This document discusses the use of biocontrol to manage aflatoxin contamination in crops in West and East Africa. It presents results from multiple field trials showing that applying atoxigenic strains of Aspergillus significantly reduces aflatoxin levels in maize and groundnuts at harvest and after storage compared to untreated controls. The biocontrol works by competitively excluding toxigenic strains, resulting in shifts in the Aspergillus population to predominately consist of the applied atoxigenic strains. No increases in total Aspergillus levels or infection were observed due to the biocontrol treatment. The approach provides an effective and sustainable solution to reducing the dangerous health impacts of aflatoxin contamination in crops in Africa

1. www.iita.orgA member of CGIAR consortium
Effective Aflatoxin
Management in
Farmers' Fields in
West and East
Africa
IITA: Bandyopadhyay, Atehnkeng, Mutegi
USDA-ARS / Univ of Arizona: Cotty,
Jaime-Garcia, Callicot, Probst
Senegal: Senghor; Burkina: Bonkoungou
Annual Meeting of the American Phytopathological Society
Minneapolis, 9-13 August, 2014

2. • Highly toxic metabolite
produced by the ubiquitous
Aspergillus flavus fungus
• The fungus resides in soil and
crop debris, infects crops and
produces the toxin in the field
and in stores
• Death, liver cancer, immune-
suppression, stunted growth
• Impacts animal productivity
• Negatively impacts trade
• Fungus carried
from field to store
• Contamination
possible without
visible signs of the
fungus
Aflatoxin Facts

3. www.iita.orgA member of CGIAR consortium
Pre-Harvest Problem
Aflatoxin
(ppb)ppb)
Peanut (n = 188) Maize (n = 241)
Distribution (% samples)
> 4 54 70
> 10 41 52
> 20 29 24
Descriptive statistics (ppb)
Minimum < LOD < LOD
Maximum 3487 838
Mean 111 33
LOD = Limit of Detection; 1 ppb
Aflatoxin in Groundnut and Maize at Harvest
Increases in store

4. www.iita.orgA member of CGIAR consortium
EPA approved 2 products
AF36
Afla-guard
Hundreds
of
Thhosands
of acres
treated
annually
in the US!
Production Room
Atoxigenic Strain Manufacturing Facility
Arizona Cotton Research & Protection Council
(Funded and Governed by the Farmers of Arizona),
Phoenix, Arizona
It Works in
Africa Too
Biocontrol Works!

5. www.iita.org
• IITA
• USDA
• AATF
• BMGF/USAID
• Doreo Partners
• National institutions
Strong Partnership
www.iita.orgA member of CGIAR consortium

6. www.iita.orgA member of CGIAR consortium
Biocontrol Principles
 In nature, some strains produce a lot
(toxigenic), and others no aflatoxin
(atoxigenic) (Donner, Soil Biol Biochem
2009)
 Atoxigenic strains are already present on
the crop (Atehnkeng et al., IJFM, 2008)
 Increase the frequency of atoxigenic
strains to competitively displace
toxigenic strains (Cotty & Bayman,
Phytopath 1993) to reduce aflatoxin
contamination .
 Atoxigenic strains can be applied without
increasing infection and without
increasing the overall quantity of A. flavus
on the crop or in the environment (Cotty,
Phytopath 1994; Atehnkeng et al., Biological
Control 2014)
0
1
2
3
4
5
6
7
8
9
0 20 40 60 80 100
AflatoxinB1(ng/gX10,000)
Isolates (%) in Applied Atoxigenic Strain
 Strains move from
field to stores
 Multiple year & crop
carry-over effect
(Jaime & Cotty,
Phytopath 2006)
 We use only native
strains

7. www.iita.orgA member of CGIAR consortium
Strain Selection Criteria
In the laboratory (~5,000 strains):
• Does not produce aflatoxin
• VCG/SSR group with
Wide geographic distribution
No toxigenic member
• Defective in >2 aflatoxin & CPA
genes
• Outcompetes toxigenic strains
After field application:
• Superior capacity to colonize,
multiply and survive in soil
• Superior frequency of isolation
from grains
• Superior capacity to reduce
aflatoxin
8-12 native strains
selected for field tests
4 native strains
formulated into
the final
product

8. Broadcast
@ 10 kg/ha 2-3 weeks
before flowering
Sporulation on moist soil
Spores
Insects
Aflasafe in 5 kg boxes
3-20
days
Wind
Soil
colonization
30-33 grains m-2
Fungal network
in killed grain
How Does aflasafe Work?

