Diagnostic activities for plant pests
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Revised version of the Pictorial Glossary of Morphological Terms in Nematology

Revised version of the Pictorial Glossary of Morphological Terms in Nematology | Diagnostic activities for plant pests | Scoop.it

The Pictorial Glossary of Morphological Terms in Nematology lists all nematological terms used in the EPPO diagnostic protocols on plant parasitic nematodes with their definitions.

A revised version has just been posted online.

http://www.eppo.int/QUARANTINE/diag_activities/EPPO_TD_1056_Glossary.pdf

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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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Regulatory and Forensic Applications of DNA Barcoding | DNA Barcoding Courses  

Regulatory and Forensic Applications of DNA Barcoding | DNA Barcoding Courses   | Diagnostic activities for plant pests | Scoop.it

A DNA Barcoding Course from the University of Guelph (Canada)

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Development of a comprehensive detection and identification molecular based system for eight pospiviroids

Development of a comprehensive detection and identification molecular based system for eight pospiviroids | Diagnostic activities for plant pests | Scoop.it
Pospiviroids cause serious economic damage to solanaceous crops, including tomato (Solanum lycopersicum), and cause symptomless infections in many horticultural plants. Symptomless infection and seed transmission are obstacles in visual inspection for plant quarantine. Therefore, in the present study, a comprehensive detection and identification system for molecular diagnosis of eight pospiviroids was developed, based on real-time reverse transcription polymerase chain reaction (RT-PCR). In this system, two real-time RT-PCR methods based on SYBR Green and TaqMan technologies were combined. First, tomato seed samples were screened for all the eight pospiviroids by the SYBR Green method using three primer pairs, including one pair of universal primers. Samples that were positive using the universal primer set were subjected to secondary screening for the identification to species level of six viroids by TaqMan real-time RT-PCR method, with specific primer and probe sets. This two-phase system could specifically detect a viroid in a sample of 400 seeds containing a single contaminated seed. The developed system could be an effective and simple tool to avoid the introduction of pospiviroids through tomato seeds.
Petter Françoise's insight:
An EPPO diagnostic protocol for the detection of pospiviroids is in preparation.
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A diagnostic tool for improved detection of Xanthomonas fragariae using a rapid and highly specific LAMP assay designed with comparative genomics

A diagnostic tool for improved detection of Xanthomonas fragariae using a rapid and highly specific LAMP assay designed with comparative genomics | Diagnostic activities for plant pests | Scoop.it

Molecular diagnostics of plant pathogens are crucial to prevent disease spread and to enhance food quality and security. A comparative genomics approach using genomes of different Xanthomonas species and pathovars was applied to identify highly specific targets in the genome of Xanthomonas fragariae, the causal agent of angular leaf spot of strawberry, listed under quarantine regulations in Europe. A reliable and sensitive loop-mediated isothermal amplification (LAMP) assay was designed using a unique marker, providing a highly specific and rapid detection technique, convenient for on-site detection. Specificity of the designed assay was tested on 37 strains from a culture collection of X. fragariae, 82 strains of other Xanthomonas species and pathovars and 11 strains of other bacterial genera isolated from strawberry leaves. A detection limit of 102 fg was achieved, approximating to 20 genome copies per reaction. When performing analyses with crude plant material, a consistent lower detection efficiency of 102 CFU mL−1 was achieved. The LAMP assay designed in this study was adapted to work on crude plant material without any prior extensive extraction steps or incubation period; moreover, it does not require advanced analytical knowledge or a fully equipped laboratory. Results were produced within 7–20 min, depending on the pathogen concentration, thus providing a high-throughput and user-friendly method for detection and screening of plant material in support of quarantine regulations.

Petter Françoise's insight:
An EPPO Diagnostic protocol erxists for this bacterium PM7/065(1) Xanthomonas fragariae and will be considered for revision
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Several subspecies and sequence types are associated with the emergence of Xylella fastidiosa in natural settings in France

Several subspecies and sequence types are associated with the emergence of Xylella fastidiosa in natural settings in France | Diagnostic activities for plant pests | Scoop.it
Xylella fastidiosa is a plant pathogenic bacterium emerging in Europe. In France its emergence has been demonstrated through interceptions of contaminated coffee plants and, in 2015, by a survey of natural settings. The first French focus of contamination was detected in 2015 in Corsica; since then, almost 300 foci have been found and nearly 30 plant species have been declared contaminated, with Polygala myrtifolia remaining the principal host, suffering from severe leaf scorch. This study reports on the diversity of X. fastidiosa identified in France in 2015. Multilocus sequence analysis/typing revealed the presence of mainly X. fastidiosa subsp. multiplex sequence types (STs) ST6 and ST7. A focus of X. fastidiosa subsp. pauca ST53 was identified in mainland France; one sample contaminated by X. fastidiosa subsp. sandyi ST76, one novel recombinant, and coinfections of different isolates in individual samples were also identified, but could not be confirmed by successive samplings, indicating limited or transient contamination. 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, the results suggest that the emergence of X. fastidiosa in France is linked to several introduction events of diverse strains from different subspecies.
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Rapid and sensitive detection of Meloidogyne mali by loop-mediated isothermal amplification combined with a lateral flow dipstick, Zhou et al. (2017) 

