Biocontrol and other solutions:Biocontrol and other solutions:

Addressing Aflatoxin in Addressing Aflatoxin in

SubSub--Saharan Africa Saharan Africa

Peter J. CottyPeter J. Cotty

Agricultural Research ServiceAgricultural Research Service

United States Department of AgricultureUnited States Department of AgricultureUnited States Department of AgricultureUnited States Department of Agriculture

School of Plant SciencesSchool of Plant Sciences

University of Arizona, TucsonUniversity of Arizona, Tucson

Ranajit BandyopadhyayRanajit Bandyopadhyay

International Institute of Tropical AgricultureInternational Institute of Tropical Agriculture

Ibadan, NigeriaIbadan, Nigeria

Biocontrol and other solutions:Biocontrol and other solutions:

Addressing Aflatoxin in Addressing Aflatoxin in

International Institute of Tropical AgricultureInternational Institute of Tropical Agriculture

Aflatoxin Contamination Occurs in Aflatoxin Contamination Occurs in Two PhasesTwo Phases

Phase I: Before Crop MaturityPhase I: Before Crop Maturity

� Developing crops become infected.

� Associated with crop damage (insect,

bird, stress). bird, stress).

� Crop may exhibit BGYF (bright-green-

yellow fluorescence).

� Favored by high temperature (night)

and dry conditions.

Aflatoxin Contamination Occurs in Aflatoxin Contamination Occurs in Two PhasesTwo Phases

Phase II: After Crop MaturityPhase II: After Crop Maturity

� Aflatoxin increases in mature crop.

� May occur before or after harvest.

� Seed is vulnerable until consumed. � Seed is vulnerable until consumed.

� Associated with high humidity in the

field, & improper crop storage or

transportation.

� Rain on the mature crop increases

contamination.

Aspergillus flavus occurs throughout warm regions. However, this fungus varies widely in several important traits.

occurs throughout warm regions. However, this fungus varies widely in several important traits.

Lo

g n

g/g

(p

pb

) Aflatoxin Production by Fungal Isolates in Liquid FermentationAflatoxin Production by Fungal Isolates in Liquid Fermentation

3

4

5

Lo

g n

g/g

(p

pb

)

54 Isolates from One Agricultural Field

1

2

Aflatoxin Production by Fungal Isolates in Liquid FermentationAflatoxin Production by Fungal Isolates in Liquid Fermentation

Isolates from One Agricultural Field

A.

A. flavus

flavus

40

50

60Field 1 Field 2

Aflatoxin Production by Aflatoxin Production by A. flavusA. flavus

Fungi Vary Across Areas in Aflatoxin

Farmers apply atoxigenic strains to reduce the average aflatoxin

fungi on farms & thus the vulnerability of crops to aflatoxin contamination

Fungi Vary Across Areas in Aflatoxin

Farmers apply atoxigenic strains to reduce the average aflatoxin

fungi on farms & thus the vulnerability of crops to aflatoxin contamination

Pe

rce

nt o

f

Pe

rce

nt o

f A

. A

.

0

10

20

30

40

1 2 3 4

Aflatoxin Produced in Culture (log ppb)Aflatoxin Produced in Culture (log ppb)

Field 2

Aflatoxin-Producing

Potential

Aflatoxin-Producing

Potential

A. flavusA. flavus from Two Fieldsfrom Two Fields

Fungi Vary Across Areas in Aflatoxin-Producing Ability

Farmers apply atoxigenic strains to reduce the average aflatoxin-producing potential of

fungi on farms & thus the vulnerability of crops to aflatoxin contamination

Fungi Vary Across Areas in Aflatoxin-Producing Ability

Farmers apply atoxigenic strains to reduce the average aflatoxin-producing potential of

fungi on farms & thus the vulnerability of crops to aflatoxin contamination

5 6

Aflatoxin Produced in Culture (log ppb)Aflatoxin Produced in Culture (log ppb)

