'Diagnostic activities for plant pests' is maintained by the Secretariat of the European and Mediterranean Plant Protection Organization (EPPO) and its aim is to share information on diagnostics.
EPPO is an intergovernmental organization created in 1951 which currently has 50 member countries. EPPO is responsible for harmonization and cooperation among the National Plant Protection Organizations (official authorities) of its member countries. EPPO helps its members in their efforts to protect plant health in agriculture, forestry and the uncultivated environment (standard-setting activities and exchange of information).
EPPO has established a programme on diagnostics since 1992
view the approved diagnostic protocols on the EPPO official website:
Plant pathogens like ‘Ca. Liberibacter’, phytoplasma, viruses and viroids cause diseases to almost all economically important plants. A simple and fast sap-mediated polymerase chain reaction (PCR) method for the detection of these pathogens infecting various plant hosts is described in the present study. Sap from selected plants was drawn aseptically on parafilm, from the mid-rib of young leaves. Depending on the type of host plant, sap was diluted to optimal concentration before PCR analysis. ‘Ca. Liberibacter’, Citrus tristeza virus (CTV) and Citrus exocortis viroid (CEVd) infecting citrus, and ‘Ca. Phytoplasma’ infecting pepper and sandal trees were tested by sap-mediated PCR. The reliability of this procedure was evaluated by comparing the findings with previously described protocols. The sap-mediated nucleic acid template preparation for PCR assay is devoid of laborious nucleic acid extraction and expensive chemicals. Hence the present method is rapid, economical and so can be employed for diagnosis of large number of plant samples
The genus Spodoptera comprises 31 species, 4 of which are listed as quarantine pests for the European Union: Spodoptera eridania (Cramer), Spodoptera frugiperda (Smith), Spodoptera littoralis (Boisduval), and Spodoptera litura (F.). In international trade, the earlier life stages (eggs and larvae) are being intercepted at point of inspection most frequently, challenging the possibilities of morphological identification. To realize a rapid and reliable identification for all stages, we developed and validated four simplex real-time polymerase chain reaction identification tests based on the mitochondrial cytochrome b gene using dual-labeled hydrolysis probes. Method validation on dilutions of extracted DNA of the target organisms showed that low levels of template (up to 0.2–100 pg) can reliably be identified. No cross-reactivity was observed with 14 nontarget Spodoptera and 5 non-Spodoptera species in the specific Spodoptera tests. The tests showed to be repeatable, reproducible (both 100%), and robust. The new Spodoptera tests have proven to be suitable tools for routine identification of all life stages of S. eridania, S. frugiperda, S. littoralis, and S. litura.
Petter Françoise's insight:
A diagnostic protocol on Spodoptera species is in development in EPPO
The first droplet-digital-PCR-based absolute quantification of “flavescence dorée” phytoplasma, agent of a quarantine phytoplasma yellows in grapevine was performed. Quantitative PCR that targets the secY gene was transferred to droplet digital PCR and used for absolute quantification of “flavescence dorée” phytoplasma, without the need for calibration curves. The sensitivity of the two assays compares well and was shown that it could be used for quantification and quality control of DNA based on in-house reference materials typically used in diagnostics and metrological laboratories. This new tool has great potential for monitoring phytoplasma kinetics, such as there relationship with the progress of an infection, and variations of the phytoplasma titer through the season and screening plants for resistance.
Petter Françoise's insight:
The EPPO standards PM 7/79(1) Grapevine flavescence dorée phytoplasma is under revision.
A new symptomatology was observed in celery (Apium graveolens) in Villena, Spain in 2008. Symptomatology included an abnormal amount of shoots per plant and curled stems. These vegetative disorders were associated with ‘Candidatus Liberibacter solanacearum’ and not with phytoplasmas. Samples from plant sap were immobilized on membranes based on the spot procedure and tested using a newly developed real-time polymerase chain reaction assay to detect ‘Ca. L. solanacearum’. Then, a test kit was developed and validated by intralaboratory assays with an accuracy of 100%. Bacterial-like cells with typical morphology of ‘Ca. Liberibacter’ were observed using electron microscopy in celery plant tissues. A fifth haplotype of ‘Ca. L. solanacearum’, named E, was identified in celery and in carrot after analyzing partial sequences of 16S and 50S ribosomal RNA genes. From our results, celery (family Apiaceae) can be listed as a new natural host of this emerging bacterium.