Rapid and sensitive detection of Meloidogyne mali by loop-mediated isothermal amplification combined with a lateral flow dipstick, Zhou et al. (2017)  | Diagnostic activities for plant pests | Scoop.it

Meloidogyne mali Itoh, Ohshima & Ichinohe, 1969 is a polyphagous root-knot nematode that infects a wide range of host plants and is subject to phytosanitary restrictions in many countries, including China. In this study, we integrated the loop-mediated isothermal amplification (LAMP) technique with visual detection by a lateral flow dipstick (LFD) and developed an accelerated LAMP-LFD method for the rapid identification of M. mali, targeting the internal transcribed spacer regions and 5.8S ribosomal DNA sequence (ITS-5.8S). The entire test could be performed within 45 min, including 35-min LAMP amplification, 5-min hybridization, and 3–5-min visualization on LFD. The detection limit of this method was 2-pg bulked M. mali gDNA per reaction, and the equivalent of 1‰ single female adult or 1% single second-stage juvenile (J2) could be detected. It is recommended that DNA is extracted from single J2 isolated from soil samples by the modified Baermann funnel method or from an adult female from galls. Based on this, the field sensitivity and specificity of this LAMP-LFD method for the detection of M. mali in field soil samples were both 100% compared to traditional microscopic observation. These results indicate that the LAMP-LFD method is rapid, sensitive, accurate, and simple, and would be applicable for the routine monitoring of phytosanitary M. mali.

Petter Françoise's insight:
This pest is considered for addition to the EPPO A2 list of pests recommended for regulation.
 
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Workshop on DNA barcoding, EPPO Headquarters, Paris, 2017-10-09/13

Workshop on DNA barcoding, EPPO Headquarters, Paris, 2017-10-09/13 | Diagnostic activities for plant pests | Scoop.it
DNA barcoding is increasingly used as a diagnostic tool in phytosanitary laboratories. The EPPO Standard PM7/129 ‘DNA barcoding as an identification tool for a number of regulated pests’ was published on 2016-11-28 and provides guidance on the use of the DNA barcoding protocols in support of the identification of a number of regulated pests and invasive plant species comparing DNA barcode regions with those deposited in public sequence databases. This workshop builds on the recommendations of the EU and Euphresco projects ‘Quarantine organisms Barcoding of Life (QBOL)’ and ‘DNA Barcoding - Optimizing and validating DNA barcoding protocols for plant pests’ and will provide information on the most recent advancements on the method and will allow potential users of DNA barcoding to be trained on tools for data analysis, that can critically influence the outcomes of DNA barcoding for pest identification.
Petter Françoise's insight:
A workshop ‘Use of DNA barcoding, from theory to practice’, organised in the framework of the Euphresco project PRACTIBAR, will be held on 2017-10-09/13 at the EPPO Headquarters, in Paris, France. A draft agenda will be posted on the EPPO website in due course. Register on-line by 2017-06-30 at: http://meeting.eppo.int/meeting.php/E5147
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Real‐time PCR assays for the detection of Heterobasidion irregulare, H. occidentale, H. annosum sensu stricto and the Heterobasidion annosum complex l Lamarche et al.

Real‐time PCR assays for the detection of Heterobasidion irregulare, H. occidentale, H. annosum sensu stricto and the Heterobasidion annosum complex l Lamarche et al. | Diagnostic activities for plant pests | Scoop.it

A set of quantitative hierarchical real-time PCR assays was developed for the detection of Heterobasidion irregulare, H. occidentale, H. annosum sensu stricto and of the entire Heterobasidion annosum complex. These assays enable specific and accurate detection and quantification of the target species from DNA extracted on airborne collected spores. Heterobasidion-specific TaqMan™ real-time PCR detection assays were designed to function under homogeneous conditions so that they may be used in 96- or 384-well plate format arrays for high-throughput testing of large numbers of samples against multiple targets. Assays were validated for (i) specificity, (ii) sensitivity and (iii) repeatability. All assays were highly specific when evaluated against a panel of pure cultures of target and phylogenetically closely related species. Sensitivity, evaluated by assessing the limit of detection (with a threshold of 95% of positive samples), was found to be between one and a hundred ITS gene region copies or one conidia count equivalent. Precision or repeatability of each assay revealed a mean coefficient of variation of 5.9%. These molecular tools are now available for rapid and reliable monitoring of one of the most significant pathogen species complex of temperate northern coniferous forest around the world.

