate a swift and sure response.

Further optimisation and validation of assays, laboratory and in-field, to be included in the Australian National Diagnostic Protocol (NDP) for the detection of Xylella species and their subtypes were undertaken in this reporting period, as well as the first draft of an Australian NDP for the detection of Xylella species and their subtypes.

Five Australian and NZ laboratories evaluated the specificity and sensitivity of four qPCR assays and one endpoint PCR assay, and the data supports their inclusion in the NDP. Two assays were designed and developed in this project and provide accurate screening assays for both Xylella species and their subspecies, and sequencing of the endpoint PCR product provides the means to differentiate Xylella taiwanensis (Xt), X. fastidiosa (Xf), and the Xf subspecies, pauca (Xfp), multiplex (Xfm) and fastidiosa (Xff). It was observed that different test reagents (PCR and LAMP) may have a minor effect on the limit of detection and one generic Xylella spp. qPCR assay may give a late false-positive amplification. Provisions to counteract these effects will be written into the NDP, including the use of multiple confirmatory assays.

All seven assays were evaluated against 179 field samples from a broad range of Xf host plants, spanning 24 genera, that were collected from across Australia (134), New Zealand (14) and other parts of the world (31). All assays except one specific Xf qPCR provided 100 per cent specificity using these samples. These results provide confidence in these laboratory and in-field diagnostics assays in an Australian and New Zealand setting and further underpin their recommended inclusion in the Australian NDP.

Key project resource acquisition continued with DNA from a further 16 Xf bacterial isolates extracted, and DNA from Xf-infected and uninfected field samples distributed to Australian laboratories.

More than 100 samples have been sourced from diverse Xf hosts across Australia. These will be used to assess and recommend diagnostic protocols to detect and identify Xf in Australian and New Zealand environments.

Diagnostic PCR assays were developed to detect and differentiate between both Xylella species (Xf and Xt). These assays were evaluated for specificity and sensitivity alongside previously published protocols. In total, 14 PCR assays for detection of Xylella species at different taxonomic levels were evaluated across a diverse panel of 81 samples from the team’s collections.

Final sensitivity data for field-based experiments were also completed, with a final list of five PCR assays for laboratory-based detection and two isothermal assays and Oxford Nanopore Technology (ONT) amplicon sequencing recommended for validation.

The evaluation of high-throughput sequencing approaches for the detection of sub-species and sequence types of Xf directly from plant tissue continued.

This project is focused on diagnostics and diagnostic capability, in preparation for an incursion of Xylella fastidiosa (Xf). The team aims to deliver the following outcomes:

  • Improved, rapid diagnostics for Xylella at the genus, species and subspecies level
  • Clear sampling guidelines for important hosts
  • In-field detection capability, ready for application by field officers
  • Validation of laboratory and field tests in an Australian environment
  • Harmonisation of testing protocols used across Australia and New Zealand via an updated National Diagnostic Protocol (NDP)
  • Ensuring that major node laboratories in Australia and New Zealand have the appropriate resources to test for Xf and are proficient in detection and identification protocols.

In year two of the program, the team reported progress across several areas:

  • Key project resources including cultures, infected plant material, DNA, genomic data and diagnostic targets have been acquired or generated, strengthening the team’s ability to design, evaluate, validate diagnostic protocols for the detection of Xf
  • Evaluation of qPCR assays for detection of Xf and its subspecies has resulted in testing recommendations to begin validation for the NDP
  • Generic isothermal tests for the detection of Xylella spp have been designed and identified via preliminary evaluation. Results indicate that four tests are highly specific to the Xylella
  • An ONT-MLST amplicon sequencing pilot study has generated some exiting results, with this technology to be explored further
  • In-field extraction procedures for use with LAMP assays were recommended and sampling advice was updated

Updates and project progress will continue to be shared with industry as available.

Details

This project is a strategic levy investment in the Hort Innovation Apple and pear, Avocado, Citrus, Cherry, Dried Grape, Nursery, Olive, Prune, Raspberry and Blackberry, Strawberry, Summerfruit and Table Grape Funds.