This document summarizes work from Project WP1 which aims to analyze the community structure of Phytophthora in UK plant nurseries and associated ecosystems. The objectives are to provide insights into Phytophthora problems to improve disease management using metabarcoding, and to model factors influencing Phytophthora richness between nurseries. Methods described include sampling plants and water from nurseries, detecting and sequencing Phytophthora DNA, and using the data for modeling. Preliminary results found various Phytophthora species in water sources, puddles, and plant roots from sampled nurseries. Ongoing work includes validating analysis methods and sequencing more samples to better understand relationships between management practices and Phytop

1. WP1 - Distribution, diversity and
management of Phytophthora in
UK plant nursery systems
Phyto-threats project meeting – 26 Apr 2018
David Cooke, Leighton Pritchard, Peter Thorpe, Eva Randall, Beatrix and Mhairi 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. 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

5. 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

6. Fine-scale sampling update
• 49 (34 Oct 17) sample sets from 15 nurseries (6 English, 1 Welsh, 8 Scottish)
• 2292 (1700 Oct 17) samples from 150 plant hosts plus
associated meta-data
• 1660 (400 Oct 2017) PCR tested for Phytophthora
– Plant roots (617)
– Water filters (193)
– Buffer associated with filter (850)
• Root wash
• Irrigation water
• Ponds/ditches/run-off capture
• Equipment washing (e.g. trolleys)
• Water blanks
• Isolations on selected samples
– 3 confirmed P. austrocedri findings
– P. cambivora on shelter belt trees

7. 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

8. 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
• 64 (27 - Oct 17) nurseries sampled to date
– 25 from Scotland
– 39 from England and Wales
• 5-10 root samples per nursery - 408 samples
• Root extractions remain to be run to provide
results later in 2018

9. 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 source of pathogen. Risk
varies from lower risk native seed to higher risk 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.

10. 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

17. Good water management

19. Good drainage to avoid mud/puddles

20. Progress since Oct 2017
• Accelerate lab testing to process sample backlog
Done – 800 buffer samples run - intensive 3 week period
in March 2018
• Run metabarcoding to identify species present
Done – 96 samples and synthetic control sequences at
different proportions run late in 2017
• Report Phytophthora findings and species to
nurseries
Done – reports on the +ve and –ve results and species
present sent in March 2018

21. PCR test results
• Data interpretation in relation to management
1660 samples

22. PCR test results - roots and water
(n = 1660)
• Data interpretation in relation to management
practices needed next

23. PCR test results - roots only
(n = 617)
• +ve rate higher as water samples includes
irrigation and controls

24. Illumina run – Nov 2017
• 250 base pair reads, 15M barcode reads = 156K reads
per sample
• Synthetic control sequences included as a test of
a) sequencing error rate (Pete to discuss)
b) indexing error to set threshold read number to
accept. To be finalised but presence of control
sequences across plate in low number suggests
threshold in 50-100 range
c) sensitivity range – 10 attograms
• Preliminary data - needs full validation against
reference sequences of 45 known cultures already set
up for a control plate due to be run in May (Leighton to
discuss)
• Example of Word file sent to nursery owner….

25. Illumina data – findings & challenges
• Good quality data – almost all Phytophthora, downy
mildew or Nothophytophthora. Low levels of
Phytopythium
• Data was not perfect – issue with contamination needs
to be examined as P agathidicida not expected to be
common in UK
• Some results unexpected - we need a means of
examining false positives
• What to do with reads with no match (unknowns)?
• Reporting results to growers is challenging - implications
for management not always clear
• Need to report summary findings to Plant Health
Committee via Jane Barbrook

26. 18/06/201827
Water sources
• Storage tank uncovered
• P. chlamydospora
• P. gonapodyides
• River water (a single sample);
• P. lacustris
• P. rubi/fragariae
• P. gallica
• P. riparia
• P. syringae
• P. chlamydospora/P. x stagnum
• P. pseudosyringae
• P. gonapodyides
• Rain water (off glasshouse roof);
• P. gonapodyides
• Suspected clade 5 sp.
• P. palmivora

27. 18/06/201828
Puddles
• A single puddle sample;
• P. syringae
• P. gonapodyides
• P. pseudosyringae
• P. bilorbang
• P. hibernalis
• P. chlamydospora/P. x stagnum
• P. gallica
• P. plurivora
• P. inundata
• P. cryptogea
• Roots

28. 18/06/201829
Root and stem samples
• Juniperus spp.;
• P. austrocedri
• P. gonapodyides
• Chamaecyparis spp.;
• P. lateralis
• P. cambivora
Delivery tested on arrival
from EU:
• Hebe;
• P. cactorum
• Buxus ;
• P. occultans/P. terminalis

29. Reference database
8 taxa 2 named
4 taxa 1 named
Clade 6 2003
Brasier et al 2003
12 taxa

30. Reference database
21 taxa 17 named
9 taxa 4 named
Clade 6 2015
Scanu et al 2015
30 taxa

31. Ongoing/future work
• Validate computational pipeline against control
species
• Complete sampling in 2018
• Run many more Illumina plates (700 samples currently
in queue)
• Data interpretation in relation to management
practices – need to collate data on practices that can
be related to Phytophthora findings