9. www.iita.orgA member of CGIAR consortium
Efficacy Trials: Data Collection
• All trials conducted in farmers’
fields on crops grown by farmers
• Aflasafe applied by farmers
• Soil sampled before treatment
and grains at harvest:
– Aspergillus population density
– Aspergillus strain profile
– Incidence of aflasafe strains
• Aflatoxin concentration in grains
at harvest and after poor storage

10. www.iita.orgA member of CGIAR consortium
Design & Analysis of Trials
• Field trial size: 0.25 to 15 ha
• Number of fields: 14 to 200
per year
• Paired plot: Each treated
field with its own
companion control field in
close vicinity
• Each farmers’ field
considered as a replicate
• Student’s t-test to compare
treatment effects

11. www.iita.orgA member of CGIAR consortium
Nigeria: Efficacy on Maize
372
0
20
40
60
80
100
120
2009 2010 2011 2012
Aflasafe™
Control
0
100
200
300
400
500
600
2009 2010 2011 2012
82 94 83 86 82 93 89 90
51 14 199 38 51 14 166 38Fields (#)
Less (%)
At Harvest After Storage
*All means of aflasafe and control pairs significantly different; Student’s t-test (P<0.05)
*
Aflatoxin(ppb)

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Crop Sample Treatment Fields
AF
mean
(ppb)*
Red
uction
(%)
Groun
dnut
Harvest
Treated 51 3.7
92
Control 51 44.0
Storage
Treated 49 15.0
86
Control 49 101.0
Maize
Harvest
Treated 17 1.7
82
Control 17 9.1
Storage
Treated 17 50.3
84
Control 17 319.0
*All means of aflasafe treated and control pairs
significantly different; Student’s t-test (P<0.05)
Efficacy trial sites
Crop sampling sites
Burkina: Efficacy of aflasafe BF01
2012

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Area Sample Treatment
Mean
Aflatox
(ppb)
Reduct.
(%)
Mean
Aflatox
(ppb)
Reduct.
(%)
Mean
Aflatox
(ppb)
Reduct.
(%)
Diourbel
Harvest
Treated 1.9
93
6.6
87
3.7
82
Control 29.7 50.1 20.3
Storage
Treated 4.4
86
2.1
91
6.9
81
Control 31.3 22.1 35.5
Nioro
Harvest
Treated 4.4
75
5.6
76
5.4
90
Control 17.6 23.1 55.7
Storage
Treated 3.5
95
2.8
94
11.5
84
Control 52.1 46.7 72.5
*All means of aflasafe treated and control pairs significantly different; Student’s t-test (P<0.05)
Senegal: Efficacy of aflasafe SN01
2010 (n=40) 2011 (n=34) 2012 (n=71)

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Basis of efficacy: species shift
Treatment
(n = 14)
Aspergillus species/strain distribution (%) – MAIZE/NIGERIA
Soil before inoculation Grain at harvest
L SBG parasiticus L SBG parasiticus
Aflasafe™ 90 aB 7 aA 3 aA 100 aB 0 bA 0 aA
Control 78 aB 15 aA 7 aA 83 bB 16 aA 0.3 aA
Means within the column with different lowercase letters are significantly different according to the t-
test at 5% level of probability. Means within the row with different uppercase letters are significantly
different according to the Fisher’s LSD test at 5% level of probability
Region Treatment
Aspergillus Colony Forming Units/g – G-nut/Senegal
2010 (n = 20) 2011 (n = 17)
Soil Kernel Soil Kernel
Diourbel
Control 2311 a 2912 a 474 a 3257 a
Aflasafe SN01 1793 a 3598 a 795 a 3965 a
Nioro
Control 228 a 3367 a 369 a 3572 a
Aflasafe SN01 120 a 3189 a 470 a 4275 a
*All means of aflasafe and control pairs significantly different; Student’s t-test (P<0.05)
Aspergillus population does not increase due to aflasafe application
No change in Aspergillus Pop.

