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INTERNATIONAL JOURNAL OF AGRICULTURE & BIOLOGY
1560–8530/2004/06–6–955–959
http://www.ijab.org
Diversity of Fungal Species Associated with Peanuts in Storage
and the Levels of Aflatoxins in Infected Samples
GACHOMO E.W.†, E.W. MUTITU
AND O.S. KOTCHONI†
1
Department of Crop Protection, Faculty of Agriculture, University of Nairobi, P.O. Box 29053 Nairobi, Kenya
†Department of Molecular Biochemistry, Institute of Plant Molecular Physiology and Biotechnology, University of Bonn,
Kirschallee 1, D–53115 Bonn, Germany
1
Corresponding author’s e–mail: skotchoni@yahoo.com
ABSTRACT
The threat of aflatoxin contamination in food commodities and its association with health risks in both animals and humans
continues to raise increasing concern over years. In this report, fungal species found in association with peanuts in storage and
their potential to produce aflatoxin in collected samples was determined. About 60 to 70% of collected peanut samples were
infected with various moulds including Rhizopus stolonifer, Fusarium sp., Aspergilus flavus, other Aspergillus sp., Penicillium
sp., Eurotium repens, Sclerotium sp., Rhizoctonia sp., and Aspergillus Parasiticus. Eurotium repens, Aspergillus Parasiticus,
and A. flavus were found to be the most patent aflatoxigenic strains. The average levels of aflatoxins detected in the seed
samples were far above 100 ppb. This level of toxicity is more than five times higher than the acceptable dosage (20 ppb: US
Standards) in edible peanuts. This report points out the health risks associated with aflatoxin contamination in edible food
commodities despite enormous efforts to control this mycotoxin. Current research efforts to control or minimize the intake of
aflatoxins especially in warmer regions of the world are hereby included.
Key Words: Aflatoxins; Food commodities; Fungal infection; Kenya; Peanuts
INTRODUCTION
Fungal infection of seeds before and after harvest
remains a major problem of food safety in most parts of
Africa. Problems associated with this infection include loss
of germination, mustiness, mouldy smell (Sauer et al., 1992;
Frisvad, 1995) and aflatoxin contamination (McAlpin et al.,
2002; Bankole & Adebanjo, 2003). These problems are
however dealt with, in most developed world where a
careful commodity screening and improved storage
conditions are provided (Ito et al., 2001; McAlpin et al.,
2002; Wilson et al., 2002). However, fungal species that
produce mycotoxins are more common in the warmer,
subtropical and tropical areas than in temperate areas of the
world. Validated methods of analysis exist but an
internationally accepted sampling plan for aflatoxin control
for each commodity is still a targeted goal despite years of
various contributions (Coker, 1989; Cunnif, 1995) and
recently a so–called update on worldwide regulation for
mycotoxin contamination was published by FAO under the
title Food and Nutrition (van Egmond, 2002). In this edition,
only 77 countries were reported to have specific regulations
for mycotoxins, 13 countries were known to have no
specific regulations, whereas no data were available for
about 50 other countries many of them in Africa (van
Egmond, 2002).
Aflatoxins are secondary metabolites produced by
some isolates of Aspergillus flavus, A. parasiticus, and
several unnamed fungi belonging to a non–classified taxon
from Africa (Ito et al., 2001). Developing grains, nuts and
nut products such as peanut butter, roasted shelled peanuts
and peanut oil are the most vulnerable to aflatoxigenic
fungal infections (Rachaputi et al., 2002). It has been a
serious concern to control the increasing incidence of fungal
infection and aflatoxin contamination of valuable
commodities. Aflatoxins are among the most potential
mutagenic and carcinogenic substances known (Bankole &
Adebanjo, 2003). Therefore, setting of internationally
agreed tolerance levels of aflatoxins in foods and feedstuffs
is of global importance. Currently aflatoxin B
1
is the major
contaminant of foods in tropical regions of Africa and this,
has been linked with hepatitis B and C infections and, to the
high incidence of liver cancer in these regions (Montalto et
al., 2002; Elegbede & Gould, 2002). The high mortality rate
of liver cancer patients in these regions indicates the
seriousness of the issue (Montalto et al., 2002). In addition,
Li et al. (2001) found that the level of aflatoxin B
1
, B
2
and
G
1
in corn were significantly higher in the area with high
incidence of human hepatocellular carcinoma, and the
average daily intake of aflatoxin B
1
from the high–risk area
was 184.1μg. Therefore, means of tackling this problem
should be a priority. Molecular characterization of microbial
genes involved in the regulation of aflatoxin biosynthesis
pathway could provide ways to produce genetically
modified organisms to permanently inhibit aflatoxin
synthesis, especially during interactions between aflatoxin–
producing fungi and plants.
