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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
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