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Gary Bending
School of Life Sciences,
University of Warwick, UK
Roots of decline? Assembly and
function of the rhizosphere
microbiome in relation to yield
decline
Penny Hirsch
Tim Mauchline
Ian Clark
Peter Mills
Grace Smith
Chris van der Gast
Anna Oliver
David Bass
Keith Norman
Sam Maycock
Gary Bending
Graham Teakle
Jay Moore
Project Partners
Penny Hirsch
Tim Mauchline
Ian Clark
Susanne Schreiter (PDRA)
Peter Mills
Grace Smith
Chris van der Gast
Anna Oliver
David Bass
Keith Norman
Sam Maycock
Gary Bending
Graham Teakle
Jay Moore
Sally Hilton (PDRA)
Sarah Thornber (RA)
Project Team
OSR yield declines with increased frequency
in rotations
£43-86 million annual loss
http://cereals.ahdb.org.uk/media/305093/g55-oilseed-rape-guide-
jan-2014-update.pdf
Bennett et al. 2012 Biol. Rev. 87, 52–71
OSR yield declines with increased frequency
in rotations
£43-86 million annual loss
http://cereals.ahdb.org.uk/media/305093/g55-oilseed-rape-guide-
jan-2014-update.pdf
OSR yield declines with increased frequency
in rotations
£43-86 million annual loss
http://cereals.ahdb.org.uk/media/305093/g55-oilseed-rape-guide-
jan-2014-update.pdf
What determines assembly of
the rhizosphere microbiome?
What do ‘good’ and ‘bad’
microbiomes look like?
How do good and bad
microbiomes interact with
the plant?
Can we engineer a ‘good’
microbiome?
WP 1 What controls assembly of the rhizosphere
microbiome at the landscape scale?
• Multiple microbial taxa
• Rotation, environment, geography
Sampling sites
Green bud stage
Sampling strategy
= 5 root, 5 rhizosphere and 5 bulk soil samples for DNA
= 15 samples/site x 37sites =555 samples
Plant Soil
Soil bulk density
Processing
Vortex Vortex Invert Invert
Pool and spin
25 ml sterile water
Processing procedure
Bulk soil
Root/Rhizoplane
Rhizosphere
Field site metadata
Rotation lengths
0
2
4
6
8
10
12
14
1 in 2 1 in 3 1 in 4 1 in 5 1 in 6 1 in 7 1 in 8 1 in 9 1 in 12 Virgin
Rotation (frequency of OSR)
Numberofsites
Soil types selected for sampling
(as defined by land managers)
11
7
6
4
2
1
1
1
1 1
Loam
Clay
Sandy Clay Loam
Brash
Clay Loam
Chalk
River Gravel
Silt
Silty clay loam
Sandy
Clay Loam,
17
Clay, 11
Silty Clay
Loam, 4
Sandy
Loam, 3
Sandy Clay
Loam, 1 Silty Clay, 1
Sandy loam, 3
Actual soil types sampled
Textural Class
Sand
2.00-
0.063m
m
Silt 0.063-
0.002mm
Clay
<0.002mm
F1 Clay Loam 48 31 21
F2 Clay 35 25 40
F3 Clay Loam 32 35 33
F3A Clay Loam 34 35 31
F4 Silty Clay Loam 16 54 30
F5 Clay 14 44 42
5C Clay 23 41 36
F5D Clay Loam 24 48 28
F6 Clay Loam 25 46 29
F7 Sandy Clay Loam 54 22 24
F8A Clay 35 25 40
F8B Clay Loam 28 38 34
F9 Clay Loam 25 42 33
F9A Silty Clay Loam 20 49 31
F10 Clay Loam 32 37 31
F10A Clay Loam 49 31 20
F11 Clay Loam 23 44 33
F12 Clay Loam 32 42 26
F13 Silty Clay Loam 20 46 34
F14A Clay Loam 33 36 31
F14C Clay 24 40 36
F15 Sandy Loam 55 27 18
F15A Clay 26 35 39
F16 Clay Loam 32 44 24
F16A Clay 24 27 49
F17 Clay 19 41 40
F18 Silty Clay Loam 19 48 33
F18A Silty Clay 16 48 36
F19 Clay 22 34 44
F20 Sandy Loam 78 12 10
F21 Clay Loam 38 32 30
F21A Clay 24 33 43
F21B Clay Loam 33 34 33
F21C Clay Loam 31 41 28
F22 Clay 23 40 37
F24 Sandy Loam 65 19 16
F25 Clay Loam 42 30 28
0
50
100
150
200
250
17
21a
3
13
9a
19
22
9
3a
16
16A
