Modulation of aflatoxin B1 production by A. flavus
Carol VERHEECKE
Supervisors: Pr. MATHIEU & Pr. LIBOZ http://lgc.cnrs.fr
11/25/2014 PhD defence

• Introduction
• Objectives
• Results
– Part 1: Impact of A w
, °C, incubation time on aflatoxins B production
– Part 2: Impact of actinomycetes impact on aflatoxins production
• Conclusion
• Perspectives
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Mycotoxins:
- are low molecular weight molecules (mostly thermostable);
- are secondary metabolites produced by filamentous fungi;
- can cause death or disease to human being or animal at a low concentration.
Citrinin T-2 & HT-2
Aflatoxins (B1, B2, G1, G2, M1)
Deoxynivalenol
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Patulin Fumonisins B1,
B2
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Bennett & Klich (2003)
3

• Food and Feed Regulations:
•
Aflatoxin B1: (EU 1881/2006 modified 03/2014)
–
Food:
Cereals for infants (0.1 µg.kg
-1 ) to humans (2 µg.kg
-1 )
– Feed (≤ 20 µg.kg
-1 )
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(Pitt et al.
, 2013)
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•
In France:
•
Current risk:
–
Deoxynivalenol, Fumonisins
•
In the Future ( 2001-2100):
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(Battilani et al., 2012).
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Discovered in 1962
AFB1 AFB2
Composed of:
•
Coumarin group
•
Bisfuran ring
•
Pentan group
AFG1 AFG2
AFB
•
Furan group
AFG 2D structures of aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1) and aflatoxin G2 (AFG2)
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HepatoCellular
Carcinoma:
2.4 to 5.5
If Hepatitis B:
7 to 10.6
Child growth retardation:
1.7 cm (8 months)
Immune System:
Reduction in immune system differentiation
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Still born and jaunice
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(Ross et al.
, 1992; Jiang et al.
, 2005;
Gong et al., 2004; Shuaib et al., 2010)
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A. mottae
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p h v c cd
5 µm
Aspergillus flavus by microscopy. c= conidies; cd= conodiophores; h= hyphae; p= phialides and v= vesicule.
(Personal data)
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AFB1 production Growth
• Water activity: a w
– 0.94 - 0.99
• Temperature
– 30 - 35°C
– 0.96 - 0.99
– 25 - 30°C
Other parameters:
Gas composition, Medium, pH, Light, Chemical compounds
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(IITA, WMF2012)
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• 1 AcetylCOA & 9 MalonylCOA
(Yu et al.
, 2004)
Norsolorinic Acid (NOR)
1
Versicolorin B
2
Sterigmatocystin
AFB1
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AflU
(NadA,
AflF)
AFG1 AFG2 AFB2
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• AflR and AflS: specific regulators
( )
AflR:
• Zinc Finger transcription factor
• Specific binding
5’- WCGSNNNSCGA-3’
(W: A ou T, S: C ou G, R: A ou G)
AflS: AflR coactivator?
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Illumination Fiber
Reflectance Fiber
Maize kernel
Fiber Optic to
Spectrometer
(Pearson et al., 2004)
Fiber Optic from Light
Source
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(= Bentonite, Biomin.net)
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Aflatoxin B1 (AFB1) risk management for a sustainable maize Food chain
WP1: Modelisation of A. flavus growth and
AFB1 production
WP2: AFB1 inhibition strategy
WP3: Characterisation in greenhouse
WP4: Impact on the maize chain and good practices
WP5: Maize chain sustainability increase
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• 1) Monitoring A. flavus entry into the French maize ecosystem (e. g. Fusarium sp.) and its impact on aflatoxins and Deoxynivalenol (DON) risks at prestorage.
• 2) Development of a biocontrol (based on actinomycetes) able to reduce (in interaction with Aspergillus sp.) AFT (AFB + AFG) contamination at field without impacting the maize microbial ecosystem
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• 1) Monitoring A. flavus entry into the French maize ecosystem (e. g. Fusarium sp.) and its impact on aflatoxins and DON risks at prestorage.
Fusarium graminearum (Deoxynivalenol)
Impact ?
Aspergillus flavus & aflatoxins production
Mycotoxins:
Maize rejection
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Prestorage
(10-40 °C; a w of 0.75-1)
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Aim: identification of a bacterial biocontrol able to grow with
A. flavus and reduce AFT accumulation in maize.
• Reduction of AFT concentration by the biocontrol (ultimate aim in planta)
• Have the least impact on the maize microbial ecosystem
• Biocontrol harmlessness
• Biocontrol survival in maize or in maize soil.
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• class of filamentous, soil born, Gram positive bacteria
• major source of secondary metabolites useful for the pharmaceutical and agricultural industries
• Actinomycetes are potential biocontrol agents
– On maize:
• Streptomyces spp. DAUFPE 11470 & 14632 (Bressan and
Figueiredo 2008)
• Endophytic Actinobacteria (Costa et al. 2013)
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Verheecke et al ., 2014
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•
aflD, aflM, aflP:
AflD: (ketoreductase)
Norsolorinic acid Averantin
AflM: (deoxygenase)
Versicolorin A
Demethylsterigmatocystin
(Yu et al.
, 2004)
Sterigmatocystin
AflP: (O-methyltransferase)
O-methylsterigmatocystin
aflR and aflS: encode regulators
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from Yu et al. 2004
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RT-qPCR analysis to monitor
Aflatoxin biosynthesis gene expression
– Gene of reference: identified thanks to geNorm
– Gene of interest:
• aflD, aflM afP: ‘structural’ genes
• aflR, aflS: ‘regulator’ genes
– A. flavus & A. parasiticus
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reduction of AFB1 concentration
ISP2 media supplemented with pure-AFB1 (5 mg.l
-1 )
Adsorption test
AFB1: 1 µg.ml
-1
Spores suspension:
10 7 spores.ml
-1
Incubation at 28 ° C for 4 days 1 to 60 minutes 30 ° C filtration HPLC (1)
HPLC (2) Water wash
AFB1 methanol Extraction &
Quantification coupled with Coring
Cell ® by HPLC
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Methanol wash HPLC (3)
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Study of additional factors:
• Mycotoxin analysis of the coinoculation tests
• Fusarium verticillioides
& fumonisins production
(Objective 1)
Transfer to prestorage:
• in vivo tests
• software development
Transfer to maize grain:
• test critical a w time and CO
2
, temperature, content
• data compilation to develop models of mycotoxins risk management
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(Objective 2)
• Mechanisms characterisation:
– Adsorption tests on the other strains
– Kinetic on gene expression study (e.g. S35, S38)
– Metabolites identification
• Prevent production: VOC? Aflatoxins repressors? …
• Reduce concentration: enzyme identification? Degradation products? …
– Transcriptome and metabolome studies
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(Objective 2) on maize:
- biocontrol production in various scales
- Test against other mycotoxins producers (e.g. Fusarium sp.)
- test in vitro and under greenhouse
At field:
- biocontrol dispersal (biocontrol characterisation)
- formulation
- harmlessness validation
- accreditation
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