International standards for the diagnosis of regulated pests

For the last 10 years, the European and Mediterranean Plant Protection Organization (EPPO) has run a European Panel on diagnostics, which has developed regional standards on diagnostic protocols. Nearly 80 such standards have now been approved, and are in active use in EPPO countries. In 2003, the Commission for Phytosanitary Measures (CPM) of FAO, in reviewing global needs for International Standards for Phytosanitary Measures (ISPMs), recognized that there is a strong interest in developing diagnostic protocols for all contracting parties to the International Plant Protection Convention (IPPC). Such protocols would support the harmonization of detection and identification procedures worldwide, contribute to greater transparency and comparability in the diagnostics for regulated pests, and assist in the resolution of disputes between trading partners. In addition, such protocols would be very useful in technical assistance programmes. In 2004, the CPM adopted a mechanism for rapid development of ISPMs in specific areas, particularly suitable for diagnostic protocols. A Technical Panel was accordingly established to develop protocols for specific pests and meets on an annual basis. A format for international diagnostic protocols was adopted in 2006 and a list of priority pests was established. In 2003, EPPO initiated a new programme on quality management and accreditation for plant pest laboratories and Standards are now also being developed in this area. In 2006, a survey of existing diagnostic capacities in EPPO member countries was undertaken and a database on diagnostic expertise was created.     

EPPO database on diagnostic expertise: http://dc.eppo.org

In 2004, the EPPO Council expressed profound concerns about the decreasing expertise in plant protection and declared a state of emergency for Plant Health (‘Madeira declaration’). As diagnostics is one of the scientific fields which are vital for sustaining sound plant health policies, a questionnaire was launched and all EPPO member countries were asked to provide information about their diagnostic expertise, focusing on regulated pests or pests which may present a risk to the EPPO region. In 2006, results of the questionnaire were analysed and compiled by the EPPO Secretariat into a new database. This database now contains detailed information (contact addresses, quality programmes, and accreditations) for 80 diagnostic laboratories from 28 EPPO member countries. More than 500 experts are now registered with details about their diagnostic expertise (pests diagnosed and methods used). The EPPO database on diagnostic expertise can be freely accessed on the Internet: http://dc.eppo.org .     

Comparison of different PCR tests to identify Monilinia fructicola

Monilinia fructicola was until very recently a regulated pest in the European Union, and EU countries were requested to monitor its presence on their territories. As accredited laboratories should use validated tests, the mycological laboratory of CRA‐PAV carried out a validation process for the multiplex based PCR test (Coté et al., 2004 ), that is one of the most widely used tests for the identification of M. fructicola, although this test is not described in the EPPO diagnostic protocol PM 7/18 (2) because the validation data were lacking. The performance characteristics of this multiplex PCR test were established according to the EPPO Standard PM 7/98 (1) and the test was compared in a collaborative study with the end point PCR test (Ioos & Frey, 2000 ), considered as the ‘standard test’. The validation data were obtained using different isolates of M. fructicola, M. laxa, M. fructigena and Monilia polystroma, as well as different fruit tissues. Four series of the DNA target at different concentration, repeated three times, were analyzed in four Italian laboratories. The results showed that the multiplex PCR detection test (Coté et al., 2004 ) was fit for diagnostic purpose, although the analytical sensitivity was significantly lower compared to the conventional PCR ‘standard test’.     

PM 7/29 (3) Tilletia indica

Specific scope

This Standard describes a diagnostic protocol for Tilletia indica. 1 It should be used in conjunction with PM 7/76 Use of EPPO diagnostic protocols.

Specific approval and amendment

This Standard was originally developed under the EU DIAGPRO Project (SMT 4‐CT98‐2252) by a partnership of contractor laboratories and interlaboratory comparison in European countries. First approved as an EPPO Standard in 2003–09. First revision approved in 2007–09. Second revision approved on 2017–11. Although this EPPO Diagnostic Standard differs in terms of format it is in general consistent with the content of the IPPC Standard adopted in 2014 on Tilletia indica (Annex 4 to 2006 ) with the following exceptions. (1) In the EPPO region, as the pest is not present, a higher confidence in the results is required, a sieve wash test should be carried out (optional in the IPPC protocol). (2) When fewer than 10 teliospores are found the options should allow testing the (<10) teliospores with conventional or real‐time PCR (this was not an option in the IPPC protocol flow chart, although it was stated that direct real‐time PCR could be used on individual teliospores in the text). (3) The method for extracting teliospores from untreated seed or grain by size‐selective sieving is slightly different based on the experience in the region (European Union test performance study). The EPPO Diagnostic Standard also includes a test for a direct real‐time PCR for use on pellets (developed in 2016). Some additional information on methods for morphological identification, from the former version of the EPPO Standard, which are not in the IPPC protocol are included in this protocol in Appendix 3 as they were considered useful by the members of the Panel on Diagnostics in Mycology.     

