1. Experiences in Controlling and Managing Ralstonia solanacearum in Scotland Planning, Sampling, Host Plants, Water and Laboratory Analysis
Karen Fraser
Bacteriology Unit Manager
Science and Advice for Scottish Agriculture (SASA)
Scotland

2. Science and Advice for Scottish Agriculture (SASA) is part of Scottish Government. Its primary role is to provide scientific services and advice in support of Scotland’s agriculture and wider environment.
It consists of a community of more than a hundred life scientists and analytical chemists, with its expertise in life science ranging from botany, plant pathology, zoology through to molecular biology.

3. The Official Authority Scotland
Scottish Government
Agriculture, Food and Rural Communities Directorate
Policy
Agriculture and Rural Development Division
Science
SASA
Operations
(Inspectorate)
Rural Payments and Inspections Division

4. Potato Production - Scotland
Highly specialised in seed potatoes
Ideal climate for growing potatoes
Free from many important potato pests and diseases
Second largest seed potato exporter in Europe to non-EU countries, 30% seed production exported outside the EU, around 65,000 export tonnes per year (10,000 ware), 50% England, 20% EU
Recognised by the EU as a high quality Community Grade Area, stricter health standards applied, produces only Pre-basic and Basic seed. Lower quality Certified seed (blue labels) is not permitted for planting in Scotland for seed or ware production (ware – seed or no more than once grown farm saved seed)
All stages of production are monitored by government officials
Seed potatoes produced and marketed in Scotland must be classified under the Seed Potato Classification Scheme (SPCS)
SASA is the Certifying Authority

5. Scottish Seed Potato Crops (2010/11) No. of producers: 250
No. of registered crops:
4,468
Area planted (ha):
11,374
No. of varieties grown:
234
Approx. tonnage marketed
264,000
of which was for non-EU export
76,000
Aberdeen
Dundee
Edinburgh
seed
ware
Scottish Ware Potato Crops (2010/11)
No. of producers:
329
Area planted (ha):
14,656

6. Scottish Seed Potato Classification Scheme
Classes of seed potatoes produced in Scotland
Nuclear Stock (SASA microplants) 
Pre-basic TC (minitubers)
Pre-basic 1 - 4
Basic Super Elite
SE 1 - 3
Basic Elite
E 1 – 3
Basic A
The Seed Potatoes (Scotland) Regulations 2000 (as amended).
Official limit to number of generations produced at each class ensuring older stocks are flushed out.
Minimum health standards for each class are maintained by inspection of the growing crop.
Growing crop officially inspected at least twice during the growing season.

7. Potato production Scotland - Nuclear stock
Seed stocks in Scotland originate from nuclear stock produced by SASA.
Ensures starting material is pathogen-free according to a programme of official testing for indigenous and EU-quarantine pathogens, including C. m. ssp. s and R. s. and Dickeya spp.

8. Potato Production Scotland Nuclear Stock
C. 700 varieties (lines) are held in the Nuclear Stock collection at SASA on behalf of the potato industry
Only officially approved laboratories multiply SASA microplant material
Microplants are grown on in peat or hydroponics/aeroponics to produce mini- tubers - protected, pest and disease free environment
All PBTC crops are monitored at SASA in post control field plots

9. Seed Potato Certification Scheme Field Requirements
Pest Freedom of Land
Free from potato cyst nematodes (Globodera rostochiensis and G. pallida), official testing of soil samples before planting
No known occurrence of wart disease (Synchytrium endobioticum)
Rotation interval
7 years for Pre-basic years for Basic seed potato crops
Separation requirements
PB crops must be grown on dedicated farms
All seed farms must plant class E seed or better

10. Seed Within the EU From Outside the EU
Only seed potatoes classified at Community Grade can be introduced into Scotland from the EU.
Movement is covered by plant health legislation (EC Plant Passport) and seed potato certification.
Provides a safeguard against introduction of pathogens such as Ralstonia solanacearum (brown rot), Clavibacter michiganensis ssp. sepedonicus (ring rot), and Potato spindle tuber viroid, which have never been found in potatoes in Scotland.
From Outside the EU
Imports of seed potatoes from outside the EU are permitted only through quarantine units.
UK Potato Quarantine Unit is based at SASA.

