Clonostachys rosea is being investigated as a biological control agent to reduce Fusarium graminearum and the accumulation of mycotoxins deoxynivalenol (DON) and zearalenone (ZEN) in wheat. Field experiments were conducted treating artificially infected maize residues with C. rosea and another antagonist. C. rosea reduced DON levels by up to 82% and ZEN by up to 93%, with oil formulations performing best. Further experiments aim to improve formulations to protect antagonists from UV light and investigate the impact on wheat varieties under natural disease pressure.

1. www.agroscope.ch I good food, healthy environment
Federal Department of Economic Affairs,
Education and Research EAER
Agroscope
Prevention and intervention strategies to control
Fusarium graminearum with fungal antagonists
A. Gimeno, A. Kägi, I. Bänziger E. Jenny, M. Leimgruber, Z. Ngamenie, D. Drakopoulos,
H. R. Forrer, B. Keller & S. Vogelgsang

2. 2Mycokey Workshop 2019
Alejandro Gimeno
WP4
Prevention in the field
WP5
Intervention strategies
Task 4.3 Biological control of F. graminearum
with antagonists on preceding crop residues
Task 5.1 Pre-harvest strategies for wheat

3. 3Mycokey Workshop 2019
Alejandro Gimeno
Primary inoculum
Diseased kernels
contaminated with
mycotoxins
Task 4.3 Biological control of F. graminearum on crop residues
Biological control agent
(BCA) =
Natural organism with ability to suppress
pathogens and/or enhance plant resistance
against diseases
Prevent disease development to reduce
accumulation of DON and ZEN
Mode of action
• Hyperparasitism
• Antibiosis
• Competition
© Jonas Lehner,
Agroscope

4. 4Mycokey Workshop 2019
Alejandro Gimeno
Primary inoculum
Diseased kernels
contaminated with
mycotoxins
Task 5.1 Pre-harvest strategies for wheat
Intervention during infection period
to reduce accumulation of DON and ZENBiological control agent
(BCA) =
Natural organism with ability to suppress
pathogens and/or enhance plant resistance
against diseases
Mode of action
• Hyperparasitism
• Antibiosis
• Competition
© Jonas Lehner,
Agroscope

5. 5Mycokey Workshop 2019
Alejandro Gimeno
*Gimeno et al., Frontiers in Microbiology (submitted)
FORMULATION
Production for experiments
Improving survival and effect
Protection against harmful UV-B
BCAs
Isolates from Switzerland for biological control of FHB
Establishment of a protocol for detection of C. rosea
from plant material (VTT collaboration)*
LABORATORY EXPERIMENTS
Suppression of perithecia production and ascospore
release on infected maize residues
Growth chamber experiments with wheat
Investigation of the mode of action of C. rosea
FIELD EXPERIMENTS
4.3 Prevention: treatment of artificially infected
maize residues
5.1 Intervention: treatment of flowering wheat heads
under semi-natural disease pressure
Research project overview
REDUCTION OF MYCOTOXINS IN WHEAT

6. 6Mycokey Workshop 2019
Alejandro Gimeno
FORMULATION
Method development and production for experiments
Improving survival and effect of the microorganism
Protection against harmful UV-B
FIELD EXPERIMENTS
4.3 Prevention: treatment of artificially infected
maize residues
5.1 Intervention: treatment of flowering wheat heads
under semi-natural disease pressure
Research project overview
REDUCTION OF
MYCOTOXINS IN WHEAT
LABORATORY EXPERIMENTS
Suppression of perithecia production and ascospore release on
infected maize residues
Growth chamber experiments with wheat
Investigation of the mode of action of C. rosea
BCAs
Isolates from Switzerland for biological control of FHB
Establishment of a protocol for detection of C. rosea
from plant material (VTT collaboration)*

7. 7Mycokey Workshop 2019
Alejandro Gimeno
Clonostachys rosea
Biological control agent
Clonostachys rosea 016*
Worldwide distribution
(https://unite.ut.ee/)
hyperparasitic antagonist of
important plant pathogens
in the rhizosphere and the
phylosphere including
Fusarium species (Jensen et
al., 2000; Xue, 2003; 2009)
Oatmeal agar
Trichoderma / Gliocladium
selective rose bengal agar
(Vargas et al. 2009)
Potato dextrose agar (Oxoid)
*Kindly provided by J. Köhl
(Wageningen University)
Schöneberg et al. (2015)

