Diagnostic activities for plant pests
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A sensitive real-time PCR assay for the detection of the two Melampsora medusae formae speciales on infected poplar leaves - Springer

A sensitive real-time PCR assay for the detection of the two Melampsora medusae formae speciales on infected poplar leaves - Springer | Diagnostic activities for plant pests | Scoop.it

A sensitive real-time PCR assay was developed to detect the presence of M. medusae in poplar leaf samples.

The test developed demonstrated a high sensitivity since it enables the reproducible detection of two M.medusae urediniospore in a mixture of 2 mg of urediniospores (ca 800 000 urediniospores) of other Melampsora species. This new real-time PCR tool should be useful for laboratories in charge of official analyses since it has many advantages over the techniques currently used to monitor this quarantine pathogen in Europe

Petter Françoise's insight:

Melampsora medusae is pest recomended for regulation by EPPO A2 List  

An EPPO standard on Melampsora medusae PM 7/93(1) was adopted in 2009

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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

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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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One-Step Detection of Monilinia fructicola, M. fructigena, and M. laxa on Prunus and Malus by a Multiplex Real-Time PCR Assay | Plant Disease l Guinet et al. 

One-Step Detection of Monilinia fructicola, M. fructigena, and M. laxa on Prunus and Malus by a Multiplex Real-Time PCR Assay | Plant Disease l Guinet et al.  | Diagnostic activities for plant pests | Scoop.it
Empty descBrown rot is an economically important fungal disease affecting stone and pome fruit orchards, as well as harvested fruit during storage and on the market. Monilinia fructicola, M. laxa, and M. fructigena are the main causal agents of this disease and each have a different regulatory status depending on regional regulations. In this study, a new multiplex tool based on real-time polymerase chain reaction was developed to detect the three pathogenic fungi in a single reaction on fruit, twigs, and flowers of Prunus and Malus spp. Species-specific primer-hydrolysis probe combinations were designed to amplify a region located in a previously described MO368 sequenced characterized amplified region marker, and used in a quadruplex format coupled with the 18S Uni universal primer-probe test in order to check the quality of the DNA template. The assay was designed and optimized with the objective to provide high performance values. Experimental data supported its sensitivity, specificity, reproducibility, and robustness. In addition, a set of quality controls was implemented to minimize the risk of false-positive and false-negative results, thus making this new test fit for use in serial analyses and reliable in the framework of official controls.ription
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This standard is currently under revision - PM 7 Monilinia.
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Fast and sensitive on-site isothermal assay (LAMP) for diagnosis and detection of three fruit tree phytoplasmas I De Jonghe et al.

Fast and sensitive on-site isothermal assay (LAMP) for diagnosis and detection of three fruit tree phytoplasmas I De Jonghe et al. | Diagnostic activities for plant pests | Scoop.it

Over the years, real-time PCR outflanked endpoint PCR in phytopathogen diagnostics, mainly because of the increase in sensitivity and timesaving aspects of the technique. However, a time consuming 16S rRNA-based nested PCR method is still the gold standard for phytoplasma diagnosis. This is also the case for phytoplasma detection in Malus, Pyrus and Prunus, the three main host plants of apple proliferation (AP), pear decline (PD) and European stone fruit yellows (ESFY) phytoplasma, respectively. The last decade, loop-mediated isothermal amplification (LAMP) (Notomi et al. 2000) is gaining a lot in significance and is also for phytoplasmas expected to become a widely used reliable diagnostic tool. High specificity and sensitivity which also requires a less stringent need for DNA purification, and the short analysis time and the limited equipment requirements makes the LAMP method a fast and affordable alternative with great point-of-care diagnostic potential. In this paper, we present a LAMP primer set for the ribosomal group 16SrX, containing the important fruit tree phytoplasmas AP, PD and ESFY. The primers were developed and validated for fast and sensitive detection and general use for diagnosis. We foresee that the LAMP technique will also have its application in on-site diagnosis of the fruit tree phytoplasmas during inspections and surveys.

Petter Françoise's insight:
A diagnostic protocol on AP, PD and ESFY was recently adopted and about to be published. The addition of this test will be considered at the next meeting of the Panel on Diagnostics in Virology and Phytoplasmology.
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Euphresco strategic Agenda 2017-2022 published

Euphresco strategic Agenda 2017-2022 published | Diagnostic activities for plant pests | Scoop.it
Euphresco is a network of organisations funding research projects and coordinating national research in the phytosanitary area. The overall goal of Euphresco is to support coordination and collaboration in the area of phytosanitary research, and to become a strong, long-term network of funders that fully incorporate existing and new members.

