Development and implementation of diagnostic tools for Xylella

BRIGIT Consortium Meetings 2020/21 – Wednesday 2 December

Xylella fastidiosa is an insect-transmitted bacterium that causes economically important diseases in a large number of hosts.

There is considerable variation of symptom development in different hosts and, in some cases, plants can remain asymptomatic for many years and still contribute to the spread of disease.

Some of the diseases caused by Xylella are established in South America and the Southeastern and Western coast regions of the US; the areas affected in Europe in countries like Italy and France have expanded despite efforts to eradicate it.

This Brigit Consortium Meeting addressed issues surrounding diagnostics and presented updates on progress made to build the UK’s capability to prevent the establishment of Xylella and to prepare to respond in case of introduction.

There were presentations from other scientists involved in diagnostics and research including colleagues from countries that, like the UK, do not have Xylella (e.g. Australia and New Zealand) and countries that have been dealing with Xylella for longer (e.g. Brazil) or shorter periods of time (e.g. Italy, France).

Harmonisation of standard laboratory diagnostics in the UK

Xylella detection predominantly relies on DNA based diagnostics.

The laboratories involved in statutory testing in the event of an outbreak worked together to ensure that samples sent to any of the laboratories would return an equivalent result.

Jennifer Cole, Jenny Tomlinson, Adam Bryning, Eleanor Jones (presenter)

Diagnostic preparedness for the threat posed by Xylella in Australia

Xylella species are exotic to Australia and X. fastidiosahas been identified as the nation’s number one plant biosecurity threat.

Work is being done to harmonise and streamline laboratory and point-of-care diagnostics for Xylella that are suitable for use in an Australian environment and for inclusion in an Australian national diagnostic protocol.

Rachel Mann (presenter), Toni Chapman, Luciano Rigano, Pragya Kant, John Webster, Monica Kehoe, Cherie Gambley, Robert Taylor, Fiona Constable, Brendan Rodoni

Diagnostics for Xylella in New Zealand

Xylella fastidiosa is absent from New Zealand and its early detection and accurate identification is fundamental to New Zealand’s biosecurity.

The Plant Health and Environment Laboratory (PHEL) is the national reference plant pest laboratory in New Zealand and is responsible for the identification of new emerging pests and diseases.

Our current diagnostic approach for testing for Xylella fastidiosa and some of our readiness activities to improve diagnostic capability.

Robert Taylor (presenter) and Luciano Rigano

Improving diagnostics for Xylella fastidiosa, a potential threat to UK trees

Xylella fastidiosa has a large host range including broadleaf tree hosts such as oak (Quercus), plane (Platanus) and elm (Ulmus) which have already been identified in Xylella affected regions in North America.

Current diagnostic process relies on DNA extraction from potentially infected plant tissue and sample processing is labour intensive and time-consuming.

In real-time PCR detection trials it was found that the sensitivity of the detection varied between tissue type and tree host.

The aim of this study is to make adjustments to current DNA extraction protocols and real-time assays to reduce sample processing time and improve overall detection sensitivity and efficiency on trees.

Sundeep Kaur (presenter), A. Lewis, C. Gorton, A. Chitty, A. Perez-Sierra, S. van der Linde

A core-genome MLST scheme for source tracing Xylella fastidiosa

The ability to trace to source any infected material in the event of an interception or outbreak can be improved through the use of genomic data.

We have used the sequences of 213 genomes in the public domain to develop a core-genome MLST (cgMLST) scheme for Xylella fastidiosa, involving more than 1,400 gene loci.

This readily-applicable scheme enables typing of future X.f. isolates at a higher resolution than afforded by traditional multilocus sequence typing (MLST), and also provides a perspective on hidden complexities within established sequence types (STs).

Sam McGreig, John Walshaw (presenter), Alan McCluskey, Karen Fraser

Socio-technological innovation for Xylella diagnostics

The development and successful implementation of diagnostic tools and approaches is dependent not only on science, but also on the extent of co-design with end users, social acceptability, implementation strategies, confidence in findings and subsequent policy and practice changes.

