REFERENCE PROTOCOL NAKTUINBOUW. Detection of Clavibacter michiganensis subsp. michiganensis in seeds of tomato

Transcription

1 REFERENCE PROTOCOL NAKTUINBOUW Detection of Clavibacter michiganensis subsp. michiganensis in seeds of tomato Protocol number: SPN-B004 Version: 9.1UK Date: 18 March 2010 Validation: Dilution plating and PCR are validated

2 1. Objective Detection and identification of Clavibacter michiganensis subsp. michiganensis in seeds by dilution plating, real-time PCR and pathogenicity assays. 2. Principle The assay can be performed on seeds (3 subsamples of seeds each) or seeds (2 subsamples of 5000 seeds each). First, seeds are soaked in buffer and then extracted with a bagmixer. Extracted bacteria are concentrated and then dilution plated on two semiselective media. Suspected are transferred to YDC. YDC suspected are screened with two complementary Cmm-specific real-time PCRs. Finally, a pathogenicity assay is performed to confirm the identity of the PCR suspected. Recovery of Cmm is monitored by spiking with a rif resistent Cmm reference strain for each subsample. In case of insufficient recovery of the spike there will be no valid test result. 3. Abbreviations Cmm Cmm1 GF-rif SCMF KB YDC TGW PCR spb spbt DNA Clavibacter michiganensis subsp. michiganensis Tris buffered semi-selective medium (Cmm1-Tris100) Growth factor medium with rifampicine SCM-fast: semi-selective medium on basis of SCM Kings B medium (non selective) Yeast-Dextrose-CaCO 3 medium (non selective) Thousand grain weight Polymerase chain reactie Steril phosfate buffer Sterile phosphate buffer with Tween20 Desoxyribonucleinezuur 4. Materials ZUM 3059 stock suspension in 25% glycerol stored in -80 C (ca. 1, cfu/ml) IPO 500, IPO 501 and IPO 542 reference strains PCR Biorad CFX 96 Interscience BagMixer 400 TaqMan 2 x gene expression mastermix (Applied Biosystems) Seedlings of tomato (S. lycopersicum cv. Moneymaker) Primer Nr. Naktuinbouw Sequence primer-collection CmmIGS-F 258a TGT CGA GGG CAT GTT GCA CG CmmIGS-R 258b GTT TCG CCT CCC CGA AG CmmIGS-Probe 258c 6FAM-TCGTCCTGTTGTGGATG-MGB CmmPat1-R 259b CTC GTA GTC ATT GAC GAG AG CmmPat2-F 259e CGA ATC AGC CCA TAT CAA C CmmPat1-Probe1 259c VIC-AGTTGTTATCCGACTTCG-MGB CmmPat1-Probe2 259d VIC-CGAAGCATTGCGGCGGTCGT-BHQ1 BAC16S-F 183a TCC TAC GGG AGG CAG CAG T BAC16S-R 183b GGA CTA CCA GGG TAT CTA ATC CTG TT BAC16S-Probe 183d 6FAM-CGT ATT ACC GCG GCT GCT GGC AC-MGB Detection of Cmm in tomato seed 2/16 SPN-B004-v9.1UK

