MSc. internship, end colloquium. Internship with NIOO_KWAW 15-August-2016

1. The influence of rare soil bacteria on plant –
herbivore interaction
Sebastian Yanore
MSc. internship, end colloquium
15-August-2016
Supervisors:
Viola Kurm, Prof. Dr. Lijbert Brussaard

2. Outline
1. About NIOO-KNAW
2. Internship project: The influence of rare soil
bacteria on plant –herbivore interaction
2

3. Brief History of NIOO-KNAW
 Three institutes of KNAW
Centre for Terrestrial
Ecology
Centre for Limnology (study
of inland waters)
Centre for Estuarine and
Marine ecology (study of
deep waters)
 The name NIOO-KNAW
was adopted in 2002
3

4. Vision of NIOO-KNAW
“ Generates basic knowledge on the ecology of
natural systems, in order to elucidate the
mechanisms underlying ecosystem functioning
and to discover new means of preserving and
enhancing the biodiversity and sustainability of
natural and managed ecosystems."
4
www.NIOO-KNAW.nl

5. Current Research themes
 Wageningen office, 2011
Seven thematic areas
– Global environmental change
– Eco-evolutionary dynamics
– Ecological epigenetics
– Microbiomes
– Chemical communication
– Disease ecology
– Restoration ecology
5

6. Research departments
• Animal Ecology
• Aquatic Ecology
• Microbial Ecology
• Terrestrial Ecology
6
Plant-soil feedback studies in the
greenhouse of NIOO-KNAW

7. The influence of rare soil bacteria
on plant –herbivore interaction
7

8. 100 species
insects
arachnids
molluscs
worms
50 species nematodes
10,000 species bacteria
500 species fungi
100 species protozoa
Soil microbes

9. Rare microbial species defined
• Soil microbes that are low in abundance in the
soils under study
• Empirical threshold:
• < 0.1 % of the soil under study
• < 0.01% of the soil under study
• < 1% of the soil under study
• Rare species may not necessarily be rare in a
global population perspective
9
Galand et al., 2009; Pegros-Alio et al., 2012; Hol et al., 2010

10. Do rare soil microbes matter?
(Hol et al., 2010)

11. 11
(Hol et al., 2010)
Reduction of rare soil microbes
modifies plant–herbivore interactions

12. Reduction of rare soil microbes
modifies plant–herbivore interactions
12(Hol et al., 2010)
Rare soil microbes reduction

13. Plant-growth promoting
rhizobacteria (PGPR)
13
• Nutrient supply
• Suppress soil-borne
pathogens
• Activate Induce
systemic resistance
(ISR) against
herbivores

14. PGPR modifies plant–herbivore
interactions
14(Pineda et al., 2011)(Hol et al., 2010)
Rare soil microbes reduction

15. How PGPR mediates the effect of
aphid feeding on Arabidopsis
(Pineda et.al 2011) 15
Rhizobacteria
induce systemic
resistance via the
Jasmonic acid
signaling pathway
in Arabidopsis

16. ...... So far
• Rare soil bacteria;
– reduction increase plant biomass
– can suppress soil-borne pathogens
– mediate plant-herbivore interaction
• Similarly, PGPR;
– mediate plant-herbivore interaction by inducing
induce systemic resistance (ISR) via the
jasmonic acid pathway
16

17. What we do not know
• Do rare soil bacteria induce ISR in
Arabiodopsis against an aphid herbivore via
the jasmonic acid pathway
17

18. Hypotheses
• H1: Rare bacteria mediates (reduces) the
negative effect of aphid feeding on
Arabidopsis
• H2: Pseudomonas fluorescens SS101 (Pf.
SS101) mediates (reduces) the negative effect
of aphid feeding on Arabidopsis
18

19. Hypotheses cont’d
• H3: Rare bacteria induce ISR in Arabidopsis
against aphid infestation via the jasmonic acid
(JA) pathway
19

