Apple Replant Disease Evolution and Rootstock Interaction ...

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

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

What we know

Rhizosphere microbiome

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

What we proposed to do

Metagenomics

Lead researcher: Dr Nicola Harrison (Root biologist)

Work package 1

What is Metagenomics?

Metagenomics is the study of genetic material recovered directly from environmental samples.

Sample Soil

Extract DNA

Amplify specific regions of DNA

Sequence those specific regions

of DNA

Relate DNA sequences to

micro-organism database

Metagenomic Pipeline

DNA Sequencer

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

ARDERI Metagenomics

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?

Questions we aim to answer

Methodology development

Progress so far…

Soil sampled in Triplicates 0.25g Soil per DNA

extraction Spare samples stored -80°C Each soil sample extracted

individually

Power soil DNA isolation kit

Reverse

… …

16S 16S

Forward

Forward Reverse …

…

Overlapping region

No overlap

Bacteria

Fungi

16S (bacteria) and ITS (fungi) regions

Tested single sample sequencing versus ‘pooled’ sample sequencing

DNA Sample 1 DNA Sample 1 DNA Sample 1

Sequence DNA Sequence DNA Sequence DNA

ITS Region

16S Region

16S Region

ITS Region

single single pooled

Pooling & Sequencing Strategy

L Aisle L Row

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

Bloc

k 1

Bloc

k 2

Bloc

k 3

T0 sampling: • 1st 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

Plot set-up - Goatham

Grass

Tree

SWD

PCA plot illustrating distinct communities of fungi between soil and insect samples

ITS: Soil and SWD PCA Plot

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: M2 = 0.022; Monte Carlo p < 0.001

ITS 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: M2 = 0.007; Monte Carlo p < 0.0001

16S samples “multiplexed” and individual NJ tree

Grass Tree

16S PCA Plot

Grass Tree

ITS PCA Plot

Grass

Tree

16S Taxa (class level)

Highlighted – Tree station

16S taxa (family and above)

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;Neocallimastigomycetes

Rozellomycota;unidentified Zygomycota;Incertae sedis unidentified;unidentified

ITS Taxa (class level)

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

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

16S Region

ITS Region

Statistical Tables

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?

Current and future work

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

Industry Inputs

Elucidation of interactions between ARD complex members

Lead researcher: Emma Tilston

(Soil / rhizosphere scientist)

Work package 2

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

Research questions

Experiment

• Factorial design comprising 4 rootstocks and 4 biocide treatments + untreated

• Three replicates

• 60 rhizotrons in total

• 15 months’ duration

• Scion: Discovery

Trees

Biocide target group

Residual active organisms () Predicted ARD

severity Nematodes Oomycetes Ascomycetes & Basidiomycetes

None, untreated *** Nematodes ** Nematodes + Oomycetes * Nematodes + Ascomycetes & Basidiomycetes * Nematodes + Oomycetes + Ascomycetes & Basidiomycetes

Biocide treatments

Soil being processed

Rootstocks being grafted

Biocides reserved / ordered

Sacrifice plants in cold storage

Rhizotron planting in early May?

Status – April 2015

2 cm

2 cm

Soil preparation

2 m3 (or 3 t) soil sieved Sieved by hand to 6 mm Sieved damp Stored covered, outside Mixed with a concrete

mixer

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

12 June

Key dates

26 October 2015

Four back-to-back rows of 15 rhizotrons

Randomised as 3 blocks

No supplementary lighting

Fertigation

Growing conditions

Num

ber o

f tre

e de

aths

Tree mortality

Identifiable causes: Graft failure, rhizotron failure (soil leakage), canker…

Other causes: Heat stress

Biocide phytotoxicity

Action: Replace dead trees, monitor and identify

improvements…

? Tree failure

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

Fan in outside wall

Original plant survives

Late failure More plants survive near the fan

Heat stresses

Rootstock M.9 M.106 M.116 G.41

Soi

l dre

nch

trea

tmen

t

Untreated 1 2 3 4 5 6 7 8 9 10 11 12

Nematicide 13 14 15 16 17 18 19 20 21 22 23 24

Nematicide + Oomyceticide

25 26 27 28 29 30 31 32 33 34 35 36

Nematicide + Fungicide

37 38 39 40 41 42 43 44 45 46 47 48

Nematicide + Oomyceticide + Fungicide

49 50 51 52 53 54 55 56 57 58 59 60

No universal phytotoxicity

M.106 appears to tolerate stress well

Original plant survives

Late failure

Biocide phytotoxicity

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

Measurements

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

Root growth

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 1st week of April

Grow-on in a polytunnel

15 months’ duration as before

Experiment (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

Measurements (revised)

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

Work package 3

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?

Questions?

Apple genotypes

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

Experimental set-up

Split plot design (row vs. aisle):

3 replicates

Experimental set-up

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

Scions: Discovery

(Dessert orchard)

W. Permain (Cider orchard)

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 All trees were tipped and lateral shoots removed o Most similar trees were paired for each rep o Girths and tree heights were recorded

Experimental set-up

L Aisle L Row

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

Bloc

k 1

Bloc

k 2

Bloc

k 3

T0 sampling: • 1st 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

Plot set-up - Goatham

Summer 2015 Planting 30th October 2015

Experimental sites

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

Bloc

k 3

Bloc

k 1

Bloc

k 2

T0 sampling: 3rd 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

Plot set-up – Heineken

Planting 14th October 2015 Summer 2015

Experimental sites

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).

Summary of progress

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

Work for 2016