11. Brown Rot: Distribution
Caused by Ralstonia solanacearum. 2 distinct divisions split into biovars. Division I (biovars 3, 4, and 5) and Division II (biovars 1 and 2). Biovar 2 race 3 main pathogen of potatoes and most commonly found in Europe.
Primarily/historically a pathogen of tropical and warm-temperate regions (has occurred in Egypt, Algeria, Israel, Morocco, Syria & Tunisia).
EU: First major outbreak - Sweden Been found in Belgium, France, Austria, Germany, The Netherlands, Spain, Greece, Hungary, Eire, Italy, Portugal, Slovakia. There have also been outbreaks in England and Wales, which have been eradicated, although a number of rivers have been designated and remain contaminated.
No outbreaks in potatoes in Scotland! Important to maintain procedures to prevent introduction. Scotland has a Contingency plan in place which is regularly reviewed and up-dated. The aim is to prevent introduction of the disease to Scotland or if found to prevent its spread within Scotland.

12. Policy and Legislation Background Publicity Surveillance and testing
CONTINGENCY PLAN FOR POTATO BROWN ROT (Ralstonia solanacearum (Smith) Yabuuchi et al.) March 2011
Introduction
Scope
Policy and Legislation
Background
Publicity
Surveillance and testing
Action on suspicion of brown rot
Action on confirmation of brown rot
Control measures

13. Provides guidance for Scottish Government staff on;
CONTINGENCY PLAN FOR POTATO BROWN ROT (Ralstonia solanacearum (Smith) Yabuuchi et al.) March 2011
Introduction
This document acts as The Scottish Government’s Eradication and Containment Plan for potato brown rot.
Forms part of the Emergency Plan for the Plant Health Service in Scotland.
Scope
Provides guidance for Scottish Government staff on;
policy and legislation
symptoms
factors relevant to prevention, detection and control
procedures for surveillance and detection
procedures for the management of any outbreak

14. Policy and Legislation
R. solanacearum EU Annex I/AII listed, a harmful organism known to occur in parts of the Community, but whose introduction and spread is prohibited.
As a significant threat to potato production the EU introduced legislation in the form of Directive 2006/63/EC (amending EC Directive 98/57/EC).
Phytosanitary measures in this Directive aim to prevent entry of the organism into the EU and if found, to determine the distribution and to contain, eradicate and control.
Requires member states to carry out surveys involving significant inspection and surveillance activities.
Annexes set out approved testing procedures and the required actions to be taken in the event of a confirmed outbreak.

15. Scotland - Controlling Introduction and Spread
Member States have freedom to take additional or stricter measures where necessary
The Plant Health (Potatoes) (Scotland) Order notification of all potatoes for planting (1 January 2007)
Increased information on origin of seed used to plant ware, location of ware crops, irrigation sources used, etc.
Testing introductions and imports
Statutory notification of all introductions/imports from certain countries

16. What can industry/growers do?
Consider where seed and potatoes are sourced from
Industry-lead voluntary ban on seed imports
~99.5% seed potatoes produced in Scotland originate from Scotland
Risk of brown rot, ring rot and Dickeya is higher with imported seed
Join the Safe Haven Scheme (Industry voluntary scheme)
Apply strict separation of seed and ware potatoes
Risks particularly high for grower/merchants
Non-returnable bags
Clean and disinfect machinery, containers, etc.
Think about how tasks are ordered: Low risk → High risk
Take care with the disposal of soil and waste

