This document summarizes the objectives, methods, and progress of a project studying Phytophthora in UK plant nursery systems. The project aims to analyze Phytophthora communities in nurseries using metabarcoding, and model variation among nurseries based on trade, management, and ecology factors. Methods include surveying nurseries, sampling at fine and broad scales, detecting and identifying Phytophthora via sequencing, and providing feedback. To date, over 1000 samples from 15 nurseries have been collected and 395 tested, finding Phytophthora in roots and irrigation water. Next steps include broad-scale sampling, resampling partner nurseries, extracting and sequencing additional samples, and estimating sequencing error rates.

1. WP1 ‐ Distribution, diversity and
management of Phytophthora in
UK plant nursery systems
Phyto‐threats project meeting – 04 May 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. Opening 2018
V&A Museum of Design Dundee

3. Spread of novel P. infestans clones
Live maps at
http://euroblight.net/pathogen‐characteristics‐and‐host‐resistance/sampling‐sites‐and‐genotype‐maps/

4. 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

5. Methods
• Questionnaire – simple (6 questions) to collect basic
data on nursery practices
• Sampling
– Fine‐scale – testing nurseries by project staff for detailed
breakdown of management problems and solutions
– Broad‐scale – sampling wider nursery selection alongside statutory
plant health testing
– OPAL – engagement with community sampling
• Phytophthora detection and metabarcoding
• Computational biology to process large sequence
datasets
• Interpretation and provision of feedback to owners
• Use of data for Community modelling

6. Field
Capture
spores on
filter
Amplify DNA of
pest/pathogen
Sequence DNA
barcode
Bioinformatics
to identify
species
in sample
Results to
inspectors &
project team
Roots
Lab Computer

7. 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

8. Work programme
• Nursery survey – questionnaires and leaflets
• Fine‐scale sampling of 10 ‘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

9. Sampling update
• 18 sample sets from 15 nurseries (6 English, 1 Welsh, 8 Scottish)
• 1009 samples – mostly in triplicate (includes blanks)
• 395 PCR tested for Phytophthora to date
– 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

10. Samples taken per nursery

11. Sample types for testing

12. Sample types for testing

13. Phytophthora findings by nursery
• Differences between nurseries emerging

14. Phytophthora findings by sample type
• More positive findings from roots
• Blank controls and irrigation water included in
filters and buffer findings

15. Types of nursery
• Water – mostly borehole; some mains water (plus rainwater)
or river water (‘filtered’)
• Plants all above‐ground in cells ‐ to all on or in ground
• Production – From holding nurseries/wholesalers importing
most stock to entire production of native plants on site
• Garden centres/nurseries, trees only, mixed
amenity/horticulture and hedgerow trees
• Generally good awareness of plant health issues but fungicide
use widespread

22. Next steps
Sampling
• Broad‐scale ‐ APHA and PMU – sample instructions
and pack drafted for 2017 sampling
• OPAL – Co‐ordinating with David Slawson and
Vanessa Barber ‐ samples to be collected with
communities via contact points in Plymouth,
Cardiff, North Wales and Glasgow
• Resample fine‐scale nurseries once results known
Sample extraction
• Extract all root samples
• Decision on buffer versus filter extraction

23. Next steps
Illumina run (May 2017)
• Selection of 176 positive findings for first Illumina
run
• 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
• Pipeline …. Pete Thorpe

24. Reproducability
Furlan et al 2016 A framework for estimating the
sensitivity of eDNA surveys Mole Ecol Res 16, 641‐54
• Clumpiness?
• Error?