GACHOMO et al. / Int. J. Agri. Biol., Vol. 6, No. 6, 2004
956
In this study, the correlation of occurring fungal
infection in stored peanuts and the level of aflatoxin content
in the collected samples was investigated in different fresh–
produce markets of Nairobi, Kenya (Eastern Africa). We
present here evidence of various identified fungal infections
and a very high detectable level of aflatoxins in the samples.
In addition, proper monitoring programs, recommendations
for minimizing the rate of aflatoxigenic fungal infection in
food commodities and the risk of toxicity to the consumers
are herein discussed.
MATERIALS AND METHODS
Reagents and chemicals. Unless stated otherwise, all
reagents and chemicals used in this work were from Sigma
and Merck’s Company, Roch (Germany).
Sample collection. Unshelled peanuts were obtained from
five fresh–produce markets within Nairobi (Kenya). The
markets were identified as V, W, X, Y and Z. Two peanut
varieties (Valencia Red: VR & Homa Bay local: HbL) were
sampled from each of the markets V, W, and X while only
one variety (VR) was available from the markets Y and Z.
An average weight of 2000 g of peanuts per variety and per
market was considered. Fungi were isolated from the
peanuts under laboratory conditions using agar and blotter
test methods according to Dhingra and Sinclair (1994).
Isolation of fungi using blotter and agar test. Under
blotter test, sterile filter papers were aseptically placed in
petri dishes and moistened with sterile distilled water to
serve as moist chambers. All experiments were carried out
under sterile conditions to avoid contamination. Eight
hundred peanuts per variety and per market were considered
for this test. Half of the seeds (400 seeds per variety) was
surface–sterilized in 3% (v/v) sodium hypochlorite for 3
min and rinsed in three changes of sterile distilled water.
The seeds were then placed on the filter papers and
incubated at room temperature (23°C±2) for 14 days. The
second half of the samples (400 seeds) were not sterilized
but were incubated under similar conditions. The results
recorded represent means (±SD) of triplicate experiments.
For agar test, the set up was similar to that of blotter
test except petri dishes containing 10 ml potato dextrose
agar (PDA) were used as moist chambers. After incubation,
colony characteristics (colour, shape) of different types of
fungi that grew were recorded. The number of seeds
infected with the same type of fungus was recorded. The
individual isolates were transferred to new PDA plates in
order to obtain pure cultures. All isolates were maintained
on PDA and kept at 4°C for further analysis. The fungal
identification was performed using microscopic
observations, identification keys and illustrated manuals
(Raper & Fennell, 1965; Klich & Pitt, 1998). Synoptic keys
were used to identify different fungal genera.
Qualitative and quantitative analysis of aflatoxins
produced by the isolates. The seeds were qualitatively
analysed for the presence of aflatoxins according to Cunnif
method adopted for aflatoxin analysis by the Association of
Official Analytical Chemists (AOAC) (Cunnif, 1995).
Basically, the method consists of an extraction phase,
followed by a column clean–up phase and finally by a
qualitative assay via a one–dimensional thin layer
chromatography (TLC), which uses a silica gel adsorbent
and an acidic solvent system as described by Kuiper–
Goodman and Scott (1989).
For the quantitative estimation of aflatoxins, scanning
densimetric analysis was carried out. The TLC plates were
scanned according to the instructions of the manufacturer
using CD 60 Desaga computer program. The program is set
up to analyse the intensity of the spots developed by TLC.
The peak areas of the samples were compared to those of
the standards to quantify the aflatoxin content in the
samples.
In order to identify the most toxigenic fungi,
uninfected peanut samples were infected with the isolated
fungi under laboratory controls and screened for aflatoxin
production after two weeks of incubation as described
above.
RESULTS
Isolation of the fungi from peanut samples. To better
isolate the various fungal species from the infected fresh
peanut samples, simple growth analyses made of agar plates
and sterile moistened filter papers were carried out. Several
fungal species became obvious from the growth media after
seven days of incubation. From the seven days onwards,
infected samples displayed fungal species, which were
obvious enough for isolation. Fig. 1 presents an illustration
of fungi screening by a blotter test showing different types
of fungi growing from the peanut samples. From these tests,
it was easy to evaluate the percentage of various fungal
occurrences in the collected fresh peanuts. Table 1 shows
sum total occurrence (in %) of fungal species in different
markets samples using both agar and blotter methods. Of the
several fungal species isolated from the peanuts, R.
stolonifer was the most predominant with 80% occurrence
followed by Fusarium sp. (45%) and Aspergillus species
(24%). The other species accounted for 5 to 10% (Table I).