17. The Safe Haven Scheme
Two seed production units represented in chain, in practice could be more.
Arrows labelled C, seed stocks leaving the Safe Haven Certification Scheme to non-certified units.
Seed stocks are NOT allowed to pass in the opposite direction.
Overseas varieties will remain available through either:
• Import of disease free plant material through existing quarantine service and multiplication through the Safe Haven certified supply chain.
• Import of seed potatoes by growers choosing not to join the Safe Haven scheme. This seed is excluded from entering the Safe Haven certified supply chain.
Additional level of security
Complete tractability of Scottish source

18. Brown Rot: Tuber Symptoms
Early stages
brownish staining of the vascular tissue
Pale bacterial ooze may exude from vascular ring
Advanced stages
necrosis and secondary rots in the surrounding tissue
Severe infections
Bacterial ooze may exude from the eyes and the heel end, causing soil and debris to adhere to the tuber

19. Brown Rot: Foliar Symptoms
Earliest symptom is wilting of the youngest leaves, wilting progressive from the top of the plant
Wilting may be initially confined to only one stem
Recovery at night when temperature drops or after irrigation
Eventually rapid, complete wilt and subsequent desiccation

20. Brown Rot: infection route
Clonal multiplication links - planting infected seed. Latent infection can pass through 1 or more field generations without causing detectable symptoms in stems and tubers
Import of potatoes for planting (ware & seed) Highest risk; seed more so
Irrigation with infested water or flooding of potato crops
Processing - Discharge of untreated waste from the handling/processing of infected imported ware potatoes. Poor separation. Infested/infected soil/debris/waste
Physical contact - Contaminated equipment/containers/transportation
Alternative hosts

21. Other Hosts Solanum dulcamara
In almost all European outbreaks associated with contaminated water, woody nightshade (Solanum dulcamara) plants with roots growing in contaminated watercourses have been found to be infected.
These plants then serve as a disease reservoir.

22. Other Hosts
Tomatoes - although there is no significant commercial production of tomatoes in Scotland, potato growers and inspectors are vigilant for any links to tomato production or packing since many pests and diseases are common to both
Pelargonium - imported cuttings
In Scotland, if any symptoms of bacterial wilt observed in the above, official horticultural inspectors will sample affected stems or whole plant and submit to SASA for testing
Solanum nigrum

23. Brown rot, Ring rot and Dickeya : Scottish Surveillance Programmes
Quarantine and Nuclear stock
Statutory EU tuber surveys seed and ware
Non-Scottish seed; pre-planting and post - harvest
Scottish watercourses
Processing plants – imported material
Growing crop inspections – stem survey for Dickeya

24. Brown rot, Ring rot and Dickeya : Tuber Surveys – Sampling Strategy
Approximately 1000 seed lots are sampled and tested annually, representing 25% of Scottish production (sampling density of 10 ha/sample) and 300 ware (67 ha/sample)
Sampling carried out by SG Official Inspectors
Samples may be taken from field or store
Routine testing of 200 tubers, as required by the Directive
This gives an 87% chance of detecting a 1% level of infection, assuming that the sample is representative of the total lot.
For higher risk stocks, 3 x200 tubers
No level of testing will eliminate risk!

25. Ring rot: Sampling strategy
In previous years 200-tuber samples were drawn from each stock and tested for ring rot & brown rot
Experience of outbreaks in England & Wales has indicated this may not be sufficient to pick up very low level infection
Approximate number of tubers tested to give a 95% probability of getting a positive result
Note: the infection level in the Welsh ring rot outbreak was estimated to be approximately 0.5%

26. Scottish Ring Rot & Brown Rot Surveys: 1994-2011

27. Scottish Brown Rot, Ring Rot & Dickeya Tuber Surveys: Sept - Feb
Stocks targeted based on risk.
Seed
Stocks grown from non-Scottish seed
Stocks from irrigated crops
Seed crops grown next to previously contaminated watercourses
1 stock from every seed grower in Scotland
Seed stocks targeted to be included in the Dickeya survey
Additional Scottish seed stocks (Inspectors to select)
Ware
1 ware stock from every seed farm producing ware
Ware production from non-Scottish stocks
Ware stocks targeted to be included in the Dickeya survey
Other Scottish ware production from non-seed producing farms

