REFERENCE PROTOCOL NAKTUINBOUW Detection of Clavibacter

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


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.


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.


Cmm1 or SCMF for

all subsamples

Suspect

colonies

No

suspected

colonies

Cmm not

detected


Cmm1 or SCMF for

all subsamples

No suspect

colonies

N.a Cmm not

detected

* N.a = Not applicable


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.


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"


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


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


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.


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


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


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


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.


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 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.


SCM* pH 7,3 Add per liter

mSCM medium (Duchefa) 32,2 gYeast extract 1,9 g

Check pH 7,3 and autoclave


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


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

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REFERENCE PROTOCOL NAKTUINBOUW Detection of Clavibacter