Field 1 = Low,

3,400 ppb

Field 1 = Low,

3,400 ppb

Field 2 = High,

54,000 ppb

Field 2 = High,

54,000 ppb

As Applied

After

Fungal Growth

As Applied

After

Fungal Growth

(ng

/g X

(n

g/g

X 1

010,,0

00

000))

4

5

6

7

8

9

Aflatoxin in Crop versus Atoxigenic IncidenceAflatoxin in Crop versus Atoxigenic IncidenceA

flato

xin

BA

flato

xin

B11

(ng

/g X

(n

g/g

X

Isolates (%) in Applied Atoxigenic StrainIsolates (%) in Applied Atoxigenic Strain

0

1

2

3

4

0 20 40

Dots Represent Values Dots Represent Values for Replicate Plotsfor Replicate Plots

Aflatoxin in Crop versus Atoxigenic IncidenceAflatoxin in Crop versus Atoxigenic Incidence

Isolates (%) in Applied Atoxigenic StrainIsolates (%) in Applied Atoxigenic Strain

60 80 100

Influences of Field Application of Atoxigenic

Aflatoxin, Infection, & Total A. flavus

Aflatoxin B1

(mg kg-1

) Infection

(%)

Treated

0.3

b

1.03 Treated 0.3 b 1.03

Control

81.8

a

0.85

Values followed by a common letter do not differ significantly

Influences of Field Application of Atoxigenic A. flavus on:

A. flavus on the Crop at Harvest

Infection

A. flavus on crop (spores/g)

Applied strain (%)

a

23,949

a

100

a a 23,949 a 100 a

a

28,949

a

7

b

Values followed by a common letter do not differ significantly.

Area Samples (#) AF36 (%)

Grayson

North17

Commercial Maize Test: North Central Texas 2008

North

GraysonSouth

16

GraysonControl

8

Means in the same column with different letters are significantly different by

Tukey’s HSD test, P < 0.001.

Aflatoxin (ppb)

AF36 (%) Mean Range

96 a 12 a 0 to 48

Commercial Maize Test: North Central Texas 2008

98 a 15 a 0 to 38

24 b 230 b 5 to 530

Means in the same column with different letters are significantly different by

Contamination is worse during dry periods.

During droughts the zone containing contaminated crops enlarges.

Aflatoxin Contamination: A Perennial Concern in Warm Climates

35°N35°N

35°S35°S Zone with PerennialContamination Risk

Contamination is worse during dry periods.

During droughts the zone containing contaminated

A Perennial Concern in Warm Climates

Zone with PerennialContamination Risk

Atoxigenic Strains from KenyaAtoxigenic Strains from Kenya

• More than 3,400 A. flavus isolates recovered from maize samples obtained between Eastern, Coast and Rift Valley

• 290 L strain isolates screened for aflatoxinmaize

• 96 identified as atoxigenic and coon maizeon maize

• 23 Strains that reduced >80% or members of VCG from more locations were further evaluated

• 12 atoxigenic VCGs (13 strains) selected for further evaluation under field conditions in Kenya

• The best atoxigenics from Kenya reduce aflatoxins in maize as good or better than the atoxigenics used in the US

Probst, C., Bandyopadhyay, R., Price, L. E., and Cotty, P. J. 2011. Identification of atoxigenic

of maize in Kenya. Plant Disease 95:212-218. Probst, C., Schulthess, F., Cotty, P.J.

development of lethal levels of aflatoxins in Kenyan maize (Zea mays). Journal of Applied Microbiology

Outbreak of an Acute Aflatoxicosis in Kenya in 2004: Identification of the Causal Agent. Applied and Environmental Microbiology

Atoxigenic Strains from KenyaAtoxigenic Strains from Kenya

isolates recovered from 263 ground maize samples obtained between 2004 and 2006 from the

L strain isolates screened for aflatoxin-producing ability on

identified as atoxigenic and co-inoculated with toxigenic stain

www.iita.org

% or members of VCG from 2 or more locations were further evaluated

strains) selected for further evaluation

The best atoxigenics from Kenya reduce aflatoxins in maize as good or better than the atoxigenics used in the US

. Identification of atoxigenic Aspergillus flavus isolates to reduce aflatoxin contamination

. Probst, C., Schulthess, F., Cotty, P.J. 2010. Impact of Aspergillus sect. Flavi community structure on the

). Journal of Applied Microbiology 108: 600-610. Probst, C., Njapau, H., Cotty, P.J., 2007

: Identification of the Causal Agent. Applied and Environmental Microbiology 73:2762-2764.