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Bars with same letter within the same
crop/year not significantly different (P<0.05)
Basis of Efficacy: Strain Shift
0
10
20
30
40
50
60
70
80
90
Soil Grain Soil Grain
2009 (n = 49) 2010 (n = 14)
Control Treated
0
10
20
30
40
50
60
70
80
90
Soil Grain Soil Grain
2009 (n = 2) 2010 (n = 16)
Proportion of 4 aflasafe™ strains in soil before treatment
and grains after harvest in control and treated fields
Aflasafestrains(%)
aa a aa a aa a aa a
b
b b
b
Carry-over of inoculum: 71, 52
and 28% after 1, 2, and 3 years

16. www.iita.orgA member of CGIAR consortium
Kenya: Efficacy of aflasafe KE01™
Area (fields) Control Treated
Reduction
(%)
Hola (n = 20) 885 20 98
Bura (n = 16) 105 7 93
Makueni (n = 15) 85 1 99
Aflatoxin (ppb)
*All means of aflasafe treated and control pairs significantly different; Student’s t-test (P<0.05)
38
20
0
88
60
33
0
10
20
30
40
50
60
70
80
90
100
Treated
Control
Fields (%) above
10 ppb in 3 areas
Fields(%)
Deadly (3,700 ppb & 2,270 ppb)
533 ppb
Hola

17. 74 ppb - Treated
1,133 ppb - Control
93.5% Reduction
Mutomo, Kitui County, Kenya: 2012 Tests
Short Rain Season Farmer Field Trials
Deadly
Average 2,750 ppb!
Range 1,790 ppb to 3,710 ppb.
Safe Food
510 ppb
Percent of Fields
TotalAflatoxins(ppb)

18. www.iita.orgA member of CGIAR consortium
Product Development in Africa
Products ready for registration
Products under testing
Strain development in progress
Senegal
Mali
Burkina
Ghana
Nigeria
Kenya
Tanzania
Mozambique
Zambia
Rwanda
Burundi
Uganda
2015
onwards
Benin
Togo
Ivory Coast
Ethiopia
South Sudan
Malawi
Sierra Leone
…………
The Gambia

19. www.iita.orgA member of CGIAR consortium
Groundnut sampling in The Gambia
(2013)
• Number of samples/region = 20 (4 villages per region; 5 samples per village) = 14
• Aflatoxin analyzed in all 140 samples
• Samples plated on semi-selective medium; 12 Aspergillus isolates / sample (excep
• Isolates characterized into species and strains; 72% L; 27% SBG and 1% parasiti
• All L-strain isolates interrogated for presence of the four aflasafe SN01 VCGs

20. www.iita.orgA member of CGIAR consortium
Aflatoxin in Groundnut in The Gambia
Regions*
Total Aflatoxins (ppb)
Mean Median Minimum Maximum
West Coast 268 19 ND 1,845
Lower River 3 ND ND 21
Upper River South 17 1 ND 208
Upper River North 5 1 ND 44
Central River
South 39 7 ND 253
Central River
North 102 13 ND 1,157
North Bank 102 23 ND 526
*Number of samples/region = 20 (4 villages per region; 5 samples per village)
Incidence