Petter Françoise's insight:
An EPPO Diagnostic protocol is in preparation for ‘Ca. L. solanacearum’.
Following clearance by the Standards Committee (SC), four draft diagnostic protocols are sent for member consultation by the IPPC Secretariat to contracting parties, national plant protection organizations (NPPOs), regional plant protection organizations (RPPOs) and international organizations.
Comments must be submitted through the IPPC contact point in the IPPC
In Europe the most devastating phytoplasma associated with grapevine yellows (GY) diseases is a quarantine pest flavescence dorée (FDp) from the 16SrV taxonomic group. The on-site detection of FDp with an affordable device would contribute to faster and more efficient decisions on the control measures for FDp. Therefore, a real-time isothermal LAMP assay for detection of FDp was validated according to the EPPO standards and MIQE guidelines. The FD LAMP assay was shown to be specific and extremely sensitive, since it detected FDp in all leaf samples that were determined to be FDp infected using quantitative real-time PCR. The whole procedure of sample preparation and testing was designed and optimised for on-site detection and can be completed in one hour. The homogenisation procedure of the grapevine samples (leaf vein, flower or berry) was optimised to allow direct testing of the crude homogenates with the LAMP assay, without the need for a DNA extraction, and was shown to be extremely sensitive.
The National Plant Diagnostic Network (NPDN) has developed into a critical component of the plant biosecurity infrastructure of the United States. The vision set forth in 2002 for a distributed but coordinated system of plant diagnostic laboratories at land grant universities and state departments of agriculture has been realized. NPDN, in concept and in practice, has become a model for cooperation among the public and private entities necessary to protect our natural and agricultural plant resources. Aggregated into five regional networks, NPDN laboratories upload diagnostic data records into a National Data Repository at Purdue University. By facilitating early detection and providing triage and surge support during plant disease outbreaks and arthropod pest infestations, NPDN has become an important partner among federal, state, and local plant protection agencies and with the industries that support plant protection.
A sensitive and reliable detection of R. solanacearum strains of different phylotypes.
Loop-mediated isothermal amplification (LAMP) assays that target 16S rRNA, fliC and egl genes were compared and evaluated as on-site applications. The assay with the best performance was that targeted to the egl gene, which shows high analytical specificity for diverse strains of the betaproteobacterium Ralstonia solanacearum, including its non-European and non-race 3 biovar 2 strains. The additional melting curve analysis provides confirmation of the test results. According to our extensive assessment, theegl LAMP assay requires minimum sample preparation (a few minutes of boiling) for the identification of pure cultures and ooze from symptomatic material, and it can also be used in a high-throughput format in the laboratory.
Petter Françoise's insight:
This test will be included in an interlaboratory comparison organized in the framework of the EUPHRESCO project Ralsto-ID
Seven pairs of primers (SSR38, SSR58, SSR114, SSR198, SSR206, SSR211 and SSR212) were found to bind only to the genome of H. pseudoalbidus, but not to the genome of H. albidus or to 52 different fungal endophytes isolated from F. excelsior and F. angustifolia. Using these seven primer pairs, H. pseudoalbidus was identified in fruiting bodies and different types of ash tissues including dead leaves, dead petioles and discoloured or non-discoloured wood. Along one twig, H. pseudoalbidus was detected at different levels of intensity, which depended on the distance from symptomatic tissue. The detection limit was 0.9–1.8 pg of genomic DNA per PCR. Of 50 analysed commercially available seedlings, six were infected with H. pseudoalbidus. Two SSR loci (SSR198 and SSR211) showed fragment length polymorphism. Our results showed that the new primers not only provide an easy and inexpensive means of detecting H. pseudoalbidus in ash tissues, but can also provide information on the genetic heterogeneity of the species.
