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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
Detection of Cmm in tomato seed 2/16 SPN-B004-v9.1UK
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 30.000 seeds (3 subsamples of 10.000 seeds each) or
10.000 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 semi-
selective media. Suspected colonies are transferred to YDC. YDC suspected colonies are screened
with two complementary Cmm-specific real-time PCRs. Finally, a pathogenicity assay is performed
to confirm the identity of the PCR suspected colonies. 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 Clavibacter michiganensis subsp. michiganensis
Cmm1 Tris buffered semi-selective medium (Cmm1-Tris100)
GF-rif Growth factor medium with rifampicine
SCMF SCM-fast: semi-selective medium on basis of SCM
KB Kings B medium (non selective)
YDC Yeast-Dextrose-CaCO3 medium (non selective)
TGW Thousand grain weight
PCR Polymerase chain reactie
sPB Steril phosfate buffer
sPBt Sterile phosphate buffer with Tween20
DNA Desoxyribonucleinezuur
4. Materials ZUM 3059 stock suspension in 25% glycerol stored in -80 °C (ca. 1,5 106 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
primer-collection
Sequence
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 3/16 SPN-B004-v9.1UK
5. Method
5.1.1 Dilution plating phase
1. Determine TGW and weigh 10.000 (2 x 5.000) or 30.000 (3 x 10.000) seeds in a stomacher bag.
2. Add per bag with 5.000 seeds 100 ml and per bag with 10.000 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 4.
5. 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 9.000 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.
5.1.2 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.
5.1.3 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 542.
18. Plate 100 µl from each spike and dilutions from reference on Cmm1, SCMF and KB.
19. Incubate plates 7 days at 28 °C.
5.1.4 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 20-50 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.
5.1.5 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 4/16 SPN-B004-v9.1UK
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 colonies.
Non spiked samples 27. Evaluate all subsamples for the presence of Cmm suspected colonies.
28. Transfer Cmm suspected colonies from all subsamples to YDC (10 colonies 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 colonies 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 colonies. Regard colonies 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, colonies could have orange, pink and
white appearance.
5.1.6 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
colonies
N.a invalid .
No* recovery on
Cmm1 and SCMF
for part of
subsamples
No suspected
colonies
N.a Cmm not
detected
Report valid result for
reduced number of seeds
(see 6)
No* recovery on
Cmm1 and SCMF
for part of
subsamples
Suspect
colonies
No
suspected
colonies
Cmm not
detected
Report valid result for
reduced number of seeds
(see 6)
No* recovery on
Cmm1 and SCMF
for part of
subsamples
Suspect
colonies
Suspected
colonies
Suspect Continue with 5.2.1.
Report later number of
seeds for which result is
valid (see 6)
≥ 1 spike colony on
Cmm1 or SCMF for
all subsamples
Suspect
colonies
Suspected
colonies Suspect Continue with 5.2.1.
≥ 1 spike colony on
Cmm1 or SCMF for
all subsamples
Suspect
colonies
No
suspected
colonies
Cmm not
detected
≥ 1 spike colony on
Cmm1 or SCMF for
all subsamples
No suspect
colonies
N.a Cmm not
detected
* N.a = Not applicable
Detection of Cmm in tomato seed 5/16 SPN-B004-v9.1UK
5.2.1 PCR on YDC suspected colonies
1. Check all YDC suspected colonies with PCR.
10.000 seeds max. 20 isolates (4 pools of 5
isolates)
30.000 seeds max 30 isolates (6 pools of 5
isolates)
2. Transfer with sterile pipette tip or toothpick YDC suspected colonies 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 10.000 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 6/16 SPN-B004-v9.1UK
5.2.2 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
< 35 ≥ 35 ≥ 35 Not detected
< 35 ≥ 35 < 35 Suspect, proceed to 5.3
< 35 < 35 ≥ 35 Suspect, proceed to 5.3
< 35 < 35 < 35 Suspect, proceed to 5.3
≥ 35 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 GF-
rif this might be a cross contamination. Check with responsible how to proceed. If there is no growth on GF-
rif 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. 25-30 °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.
Conditions (both PCR’s)
Step Temp. time
Hold 95°C 10'00"
95°C 0'15" 40 cycli
60°C 1'00"
Detection of Cmm in tomato seed 7/16 SPN-B004-v9.1UK
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.
Suspected colonies
Symptoms visible
Verification PPS
Concentration/dilution plating
YDC
PCR suspected colonies
PCR
suspected
YDC suspected colonies
Pathogenicity assay
Cmm confirmed presence
Cmm not detected
Isolate to PPS
Cmm suspected occurrence
Grow colonies
Plating on GF-rif-rif
Not confirmed
yes
yes
yes
no
no
no
Cross contamination?
yes
ja
confirmedno
no
recovery of spike
yes
Assay not valid
Indicate in result number of seeds if no recovery of spike in all subsamples
no
Detection of Cmm in tomato seed 8/16 SPN-B004-v9.1UK
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
1 subsample This result is valid for 10,000 seeds 30,000
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. 1998. Semi selective agar medium for isolation of Clavibacter michiganensis
subsp. michiganensis from tomato seed. Phytopathology 77:121-126.