Petter Françoise's insight:
Heterobasidion irregulare was added to the EPPO List of pests recommended for regulation in 2015. The preparation of a diagnostic protocol is on the works programme of the Panel on Diagnostics in Mycology.
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Detection of plant pathogens using real‐time PCR: how reliable are late Ct values? l  Grosdidier et al. 

Detection of plant pathogens using real‐time PCR: how reliable are late Ct values? l  Grosdidier et al.  | Diagnostic activities for plant pests | Scoop.it

Effective detection of pathogens from complex substrates is a challenging task. Molecular approaches such as real-time PCR can detect pathogens present even in low quantities. However, weak real-time PCR signals, as represented by high cycle threshold (Ct) values, may be questionable. Therefore, setting a reliable Ct threshold to declare a positive reaction is important for specific detection. In this study, five methods were assessed for their performance in determining a Ct cut-off value. These methods were based on the widely used probability of detection (POD) or receiver-operating characteristic (ROC) approaches. Two important forest pathogens, Hymenoscyphus fraxineus and Fusarium circinatum, were used to set up three experimental frameworks that combined two types of substrates (seed lots and spore traps) and different PCR machines. The ROC-based method emerged as the most complete and flexible method under various experimental conditions. It was demonstrated that the ROC method leads to a cut-off value below which late Ct results can reliably be considered indicative of positive test results. This cut-off value must be determined for each experimental approach used. The method based on the distribution of a previously determined set of Ct values corresponding to false-positives appeared to be better adapted to detecting false-negative results, and thus useful for testing potentially invasive pathogens.

Petter Françoise's insight:
Interpreting late Ct values can indeed be a challenge. A workshop on setting Ct cut-off values for real-time PCR was organized by EPPO in 2013: http://archives.eppo.int/MEETINGS/2013_conferences/cut-off_values.htm.

An article was also published on this topic in the EPPO Bulletin Chandelier A, Planchon V, Oger R, 2010. Determination of cycle cut off in real-time PCR for the detection of regulated plant pathogens. EPPO Bulletin 40 ,52–8.
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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.

Petter Françoise's insight:
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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Santé des plantes : de l’observation au diagnostic

Santé des plantes : de l’observation au diagnostic | Diagnostic activities for plant pests | Scoop.it
L'excellence de l'enseignement supérieur pour des cours en ligne, gratuits et ouverts à tous
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EPPO Workshop on Flexible Scope 

EPPO Workshop on Flexible Scope  | Diagnostic activities for plant pests | Scoop.it
Access the presentations and read a summary the main conclusions and recommendations of the Workshop
Petter Françoise's insight:
An excellent Workshop which allowed sharing experience in the region. A revision of our quality assurance standards has now been launched 
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Filling the gaps in diagnostics of Pepino mosaic virus and Potato spindle tuber viroid in water and tomato seeds and leaves

Filling the gaps in diagnostics of Pepino mosaic virus and Potato spindle tuber viroid in water and tomato seeds and leaves | Diagnostic activities for plant pests | Scoop.it
Waterborne and seedborne Pepino mosaic virus (PepMV) and Potato spindle tuber viroid (PSTVd) pose serious threats to tomato production due to seed transmission and mechanical transmission, coupled with their long-term stability outside the host plant. Therefore, rapid and sensitive diagnostic procedures are needed to prevent the spread of these quarantine pathogens. In particular, water and seed contamination are difficult to detect and confirm without efficient concentration methods. This study presents procedures that improve detection of PSTVd from tomato seeds and leaf tissue, and PepMV from water and tomato leaf tissue. For efficient concentration of PepMV from water samples, a procedure was optimized using convective interaction media monolithic chromatography columns, which provides concentration by three orders of magnitude. For concentration of PSTVd from seed extracts, an easy-to-use and efficient method was developed based on RNA binding to positively charged anion-exchange resin beads that provides up to 100-fold more sensitive detection in comparison with procedures without a concentration step. This thus allows confirmation of RT-qPCR results with sequencing of RT-PCR products in samples with low viroid levels. In addition, reverse-transcription loop-mediated isothermal amplification assays for detection of PSTVd and PepMV were optimized and adapted to both laboratory and on-site testing requirements. This allows rapid detection of these pathogens in crude leaf homogenates, in under 30 min. These procedures of concentration and detection are shown to be efficient and to fill the gaps in diagnostics of PepMV and PSTVd, especially when these pathogens are present at low levels in difficult matrices such as water and seeds.
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Assessment of the genetic diversity of Xylella fastidiosa in imported ornamental Coffea arabica plants

