Biodiversity of Toxigenic Fungi and its Implications in Disease Management

International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org

Biodiversity of Toxigenic Fungi and its

Implications in Disease Management

Ranajit BandyopadhyayIITA, Ibadan


Outline

• Food systems

• Fusarium diversity

• Aspergillus diversity

• Aflatoxin risk in

cereals

• Management

• Conclusions


Food Systems

Large Scale and Regulated

– Developed countries

– Trade based

– Advanced infrastructure

– Capital intensive

Tim Williams, Peanut CRSP

Small Scale and Unregulated

– Developing countries

– Informal markets

– Subsistence

– High food insecurity



Major Classes of Mycotoxins

• Aflatoxins: Aspergillus flavus, A.

parasiticus

• Fumonisins: F. verticillioides etc.

• Trichothecenes: Fusarium spp,

Stachybotrys

• Zearalenone: F. graminearum

• Ochratoxins: Penicillium verrucosum, A.

ochraceous


Mycotoxin and Trade

• Protect consumers from undue exposure

• Promote regional and international trade• Stringent regulatory standards in

importing countries (e.g., EU)

• Rapid Alert Reporting System globally reports food safety issues (on internet) –poor country image

• To promote regional and international trade

• To encourage national development of agro-based economies

• To protect consumers from economic exploitation

• Impractical in local systems


Mycotoxins and Health

Death

Contributes to 40% of DALYs

Impairs growth and development of children

Suppress immune system

Aflatoxin interacts with HBV

• 30 times more potent in HBV+ people

• 5-60 times higher cancer risk

May impede uptake and utilization of micronutrients in human systems

Associated with Kwashiorkor in children, oesophageal cancer in humans, neural tube defect

Also affect animal health and productivity


Confounding Toxicological Effects

• Poisoning modes

– Acute poisoning

• Clear symptoms

– Chronic exposure

• Indirect symptoms usually attributed to other agents

– Immune suppression -> infectious diseases

– Nutritional interference -> vitamin deficiencies

– Developmental interference

– Cumulative exposure

• Genetic and carcinogenic

• Medical professionals need better information


Wrong Emphasis on Aflatoxin and

Human Disease (Gong et al)

Health effect

Possible

deaths (No.)

Relative public

attention

Biological weapon 0 (?) Very high

Acute aflatoxicosis 100’s High

Hepatocellular

carcinoma

10,000’s Medium

Growth impairment/

immunosuppression

100,000’s (?) Low/None


Aflatoxin (ng/g)

Contamination in West Africa

• Peanut paste: 3278 – Ghana

• Peanut sauce: 943 – Ghana

• Leaf sauce: 775 – Gambia

• Maize dough: 313 – Ghana

• Kenkey: 524 – Ghana

• Cashew paste: 366 – Ghana

• Peanut oil: 500 – Nigeria

• Yam flour: 7600 – Nigeria

• Local beer: 135 - Nigeria

• Maize: 4000 – Benin

• Peanut: 216 – Ghana

• Sorghum: 80 – Ghana

• Millet: 200 – Nigeria

• Tiger nuts: 120 – Nigeria

Primary products

Food products

MTL: 20 ng/g


Aflatoxin-albumin adducts (pg AFB1-lysine eq./mg albumin)

<5 5-25 26-100 >100

Nu

mb

er

of

indiv

idua

ls (

%)

0

20

40

60

80

100 Gambia (n = 950)

Benin (n = 479)

USA (n = 48)

Europe (n = 74)

Aflatoxin Exposure in Africa,

Europe & USA


Fusarium species

Courtesy: Leslie & Summerell


Fusarium – A General Division

• Three broad groups

• “Dark red” pigment producers

– F. graminearum, F. culmorum, F. poae, F. sporotrichioides, etc.

– Trichothecenes and zearalenone

– Not common in Africa

• F. solani species complex

– Not strictly toxin producers, but can turn plant host defense compounds into toxins

– Most important cause of direct human infections

• Gibberella fujikuroi species complex important in Africa

– Fumonisins are the most prominent toxin


Fusarium Diversity and Toxins

• Several Fusarium species

and types of toxins occur

on cereals.