We will draw on a large-scale research project in which we previously explored the socio-technological innovation of several novel detection approaches for plant pests and pathogens.

We will then consider how these lessons apply to the development and implementation of diagnostics for Xylella, considering our ongoing research within BRIGIT.

Rehema White (presenter), Mariella Marzano, Althea Davies, Chris Pollard, Glyn Jones

Field deployable technologies for Xylella fastidiosa detection

Surveillance and detection for Xylella fastidiosa relies on targeted laboratory methods however, there are technologies available which have been developed for application in the field.

An assessment of the performance from a range of platforms will be discussed along with the potential for deployment to outbreak sites.

Sioban Ostoja-Starzewska, Jayne Hall, Jenny Tomlinson, Eleanor Jones, Steven Bryce (presenter)

Identification of pathogenicity factors and first detection of Xylella in Colombia

Understanding how Xylella fastidiosa works at a molecular level could lead to the development of targeted treatment plans.

We will describe the work that led to the detection of X. fastidiosa in coffee plants in Colombia, where no serious outbreak of the disease has been reported so far.

Louisse P. Mirabueno (presenter), Valeska Villegas-Escobar, Glyn A. Barrett, Robert W. Jackson, Emma Cascant López, Michelle Hulin

Diagnostics of Xylella fastidiosa in Italy

The talk covers:

  1. The diagnostic workflow officially adopted in Apulia based on serological and molecular approaches carried out at a two-tier level of diagnostic assays by independent laboratories
  2. The latest updates on the diagnostic tools for the detection and identification of Xylella fastidiosa in Apulia including the efforts made in the framework of H2020 projects (POnTE and XF-ACTORS) for the validation of official procedures for X. fastidiosa through inter-laboratory tests

Giuliana Loconsole (presenter)

Current situation in France regarding diagnostics of Xylella fastidiosa

The talk covers:

  1. The French official surveillance and diagnostic process
  2. The evaluation and validation of alternative methods for Xylella fastidiosa detection and identification of the subspecies
  3. An update of the situation in France with a focus on the new outbreak in Occitanie area

Valérie Olivier (presenter), Bruno Legendre, Virginie Juteau, Aurélie Forveille, Christèle Dousset, Sandrine Paillard, Amandine Cunty and Françoise Poliakoff

Diagnostics of Xylella fastidiosa in Brazil

The presence of Xylella fastidiosa (Xf) in citrus plants causing Citrus Variegated Chlorosis (CVC) was firstly reported in Brazil in the 80’s becoming endemic nowadays.

The first diagnosis toll used was based on serology (Dot blot) by polyclonal antibodies.

By the middle of the 90’s the PCR was introduced as a tool for the diagnosis of this pathogen. By 2005’s the qPCR methodology based on TaqMan chemistry was introduced as a golden pattern for Xf diagnosis which has been used until now.

Routine use of qPCR for diagnosis of X. fastidiosa by analysing approximately 1,500 samples / month shown us that is necessary a careful interpretation for the cycle threshold (CT) values exported by the qPCR system, making necessary the use of Multicomponent Plot tolls to release the results.

Helvécio Coletta-Filho (presenter)

Understanding the distribution and colonisation of Xylella fastidiosa in plants

The colonisation rate following infection of a host depends on many factors including the environmental conditions and the host genetics.

The aim of our studies is to understand spread, latency and symptom development in key high-risk plant species for the UK.

This will inform how we approach sampling and interpret results of diagnostic tests.

Adam Bryning, Jenny Cole, Eleanor Jones, A. Lloyd, M. Dickinson, John Elphinstone, Joana Vicente (presenter)

Xylella fastidiosa diagnostics in the United States

Xylella fastidiosa is endemic in some regions of the US; in particular, Pierce disease causes significant damage every year in raisin, table and wine grapevines in California.

In contrast, the subspecies pauca that causes Citrus Variegated Chlorosis is not present in the country and measures are in place to avoid the introduction of this disease.

A range of diagnostic tools are routinely used from observation of external and internal symptoms to molecular methods, and these tools are used to support a ‘Start Clean, Stay Clean’ programme.

Caroline Roper (presenter)