PM 7/87 (2) Ditylenchus destructor and Ditylenchus dipsaci

Specific scope

This Standard describes a diagnostic protocol for Ditylenchus destructor and Ditylenchus dipsaci. 1 This Standard should be used in conjunction with PM 7/76 Use of EPPO diagnostic protocols. Terms used are those in the EPPO Pictorial Glossary of Morphological Terms in Nematology. 2

Specific approval and amendment

Approved in 2008‐09. This revision was prepared on the basis of the IPPC Diagnostic Protocol adopted in 2015 on D. dipsaci and D. destructor (Annex 8 to ISPM 27 Diagnostic protocols for regulated pests). The EPPO Diagnostic Protocol is consistent with the text of the IPPC Standard for morphological identification for this species. For comparison with other species the IPPC table includes Ditylenchus africanus whereas the EPPO table includes Ditylenchus convallariae due to the different distribution of the species. The molecular tests for which there is experience in the EPPO region are described in full in the appendices (some of these are additional tests to those in the IPPC protocol). Reference is given to the IPPC protocol for tests for which there is little experience in the EPPO region. DNA barcoding is also included. Revision approved in 2017‐04.     

Vergleich von Nachweismethoden für Kartoffelzystennematoden (Globodera spp.) im Hinblick auf eine Qualitätssicherung von Diagnosen geregelter Schadorganismen

Testing for PCN is indispensable in EC seed potato production, to avoid a further spread or introduction of this pest. Till now methods for sampling and detection are varying between member states and even within Germany. Therefore in order to obtain comparable results within a new PCN directive the procedure will be regulated by EPPO standard diagnostic protocols, like it is the case for testing Globodera spp. resistance of potato varieties. The Bavarian plant protection service hopes, that beside cyst extraction by flotation the bioassay will be part of the new EPPO standard. The bioassy – in contrast to PCR methods – is well established and allows a reliable discrimination not only of PCN species but also of races and pathotypes. Results of a long-time survey demonstrate clearly, that bioassay and cyst extraction yield comparable results provided quality assurance criteria are met.     

DNA barcoding as an identification tool for selected EU‐regulated plant pests: an international collaborative test performance study among 14 laboratories

DNA barcoding protocols for selected EU‐regulated arthropods, bacteria, fungi, nematodes and phytoplasmas were developed within the Quarantine organisms Barcoding of Life (QBOL) project financed by 7th framework program of the European Union. DNA barcodes generated with the developed protocols were stored in the Q‐bank database. An test performance study (TPS) was set up involving 14 participating laboratories to validate the use of the developed protocols as a diagnostic tool and to identify possible difficulties in the use of the protocols and Q‐bank. This paper describes the steps that were used to set up the TPS, to validate the protocols and to identify difficulties. TPS data shows that the developed tests are very robust and produce highly reproducible results. Participants managed to define good consensus sequences which allowed them to correctly identify their samples using Q‐bank in 78% of all cases. Q‐bank outperformed NCBI and BOLD in terms of diagnostic sensitivity and diagnostic specificity for all organism groups. Using general qualifiers, performance criteria and feedback from TPS participants, difficulties in the set‐up of the TPS, the use of the protocols and databases, and the proficiency of participants were identified/evaluated and recommendations for future work were made. The developed DNA barcoding protocols and Q‐bank have proven to be useful tools in support of the identification of selected EU‐regulated plant pests and pathogens on the desired taxonomical level.     

