Diagnostic activities for plant pests
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Diagnostic activities for plant pests
Information on diagnostic activities on pests and diseases of plants follow the activities of EPPO Panels on diagnostics
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3rd Workshop for Heads of Laboratories Rome, 2015-09-08/11

3rd Workshop for Heads of Laboratories Rome, 2015-09-08/11 | Diagnostic activities for plant pests | Scoop.it
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View the presentations and read the summary and conclusions of the Workshop

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A real-time TaqMan PCR assay to discriminate between pathotype 1 (D1) and non-pathotype 1 (D1) isolates of Synchytrium endobioticum - Springer

A real-time TaqMan PCR assay to discriminate between pathotype 1 (D1) and non-pathotype 1 (D1) isolates of Synchytrium endobioticum - Springer | Diagnostic activities for plant pests | Scoop.it
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The EPPO Diagnostic Protocol on Synchytrium endobioticum is under revision and will be studied at the next meeting of the PAnel on DIagnostics in Mycology in November 2015.

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Development and Validation of LNA-Based Quantitative Real-Time PCR Assays for Detection and Identification of the Root-Knot Nematode Meloidogyne enterolobii in Complex DNA Backgrounds

Development and Validation of LNA-Based Quantitative Real-Time PCR Assays for Detection and Identification of the Root-Knot Nematode Meloidogyne enterolobii in Complex DNA Backgrounds | Diagnostic activities for plant pests | Scoop.it
Phytopathology, Ahead of Print.
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An EPPO diagnostic protocol on Meloidogyne enterolobii PM 7/103 is under revision.  

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Two revised EPPO diagnostic protocols published in the EPPO Bulletin

Two revised EPPO diagnostic protocols published in the EPPO Bulletin | Diagnostic activities for plant pests | Scoop.it

PM 7/46 (3) Lecanosticta acicola (formerly Mycosphaerella dearnessii),Dothistroma septosporum

PM 7/048 (3) Plenodomus tracheiphilus 

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Two revised EPPO diagnostic protocols published and available freely online.

 

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9th Meeting of the Panel on Diagnostics in Nematology

9th Meeting of the Panel on Diagnostics in Nematology | Diagnostic activities for plant pests | Scoop.it

Read a short report of the 9th Meeting of the Panel on Diagnostics in Nematology 

Petter Françoise's insight:

Keep updated with the EPPO programme of preparation of diagnostic protocols in nematology

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3rd Meeting of the Panel on Diagnostics in Entomology

3rd Meeting of the Panel on Diagnostics in Entomology | Diagnostic activities for plant pests | Scoop.it

Short report of the 3rd Meeting of the Panel on Diagnostics in Entomology. 

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Keep updated with the EPPO programme of preparation of diagnostic protocols in entomology

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Comparison of different PCR tests to identify Monilinia fructicola - Riccioni & Valente 2015

Comparison of different PCR tests to identify Monilinia fructicola - Riccioni & Valente 2015 | Diagnostic activities for plant pests | Scoop.it
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’.
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An EPPO Protoocl on M fructicola is under revision

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Detection of the Quarantine Species Thrips palmi by Loop-Mediated Isothermal Amplification

Detection of the Quarantine Species Thrips palmi by Loop-Mediated Isothermal Amplification | Diagnostic activities for plant pests | Scoop.it
Thrips palmi (from the order Thysanoptera) is a serious insect pest of various crops, including vegetables, fruits and ornamental plants, causing significant economic losses. Its presence constitutes a double threat; not only does T. palmi feed on the plants, it is also a vector for several plant viruses. T. palmi originated in Asia, but has spread to North and Central America, Africa, Oceania and the Caribbean in recent decades. This species has been sporadically noted in Europe and is under quarantine regulation in the European Union. For non-specialists its larval stages are indistinguishable morphologically from another widespread and serious insect pest Frankliniella occidentalis (a non-quarantine species in the European Union) as well as other frequently occurring thrips. In this study, we have developed a loop-mediated isothermal amplification protocol to amplify rDNA regions of T. palmi. The results were consistent whether isolated DNA or crushed insects were used as template, indicating that the DNA isolation step could be omitted. The described method is species-specific and sensitive and provides a rapid diagnostic tool to detect T. palmi in the field.
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An IPPC protocol has been published in this pest 

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LAMP assay and rapid sample preparation method for on-site detection of flavescence dorée phytoplasma in grapevine - Kogovšek - 2014 - Plant Pathology - Wiley Online Library