38 Farms in 2 Zones Were Treated in 201038 Farms in 2 Zones Were Treated in 2010

Farmers threshing groundnut

Farmers treating groundnut fields with AflaSafe

Aflatoxin Reduction: 87% Aflatoxin Reduction: 87% at harvestat harvest

Senegal

38 Farms in 2 Zones Were Treated in 201038 Farms in 2 Zones Were Treated in 2010

www.iita.orgFarmers treating groundnut fields with AflaSafe

NigeriaNigeria

Zone

Number of

farmers

Mean B

AflaSafe™

Maigana 22 2.1

Aflatoxin Reduction in Maize after Aflatoxin Reduction in Maize after

AflaSafe™ Application in Kaduna AflaSafe™ Application in Kaduna

Maigana 22 2.1

Pampaida 10 3.6

Lere 9 5.1

Birnin-Gwari 10 2.3

Mean 3

Mean B-aflatoxin (ng/g)

Reduction

(%)AflaSafe™ Control

2.1 9.8 78

Aflatoxin Reduction in Maize after Aflatoxin Reduction in Maize after

AflaSafe™ Application in Kaduna AflaSafe™ Application in Kaduna

2.1 9.8 78

3.6 27.2 87

5.1 11.8 57

2.3 12.6 82

3.3 15.4 79

Aflatoxin reduction in Aflatoxin reduction in

after poor storage in Nigeriaafter poor storage in Nigeria

600

800

Afl

ato

xin

(p

pb

) Control

Treated

85% 82%

% reduction in aflatoxin content in treated fields over control

0

200

400

600

Birnin Gwari Lere

Afl

ato

xin

(p

pb

)

Treated

96

14

271

49

Aflatoxin reduction in Aflatoxin reduction in 4 4 zones zones 4 4 months months

after poor storage in Nigeriaafter poor storage in Nigeria

646

99% 95%

% reduction in aflatoxin content in treated fields over control

Maigana Pampaida

271

4917

171

9

Farmers in Nigeria treat maize and groundnut fields with AflaSafe

Aflatoxin reduction at harvest:Aflatoxin reduction at harvest:

2009: 80%2009: 80%

56 56 to to 7373% carry% carry--over of over of

inoculum one year after inoculum one year after

application application

Farmers in Nigeria treat maize and groundnut fields with AflaSafe

Aflatoxin reduction at harvest:Aflatoxin reduction at harvest:

2009: 80%2009: 80% 2010: 89%2010: 89%

www.iita.org

60%

80%

100% Treated

Adjacent

Incidence of AF36 within Aspergillus flavus

Prior to and One Year After Application

Incid

en

ce o

f A

F36

= 1997 = 1996

0%

20%

40%

Barkley

Sharp

Stuhr

Barkley-N

Sharp-204W

Stuhr-3W

Barkley-29

Incid

en

ce o

f A

F

Diagonal

Aspergillus flavusCommunities

Prior to and One Year After Application

1996 =1997 & 1996 differ

Fungi Resident in the Soil

Barkley-29

Barkley-95N

Barkley-95S

Sharp-205W

Sharp-201

MV: 0.3 mi-SE

YV: 0.6 mi-NE

MV: 0.7 mi-E

MV: 1.2 mi-W

Diagonal

Other

0%

25%

50%

75%

100%

0%

25%

50%

75%

100%

Percent of the A. flavus Soil Community Composed of the Applied Atoxigenic Strain & the Highly Toxigenic S Strain

