Development of biocontrol product (Aflasafe) for maize and groundnut in Rwanda

www.iita.org A member of CGIAR consortium

Development of biocontrol product (Aflasafe) for maize and groundnut in Rwanda

Joseph Atehnkeng, IITA

For the team


Occurrence of aflatoxin in crops Biocontrol technology Product development for Rwanda Experiences in Nigeria Conclusion

Outline


Joseph Atehnkeng, IITA

Research experience continues

Aspergillus in

maize Healthy maize cobs

Aspergillus in groundnut

Aflatoxin contamination in grains

Chronic exposure

Liver cancer

Immunosupression

Underweight

Stunting

Infants & young children

most affected

Acute exposure

Death (eg. Kenya in 2004)

Unreported cases,

UNKNOWN

Farm animals

Reduced feed consumption

Reduced reproductivity

Immune suppression

Decreased milk and egg

production

Retarded growth and

development

CONSEQUENCE?

Reduction in farmers’ incomes

Effects on human & animal health and Trade

Trade losses (US$ 450

million/annum) in Africa

due to aflatoxin contamination

Aflatoxin Contamination Occurs in Two Phases

Phase I: Before Crop Maturity

Developing crops become infected.

Associated with crop damage (insect, bird, stress).

Favored by high temperature (night) and dry conditions.

Phase II: After Crop Maturity

Aflatoxin increases in mature crop.

Seed is vulnerable until consumed.

Rain on the mature crop increases contamination.

Associated with high humidity in the field & store, insect

damage, and improper crop storage or transportation.

6

History of Acute Aflatoxicoses Outbreaks

Taiwan

1967

Uganda

1970

Western India

1974

Kenya

1982

2002

2004

2005

2006

2007

2008

Malaysia

1988

35°N

35°S

5 billion people

exposed --CDC

Aflatoxin Contamination: A Perennial Concern in

Warm Climates

Contamination is worse during dry

periods.

During droughts the zone containing

contaminated crops enlarges.

Management Strategies

Pre harvest

• Awareness

• Host plant resistance

• Insect control

• Irrigation

• Biological control

Post harvest

• Time of harvest

• Sorting and processing

• Grain drying method

• Storage structure

• Insect control

• Hygiene and sanitation

www.iita.org

AF36

Aflaguard

Biocontrol WORKS

In 100’S of thousands of acres in

the US!

IT WORKS In Africa

TOO!

Aflasafe

www.iita.org A member of CGIAR consortium Contract Review , 04 October , 2013

Naturally, some strains produce aflatoxins (toxigenic)

while others do not (atoxigenic)

Atoxigenic strains co-exist with toxigenic relatives and

present in soil and on crop

Increase the frequency of atoxigenic strains & shift the

population profile

Consequently, reducing aflatoxin contamination

Application of atoxigenic strains can be done without

increasing infection and without increasing the overall

quantity of A. flavus on the crop or the environment

Strains protect crop from field to store

Multiple year & multiple crop benefit

Only native strains used.

0

20

40

60

80

100

Natural BiocontrolF

req

ue

nc

y (

%)

AT

OX

IGE

NIC

TO

XIG

EN

IC

Biocontrol principle

Product: Aflasafe

Mixture of 4 native atoxigenic strains

Nigeria

www.iita.org

Farmers treating maize and groundnut fields with Aflasafe in Nigeria

MAIZE: Aflatoxin reduction (%)

Stage 2009 2010 2011 2012

Harvest 82 94 83 93

Storage 92 93 x x

PEANUT: Aflatoxin reduction (%)

Stage 2009 2010 2011

Harvest - 95 82

Storage 100 80 x

Results from 482

on-farm trials

71% and 52% carry-over of

inoculum 1 & 2 years after

application

www.iita.org www.iita.org A member of CGIAR consortium

Time of aflasafe application


Nsengiyumva et al., 2012 https://www.linkedin.com/pub/nsengiyumva-prosper/7b/887/a04.

Prevalence of aflatoxin in Rwanda

• Analysed eight samples of maize flour collected from four markets in

Kigali and were from two origins, industrial and non industrial.

• The average contents in industrial maize flour ranged from (0.9-3.1

ppb) while for traditional maize flour, aflatoxin level was very high with

91.2 ppb.

• Also collected peanuts from three different markets in Kigali.

• Highly variable aflatoxin contamination levels were found in three

markets samples 0.13, 22.58 and >35.95 ppb respectively for the

three markets.

• This study showed that the total aflatoxins in industrial maize flour is

much lower than the regulated maximum amount allowed in foodstuffs

in Rwanda


• Evaluated different crop samples for the prevalence of

mycotoxins in Rwanda.

• Data obtained reveal that peanuts and maize were the

most contaminated with mycotoxins at the incidence rate

of 85 and 80% respectively.