8. 8Mycokey Workshop 2019
Alejandro Gimeno
Clonostachys rosea
Biological control agent
Clonostachys rosea 016*
Worldwide distribution
(https://unite.ut.ee/)
hyperparasitic antagonist of
important plant pathogens
in the rhizosphere and the
phylosphere including
Fusarium species (Jensen et
al., 2000; Xue, 2003; 2009)
Oatmeal agar
Trichoderma / Gliocladium
selective rose bengal agar
(Vargas et al. 2009)
Potato dextrose agar (Oxoid)
p < 0.000
Gimeno et al. (in preparation)
*Kindly provided by J. Köhl
(Wageningen University)
Control Clonostachys rosea 016

9. 9Mycokey Workshop 2019
Alejandro Gimeno
FORMULATION
Protection against
harmful UV-B
Protection against
harmful UV-B
UV-B dose rate (kJ m-2)
0.0 7.1 10.7 14.3 17.8 21.4
0
20
40
60
80
100
Relativegermination(%)
* BSc. thesis M. Leimgruber
*
*
24 h; 20 ± 2°C
UV-B: 2000 mW m-2
120 min (total 17.8 kJ m-2)

10. 10Mycokey Workshop 2019
Alejandro Gimeno
FORMULATION
Protection against
harmful UV-B
Protection against
harmful UV-B
 24 h; 20 ± 2°C
 UV-B: 2000 mW m-2
 7.14 kJ m-2 h-1
UV-B dose rate (kJ m-2)
0.0 7.1 10.7 14.3 17.8 21.4
0
20
40
60
80
100
Relativegermination(%)
* BSc. thesis M. Leimgruber
Costa et al. 2012
Costa et al. 2016
*
*

11. 11Mycokey Workshop 2019
Alejandro Gimeno
0
20
40
60
80
100
a
b
ffff
cc
c
d
e
Control +
Control -
Germinationrate(%)
6%
96%
-90%
Screening UV-protective substances
BSc. thesis M. Leimgruber
24 h; 20 ± 2°C
UV-B: 2000 mW m-2
120 min (total 17.8 kJ m-2)
Control -
Control +
Clonostachys rosea SHA77.3 (CH)

12. 12Mycokey Workshop 2019
Alejandro Gimeno
0
20
40
60
80
100
a
b
ffff
cc
c
d
e
Control +
Control -
6%
96%
Screening UV-protective substances
Vegetable oils
± UVB absorber (ethyl-hexyl methoxycinnamate)
Rapeseed oil
Olive oil
Sunflower oil
Rapeseed oil + absorber (Escalol 557)
Olive oil + absorber (Escalol 557)
Telmion
Tween80
Sunflower oil + absorber (Escalol 557)
BSc. thesis M. Leimgruber
Germinationrate(%)
5% Olive oil

13. 13Mycokey Workshop 2019
Alejandro Gimeno
0
20
40
60
80
100
a
b
ffff
cc
c
d
e
Control +
Control -
6%
96%
Rapeseed oil
Olive oil
Sunflower oil
Rapeseed oil + Escalol
Olive oil + Escalol
Telmion
Tween80
Field Formulation
•Sunflower oil (70%)
•Escalol 557 (10%)
•Surfactants (20%)
Sunflower oil + Escalol
BSc. thesis M. Leimgruber
Germinationrate(%)
5%
5%
92%

14. 14Mycokey Workshop 2019
Alejandro Gimeno
4.3 Biological control of F. graminearum on crop residues
Field experiments 2016-2018

15. 15Mycokey Workshop 2019
Alejandro Gimeno
Field experiments 2016-2018
Fusarium graminearum infected
maize residues were treated with
antagonist suspensions [107/ml]
and incubated
Laboratory
• Simulated disease pressure of maize – wheat rotation• Simulated disease pressure of maize – wheat rotation
Experimental designExperimental design

