Apple Replant Disease Evolution and Rootstock Interaction (ARDERI):

Apple Replant Disease Evolution and Rootstock Interaction (ARDERI): Understanding the relationships among host, pathogen(s) and soil microbes Project leader: Prof. Xiangming Xu November 2015 Partner meeting

Replant disease Mark Mazzola

What we know Causal agents: four principal genera - Cylindrocarpon, Rhizoctonia, Phytophthora and Pythium relative dominance varies from site to site Nematodes can exacerbate ARD Rootstock genotypes differ in their response to ARD Soil microbial community affects ARD development New research paradigm microbiome, pioneered in human disease epidemiology the collective genomes of the microorganisms that reside in an environmental niche (synonymous to microbiota) new tools to characterise microbiome

Rhizosphere microbiome

What we proposed to do WP1: who are there? what are related to specific host genotypes and ARD? what genes are expressed WP2: how do ARD components interact in determining ARD development WP3: how fast do host genotypes change rhizosphere microbiome

Work package 1 Metagenomics Lead researcher: Dr Nicola Harrison (Root biologist)

What is Metagenomics? Metagenomics is the study of genetic material recovered directly from environmental samples. Metagenomic Pipeline DNA Sequencer Sample Soil Extract DNA Amplify specific regions of DNA Sequence those specific regions of DNA Relate DNA sequences to micro-organism database

ARDERI Metagenomics We will use an amplicon-based metagenomics approach already established at EMR to characterize each sample Use MiSeq to sequence each sample using primer sets to amplify regions in: 16S for bacteria ITS for fungi, nematode and oomycetes incorporating barcode tagging to enable multiplexing of samples

Questions we aim to answer Do M9 rootstocks recruit different microorganism communities dependent upon soil type/location? Do different microorganism communities perform similar functions such as cell wall degradation in ARD sites? Do different rootstocks recruit different communities of microorganisms?

Progress so far Methodology development

Power soil DNA isolation kit Soil sampled in Triplicates 0.25g Soil per DNA extraction Spare samples stored -80 C Each soil sample extracted individually

16S (bacteria) and ITS (fungi) regions 16S 16S Bacteria Forward Reverse Overlapping region Forward Reverse Fungi No overlap

Pooling & Sequencing Strategy Tested single sample sequencing versus pooled sample sequencing DNA Sample 1 DNA Sample 1 DNA Sample 1 single single pooled 16S Region ITS Region 16S Region ITS Region Sequence DNA Sequence DNA Sequence DNA

Plot set-up - Goatham L Aisle L Row T0 sampling: 1 st of May 2015 3x 10cm-cores per location 6 controls taken from grass area near hedge 150 samples in total Land preparation agreed: No land inversion Stakes for planting holes Block 3 Block 2 Block 1 09 M.M.106 01 M.M.106 10 M.9 02 M.9 11 M.27 03 M.27 12 AR295-6 04 AR295-6 13 M.26 05 M.26 14 M.116 06 M.116 15 G.41 07 G.41 16 G.16 08 G.16 25 G.41 17 G.41 26 AR295-6 18 AR295-6 27 M.M.106 19 M.M.106 28 M.9 20 M.9 29 M.116 21 M.116 30 G.16 22 G.16 31 M.27 23 M.27 32 M.26 24 M.26 41 M.9 33 M.9 42 M.116 34 M.116 43 M.26 35 M.26 44 G.16 36 G.16 45 AR295-6 37 AR295-6 46 M.27 38 M.27 47 M.M.106 39 M.M.106 48 G.41 40 G.41 Non-ARD ARD

ITS: Soil and SWD PCA Plot PCA plot illustrating distinct communities of fungi between soil and insect samples Grass Tree SWD

ITS Samples multiplexed and individual NJ tree ID 16S-ITS ID ITS condition S1 S17 Tree S2 S18 Tree S3 S19 Grass S4 S20 Grass S5 S21 Grass S6 S22 Tree S7 S23 Tree S8 S24 Grass S9 S25 Tree S10 S26 Tree S11 S27 Grass S12 S28 Grass S13 S29 Grass S14 S30 Tree S15 S31 Tree S16 S32 Grass = Tree Station = Grass Aisle Beta diversity: M 2 = 0.022; Monte Carlo p < 0.001

16S samples multiplexed and individual NJ tree ID 16S-ITS ID 16S Condition S1 S33 Tree S2 S34 Tree S3 S35 Grass S4 S36 Grass S5 S37 Grass S6 S38 Tree S7 S39 Tree S8 S40 Grass S9 S41 Tree S10 S42 Tree S11 S43 Grass S12 S44 Grass S13 S45 Grass S14 S46 Tree S15 S47 Tree S16 S48 Grass = Tree Station = Grass Aisle Beta diversity: M 2 = 0.007; Monte Carlo p < 0.0001

