This document summarizes the objectives, methods, and progress of Work Package 1 of the Phyto-threats project, which aims to analyze the distribution, diversity, and management of Phytophthora in UK plant nursery systems using metabarcoding. The objectives are to analyze Phytophthora communities in nurseries and associated ecosystems using metabarcoding, and model factors influencing community variation. Methods include sampling nurseries and processing samples for Phytophthora detection and metabarcoding. Several Illumina runs have been performed and preliminary results show detection of various Phytophthora clades. Ongoing work includes further sample processing, validation, and linking nursery data with management factors for community modelling.

1. WP1 - Distribution, diversity and
management of Phytophthora in
UK plant nursery systems
Phyto-threats project meeting – 20 Nov 2018
David Cooke, Leighton Pritchard, Peter Cock, 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

3. Methods
• Sampling – fine-scale, broad-scale and community
• Phytophthora detection and metabarcoding
• Computational biology - process metabarcode data
• Interpretation and provision of feedback to owners
• Use of data for Community modelling

4. 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

5. Fine-scale sampling update
• 59 (49 May 18) sample sets from 15 nurseries (6 English, 1
Welsh, 8 Scottish)
• 4016 (2292 May 18) samples plus associated meta-
data
• 2165 (1660 May 2018) PCRed for Phytophthora
– Plant roots 1126 on 150+ plant hosts (JHI & FR)
– Water filters (309)
– Buffer associated with filter (703)
• Root wash
• Irrigation water
• Ponds/ditches/run-off capture
• Equipment washing (e.g. trolleys)
• Water blanks

6. OPAL
• Co-operation with David Slawson
and Vanessa Barber – community
sampling and engagement in
particular areas of recent planting/
regeneration
• Sample kits provided to 5 volunteers
(Nov 2017)
• Training course/video made to
inform volunteers
• 26 samples provided to date

7. Broad-scale sampling
• 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
• 101 (64 – May 18) nurseries sampled to date
– 36 from Scotland
– 65 from England and Wales
• 5-10 root samples per nursery - 653 samples
• Root extractions underway – only 18 samples run to
date
• Top hosts - Rhododendron 40, Viburnum 40, Camelia 23,
Pieris 22, Juniper 19, Chamaecyparis 17, Hebe, Fagus,
Olea, Taxus, Prunus

8. Root extraction optimisation
• Attempt to automate with QIAGEN kit/robot - failed

9. Root extraction info
• Root extractions underway at Hutton and FR

10. PCR test results (all n = 2162)
• 1600 samples still to process

11. Illumina run #1 – Nov 2017
• 250 base pair reads, 15M barcode reads = 156K reads
per sample
• Synthetic control sequences included as a test of
a) technical error rate (Leighton to cover)
b) sensitivity range – 10 attograms 10-18g (weight of a virus)
• Preliminary data - needs full validation against
reference sequences of 45 cultures of known species

12. Illumina plates run to date
• V2 Nano kit 1M
– IGB, GSB, SRP & ECN 20160601
– FR Raintraps 42 PhytoThreats root samples 20180709
• V2 Standard 15 M
– #1 PhytoThreats buffers, filters, roots 20171107 – reported to nurseries
– #2 WP1.3 IGB 20180508 – 34 known species, many unknown & DMs
– #3 Control species, cycle no. controls 20180723 – LP to cover
– #4 SG Survey Soils (68) and PhytoThreats roots (27) 20180814
– #5 PhytoThreats - roots 20180828 (Edin Genomics)
– #6 192 A & D PhytoThreats buffers, roots 201810xx (Edin Genomics)
• Synthetic controls added in each case
• Run through provisional pipeline (MetaPy)

13. Raintrap FR plate examples
• N05 Douglas fir Pseudotsuga menziesii Root samples 2 reps
• Clade 8a P. cryptogea complex 16.3K reads
• Clade 8a P. uliginosa/europaea 3.8K reads
• Clade 1 P. pseudotsugae/cactorum 1.7K reads

14. Raintrap FR plate examples
• N14 field samples of Douglas fir, Noble Fir, Grand Fir,
Oak, Beech & Juniper
• Clade 6 P. megasperma & gonapodyides 50K
• Clade 8a P. cryptogea complex 25K
• Clade 8d P. obscura 1K
• Clade 12 P. quercina 7.5K
• Downy mildews
• N15 similar species

15. #4 plate examples
• N10 samples of Pieris japonica
• Clade 7 P. cambivora/formosa/alni/europaea
39K
• Clade 7 P. cinnamomi 19K
• Clade 2 P. citricola complex 3K

16. #4 plate examples
• N10 Choisya dewitteana (prone to root rot)
• Clade 1 P. nicotianae 42K

17. #4 plate examples
• N10 Pachysandra variegata (prone to root rot)
• Clade 2 P. occultans/citrophthora/terminalis 53K
• Clade 2 P. citricola/pachypleura/plurivora/multivora 2K
• Clade 8 P. cryptogea/pseudocryptogea 4.6K

18. Community modelling – Linking with WP3
 Nursery features
 Size/scale
 Type of trade
 Distributor
 Propagator
 Amenity
 Forestry
 Mixed
 Management practices
 Phytosanitary practices
 Water supply
 Staff Training
 Use of fungicides
 Disease management
 Suppliers
 Seed
 Plants
 Compost
 Location
 Connections by road and rivers
 Surrounding vegetation
 Phytophthora diversity data
 Community richness and composition
 Traits of the species
 Link to nursery and management

19. Ongoing work
 Complete sample processing, PCR and barcoding
 Re-run samples where contamination suspected
 Validate the data with new classifier (Leighton &
Peter)
 Harmonise database and link to Illumina data
 Feedback data to;
 Nursery owners
 CEH
 Plant Health Committee
 Link nursery data to that from natural ecosystems
 Strengthen links with other eDNA communities