• The highest aflatoxin contamination was in the Western

province with a mean of 829.3 ng/g and range of 1.3 –

3219.6 ng/g followed by

• peanut from the same region with a mean of 401.5 and

range of 3.2 – 1755.8 ng/g.

Felicie Nyinawabali (2013),

Prevalence of aflatoxin in Rwanda


www.iita.org A member of CGIAR consortium Date: 02 – May - 2013

Atoxigenic Strain Identification

Collection/characterization Toxin assay

Field

efficacy

test

Lab

competition

assay

VCG/DNA characterization

cnx nia-D

Unknown 2

+


GOAL: To develop aflatoxin bio-control product for the mitigation of aflatoxins in maize & groundnuts in Rwanda, using native atoxigenic strains of Aspergillus flavus

Specific objectives:

Determine the prevalence of aflatoxin contamination in maize and groundnuts in Rwanda

Determine the distribution and aflatoxin-producing profiles of the etiologic agents

Identify and select atoxigenic strains of Aspergillus flavus native to target regions for large scale aflatoxin mitigation in maize and groundnuts in Ghana.

Evaluate the efficacy of the atoxigenic strains in reducing aflatoxin contamination in- vitro, and under field conditions on farmers’ fields in aflatoxin-prone regions in Ghana.

Research objectives

Data on aflatoxin prevalence grouped into four categories based on their

aflatoxin content as recognized by different regulatory bodies: samples with no

detectable aflatoxins (Limit of detection = 0.1 μg/kg),

Sample processing Developed TLC plate

Extraction

Densitometry

Aflatoxin analysis


Province Districts Number of samples Mean Aflatoxin

(ng/g)

Range

Eastern

Bugesera 5 6.2 4.1-18.9

Gatsibo 15 5.7 0.0 -15.4

Kirehe 15 5.3 0.0 -14.2

Nyagatare 15 6.2 2.3 -9.4

Northern

Burera 15 9.6 4.6 -18.9

Gakenke 5 5.6 4.2 - 8.3

Musanze 15 9.6 0.0 -15.0

Rurindo 5 11.4 4.5 -15.3

Southern

Gisagara 5 6.9 2.9 -12.2

Kamonyi 5 4.4 2.6 -7.8

Mahunga 5 9.4 4.2-2

Ruhango 5 6.9 1.7-12.6

Western

Karongi 15 4.1 0.0 -7.1

Nyabihu 15 5.2 0.0 -8.5

Nyamasheke 5 6.4 1.5 -12.6

Rusizi 15 5.0 0.0-9.8

Rutsiro 15 8.5 2.7-15.2

Aflatoxin concentration (ng/g) in maize in

each province


Region Crop <4 <10 <20

Eastern

Maize

24.0 94.0 100.0

Northern 2.5 62.5 100.0

Southern 25.0 85.0 100.0

Western 32.3 93.8 100.0

Proportion (%) of samples that met each

cut off

<4 = EU/Nestle acceptable limit, <10 = World Food Program acceptable

limit, <20 = US regulation limit, >20 = unacceptable level of aflatoxin.


Isolation & identification of Aspergillus sect. Flavi

L - strain S - morphotype

A. tamarii A. parasiticus

31 oC

3 days

31 oC

5 – 7 days

++++ +/-

- ++++

Silica save

H2O save


Laboratory selection for atoxigenics

Extraction TLC

Positiv

e

Extraction

TLC Negative

Single spore twice

Atoxigenic L

strain on silica

H2O save Healthy maize

Colonized grains

31 oC

7 days

≈ 1.0 x 106 spores

Genetic &

molecular

analysis

Densitometry


Crop Provinces Number of

samples

Aspergillus species/strain distribution

(%)

L % S % P % T %

Groundnut Eastern 16 9.3b 26.1a 60.5a 4.1a

Southern 19 26.3a 11.4a 52.2a 10.0a

Maize

Eastern 55 63.7a 12.3a 17.3a 6.9a

Southern 35 73.0a 10.5a 13.8a 2.8a

Western 20 69.5a 14.4a 12.9a 3.2a

Northern 65 79.3a 8.9a 11.3a 0.5a

Strain distribution in maize and groundnut

samples

We examined 1261 isolates


Proportion of Toxigenic and Atoxigenic

strains obtained in each District


Proportion of Toxigenic and Atoxigenic

strains obtained in each province


A total of 379 atoxigenic strains was obtained

after strain characterization and are further

undergoing molecular characterization at

Cotty laboratory, University of Arizona to

select the best 8-12 isolates for field

evaluation.

Progress so far

• Criteria

• Atoxigenic

• Well distributed

• Highly competitive in lab and field

• Must not be related to and toxin producing family.

• Stability in the field

VCG/DNA characterization

cnx nia-D

Unknown 2

+


What is going on in Nigeria?