Petter Françoise's insight:
An EPPO DP on Hymenoschyphus pseudoalbidus was approved in 2013
A new root-knot nematode parasitising vegetables, flowers and fruits in Brazil, Iran and Chile, is described as Meloidogyne luci n. sp. The female has an oval to squarish perineal pattern with a low to moderately high dorsal arc and without shoulders, similar to M. ethiopica. The female stylet is robust and 15-16 μm long; the distance from the dorsal pharyngeal gland orifice to the stylet base (DGO) is 3-4 μm. Males have a high, rounded head cap continuous with the body contour. The labial disc is fused with the medial lips to form an elongated lip structure. The head region is not marked by incomplete annulations. Male stylet robust, 20.8-23.0 μm long with rounded knobs; the DGO is 2.5-4.5 μm. The stylet of second-stage juveniles (J2) is 12.0-13.5 μm long and the DGO to the stylet base is 2.3-3.3 μm. The J2 tail is conoid with finely rounded terminus and is 40.0-48.5 μm long. Biochemically, the esterase phenotype L3 (Rm: 1.05, 1.10, 1.25) is unique and is the most useful character to differentiate M. luci n. sp. from all other Meloidogyne species. Reproduction is by mitotic parthenogenesis (2n = 42-46 chromosomes). In a differential host test, the population from Lavandula spica, Caxias do Sul, RS State, Brazil, reproduced on tomato cv. Rutgers, tobacco cv. NC95 and pepper cv. California Wonder. No reproduction occurred on watermelon cv. Charleston Gray, cotton cv. Deltapine 61 or peanut cv. Florunner. In Neighbour-Joining analyses of ITS and D2-D3 rRNA sequences, populations of M. luci n. sp. from Brazil, Chile and Iran clustered together and were clearly separated from other Meloidogyne spp., thus confirming that all three populations are very similar and conspecific.
Huanglongbing (HLB) disease is seriously threatening and/or damaging the citrus industry worldwide. Accurate detection of the three species associated with HLB disease, ‘Candidatus Liberibacter asiaticus’, ‘Candidatus Liberibacter africanus’ and ‘Candidatus Liberibacter americanus’, is essential for the preventive control of the disease. Real-time PCR is a useful tool for bacterial detection. However, nucleic acid purification steps limit the number of samples that can be processed by PCR. Universal detection of ‘Ca. Liberibacter’ species was achieved by direct tissue-printing and spotting of plant leaf petiole extracts or squashing of individual psyllids onto paper or nylon membranes. Primers were designed and used with TaqMan chemistry for accurate detection of the bacterium in immobilized targets (prints of 10 overlapping leaf pedicels per tree, or squashed single vectors), by extraction with water and direct use for real-time PCR. This simplified method was validated and could detect HLB-liberibacters in 100% of leaves with symptoms and 59% of symptomless leaves collected from HLB-infected trees. The use of direct assays as template showed good agreement with use of purified DNA (κ = 0·76 ± 0·052). The squash assay allowed detection of the bacterium in 40% of mature Diaphorina citri that fed on leaves of HLB-infected trees with or without symptoms. A commercial ready-made kit based on this technology showed 96% accuracy in intra-laboratory performance studies. The simplified direct methods of sample preparation presented herein can be effectively adopted for use in rapid screening of HLB agents in extensive surveys, certification schemes or for epidemiological and research studies.
Petter Françoise's insight:
An EPPO Diagnostic Protocol on HLB is about to be adopted and includes these tests!
Here we report on the first assessment of droplet digital PCR (ddPCR) for detection and absolute quantification of two quarantine plant pathogenic bacteria that infect many species of the Rosaceae and Solanaceae families: Erwinia amylovora and Ralstonia solanacearum. An open-source R script was written for the ddPCR data analysis. Analysis of a set of samples with known health status aided the assessment and selection of different threshold settings (QuantaSoft analysis, definetherain pipeline and manual threshold), which led to optimal diagnostic specificity. The interpretation of theE. amylovora ddPCR was straightforward, and the analysis approach had little influence on the final results and the concentrations determined. The sensitivity and linear range were similar to those for real-time PCR (qPCR), for the analysis of both bacterial suspensions and plant material, making ddPCR a viable choice when both detection and quantification are desired. With the R. solanacearum ddPCR, the use of a high global threshold was necessary to exclude false-positive reactions that are sometimes observed in healthy plant material. ddPCR significantly improved the analytical sensitivity over that of qPCR, and improved the detection of low concentrations of R. solanacearum in potato tuber samples. Accurate and rapid absolute quantification of both of these bacteria in pure culture was achieved by direct ddPCR. Our data confirm the suitability of these ddPCR assays for routine detection and quantification of plant pathogens and for preparation of defined in-house reference materials with known target concentrations.