The Aspergillus species isolated were Aspergillus flavus, A.
parasiticus, A. niger, A. ochraceous, and the Fusarium
species isolated include F. oxysporum, F. equiseti and F.
torulosum (results not shown). The highest occurrence of
Aspergillus species (A. parasiticus, A. flavus & the other
Aspergilli) was in the markets V and X. Relatively high
percentage of occurrence for Rhizopus sp. and Fusarium sp.
was reported in all the samples collected, while the
occurrence of Penicillium sp. and Aspergillus sp. was
moderate and that of Eurotium repens, Sclerotium sp., and
Rhizoctonia sp. was low (Table I).
To ascertain whether the fungal infection is on the
surface or within the peanut seeds, the collected samples
were surface–sterilized prior to incubation, and a
AFLATOXIN CONTAMINATION OF FRESH PEANUTS / Int. J. Agri. Biol., Vol. 6, No. 6, 2004
957
comparative analysis was carried out with the non–sterilized
sample cultures. Fig. 2 shows the rates of occurrence of
different fungi on the surface–sterilized and non–sterilized
peanut samples. Rhizopus stolonifer and Fusarium sp. had
the highest occurrence rate in both surface–sterilized and
non–sterilized peanuts but this occurrence was higher in the
non–sterilized samples. Aspergillus sp. (A. flavus, A.
parasiticus & the other Aspergilli) had markedly higher
incidence of occurrence in the surface–sterilized samples
than the non–sterilized ones. A similar pattern of occurrence
was observed for Eurotium repens, Sclerotium sp. and
Rhizoctonia sp. (Fig. 2). However, the occurrence rate of
Penicillium sp. was found to be almost equal (10%) in both
surface–sterilized and non–sterilized samples.
Detection and estimation of aflatoxins in peanut
samples. All samples were tested for aflatoxin contents. We
carried out also in vitro fungal infection experiments of
healthy seeds in order to identify the aflatoxigenic strains
from the isolated fungal population. Fig. 3 shows the levels
of aflatoxins in the peanuts from the sampling locations
(Fig. 3a) coupled with the identification of the most
aflatoxigenic fungi in the samples (Fig. 3b). Peanut VR was
found to contain high levels of aflatoxins B
1
, B
2
and traces
of aflatoxin G
1
. The peanut HbL however had relatively
lower detectable levels of aflatoxin B
1
and B
2
(Results not
shown). The aflatoxin contamination of the samples was
generally associated with the isolation of strains such as
Aspergillus flavus, Aspergillus parasiticus, and Eurotium
repens. Fig. 3b shows the estimation of aflatoxins produced
by the different toxigenic fungi. Among the species of
Aspergillus, A. flavus produced 151.26 ppb and 130.5 ppb
of aflatoxin B
1
and B
2
, respectively, while A. parasiticus
produced 159.3 ppb and 110.9 ppb of aflatoxin B
1
and B
2
respectively. Both Aspergillus sp. produce traces of
aflatoxin G
1
(4.0 ppb) as shown in Fig. 3b. Eurotium repens
on the other hand produced larger amounts of aflatoxin B
1
(160, 19 ppb), aflatoxin B
2
(140 ppb) and aflatoxin G
1
(75.26 ppb) and considered therefore as the most
aflatoxigenic fungus in this work.
DISCUSSION
Interest in aflatoxin contamination of food and
feedstuff arose from its association with disease and
mortality in humans and animals. Up to date, practical
strategies to control this mycotoxin are still under
investigation. Mycotoxins of the greatest concern are
aflatoxins, ochratoxin A, and fumonisins produced by
Aspergillus sp., Penicillium sp. and Fusarium sp.,
respectively (Bullerman, 2002). These toxins are a major
threat for public health particularly in warmer and tropical
regions of the world, like in Africa where proper and
accurate screening methods are lacking. We point out in this
report, the ever–permanent concern of aflatoxin
contamination for rural and urban communities of Africa.