• Risk of toxin based on

Fusarium species present

• An understanding of

species diversity can help

in toxin risk management

• Species and diversity

depend on crops, but least

understood in Africa

Fusarium spp. FB1 FB2 MON DON ZEA T2 NIV

F. verticillioides FB1 FB2

F. proliferatum FB1 FB2 MON

F. thapsinum MON

F. graminearum DON ZEA NIV

F. sporotrichioides T2


Gibberella fujikuroi Species Complex

• Most common problem in Africa, especially if F.

oxysporum included here

• May be problematic on maize, millet and sorghum

• Most prominent toxin is fumonisins, but other

potential problems include moniliformin, fusaproliferin

and beauvericin

• At one time all were called “F. moniliforme”

• May produce a compound that can be confused with

zearalenone in TLC analyses


Fusarium spp. from Sorghum

• Common

F. thapsinum dominates followed by F. andiyazi and F. proliferatum with occasional other species

• Egypt

F. proliferatum – 52%; F. thapsinum – 26%; F. verticillioides– 17%; F. sp. nov – 5%

• West Africa

F. thapsinum dominates followed by F. andiyazi and numerous unidentified species

• Dominant species varies by location

• Many unidentified species with undescribed toxigenic potential

• Presence of fumonisins and moniliformin confirmed


Fusarium Diversity on Maize

in Ghana

• Data from two AEZ; more than 650 isolates

• All grains yielded at least one isolate

• 95% were F. verticillioides

• F. proliferatum 3%, a third species 1%, and six other species represented by a single isolate each

• 51 clones containing 2-4 isolates each of F. verticillioides recovered representing ~ 20% of the total population

• Both sexual and asexual reproduction important in this fungal population

• Fumonisin contamination most prevalent and risk is high (100+ ppm)

Leslie and Bandyopadhyay (2005) Phytopathology 95:S58


Fumonisin (µg/g) in Selected Elite Lines

Artificially inoculated with F. verticillioides

Inbred lines 2003 2004

(1368/S.A. Pub Lines36/1368)-2-2-2-B 2.5 0.8

(CIM 116 x TZMi 302 x CIM 116)-2-2-B 3.3 3.7

KU1414xICAL 36-1xKU1414-6-1-B 4.7 3.5

Obantapa-9-3-1-1-B 5.9 6.5

Obantapa-33-5-1-B 5.9 4.7

Obantapa-31-1-1-B 5.9 0.3

PIONEER SEEDS-26-2-1-B 32.4 37.1

((KU1414 x 9450) x 9450)-24-2-1-B 43.5 25.4

P43SRC9FS100-1-1-8-#1-B1-4-B 55.0 21.6

1368xINV 534-1x1368-7-1-B 62.0 58.6

4205 63.5 99.2

1368xICAL 224-1x1368-2-2-B 87.2 25.0

Mean 28.9 17.1

SE 4.2 3.3

Afolabi et al. (2007) Plant Disease 91:279.


Status Report

• Most populations are diverse and are outbred

• Sexual stages not commonly encountered in the field

• Many plants are multiply infected

• Fusarium spp. from sorghum and millets in Africa are unique and largely undescribed

• Basic fungal taxonomy and mycotoxin profiling remains to be done

• Fusarium toxins are a clear problem on maize, less so on other crops

• Best plant pathogens are not the best toxin producers

• The African picture often is different from from Europe or North America

• Subsistence farm scenario may be of critical scientific & practical importance


A

F

L

A

T

O

X

I

N


Biological Control of Aflatoxin

Ability to produce aflatoxin in A. flavus

strains varies

Some strains produce a lot (toxigenic), and

others no aflatoxin (atoxigenic)

Competitive exclusion (one strain competing

to exclude another) as biocontrol principle in

practice in the US

Diversity studies in Aspergillus section Flavi

conducted in Nigeria to identify atoxigenic

strains for biocontrol

Toxigenic

AtoxigenicBandyopadhyay et al. (2006) Tropentag


Selection Criteria for Biocontrol

Agent

• Atoxigenic strains evaluated for a set of selection criteria:– Should not produce aflatoxin

– Ensure that the candidate strains belong to unique groups that are unable to produce toxigenic progenies in the natural environment.

– Propensity to multiply, colonize and survive so that few reapplications will be required once the atoxigenic strains are introduced.