3 5. Method Dilution plating phase 1. Determine TGW and weigh (2 x 5.000) or (3 x ) seeds in a stomacher bag. 2. Add per bag with seeds 100 ml and per bag with seeds 150 ml cold (4 C) spbt. Check: All seeds must be emerged in buffer during soaking. 3. Incubate seeds overnight at 4 C. 4. Extract sub samples for 4 minutes with Interscience BagMixer at setting Transfer 50 ml of extract to centrifugation tubes. 6. Centrifuge the extract for 1 minute at 1000 g. Transfer supernatant carefully to new tube and centrifuge for 20 minutes at g at 4ºC. 7. Carefully remove supernatant and resuspend pellet in ca. 3 ml 0,07 M spb. 8. Transfer 2 x 1 ml per subsample to two tubes. Use one tube for dilution plating without spike and one tube for dilution plating with spike (see 5.2). 9. Prepare for non-spiked version 10x and 100x dilutions with 0,07 M spb for each subsample. 10. Keep extracts and dilutions at room temperature till plating. 11. Transfer in duplicate 100 µl of concentrated extracts and once 1 x 100 µl of the 10x and 100x dilution to Cmm1 and SCMF. 12. Spread the extract over plates and incubate upside down for 7 days at 28 ºC Spiking of subsamples 13. Transfer one tube with the Cmm reference strain ZUM 3059 stock suspension from -80 C to the bench and defrost slowly. Dilute with spb to working dilution of ca. 4,000 CFU/ml. 14. Add per sub sample 100 µl of the diluted ZUM 3059 spike to the tube with 1ml of concentrated extract. 15. Transfer 100 µl of spiked extracts and plate on semi-selective media (Cmm1 and SCMF). Incubate plates up side down for 7 days at 28 C Control spike and reference Additional control per set of samples. 16. Add in triplicate 100 µl of spike dilution of ZUM 3059 to 1 ml spb. 17. Make 10-2x, 10-4x, 10-6x en 10-8x.dilutions from reference cultures Cmm 500, 501 and Plate 100 µl from each spike and dilutions from reference on Cmm1, SCMF and KB. 19. Incubate plates 7 days at 28 C Evaluation of spike and references strains 20. Evaluate first reference plates after 7 days. 21. Check whether reference strains (see 5.3.3) grow on two selective media (Cmm1, SCMF). See photos 1A and 1B. 22. Then evaluate spike control plates on Cmm1 and SCMF. Determine whether recovery is similar between triplicates (about CFU/plate on SCMF medium). Declare test invalid when the recovery in the spike control is too low (<1 CFU per plate). 23. Note cfu per plate in the data sheet Evaluation samples and transfer to YDC Note that the colony morphology of Cmm is highly variable and influenced by the medium and saprophytes. Detection of Cmm in tomato seed 3/16 SPN-B004-v9.1UK

4 Spike 24. First evaluate the recovery of the Cmm spike for each subsample on Cmm1 and SCMF. 25. For a valid evaluation of the dilution plating at least 1 clearly suspected Cmm colony should be visible on at least one of the two semi-selective media (Cmm1 or SCMF). 26. Note per subsample and per medium the number of suspected Cmm. Non spiked samples 27. Evaluate all subsamples for the presence of Cmm suspected. 28. Transfer Cmm suspected from all subsamples to YDC (10 per subsample). Note that colony morphology on both media is highly variable and that lookalikes could reduce the detection chance. Therefore transfer high numbers of suspected to reduce the chance of false negative results. 29. Check whether there is logical distribution of the target pathogen over the different dilutions. 30. For subsamples with no recovery of the spike. Check rapidly for the presence of Cmm suspected. Regard that resemble Cmm as suspected and proceed with test. 31. Transfer ZUM 3059 and the 3 reference strains to YDC as positive controls. 32. Incubate plates upside down for 2-3 days at 28ºC. 33. Check positive controls and evaluate whether putative Cmm isolates remain suspected. Note that colour on YDC is highly variable. Apart from yellow, could have orange, pink and white appearance Evaluation dilution plating Decision matrix for dilution plating and morphology on YDC Spike recovery subsampless YDC Test result Remarks No* recovery on Cmm1 and SCMF for all 3 subsamples No suspect N.a invalid. No* recovery on Cmm1 and SCMF for part of subsamples No* recovery on Cmm1 and SCMF for part of subsamples No* recovery on Cmm1 and SCMF for part of subsamples 1 spike colony on Cmm1 or SCMF for all subsamples 1 spike colony on Cmm1 or SCMF for all subsamples 1 spike colony on Cmm1 or SCMF for all subsamples No suspected Suspect Suspect Suspect Suspect No suspect N.a No suspected Suspected Suspected No suspected N.a Cmm not detected Cmm not detected Report valid result for reduced number of seeds (see 6) Report valid result for reduced number of seeds (see 6) Suspect Continue with Report later number of seeds for which result is valid (see 6) Suspect Continue with Cmm not detected Cmm not detected * N.a = Not applicable Detection of Cmm in tomato seed 4/16 SPN-B004-v9.1UK

