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2013 Member consultation on draft ISPMs opened including two diagnostic protocols

2013 Member consultation on draft ISPMs opened including two diagnostic protocols | Diagnostic activities for plant pests | Scoop.it

Two diagnostic protocols have been posted on 2013-07-01 for IPPC member consultation

2004-011: Draft Annex to ISPM 27:2006 - Xanthomonas citri subsp. citri

2006-022: Draft Annex to ISPM 27:2006 - Potato spindle tuber viroid

 

All comments must be submitted by the IPPC contact point using the OCS (http://ocs.ippc.int/index.html). Consult the IPPC website for more information.

 

The consultation ends on the 1st od December 2013

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The EPPO Panels on Diagnostics and the relevant specialized Panels will srutinize these texts and prepare EPPO draft comments if needed. 

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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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EPPO and Diagnostics for plant pests of quarantine significance

EPPO and Diagnostics for plant pests of quarantine significance | Diagnostic activities for plant pests | Scoop.it

 

'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:

http://archives.eppo.int/EPPOStandards/diagnostics.htm

 

On this website, a page dedicated to EPPO activities on diagnostics is also available at: http://www.eppo.int/QUARANTINE/diagnostic_activities.htm

Petter Françoise's insight:

The EPPO Secretariat maintains other scoop.it magazines onPest Risk Analysis (http://www.scoop.it/t/pest-risk-analysis) Pest Alerts (http://www.scoop.it/t/pest-alerts), Invasive Alien Plants (http://www.scoop.it/t/invasive-alien-plants), Video of plant pests (http://www.scoop.it/t/pests-on-videos), Communication on pests (http://www.scoop.it/t/communication-and-citizen-sciences-on-pests-and-invasive-alien-species)

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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.
Petter Françoise's insight:

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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Best Wishes

Best Wishes | Diagnostic activities for plant pests | Scoop.it

LE SECRÉTARIAT DE L’OEPP VOUS SOUHAITE UNE BONNE ANNEE 2015

EPPO SECRETARIAT WISHES YOU A HAPPY NEW YEAR 2015

СЕКРЕТАРИАТ ЕОКЗР ЖЕЛАЕТ ВАМ СЧАСТЛИВОГО 2015 ГОДА

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The Top 10 oomycete pathogens in molecular plant pathology - Kamoun et al. 2014

The Top 10 oomycete pathogens in molecular plant pathology - Kamoun et al. 2014 | Diagnostic activities for plant pests | Scoop.it

Oomycetes form a deep lineage of eukaryotic organisms that includes a large number of plant pathogens which threaten natural and managed ecosystems. We undertook a survey to query the community for their ranking of plant-pathogenic oomycete species based on scientific and economic importance. In total, we received 263 votes from 62 scientists in 15 countries for a total of 33 species. The Top 10 species and their ranking are: (1) Phytophthora infestans; (2, tied) Hyaloperonospora arabidopsidis; (2, tied) Phytophthora ramorum; (4)Phytophthora sojae; (5) Phytophthora capsici; (6) Plasmopara viticola; (7) Phytophthora cinnamomi; (8, tied) Phytophthora parasitica; (8, tied) Pythium ultimum; and (10) Albugo candida. This article provides an introduction to these 10 taxa and a snapshot of current research. We hope that the list will serve as a benchmark for future trends in oomycete research.

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After viruses (2011), fungi (2012), bacteria (2012),  nematodes (2013), now the oomycetes 

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Development and Validation of Real-Time PCR Tests for the Identification of Four Spodoptera Species Van De Vossenberg, & Van Der Straten (2014)

Development and Validation of Real-Time PCR Tests for the Identification of Four Spodoptera Species  Van De Vossenberg, & Van Der Straten (2014) | Diagnostic activities for plant pests | Scoop.it
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:

An EPPO diagnostic protocol on Spodoptera species is in development and includes this real-time PCR test. It will be sent for EPPO member country consultation shortly.

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Detection, Diversity, and Population Dynamics of Waterborne Phytophthora ramorum Populations Eyre & Garbelotto 2014

Detection, Diversity, and Population Dynamics of Waterborne Phytophthora ramorum Populations Eyre & Garbelotto 2014 | Diagnostic activities for plant pests | Scoop.it

Information on detection of P. ramorum 

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An EPPO diagnostic protocol exist on P. ramorum and is considered for revision

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3rd Meeting of the Panel on Diagnostics in Virology and Phytoplasmology

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

read a short report of the last Panel! 