16. 16Mycokey Workshop 2019
Alejandro Gimeno
Field experiments 2016-2018
Fusarium graminearum infected
maize residues were treated with
antagonist suspensions [107/ml]
and incubated
Laboratory
Simulated disease pressure of maize – wheat rotation
Experimental designExperimental design
treated maize stalks were
distributed in the plots (80 per plot)
Field
Split plot design with two winter
wheat cultivars (Levis, Forel)
Treatment of artificially infected maize residues:
Biological control agent
C. rosea
strain 016
T. atrobrunneum
strain ITEM908*
Unformulated (Conidia from PDA) 107 conidia ml-1 107 conidia ml-1
Wettable powder (WP) 106 – 107 CFU ml-1 -
Sunflower oil + UVB protection (Oil) 5 vol% emulsion 5 vol% emulsion
*Fanelli et al. 2018

17. 17Mycokey Workshop 2019
Alejandro Gimeno
Field experiments 2016-2018
Fusarium graminearum infected
maize residues were treated with
antagonist suspensions [107/ml]
and incubated
Laboratory
Simulated disease pressure of maize – wheat rotation
Experimental designExperimental design
treated maize stalks were
distributed in the plots (80 per plot)
Field
Split plot design with two winter
wheat cultivars (Levis, Forel)
Treatment of artificially infected maize residues:
Biological control agent
C. rosea
strain 016
T. atrobrunneum
strain ITEM908*
Unformulated (Conidia from PDA) 107 conidia ml-1 107 conidia ml-1
Wettable powder (WP) 106 – 107 CFU ml-1 -
Sunflower oil + UVB protection (Oil) 5 vol% emulsion 5 vol% emulsion
*Fanelli et al. 2018
Do treatments with BCAs on infected maize crop residues
reduce the accumulation of DON and ZEN?
 Differences between the antagonists?
 Formulations?
 Wheat varieties?
Do treatments with BCAs on infected maize crop residues
reduce the accumulation of DON and ZEN?
 Differences between the antagonists?
 Formulations?
 Wheat varieties?

18. 18Mycokey Workshop 2019
Alejandro Gimeno
61 61-65 65-69 69
Zadok’s growth scale
+61 61-65 65-69 69
Growth stage:
+61 61-65 65-69 69
Growth stage:
F.graminearumcfu
F.graminearumcfu
F.graminearumcfu
F. graminearum
Control
Co-formulants
WP, Oil
C. rosea 016
PDA, WP, Oil
T. atrobrunneum
ITEM908
PDA, Oil Gimeno et al. (in preparation)
Total cfus: 17’147 (2017) < 23’862 (2018) (+39%)
Peaks: End of flowering (GS 65–69) in 2017
Full flowering stage in 2018 (GS 61–65)H.R.Forrer
D. Drakopoulos (Foto)

19. 19Mycokey Workshop 2019
Alejandro Gimeno
Deoxynivalenol
Fusarium graminearum 92%
Fusarium poae 5%
Microdochium sp. 3%
/ 96%
/ 2%
/ 2%
2016/17 2017/18
Treatment 2016/17
mean DON
reduction%
2017/18
mean DON
reduction%
2016/17
mean ZEN
reduction%
2017/18
mean ZEN
reduction%
No inoculum
90.8 95.7 87.6 94.5
Control
- - - 0.0
016 PDA
65.0 90.3 54.8 93.8
016 WP
81.5 81.7 78.1 91.8
016 PDA+Oil
68.3 85.0 52.1 91.1
016 WP+Oil
69.5 85.2 59.0 89.0
ITEM908 PDA
-30.2 72.3 -157.1 79.5
ITEM908 PDA+Oil
-53.1 79.7 -135.2 84.9
WP Formulation
-8.1 -29.3 -53.2 -26.7
Oil Formulation
-8.7 -0.9 -86.0 31.5
Forel Levis Forel Levis
2016/17
treatment: p<0.001; variety: n.s.; treatment×variety: n.s.
2017/18
treatment: p<0.001; variety: p<0.001;
treatment×variety: n.s.
WP4
C. rosea 016 reduced deoxynivalenol (DON) up
to 82% in 2016/17 and up to 90% in 2017/18
Unpublished data ©
Agroscope