20. Hypotheses
ISRgenerelatedexpression
Increasing dilution step
+ P. fluorescens SS101
Rare bacteria
20
H3:

21. Materials & methods
Experimental soil -- CLUE fields (Mossel, Ede, The
Netherlands)
21
52°04′N 05°45′E

22. Soil treatment
• 180kg soil(0-20cm) sampled outside
the fenced area
• Gama sterilization (> 25 kG ray)
• 10 soil samples (35g each) from 10
plots within the CLUE field
• 6 serial dilution treatments (KH2PO4
buffer, pH = 6.5)
• 180 kg soil subdivided into 60 bags
(3kg each)
• Incubated for 25 weeks
• + Pseudomonas fluorescens SS101 /-
Pseudomonas fluorescens SS101
22

23. Plant and Insect material
23
• Wild- type
Arabidopsis thaliana
• Seeds were sterilised
and veneralised
before sowing in MS
plant agar
• Generalist green
peach aphid (Myzus
Persicae)

24. Experimental setup
24
In summary,
6 dilution treatments × 2 Pf.SS101(+/-) × 10 Replicates + 6 dilution treatments × 2
Aphids(+/-) × 20 Replicates = 360 pots
Treatments Pseudomonas Aphids Replicates
CTRL: sterile soil 10
Full community 10
10^2 10
10^4 10
10^6 10
10^8 10
CTRL: sterile soil Pseudomonas 10
Full community Pseudomonas 10
10^2 Pseudomonas 10
10^4 Pseudomonas 10
10^6 Pseudomonas 10
10^8 Pseudomonas 10
CTRL: sterile soil aphids 20
Full community aphids 20
10^2 aphids 20
10^4 aphids 20
10^6 aphids 20
10^8 aphids 20
CTRL: sterile soil Pseudomonas aphids 20
Full community Pseudomonas aphids 20
10^2 Pseudomonas aphids 20
10^4 Pseudomonas aphids 20
10^6 Pseudomonas aphids 20
10^8 Pseudomonas aphids 20

25. Aphids’ infestation
• + Aphids after 6 weeks
1 newborn aphid/clip
cage/plant
6 additional
aphids/per plant
25

26. Measurements and observations
26
24hrs after aphids’ infestation On week 8

27. Statistics
27
• Linear mixed model (lme4)
• Penalized quasilikelihood (PQL) couple
with the General linear mixed model
(GLMM) function (glmmPQL)
• All analyses were performed in R
Statistical package version 3.3.1 (R Core
Team, 2016)

28. Results and Discussions:
Dilution treatments affected Arabidopsis
biomass
28
P = 0.014 P = 0.0019
p = 0.0001

29. Ahids affected Arabidopsis
biomass
29
0
0.05
0.1
0.15
0.2
0.25
0.3
Root Shoot Total
TotalBiomass(gdw)
P < 0.000
3.2% P < 0.000
4.7%
P < 0.000
8%
No aphids
Aphids

30. Dilution treatments mediated the
effect of aphids on Arabidopsis
biomass
30
H1: Rare
bacteria
mediates the
negative effect
of Aphids on
Arabidopsis

31. Pf. SS101 affected Arabidopsis
biomass
31
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
0.2
Root biomass Shoot biomass Total biomass
Totalbiomass(gdw)
P = 0.0027
P = 0.024

32. Pf. SS101 inoculation mediated the
effect of aphids on Arabidopsis
32
0
0.05
0.1
0.15
0.2
0.25
Totalbiomass(gdw)
H2: Pseudomonas
fluorescens SS101
(Pf. SS101) mediates
the negative effect
of aphids on
Arabidopsis

33. Dilution treatments Affected Pf.
SS101 recovery
33
0
1
2
3
4
5
6
7
8
9
Pf.SS101recovered(CFU×104g-1soil)
a
d
b
ab
c
ab

34. Conclusions and recommendations
• Our results points to the importance of rare
bacteria in plant production
• We did not find rare bacteria mediated effect of
myzus on Arabidopsis
• The effectiveness of Pf. SS101 decreases with
increasing microbial abundance and diversity
• To improve the effectiveness of Pf. SS101,I
recommend a direct inoculation of the roots or
seeds
34

35. Acknowledgement
Viola Kurm , NIOO-KNAW
Gera Hol, NIOO-KNAW

36. 36
Questions
and
comments
are
welcome!