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Tissue-print and squash real-time PCR for direct detection of ‘Candidatus Liberibacter’ species in citrus plants and psyllid vectors - Bertolini et al- 2014

Tissue-print and squash real-time PCR for direct detection of ‘Candidatus Liberibacter’ species in citrus plants and psyllid vectors - Bertolini et al- 2014 | Diagnostic activities for plant pests | Scoop.it

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.

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An EPPO Diagnostic Protocol on HLB is about to be adopted and includes these tests!

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Optimising droplet digital PCR analysis approaches for detection and quantification of bacteria: a case study of fire blight and potato brown rot -Dreo et al 2014

Optimising droplet digital PCR analysis approaches for detection and quantification of bacteria: a case study of fire blight and potato brown rot -Dreo et al 2014 | Diagnostic activities for plant pests | Scoop.it

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.

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Rapid diagnostic detection of plum pox virus in Prunus plants by isothermal AmplifyRP® using reverse transcription-recombinase polymerase amplification Zhang et al 2014

Rapid diagnostic detection of plum pox virus in Prunus plants by isothermal AmplifyRP® using reverse transcription-recombinase polymerase amplification Zhang et al 2014 | Diagnostic activities for plant pests | Scoop.it

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.

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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.
Petter Françoise's insight:

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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Quantification of viable eggs of the potato cyst nematodes (Globodera spp.) using either trehalose or RNA-specific Real-Time PCR Beniers et al 2014

Quantification of viable eggs of the potato cyst nematodes (Globodera spp.) using either trehalose or RNA-specific Real-Time PCR Beniers et al 2014 | Diagnostic activities for plant pests | Scoop.it

Two novel methods for the quantitative estimation of the number of viable eggs of the potato cyst nematodes (Globodera pallida and G. rostochiensis) were tested and compared with visual inspection. One is based on the loss of membrane integrity upon death and uses trehalose (a disaccharide) as a marker, the second test exploits the rapid degeneration of mRNA upon decease with a RNA-specific Real-Time Polymerase Chain Reaction (RT-PCR) assay. The viability of eggs in suspensions with different numbers of eggs was determined morphologically and was compared with both trehalose and elongation-factor-1-alpha (EF1α) mRNA measurements. 

Petter Françoise's insight:

The EPPO Diagnostic Protocol on Globodera species is under revision. The tests described in this article will be propsoed for addition. It is expected that a revised version will be sent for Country Consultation in Spring 2015 

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The Fitness for Purpose of Analytical Methods (2014)

The Fitness for Purpose of Analytical Methods (2014) | Diagnostic activities for plant pests | Scoop.it

This guide was first issued in 1998. Since the release of the first edition, however, there have been many changes in terminology, working practices, reference documents and requirements. This second edition, produced by the Eurachem Method Validation Working Group, forms a thorough revision of the 1998 edition.

This second edition accommodates the main changes in international standards and practice. The new edition also includes notes on some aspects of validation that are specific to qualitative test methods.

Petter Françoise's insight:

EPPO has developed Standards on Quality Assurance including  PM 7/98(2)Specific requirements for laboratories preparing accreditation for a plant pest diagnostic activity

http://onlinelibrary.wiley.com/doi/10.1111/epp.12118/pdf

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Q-collect Workshop for collections and users of biological materia Kleinmachnow, DE, 2014-11-27/2

Q-collect Workshop for collections and users of biological materia  Kleinmachnow, DE, 2014-11-27/2 | Diagnostic activities for plant pests | Scoop.it
Petter Françoise's insight:

Read the web-report of the Q-collect Workshop!

