Work package 7 - Progress summary On-Site Confirmation and Monitoring

1. Work package 7 - Progress summary On-Site Confirmation and Monitoring Partners involved: PRI, Fera, ACW, NIB, UNIBO, CIP, CAIQ, Qlinea and Optisense WP Coordinator: PRI 12th June, 2012 Q-detect

2. Overview of work for Task 7 Task 7.1 DNA extractions Task 7.2 Generation of protocols for isothermal, singleplex and multiplex amplification of targets Task 7.3 Generation of devices for multiplex detection on-site Task 7.4 Evaluation of promising on-site detection systems for monitoring Task 7.5 Implementation of procedure on user-friendly detection system

3. Confirmation and Monitoring DNA/RNA extractions on different substrates performed in the field and/or on-site are: White flies viruses from traps Bacterial pathogens from plant material Potato pathogens from tuber and leaves Phytophthoraand plant pathogenic Fungi DNA/RNA Extraction Criteria: - Easy - Fast - Efficient - Cheap

4. DNA/RNA extractions (summary): • QiagenDNeasy/RNeasy extraction kids have been successfully used for: • Viruses (ssRNA) and (ssDNA), bacteria, fungi and nematodes • Boiling of infected plant tissue (leafs, shoots, blossoms) • Epicentre DNA/RNA extraction procedure • Applicable for ssRNA and ssDNA viruses • Efficient recovery • LFD based DNA/RNA extractions: • Applicable for different substrates • (RNA and DNA viruses has to be tested) • Recovery has to be improved

5. Vector of Q-viruses: • Bemisia tabaci • Trialeurodes vaporariorum Confirmation and Monitoring • DNA/RNA extractions on different substrates to be performed in the field and/or on-site White flies viruses from traps Bacterial pathogens from plant material Potato pathogens from tuber and leaves Phytophthora and plant pathogenic Fungi

6. Confirmation and Monitoring • DNA/RNA extractions on different substrates to be performed in the field and/or on-site White flies viruses from traps Bacterial pathogens from plant material Potato pathogens from tuber and leaves Phytophthora and plant pathogenic Fungi Whitefly vectored viruses Crini viruses (ssRNA): Tomato chlorosis virus, (TOCV) Tomato infectious chlorosis virus, (TICV) Cucurbit yellow stunting disorder virus, (CYSDV) Potato yellow vein virus, (PYVV) Begomovirus (ssDNA): Tomato yellow leaf curl virus, (TYLCV) Cotton leaf curl virus (CLCuV) Field validation can be performed Whitefly vectored viruses Crini viruses (ssRNA): Tomato chlorosis virus, (TOCV) Tomato infectious chlorosis virus, (TICV) Cucurbit yellow stunting disorder virus, (CYSDV) Potato yellow vein virus, (PYVV) Begomovirus (ssDNA): Tomato yellow leaf curl virus, (TYLCV) Cotton leaf curl virus (CLCuV)

7. Confirmation and Monitoring • DNA/RNA extractions on different substrates to be performed in the field and/or on-site • White flies viruses from traps • Bacterial pathogens from plant material • Potato pathogens from tuber and leaves • Phytophthora and plant pathogenic Fungi Erwinia amylovora Fire blight of pome fruit (Spiraeoidea) Xanthomonas arboricola pv. pruni Bacterial spot of stone fruit (Prunus) Pseudomonas syringae Symptoms of Psa

8. XAP Ring test Confirmation and Monitoring ACW Wädenswil, CITA Zaragoza, IVIA Valencia, APR Lisse Inter- Laboratory concordance

9. Confirmation and Monitoring • DNA/RNA extractions on different substrates to be performed in the field and/or on-site • White flies viruses from traps • Bacterial pathogens from plant material • Potato pathogens from tuber and leaves • Phytophthora and plant pathogenic Fungi Ralstonia solanacearum Clavibacter michiganensis ssp. Sepedonicus PSTVd (Potato Spindle Tuber Viroid)

10. Confirmation and Monitoring LAMP target genes for Ralstonia solanacearum:fliC. and 16S rRNA • 16S rRNA: good sensitivity and specificity on control strains, but shows cross reactivity with some potato extracts(soil bacteria?). • Egl (endogluconase):in silico covers all RS isolates, work in progress.

