PM 7/39 (2) Aphelenchoides besseyi

Specific scope

This Standard describes a diagnostic protocol for Aphelenchoides besseyi. This Standard should be used in conjunction with PM 7/76 Use of EPPO diagnostic protocols. Terms used are those in the EPPO Pictorial Glossary of Morphological Terms in Nematology a .

Specific approval and amendment

Approved in 2003‐09. Revised in 2017‐04. This revision was prepared on the basis of the IPPC Diagnostic Protocol adopted in 2016 on Aphelenchoides besseyi, Aphelenchoides fragariae and Aphelenchoides ritzemabosi (Annex 17 to ISPM 27; IPPC, 2016 ). The EPPO Diagnostic Protocol only covers A. besseyi. It differs in terms of format but it is consistent with the content of the IPPC Standard for morphological identification for this species. With regard to molecular methods, one real‐time PCR test available in the region is included as well as DNA barcoding.     

PM 7/29 (3) Tilletia indica

Specific scope

This Standard describes a diagnostic protocol for Tilletia indica. 1 It should be used in conjunction with PM 7/76 Use of EPPO diagnostic protocols.

Specific approval and amendment

This Standard was originally developed under the EU DIAGPRO Project (SMT 4‐CT98‐2252) by a partnership of contractor laboratories and interlaboratory comparison in European countries. First approved as an EPPO Standard in 2003–09. First revision approved in 2007–09. Second revision approved on 2017–11. Although this EPPO Diagnostic Standard differs in terms of format it is in general consistent with the content of the IPPC Standard adopted in 2014 on Tilletia indica (Annex 4 to 2006 ) with the following exceptions. (1) In the EPPO region, as the pest is not present, a higher confidence in the results is required, a sieve wash test should be carried out (optional in the IPPC protocol). (2) When fewer than 10 teliospores are found the options should allow testing the (<10) teliospores with conventional or real‐time PCR (this was not an option in the IPPC protocol flow chart, although it was stated that direct real‐time PCR could be used on individual teliospores in the text). (3) The method for extracting teliospores from untreated seed or grain by size‐selective sieving is slightly different based on the experience in the region (European Union test performance study). The EPPO Diagnostic Standard also includes a test for a direct real‐time PCR for use on pellets (developed in 2016). Some additional information on methods for morphological identification, from the former version of the EPPO Standard, which are not in the IPPC protocol are included in this protocol in Appendix 3 as they were considered useful by the members of the Panel on Diagnostics in Mycology.     

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

Specific scope

This Standard describes the use of 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 publically available sequence databases. 1 It should be used in conjunction with PM 7/76 Use of EPPO diagnostic protocols.

Specific approval and amendment

2016‐09     

PM 7/40 (4) Globodera rostochiensis and Globodera pallida

Specific scope

This Standard describes a diagnostic protocol for Globodera rostochiensis and Globodera pallida. 1 Terms used are those in the EPPO Pictorial Glossary of Morphological Terms in Nematology. 2 This Standard should be used in conjunction with PM 7/76 Use of EPPO diagnostic protocols.

Specific approval and amendment

Approved as an EPPO Standard in 2003‐09. Revisions approved in 2009‐09, 2012‐09 and 2017‐02.     

Occurrence of Citrus psorosis virus in Campania, southern Italy

Citrus psorosis virus (CPsV), genus Ophiovirus, is associated with a severe disease of citrus worldwide. Double antibody sandwich (DAS) ELISA using a polyclonal antiserum, and triple antibody sandwich (TAS) ELISAs, employing the IgG monoclonal antibody (mab) 13C5, and the IgM mab 2A3, were used to detect CPsV in orchards of different citrus varieties in Campania, southern Italy. TAS ELISA with 13C5 detected all the infections detected by DAS ELISA. Overall, 14% of trees younger than 15 years were positive, but only 1% of older trees, suggesting that infected propagating material has been increasingly used in recent years, in the absence of certification. Highest infection rates were in younger trees of sweet orange (22.8%) and clementine (18.6%). CPsV could easily be detected at all seasons of the year tested (June–January); these and earlier results indicate that TAS ELISA using 13C5 is a sensitive, broad-spectrum and reliable diagnostic method useful for routine tests and certification programmes. Of 44 field isolates responding strongly to DAS ELISA and 13C5-TAS ELISA, mab 2A3 gave similar results with 29 isolates, but gave low values with the others, thus providing a degree of differentiation among isolates. To confirm that the ELISA tests were indeed detecting CPsV, samples of 42 ELISA-positive plants were analysed by ISEM in a blind test, and in 38 of these, characteristic virus particles were clearly seen. Although CPsV was frequently and consistently detected in the area sampled, bark scaling symptoms were not seen: possible reasons for this are discussed.     