28. Bacteriology Laboratory, Quarantine Facility,
Laboratory Testing
Directive requires a 3-stage testing procedure comprising of an initial screening test
If initial results are suspect/positive,
further diagnostic testing
Final confirmatory pathogenicity test
Validated methods - SMT Ring Tests
Bacteriology Laboratory, Quarantine Facility,
SASA
EUPHRESCO Project EUPH03 Interlaboratory test on detection of C. m. ssp. s and R. s. in potato tubers. Verify performance of official tests in the EU methods 2006/56/EC and 2006/63/EC

29. Processing tubers for ring rot, brown rot and Dickeya testing
- laboratory testing required for latent infection
test based on a 200 tuber sample
- core taken from the heel end of each tuber
- bacterial extraction
- visual examination of cut tubers

30. Ralstonia solanacearum: Diagnostics
Core screening test is selective isolation
Plated onto semi-selective SMSA media following serial dilution
Sensitivity 103 cfu/ml
Incubation 3-6 days 28oC
Typical/suspect colonies
Purification and further testing

31. Ralstonia solanacearum: further testing – Conventional PCR
Seal et al. (1993) Primers OLI-1/Y2 expected amplicon size from R.s. DNA = 288 bp
(Pastrik 2002) Primer Rs-1-F Primer Rs-1-R expected amplicon size from R.s. DNA = 718 bp
(Pastrik and Maiss 2000) PCR using Invitrogen extraction and primers PS-1/PS-2
expected amplicon size from R.s. DNA = 553 bp

32. Ralstonia solanacearum: further testing – Real time PCR
Fluorogenic DNA probes
Fluorescent reporter and quencher dye
During amplification probe digested by Taq polymerase separating the dyes resulting in an increase in reporter fluorescence
Repeated PCR cycles – exponential amplification of PCR product corresponding increase in measurable fluorescence intensity
Facilitates quantification of amount of sample DNA
(Weller et al 2000)
Real-time PCR TaqMan® assay, Typical amplification plot showing increase in fluorescence from target DNA

33. Immunofluorescence
Core screening test C. m. ssp. s, immunofluorescent antibody staining
Proven robustness to achieve required thresholds, sensitivity 103 Cfu/ml
Cells targeted with an antibody and fluorescent labeled conjugate
Viewed under fluorescent microscopy, typical morphology, staining coverage, cell numbers
Controls
Loewe validated antibody for R. s.

34. Tomato Plant Bioassay Final confirmatory, pathogenicity test
Inoculated into stem via syringe method (Janse 1988), controls
Check for typical symptoms of the disease
Up to 28 days incubation at 28oC
Extraction, plating, strain characterisation

35. Strain Characterisation
Biovar Determination
PCR Methods
Biovar
Utilization of: I II
III IV V
Maltose - +
Lactose
Cellobiose
Mannitol
Sorbitol
Dulcitol
primer set RS1/RS2 to discriminate Divisions I and II of R. solanacearum

36. Surveillance of Scottish Watercourses for Ralstonia solanacearum
Co-ordinated in accordance with
Commission Directive 2006/63/EC
- Watercourses identified as a source of irrigation
Every irrigation source tested over a
3-year rolling programme
- Rivers at risk
Watercourses of the Tay River system
Surveys performed during Aug/Sept
3 x 250ml samples, selective plating

37. Surveillance of Scottish Watercourses for Ralstonia solanacearum
Most watercourses surveyed are on the East coast - 90% potato production
2000 survey revealed parts of the Tay river system in Perthshire contaminated with Ralstonia solanacearum
Extensive epidemiology study to determine the extent of the infection
Irrigation ban placed on the contaminated watercourses in 2000

