WP1 - Distribution, diversity and management of Phytophthora in UK plant nursery systems

Phyto-threats project meeting – 05 Oct 2016

David Cooke, Leighton Pritchard & Peter ThorpeAna Perez, Sarah Green, Beatrice Henricot, Debbie Frederickson Matika - Forest ResearchTim Pettit - University of WorcesterBethan Purse - CEHJane Barbrook - APHAAlexandra Schlenzig - SASA

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

Methods

• Questionnaire – simple (6 questions) to collect basic data on nursery practices

• Sampling nurseriesFine-scale – testing 10 nurseries by project staff for detailed

breakdown of management problems and solutionsBroad-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

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

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

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

APHAApprox 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

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

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 roburChamaecyparis 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 baccataIlex aquifolium Viburnam tinus

Juniper Old Gold Viburnum davidii

Juniper tamariscifolia Viburnum tinus

Juniperus squamata 'blue carpet' Xanthocyparis vietnamensisJuniperus virginiana

Lavandula angustifolia Hidcote'

Photinia fraseri 'red robin'Pinus armandii

Pinus sylvestris

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

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

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

Reproducability

Furlan et al 2016 A framework for estimating the sensitivity of eDNA surveys Mole Ecol Res 16, 641-54

• Clumpiness?• Error?

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

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