This document summarizes objectives and methods for a project studying Phytophthora distribution in UK plant nurseries. The project aims to (1) analyze Phytophthora community structure in nurseries using metabarcoding and (2) model factors influencing community richness. Methods include surveying nurseries, sampling plants/water from 10 partner nurseries and 50 broader nurseries, detecting and sequencing Phytophthora DNA, and analyzing results to advise nurseries. Initial sampling of 4 nurseries yielded 316 samples and 40 positive PCR results, showing variability between replicates and hosts. Future plans include continued sampling and sequencing to provide feedback to nurseries.

1. WP1 - Distribution, diversity and
management of Phytophthora in
UK plant nursery systems
Phyto-threats project meeting – 05 Oct 2016
David Cooke, Leighton Pritchard & Peter Thorpe
Ana Perez, Sarah Green, Beatrice Henricot, 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

3. Methods
• Questionnaire – simple (6 questions) to collect basic
data on nursery practices
• Sampling nurseries
– Fine-scale – testing 10 nurseries by project staff for detailed
breakdown of management problems and solutions
– Broad-scale – sampling wider nursery selection alongside statutory
plant health testing
• Phytophthora detection and metabarcoding
• Computational biology to process large sequence
datasets
• Interpretation and provision of feedback to owners
• Use of data for Community modelling

4. 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

5. Sampling – practical issues
• Hosts?
• plant parts?
• Water flowing through pots?
• How many samples per batch?
• Symptomatic or asymptomatic?
• Critical control points and
contamination hazards (Parke et al
2012)
• Water supply – source and run-off
• Balance between time available and
need for detail
• Nursery visits arranged

6. 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

7. Sampling update
• 5 sample sets from 4 nurseries (1 English & 3
Scottish)
• 316 samples – mostly in triplicate
• 228 PCR tested for Phytophthora to date
– Plant roots (96) range of 35 hosts
– Water filters (106)
– Buffer associated with filter (88)
• Washed through plants
• Borehole
• Ponds/ditches
• Equipment washing (e.g. trolleys)
• Water blanks

8. Host list
• Mix of known
Phytophthora hosts
• Others selected based on
symptoms
• Some randomly selected
or requested by owner
Acer pseudoplatanus Populus tremula
Betula pubescens Quercus ilex
Buxus sempervirens Quercus robur
Chamaecyparis lawsoniana Rhododendron sp
Cornus alba Rhododendron sanguineum
Crinodendron hookerianum Rosa canina
Cryptomeria japonica Sequoia sempervirens
Cupressocyparis castlewellan Sequoiadendron giganteum
Cupressocyparis leylandii Sorbus aucuparia
Fagus Sylvatica Syringa vulgaris
Hebe rakaiensis Taxus baccata
Ilex aquifolium Viburnam tinus
Juniper Old Gold Viburnum davidii
Juniper tamariscifolia Viburnum tinus
Juniperus squamata 'blue carpet' Xanthocyparis vietnamensis
Juniperus virginiana
Lavandula angustifolia Hidcote'
Photinia fraseri 'red robin'
Pinus armandii
Pinus sylvestris

13. Bulk sampling
• Plants tested straight off
truck from continent
• Hebe rakaiensis roots and
filters +ve
• Drainage channel also
sampled in holding house
full of plants
• Puddles on site

14. PCR results
• n = 228
• +ve = 40 (not yet enough for a plate)
• Higher proportion of filters +ve (considering many blanks included)

15. Reproducability
• Replicates from same batch giving different
results
• Re-testing of a batch of buffer sample DNA
yielded some inconsistencies (technical reps)
• Working on PCR control (plant DNA) and
optimising DNA dilution
• Further work to understand cause

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

17. Lessons to date
• Time consuming
• Nurseries vary/ no ‘one size fits all’ approach
• Great host diversity
• Nurseries working to a high standard and staff very supportive

18. Future plans
• Continue sampling in Scotland (Oct)
• Commence sampling in England (Beatrice and Tim - Nov)
• Complete Illumina plate with controls (Nov 2016)
• Send sampling protocols to APHA and SASA for comment
and completion (Oct)
• Arrange sampling packs in SAEs for dispatch (Oct)
• Co-ordinate with Dave Slawson on OPAL sampling (Oct)
• Continue work on metabarcode pipeline