PM 7/100(1): Rep‐PCR tests for identification of bacteria

Specific scope

This standard describes how to perform rep‐PCR tests for identification of bacterial isolates.

Specific approval and amendments

Approved as an EPPO Standard in 2010‐09.     

PM 7/28 (2) Synchytrium endobioticum

Specific scope

This Standard describes a diagnostic protocol for Synchytrium endobioticum. 1 This Standard should be used in conjunction with PM 7/76 Use of EPPO diagnostic protocols.

Specific approval and amendment

Approved in 2003‐09. Revision approved in 2017‐06.     

PM 7/113 (1) Pepino mosaic virus

Specific scope

This standard describes a diagnostic protocol for detection and identification of Pepino mosaic virus in all plant parts, particularly on tomato seeds ¹ .

Specific approval and amendment

Approved in 2012‐09.     

PM 7/2 (2) Tobacco ringspot virus

Specific scope

This Standard describes a diagnostic protocol for Tobacco ringspot virus 1 This Standard should be used in conjunction with PM 7/76 Use of EPPO diagnostic protocols.

Specific approval and amendment

First approved in 2000‐09. Revision approved in 2017‐03     

PM 7/36 (2) Diabrotica virgifera virgifera

Specific scope

This Standard describes a diagnostic protocol for Diabrotica virgifera virgifera. This Standard should be used in conjunction with PM 7/76 Use of EPPO diagnostic protocols.

Specific approval and amendment

Approved in 2003‐09. Revised in 2017‐02.     

PM 7/21 (2) Ralstonia solanacearum,R. pseudosolanacearum and R. syzygii (Ralstonia solanacearum species complex)

Specific scope

This Standard describes a diagnostic protocol for Ralstonia solanacearum, Ralstonia pseudosolanacearum and Ralstonia syzygii, i.e. phylotype/sequevar strain in the Ralstonia solanacearum Species Complex (RSSC). 1 It should be used in conjunction with PM 7/76 Use of EPPO diagnostic protocols.

Specific approval and amendment

Approved in 2003‐09. First revised in 2018‐02.     

PM 7/62 (2) ‘Candidatus Phytoplasma mali’, ‘Ca. P. pyri’ and ‘Ca. P. prunorum’

Specific scope

This Standard describes a diagnostic protocol for ‘Candidatus Phytoplasma mali’, ‘Ca. P. pyri’ and ‘Ca. P. prunorum’. This Standard should be used in conjunction with PM 7/76 Use of EPPO diagnostic protocols

Specific approval and amendment

Approved as PM 7/62 Candidatus Phytoplasma mali and PM 7/63 Ca. P. pyri in 2006. Revised in 2017‐02 as a single Standard as PM 7/62 (2) with the addition of ‘Ca. P. prunorum’.     

PM 7/24 (3) Xylella fastidiosa

Specific scope

This Standard describes a diagnostic protocol for Xylella fastidiosa. 1 It should be used in conjunction with PM 7/76 Use of EPPO diagnostic protocols.

Specific approval and amendment

First approved in 2004‐09. Revised in 2016‐09 and 2018‐04. 2     

PM 7/87 (2) Ditylenchus destructor and Ditylenchus dipsaci

Specific scope

This Standard describes a diagnostic protocol for Ditylenchus destructor and Ditylenchus dipsaci. 1 This Standard should be used in conjunction with PM 7/76 Use of EPPO diagnostic protocols. Terms used are those in the EPPO Pictorial Glossary of Morphological Terms in Nematology. 2

Specific approval and amendment

Approved in 2008‐09. This revision was prepared on the basis of the IPPC Diagnostic Protocol adopted in 2015 on D. dipsaci and D. destructor (Annex 8 to ISPM 27 Diagnostic protocols for regulated pests). The EPPO Diagnostic Protocol is consistent with the text of the IPPC Standard for morphological identification for this species. For comparison with other species the IPPC table includes Ditylenchus africanus whereas the EPPO table includes Ditylenchus convallariae due to the different distribution of the species. The molecular tests for which there is experience in the EPPO region are described in full in the appendices (some of these are additional tests to those in the IPPC protocol). Reference is given to the IPPC protocol for tests for which there is little experience in the EPPO region. DNA barcoding is also included. Revision approved in 2017‐04.     