In this study, the innovative development of two AmplifyRP® tests is reported for a rapid isothermal detection of PPV using reverse transcription-recombinase polymerase amplification. In an AmplifyRP® test, all specific recombination and amplification reactions occur at a constant temperature without thermal cycling and the test results are either recorded in real-time with a portable fluorescence reader or displayed using a lateral flow strip contained inside an amplicon detection chamber. The major improvement of this assay is that the entire test from sample preparation to result can be completed in as little as 20 min and can be performed easily both in laboratories and in the field. The results from this study demonstrated the ability of the AmplifyRP® technique to detect all nine PPV strains (An, C, CR, D, EA, M, Rec, T, or W). Among the economic benefits to pathogen surveys is the higher sensitivity of the AmplifyRP® to detect PPV when compared to the conventional ELISA and ImmunoStrip® assays. This is the first report describing the use of such an innovative technique to detect rapidly plant viruses affecting perennial crops.
The specific objective of this pilot study was to assess if the thrips specimens processed to be observed under scanning electron microscopy (SEM) can be further utilized for DNA based assays. Larvae and adults of S. dorsalis were subjected to traditional morphological identification using high resolution SEM prior to their DNA extraction. Sequence results of both mtCO1 and ITS rDNA of individual larva and adult S. dorsalis were found to be in agreement with the taxonomic identification conducted using SEM and each result confirmed the other technique. Our results suggest that steps involved during specimen preparation and observation under SEM does not impact DNA analysis of the sample. The two techniques together could be used for the correct identification of various thrips species using the same specimen. The proof of concept was further tested on three other important thrips species Frankliniella occidentalis (Pergande), F. schultzei(Trybom) and Thrips palmi Karny, which confirmed the robustness of the technique.
The Technical Panel on Diagnostic Protocol (TPDP) met from 07 to 12 July 2014 at the European and Mediterranean Plant Protection Organization (EPPO) Headquarters in Paris, France.
The meeting’s agenda was designed to cover the revision of 7 draft diagnostic protocols (DPs) which were submitted to the expert consultation on IPP this year, discussion of the status of all 31 DPs on the work programme and the TPDP working procedures and related documents, such as the Instruction to Authors. The TPDP also started to develop a gap and a SWOT analysis.
The panel thanked all the DP authors involved in the drafting groups. During the revision of the DPs, the meeting had the presence of two authors: Mr Dominique COLLINS (UK) for draft DP on Genus Liriomyza (2006-017) and Mr Thomas PRIOR (UK) for draft DP on Xiphinema americanumm (2004-025).
The meeting achieved several important outcomes and specific plans for this and the upcoming year. In looking toward 2015, the TPDP expects that 8 draft DPs will be sent out for member consultation, divided in the two consultation periods, and 5 draft DPs to the SC for approval for adoption. It was noted that 4 draft DPs are currently under member consultation and 2 draft DPs under theDP notification period.
More information on the TPDP activities can be found here.
The ectoparasitic dagger nematodes Xiphinema index and X. diversicaudatum, often at low numbers in the soil, are vectors of grapevine nepoviruses, which cause huge agronomical problems for the vineyard industry. This study reports a method, based on real-time PCR, for the specific detection of these species and of the closely related non-vector species X. vuittenezi and X. italiae. Specific primers and TaqMan probes were designed from the ribosomal DNA internal transcribed spacer 1 (ITS1), enabling the specific detection of single individuals of each of the X. index, X. diversicaudatum, X. italiae and X. vuittenezi species from the general nematode population. This specificity of detection and absence of false positive reaction was confirmed in samples of each species mixed with the three otherXiphinema species or mixed with nematodes representative from other genera (non-plant-parasitic Dorylaimida, Longidorus sp.,Meloidogyne spp., Globodera spp. and Pratylenchus sp.). The method was shown to be valid for the relative quantification of X. indexnumbers through its use, from crude nematode extracts of soil samples, in a greenhouse assay of grapevine accessions ranging from highly susceptible to resistant. As an alternative to time-consuming microscopic identification and counting, this real-time PCR method will provide a fast, sensitive and reliable diagnostic and relative quantification technique for X. index nematodes extracted from fields or controlled conditions.