Aflatoxin content above 20 ppb in peanuts is considered
very dangerous for human health worldwide (Coker, 1989;
Cunnif, 1995; Wilson et al., 2002). According to the Kenya
Bureau of Standards, the total levels of aflatoxin content in
peanuts intended for human consumption should not exceed
20 ppb, but in this report, the detection levels of aflatoxins
in peanut samples were found to be about four to five times
higher than the acceptable 20 ppb value (Fig. 3a, b). The
presence of aflatoxigenic fungi in surface–sterilized samples
(Fig. 2) demonstrates that a simple clean–up precaution
before consumption would never safeguard the consumers
from the risk of contamination. Using this study as an
example, probably several other food commodities
Fig. 1. In vitro screening of different fungal infections
in peanut samples using blotter test; (a): Penicillium
sp., (b): Fusarium sp., (c): Aspergillus sp.
b
a
c
Fig. 2. Detection of fungal infections on surface-sterilized and non-
sterilized peanut samples: R. stol = Rhizopus stolonifer, Fus sp =
Fusarium sp., A. para = Aspergillus parasiticus, A. flav = Aspergillus
flavus, Asp sp = other Aspergillus species including A. niger and A.
ochraceous, Peni sp = Penicillium sp., E. rep = Eurotium repens, Scle sp
= Sclerotium sp., Rhiz sp = Rhizoctonia sp.
0
10
20
30
40
50
60
70
80
90
100
Non-ster.
Ste r .
R
.
s
t
o
l
F
u
s
s
p
A
.
p
a
r
a
A
.
f
l
a
v
A
s
p
s
p
P
e
n
i
s
p
E
.
r
e
p
S
c
l
e
s
p
R
h
i
z
s
p
Fungal occurancerate (in %)
GACHOMO et al. / Int. J. Agri. Biol., Vol. 6, No. 6, 2004
958
susceptible to aflatoxin contamination, such as cereal grains
and tree nuts in the region although not investigated in this
study, may contain high level of aflatoxins as well. The
aflatoxin quantitative methods used here were not accurate
above 100 ppb. Therefore, we were not able to ascertain the
aflatoxin quantities exceeding 100 ppb in the samples (Fig.
3). However, these data were more than convincing to draw
a conclusive remark i.e. consumers of fresh peanuts (non–
processed) in Africa are exposed to the risk of high
mycotoxin intake. The contamination may also result
indirectly from consumption of animal products such as
milk from livestock exposed to contaminated feed (Bankole
& Adebanjo, 2003). These are broadening effects of
aflatoxin contamination that one should take into account
for accurate risk evaluation of aflatoxin contamination in a
given region.
Several contributions about aflatoxin detoxification
using dietary clay and isothermal adsorption of aflatoxin
contamination have been documented (Grant & Phillips,
1998; Phillips, 1999). However, it is far much better to
minimize or avoid contamination of products if possible,
rather than to depend on detoxification. In addition
biological control processes such as competitive exclusions
of toxigenic fungi by use of different Aspergillus mutants
are of tremendous contributions to the control of aflatoxin
accumulation both in pre– and post–harvest seeds (Wilson
et al., 1986; Cotty & Bayman, 1993). However, several of
such controls are expensive for farmers to implement
profitably and accurately especially in developing world.
Cotty and Bayman (1993) reported that atoxic Aspergillus
species competed successfully with toxic isolates in a mixed
culture condition, but the competition mechanism is still not
well elucidated. Some atoxic aflatoxigenic fungi may be
potential producers of several other toxins, which might be
harmful both to humans and animals. For these reasons
more information should be rather generated about the
storage conditions repressing the aflatoxin contamination
worldwide and especially in regions where farmers are still
holding tightly to the traditional methods of storage. In other
words, resources should be oriented into making scientific
findings more adaptable for the traditional farmers.
Developing post–harvest strategies for sorting or any
other aflatoxin control measures in warmer, tropical and
subtropical regions should be therefore highly welcomed.
Aflatoxin formation in peanuts is favoured by prolonged
period of drought associated with soil–elevated temperature
(Wilson et al., 2002; Rachaputi et al., 2002; Bankole &
Adebanjo, 2003). Irrigation of the peanuts while still in the
fields especially in warmer and tropical regions of the world
could be therefore an effective option in reducing aflatoxin
contamination. It was also suggested that late season
irrigations could increase soil moisture and decrease soil
Fig. 3. Estimation of aflatoxins in peanut samples per markets (a) and identification of aflatoxigenic fungal species (b): A. para = Aspergillus
parasiticus, A. flav = Aspergillus flavus, E. rep = Eurotium repens: —: Limit of estimation accuracy, *: Aflatoxin content above limit of accuracy.
