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