Euphresco members are presented here
Petter Françoise's insight:
Read in particular Euphresco strategic Priority 5: Find your enemy new diagnostic technologies in plant health
the following objectives have been agreed:
to understand the biological significance of a positive molecular diagnosis
to develop and validate high-throughput DNA extraction methods 
to understand mixed infections through metagenomic analysis 
to test and validate the use NGS (e.g. whole genome sequencing, metagenomics, deep sequencing, typing by sequencing) for routine diagnostics
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Successful organization of the NIB Proficiency Test Round 2016-02 in Plant Health

Successful organization of the NIB Proficiency Test Round 2016-02 in Plant Health | Diagnostic activities for plant pests | Scoop.it
Successful organization of the NIB Proficiency Test Round 2016-02 in Plant Health
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Thanks to the laboratories in the EPPO region that organize proficiancy tests and test performance studies
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Tecia solannivora also found in Asturias

Tecia solannivora also found in Asturias | Diagnostic activities for plant pests | Scoop.it
La polilla guatemalteca, que llegó en 2015 a Galicia desde Canarias y sólo actúa a nivel del mar, ha sido detectado en 31 municipios gallegos y 11 asturianos, que critican la inacción del Gobierno central

Via Anne-Sophie Roy
Petter Françoise's insight:
EPPO has developed a diagnostic protocol for this pest

PM 7/72 


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Anne-Sophie Roy's curator insight, March 1, 6:08 AM
In mainland Spain, after Galicia, Tecia solanivora has recently been found in Asturias.
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Assessment of the genetic diversity of Xylella fastidiosa in imported ornamental Coffea arabica plants Bergsma-Vlami et al. 2017

Assessment of the genetic diversity of Xylella fastidiosa in imported ornamental Coffea arabica plants Bergsma-Vlami et al. 2017 | Diagnostic activities for plant pests | Scoop.it

A study has been performed in order to assess the presence of Xylella fastidiosa in imported ornamental plants, among them Olea europeae, Coffea arabica, and Nerium oleander.

Petter Françoise's insight:
A diagnostic protocol has been developed - PM7/24.
http://onlinelibrary.wiley.com/doi/10.1111/epp.12327/epdf
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Isolation of Xylella fastidiosa from plant stem tissues - PONTEPROJECT

Isolation of Xylella fastidiosa from plant stem tissues - PONTEPROJECT | Diagnostic activities for plant pests | Scoop.it
A video showing the procedure to isolate Xylella fastidiosa from host plant stem tissues. The video was made in the laboratories of CNR-IPSP, Bari, Italy.
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Another video for the isolation of Xylella fastidiosa from plant material (see previous soop it for the one prepared by Anses (FR) 
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Isolation of the bacterium Xylella fastidiosa from plant samples Anses LSV Angers

This video presents the preparation of plant samples for the isolation of Xylella fastidiosa and gives some tips.
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EPPO Bulletin - Volume 46, Issue 3 - December 2016 - Wiley Online Library

EPPO Bulletin - Volume 46, Issue 3 - December 2016 - Wiley Online Library | Diagnostic activities for plant pests | Scoop.it
EPPO Standards – Diagnostics – were published in December 2016: 

PM 7/127 (1) Acidovorax citrulli 

PM 7/128 (1) Xanthomonas axonopodis pv. allii 

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

PM 7/130 (1) Guidelines on the authorization of laboratories to perform diagnostic activities for regulated pests 

PM 7/24 (2) Xylella fastidiosa 

Addendum - PM 7/4 (3) Bursaphelenchus xylophilus
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EPPO Standards – Diagnostics – published in December 2016
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Rapid loop-mediated isothermal amplification assays for grapevine yellows phytoplasmas on crude leaf-vein homogenate has the same performance as qPCR

Rapid loop-mediated isothermal amplification assays for grapevine yellows phytoplasmas on crude leaf-vein homogenate has the same performance as qPCR | Diagnostic activities for plant pests | Scoop.it
A fluorescence-based real-time loop-mediated isothermal amplification (LAMP) assay for ‘Candidatus Phytoplasama solani’ (Bois noir phytoplasma; BNp) detection was developed and optimised for rapid lab
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10th Meeting of the Panel on Diagnostics in Nematology

10th Meeting of the Panel on Diagnostics in Nematology | Diagnostic activities for plant pests | Scoop.it
Read a summary on the main issues discussed during the last meeting of the EPPO Panel on Diagnostics in Nematology
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Euphresco project VirusCollect – fulfilling the need for a common collection of plant viruses and viroids for reference l Roenhorst et al. 