16S PCA Plot Grass Tree

ITS PCA Plot Grass Tree

16S Taxa (class level) Grass Tree

16S taxa (family and above) Highlighted Tree station

ITS Taxa (class level) Grass Tree Taxonomy No blast hit Ascomycota;Dothideomycetes Ascomycota;Eurotiomycetes Ascomycota;Incertae sedis Ascomycota;Lecanoromycetes Ascomycota;Leotiomycetes Ascomycota;Orbiliomycetes Ascomycota;Pezizomycetes Ascomycota;Saccharomycetes Ascomycota;Sordariomycetes Ascomycota;Taphrinomycetes Ascomycota;unidentified Basidiomycota;Agaricomycetes Basidiomycota;Agaricostilbomycetes Basidiomycota;Cystobasidiomycetes Basidiomycota;Exobasidiomycetes Basidiomycota;Microbotryomycetes Basidiomycota;Tremellomycetes Basidiomycota;Ustilaginomycetes Basidiomycota;Wallemiomycetes Basidiomycota;unidentified Chytridiomycota;Chytridiomycetes Chytridiomycota;unidentified Glomeromycota;Glomeromycetes Neocallimastigomycota;Neocallimastigomy cetes Rozellomycota;unidentified Zygomycota;Incertae sedis unidentified;unidentified

Statistical Tables 16S Region Taxa Class p Tree station Grass Aisle Pedosphaerales (o) Pedosphaerae 1.2E-30 Chthoniobacteraceae (f) Spartobacteria 3.6E-27 Alcaligenaceae (f) Betaproteobacteria 2.0E-26 Chthoniobacteraceae (f) Spartobacteria 1.0E-23 Chthoniobacteraceae (f) Spartobacteria 2.6E-22 Chthoniobacteraceae (f) Spartobacteria 9.2E-22 Nitrosovibrio (g) Betaproteobacteria 1.3E-27 Hyphomonadaceae (f) Alphaproteobacteria 3.3E-27 Hyphomonadaceae (f) Alphaproteobacteria 5.4E-24 Betaproteobacteria (c) Betaproteobacteria 2.7E-23 ITS Region Taxa Class P Tree station Grass Aisle Ascochyta fabae (s) Dothideomycetes 5.92E-27 Glomeraceae (f) Glomeromycetes 1.61E-19 Helotiales (o) Leotiomycetes 1.66E-18 Glomeraceae (f) Glomeromycetes 2.74E-18 Glomeraceae (f) Glomeromycetes 3.77E-18 Rhodotorula lamellibrachiae (s) Microbotryomycetes 4.42E-17 Otospora bareae (s) Glomeromycetes 1.03E-16 Verticillium isaacii (s) Sordariomycetes 1.53E-16 Ascomycota (p) unidentified 2.62E-16 Kregervanrija fluxuum (s) Saccharomycetes 5.56E-16

Current and future work To test our developed algorithms we will prepare and sequence a mock community DNA sample We need to judge which is the best method: Without a sample with known biological content we can t do this. This will allow us to fine tune the parameters used in the algorithm to best define soil microbial communities. Technical questions we are working on: How to handle ITS1 and ITS2 ITS1 = forward single read, ITS2 = reverse single read or combine the two reads?

Industry Inputs Nurseries and farms: Identify established orchard and stool beds for soil sampling for M9 rootstocks Identify established stool beds for soil sampling for a selection of rootstocks For each rootstock-site combination: a minimum of three samples need to be taken from each rhizosphere soil location, from six locations within the stool bed/orchard row (18 samples per site) In addition, soil samples need to be taken from field margins as controls

Work package 2 Elucidation of interactions between ARD complex members Lead researcher: Emma Tilston (Soil / rhizosphere scientist)

Research questions H1) Two groups of microbes (water moulds and true fungi) act additively to cause ARD H2) ARD-related root rot is more severe if root lesion nematodes are present as well as ARD microbes H3) Rootstock vigour and architecture modify root system responses to ARD

Experiment Factorial design comprising 4 rootstocks and 4 biocide treatments + untreated Three replicates 60 rhizotrons in total 15 months duration

Trees Scion: Discovery

Biocide treatments Biocide target group Residual active organisms ( ) Nematodes Oomycetes Ascomycetes & Basidiomycetes Predicted ARD severity None, untreated *** Nematodes ** Nematodes + Oomycetes * Nematodes + Ascomycetes & * Basidiomycetes Nematodes + Oomycetes + Ascomycetes & Basidiomycetes

Status April 2015 Soil being processed Rootstocks being grafted Biocides reserved / ordered Sacrifice plants in cold storage Rhizotron planting in early May?

Soil preparation 2 m 3 (or 3 t) soil sieved Sieved by hand to 6 mm Sieved damp Stored covered, outside 2 cm Mixed with a concrete mixer 2 cm

Key dates Soil processed: Rootstocks grafted: April - May 10 and 13 April Rhizotrons filled with soil: 18 May 3 June Biocides applied: Rhizotrons planted: 5 June 10 11 June Rhizotrons transferred to glasshouse: 12 June

Growing conditions Four back-to-back rows of 15 rhizotrons Randomised as 3 blocks No supplementary lighting Fertigation 26 October 2015

Number of tree deaths Tree mortality

Tree failure Identifiable causes: Graft failure, rhizotron failure (soil leakage), canker? Heat stress Biocide phytotoxicity Other causes: Action: Replace dead trees, monitor and identify improvements

