3rdMycoKey TechnologicalWorkshop held at Agroscope in Zurich Reckenholz

1. Biological control of Aspergillus flavus
in maize with atoxigenic strains
in Europe
Paola Battilani, Antonio Logrieco
paola.battilani@unicatt.it

2. Gary Payne, N. C. State University
Aspergillus flavus
&
aflatoxins
Stress
Tmin Tmax Topt aw min
Growth 12 48 36-38 0.78
AF 20 30 25-30 0.78

3. § The worldwide experience suggests that the prevention of
aflatoxin contamination can count on few tools, the only
effective preventive intervention is biocontrol with
Aspergillus flavus non-toxigenic strains
§ The objective of this technique is to replace the aflatoxin-
producing fungi with fungi of the same species that do
not produce these metabolites
Aspergillus flavus and aflatoxins

4. Genetic groups limit the possibility of gene exchange
A. flavus, complex community
§ Toxigenic and atoxigenic strains coexist
§ There are several genetic groups
Peter Cotty

5. Biocontrol A. flavus
Toxigenic strains
Atoxigenic strains
Competitive exclusion

6. A. flavus and aflatoxin control
worldwide experience
§ Atoxigenic isolates of A. flavus as biocontrol agents to displace
aflatoxigenic fungi
§ Pioneered in the US, with 2 atoxigenic genotypes registered
(US-EPA)
• Aspergillus flavus AF36, developed by USDA-ARS, first product
registered, produced and distributed by a farmer governed and
financed organization
• Afla-Guard® produced and distributed by Syngenta
§ Developed in African nations Aflasafe (USDA-ARS, IITA, M&B
Gates Foundations et al), trade mark authorized in Nigeria,
The Gambia, Senegal, Kenia, Burkina Faso, Ghana, Tanzania,…

7. Aflatoxins in Italy
First outbreak, milk contamination with aflatoxin M1
(October 2003)

8. • Autoctonous fungi
• Atoxigenic (confirmed by gene absence/mutation)
• Strong competitors with toxigenic strains
• Effective in field conditions
Aim of the candidate BCA selection

9. North Italy,
138 strains ,
2003-2010
!
Strain collection & characterisation
Toxigenic
54%
Atoxigenic
46%
74
strain
64
strains
Atoxigenic
25
Toxigenic
23
VCG VCG
Toxigenicity
(AF & CPA)
&
Genetic
groups (VCG)
Mauro et al., 2013

10. Best Italian strains
In vitro trialsAFLAGARDItalian strains
Mauro et al., 2015

11. 60
70
80
90
100
PR RO2 VR1 VR2
ReductionAFB1(%)
Location
0
40
80
120
160
RO1 RO3 MN1 MN2 PR RO2 VR1 VR2
AflatoxinB1(µg/kg)
Location
BCA Control
Ø 8 fields, 6 places
Ø 1 ha control/1 ha treated
Ø 10 kg/ha BCA, 2 strains
In field efficacy – year 2012
Mean reduction=92,3%
Mauro et al., 2018

12. 0
20
40
60
80
100
PR RO2 VR1 VR2
VCG(%)
Location
Other IT019 IT006
BCA
VCG recovery
Mauro et al., 2018

13. Steps towards field application…
§ Strain deposit: MUCL 54911 (May 2013)
§ Patent deposit

14. § Aspergillus flavus MUCL54911
§ AF-X1 is a biocontrol agent
§ 2.1 Compound classification It is classified as not dangerous according to
Directives 67/548/CEE and 1999/45/CE and/or Regulation CE 1272/2008 (CLP
and following updates
§ AF-X1 is devitalised sorghum, coated with spores
of A. flavus MUCL54911
§ The dossier is under evaluation for the final
approval, AF-X1 is now applied with temporary
authorisation to be renewed annually
The European product, AF-X1

15. Growth
BBCH 33-39, after tillage
Field treatment
Distribution with
fertiliser spreader

16. § The maize was intended for feed for dairies involved with
cheese production
§ Cheese makers require milk to contain < 0.050 ug/kg
§ Maize producers hired the lab to perform the analyses
§ The aflatoxin results reported here were the basis for
accepting or rejecting the maize
§ Rejection resulted in economic loss for the farmers
AF-X1 Field application 2016

17. § Geographic area: north Italy
§ Applied in ~ 15000 ha of maize commercial fields
AF-X1 Field application 2016

18. § Maize analysed~ 1.4 milion tons
§ Fields treated with AF-X1 = around 16%
§ AF mean reduction = 94%
Results 2016 - summary
No impact on other mycotoxins

19. 2003 - 2010 2011 2012 2013 2014 2015 2016 2017 2018
Collection of isolates
Strain characterisation Field trials
Candidate
BCA selection
Strain deposit
Patent deposit OK
Open field
treatment
DOSSIER
AF-X1, the history
Commercial product approval expected in 2020

20. Phytosanitary area
South Europe
• Italy
• Bulgaria
• France
• Spain
• Portugal
• Greece
• Malta
• Cyprus
Area of registration

21. MycoKey – enlargement
at European level
236 isolates of A. flavus,
(2012-2016)
>300
isolates of
A. flavus,
(2016)

22. Good evening, I am a technician who works in the province of Treviso and Venice, ...
I wanted to thank you for the work you have done and inform you that thanks to
several small fertilizer spreaders that the contractors in my area have equipped with,
we are doing a good job. I don't know if it can be defined as a "district" treatment, as
you wish, but we are on the right track ....
The voice of farmers

23. Thanks for your time
Giorni P., Pietri A.
AFLARID
MIPAF
Prof. G. Piva
AGRISYSTEM
Antonio Mauro
P. J. Cotty