20. 20Mycokey Workshop 2019
Alejandro Gimeno
Zearalenone WP4
Fusarium graminearum 92%
Fusarium poae 5%
Microdochium sp. 3%
/ 96%
/ 2%
/ 2%
2016/17 2017/18
Treatment 2016/17
mean DON
reduction%
2017/18
mean DON
reduction%
2016/17
mean ZEN
reduction%
2017/18
mean ZEN
reduction%
No inoculum
90.8 95.7 87.6 94.5
Control
- - - 0.0
016 PDA
65.0 90.3 54.8 93.8
016 WP
81.5 81.7 78.1 91.8
016 PDA+Oil
68.3 85.0 52.1 91.1
016 WP+Oil
69.5 85.2 59.0 89.0
ITEM908 PDA
-30.2 72.3 -157.1 79.5
ITEM908 PDA+Oil
-53.1 79.7 -135.2 84.9
WP Formulation
-8.1 -29.3 -53.2 -26.7
Oil Formulation
-8.7 -0.9 -86.0 31.5
Forel Levis Forel Levis
2016/17
treatment: p<0.001; variety: p<0.001 ;
treatment×variety: n.s.
2017/18
treatment: p<0.001; variety: p<0.001 ;
treatment×variety: p<0.001
Zearalenone (ZEN) was reduced by C. rosea 016
up to 80% in 2016/17 and 90% in 2017/18.
Unpublished data ©
Agroscope

21. 21Mycokey Workshop 2019
Alejandro Gimeno
5.1 Pre-harvest strategies for wheat
Field experiments 2016-2018

22. 22Mycokey Workshop 2019
Alejandro Gimeno
Fusarium graminearum infected
maize residues were treated with
antagonist suspensions [107/ml]
and incubated
Laboratory
Task 5.1 Pre-harvest strategies for wheat
treated maize stalks were
distributed in the plots (80 per plot)
Field
Split plot design with two winter
wheat cultivars (Levis, Forel)
Active ingredient Formulation Application timing and density
Water
- /
Emulsion [5%vol]
GS 59-61; 600 l/ha
Clonostachys rosea strain 016
Suspension in water /
Emulsion [5%vol]
GS 59-61; 107 cfu/ml; 600 l/ha
Clonostachys rosea strain NBB2.9
Suspension in water /
Emulsion [5%vol]
GS 59-61; 107 cfu/ml; 600 l/ha
Clonostachys rosea strain SHA77.3
Suspension in water /
Emulsion [5%vol]
GS 59-61; 107 cfu/ml; 600 l/ha
Trichoderma atrobrunneum ITEM908 Suspension in water GS 59-61; 107 cfu/ml; 600 l/ha
Prothioconazole Emulsion (Proline ®) BBCH 59-61; 0.8l/ha ; 600 l/ha
Winter wheat variety Category FHB resistance
‘Levis’ Kl. II low
‘Forel’ Kl. I medium to low

23. 23Mycokey Workshop 2019
Alejandro Gimeno
WP5
Fusarium graminearum infected
maize residues were treated with
antagonist suspensions [107/ml]
and incubated
Laboratory
Task 5.1 Pre-harvest strategies for wheat
treated maize stalks were
distributed in the plots (80 per plot)
Field
Split plot design with two winter
wheat cultivars (Levis, Forel)
Active ingredient Formulation Application timing and density
Water
- /
Emulsion [5%vol]
GS 59-61; 600 l/ha
Clonostachys rosea strain 016
Suspension in water /
Emulsion [5%vol]
GS 59-61; 107 cfu/ml; 600 l/ha
Clonostachys rosea strain NBB2.9
Suspension in water /
Emulsion [5%vol]
GS 59-61; 107 cfu/ml; 600 l/ha
Clonostachys rosea strain SHA77.3
Suspension in water /
Emulsion [5%vol]
GS 59-61; 107 cfu/ml; 600 l/ha
Trichoderma atrobrunneum ITEM908 Suspension in water GS 59-61; 107 cfu/ml; 600 l/ha
Prothioconazole Emulsion (Proline ®) BBCH 59-61; 0.8l/ha ; 600 l/ha
Winter wheat variety Category FHB resistance
‘Levis’ Kl. II low
‘Forel’ Kl. I medium to low
Does foliar application of fungal antagonists at early flowering
protect against FHB and mycotoxin accumulation?
 Differences between local and foreign C. rosea?
 Benefits of UV-B protection?
 Differences between the wheat varieties?
Does foliar application of fungal antagonists at early flowering
protect against FHB and mycotoxin accumulation?
 Differences between local and foreign C. rosea?
 Benefits of UV-B protection?
 Differences between the wheat varieties?