LAMP assay and rapid sample preparation method for on-site detection of flavescence dorée phytoplasma in grapevine - Kogovšek - 2014 - Plant Pathology - Wiley Online Library | Diagnostic activities for plant pests | Scoop.it
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 LAMP assay was shown to be specific and extremely sensitive, because 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 optimized for on-site detection and can be completed in one hour. The homogenization procedure of the grapevine samples (leaf vein, flower or berry) was optimized to allow direct testing of crude homogenates with the LAMP assay, without the need for DNA extraction, and was shown to be extremely sensitive.
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A revision of the EPPO Protocol on Flavescence dorée is in preparation.

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Evaluation of a real-time PCR and a loop-mediated isothermal amplification for detection of Xanthomonas arboricola pv. pruni in plant tissue samples

Real-time PCR and LAMP were suitable for reliable, sensitive X. arboricola pv. pruni detection.

The first technique was slightly more accurate than LAMP, depending upon sample preparation.

Critical data to support recommendations for practical use and phytosanitary inspection guidelines are provided.
Petter Françoise's insight:

A revision of the EPPO protocol onX. arboricola pv. pruni is in preparation and hsould be presented at the next Panel on Diagnostics in Bacteriology in October 2015

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One-step multiplex quantitative RT-PCR for the simultaneous detection of viroids and phytoplasmas of pome fruit trees Malandraki et al 2015

One-step multiplex quantitative RT-PCR for the simultaneous detection of viroids and phytoplasmas of pome fruit trees Malandraki et al 2015 | Diagnostic activities for plant pests | Scoop.it
A one-step multiplex real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) based on TaqMan chemistry was developed for the simultaneous detection of Pear blister canker viroid and Apple scar skin viroid along with universal detection of phytoplasmas, in pome trees. Total nucleic acids (TNAs) extraction was performed according to a modified CTAB protocol. Primers and TaqMan MGB probes for specific detection of the two viroids were designed in this study, whereas for phytoplasma detection published universal primers and probe were used, with the difference that the later was modified to carry a MGB quencher. The pathogens were detected simultaneously in 10-fold serial dilutions of TNAs from infected plant material into TNAs of healthy plant up to dilutions 10−5 for viroids and 10−4 for phytoplasmas. The multiplex real-time assay was at least 10 times more sensitive than conventional protocols for viroid and phytoplasma detection. Simultaneous detection of the three targets was achieved in composite samples at least up to a ratio of 1:100 triple-infected to healthy tissue, demonstrating that the developed assay has the potential to be used for rapid and massive screening of viroids and phytoplasmas of pome fruit trees in the frame of certification schemes and surveys.
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Welcome to Forest Phytophthoras of the World | Forest Phytophthoras of the World

Welcome to Forest Phytophthoras of the World | Forest Phytophthoras of the World | Diagnostic activities for plant pests | Scoop.it
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New and revised EPPO Standards on Diagnostics available in the December issue of the EPPO Bulletin

New and revised EPPO Standards on Diagnostics available in the December issue of the EPPO Bulletin | Diagnostic activities for plant pests | Scoop.it

PM 7/76 (3) Use of EPPO diagnostic protocols


PM 7/014 (2): Ceratocystis platani


PM 7/85 (2) Plasmopara halstedii

 

PM 7/120 (1) Pseudomonas syringae pv. actinidiae 

 

PM 7/121 (1) ‘Candidatus Liberibacter africanus’, ‘Candidatus Liberibacter americanus’ and ‘Candidatus Liberibacter asiaticus’


PM 7/122 (1) Guidelines for the organization of interlaboratory comparisons by plant pest diagnostic laboratories

 

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Thanks to experts from the EPPO region for their hard work!

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2nd Q-collect project Workshop

2nd Q-collect project Workshop | Diagnostic activities for plant pests | Scoop.it
A Workshop for plant pest collections and for users of biological material -
Rome, 2015-09-08/09
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Read a summary of the meeting and conclusions. 

A white paper on collections was also finalized during this meeting. 

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Detection of Citrus leprosis virus C using specific primers and TaqMan probe in one-step real-time reverse-transcription polymerase chain reaction assays

Detection of Citrus leprosis virus C using specific primers and TaqMan probe in one-step real-time reverse-transcription polymerase chain reaction assays | Diagnostic activities for plant pests | Scoop.it
Citrus leprosis virus C
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LepIntercept: an identification resource for intercepted Lepidoptera larvae

LepIntercept: an identification resource for intercepted Lepidoptera larvae | Diagnostic activities for plant pests | Scoop.it
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A useful ressource!

