In 2017, Hort Innovation commissioned SARDI to develop protocols for large-scale testing of potato leave, tuber and stem samples for area-wide surveillance of Candidatus Liberibacter solanacearum (CLso), the bacterium that causes ‘zebra chip’ disease in potatoes. A follow-up project in 2019/20 focused on developing a practical tuber sampling protocol that could be used by industry. Potatoes Australia spoke with Danièle Giblot-Ducray about the outcome of the projects.

In February 2017, tomato-potato psyllid (TPP) was detected in Western Australia. TPP is a destructive pest that can cause devastating losses for potato growers and the wider industry, and can vector Candidatus Liberibacter solanacearum (CLso).

Since then, the Western Australian Department of Primary Industries and Regional Development has been monitoring for CLso; fortunately, the bacterium has not been detected.

Early detection of CLso-infected seed lots is important to manage the risk of production losses. TPP can spread the bacterium from low numbers of infected plants to infect whole crops. A practical method of sampling seed lots was needed to support industry manage the risk if incursions are detected.

In response to the Western Australian TPP incursion, Hort Innovation commissioned a series of projects to accurately and reliably detect CLso to support surveillance and certification activities:

• Diagnostic capability to detect Candidatus Liberibacter solanacearum (CLso) (PT17000), undertaken by Agriculture Victoria (featured in the June/July 2019 edition of Potatoes Australia). The project aimed at more accurately detecting CLso, by determining how CLso is distributed in a potato plant and the best part of the plant to test.
• Developing and implementing a high throughput diagnostic test for Candidatus Liberibacter solanacearum (CLso) (PT17000), undertaken by Department of Primary Industries and Regions research division, South Australian Research and Development Institute (SARDI). The project aimed at developing high throughput diagnostics for CLso; that is, large-scale, fast turnaround testing.
• Sampling for Candidatus Liberibacter solanacearum (CLso) (PT19001), also undertaken by SARDI. This project looked at developing a practical tuber sampling protocol. The aim was to deliver a fully operational surveillance capability for CLso.

These projects were strategic levy investments under the Hort Innovation Fresh Potato and Potato Processing Funds.

External assistance

External assistance was key to the success of the SARDI research.

For PT17000, the SARDI project team worked closely with researchers in the United States, who provided potato plant material infected with CLso. The freeze-dried material was imported using the appropriate quarantine channels, and therefore posed no risk to the Australian horticulture industry.

For PT19001, the SARDI team enlisted the help of a group of growers to evaluate the sampling protocol being developed and ensure practicality.

Figure 1. Tuber cores stored on desiccant just after collection (left) and after 7 days (right). Images supplied by SARDI.

Towards large scale testing for CLso

“One of the recommendations from the National Diagnostic Protocol for CLso is to test 60 composites of five leaf midribs from a paddock, glasshouse or seed lot to get an appropriate diagnosis. This is a slow protocol and difficult to scale up,” SARDI project team leader Dr Danièle Giblot-Ducray explained.

One of the features of the SARDI Molecular Diagnostic Centre (MDC) facilities is the ability to process and extract DNA from large samples.

“The aim of the project was to investigate whether our system could be used to scale up testing for CLso by processing large composites, while maintaining the sensitivity of detection,” Dr Giblot-Ducray said.

The Agriculture Victoria and SARDI projects both demonstrated that the best parts of the potato plant to test for CLso detection were specific sections of the stems and tubers and not the leaves (as previously thought). This was a vital finding.

“With the SARDI protocol, we have improved sensitivity 10-fold and can process composite samples of 50 stem or tuber sections. This significantly increases surveillance throughput and reduces costs,” Dr Giblot-Ducray said.

Additionally, SARDI designed a new, more specific DNA test to more accurately detect CLso.

“We recommend that anyone looking to detect CLso use our assay, which is available under MTA to any biosecurity lab in Australia.”

A new diagnostic service for CLso

Dr Giblot-Ducray outlined that before rolling out the new protocol as a service to industry, more work was needed to ensure samples submitted for testing were not deteriorating during transport and remained of a suitable quality for DNA-based testing.

“Collecting tuber samples rather than stem had been industry’s preference. However, tuber tissue is highly prone to rapid decay, so we needed a method to ensure samples arrive at the laboratory in good condition,” Dr Giblot-Ducray said.

With this in mind, the SARDI team investigated a range of preservation methods; calcium carbonate-based desiccant proved to be the most efficient and practical to preserve tuber samples (Figure 1). The team also designed a sampling device to assist with sample collection (Figure 2).

“In order to facilitate uptake by the industry, we needed an easy and efficient system to collect the tuber samples. The industry input was invaluable in helping improve the sampling tool.” Dr Giblot-Ducray added.

Figure 2 (above and below). Customised sampling device for CLso surveillance.

Preparation is key

According to Dr Giblot-Ducray, now that TPP is established in Western Australia, the bacterium is more likely to arrive. Therefore, it is important that Australia has a standardised surveillance system.

“The SARDI lab is now ready to start testing and we are encouraging industry to implement a surveillance program,” Dr Giblot-Ducray said.

“Following an upgrade of the SARDI facilities, we can process composite samples from up to 70 paddocks or seed lots per day if required, which should be more than enough to cope with peak demand from an industry-wide surveillance program or seed certification program.”

Coring devices and sampling instructions are available from SARDI upon request. A video demonstrating the sampling protocol should be available from the Hort Innovation website shortly.

Find out more

Please contact Dr Danièle Giblot-Ducray at daniele.giblot-ducray@sa.gov.au.

These projects have been funded by Hort Innovation using the fresh potato and potato processing research and development levies and contributions from the Australian Government.

Project Numbers: PT17000 and PT19001

This article features in the autumn 2021 edition of Potatoes Australia. Click here to read the full publication.