21. www.iita.orgA member of CGIAR consortium
Aflatoxin in Groundnut in The
Gambia
Regions*
Samples (%) with Total Aflatoxins (ppb)
ND → 4 >4 → 20 >20 → 100 >100
West Coast 40 10 10 40
Lower River 85 10 5 0
Upper River South 60 25 10 5
Upper River North 85 5 10 0
Central River
South 45 30 10 15
Central River
North 35 25 20 20
North Bank 40 10 20 30
*Number of samples/region = 20 (4 villages per region; 5 samples per village)
Severity

22. www.iita.orgA member of CGIAR consortium
Aflasafe SN01 VCG is Native in The
Gambia
Aflasafe
SN01 VCG
Gambian members of
VCG
Present in
Locations/Region
sNumber Name
M2-7 5
GMG 72-8 Upper River South
GMG 72-9 Upper River South
GMG 20-10 West Coast
GMG 87-9 Upper River North
GMG 105-14 Central River North
MS14-19 2
GMG 35-7 Lower River
GMG 107-1 Central River North
M21-11 1 GMG 72-12 Upper River South
SS19-14 4
GMG 72-3 Upper River South
GMG 72-4 Upper River South
GMG 72-6 Upper River South
GMG 72 - 7 Upper River South

23. www.iita.orgwww.iita.orgA member of CGIAR consortium R4D week, 25 – 30 Nov.
Integrated approach to manage aflatoxins in
crops

24. AgResults Aflasafe Pilot -- 2013
www.iita.orgA member of CGIAR consortium
Some key statistics
• Number of implementers: 4
• Number of farmers: 1,015
• Treated area: 1,457 ha
• Average productivity: 4.3 tons/ha
• Maize aggregated for sale: 2,031 tons
• Samples with <4 ppb AF (n = 660): 99%
• Samples with >70% aflasafe strains
(n = 88): 65% to 100%
• Return on investment: Up to 510%
• Aflasafe maize kept for family (n = 60): 46%

25. www.iita.orgA member of CGIAR consortium
Aflasafe Return on Investment
Quantity
sold
(tons)
Premium
(%)
Premium
(USD)
Aflasafe
cost
(USD)
Finance
cost
(USD)
Net
profit
(USD)
Seasonal
RoI
AgResults
Premium
(USD)
Total
Profit
(USD)
Total
RoI
120.0 7.5% 3,000 836 146 2,017 241% 1,800 3,817 456%
150.0 7.5% 3,750 1,046 183 2,521 241% 2,250 4,771 456%
96.0 3.6% 1,200 669 117 414 62% 1,440 1,854 277%
128.0 13.2% 5,600 892 156 4,552 510% 1,920 6,472 725%
32.0 7.5% 800 223 39 538 241% 480 1,018 456%
30.1 1.8% 188 210 37 -58 -28% 452 393 187%

26. www.iita.orgA member of CGIAR consortium
Scaling-Out
• Nigeria: AgResults (260,000 t)
• Senegal: Area-wide treatment in
2013; about 8 tons used
• Kenya: Government buy-in;
excellent support
• Zambia: Large-scale efficacy tests
and demonstration of product
value with private sector (12 t)
• Need for business plan,
manufacturing capacity,
marketing and distribution
strategies
• Critical role of PACA and RECs

27. www.iita.orgA member of CGIAR consortium
Aflasafe Manufacturing Facility
Large-scale: capacity 5 tons/hour

28. • Aflatoxins in food and feed pervasive
in Africa
• Biological control, as the foundation,
with other practices can dramatically
reduce aflatoxin contamination and
improve food safety and security
• Efforts underway to pilot
commercialization of aflatoxin
biocontrol and develop regional
strains
• The pilots need to be up-scaled and
efforts to improve efficacy needs a
fillip for wide-spread impact on
health and trade in Africa
Summary

29. IITA
Tucson
USDA/ARS
IITA, USDA, & Doreo have Teamed up to Bring
Aflatoxin Prevention to Africa
Made Possible by Many National Partners in Ministries, Industry, and on the Farm
Nigeria
For more information about aflatoxin biocontrol for Africa, check out: www.aflasafe.com