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Jorge Luiz's curator insight, August 3, 2015 6:27 AM

A useful ressource!

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Improved morphological key to the species of the xylophilus group of the genus Bursaphelenchus Fuchs, 1937 - Braasch & Schönfeld 2015

Improved morphological key to the species of the xylophilus group of the genus Bursaphelenchus Fuchs, 1937 - Braasch & Schönfeld 2015 | Diagnostic activities for plant pests | Scoop.it
Correct identification of the pine wood nematode Bursaphelenchus xylophilus becomes more difficult, as more related or similar species are described. Spread of the nematode is being monitored worldwide, and in view of the fact that several new Bursaphelenchus species have been described, identification keys should be adjusted accordingly. Based on the identification key of the xylophilus group published by Braasch (2008) and the revised intrageneric grouping of Bursaphelenchus by Braasch et al. (2009), an improved dichotome morphological key including newly described species (B. paraluxuriosae, B. populi, B. macromucronatus, B. firmae, B. koreanus and B. gillanii) is presented. The recognition of the quarantine pest B. xylophilus using morphological characteristics in laboratories of the National Plant Protection Services is facilitated by a simplified key
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Detection and identification of Xanthomonas arboricola pv. pruni from symptomless plant material: results of an Italian test performance study - Loreti et al 2015 -

Detection and identification of Xanthomonas arboricola pv. pruni from symptomless plant material: results of an Italian test performance study - Loreti et al 2015 - | Diagnostic activities for plant pests | Scoop.it
anthomonas 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.
Petter Françoise's insight:

The EPPO protocol on Xanthomonas arboricola pv. pruni is under revision

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Detection of PSTVd and TCDVd in seeds of tomato using real-time RT-PCR - Bakker et al. 2015

Detection of PSTVd and TCDVd in seeds of tomato using real-time RT-PCR - Bakker et al. 2015 | Diagnostic activities for plant pests | Scoop.it
Worldwide outbreaks of pospiviroids in potato and tomato have increased the need for a reliable test for the detection of pospiviroids in seeds. This study describes the development and validation of a sensitive and fast test for the detection of Potato spindle tuber viroid (PSTVd) and Tomato chlorotic dwarf viroid (TCDVd) in tomato seeds. The test is based on RNA isolation using a commercial kit and is suitable for routine application. The test is able to detect one PSTVd or TCDVd contaminated seed in sub samples of 1000 seeds and results were both repeatable and reproducible.
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The EPPO Protocol on PSTVd is under revision and a standard on generic detection of pospiviroids is in preparation.

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A quantitative hybridization approach using seventeen DNA markers for identification and clustering analysis of Ralstonia solanacearum - Albuquerque - Plant Pathology - Wiley Online Library

A quantitative hybridization approach using seventeen DNA markers for identification and clustering analysis of Ralstonia solanacearum - Albuquerque - Plant Pathology - Wiley Online Library | Diagnostic activities for plant pests | Scoop.it
Ralstonia solanacearum (Rs) is a quarantine phytopathogenic bacterium accountable for heavy economic losses worldwide. Monitoring and eradication programs required for this pathogen are dependent on the availability of time- and cost-efficient detection and typing methods. However, members of the Rs species complex are characterized by a high phenotypic and genetic diversity, which requires improved diagnostics methods.

The currently available full genome sequences of several Rs strains allow for the selection of novel specific DNA markers using comparative genomics tools. In this work, seventeen novel markers were selected based on Rs-specific protein domains and thoroughly validated for specificity and stability, both in silico and using “wet lab” assays. PCR- and hybridization-based validation assays revealed that the DNA regions selected as markers were unevenly distributed amongst the tested strains, with nine markers present throughout the species complex. The distribution of the remaining eight markers was highly variable between the different analyzed strains and allowed to obtain strain-specific dot blot hybridization patterns particularly informative for typing. The average probability value of each strain being positive for each of the seventeen markers was calculated by an algorithm and used to obtain a dendrogram representing the hierarchical clustering analysis of Rs, according to the similarity of their hybridization patterns. We believe that this method can be a robust contribution for the straightforward genotyping of members from the Rs species complex. Furthermore, this quantitative hybridization approach would allow to construct databases increasingly informative to determine new Rs genotypes and infer epidemiological patterns.
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The EPPO Diagostic protocol for Ralstona solanacearum is currently under revision

