Transcript Streptomyces-Aspergillus interactions: Impact on - e

Modulation of aflatoxin B1 production
by A. flavus
Carol VERHEECKE
Supervisors: Pr. MATHIEU & Pr. LIBOZ
http://lgc.cnrs.fr
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Plan
• Introduction
• Objectives
• Results
– Part 1: Impact of Aw, °C, incubation time on aflatoxins B
production
– Part 2: Impact of actinomycetes impact on aflatoxins
production
• Conclusion
• Perspectives
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Introduction
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.
Zearalenon
Citrinin
T-2 & HT-2
Aflatoxins (B1, B2, G1, G2, M1)
Deoxynivalenol
Patulin
Ochratoxin A
Fumonisins B1, B2
Bennett & Klich (2003)
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Introduction
• 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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Introduction
• In France:
• Current risk:
– Deoxynivalenol, Fumonisins
• In the Future ( 2001-2100):
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(Battilani et al., 2012).
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Aflatoxins: presentation
Discovered in 1962
AFB1
AFB2
AFG1
AFG2
Composed of:
• Coumarin group
• Bisfuran ring
• Pentan group
AFB
• Furan group
AFG
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2D structures of aflatoxin B1 (AFB1), aflatoxin B2 (AFB2),
aflatoxin G1 (AFG1) and aflatoxin G2 (AFG2)
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Aflatoxins: toxicity
- 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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Aflatoxins: producers
p
h
v
c
A. mottae
cd
5 µm
Aspergillus
flavus
by
microscopy. c= conidies; cd=
conodiophores; h= hyphae; p=
phialides and v= vesicule.
(Personal data)
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Aflatoxins: abiotic parameters
Growth
AFB1 production
• Water activity: aw
– 0.94 - 0.99
– 0.96 - 0.99
• Temperature
– 30 - 35°C
– 25 - 30°C
Other parameters:
Gas composition, Medium, pH, Light, Chemical compounds
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Aflatoxins: biotic parameters
(IITA, WMF2012)
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Aflatoxins: Biosynthesis
• 1 AcetylCOA & 9 MalonylCOA
Norsolorinic Acid (NOR)
Versicolorin B
1
2
Sterigmatocystin
(Yu et al., 2004)
AFB1
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AflU
(NadA,
AflF)
AFG1 AFG2
AFB2
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Aflatoxins: Regulation
• 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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(Alkhayyat & Yu, 2014)
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Aflatoxins: reduction
Illumination Fiber
Reflectance Fiber
Maize
kernel
Fiber Optic to
Spectrometer
Fiber Optic
(Pearson et al., 2004) from Light
Source
(= Bentonite, Biomin.net)
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Aflafree project 2011- 2015
Aflatoxin B1 (AFB1) risk management for a
sustainable maize Food chain
WP1: Modelisation of A. flavus growth and
WP2: AFB1 inhibition strategy
AFB1 production
WP3: Characterisation
WP4: Impact on the maize chain
in greenhouse
and good practices
WP5: Maize chain sustainability increase
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Objectives
• 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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Objectives 1: Context
• 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
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Mycotoxins:
Maize rejection
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Prestorage
(10-40°C; aw of 0.75-1)
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Objectives 2: Context
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
–
Maize in greenhouse
(Daniel Caron)
•
Actinobacteria
(ISP2 medium)
Why actinomycetes?
On maize:
•
Streptomyces spp. DAUFPE 11470 & 14632 (Bressan and
Figueiredo 2008)
•
Endophytic Actinobacteria (Costa et al. 2013)
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Methodology:
Screening actinomycetes isolates
Verheecke et al., 2014
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Prevention methodology
• aflD, aflM, aflP:
AflD: (ketoreductase)
Norsolorinic acid
Averantin
AflM: (deoxygenase)
Versicolorin A
Demethylsterigmatocystin
AflP: (O-methyltransferase)
Sterigmatocystin
O-methylsterigmatocystin
(Yu et al., 2004)
aflR and aflS: encode regulators
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Prevention methodology
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
from Yu et al. 2004
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Curative methodology:
reduction of AFB1 concentration
ISP2 media supplemented with
pure-AFB1 (5 mg.l-1)
Adsorption test
AFB1: 1 µg.ml-1
Spores suspension:
107 spores.ml-1
Incubation
at 28°C for 4 days
AFB1 methanol Extraction &
Quantification coupled with Coring
Cell® by HPLC
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1 to 60 minutes 30°C
filtration
HPLC (1)
Water wash
HPLC (2)
Methanol wash
HPLC (3)
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Perspectives (Objective 1)
Study of additional
factors:
Transfer to
prestorage:
• Mycotoxin analysis of
the coinoculation tests
• Fusarium verticillioides
& fumonisins
production
• in vivo tests
• software
development
Transfer to maize grain:
• test critical aw, temperature,
time and CO2 content
• data compilation to develop
models of mycotoxins risk
management
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Perspectives (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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Perspectives (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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