Long-term Influences Suggest Area-wide Programs may be EffectiveLong-term Influences Suggest Area-wide Programs may be EffectiveLong-term Influences Suggest Area-wide Programs may be EffectiveLong-term Influences Suggest Area-wide Programs may be EffectiveLong-term Influences Suggest Area-wide Programs may be EffectiveLong-term Influences Suggest Area-wide Programs may be Effective

Applied Atoxigenic Strain & the Highly Toxigenic S Strain

= % Atoxigenic Strain

1996 1997

COTTONCOTTON

M J J A S O N D J F M A M J J A S O N

Application

WHEATWHEAT

0%

25%

50%

75%

100%

0%

25%

50%

75%

100%

Soil Community Composed of the Applied Atoxigenic Strain & the Highly Toxigenic S Strain

wide Programs may be Effectivewide Programs may be Effectivewide Programs may be Effectivewide Programs may be Effectivewide Programs may be Effectivewide Programs may be Effective

Yuma Valley, Arizona

Applied Atoxigenic Strain & the Highly Toxigenic S Strain

= % Atoxigenic Strain = % S strain

1998 1999

MELONSMELONSWHEATWHEAT

D J F M A M J J A S O N D J F M A M J

100%

77%74%

40%

60%

80%

100%

Pe

rce

nt

of

As

pe

rgillu

s f

lav

us

Grayson County: Carry Over to the Second Year Crop

0% 0% 0%0%

20%

Pa

rha

m

So

il

Ru

the

rfo

rd

So

il

Pa

rha

m

Co

rn

2008 2008 2008

Pe

rce

nt

of

82%

53%

96%

Grayson County: Carry Over to the Second Year Crop

= AF36

= S Strain

0% 1% 17% 11%

Ru

the

rfo

rd

Co

rn

Pa

rha

m

Co

rn

Ru

the

rfo

rd

Co

rn

2008 2009 2009

There are many atoxigenic strainsThere are many atoxigenic strainsSelect strains best adapted to rotations, ecosystems, & climates

Crops are infected by complex communities of diverse fungiCrops are infected by complex communities of diverse fungiWe can influence aflatoxin-producing ability of fu

through crop rotations, agronomic practice, and by applying atoxigenic strains

Atoxigenics are Already Present on the CropAtoxigenics are Already Present on the CropJust increasing the frequency of endemic strains & natural interference with contaminationJust increasing the frequency of endemic strains & natural interference with contamination

Treatments have LongTreatments have Long--Term Influences & Cumulative BenefitsTerm Influences & Cumulative Benefits

More than One Crop May Benefit From the Same StrainMore than One Crop May Benefit From the Same Strain

Atoxigenic Strains can be Applied Without Increasing InfectionAtoxigenic Strains can be Applied Without Increasing Infectionand without increasing the overall quantity of A. flavus

There are many atoxigenic strainsThere are many atoxigenic strainsSelect strains best adapted to rotations, ecosystems, & climates

Crops are infected by complex communities of diverse fungiCrops are infected by complex communities of diverse fungif fungal communities resident in production areas

through crop rotations, agronomic practice, and by applying atoxigenic strains

Atoxigenics are Already Present on the CropAtoxigenics are Already Present on the CropJust increasing the frequency of endemic strains & natural interference with contaminationJust increasing the frequency of endemic strains & natural interference with contamination

Term Influences & Cumulative BenefitsTerm Influences & Cumulative Benefits

More than One Crop May Benefit From the Same StrainMore than One Crop May Benefit From the Same Strain

Atoxigenic Strains can be Applied Without Increasing InfectionAtoxigenic Strains can be Applied Without Increasing InfectionA. flavus on the crop & throughout the environment

Ranajit Bandyopadhyay, Plant PathologistInternational Institute of Tropical AgricultureIbadan, Nigeria

Ranajit Bandyopadhyay, Plant Pathologist