Euphresco project VirusCollect – fulfilling the need for a common collection of plant viruses and viroids for reference l Roenhorst et al.  | Diagnostic activities for plant pests | Scoop.it

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.

Petter Françoise's insight:
Availability of reference material is an issue in plant health. The Euphresco project is a useful initiative in virology and complementary to the EU funded project Q-collect which ended in 2015.
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Further Characterization of Genetically Distinct Groups of Acidovorax citrulli Strains | Phytopathology l Zivanovic & Walcott

Further Characterization of Genetically Distinct Groups of Acidovorax citrulli Strains | Phytopathology l Zivanovic & Walcott | Diagnostic activities for plant pests | Scoop.it
Bacterial fruit blotch of cucurbits (BFB) is caused by the gram-negative bacterium Acidovorax citrulli, whose populations can be distinguished into two genetically distinct groups, I and II. Based on visual assessment of BFB severity on cucurbit seedlings and fruit after inoculation under greenhouse conditions, group I A. citrulli strains have been reported to be moderately to highly virulent on several cucurbit hosts, whereas group II strains have exhibited high virulence on watermelon but low virulence on other cucurbits. Additionally, group I strains are recovered from a range of cucurbit hosts, while group II strains are predominantly found on watermelon. The goal of this research was to develop tools to characterize and rapidly distinguish group I and II A. citrulli strains.
Petter Françoise's insight:
A diagnostic protocol has been developed - PM7 Acidovorax citrulli.
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Development of a TaqMan probe-based insulated isothermal PCR (TiiPCR) for the detection of Acidovorax citrulli, the bacterial pathogen of watermelon fruit blotch l Wu et al.

Development of a TaqMan probe-based insulated isothermal PCR (TiiPCR) for the detection of Acidovorax citrulli, the bacterial pathogen of watermelon fruit blotch l Wu et al. | Diagnostic activities for plant pests | Scoop.it

Watermelon (Citrullus lanatus) is an important crop of the Cucurbitaceae family in fruit production worldwide. During its production, bacterial fruit blotch (BFB) caused by Acidovorax citrulli (Acidovorax avenae subsp. citrulli) is an important limiting factor on the volume and value of crops. This pathogen is known as a seed-borne pathogen, and the infested seeds can be a primary source of inoculum. Hence, a rapid and sensitive method for detecting A. citrulli on seeds would be an important tool in the management of BFB. In this study, we sought to develop a method to detect A. citrulli bacterial cells based on a TaqMan probe-based insulated isothermal PCR (TiiPCR) assay. Firstly, the specific primers and probe were designed based on a specific DNA fragment from the genome of A. citrulli. Then, PCR amplification was performed with the plasmid DNA to adjust the components of the PCR reagents, such as the concentrations of primers, magnesium chloride, and Taq DNA polymerase. Results revealed that 10 copies of plasmid DNA were detectable within the modified reagents by TiiPCR. Moreover, 10 bacterial cells in each reaction tube were detectable at a 100 % detection rate in this condition with a fluorescent signal intensification over 1.8. Based on these results, we concluded that a specific, rapid, and sensitive method based on TiiPCR had been successfully developed to detect bacterial cells of A. citrulli.

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A diagnostic protocol for Acidovorax citrulli was adopted in 2016.
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Development and validation of a simplified morphological identification key for larvae of tephritid species most commonly intercepted at import in Europe l Balmès & Mouttet 2017

Fruit flies (Diptera, Tephritidae) are amongst the most important pests on fruits and vegetables worldwide. All non-European Tephritidae are listed in Annex I/A1 of Directive 2000/29/EC but only a limited number of species are regularly intercepted in Europe. This study presents a morphological identification key, supported by molecular analysis, for the third-instar larvae of 10 commonly intercepted taxa. The key proved to be sensitive and specific. However, the evaluation of its reproducibility emphasizes the importance of the operator's taxonomic expertise and questions the relevance of criteria under which a morphological method can be validated. This morphological identification key provides a rapid and economic diagnostic tool for identifying tephritid larvae. It is not intended to cover all species but allows for an initial diagnosis to be made. Given the current difficulty in identifying most immature stages of insects, the approach presented here could be used to develop some morphological identification tools for other species of concern. 

Petter Françoise's insight:
Several diagnostic protocols on fruit flies have been published by EPPO:
PM7/104 Ceratitis capitata 
PM7/105 Ceratitis cosyra 
PM7/107 Rhagoletis completa 
PM7/114 Bactrocera zonata 

They are all freely available at https://gd.eppo.int/standards/PM7/.

