WP1 Phytophthora distribution, diversity and management in UK nursery systems

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