11. RT-LAMP on PSTVd Confirmation and Monitoring • Specificity for PSTVd; TPMVd, CSVd and CLVd discriminated by Tm

12. Confirmation and Monitoring BursaphelenchusxylophilusPine Wood Nematode (PWN) • DNA/RNA extractions on different substrates to be performed in the field and/or on-site • LAMP assay design on ITS region of B. xylophilus

13. Confirmation and Monitoring LAMP assay design for the vector insect LAMP assay design on COI gene of Monochamusgalloprovincialis(positive control for LAMP assay on PWN)

14. Confirmation and Monitoring Detection of blue-stain fungi (Ophiostoma clavatumor Ophiostoma brunneo-ciliatum) associated to Ips acuminatus using LAMP technology Ips acuminatus on Pinus sylvestris

15. Confirmation and Monitoring • LAMP assay development: • O. clavatum and O. brunneo-ciliatum primers were designed on the β-tubulin gene • Ips acuminatus primers were designed in the COX I gene • The blue-stain fungus O. clavatum is associated toIps acuminatus in the Italian Alps • LAMP technology can be useful also for ecological and biological studies

16. Monitoring of LAMP products (first line screening) • Multiplex and simplex amplification • Easy • Fast • Sensitive • Applicable for DNA and RNA • On-site

17. Detection The detection system needs to be adjusted for field inspectors who might not necessarily have laboratory-work experiences, while the given result has to be unambigous. The most promising detection systems include: 1)Turbidity2) Fluorescence Positive reaction is green Positive reaction is turbid

18. Detection 3) LFD4) Array-tubes On-site diagnostics device (Forsite Diagnostics Ltd.). Micro reaction vial with integrated biochip (Alere Technologies GmbH).

19. Combined amplification and detection To simplify the procedure, amplification and detection can be combined in a single system: Genie II/III • 16/8-well device with touch screen to follow DNA amplification in real-time • Stand-alone operation without PC • Internal rechargeable Li-Po battery • (OptiSense Ltd.) • .

20. Combined amplification and detection • ISO-001 standard mastermix (GspSSD) • GspMand GspM2.0 polymerases • TIN-001 DNA polymerase • ‘Lyse & LAMP’ • Lyse in Potassium Hydroxide solution (0.5 Molar) • Dispense directly into reaction tube • Mastermix buffer modified with lower pH • Method being used by researchers in crop science • Plant leaves used directly Licensing • Q-Detect assays discussed at meeting held with Eiken Master Mix and Enzymes

21. Combined amplification and detection To simplify the procedure, amplification and detection can be combined in a single system: Microfluidics Microfluidics allow multiple reactions combined with detection (PRI)

22. Extraction DNA / RNA First line screening (monitoring via fast semi specific method) - + Χ Second line screening (confirmation via target specific detection methods) Specific target detection

23. Multiplex TYLCV, TOCV and TICV Combination of TICV, ToCV and TYLCV LAMP primer sets • Multiplex LAMP with biotinylatednucleotides can be used for First and Second line screening

24. Ligation based Univ-LAMP • After ligation biotinylated LAMP amplicon can efficiently and specifically be detected with Luminex

25. Developed Isothermal methods LAMP • LAMP method for Ralstonia solanacearum • RT-LAMP for PSTVd detection • LAMP method for E. amylovora and X. arboricola. prun. are developed and validated • LAMP assay for B. xylophilus developed and validated • LAMP assays in white flies for TICV, TOCV, CYSDV and PYVV (Crini viruses) and TYLCV, CLCuV (Begomoviruses) have been developed. Validation is in progress. • LAMP assays designed for quarantineL. huidobrensis, L. sativae, L. trifolii (vegetable leafminer). Also assays in development for TCDVd, CSNV

26. Contact end users • Engage with end users to ensure your deliverables are fit for purpose and work with other WP to ensure synergies are created. • PWN assays developed by University of Padova utilised on trapped monchamus (linking WP7 and WP4). Assays also requested by Australian Inspector who attended EPPO workshop • PRI developed suite of assays for whitefly viruses to enable testing of trapped whitefly (linking WP7 and WP4) • PRI have planned a demonstration for Dutch NPPO. • ACW talking to Swiss NPPO about needs (list of pest elaborated), they plan to implement LAMP pipeline • Ferahave an implementation project with UK inspectors, plan to implement LAMP/Genie at Heathrow airport.

27. Dissemination • Develop and implement a dissemination plan for information and experience gained from WP7. • EPPO workshop should be more hands on – need to develop a programme. • Work with CIP/CAIQ to deliver training in China and Peru • Good dissemination already achieved with papers, TV shows, videos, newspapers, web site etc. • ACW working with AQIS in Melbourne have validated assays on Australian isolates and AQIS plan to implement LAMP

28. Follow up • How you plan for the deliverables and knowledge gained in the q-detect project to be utilised after the project finishes. • Exploitation plan for simplex LAMP well established • Optisense already started negotiation of license with Eiken • Partners starting to validate assays to EPPO standard • Large number of other assays under development.