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     

The plant viruses and viroids database and collections of Q‐bank

The availability of characterised reference isolates of plant pathogens is of crucial importance for research and diagnostic laboratories. The Q‐bank Plant Viruses and Viroids database contains data and information on plant viruses and viroids, with the unique feature that it is linked to specimens present in publicly available physical collections. The Q‐bank database aims to share data and information on the virus and viroid species and their availability between research and diagnostic laboratories. Currently the database focuses on regulated virus species. In future more plant viruses and viroids will be included to provide a comprehensive data and information system. The curators invite virologists worldwide to participate in this international initiative by making their data and isolates available via Q‐bank ( http://www.q-bank.eu ).     

Q‐collect: results of a European Union project to improve the status of reference collections important for plant health

The ability of National Plant Protection Organizations (NPPOs) and their affiliated laboratories to quickly and reliably detect and identify organisms is critical for the effective performance of phytosanitary measures. Plant pest diagnostics is also essential to support the phytosanitary certification of consignments of plants and plant products exported from the EU. Access to reference material has been identified as a limitation on the ability of laboratories to develop and validate diagnostic tests and ensure the reliability of diagnostics. The EU FP7 Q‐collect project worked to establish the state of the art of current phytosanitary collections, to identify gaps and propose minimum quality standards, to facilitate access to specimens and to design and build networks of reference collections. The main results of the project are presented in this paper.     

Euphresco project VirusCollect – fulfilling the need for a common collection of plant viruses and viroids for reference

The availability and accessibility of suitably characterized plant virus and viroid isolates for reference is vital for research and diagnostic laboratories. To ensure the long‐term availability of isolates and reference materials, there is a need for international collaboration. The Euphresco (European Plant Health Research Coordination) project VirusCollect aimed to establish a platform to link collections of viruses and viroids maintained by individual institutions via Q‐bank ( http://www.q-bank.eu/ ), a database on plant pests and diseases. Within the VirusCollect project, standard operating procedures were developed and implemented by the participating laboratories to guarantee the quality of isolates and data. In addition, over 135 virus and viroid isolates of phytosanitary and/or economic importance were added to Q‐bank, which now provides links to over 500 virus isolates of almost 100 species, in addition to basic information on many more plant viruses. VirusCollect has enabled the first step in collaboration between curators and standardization of maintenance of virus collections. The project established the basis for improving the quality of individual collections and the layout of Q‐bank as a platform to share data and information. The follow‐up project, VirusCollect II, enables further international collaboration to ensure future access to reliable collections of plant viruses and viroids.     

QBOL: a new EU project focusing on DNA barcoding of Quarantine organisms

In 2009 a new three year EU funded project (QBOL) started on DNA barcoding of important plant pests. An international consortium of 20 partners (universities, research institutes, and phytosanitary organizations) from around the world, coordinated by Plant Research International (Wageningen, the Netherlands), will collect DNA barcodes from many plant pathogenic quarantine organisms, store these sequences in a database accessible over the internet, develop a DNA bank and train end-users. All these activities should help National Plant Protection Services in the correct identification and detection of plant pathogenic quarantine organisms.     

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.     

DNA barcoding: a new module in New Zealand's plant biosecurity diagnostic toolbox

Molecular methods for identification of high risk pests and pathogens have been employed for more than a decade to supplement standard diagnostic protocols. However, as the volume of traded goods continues to increase so does the breadth of taxa that diagnosticians need to deal with. Keeping pace by introducing more molecular tests that are typically species-group specific is not an efficient way to progress. Since 2005 classical DNA barcoding using cytochrome oxidase I sequence has been employed routinely in New Zealand for the highest risk insect species (fruit flies and lymantriid moths). Subsequently a broader range of pests have been considered. Case studies are presented here for three important lepidopteran pests, Lymantria mathura (pink gypsy moth), Conogethes punctiferalis (yellow peach moth) and Hyphantria cunea (fall web worm), as well as a trial to identify miscellaneous border interceptions. While the data support the effectiveness of DNA barcoding for border diagnostics, they also raise issues around cryptic species identification and potential species discovery that could impact on operational biosecurity systems.     

Development of an EU protocol for the detection and diagnosis of Potato spindle tuber pospiviroid*

The work described here formed part of the EU SMT DIAGPRO project, to develop diagnostic protocols for 18 regulated pests. The Potato spindle tuber pospiviroid (PSTVd) protocol was developed primarily for testing in vitro‐ and glasshouse‐grown potato plants for the purposes of post‐entry quarantine and the production of pathogen‐tested nuclear stock. After a performance audit of methods used by 12 laboratories in Europe and America by ring testing, four methods were chosen for multilaboratory validation. For most laboratories, the detection limits were 10–20 mg of PSTVd‐infective tissue for R‐PAGE; 0.25–0.5 mg for DIG‐probe; 0.062 mg for RT‐PCR; and 0.0155 mg for TaqMan (this was the lowest weight of infective tissue tested). Some laboratories were able to extend the detection limit to 0.0155 mg for DIG‐probe and RT‐PCR. The DIG‐probe and R‐PAGE are recommended as primary detection methods, with confirmation of viroid presence by any of the four validated detection methods. Specific diagnosis requires the viroid to be sequenced. Other methods may be used for primary detection, providing that they preferably detect all PSTVd isolates and other Pospiviroids that have the potential to infect potato, and detect viroid in at least 1/10 of the tissue weight normally tested per plant.     

