Biodiversity of Toxigenic Fungi and its Implications in Disease Management

Biodiversity of Toxigenic Fungi and its
Implications in Disease Management

Ranajit Bandyopadhyay
IITA, Ibadan
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org

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

 Small Scale and Unregulated
– Developing countries
– Informal markets
– Subsistence
– High food insecurity Tim Williams, Peanut CRSP

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)

Possible Relative public
Health effect deaths (No.) attention

Biological weapon 0 (?) Very high

Acute aflatoxicosis 100’s High

Hepatocellular 10,000’s Medium
carcinoma
Growth impairment/ 100,000’s (?) Low/None
immunosuppression


Aflatoxin (ng/g)
Contamination in West Africa

Food products
• Peanut paste: 3278 – Ghana
Primary products • Peanut sauce: 943 – Ghana
• Leaf sauce: 775 – Gambia
• Maize: 4000 – Benin
• Maize dough: 313 – Ghana
• Peanut: 216 – Ghana
• Kenkey: 524 – Ghana
• Sorghum: 80 – Ghana
• Cashew paste: 366 – Ghana
• Millet: 200 – Nigeria
• Peanut oil: 500 – Nigeria
• Tiger nuts: 120 – Nigeria
• Yam flour: 7600 – Nigeria
MTL: 20 ng/g • Local beer: 135 - Nigeria

Aflatoxin Exposure in Africa,
Europe & USA
100 Gambia (n = 950)
Benin (n = 479)
USA (n = 48)
Europe (n = 74)
Number of individuals (%)

80

60

40

20

0
<5 5-25 26-100 >100

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

Fusarium species

Courtesy: Leslie & Summerell International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org

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 • An understanding of
and types of toxins occur species diversity can help
on cereals. in toxin risk management
• Risk of toxin based on • Species and diversity
Fusarium species present 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
Toxigenic
 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
Bandyopadhyay et al. (2006) Tropentag Atoxigenic

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
J. Atehnkeng

Wet

Dry

M. Donner

 Derived Savanna & Southern Guinea Savanna – 5 districts
 Northern Guinea Savanna – 2 locations
 5 locations / state; 50 to 124 isolates / location; n = 4414

Atoxigenic Strain Identification
Toxin assay -
Unknown

Strain characterization cnx nia-D

VCG

Field Field release
Unknown
Competition assays
cnx
nia-D

Lab +

Distribution of Aspergillus in
Nigerian Soils & Grains
Soil Grain L-strain
S-strain
A. tamarii
A. parasiticus

1% 0.1%
88% 97.9%
5%
0.6% 1.4%
6%

 L-strains more predominant than S-strains
 Low frequency of A. parasiticus Atehnkeng et al. (in press)
Int. J. Food Microbiol.

Aspergillus Strains in Grain
600

500 L-strain

S-strain

A. parasiticus
400
A. tamarii
Strains / location

300

200

100

0
o

ti
a
ja

di

a
a
a

ga

a
f ia
a
h

j

ki

un
id

ri
w

n

ko
bu

ur
os

La

Za
an
in

E
ok

B

ad
ak

Lo
A
M
om

do
kw
M

M

K
A
A
gb
O

Districts


Proportion of Toxigenic and
Atoxigenic Strains in Grain
90
80
70
60
Percentage

50 Toxigenic
40 Atoxigenic
30
20
10
0
M fia

na
Ka ia
ga
da

Ak uja

ho
wa

Ad oja
na

it i
Lo i
d

r
Ek
ur
La

Za
an
Bi

du
in

os
Ab
ok

k
ak
M

w

om

o
M

gb
O

Districts


Atoxigenic Performance in Mixtures
Grain Inoculation Studies
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 8567 --
(Control)
LSD (α = 0.05) 462.6 --

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 AfB1 (ng/g) in
inner ring outer ring
Strains Alone Mixture Alone Mixture
La3279 0.0 9.4 0.2 1.1 Inner ring
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 x
Ak3020 0.6 264.4 0.0 15.9
La3108 0.0 272.1 0.0 1.9
La3228 5054.0 253.5
(Control) Outer ring
LSD ---- 656.7 ---- 120.1 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
100
-
Unknown
80

cnx nia-D
60

VCG
40

20

0
Ab2216 La3228 La3279 La3303 Ka16127 +
Isolate


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
Collaboration:
dissemination activities • USDA-ARS
• Scope for impact • Univ. of Bonn
• Univ. of Ibadan

Nigeria


Relative Aflatoxin Risk of Cereals
in Africa
M. Kumar
• 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.

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

CFU per Aspergillus -- % of total (range)
Crop* g grain
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
• 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)

Aflatoxin (ng/g) Samples
Exposure
> 20 ppb
(ng/kg
aflatoxin
Crop Mean SD Median Range bw/day)
(%)

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

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


P. Cotty, R. Sikora, S. Kiewnick J. Leslie P
E
O
P
L
E

C. Afolabi
A. Menkir K. Hell P. Ojiambo

Team


Biodiversity of Toxigenic Fungi and its Implications in Disease Management

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