By David Cooke, JHI part of WP1 of Phytothreats project

1. WP1 - Distribution, diversity and
management of Phytophthora in
UK plant nursery systems
Phyto-threats project meeting – 03 Oct 2017
David Cooke, Leighton Pritchard, Peter Thorpe, Eva Randall & Beatrix Clark
Ana Perez, Sarah Green, Debbie Frederickson Matika - Forest Research
Tim Pettit - University of Worcester
Bethan Purse - CEH
Jane Barbrook - APHA
Alexandra Schlenzig - SASA

2. Objectives
• WP1 objective I – Using metabarcoding to analyse
community structure in nurseries and associated
ecosystems
– Providing a detailed insight into Phytophthora problems to improve
disease management and advise ‘best practice’
• WP1 objective II – Phytophthora community
modelling
– Seeking explanations for variation in Phytophthora community
richness among nurseries – trade, management and ecology

4. Methods
• Questionnaire – simple (6 questions) to
collect basic data on nursery practices
• Sampling – at many scales
• Phytophthora detection and metabarcoding
• Computational biology to process large
sequence datasets
• Interpretation and provision of feedback to
owners
• Use of data for Community modelling

5. Field
Capture
spores on
filter
Sequence DNA
barcode
Computational
biology
Validation of
species
in sample
Results to
nurseries &
project team
Roots
Lab Computer
Phytophthora-
specific DNA
amplification

6. Sampling – practical issues
• Hosts – mix of known and unknown
• plant parts – mostly roots
• Water flowing through pots – yes
but slow
• Symptomatic or asymptomatic? both
• Control points and contamination
hazards (Parke et al 2012 Plant Disease)
• Water supply – source and run-off
• Balance between time available and
need for detail

7. Work programme
• Nursery survey – questionnaires and leaflets
• Fine-scale sampling of 15 ‘partner nurseries’
– Critical control points sampled over three years, feedback provided
and the effect of mitigations examined
• Broad-scale sampling as part of statutory testing by
PMU and APHA
– Approx 200 samples from 50 nurseries/garden centres England &
Wales and 25 in Scotland to be sampled twice
• OPAL project – co-operation with David Slawson and
staff associated with this project – community
sampling and engagement in particular areas of
recent planting/ regeneration

8. Fine-scale sampling update
• 34 sample sets from 15 nurseries (6 English, 1 Welsh, 8 Scottish)
• 1700 samples plus associated meta-data
• >400 PCR tested for Phytophthora
– Plant roots (93) range of 35 hosts
– Water filters (132)
– Buffer associated with filter (170)
• Washed through plants
• Borehole
• Ponds/ditches
• Equipment washing (e.g. trolleys)
• Water blanks
• Isolations on selected samples
– 3 confirmed P. austrocedri findings
– P. cambivora on shelter belt trees

9. OPAL
• Co-operation with David Slawson
and Vanessa Barber – community
sampling and engagement in
particular areas of recent planting/
regeneration
• Sample kits provided to 4 volunteers
(June 2017)
• Training course/video made to
inform volunteers
• 10 samples provided to date

12. Work programme
• Broad-scale sampling as part of statutory
testing by PMU and APHA
– Target 50 nurseries/garden centres England & Wales
and 25 in Scotland to be sampled in 2017 and 2018
• 27 nurseries sampled to date
– 11 from Scotland
– 16 from England and Wales
• 5-10 root samples per nursery

13. Next steps
Illumina runs (Oct 2017)
• 250 base pair reads, 15M barcode reads = 156K
reads per sample
• Synthetic control sequences generated to be
included in Illumina run as a test of a) sequencing
error rate, b) indexing error c) sensitivity range
• Sample loss during clean-up stage delayed first run
2 weeks ago
• Reference database – current work
• Pipeline …..Pete Thorpe

15. Phytophthora findings by nursery
• Preliminary results suggest differences in practice may influence
findings
+ ve
- ve

16. Phytophthora findings by sample type
• More positive findings from roots
• Blank controls and irrigation water included water tests

17. Observations while sampling
• Material coming onto site
o Water – borehole or mains water best - covers for tanks
important. Local streams or lakes used in some cases –
awareness of origins of water and upstream plant disease
o Compost – not testing but no current concerns
o Planting material – highest risk, native seed, cuttings, trade
from EU or third countries
o Visitors/staff/vehicles – Biosecurity implementation varies.
Isolated concrete pad for all delivery and dispatch avoids
traffic in production areas. Mud a problem in some nurseries.
Footbaths and vehicle washing serves to increase awareness.

18. Observations while sampling
• Dissemination on site
o Plant-to-plant spread least in cells raised above ground
o Puddles commonly positive – improve drainage
o Mud spreads inoculum around site
o Infection in shelter belt trees
o ‘hospital / recovery’ areas in some nurseries generally
not a good idea. Rapid disposal optimal.
o Quarantine new plant material if possible

19. Next steps
• Accelerate lab testing to process sample backlog
• Report Phytophthora findings to nurseries
• Run metabarcoding to identify species present
• Test and validate new computational biology
platform
• Report specific species finding to nurseries
• Data interpretation in relation to management