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Transmission of ‘Candidatus Liberibacter solanacearum’ in carrot seeds - Bertolini - 2014 - Plant Pathology - Wiley Online Library

Transmission of ‘Candidatus Liberibacter solanacearum’ in carrot seeds - Bertolini - 2014 - Plant Pathology - Wiley Online Library | Diagnostic activities for plant pests | Scoop.it
A protocol for the specific detection and quantification of ‘Candidatus Liberibacter solanacearum’ in carrot seeds using real-time PCR was developed. The bacterium was detected in 23 out of 54 carrot seed lots from 2010 to 2014, including seeds collected from diseased mother plants. The average total number of ‘Ca. L. solanacearum’ cells in individual seeds ranged from 4·8 ± 3·3 to 210 ± 6·7 cells per seed from three seed lots, but using propidium monoazide to target live cells, 95% of the cells in one seed lot were found to be dead. Liberibacter-like cells were observed in the phloem sieve tubes of the seed coat and in the phloem of carrot leaf midrib from seedlings. The bacterium was detected as early as 30 days post-germination, but more consistently after 90 days, in seedlings grown from PCR positive seed lots in an insect-proof P2 level containment greenhouse. Between 12% and 42% of the seedlings from positive seed lots tested positive for ‘Ca. L. solanacearum’. After 150 days, symptoms of proliferation were observed in 12% of seedlings of cv. Maestro. ‘Candidatus Liberibacter solanacearum’ haplotype E was identified in the seeds and seedlings of cv. Maestro. No phytoplasmas were detected in seedlings with symptoms using a real-time assay for universal detection of phytoplasmas. The results show that to prevent the entry and establishment of the bacterium in new areas and its potential spread to other crops, control of ‘Ca. L. solanacearum’ in seed lots is required.
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An EPPO diagnostic protocol on Candidatus Liberibacter solanacearum is in preparation

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Expert Consultation on Draft Diagnostic Protocols (ECDP) - International Plant Protection Convention

This year, experts worldwide are invited to submit comments on five different draft diagnostic protocols (DPs) under the work programme of the technical panel on diagnostic protocols (TPDP). The schedule for the Expert consultation and the tentative list of the draft DPs to comment on are as following:
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If you are an expert on the pests concerned , please participate your input is needed.

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Potential of Ypt1 and ITS gene regions for the detection of Phytophthora species in a lab-on-a-chip DNA hybridization array - König - Plant Pathology - Wiley Online Library

Potential of Ypt1 and ITS gene regions for the detection of Phytophthora species in a lab-on-a-chip DNA hybridization array - König - Plant Pathology - Wiley Online Library | Diagnostic activities for plant pests | Scoop.it

A novel DNA-chip hybridization assay that uses the ras related GTP-binding protein 1 gene (Ypt1) was developed for the identification of several devastating Phytophthora species. The hybridization was conducted in a portable microfluidic lab-on-a-chip (LoC) device for fast and accurate detection of 40 Phytophthora spp., two Pythium and one Phytopythium sp.. Moreover, the functionality of the Ypt1 region was examined in comparison to an array for the internal transcribed spacer (ITS) region by in silico modeling. The difference in species-specific capture probe sequences was lower for the ITS than for the Ypt1 region. While ITS-probes of Phytophthora ramorum (P. ramorum),P. fragariae and P. lateralis cross-reacted with up to 11 non-target species, Ypt1-probes were specific except for P. fragariae/P. rubi. First analyses of artificially inoculated Rhododendron leaves successfully demonstrated the usability of the respective capture probes for theYpt1 and the ras-related plant protein Rab1a gene region. The on-chip hybridization enabled the detection of up to 1 pg μL−1 target DNA depending on the species examined. Due to the complementarity of ITS and Ypt1 genetic features, we recommend the use of multiple loci to identify targets of different taxonomic rank.

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CBD and its Global Taxonomy Initiative is calling for experts to the Training Course on Rapid Identification of Invasive Alien Species | International Plant Protection Convention

CBD and its Global Taxonomy Initiative is calling for experts to the Training Course on Rapid Identification of Invasive Alien Species | International Plant Protection Convention | Diagnostic activities for plant pests | Scoop.it
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