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.     

世界主要储粮书虱环介导等温扩增试剂盒研发与应用

为利用分子生物学技术快速准确鉴定国内外主要储粮书虱,基于环介导等温扩增 （loop-mediated isothermal amplification, LAMP）技术拟研发一款世界主要储粮书虱LAMP试剂盒,利用该LAMP试剂盒对10种储粮书虱的DNA粗提液进行测试,并结合DNA条形码技术对采自中国山东省和内蒙古自治区粮库的未知种类储粮书虱成虫样品进行鉴定。结果显示,本试验研发的LAMP试剂盒可准确鉴定10种储粮书虱;使用LAMP试剂盒和DNA条形码技术鉴定未知种储粮书虱样品的结果一致,即山东省储粮书虱样品为嗜虫书虱 Liposcelis entomophila,内蒙古自治区储粮书虱样品为啮书虱 L. corrodens,表明本试验研发的世界主要储粮书虱LAMP试剂盒可以快速、精准完成主要储粮书虱的鉴定,且操作便捷,可为海关口岸和基层仓储保护部门书虱鉴定工作提供技术支持。     

Q‐bank,a database with information for identification of plant quarantine plant pest and diseases

This paper describes the database Q‐bank ( www.q-bank.eu ). This freely accessible database contains data on plant pathogenic quarantine organisms to allow fast and reliable identification. Development of accurate identification tools for plant pests is vital to support European Plant Health Policies. Council Directive 2000 ? 29 ?EC lists approximately 300 entries representing a large number of species (e.g. non‐European Tephritids contains approximately 3500 species) for which protective measures, against introduction and their spread within the European Community, need to be taken. The risk of introduction of pests into the European Union is increasing because of the increase in the volumes, commodity types and origins of trade, the introduction of new crops, the continued expansion of the EU, the numbers of international travellers and the impact of climate change. Identifying pests (in particular new emerging pests) requires staff with specialised skills in all disciplines (mycology, bacteriology etc.), which is only possible within large centralised laboratory facilities. Expertise in taxonomy, phytopathology and other fields in plant health, which are vital for sustaining sound public policy on phytosanitary issues, are under threat. Sharing knowledge on regulated and non‐regulated pests is necessary to manage a cost‐effective and efficient plant health system in the context of expanding globalisation of trade in plant material.     

Test performance study of isothermal amplification tests for the detection of Grapevine flavescence dorée phytoplasma and ‘Candidatus Phytoplasma solani’

This test performance study (TPS) was carried out on DNA samples from grapevine, clematis, fungi and bacteria to compare and validate loop‐mediated isothermal amplification (LAMP) tests for detection of Grapevine flavescence dorée phytoplasma and ‘Candidatus Phytoplasma solani’ (Grapevine Bois noir phytoplasma). Two LAMP tests, for Grapevine flavescence dorée phytoplasma and ‘Candidatus Phytoplasma solani’ (as developed by Kogov?ek and colleagues), with proven applicability for rapid laboratory or on‐site detection were included in this study. They were performed in 10 laboratories. In addition, the commercial Qualiplante/Hyris isothermal amplification test for Grapevine flavescence dorée phytoplasma was performed in three laboratories. The accuracy of the three tests was shown to be over 98%. Moreover, the high accuracy of these tests, which used different devices across different laboratories, confirmed their reproducibility.     

Rapid preparation of Fusarium DNA from cereals for diagnostic PCR u sing sonification and an extraction kit

Several PCR methods have recently been developed for Fusarium analysis in pure cultures. To use these new techniques in mycological studies and in industrial quality control, a protocol was set up for the rapid preparation of fungal DNA from cereals. An ultrasonification probe (sonotrode) and a lysis buffer were used for mechanical lysis of mycelia from infected grains. Following ultrasonification, DNA was isolated using a commercially available kit. DNA preparation was completed within 5 min per sample. The method resulted in DNA of sufficient quality and quantity for diagnostic PCR. Group- and species-specific primers were used to detect DNA of Fusarium graminearum and F. culmorum in species-specific assays as well as trichothecene-producing Fusarium spp. in a group-specific system. A minimum of one F. graminearum -infected grain added to an uninfected 40 g wheat sample was detectable with a species-specific PCR. The PCR signals produced with primers specific for the tri5 gene of trichothecene-producing Fusarium spp. and with primers for the detection of F. graminearum (gaoA) were in accordance with corresponding concentrations of deoxynivalenol (DON) found in samples by HPLC analysis. The speed of the protocol developed may promote the use of PCR in routine applications in an agro-industrial context.     