12a
1
10
21b
21
14c
18
11
24
14a
8b
4
15
18a
15a
20
8a
10a
7
25
2
5d
5c
21c
5
metresabovesealevel
Farm
Elevation
0
10
20
30
40
50
60 F17
F9
F13
F9A
F16
F16A
F10A
F22
F21A
F6
F14C
F14A
F18A
F10
F5
F12
F8A
F21
F15A
F18
F11
F3
F3A
F5C
F1
F4
F19
F21C
F2
F5D
F24
F20
F15
F21B
F8B
F25
F7
%Water
Farm
Average soil moisture content
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
F20
F7
F12
F25
F5
F2
F24
F5c
F18
F5d
F8a
F10a
F11
F15
F3a
F21b
F4
F21c
F3
F1
F8b
F14c
F18a
F19
F15a
F10
F21a
F6
F22
F13
F14a
F16
F9a
F9
F21
F16a
F17
Bulkdensity(g/cm3)
Farm
Bulk Density
0
1
2
3
4
5
6
7
8
9
VarietyNo.sites
0
50
100
150
200
F16
F25
F13
F22
F8a
F3a
F17
F14c
F4
F5c
F20
F9a
F5
F21b
F18
F3
F6
F14a
F5d
F18a
F2
F21c
F12
F10
F1
F7
F10a
F11
F21a
F21
F16a
F9
F24
F15a
F8b
F19
F15
Dryweight(g)
Farm
Average plant dry weight
Wheat, 18Barley, 13
Fallow, 3 Rye, 1
Previous crop
Sowing date
%sitessown
0
20
40
60
80
100
7/30/2014 8/4/2014 8/9/2014 8/14/2014 8/19/2014 8/24/2014 8/29/2014 9/3/2014 9/8/2014 9/13/2014
Soil analysis ongoing…..
Amplicon sequencing
• 2x300 bp MiSeq analysis
• Bacterial, fungal and microbial eukaryote communities
Follow up with further specific taxa
• Which DNA extraction kit to use?
FastDNA™ SPIN Kit for Soil (MP Biomedicals)?
PowerSoil® DNA Isolation Kit (MO-BIO)?
Root
0
20
40
60
80
100
120
NR PR WR NRh PRh WRh NS PS WS No
wash
No
wash
0.5 g
µg/mlDNA(100ul
volume)
MoBio
0
20
40
60
80
100
120
NR PR WR NRh PRhWRh NS PS WS
µg/ml(totalvolume
100µl) FastDNA
Rhizosphere Bulk soil
0
20
40
60
80
100
120
NR PR WR NRh PRh WRh NS PS WS
Griffiths
Root Rhizosphere Bulk soil
Root Rhizosphere Bulk soil
DNA extraction
ITS CEH 33K
Resemblance: S17 Bray Curtis similarity
KitSoil type2
FP1
FP2
FP3
MB1
MB2
MB3
2D Stress: 0.01
ITS
0
100
200
300
400
1 2 3
FP
MB
No.OTUs
Soil type
WP2 Functional responses of the
rhizosphere microbiome to rotation
• Different rotation frequencies of OSR
• Trials at Warwick Crop Centre, Harper-Adams and Rothamsted
• Metatranscriptome analysis
• Cross-talk between plant roots and microbial community
• Pathways associated with host defence, nutrient uptake and
assimilation
WP 2 Structure and function of the microbiome
Rotation 2014-15 2015-16 2016-17 2017-18
Continuous OSR Wheat OSR OSR OSR
Alternate OSR Wheat OSR Wheat OSR
Virgin OSR Wheat Wheat Wheat OSR
Rotation 2013-14 2014-15 2015-16 2016-17
Rothamsted
White horse plot 2 –
4/6/2015
Rothamsted
Scout plot 2 – 4/6/2015
WP3 Rhizosphere engineering via crop genotype
• Contrasting varieties grown in rotation
• Microbiome assembly
• Metatranscriptome profiling
2 x 6 m plots
WP4 Rhizosphere engineering via soil management
• Furzefield site at Rothamsted
• Cropped continuously with OSR since 1991
• Yields have declined by 30 %
Rotation 2013-2014 2014-2015 2015-2016
1. Continuous OSR OSR OSR OSR
2. FYM OSR FYM/OSR OSR
3. Biosolids OSR Biosolids/OSR OSR
4. Fallow OSR Fallow OSR
5. Legume break OSR Faba beans OSR
6. Oat break OSR Oats OSR
7. Wheat break OSR Wheat OSR
Impact activities
Steering group
Paul Gosling/ Amanda Bennett, HGCA
Andrew Richards, Agrii
Mark Nightingale, Elsoms
Peter Scott, Origin fertilisers
Richard Elsdon, United Oilseeds
First meeting 17th November 2015 (Warwick)
Project Newsletter

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