Assessment of the genetic diversity of Xylella fastidiosa in imported ornamental Coffea arabica plants | Diagnostic activities for plant pests | Scoop.it
A study was performed in order to assess the presence of Xylella fastidiosa in imported ornamental plants, among them Olea europaea, Coffea arabica and Nerium oleander. Positive results were only obtained from C. arabica, where 15 plant samples tested positive for X. fastidiosa by PCR, nine from Costa Rica and six from Honduras. Transmission electron microscopy observations indicated that rod-shaped bacterial cells exhibiting the characteristics of X. fastidiosa cells were present in the xylem vessels of leaf petioles obtained from the infected C. arabica plants. Diversity of X. fastidiosa in C. arabica plants was assessed through a multilocus sequence typing (MLST) analysis of seven housekeeping genes (leuA, petC, lacF, cysG, holC, nuoL and gltT) and compared with X. fastidiosa infecting different host plants worldwide. Based on this MLST analysis, the prevalence of different sequence types (STs) of X. fastidiosa in the C. arabica ornamental plants was demonstrated and related to different X. fastidiosa subspecies, underlining the risk of introducing additional genetic diversity for X. fastidiosa to Europe. ST53, related to X. fastidiosa subsp. pauca, was frequently found in these C. arabica samples. A second ST related to X. fastidiosa subsp. pauca, ST73, has been assessed in coinfection with ST53 in one individual plant. Additionally, ST72 and ST76, related to X. fastidiosa subsp. fastidiosa, have been recorded. Next to these previously described STs, a novel ST, namely ST77 has been revealed, related to X. fastidiosa subsp. fastidiosa. Isolation of X. fastidiosa from leaf petioles and midribs of infected C. arabica plants was successfully performed only after the application of an additional ultrasonication step during the extraction procedure. Based on this approach, a number of X. fastidiosa isolates were obtained and further characterized.
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A field based detection method for Rose rosette virus using isothermal probe-based Reverse transcription-recombinase polymerase amplification assay, Babu et al. (2017)

A field based detection method for Rose rosette virus using isothermal probe-based Reverse transcription-recombinase polymerase amplification assay, Babu et al. (2017) | Diagnostic activities for plant pests | Scoop.it
Rose rosette disease, caused by Rose rosette virus (RRV; genus Emaravirus) is a major threat to the rose industry in the U.S. The only strategy currently available for disease management is early detection and eradication of the infected plants, thereby limiting its potential spread. Current RT-PCR based diagnostic methods for RRV are time consuming and are inconsistent in detecting the virus from symptomatic plants. Real-time RT-qPCR assay is highly sensitive for detection of RRV, but it is expensive and requires well-equipped laboratories. Both the RT-PCR and RT-qPCR cannot be used in a field-based testing for RRV. Hence a novel probe based, isothermal reverse transcription-recombinase polymerase amplification (RT-exoRPA) assay, using primer/probe designed based on the nucleocapsid gene of the RRV has been developed. The assay is highly specific and did not give a positive reaction to other viruses infecting roses belonging to both inclusive and exclusive genus. Dilution assays using the in vitro transcript showed that the primer/probe set is highly sensitive, with a detection limit of 1 fg/μl. In addition, a rapid technique for the extraction of viral RNA (< 5 min) has been standardized from RRV infected tissue sources, using PBS-T buffer (pH 7.4), which facilitates the virus adsorption onto the PCR tubes at 4 °C for 2 min, followed by denaturation to release the RNA. RT-exoRPA analysis of the infected plants using the primer/probe indicated that the virus could be detected from leaves, stems, petals, pollen, primary roots and secondary roots. In addition, the assay was efficiently used in the diagnosis of RRV from different rose varieties, collected from different states in the U.S. The entire process, including the extraction can be completed in 25 min, with less sophisticated equipments. The developed assay can be used with high efficiency in large scale field testing for rapid detection of RRV in commercial nurseries and landscapes.
Petter Françoise's insight:
Rose rosette virus is on the EPPO Alert List and a PRA will be prepared by the end of 2017.
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Development of a Sensitive and Specific Polyclonal Antibody for Serological Detection of Clavibacter michiganensis subsp. sepedonicus Przewodowski & Przewodowska (2017)