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

Specific scope

This Standard describes a diagnostic protocol for Xanthomonas axonopodis pv. allii. 1 This Standard should be used in conjunction with PM 7/76 Use of EPPO diagnostic protocols.

Specific approval and amendment

First approved in 2016‐09.     

From laboratory to point of entry: development and implementation of a loop‐mediated isothermal amplification (LAMP)‐based genetic identification system to prevent introduction of quarantine insect species

BACKGROUND

Rapid genetic on‐site identification methods at points of entry, such as seaports and airports, have the potential to become important tools to prevent the introduction and spread of economically harmful pest species that are unintentionally transported by the global trade of plant commodities. This paper reports the development and evaluation of a loop‐mediated isothermal amplification (LAMP)‐based identification system to prevent introduction of the three most frequently encountered regulated quarantine insect species groups at Swiss borders, Bemisia tabaci, Thrips palmi and several regulated fruit flies of the genera Bactrocera and Zeugodacus.

RESULTS

The LAMP primers were designed to target a fragment of the mitochondrial cytochrome c oxidase subunit I gene and were generated based on publicly available DNA sequences. Laboratory evaluations analysing 282 insect specimens suspected to be quarantine organisms revealed an overall test efficiency of 99%. Additional on‐site evaluation at a point of entry using 37 specimens performed by plant health inspectors with minimal laboratory training resulted in an overall test efficiency of 95%. During both evaluation rounds, there were no false‐positives and the observed false‐negatives were attributable to human‐induced manipulation errors. To overcome the possibility of accidental introduction of pests as a result of rare false‐negative results, samples yielding negative results in the LAMP method were also subjected to DNA barcoding.

CONCLUSION

Our LAMP assays reliably differentiated between the tested regulated and non‐regulated insect species within <1 h. Hence, LAMP assays represent suitable tools for rapid on‐site identification of harmful pests, which might facilitate an accelerated import control process for plant commodities. © 2018 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.     

Enzyme-linked immunosorbent assays for study of serological relationships and detection of three luteoviruses

Enzyme-linked immunosorbent assay (ELISA) systems were used to examine serological relationships and to detect three luteoviruses; a vector-non-specific strain of barley yellow dwarf virus from Illinois (BYDV-PAV-IL), a strain of beet western yellows virus from California (BWYV-CA) and a dwarfing strain of soybean dwarf virus from Japan (SDV-D). Indirect ELISA (IND-ELISA) systems detected distant reciprocal serological relationships among all three viruses. This is the first report of a serological relationship between a vector-non-specific strain of BYDV and any strain of SDV. Double antibody sandwich ELISA (DAS-ELISA) systems detected the purified homologous viruses at concentrations as low as 1.6 ng/ml. hut did not detect heterologous viruses as concentrated as 800 ng/ml. In contrast, when DAS-ELISA systems were used for detection of the three viruses in sap extracts from infected plants some weak but significant (P=0.05 ) heterologous reactions occurred. The BYDV-PAV-IL DAS-ELISA system usually detected BWYV-CA and sometimes detected SDV-D; the BWYV-CA DAS-ELISA system never detected BYDV-PAV-IL and rarely detected SDV-D; the SDV-D DAS-ELISA system sometimes detected BYDV-PAV-IL and consistently detected BWYV-CA.     

PM 7/40(2): Globodera rostochiensis and Globodera pallida

Specific scope

This standard describes a diagnostic protocol for Globodera rostochiensis and Globodera pallida ¹ 1 Use of brand names of chemicals or equipment in these EPPO Standards implies no approval of them to the exclusion of others that may also be suitable.
.

Specific approval and amendment

This Standard was developed under the EU DIAGPRO Project (SMT 4‐CT98‐2252) by partnership of contractor laboratories and intercomparison laboratories in European countries. Approved as an EPPO Standard in 2003–09. Revision approved in 2009–09.

     

PM 9/5 (2) Synchytrium endobioticum

Specific scope

This Standard describes the procedures for official control of Synchytrium endobioticum.

Specific approval and amendment

First approved in 2006‐09. Revised in 2017‐09 (editorial changes).     

PP 1/302 (1) Ceratitis capitata – foliar application

Specific scope

This Standard describes the conduct of trials for the efficacy evaluation of foliar insecticides against Ceratitis capitata on citrus and top fruits. For bait spray application see EPPO Standard PP 1/301. Together they replace EPPO Standard PP 1/106 Ceratitis capitata.

Specific approval and amendment

First approved in 2017‐09.