Detection and identification of Xanthomonas arboricola pv. pruni from symptomless plant material: results of an Italian test performance study

Xanthomonas arboricola pv. pruni, the causal agent of bacterial spot disease of stone fruits, is a regulated quarantine pathogen in the European Union, listed as an A2 pest by the European and Mediterranean Plant Protection Organization (EPPO). Because detection and identification of this pathogen is key for its management and to ensure the production of pest free propagation material, it should be based on reliable tests, in particular when dealing with symptomless material. The current EPPO diagnostic Standard (PM 7/64) does not provide specific molecular methods for detection of this pest. The present paper summarizes the results of a test‐performance study (TPS) to validate, at a national level, a detection procedure for this bacterium. A working group was established in order to evaluate the performance criteria for tests included in the current EPPO Standard, and for a conventional PCR. On the basis of the obtained performance criteria, a diagnostic procedure was elaborated and then applied to perform an inter‐laboratory comparison. Screening tests for the detection of the bacterium on symptomless plant material based on IF and/or PCR were proposed, in parallel with isolation on agar media. For identification two methods were suggested: a molecular test or IF. This paper reports on the results of the TPS and proposes a flow diagram for the detection and identification of X. arboricola pv. pruni.     

Quality control in bioassays used in screening for plant viruses

Test plants are often used for broad screening for plant viruses. Mechanical inoculation of a series of test plants enables generic detection of mechanically transmitted viruses in only 1 assay. Moreover, such an assay is suitable for known as well as unknown viruses and their variants. However, in comparison to serological and molecular methods, quality control in bioassays is almost never addressed. The system of positive and negative controls, blind samples and proficiency tests is applicable, provided that a broader interpretation of positive and negative controls is used. For validation, performance criteria can only be determined for individual viruses. However, results can often be extrapolated. Sensitivity is addressed by dilution and expressed as a relative value. Specificity has to consider the virus species and the plant species to be tested. Selectivity mostly depends on the plant species tested, because some hosts contain components that inhibit transmission. Repeatability and reproducibility, determined for a limited number of samples, appears high, as also substantiated by the authors' experience. This paper details how EPPO Standards on quality control were implemented by the National Plant Protection Organization of the Netherlands (NPPO‐NL). This information will be of use for other laboratories that wish to introduce quality control in bioassays for virus testing.     

PM 7/129 (1) DNA barcoding as an identification tool for a number of regulated pests

Specific scope

This Standard describes the use of DNA barcoding protocols in support of the identification of a number of regulated pests and invasive plant species comparing DNA barcode regions with those deposited in publically available sequence databases. 1 It should be used in conjunction with PM 7/76 Use of EPPO diagnostic protocols.

Specific approval and amendment

2016‐09     

International and regional standards for diagnosis of regulated pests*

For the last 8 years, EPPO has run a European Panel on diagnostics, which has developed regional standards on diagnostic protocols. Nearly 60 such standards have now been approved, and are in active use in EPPO countries. In 2004, the Interim Commission for Phytosanitary Measures (ICPM) of FAO, in reviewing global needs for International Standards for Phytosanitary Measures (ISPMs), recognized that there is a strong interest in developing diagnostic protocols for all IPPC contracting parties. Such protocols would support the harmonization of detection and identification procedures worldwide, contribute to greater transparency and comparability in the diagnostics of regulated pests, and assist in the resolution of disputes between trading partners. In addition, such protocols would constitute a very good material for technical assistance. In 2004, the ICPM adopted a mechanism for more rapid development of ISPMs in specific areas, suitable particularly for diagnostic protocols. A Technical Panel to develop protocols for specific pests was also established and met for the first time in September 2004. A format for international diagnostic protocols was prepared, together with a list of priority pests.     

Validation of morphological keys for identification of Bursaphelenchus xylophilus (Nematoda,Parasitaphelenchidae) to group and species level

Official analyses undertaken in the framework of an official survey, or import controls need reliable results. This can be achieved by using validated methods. For morphological tests, this validation process is rarely illustrated despite guidance provided in EPPO PM 7/98 (1) Specific requirements for laboratories preparing accreditation for a plant pest diagnostic activity. This paper presents validation for the morphological identification of Bursaphelenchus xylophilus, with the evaluation results of published identification keys and of internally designed identification keys at B. xylophilus group and species levels. For published identification keys some criteria were shown not to be reliable for routine use: excretory pore position and number of caudal papillae. The key designed in the laboratory for xylophilus group identification was shown to be sensitive and specific when one male and one female were observed. The key designed for B. xylophilus species identification is sensitive, specific and reproducible if only one female of B. xylophilus is observed. The tools designed were validated as simple and reliable for routine analysis. The advantages and limitations of the validation process for morphological tools are discussed for process improvement.     