5 5.2.1 PCR on YDC suspected 1. Check all YDC suspected with PCR seeds max. 20 isolates (4 pools of 5 isolates) seeds max 30 isolates (6 pools of 5 isolates) 2. Transfer with sterile pipette tip or toothpick YDC suspected from YDC medium to tube with 1,0 ml fresh 5 mm NaOH. Pool max. 5 suspected isolates per subsample and vortex. 3. Include positive (ZUM 3059, IPO 500, IPO 501 and IPO 542) and negative controls. 4. Incubate tubes 10 minutes at 100 C and then put on ice. 5. Centrifuge 1 minute at g. 6. Add 2 µl from the supernatant to 23 µl PCR-mix. Perform two PCR s for each pool: qpcr Cmm-IGS: Master-mix components 1 reaction...x PCR water 8 µl µl 2 x gene expression mastermix 12.5 µl µl 10 µm CmmIGS-F (258a) 0.75 µl µl 10 µm CmmIGS-R (258b) 0.75 µl µl 10 µm CmmIGS-Probe (258c) 1 µl µl Subtotal 23 µl Sample 2 µl Total 25.0 µl qpcr-pat1 BAC: Master-mix componenten 1 reaction...x PCR water 4,5 µl µl 2 x gene expression mastermix 12,5 µl µl 10 µm CmmPat1-R (259b) 0,75 µl µl 10 µm CmmPat2-F (259e) 0,75 µl µl 10 µm CmmPat1-Probe (259c) 1,0 µl µl 10 µm CmmPat1-Probe2 (259d) 1,0 µl µl 10 µm BAC16S-F (183a) 1,0 µl µl 10 µm BAC16S-R (183b) 1,0 µl µl 10 µm BAC16S-Probe (183d) 0,5 µl µl Subtotal 23 µl Sample 2 µl Total 25,0 Remark: Include always a blanco as control. Detection of Cmm in tomato seed 5/16 SPN-B004-v9.1UK

6 Conditions (both PCR s) Step Temp. time Hold 95 C 10'00" 40 cycli 95 C 0'15" 60 C 1'00" Evaluation PCR Check whether positive and negative controls give expected result. Repeat PCR when internal control (UP- BAC) does not react (Ct > 35). Analyse individual isolates from positive pools with both PCR s to determine whether one or more isolates were positive (Ct < 35). Use only PCR positive isolates (one or both PCR s) in pathogenicity assay. In case of doubt perform pathogenicity assay. Decission matrix PCR BAC-PCR Pat 1 IGS Cmm < Not detected < < 35 Suspect, proceed to 5.3 < 35 < Suspect, proceed to 5.3 < 35 < 35 < 35 Suspect, proceed to Test invalid 5.3 Plating on GF-rif Plate PCR suspected isolates from samples and the reference strains (ZUM3059 ( if resistant spike!!), IPO500, IPO501 and IPO542) on GF-rif and GF medium without rif. Suspected and reference isolates (except for the ZUM3059) should have restricted growth on GF-rif. If a suspected isolates does grow on GFrif this might be a cross contamination. Check with responsible how to proceed. If there is no growth on GFrif the isolates is PCR suspected. Send isolates to PD for verification and perform pathogenicity assay (5.4). 5.4 Pathogenicity assay 1. Use for the pathogenicity assay a maximum of 6 or 9 PCR suspected isolates for the 10,000 seeds and 30,000 seeds assay, resp. 2. Inoculate per suspected isolate 2 seedlings of the variety moneymaker with 2-4 real leaves. 3. Dip sterile toothpick in suspected colony and inoculate the stem of the seedling between cotyledon and the first real leaf. Include positive controls (ZUM3059, IPO 500, IPO 501 en IPO 542) and a negative control. Place samples, positive controls and negative controls in different trays to minimise chance of cross contamination. 4. Incubate plants in open trays in the quarantine greenhouse at ca C. 5. Evaluate for symptoms after 7 and 14 days (fig. 4). 6. For valid results the controls should react as expected. Typical symptoms are wilting and the formation of cankers. Note that the virulence of Cmm is highly variable and therefore judge plants symptomatic when the symptoms are limited. Detection of Cmm in tomato seed 6/16 SPN-B004-v9.1UK

7 6. Evaluation and interpretation The presence of avirulent Cmm isolates on seed is known. Avirulent and virulent Cmm s can coexist on seed lots and therefore PCR positive seed lots but pathogenicity negative seed lots should be regarded as high risk. Concentration/dilution plating recovery of spike no Assay not valid yes Indicate in result number of seeds if no recovery of spike in all subsamples Suspected no YDC yes YDC suspected no PCR yes PCR suspected no yes Plating on GF-rif Grow yes Cross contamination? no suspected Isolate to PPS Pathogenicity assay Verification PPS Symptoms visible no confirmed Not confirmed ja Cmm confirmed presence Cmm suspected occurrence Cmm not detected Detection of Cmm in tomato seed 7/16 SPN-B004-v9.1UK