Q-collect is a EU FP7 funded project which started on the 1st of October 2013 and aims to improve the status of reference collections important to plant health

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Detection and quantification of airborne inoculum of Hymenoscyphus pseudoalbidus using real-time PCR assays - Chandelier - et al. 2014

Detection and quantification of airborne inoculum of Hymenoscyphus pseudoalbidus using real-time PCR assays - Chandelier - et al. 2014 | Diagnostic activities for plant pests | Scoop.it

 A method based on real-time polymerase chain reaction (PCR) and the use of rotating-arm spore traps was developed for quantifying airborne Hymenoscyphus pseudoalbidus ascospores. The method was sensitive and reproducible, and the collection efficiency was 10% of the spores present in the air. The temporal ascospore dispersal pattern was studied over 3 years by collecting spores every 15 days for a 24 h air-sampling period during the ash-growing season. The highest production was detected from the end of June to the beginning of September. The overall ascospore production did not differ significantly among stands within a specific year but there were differences from year to year. There was a positive correlation between air temperature and the number of ascospores trapped, with most of the positive samples being observed at temperatures above 12°C. The vertical profile of ascospore dispersal showed a strong decrease in ascospore density within a height of 3 m, regardless of date of collection. An analysis of the spore traps installed at increasing distances from an infected stand showed that most of the ascospores were deposited downwind within 50 m of the stand. These data are discussed in context of the epidemiology of the disease.

Petter Françoise's insight:

A Diagnostic protocol on Hymenoscyphus pseudoalbidus was adopted in 2013

http://onlinelibrary.wiley.com/doi/10.1111/epp.12061/pdf 

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Detection of living Bursaphelenchus xylophilus in wood, using reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) - Leal et al - 2014

Detection of living Bursaphelenchus xylophilus in wood, using reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) - Leal et al - 2014 | Diagnostic activities for plant pests | Scoop.it

Pinewood nematode (PWN), Bursaphelenchus xylophilus, the causal agent of pine wilt disease, is an inhabitant of native pine species of North America, where its presence has minor impact. In contrast, the introduction of this nematode to forests in Asia and Europe has devastated some pine stands and is recognized as a pest of significant phytosanitary concern by the National Plant Protection Organizations of several countries. The ability to detect PWN in internationally traded wood products is crucial to reduce the spread of this organism. Currently, the majority of molecular techniques for the detection of PWN rely on the presence of genomic DNA and thus fail to differentiate between living and dead PWN. The detection of dead nematodes could lead to unnecessary trade disruption. Therefore, accurate techniques for the detection of and differentiation between living and dead PWN are critical. We have developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay, which specifically identifies living PWN in wood by detecting the presence of mRNA encoding an expansin gene as a viability marker. This diagnostic method was found to be more sensitive, faster and less dependent on expensive laboratory equipment than PCR. In addition, unlike PCR, it allows for simple colour detection of amplification products. This method will help resolve disputes over the detection of PWN by clarifying whether it originates from live or dead organisms. Where approved treatments are implemented, unnecessary trade disruption will be avoided, thus protecting market access of wood products from PWN-infested areas.

Petter Françoise's insight:

This test will be evaluated for inclusion in the EPPO Diagnostic Protocol on PWN

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Affordable and reliable plant sap-mediated template preparation for the detection of various phytopathogens by PCR assay - Sahana et al. 2014

Affordable and reliable plant sap-mediated template preparation for the detection of various phytopathogens by PCR assay - Sahana et al. 2014 | Diagnostic activities for plant pests | Scoop.it

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

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Development and Validation of Real-Time PCR Tests for the Identification of Four Spodoptera Species: S. eridania, S. frugiperda, S.littoralis, and S. litura (Lepidoptera: Noctuidae) B.T.L.H. Van De...

Development and Validation of Real-Time PCR Tests for the Identification of Four Spodoptera Species: S. eridania, S. frugiperda, S.littoralis, and S. litura (Lepidoptera: Noctuidae) B.T.L.H. Van De... | Diagnostic activities for plant pests | Scoop.it

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

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New adopted Diagnostic Protocols on Phyllosticta citricarpa (McAlpine) Aa on fruit and Xanthomonas citri subsp. citri | International Plant Protection Convention

New adopted Diagnostic Protocols on Phyllosticta citricarpa (McAlpine) Aa on fruit and Xanthomonas citri subsp. citri | International Plant Protection Convention | Diagnostic activities for plant pests | Scoop.it

Two new IPPC protocols have been adopted. 

EPPO will adjust its dignostic protocols for these pests. 

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