Two diagnostic protocols on Dacus ciliatus and Zeugodacus cucurbitae have recenty been sent for country consultation, and the EPPO Secretariat hopes that it will be possible to finalize them for adoption this year.
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PCR Multiplexes Discriminate Fusarium Symbionts of Invasive Euwallacea Ambrosia Beetles that Inflict Damage on Numerous Tree Species Throughout the United States | Plant Disease | Short et al. 

PCR Multiplexes Discriminate Fusarium Symbionts of Invasive Euwallacea Ambrosia Beetles that Inflict Damage on Numerous Tree Species Throughout the United States | Plant Disease | Short et al.  | Diagnostic activities for plant pests | Scoop.it
Asian Euwallacea ambrosia beetles vector Fusarium mutualists. The ambrosial fusaria are all members of the ambrosia Fusarium clade (AFC) within the Fusarium solani species complex (FSSC). Several Euwallacea–Fusarium mutualists have been introduced into nonnative regions and have caused varying degrees of damage to orchard, landscape, and forest trees. Knowledge of symbiont fidelity is limited by current identification methods, which typically requires analysis of DNA sequence data from beetles and the symbionts cultured from their oral mycangia. Here, polymerase chain reaction (PCR)-based diagnostic tools were developed to identify the six Fusarium symbionts of exotic Euwallacea spp. currently known within the United States. Whole-genome sequences were generated for representatives of six AFC species plus F. ambrosium and aligned to the annotated genome of F. euwallaceae. Taxon-specific primer-annealing sites were identified that rapidly distinguish the AFC species currently within the United States. PCR specificity, reliability, and sensitivity were validated using a panel of 72 Fusarium isolates, including 47 reference cultures. Culture-independent multiplex assays accurately identified two AFC fusaria using DNA isolated from heads of their respective beetle partners. The PCR assays were used to show that Euwallacea validus is exclusively associated with AF-4 throughout its sampled range within eastern North America. The rapid assay supports federal and state agency efforts to monitor spread of these invasive pests and mitigate further introductions.
Petter Françoise's insight:
Euwallacea fornicatus sensu lato and Fusarium euwallaceae have recently been added to the EPPO A2 list of pests recommended for regulation.
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Several subspecies and sequence types are associated with the emergence of Xylella fastidiosa in natural settings in France Denance et al. 2017

Several subspecies and sequence types are associated with the emergence of Xylella fastidiosa in natural settings in France Denance et al. 2017 | Diagnostic activities for plant pests | Scoop.it

Xylella fastidiosa is a plant pathogenic bacterium emerging in Europe. In France its emergence has been evidenced through interceptions of contaminated coffee plants and in 2015 by the survey of natural settings. The first French contaminated focus was detected in 2015 in Corsica; then almost 300 foci and nearly 30 plant species were declared contaminated, with Polygala myrtifolia remaining the principal host suffering from severe leaf scorches. We report on the diversity of X. fastidiosa identified in France in 2015. Multilocus sequence analysis/typing revealed the presence of mainly X. fastidiosa subsp. multiplex ST6 and ST7. A focus of subspecies pauca ST53 was identified in mainland France; one sample contaminated by X. fastidiosa subsp. sandyi ST76, one novel recombinant, and co-infections of different isolates in individual samples were also identified, but could not be confirmed by successive samplings indicating limited or transient contaminations. Koch's postulates were fulfilled for two isolates of X. fastidiosa subsp. multiplex on P. myrtifolia one being ST6 and the other ST7. Comparative genomics of the genome sequences of three French isolates (one ST6 and two ST7), with available sequences revealed that unlike the American Dixon strain, the French ST6 and ST7 strains are devoid of a plasmid encoding a complete type IV secretion system. Other differences regarding phage sequences were highlighted. Altogether, our results suggest that the emergence of X. fastidiosa in France is linked to several introduction events of diverse strains from different subspecies.

Petter Françoise's insight:
A diagnostic protocol has been developed - PM 7/24. 
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A Framework for the Evaluation of Biosecurity, Commercial, Regulatory, and Scientific Impacts of Plant Viruses and Viroids Identified by NGS Technologies | Virology  | Massart et al 2017

A Framework for the Evaluation of Biosecurity, Commercial, Regulatory, and Scientific Impacts of Plant Viruses and Viroids Identified by NGS Technologies | Virology  |  Massart et al 2017 | Diagnostic activities for plant pests | Scoop.it
Recent advances in high-throughput sequencing technologies and bioinformatics have generated huge new opportunities for discovering and diagnosing plant viruses and viroids. Plant virology has undoubtedly benefited from these new methodologies, but at the same time is also faced with substantial bottlenecks, namely the biological characterization of the newly discovered viruses and the analysis of their impact at the biosecurity, commercial, regulatory and scientific levels. This paper proposes a scaled and progressive scientific framework for efficient biological characterization and risk assessment when a previously known or a new plant virus is detected by next generation sequencing (NGS) technologies. Four case studies are also presented to illustrate the need for such a framework, and to discuss the scenarios.
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Use of Nested and Real-Time PCR for the Detection of Ceratocystis fagacearum in the Sapwood of Diseased Oak Species in Minnesota Yang & Juzwik 2017

