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Improved detection of Xanthomonas fragariae and X. arboricola pv fragariae in UK strawberry imports - PH0309


Xanthomonas fragariae (Xf) is a quarantine pathogen of strawberries, causing angular leaf spot. It has apparently spread within the EU in recent years. The UK remains free of the pathogen, but recent interceptions suggest there are increased risks to UK production. Although there is no detailed cost benefit analysis, the costs of maintaining freedom will almost certainly be less than the costs of having to control the disease once present. Xf is a particularly difficult disease to diagnose since it is extremely difficult to culture on agar media. The pathogen is also difficult to detect, especially when present in asymptomatic latent infections. Xanthomonas arboricola pv. fragariae (Xaf) is a recently described pathogen causing a new disease of strawberry, bacterial leaf blight . Currently it is known only from Italy. The risks to UK production are not known. Diagnosis is not regarded to be as difficult as for Xf.

Current methods for diagnosis of Xf have been validated in a recent EC DIAGPRO project but they are still regarded as being unreliable especially for latent infections. CSL has recently pioneered real-time PCR technology for simultaneous detection, identification and quantification of a wide range of plant pathogens, including bacteria. This second generation PCR technology has revolutionised routine diagnostics. Such assays are more robust, more cost-effective and are readily translated into high-throughput screening tests. This proposed project thus aims to develop and fully validate real-time PCR methods for reliable detection and differentiation of Xf and Xaf. The overall aim will be to providing reliable diagnostic tools which will assist in excluding both pathogens, thus protecting the UK-free status. The assays will also be used to assess the risks of introduction in imported planting material. Additional research is also planned to examine the pathogenesis of Xaf so that Defra can be alerted to the risks of entry of this new pathogen and and the potential threat it poses to UK production, relevant to that posed by Xf, can be more effectively assessed.

The problem
Xanthomonas fragariae causes bacterial angular leaf spot of strawberry and is listed as a quarantine organism in the EPPO region (A2) and in EU plant health legislation (Council Directive 2000/29/EC Annex IIAII). It is a potentially serious and insidious disease, first reported in the USA (Kennedy and King, 1962a). Natural hosts are Fragaria chiloensis cultivars and hybrids, F.vesca and F.virginiana. From the USA it probably spread, in infected planting material, to Australia, New Zealand and Brazil. By the late 1980’s, it had spread to several European countries including Greece, Italy and Portugal. There have been few reports of serious losses but further findings of the disease, or interceptions of the pathogen in planting material, in other EU member states (including Belgium, France, Germany, The Netherlands, Spain and Switzerland) have raised awareness of the pathogen in recent years. The UK is considered free although there have been several potential interceptions. In some cases these were not confirmed because of the difficulties of detecting this insidious pathogen.

Xanthomonas arboricola pv. fragariae causes bacterial leaf blight of strawberry. It is a newly described bacterial pathogen of strawberries of which little is known, especially concerning risks to the UK industry. It has been recently identified in Italy and there is also some evidence that this pathogen may have been confused with X. fragariae elsewhere in Southern Europe.

Several official EU plant health laboratories (including CSL) recently conducted ring tests in an EC-funded project (DIAGPRO) to validate available detection methods for X. fragariae. Whilst several test methods, including immunofluorescence microscopy and polymerase chain reaction (PCR) assays proved useful for diagnosis of symptomatic plants, isolation of the pathogen was unreliable due to its fastidious growth habit. Detection of the pathogen in asymptomatic plant material using the best available methods was also found to be unreliable. The best available methods have been proposed as an EPPO diagnostic protocol, but it is realised that they have limitations.

This project proposes to exploit existing expertise and resources at the Central Science Laboratory (CSL) for the development and delivery of robust and reliable molecular diagnostic methods which can ensure that UK strawberry production is maintained free from both X. fragariae and X arboricola pv. fragariae. The selected methods will be fully validated using plant material infected under quarantine conditions and on healthy production plant material supplied by industry collaborators. Improved speed, robustness, reliability and cost-effectiveness of the new assays will be demonstrated in comparative trials over existing PCR (Roberts et al.,1996; Pooler et al., 1996; Goncalves & Rosato, 2002) and other current EPPO standard methods (DIAGPRO, 2003). The opportunities presented in this proposed project are:
• Development of robust cost-effective methods that will reliably detect and differentiate both pathogens and distinguish them from non-pathogenic Xanthomonas spp.
• Improved understanding of the pathogenicity and biology of both pathogens and increased awareness of the current risks of their introduction in planting material.
• Increased confidence in the maintenance of national freedom from the pathogens, and associated production cost savings in comparison with the cost of general control measures should the pathogen enter and become widely established.

Need for the project
1. Xf is an EPPO A2 listed quarantine organism and is under statutory control in the EU (Directive 2000/29/EC Annex IIAII). As a newly described pathogen, Xaf has been placed on the EPPO Alert List pending a full assessment of the phytosanitary risk it poses.
2. There are significant imports of breeding and propagating material from the EU to the UK. As the pathogen continues to spread on the EU, the risks of entry and establishment increase. Indeed, there have been recent interceptions of Xf in material destined for planting in the UK.
3. Current testing methods are difficult to use reliably, are often difficult to interpret and are fairly costly and slow. They are also severely limited in their ability to detect latent infections which are characteristic of the pathogen on strawberry. Significant investment at CSL has led to the development of real-time PCR technology which has the capacity for highly specific and sensitive detection of plant pathogens with a sample throughput high enough for the routine screening of strawberry plants, even for the low pathogen populations expected in latently infected tissues.
4. Further expertise at CSL in automated CTAB, silica-based and magnetic DNA extraction and purification methods tissues, developed through past research on other strawberry pathogens (including Phytophthora fragariae var. fragariae, P. cactorum and Colletotrichum acutatum), will further facilitate the development of real-time PCR assays.
5. Reliable detection methods will allow for the first time, early-warning of possible pathogen introductions into UK production systems, increased phytosanitary security and protection of the UK-free status.

i) Overall aim of the project

To increase the reliability of excluding Xanthomonas fragariae and Xanthomonas arboricola pv fragariae from the UK production system by developing and validating robust, cost-effective diagnostic methods and improving general knowledge on the current geographic distribution and ease of spread of the pathogens.

ii) Specific objectives

1. Develop real-time PCR assays for both pathogens.
2. Validate these against existing technologies.
3. Establish standard operating procedures for an improved testing service.
4. Carry out some initial pathogenesis studies on Xaf to familiarise symptom development and to form the basis of an initial risk assessment.
Project Documents
• Final Report : Improved detection of Xanthomonas fragariae and X. arboricola pv fragariae in UK strawberry imports   (314k)
Time-Scale and Cost
From: 2004

To: 2006

Cost: £40,000
Contractor / Funded Organisations
Central Science Laboratory
Alien Species              
Plant diseases              
Plant health              
Plants and Animals              
Fields of Study
Plant Health