3. ISHI-veg seed testing manual. “Method for the detection of Clavibacter michiganensis subsp. michiganensis on
tomato seeds” version 3 January 2008.
http://www.worldseed.org/cms/medias/file/FocalPoints/PhytosanitaryMatters/SeedHealthTesting/ISHI-
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.257-266
5. PT report.
6. Koenraadt, H., van Vliet, A, Neijndorff, N. and Woudt, B. 2009. 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 07-07-0 - Versie 2 of 30-03-04 - Reference protocol changed to protocol Naktuinbouw. - Versie 3 of 16-11-2006
Several minor changes in tekst - Versie 4 van 01-12-2007 PCR protocol adaptation - Versie 5 of 01-01-2008
Cosmetic changes. - Versie 6, 7 en 8 van 02-02-09 Diverse teditorial changes and adaptation to MMS - Versie 9 of 07-02-2010
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 9/16 SPN-B004-v9.1UK
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 colonies. Often the colony shape becomes more irregular with the increase of size. Size of the colonies is rather variable as well as grey colour. In case of slow growth the colonies are almost black while in fast growing colonies 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 colonies. Size of the colonies is rather variable as well as the colour. In general colonies are yellow but pink, orange and white colonies do exist. Transfer if present many suspected mucoid colonies 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 colonies does increase considerably.
Detection of Cmm in tomato seed 10/16 SPN-B004-v9.1UK
Fig. 1C. Examples of morphology of colonies 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 colonies.
Fig. 2A. Cmm reference colonies (IPO500, IPO501, IPO 542) on YDC after 3 days of growth at 28 °C.
Typical is the very mucoid nature of the colonies. 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 colonies have a yellow colour
but orange-yellow, pink and even white colonies have been found. Therefore the colour appears less typical
than the low viscosity of the colonies.
IPO 542
IPO 500
IPO 501
Detection of Cmm in tomato seed 11/16 SPN-B004-v9.1UK
Fig. 2B. Examples of colony morphology on YDC after 3 days of incubation. Two colonies 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 12/16 SPN-B004-v9.1UK
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 13/16 SPN-B004-v9.1UK
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 14/16 SPN-B004-v9.1UK
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
MgSO4 • 7H2O 0,25 g
LiCl 5,0 g
Yeast extract 2,0 g
NH4Cl 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 10 mg
Nalidixic acid 28 mg
Nystatine 100 mg Note: pH is critical and do not adjust the pH.
GF-rif (50 mg/l) Add per liter:K2HPO4 0,40 g
MgSO4 . 7H2O 0,05 g
NaCl 0,1 g
NH4H2PO4 0,5 g
FeCl3 * 0,01 g
glucose (watervrij) 1,0 g
gist extract (SIGMA) 3,0 g
Agar 15,0 g
Adjust pH 7,2 and autoclave
Add after cooling down to ca. 45 °C
Add 50 mg of rifampicine**
*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 15/16 SPN-B004-v9.1UK
SCM* pH 7,3 Add per liter
mSCM medium (Duchefa) 32,2 gYeast extract 1,9 g
Check pH 7,3 and autoclave
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 (per liter)
Agar 18,0 gK2HPO4 2,0 g
KH2PO4 0,5 g
MgSO4 water freej 0,122 g
H3BO3 1,5 g
Yeast extract 0,1 g
Sucrose 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) (per liter)
Proteose 20 g
K2HPO4 1,5 g
MgSO4 0,73 g
Agar 15 g
Detection of Cmm in tomato seed 16/16 SPN-B004-v9.1UK
YDC medium Yeast extract (SIGMA) 10,0 g
CaCO3 20,0 g
demiwater 870 ml
Agar (Sigma) 15,0 g CHECK pH = 6,9 and autoclave 15 minutes at 121 °C Autoclave separately: 20,0 g glucose (anhydrous) in 100 ml demi water
After cooling to ca. 50 oC add glucose to bottle with autoclaved and cooled YDC. Mix well before and during pouring of medium in plates.
sPB Add per liter:
Na2HPO4 x 12H2O 19,57 g
KH2PO4 1,65 g
Na2S2O3 0,5 g Controleer pH 7,4
autoclave 15 minuten at 121 °C
sPBt Add per liter:
Na2HPO4 x 12H2O 19,57 g
KH2PO4 1,65 g
Na2S2O3 0,5 g Check pH 7,4
autoclavere 15 minuten at 121 °C
Add sterile Tween 20 (10% opl.) 2 ml