Performance of diagnostic tests for the detection and identification of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">actinidiae</Emphasis> (Psa) from woody samples

The aim of this study was to characterise the performance of new molecular methods for the detection and identification of Pseudomonas syringae pv. actinidiae (Psa) and to provide validation data in comparison to the assays mentioned in official diagnostic protocols and being currently used. Eleven molecular tests for the Psa detection were compared in an inter-laboratory comparison where each laboratory had to analyse the same panel of samples consisting of thirteen Psa-spiked kiwifruit wood extracts. Laboratories had to perform also isolation from the wood extracts. Data from this interlaboratory test performance study (TPS) was statistically analysed to assess the performance of each method. In order to provide complete validation data, both for detection and identification, this TPS was supplemented by a further study of identification from pure culture of phylogenetically closely related Pseudomonas spp., Psa, and bacterial strains associated with kiwifruit. The results of both these studies showed that simplex-PCRs gave good results, whereas duplex-PCR and real-time PCR were the most reliable tools for detection and identification of Psa. Nested and multiplex-PCR gave false-positive results. The use of the most reliable detection test is suggested for routine analyses, but when Psa-free status needs to be accurately assessed, it is recommended that at least two detection tests are used. This work provides a wide comparison of the available diagnostic methods, giving new information for a possible revision of the official diagnostic protocols (e.g. European and Mediterranean Plant Protection Organization (EPPO) protocol PM7/120 for the detection of Psa).     

Diagnostic methods for phytopathogenic bacteria of stone fruits and nuts in COST 873

Different diagnostic methods used or developed in the EU-COST ² 873 project 'Bacterial diseases of stone fruits and nuts' are presented. The methods concern detection and identification of the plant pathogenic bacteria Xylella fastidiosa (EPPO A1 list), Xanthomonas arboricola pv corylina , X.a . pv. pruni , Pseudomonas syringae pv. persicae (A2 list pathogens), Agrobacterium tumefaciens , Brenneria nigrifluens and B. rubrifaciens , P. amygdali , P. avellanae , P.s. pv . avii , P.s. pv. morsprunorum , P.s . pv. syringae , X.a . pv. juglandis . Furthermore, a recently described xanthomonas species (proposed name X. translucens pv . pistachiae ), causing a new disease on pistachio, viz. Pistachio decline, in Australia and the recently renamed Xanthomonas citri pv. mangiferaeindicae on mango are included. The methods range from classical ones such as nutritional tests, use of (semi-)selective media, PCR, fatty acid analysis, serology and pathogenicity tests as well as (more) modern ones such as free flow capillary electrophoresis, real-time PCR, rep-PCR, fAFLP and sequencing of open reading frames (ORFs) and/or housekeeping genes such as gyrB and rpoD . The usefulness of these methods are outlined and reference made to publications where they were successfully used. Development of useful (molecular) tools are also indicated. Whole genome sequencing has been performed for Pantoea agglomerans (a relevant biocontrol agent) by the Swiss laboratory and initiated for X. a . pv. pruni by the French laboratory in cooperation with the Swiss and Italian laboratories and development of a microarray test has been initiated by the Swiss laboratory, Details of meetings and training programmes throughout the region are elaborated.     

Results and experiences from the first EU proficiency test for the detection of Phytophthora ramorum1

The Crop Protection unit of the Institute for Agricultural and Fisheries Research offers plant disease diagnostic services through its ‘Diagnostic Centre for Plants’ (ILVO‐DCP). ILVO‐DCP has requested accreditation (ISO 17025) for a number of diagnostic detection methods in bacteriology, mycology, entomology and nematology. Accreditation forms an essential part of quality control programs such as ISO17025, which can in part be realized by proving the laboratory's competence in inter‐laboratory proficiency or ring tests. In 2006, ILVO‐DCP organized such a proficiency test for the detection of Phytophthora ramorum. The protocol was developed using the standard ILAC‐G13:2000, and defined rules for participation, sample preparation and transport, communication, fraud prevention and reporting. Eight European diagnostic centres participated in the proficiency test including ILVO‐DCP. Each participant received one set of 10 coded samples, each sample consisting of either leaves or stems that were artificially inoculated with either the target or an alternative organism. Participants could use one or more methods listed in the EPPO diagnostic protocol PM 7/66. They had to report within a specific timeframe and received a detailed report of their performance. The success rate of the proficiency test was 100%. This paper lists some of the experiences gained from organising this type of proficiency test.     