8 The test result will be adjusted when the spike is not recovered in all subsamples: # of seeds in assay Spike recovered in: Additional sentence in attest 10,000 1 subsample This result is valid for 5,000 seeds 30,000 1 subsample This result is valid for 10,000 seeds 2 subsamples This result is valid for 20,000 seeds 7. References 1. ISTA handbook on seed health testing. Working sheet no. 67. Eds. Bolkan, H.A., Waters, C.M., and Fatmi, M. 2. Fatmi, M. and Schaad, N.W Semi selective agar medium for isolation of Clavibacter michiganensis subsp. michiganensis from tomato seed. Phytopathology 77: ISHI-veg seed testing manual. Method for the detection of Clavibacter michiganensis subsp. michiganensis on tomato seeds version 3 January Veg/Tomato_Clavibacter_michiganensis_subsp_michiganensis.pdf 4. Nadkarni, et al. (2002) Determination of bacterial load by real-time PCR using a broadrange (universal) probe and primers set. Microbiology,148,p PT report. 6. Koenraadt, H., van Vliet, A, Neijndorff, N. and Woudt, B Improvement of semi-selective media for the detection of Clavibacter michiganensis subsp. michiganensis in seeds of tomato Phytopathology 99:S66 8. History and revisions - Version 1 of Versie 2 of Reference protocol changed to protocol Naktuinbouw. - Versie 3 of Several minor changes in tekst - Versie 4 van PCR protocol adaptation - Versie 5 of Cosmetic changes. - Versie 6, 7 en 8 van Diverse teditorial changes and adaptation to MMS - Versie 9 of Diverse critical adaptations: A) Introduction of new improved semi-selective media. B) Adaptation spike protocol from the spike of one cocktail subsample to spike of each individual subsample C) Explicit description to dilution plate rapidly after stomaching D) Use of alternative bagmixer with higher efficacy of extraction in comparison with stomacher. E) Use of spike for additional control of media. F) Addition of photographs. G) Introduction of qpcr with improved primer-probe sets. H) Removal of long term storage of seed extracts at -80 C. - version 9.1 Several cosmetic changes in text and addition of flow Detection of Cmm in tomato seed 8/16 SPN-B004-v9.1UK

9 9. Appendices Fig. 1A. Examples of variable morphology of Cmm reference strains (IPO500, IPO501, IPO 542) on SCM fast medium after 7 days incubation at 28 C. Typical are the mucoid nature of the. Often the colony shape becomes more irregular with the increase of size. Size of the is rather variable as well as grey colour. In case of slow growth the are almost black while in fast growing the colour is light gray due to presence of tellurite. Fig. 1B. Examples of variable morphology of Cmm reference strains (IPO500, IPO501, IPO 542) on Cmm1Tris100 medium after 7 days of incubation at 28 C. Typical are the mucoid nature of the. Size of the is rather variable as well as the colour. In general are yellow but pink, orange and white do exist. Transfer if present many suspected mucoid to YDC to increase chance of detection. Note that the colony size was relatively limited since the suspension was made in PB. In sample with seed extract the size of the does increase considerably. Detection of Cmm in tomato seed 9/16 SPN-B004-v9.1UK

10 Fig. 1C. Examples of morphology of of the Cmm spike (ZUM3059) in presence of saprophytic bacteria on SCM fast en Cmm1Tris100 after 7 days of incubation at 28 C. De red arrows indicate the position of suspected Cmm. IPO 542 IPO 501 IPO 500 Fig. 2A. Cmm reference (IPO500, IPO501, IPO 542) on YDC after 3 days of growth at 28 C. Typical is the very mucoid nature of the. After about 5 days of incubation on YDC bacterial slime will start to fall down onto the lid, the speed of growth can be variable. Most have a yellow colour but orange-yellow, pink and even white have been found. Therefore the colour appears less typical than the low viscosity of the. Detection of Cmm in tomato seed 10/16 SPN-B004-v9.1UK

11 Fig. 2B. Examples of colony morphology on YDC after 3 days of incubation. Two have a suspected Cmm identity (red arrows). The other isolates have a too dry or too flat appearance. A B Detection of Cmm in tomato seed 11/16 SPN-B004-v9.1UK