24. 24Mycokey Workshop 2019
Alejandro Gimeno
2016 – 2018 Deoxynivalenol WP5
F. graminearum
Control
Co-formulants Oil
High disease pressure from the
primary inoculum
Co-formulants of the oil formula
showed no significant effects on the
accumulation of DON

25. 25Mycokey Workshop 2019
Alejandro Gimeno
2016 – 2018 Deoxynivalenol WP5
F. graminearum
Control
Co-formulants Oil T. atrobrunneum
ITEM908
C. rosea 016,
SHA77.3,
NBB2.9
No significant effects when spores
where applied in water suspension
(016; NBB2.9; SHA77.3; ITEM908)

26. 26Mycokey Workshop 2019
Alejandro Gimeno
2016 – 2018 Deoxynivalenol WP5
F. graminearum
Control
Co-formulants Oil T. atrobrunneum
ITEM908
C. rosea 016,
SHA77.3,
NBB2.9
C. rosea 016,
SHA77.3,
NBB2.9
+ oil
formulation
016 and SHA77.3 (Swiss isolate) in
combination with the oil formula
reduced DON between 30-54%
UV-B protection

27. 27Mycokey Workshop 2019
Alejandro Gimeno
2016 – 2018 Deoxynivalenol WP5
F. graminearum
Control
Co-formulants Oil T. atrobrunneum
ITEM908
C. rosea 016,
SHA77.3,
NBB2.9
C. rosea 016,
SHA77.3,
NBB2.9
+ oil
formulation
016 and SHA77.3 (Swiss isolate) in
combination with the oil formula
reduced DON between 30-54%
Prothioconazole Emulsion (Proline ®) BBCH 59-61; 0.8l/ha ; 600 l/ha
UV-B protection

28. 28Mycokey Workshop 2019
Alejandro Gimeno
2016 – 2018 Zearalenone WP5
F. graminearum
Control
Co-formulants Oil T. atrobrunneum
ITEM908
C. rosea 016,
SHA77.3,
NBB2.9
C. rosea 016,
SHA77.3,
NBB2.9
+ oil
formulation
Fungicide
No reductions in ZEN contentsNo reductions in ZEN contents

29. 29Mycokey Workshop 2019
Alejandro Gimeno
… in conclusion

30. 30Mycokey Workshop 2019
Alejandro Gimeno
4.3 Biological control of F. graminearum on crop residues
 C. rosea 016 reduced ascospore dispersal and DON and ZEN by up to 80-90%
 T. atrobrunneum ITEM908 inconsistent between experiments with
reductions of DON and ZEN by up to 80 or 90%
5.1 Pre-harvest strategies for wheat
 C. rosea 016 and SHA77.3 (Swiss isolate) in combination with
oil-based UV-B protection reduced DON between 30-54%
 ZEN was not reduced
 Strain-specific differences

31. 31Mycokey Workshop 2019
Alejandro Gimeno
Agroscope
Research group for Ecological plant
protection in arable crops
Susanne Vogelgsang (Supervisor)
Irene Bänziger
Andreas Kägi
Eveline Jenny
Dimitrios Drakopoulos
Hans-Rudolf Forrer
Miriam Leimgruber
Zacharie Ngamenie
Zivis and Interns
Agroscope Feldequipe
Research group Plant-Soil Interactions
Claire Stanley
Research group Phytopathology of fruit and
vegetable crops
Florian Freimoser
Acknowledgements
Funded by: University of Zürich
Beat Keller (Academic supervisor)
MycoKey partners
Claudio Altomare (CNR ISPA)
Jürgen Köhl (Wageningen University)
Adjuvants Plus Inc., Canada
Agrifutur S.r.l., Italy
VTT Technical Research
Institute Finland
Arja Laitila
Elina Sohlberg
Tiina Pakula
Jenni Limnell
Tuija Sarlin

32. 32Mycokey Workshop 2019
Alejandro Gimeno
Thank you for your attention
Alejandro Gimeno
alejandro.gimeno@agroscope.admin.ch
Agroscope good food, healthy environment
www.agroscope.admin.ch
Photo A Kägi