Ensuring validation in diagnostic testing is fit for purpose: a view from the plant virology laboratory

Plant diagnostic laboratories are facing increasing demands for accreditation of their testing activities from both private and public organizations. For diagnostics, validating tests for the intended use is one of the key elements of accreditation. Thus far, validation has mainly focused on tests with a fixed scope, i.e. a test for organism X in matrix Y by method Z, suitable for the analysis of targeted samples. In many laboratories, however, targeted samples only form a minority of the samples. The majority consists of symptomatic (non‐targeted) samples including a huge variety of pest, host and matrix combinations. Since it is impossible to validate all tests used for the analysis of these non‐targeted samples, ensuring the reliability of the final results needs a different approach. This paper describes such an approach for diagnostic testing in plant virology, where the extent of validation of a test is determined based on the risks of a false result within the context of the diagnostic process. Examples illustrate how this risk‐based system approach relies on a tight inter‐relationship of validation, expertise and quality assurance within the diagnostic laboratory.     

Method for honeybee brood feeding tests with insect growth-regulating insecticides

A method is proposed for testing the side-effects of plant protection products on honeybee brood, particularly aimed at products with insect growth-regulating properties. It is intended to complement the EPPO guideline on test methods for evaluating the side-effects of plant protection products on honeybees and to be used in the framework of the EPPO/CoE decision-making scheme on environmental risk assessment.     

Side-effects of 107 pesticides on the whitefly parasitoid Encarsia formosa, studied and evaluated according to EPPO guideline no. 142

The side-effects of plant protection products on beneficial arthropods have been studied by the Netherlands Plant Protection Service since 1974. Laboratory test methods were developed in the context of IOBC/WPRS for Encarsia formosa , a natural enemy of glasshouse whitefly, Trialeurodes vaporariorum. These methods were elaborated by EPPO into a sequential decision-making scheme and published as an official EPPO guideline in 1989. The scheme includes a residual toxicity test on adults, a direct contact test on pupae, a persistence test on adults, all in the laboratory, and a field test. Following this guideline, the Netherlands Plant Protection Service tested and evaluated 107 pesticides at 307 concentrations over 10 years. Test details and complete test results are reported, including the risk assessments according to the EPPO scheme and according to the IOBC/WPRS conventions. These results are further summarized per type of pesticide, kind of test and risk classification. The efficiency of the scheme in classifying pesticide concentrations for risk to E. formosa is analysed. The scheme was found to be reasonably practical and efficient except for pesticides in the range between safe and hazardous. Suggestions for improvement are given. Also the possible need for changing the original IOBC/WPRS-criterion for harmlessness (effect < 50%) is discussed. No reasons for lowering the criterion to 30% were found. The decision-making scheme fits into the general approach for environmental risk assessment of plant protection products, developed recently by EPPO and the Council of Europe.     

EPPO Study on the Risk of Imports of Plants for Planting: description and details of the first outcomes

EPPO member countries requested that a study be conducted to identify and better address the risks presented by the trade of plants for planting, which has led to numerous introductions of pests into the EPPO region in recent years. Concerns were raised about the efficacy of the current plant health systems in place in the EPPO region to deal with the risks presented by plants for planting. The EPPO Study on the Risk of Imports of Plants for Planting was launched by the EPPO Council in 2010. The first part of the Study was completed in spring 2012. It was published as EPPO Technical Document 1061 ( http://www.eppo.int/QUARANTINE/EPPO_Study_on_Plants_for_planting.pdf ). Examples of pest outbreaks in the EPPO region suspected to be caused by international trade of plants for planting were analyzed. This analysis identified characteristics of the pest/crop/trade patterns associated with the risks of importing pests. These characteristics are described as criteria that are intended to be used in a screening process to enable identification of commodities that require an assessment prior to import in the EPPO region. The further elaboration of the screening process is briefly outlined.