– Environmental safety

– Field efficacy

• Select appropriate strains for further tests

• Protocols for mass multiplication


Areas Sampled

Derived Savanna & Southern Guinea Savanna – 5 districts

Northern Guinea Savanna – 2 locations

5 locations / state; 50 to 124 isolates / location; n = 4414

Dry

Wet

J. Atehnkeng

M. Donner


Atoxigenic Strain Identification

Strain characterization

cnxnia-D

Unknown

Unknown

nia-Dcnx

-

+

VCG

Toxin assay

Field

Competition assays

Lab

Field release


L-strain

S-strain

A. tamarii

A. parasiticus

Distribution of Aspergillus in

Nigerian Soils & Grains

L-strains more predominant than S-strains

Low frequency of A. parasiticus

Soil Grain

88%

6%

5%

1%97.9%

0.6% 1.4%

0.1%

Atehnkeng et al. (in press)

Int. J. Food Microbiol.


0

100

200

300

400

500

600

Ogbom

osho

Mokw

aBid

a

Min

na

Abuja

Akwanga

Lafia

Makurd

i

Lokoja

Ado Ekiti

Zaria

Kaduna

Districts

Str

ain

s / l

oca

tio

n

L-strain

S-strain

A. parasiticus

A. tamarii

Aspergillus Strains in Grain


Proportion of Toxigenic and

Atoxigenic Strains in Grain

0

10

20

30

40

50

60

70

80

90

Mok

wa

Bida

Minna

Abu

ja

Akw

anga

Ogb

omos

hoLa

fia

Mak

urdi

Loko

ja

Ado

Ekiti

Zaria

Kad

una

Districts

Perc

en

tag

e

Toxigenic

Atoxigenic


Strains AfB (ng/g) Reduction (%)

La3279 17 99.8

Og0222 110 98.7

La3303 26 99.7

Ab2216 303 96.5

Lo4216 147 98.3

La3304 44 99.5

Ak3020 294 96.6

La3108 153 98.2

La3228 (Control)

8567 --

LSD (α = 0.05) 462.6 --

Atoxigenic Performance in Mixtures

Grain Inoculation Studies


Field Testing of Atoxigenics

• 24 atoxigenic from 6 locations

plus 4 toxigenic strains field-

tested in Ibadan

• Inoculation of atoxigenics alone

and in combination with a

toxigenic strain

• Toxin production in cobs and

competition between toxigenic

and atoxigenics evaluated.


Atoxigenic Performance in Mixtures

Cob Inoculation Studies

AfB1 (ng/g) in inner ring

AfB1 (ng/g) in outer ring

Strains Alone Mixture Alone Mixture

La3279 0.0 9.4 0.2 1.1

La3303 0.0 12.1 0.5 2.9

Ab2216 6.6 126.5 9.7 92.4

Lo4216 0.3 141.9 9.7 3.3

La3304 0.8 233.5 0.0 7.2

Ak3020 0.6 264.4 0.0 15.9

La3108 0.0 272.1 0.0 1.9

La3228 (Control)

5054.0 253.5

LSD ---- 656.7 ---- 120.1

x

Inner ring

Outer ring

X = inoculation site

Atehnkeng et al. (submitted) Food Add. Cont.


Field Release of Atoxigenic

Strains

• One toxigenic and four atoxigenic

strains released together in field;

with controls

• Grain harvested after maturity

• Recovery of released strains

tested by VCG complementation

• Atoxigenic La3279 most frequently

recovered (up to 97%)

• Further analysis is in progress


Frequency of Isolate Recovery

from Maize after Field Release

Isolate

Ab2216 La3228 La3279 La3303 Ka16127

0

20

40

60

80

100

Unknown

nia-Dcnx

-

+

VCG


On-Station Field Release Trials

2007

• 4 strains

• 0.75 ha

• Zaria

• Mokwa

• Ibadan

• Ikenne


Outlook for Aflatoxin Biocontrol

• Research to develop atoxigenic

strains is resource intensive

• Need 4-6 years for large-scale

use of biocontrol agents

• Linkage needed with other

organizations for safety,

registration, mass production,

marketing & other downstream

dissemination activities

• Scope for impact

Collaboration:

• USDA-ARS

• Univ. of Bonn

• Univ. of Ibadan


Nigeria


Relative Aflatoxin Risk of Cereals

in Africa

• Sorghum and pearl millet traditional cereals

• Nigerian maize area – 1980: 465,000 ha; 2004:

4.5 million ha

• Maize introduced and replacing in marginal

areas

• Mycotoxin risk higher in marginal areas

• Objective: Compare Aspergillus contamination

and aflatoxin levels in maize, sorghum and pearl

millet grown side-by-side by subsistence farmers Bandyopadhyay et al. 2007. Food Add. Cont. 24:1109.

M. Kumar


Method

• 14 farmers’ fields in Northern and Southern Guinea

Savanna

• Maize, sorghum and pearl millet grown by farmers in

adjacent plots

• Harvested at maturity by farmers

• Samples brought to lab in Ibadan

• Analysis: aflatoxin by ELISA, frequency of S (more

toxic) and L (less toxic) strains of A. flavus.

• Exposure calculated based on historic cereal

consumption data


Fungal Genus (%) on Grains

Crop Sample size Aspergillus Fusarium

Maize 23 17.7 a 47.3 a

Pearl millet 7 1.9 b 26.1 b

Sorghum 40 4.2 b 26.4 b

• Maize 4 & 9 fold more likely to be contaminated with

Aspergillus than sorghum & pearl millet

• Maize 1.8 fold more likely to be contaminated with Fusarium

than sorghum & pearl millet


Aspergillus in Cereal Grains

Crop*

CFU per

g grain

Aspergillus -- % of total (range)

L-strain S-strain parasiticus

Maize 9869 83-100 0-17 0-15

Sorghum 2390 83-100 0-12 0-4

*Sample size: 13 of each crop grown in same location


Aflatoxin Exposure from Cereals

Crop

Aflatoxin (ng/g) Samples

> 20 ppb

aflatoxin

(%)

Exposure

(ng/kg

bw/day)Mean SD Median Range

Maize 36 100 4.2 1.1 – 480 17 226.8

Sorghum 8.8 14 5.0 1.6 – 90 5 55.5

Pearl millet 4.6 1.8 4.4 2.6 – 8.1 0 29.0

Afla-Safe 20 - - - 126.0

• Maize could have up to 24-fold MTL, sorghum 4.5-fold

• Risk from sorghum is 4-fold less, and pearl millet 8-fold less

than maize (consumption: 138 kg/year; BW: 60 Kg)


Mycotoxin Management

Strategies• Awareness

• Host plant resistance

• Biological control

• Time of harvest

• Grain drying method

• Storage structure

• Storage form

• Sorting and processing

• Insect control


Mycotoxin Management R-4-D at IITA

Development and dissemination of mycotoxin

management strategies (BMZ)

Breeding for resistance (US-FAS; USDA)

Biocontrol of aflatoxin through competitive

exclusion (BMZ; collaboration with USDA, Univ. of

Bonn)

Awareness campaign to sensitize the population on

aflatoxin risk (Rotary International)

Intervention study for the reduction of aflatoxin and

impact of nutrition on the toxin (BMZ)

Fusarium species diversity and mycotoxin profile in

food baskets (cereals/legumes) in Burkina Faso,

Ghana, Nigeria and Cameroon (USAID; KSU, CNR,

IRAD, SARI, INERA)

Training & information exchange (USAID, BMZ, EU)


Summary

• Mycotoxins in food and feed pervasive in Africa

• Negative impact overlooked – chronic, unseen

• Fungal diversity studies can help in targeting intervention strategies – technical and diet based

• Maize riskier than sorghum and pearl millet for aflatoxin contamination

• Institutions related to food safety very weak.

• Aflatoxin received most attention; studies needed on other mycotoxins as well.

• More work required on mycology to explain toxin data


Aflatoxin Tested Pet Food

in Nairobi


PEOPLE

P. Cotty, R. Sikora, S. Kiewnick J. Leslie

P. Ojiambo

C. Afolabi

A. Menkir K. Hell


Team


http://mycoglobe.ispa.cnr.it/

Technology

Biodiversity of Toxigenic Fungi and its Implications in Disease Management