„Experience with conducting agricultural research

• 2003-06: Strain collection and characterization, atoxigenic strain identification (BMZ)

• 2007-08: on-station efficacy testing (IITA)

• 2009-10: provisional registration, partnerships, on-farm efficacy testing (Mycored, AATF)

• 2010-11: Advocacy, training, awareness campaign, market linkages (Mycored, AATF)

• 2011 / 2012: Large scales testing (BMGF)

• 2012/2017: AgResults (G20)

Nigeria Aflatoxin Biocontrol: Genesis and Time Line

www.iita.org A member of CGIAR consortium R4D week, 25 – 30 Nov. 2013

Integrated approach to manage aflatoxins in crops


You are what you eat

Food Quality

31

14

14 tons inoculum produced in 2011 for deployment


Aflasafe Plan & Plant

Capacity: 5 tons/hour

www.iita.org www.iita.org A member of CGIAR consortium Contract Review , 04

October , 2013

Training materials

Aflatoxin

management

Video


Current Practices


Drying Maize


Safe Moisture Content


Sanitation

www.iita.org www.iita.org A member of CGIAR consortium 38

•Target Group

• Farmers

• Extension agents

• Media houses

• National bodies eg NAERLS, ADP,

NAFDAC

• Community leaders

•National policy /decision makers •Agriculture •Trade •Health


Babban Gona Pilot

www.iita.org A member of CGIAR consortium Mycored Europe, 28 May, 2013

Willingness to Pay

www.iita.org Mycored Europe, 28 May, 2013 A member of CGIAR consortium Mycored Europe, 28 May, 2013

100% 99%

83%

60%

25%

19%

34% 31%

18%

12%

5% 4% 0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Farmers who have used Aflasafe (n=246) Farmers who have not used Aflasafe (n=119)

Target Farm Gate Price

Range

Poultry Feeding Study

$3,200 net

profit from

10,000 birds

in 8 weeks

www.iita.org Mycored Europe, 28 May, 2013 A member of CGIAR consortium

Aflasafe maize feed Toxic maize feed

G-20 AgResults Aflasafe


• Pull mechanism – Aflasafe is one of the first three pilots

• Provides incentives after demonstrating adoption

• Private sector driven, but focused on smallholder groups

• Implementers provide credit, inputs and technical services to increase yield

• Aflasafe purchased at cost to improve quality

• Maize tested for aflasafe strains; if present in large frequency, the implementers incentivized with $18.75/ton maize

• Implementers negotiate maize sale at premium

• Project provides aflatoxin awareness, training of implementers, and identifies potential market linkages

• Target: 200,000 ha in 4 years


Cleaning and Branding


Implementer Participating Farmers (No.)

Samples Submitted

(No.)

Aflatoxin Concentration (ng/g)

Mean Range Samples above 20 ng/g (No.)

Samples above 20 ng/g (%)

Babban Gona

621 521 0.3 ND – 21.6 1 0.2

Kaduna CADP

190 108 1.4 ND – 69.6 2 1.8

Kano CADP 182 20 2.1 ND – 27.1 1 5.3

Maslaha 22 11 0.4 ND – 0.7 0 0

Total 1015 660 1.1 ND – 29.8 4 0.7

Samples exhibited consistently low levels of Aflatoxin

42

The introduction of Aflasafe caused reduced Aflatoxin levels during Pilot Year 0. Nevertheless, extreme outliers leave some questions (e.g. a Kaduna CADP sample at 69.6ng/g and a Kano CADP sample at 27.1ng/g). These results could extend from mistimed application, or heavy rainfall right after application.


Conclusion • Awareness and training are key to understanding the dangers

posed by aflatoxin.

• The aflatoxin concentration in maize samples collected during the

survey were within the US acceptable limits for human

consumption.

• The L-strains of Aspergillus flavus were the most occurring of the

Aspergillus species obtained during isolation.

• The presence of high aflatoxin producers like S-morphotypes and

A. parasiticus was higher in groundnut than maizet samples.

• The proportion of aflatoxin producers was higher in 16 of the 18

districts. However a total of 379 atoxigenic strains was obtained

after strain characterization and are further undergoing molecular

characterization at Cotty laboratory, University of Arizona to

select the best 8-12 isolates for field evaluation.

Ibadan 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

http://www.entwicklung.at/en/

www.iita.org

75

94 100

0 3

39

73

27

0

20

40

60

80

100

120

<4 <10 <20 >20

Nestlé/EU WFP/Nigeria US Unsafe

Maximum allowable aflatoxin level (ng g-1)

Farm

ers

' fi

eld

s (

%)

Treated

Control

Different Levels of aflatoxin

in AflaSafe™ treated and

untreated fields at harvest

Science

Development of biocontrol product (Aflasafe) for maize and groundnut in Rwanda