38. Tay Water System Lunan Burn River Isla River Tay
High proportion of Infected Solanum Dulcamara plants growing in vicinity of Lunan burn, 90% infected, source of bacterium in water
Ware crops irrigated from the watercouse in 2000, seed and ware crops grown in the vicinity sampled and tested – no R.s. detected. All crops grown in the vicinity to date targeted as part of the tuber surveys
Concluded that R. solanacearum infection confined to the watercourses
Carsie Sewage works was thought to be the original source, no R. s. detected in effluent

39. Eradicating S. dulcamara from Tay river system
Eradication programme 2001 to 2003
Glyphosate herbicide treatment applied to plants with roots in or 1 meter from water. Applied twice in growing season June and Sept kill any plants missed and re-growth
The effectiveness of the exercise assessed
by monitoring numbers of bacterium in the water throughout the growing season
AUG cfu/ml
AUG cfu/ml was detected
Close monitoring of watercourses - 6 sample sites 4 x 250mls – continued throughout summer at fortnightly intervals until 2006, then monthly until 2011
No Ralstonia contamination has been detected in the Tay river system since 2001

40. Eradication of R. solanacearum from Tay river system
In 2004 large stretch of Tay banks were checked for S. dulcamara. All plants found tested negative for R. s.
The irrigation ban was lifted from the Lunan Burn & River Isla on 1 April, 2004 and from the River Tay on 1 April, 2005.
River Tay, Perthshire, Scotland
Although S. dulcamara is beginning to re-establish along banks. Any plants found in the vicinity of the sampling points tested negative for R. s. to date.
Absence of the bacterium in all samples studied from 2002 – 2011 and demonstrated reduction of contamination levels in 2001 suggests the spraying exercises of have been successful in eradicating R. solanacearum contamination from Tay River system.
River system will be incorporated into the annual survey of Scottish watercourses 2012.

41. Development work - following contamination of Tay River System
Flexible Fund Project;
Ralstonia solanacearum Contamination of Scottish Rivers: Identifying the Risks to High-Grade Seed Production 2003 – 2006
Collaborative project between SASA, CSL & SCRI
Study epidemiology of R. solanacearum in a Scottish setting
Based on the findings “Rivers at Risk” have been incorporated into the annual Surveys
PhD - Studies on the genetic variation between isolates of the potato brown rot pathogen Ralstonia solanacearum

42. Action and Control – suspect
Advisable to start action prior completion of bioassay
To minimise risk of further spread “notice” served on premises which prohibits
- planting of any tubers
- movement of potato plants and tubers on/off the premises
Associated stocks (sister stocks, grown on the same farm etc.) sampled at a higher rate to detect low levels of infection
- 20 x200 tubers
Trace source - investigate other potential links including;
other potato material which has been on the premises
shared equipment or contractors
any machinery, vessel, store, packaging etc

43. Ring rot: Trace-back 2004 outbreak
1998
1999
2000
2001
2002
2003
Herefordshire
Ware
Herefordshire
Ware
2004

44. Action and Control – confirmed
Statutory notice served confirming the presence of brown rot
Investigate extent of contamination and possible spread
Creation of a demarcated zone
EU notification
Control Measures
- Disposal and disinfection of contaminated/probably contaminated items
- Cropping and related restrictions on affected fields/premises
within demarcated zone for several years - as specified in the directive

45. Summary
Fortunately Scotland has not had to deal with an outbreak of Ralstonia solanacearum in tubers.
Protected area within the UK, Safe Haven Scheme, closed system, little imported material, local legislation.
Effective eradication of Ralstonia from the Tay river system. Rivers in Scotland generally cold and fast flowing not favourable for S. dulcamara growth.
Continued extensive monitoring programmes in Scotland for R. solanacearum, C. m. ssp. s and Dickeya spp.