Heat stresses Original plant survives Late failure More plants survive near the fan Fan in outside wall 14 3 29 11 16 25 31 57 28 2 20 58 27 50 8 34 39 52 55 21 13 42 32 23 49 41 43 48 51 5 18 10 38 54 44 35 26 56 19 24 40 53 12 9 1 17 22 47 37 7 45 36 15 6 60 46 4 30 33 59

Biocide phytotoxicity Original plant survives Untreated Rootstock M.9 M.106 M.116 G.41 1 2 3 4 5 6 7 8 Late failure 9 10 11 12 13 14 15 16 No universal phytotoxicity M.106 appears to tolerate stress well Soil drench treatment Nematicide Nematicide + Oomyceticide Nematicide + Fungicide Nematicide + Oomyceticide + Fungicide 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Measurements Digital imaging (monthly) Lesions and necrosis Root growth rate Turnover and branching Terminal, destructive sampling Hydraulic conductivity of root system Root mass Root system architecture Leaf nutrient content Molecular profiling of rhizosphere biota

Root growth Rhizotron 10, an original M.106 in untreated soil Few rhizotrons have visible root systems Problems with soil sticking to the removable cover 5 cm

Experiment (revised) Basic design unchanged, 4 rootstocks and 4 biocide treatments + untreated Five or six replicates (100 or 120 pots in total) Use deep, square rose pots January: collect and sieve soil (10 mm) Graft trees at the end of January Transplant into pots of treated soil no later than 1 st week of April Grow-on in a polytunnel 15 months duration as before

Measurements (revised) Terminal, destructive sampling Lesions and necrosis Hydraulic conductivity of root system Root mass Root system architecture Leaf nutrient content Molecular profiling of rhizosphere biota

Work package 3 Rootstock-related soil microbiota changes over time Lead researcher: Felicidad Fernández (Rootstock breeder and geneticist)

Questions? Is the severity of ARD in newly planted apple trees greater if the previous rootstock genotype was highly susceptible to ARD? Is ARD less severe if rootstock genotypes with contrasting traits follow each other in a rotation-style planting system?

Apple genotypes

Experimental set-up Two intensive orchards: Dessert orchard: o Previously planted with M.9 o AC Goatham & Son (Sutton Valence, Kent) o Soil type: Questionnaire to be completed Cider orchard: o Previously planted with M.M.106 (Reps 1 & 2, Rep 3 TBC) o Bulmers (Winchenford, Worcester) o Soil type: Questionnaire to be completed

Split plot design (row vs. aisle): 3 replicates Experimental set-up

Experimental set-up Scions: Discovery (Dessert orchard) W. Permain (Cider orchard) Rootstock sources: D. L. (France): o M.9 o M.26 o M.M.106 o M.116 o G.16 o G.41 o AR295-6 F.P. Matthews (UK): o M.27 Different sources and sizes!

Experimental set-up Measures to harmonize starting material for planting All rootstocks were washed prior to grafting Trees were potted up in the same substrate Trees were grown in pots for 7 months (Apr Oct) in a polytunnel at EMR. Spare trees were produced of each genotype to allow us to choose the most consistent set at plating time In October, prior to planting: o o o All trees were tipped and lateral shoots removed Most similar trees were paired for each rep Girths and tree heights were recorded

Experimental sites Summer 2015 Planting 30 th October 2015

Plot set-up Heineken T0 sampling: 3 rd of June 2015 3x 10cm-cores per location 6 controls taken from grass area near hedge 150 samples in total Land preparation agreed: No land inversion Block 3 Block 1 Block 2 L Aisle L Row 32 M.26 24 M.26 31 M.27 23 M.27 30 G.16 22 G.16 29 M.116 21 M.116 28 M.9 20 M.9 27 M.M.106 19 M.M.106 26 AR295-6 18 AR295-6 L Aisle L Row 25 G.41 17 G.41 48 G.41 40 G.41 16 G.16 08 G.16 47 M.M.106 39 M.M.106 15 G.41 07 G.41 46 M.27 38 M.27 14 M.116 06 M.116 45 AR295-6 37 AR295-6 13 M.26 05 M.26 44 G.16 36 G.16 12 AR295-6 04 AR295-6 43 M.26 35 M.26 11 M.27 03 M.27 42 M.116 34 M.116 10 M.9 02 M.9 41 M.9 33 M.9 09 M.M.106 01 M.M.106 Non-ARD ARD Non-ARD ARD

Experimental sites Summer 2015 Planting 14 th October 2015

Summary of progress Pre-planting sampling carried out (May 2015) Trees grafted and raised at EMR (April October) Trees planted at both sites (October 2015) Meta-genomic analysis of T0 soil samples started: DNA from 120/150 samples from the site in Kent has been extracted 40 samples have been used to optimised pooling strategy for sequencing (WP1) Spare trees for every scion-genotype combination retained to replace in 2016 (if necessary).

Work for 2016 Continue metagenomic analysis of samples at T0 Check tree survival at bud-break and replace any dead trees Sample soil around newly planted trees in May 2016 (T1) Measure tree growth (pruning weight) and girth in December 2016