Image‐driven electronic identification keys for invasive plant species in the Netherlands

For several years work has been ongoing to compile information to facilitate the identification of non‐native plants that (potentially) pose a threat to the biodiversity of the ecozone comprising Northern Germany, the Netherlands, Belgium and North‐Western France. Such identification information may also be used for species that are regulated by other countries and are likely to be present as contaminants in commercial exports originating from the Netherlands. This information system comprises a set of interactive image‐driven identification keys for invasive plants at various growth stages for use in a range of different situations: seed contaminants in bird feed, weeds in bonsai plants, seedling identification, identification keys of invasive terrestrial plants and invasive aquatic plants. The advantage of image‐driven identification keys using a multiple entry system lies in limiting misunderstandings in terminology and failure in identification, in the absence of certain characters, as is sometimes the case in dichotomous keys. The interactive keys are linked to the species information in the Q‐bank database, including datasheets, worldwide distribution maps, specimen level information, barcodes of selected species, etc. This information is now available at http://www.q-bank.eu/Plants/     

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.     

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.     

International diagnostic protocols for regulated plant pests

The development and use of international standards for phytosanitary measures provides a framework for establishing actions to protect plant health that are harmonized and science-based. Standards for diagnostic processes are being developed to harmonize detection and identification procedures to enable safe movement and trade of plants and plant products. The aim is to increase the transparency of diagnostic processes and to foster trust and mutual recognition of test results. Diagnostic protocols may also assist in dispute resolution between trading partners and contribute to technical assistance programmes. The Interim Commission on Phytosanitary Measures in 2003 identified the development of international standards for diagnostic procedures for regulated pests as a priority. An ongoing 'Technical Panel on Diagnostic Protocols' was established in 2004 to manage the development of protocols for such pests. The panel was involved in the development of a standard that clarifies the purpose and use of international diagnostic protocols and describes the requirements for them and their development. After consultation the 'Diagnostic Protocols for Regulated Pests' standard (ISPM No. 27) was adopted in 2006 by the Commission on Phytosanitary Measures. Draft diagnostic protocols are in development for over 30 pests, and a draft protocol for Thrips palmi has been sent for international consultation.     

Panel of real‐time PCRs for the multiplexed detection of two tomato‐infecting begomoviruses and their cognate whitefly vector species

A new approach for the simultaneous identification of the viruses and vectors responsible for tomato yellow leaf curl disease (TYLCD) epidemics is presented. A panel of quantitative multiplexed real‐time PCR assays was developed for the sensitive and reliable detection of Tomato yellow leaf curl virus‐Israel (TYLCV‐IL), Tomato leaf curl virus (ToLCV), Bemisia tabaci Middle East Asia Minor 1 species (MEAM1, B biotype) and B. tabaci Mediterranean species (MED, Q biotype) from either plant or whitefly samples. For quality‐assurance purposes, two internal control assays were included in the assay panel for the co‐amplification of solanaceous plant DNA or B. tabaci DNA. All assays were shown to be specific and reproducible. The multiplexed assays were able to reliably detect as few as 10 plasmid copies of TYLCV‐IL, 100 plasmid copies of ToLCV, 500 fg B. tabaci MEAM1 and 300 fg B. tabaci MED DNA. Evaluated methods for routine testing of field‐collected whiteflies are presented, including protocols for processing B. tabaci captured on yellow sticky traps and for bulking of multiple B. tabaci individuals prior to DNA extraction. This work assembles all of the essential features of a validated and quality‐assured diagnostic method for the identification and discrimination of tomato‐infecting begomovirus and B. tabaci vector species in Australia. This flexible panel of assays will facilitate improved quarantine, biosecurity and disease‐management programmes both in Australia and worldwide.     

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).     

From laboratory to point of entry: development and implementation of a loop‐mediated isothermal amplification (LAMP)‐based genetic identification system to prevent introduction of quarantine insect species

BACKGROUND

Rapid genetic on‐site identification methods at points of entry, such as seaports and airports, have the potential to become important tools to prevent the introduction and spread of economically harmful pest species that are unintentionally transported by the global trade of plant commodities. This paper reports the development and evaluation of a loop‐mediated isothermal amplification (LAMP)‐based identification system to prevent introduction of the three most frequently encountered regulated quarantine insect species groups at Swiss borders, Bemisia tabaci, Thrips palmi and several regulated fruit flies of the genera Bactrocera and Zeugodacus.

RESULTS

The LAMP primers were designed to target a fragment of the mitochondrial cytochrome c oxidase subunit I gene and were generated based on publicly available DNA sequences. Laboratory evaluations analysing 282 insect specimens suspected to be quarantine organisms revealed an overall test efficiency of 99%. Additional on‐site evaluation at a point of entry using 37 specimens performed by plant health inspectors with minimal laboratory training resulted in an overall test efficiency of 95%. During both evaluation rounds, there were no false‐positives and the observed false‐negatives were attributable to human‐induced manipulation errors. To overcome the possibility of accidental introduction of pests as a result of rare false‐negative results, samples yielding negative results in the LAMP method were also subjected to DNA barcoding.

CONCLUSION

Our LAMP assays reliably differentiated between the tested regulated and non‐regulated insect species within <1 h. Hence, LAMP assays represent suitable tools for rapid on‐site identification of harmful pests, which might facilitate an accelerated import control process for plant commodities. © 2018 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.