0
20
40
60
80
100
120
140
VWX Y Z
Af la B 1
Af la B 2
Af la G 1
*
*
*
*
A
f
l
a
t
o
x
i
n
c
o
n
t
e
n
t
(
p
p
b
)
a
0
20
40
60
80
100
120
140
VWX Y Z
Af la B 1
Af la B 2
Af la G 1
*
*
*
*
0
20
40
60
80
100
120
140
VWX Y Z
Af la B 1
Af la B 2
Af la G 1
*
*
*
*
A
f
l
a
t
o
x
i
n
c
o
n
t
e
n
t
(
p
p
b
)
a
0
20
40
60
80
100
120
140
160
180
A. f lav A. para E . rep
Afla B1
Afla B2
Afla G1
*
*
*
*
*
*
A
f
l
a
t
o
x
i
n
c
o
n
t
e
n
t
(
p
p
b
)
b
0
20
40
60
80
100
120
140
160
180
A. f lav A. para E . rep
Afla B1
Afla B2
Afla G1
*
*
*
*
*
*
0
20
40
60
80
100
120
140
160
180
A. f lav A. para E . rep
Afla B1
Afla B2
Afla G1
*
*
*
*
*
*
A
f
l
a
t
o
x
i
n
c
o
n
t
e
n
t
(
p
p
b
)
b
Table 1. Occurring rate of fungi isolated from peanut samples per market
Identification rate of Fungal species (%) in peanut samples per market
Fungal species V W X Y Z
Rhizopus stolonifer
Fusarium sp.
Aspergillus parasiticus
Aspergillus flavus
Aspergillus sp.
Penicillium sp.
Eurotium repens
Sclerotium sp.
Rhizoctonia sp.
80 ± 9
50 ± 4
15 ± 4
3 ± 0.5
50 ± 6
10 ± 2
5 ± 1
25 ± 2
2 ± 0
90 ±10
40 ± 6
5 ± 1
5 ± 1
20 ± 3
11 ± 2
2 ± 0
30 ± 7
2 ± 0
85 ± 7
20 ± 3
3 ± 1
50 ± 5
40 ± 5
7 ± 2
3 ± 1
5 ± 0
2 ± 0
82 ± 6
12 ± 2
2 ± 0
5 ± 0
10 ± 1
5 ± 1
2 ± 0
2 ± 0
10 ± 1
88 ± 9
30 ± 2
2 ± 0
3 ± 0
30 ± 3
3 ± 0
2 ± 0
2 ± 0
2 ± 0
Aspergillus sp. including other Aspergillus such as A. niger and A. ochraceous. Data represent the mean value of triplicate experiments (± SD).
AFLATOXIN CONTAMINATION OF FRESH PEANUTS / Int. J. Agri. Biol., Vol. 6, No. 6, 2004
959
temperature and thereby be used as a promising way to
lower aflatoxin content in mature seeds (Wilson et al.,
2002). Moreover, sorting the peanuts to remove damaged
seeds before storage could also be a fairly effective and
cheaper way to control aflatoxin contamination. Udoh et al.
(2000) demonstrated that aflatoxin contamination of stored
commodities in five agro–ecological zones of Nigeria
(West–Africa) was strictly related to storage practices. Seed
integrity could therefore be maintained by observing proper
storage conditions. It is imperative to avoid a long–term
storage system in warmer regions. As demonstrated by Goel
and Sheoran (2003) in stored cottonseeds, after 18 months
of storage, the germination ability of the seeds decreased
and the membrane deterioration increased with storage
period, which could lead to a high probability of fungal
infection.
On the other hand, several other mycotoxins such as
ochratoxin A, and other potent toxic metabolites (not
investigated in this study) are also thought to be present in
the collected samples. The reason being that A. flavus, A.
parasiticus, A. ochraceus, A. niger (all isolated in this study)
were known as source of ochratoxins, cyclopiazonic acid,
patulin, sterigmatocystin, gliotoxin, citrin production
(Wilson et al., 2002). Penicillium species isolated also from
the collected samples are reported to produce cyclopiazonic
acid, ochratoxins and sterigmatocystin (Wilson et al., 2002).
The principal reason for such recurring situation is mainly
the lack of awareness in these regions. For the safety of
human food and the welfare of consumers, it is imperative
to educate the population on the danger of aflatoxin
contamination and to screen for all possible mycotoxin
contaminants in any given commodity before allowing it to
be marketed. These screening steps should strictly receive a
higher priority over any economical aspects. It is in this
respect that the danger and risks of toxicity could be greatly
minimized in years to come. The ever–present health risks
to which the unsuspecting and ignorant public (especially in
most of Africa regions) is exposed to is here clearly evident.
The need for interdisciplinary cooperation involving
governments, non–governmental organizations and
scientists in this area in order to establish monitoring and
regulatory risk management procedures has never been
timelier.
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(Received 01 April 2004; Accepted 20 September 2004)