Development of a Sensitive and Specific Polyclonal Antibody for Serological Detection of Clavibacter michiganensis subsp. sepedonicus Przewodowski & Przewodowska (2017) | Diagnostic activities for plant pests | Scoop.it
The quarantine bacterium Clavibacter michiganensis subsp. sepedonicus (Cms) causes bacterial ring rot (BRR) in potato but is difficult to detect, hampering the diagnosis of this disease. ELISA immunoassays have not been widely used to detect Cms because commercially available anti-Cms antibodies detect mainly EPS-producing bacteria and can fail to detect strains that do not produce EPS. In the current study, we developed a new type of polyclonal antibody that specifically detects Clavibacter michiganensis subsp. sepedonicus bacteria irrespective of their EPS level. We first found that the presence of bacterial EPS precluded quantitative measurement of bacteria by currently available immunoenzymatic methods, but that washing Cms cells with acidic and basic buffers to remove EPS before analysis successfully standardized ELISA results. We used a mix of three strains of Cms with diverse EPS levels to generate antigen for production of antibodies recognizing Cms cells with and without an EPS layer (IgG-EPS and IgG-N-EPS, respectively). The resulting IgG-N-EPS recognized almost all Cms strains tested in this work regardless of their mucoidal level. The availability of this new antibody renders immunological diagnostics of Cms more sensitive and reliable, as our newly developed antibodies can be used in many type of immunoassays. This work represents an important step forward in efforts to diagnose and prevent the spread of BRR, and the methods and solutions developed in this work are covered by six Polish, one European and one US patents.
Petter Françoise's insight:
A revision of the EPPO diagnostic protocol  on Cms is in progress

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Workshop on the use of Next Generation Sequencing technologies for plant pest diagnostics, Bari (IT), 2017-11-22/23

Workshop on the use of Next Generation Sequencing technologies  for plant pest diagnostics, Bari (IT), 2017-11-22/23 | Diagnostic activities for plant pests | Scoop.it
High-throughput sequencing technologies, namely Next Generation Sequencing (NGS), have open the way to fast and cheap analysis of genomes. Applications of NGS technologies for plant pest diagnostics are being developed. These can be used for the detection of known pests as well as a tool to detect “unknowns”. These technologies can, at the same time, increase knowledge on pest diversity (e.g. genomics, population biology, epidemiology). The Workshop will provide basic information on these technologies and about their most recent advances and give the opportunity to discuss its applicability for plant pest diagnostics. The target audience of this workshop are current and potential users of NGS with a focus on application in plant health as well as plant health risk regulators.
Petter Françoise's insight:
A Workshop on the use of NGS technologies for plant pest diagnostics will be organized jointly by the European Cooperation in Science and Technology (COST) action DIVAS, European Phytosanitary Research Coordination Network (Euphresco) and EPPO from the 22nd to the 23rd of November 2017 in Bari, Italy. Register on-line by 2017-06-30 at: http://meeting.eppo.int/meeting.php/N5133
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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 l  Kogovšek et al.

Rapid loop-mediated isothermal amplification assays for grapevine yellows phytoplasmas on crude leaf-vein homogenate has the same performance as qPCR l  Kogovšek et al. | 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 laboratory and on-site BNp detection. This assay is highly specific, rapid and as sensitive as qPCR. It was validated according to European and Mediterranean Plant Protection Organisation recommendations. In addition, 286 grapevine leaf samples from the 2015 growing season were tested with this new real-time LAMP assay and an assay previously developed for detection of Flavescence dorée phytoplasma (FDp). These LAMP assays for detection of both BNp and FDp used without any DNA extraction step, which is a required step for qPCR analysis, were comparably effective to qPCR, and positive results were obtained in less than 35 min.

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An illustration of the use of the EPPO PM 7/98 Specific requirements for laboratories preparing accreditation for a plant pest diagnostic activity used by laboratories for validation of a test.
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EPPO Bulletin - Volume 47, Issue 1 - April 2017 - Wiley Online Library

EPPO Bulletin - Volume 47, Issue 1 - April 2017 - Wiley Online Library | Diagnostic activities for plant pests | Scoop.it
Petter Françoise's insight:
April issue of the EPPO Bulletin is now online. It includes 15 original articles from across the EPPO region and 2 revised diagnostic Standards: 

PM 7/76 (4) Use of EPPO diagnostic protocols (pages 7–9)

PM 7/109 (2) Epitrix cucumeris, Epitrix papa, Epitrix subcrinita, Epitrix tuberis (pages 10–17)
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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
Petter Françoise's insight:
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
Petter Françoise's insight:
Thanks to the laboratories in the EPPO region that organize proficiancy tests and test performance studies
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