Knowing the population structure of a pathogen is fundamental for developing reliable phytosanitary legislation, detection techniques, and control strategies based on the actual aggressiveness and distribution of the pathogen. Currently, four populations of Pseudomonas syringae pv. actinidiae (Psa) have been described: Psa 1, Psa 2, Psa 3 and Psa 4. However, diagnostic assays specific for Psa populations do not detect Psa 4, the less virulent (LV) strains isolated in New Zealand. Similarly, multilocus sequence typing (MLST) of housekeeping genes, or broad Psa strain genome comparisons, revealed that Psa 4-LV strains clustered separately from other Psa populations. In order to examine whether the placement of Psa 4 in the pathovar actinidiae was appropriate, various tests were carried out. It was shown that the Psa 4-LV strains induced leaf and shoot wilting in Prunus cerasus, extensive necrotic lesions in Capsicum annuum fruits, and no significant symptoms in Actinidia deliciosa. Moreover, repetitive-sequence PCR fingerprinting, type III secretion system effector protein genes detection and colony morphology clearly indicated the distinctiveness of Psa 4-LV strains from the other three Psa populations. Rep-PCR molecular typing revealed a high similarity of the Psa-LV strains with members of P. avellanae species. The Psa-LV strains, most probably, belong to a new, still unnamed pathovar. In summary, it was concluded that the Psa 4-less virulent strains isolated in New Zealand do not belong to the pathovar actinidiae, and, consequently, three Psa populations pathogenic to Actinidiaspp. should currently include: Psa 1, Psa 2 and Psa 3.
Petter Françoise's insight:
A Diagnostic Protocol on Pseudomonas syringae pv. actinidiae is in preparation and will be presented for adoption in September 2014. A copy of this draft is available upon request to firstname.lastname@example.org
Citrus Huanglongbing (HLB) is the most devastating bacterial citrus disease worldwide. Three Candidatus Liberibacter species are associated with different forms of the disease: Candidatus Liberibacter asiaticus, Candidatus Liberibacter americanus and Candidatus Liberibacter africanus. Amongst them, Candidatus Liberibacter asiaticus is the most widespread and economically important. These Gram-negative bacterial plant pathogens are phloem-limited and vectored by citrus psyllids. The current management strategy of HLB is based on early and accurate detection of Candidatus Liberibacter asiaticus in both citrus plants and vector insects. Nowadays, real time PCR is the method of choice for this task, mainly because of its sensitivity and reliability. However, this methodology has several drawbacks, namely high equipment costs, the need for highly trained personnel, the time required to conduct the whole process, and the difficulty in carrying out the detection reactions in field conditions.
A real-time TaqMan RT-PCR assay was developed for the rapid and sensitive detection of Tomato ringspot virus (ToRSV), an important plant virus which infects a wide range of fruit and ornamental crops. Primers and a probe were designed based on the highly conserved 3′-untranslated region (UTR) sequences of ToRSV, to amplify a 182 bp fragment of this region of RNA-1 and RNA-2. The assay was demonstrated to reliably amplify all ToRSV isolates tested. The detection limit was estimated to be about 12 copies of the ToRSV target region. No amplification was observed from the RNA of other nepoviruses or healthy host species. A comparison with a published conventional RT-PCR and a SYBR-based qRT-PCR indicated that both of the published assays lacked reliability and sensitivity, as neither were able to amplify all ToRSV isolates tested, and both were approximately 1000 times less sensitive than the novel TaqMan real-time assay. This TaqMan real-time assay was tested using four different reagent kits and was shown to be robust and stable, with no significant differences in sensitivity between kits. It is expected that the implementation of this TaqMan real-time RT-PCR assay will facilitate efficient phytosanitary certification of nursery stock requiring testing for ToRSV by regulatory agencies, and will also have wider uses for the general detection of ToRSV in a range of hosts.
Petter Françoise's insight:
An EPPO DP PM 7/49(1)Tomato ringspot nepovirus was adopted in 2004 and is under revision
The yam nematode, Scutellonema bradys, which can cause dry rot disease of yam (Dioscorea spp.), was recorded for the first time from Costa Rica in four species of yam occurring in the Atlantic and north regions. Morphometric measurements from two populations from each region using ten female and 11 male characters corresponded with previous descriptions of this species. Canonical discriminant analysis of the female morphometric data separated the populations by region, whereas no separation by region was evident using the male data. Analysis of DNA sequences from the ITS region indicated that populations from Costa Rica were monophyletic with S. bradys from West Africa and clearly distinct from other Scutellonema species. No genetic separation by geographic region or Dioscorea species host was observed between Costa Rica populations. Species-specific primers were developed from the ITS region and supported the identity of 17 populations from 15 locations in Costa Rica as S. bradys: 14 populations from D. alata (greater or water yam) and one each from D. trifida (white yampee), D. cayenensis (yellow yam) and D. rotundata (white yam). Yam production in Costa Rica began in the Atlantic region, where the yam nematode was likely introduced from the Caribbean, progressively spreading to other locations through the use of infected vegetative planting material.