12 Fig. 3. Growth of Cmm strains of GF medium with 50 ppm rifampicin (right side) and without rifampicin (left side). A. rifampicin sensitive Cmm strain IPO 542 B: rifampicin resistant Cmm strain ZUM3059. Potential cross contamination of samples with the spike ZUM3059 must be evaluated to prevent false positives Detection of Cmm in tomato seed 12/16 SPN-B004-v9.1UK

13 Fig. 4. Symptoms by Cmm in the pathogenicity assay. Upper photo s show canker formation at side of inoculation. Lower photo shows one-sided wilting due to plugging of veins and the typical splitting of stem. Avirulent isolates cause small canker and no systemic wilting. Systemic wilting is dependent on RH and more clear during dry and warm conditions. Detection of Cmm in tomato seed 13/16 SPN-B004-v9.1UK

14 9. Appendix Cmm1 (Cmm1tris100) ph 7.7 Add per liter : Sucrose 10,0 g Trizma base (Tris base) 3,32 g TrisHCl 11,44 g MgSO 4 7H 2 O 0,25 g LiCl 5,0 g Yeast extract 2,0 g NH 4 Cl 1,0 g Casein acid hydrolysate (Casamino acids) 4,0g Agar 15,0 g check ph* and autoclave Add after cooling down to ca. 45 C Polymyxin B sulfate Nalidixic acid Nystatine Note: ph is critical and do not adjust the ph. 10 mg 28 mg 100 mg GF-rif (50 mg/l) K 2 HPO 4 MgSO 4. 7H 2 O NaCl NH 4 H 2 PO 4 FeCl 3 * glucose (watervrij) gist extract (SIGMA) Agar Adjust ph 7,2 and autoclave Add after cooling down to ca. 45 C Add 50 mg of rifampicine** Add per liter: 0,40 g 0,05 g 0,1 g 0,5 g 0,01 g 1,0 g 3,0 g 15,0 g *FeCl3 is hygroscopic. Add directly after weighing. ** As an alternative add rifampicine to plates with GF medium. Add in laminar flow 200 l of a 4150 g/ml stock rifampicine and spread with a sterile glass rod. Dry plates for at least 15 minutes. Detection of Cmm in tomato seed 14/16 SPN-B004-v9.1UK

15 SCM* ph 7,3 mscm medium (Duchefa) Yeast extract Check ph 7,3 and autoclave Add per liter 32,2 g 1,9 g Add after cooling down to ca. 45 C Nalidixic acid 20 mg Trimetroprim 80 mg 0,1 g nicotinic acid/50 ml demiwater 50 ml Nystatin 100 mg Potassium tellurite (1%) 1,0 ml Composition mscm medium Agar K 2 HPO 4 KH 2 PO 4 MgSO 4 water freej H 3 BO 3 Yeast extract Sucrose (per liter) 18,0 g 2,0 g 0,5 g 0,122 g 1,5 g 0,1 g 10,0 g Kings B medium Add per liter: KB-medium (Duchefa) 37,23 g 50% glycerol 20 ml Check and adjust ph 7,5 Autoclave Composition KB medium (Duchefa) Proteose K2HPO4 MgSO4 Agar 20 g 1,5 g 0,73 g 15 g (per liter) Detection of Cmm in tomato seed 15/16 SPN-B004-v9.1UK

16 YDC medium Yeast extract (SIGMA) CaCO 3 demiwater Agar (Sigma) CHECK ph = 6,9 and autoclave 15 minutes at 121 C 10,0 g 20,0 g 870 ml 15,0 g Autoclave separately: 20,0 g glucose (anhydrous) in 100 ml demi water After cooling to ca. 50 o C add glucose to bottle with autoclaved and cooled YDC. Mix well before and during pouring of medium in plates. spb Na 2 HPO 4 x 12H 2 O KH 2 PO 4 Na 2 S 2 O 3 Controleer ph 7,4 autoclave 15 minuten at 121 C Add per liter: 19,57 g 1,65 g 0,5 g spbt Na 2 HPO 4 x 12H 2 O KH 2 PO 4 Na 2 S 2 O 3 Check ph 7,4 autoclavere 15 minuten at 121 C Add sterile Tween 20 (10% opl.) Add per liter: 19,57 g 1,65 g 0,5 g 2 ml Detection of Cmm in tomato seed 16/16 SPN-B004-v9.1UK