Use of Nested and Real-Time PCR for the Detection of Ceratocystis fagacearum in the Sapwood of Diseased Oak Species in Minnesota Yang & Juzwik 2017 | Diagnostic activities for plant pests | Scoop.it
Oak wilt caused by Ceratocystis fagacearum is a significant disease of Quercus spp. in the eastern United States. Early and accurate detection of the pathogen is particularly important when disease control is planned. Nested and real-time polymerase chain reaction (PCR) methods utilizing fungal DNA extracted from sapwood drill shavings of red, bur, and white oak at different stages of disease development were compared with culture-based detection from sapwood. The pathogen was detected in all (n = 3) actively wilting branches of each of nine red oak trees using all three methods. The lowest detection rate (33% of assayed branches; 6 of 8 trees) for actively wilting branches was found for white oak using isolation while nested PCR had a branch detection rate of 100% (8 of 8 trees) and real-time PCR of 87% (8 of 8 trees) for the same samples. For both bur and white oak, the pathogen was not detected by isolation in branches over 1 year after their death but was detected using both PCR methods. Only the PCR assays detected the fungus in sapwood samples underlying remnants of sporulation mats (n = 21; 90%, nested and 62%, real-time) on red oak. These PCR methods offer several significant improvements for laboratory-based detection methods of C. fagacearum.
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4th meeting of the EPPO Panel on Diagnostics in Entomology
Paris, 2016-11-29/12-01

4th meeting of the EPPO Panel on Diagnostics in Entomology<br/>Paris, 2016-11-29/12-01 | Diagnostic activities for plant pests | Scoop.it

Read a short report of the  4th meeting of the EPPO Panel on Diagnostics in Entomology
Paris, 2016-11-29/12-01

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Free online training course on classification and identification of Arthropods and Nematodes opening soon!

Free online training course on classification and identification of Arthropods and Nematodes opening soon! | Diagnostic activities for plant pests | Scoop.it
This MOOC (in French and English) will deliver 5 weeks of course material and other pedagogical activities covering the following topics: Classification of arthropods and nematodes Collection and trapping methods Morphological and molecular identification methods Application of these integrative concepts for agroecosystem management through case studies
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Quarantine Regulations and the Impact of Modern Detection Methods - Annual Review of Phytopathology, 54(1):189

Producers worldwide need access to the best plant varieties and cultivars available to be competitive in global markets. This often means moving plants across international borders as soon as they are available. At the same time, quarantine agencies are tasked with minimizing the risk of introducing exotic pests and pathogens along with imported plant material, with the goal to protect domestic agriculture and native fauna and flora. These two drivers, the movement of more plant material and reduced risk of pathogen introduction, are at odds. Improvements in large-scale or next-generation sequencing (NGS) and bioinformatics for data analysis have resulted in improved speed and accuracy of pathogen detection that could facilitate plant trade with reduced risk of pathogen movement. There are concerns to be addressed before NGS can replace existing tools used for pathogen detection in plant quarantine and certification programs. Here, we discuss the advantages and possible pitfalls of this technology for meeting the needs of plant quarantine and certification.
Petter Françoise's insight:
A very good article on NGS , plant quarantine and certification
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Short report of the last Panel on Diagnostics in Bacteriology 2016-06-01/02

Short report of the last Panel on Diagnostics in Bacteriology 2016-06-01/02 | Diagnostic activities for plant pests | Scoop.it
Read a short report of the last Panel meeting in June where the three diagnostic protocols were finalized and later proposed for adoption. All three protocols are now approved and the one on Xylella fastidiosa is available through the EPPO Global Database. 
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EPPO Bulletin - Revised protocol on Xylella fastidiosa (PM 7/24 (2))

EPPO Bulletin - Revised protocol on Xylella fastidiosa (PM 7/24 (2)) | Diagnostic activities for plant pests | Scoop.it
The revised protocol on Xylella fastidiosa (PM 7/24 (2))  has been published and is available as early view. It should be noted that as more experience with the diagnostic of Xylella fastidiosa will be gathered in the coming months, the EPPO Secretariat intends to schedule a review of the Protocol at the next Panel on Diagnostics in Bacteriology in 2017-05.
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