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Isolation and characterisation of aflatoxigenic Aspergillus species from maize and soil samples from selected counties of Kenya

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  • Tom Mboya University College

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Aflatoxin contamination of maize put the health and well-being of Kenyan people at risk, primarily children. Aflatoxigenic fungi can infect grains from pre-harvest stages in the field to post-harvest stages in the stores. The aim of this study was to isolate and characterize aflatoxigenic Aspergillus spp. from maize and soil samples from selected counties of Kenya: Makueni, Nyeri, Bungoma, Uasin Gishu and Siaya. Isolation was done using the direct plating technique of surface-sterilized grains on Czapek Dox Agar medium and plating of serially diluted soil samples. Aspergillus colonies were purified and identified using colony growth characteristics, colony colour on PDA media and microscopic characterisation. In total, 174 Aspergillus isolates were obtained, where 82.3% came from maize samples while 17.7% were from soil samples. Makueni County had the highest number of Aspergillus isolates at 58.1%, Nyeri 12.6%, Uasin Gishu 10.3%, Siaya 10.3% and Bungoma 8.6%. The characterization process identified 10 different Aspergillus spp.; 78.5% were Aspergillus flavus, 8.0% A. versicolor, 3.4% A. parasiticus, 2.3% A. clavatus, 2.3% A. sydowii, 2.3% A. fumigatus, 1.1% A. glaucus, 1.1% A. nidulans, 0.6% A. candidus and 0.6% A. wentii. The results evidence that maize grains and fields in the various counties are highly contaminated with aflatoxigenic Aspergillus species. Key words: Aflatoxin, Aspergillus, maize.
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Vol. 7(34), pp. 4379-4388, 23 August, 2013
DOI: 10.5897/AJMR2013.5846
ISSN 1996-0808 ©2013 Academic Journals
http://www.academicjournals.org/AJMR
African Journal of Microbiology Research
Full Length Research Paper
Isolation and characterisation of aflatoxigenic
Aspergillus species from maize and soil samples from
selected counties of Kenya
Benard Omondi Odhiambo
1
*, Hunja Murage
2
and
Isabel Nyokabi Wagara
1
1
Egerton University, P.O Box 536-20107 Egerton, Njoro, Kenya.
2
Jomo Kenyatta University of Agriculture and Technology P.O Box 62000-00200 Nairobi, Kenya.
Accepted 6 August, 2013
Aflatoxin contamination of maize put the health and well-being of Kenyan people at risk, primarily
children. Aflatoxigenic fungi can infect grains from pre-harvest stages in the field to post-harvest
stages in the stores. The aim of this study was to isolate and characterize aflatoxigenic Aspergillus spp.
from maize and soil samples from selected counties of Kenya: Makueni, Nyeri, Bungoma, Uasin Gishu
and Siaya. Isolation was done using the direct plating technique of surface-sterilized grains on Czapek
Dox Agar medium and plating of serially diluted soil samples. Aspergillus colonies were purified and
identified using colony growth characteristics, colony colour on PDA media and microscopic
characterisation. In total, 174 Aspergillus isolates were obtained, where 82.3% came from maize
samples while 17.7% were from soil samples. Makueni County had the highest number of Aspergillus
isolates at 58.1%, Nyeri 12.6%, Uasin Gishu 10.3%, Siaya 10.3% and Bungoma 8.6%. The
characterization process identified 10 different Aspergillus spp.; 78.5% were Aspergillus flavus, 8.0% A.
versicolor, 3.4% A. parasiticus, 2.3% A. clavatus, 2.3% A. sydowii, 2.3% A. fumigatus, 1.1% A. glaucus,
1.1% A. nidulans, 0.6% A. candidus and 0.6% A. wentii. The results evidence that maize grains and
fields in the various counties are highly contaminated with aflatoxigenic Aspergillus species.
Key words: Aflatoxin, Aspergillus, maize.
INTRODUCTION
Aflatoxin is one of the most famous mycotoxins produced
by several species of Aspergillus including A. flavus and
A. parasiticus in a wide variety of agricultural commo-
dities including grains (maize), legumes and nuts (Patten,
1981). The main aflatoxin producing fungi A. flavus, A.
parasiticus and A. nomius can infect maize from pre-
harvest stages in the field to post-harvest stages in the
stores. Species of Aspergillus are almost ubiquitously
present in soils of tropical areas (Ranajit et al., 2005).
Although, most species of Aspergillus are not of much
consequence in agriculture, some species are found in
plant products, particularly oil-rich seeds. Contamination
of seeds with highly poisonous aflatoxins results from the
presence of toxigenic strains of four species of
Aspergillus: A. flavus, A. parasiticus, A. nomius and A.
bombycis, each producing a combination of different
types of aflatoxins (Ranajit et al., 2005).
In Africa, aflatoxins have an impact on human and
*Corresponding author. E-mail: benodhy@gmail.com. Tel: +254-727-606-862.
Abbreviations: PDA, Potato dextrose agar; CZ, Czapek Dox Agar; ANOVA, analysis of variance.
4380 Afr. J. Microbiol. Res.
animal health and on trade. Aflatoxin has been reported
to be associated with the exacerbation of the energy
malnutrition syndrome in children and vitamin A
malnutrition in animals. In various animal models, in
addition to being hepatotoxic, aflatoxin causes significant
growth faltering and is strongly immune-suppressive at
weaning (Wild et al., 1992). Similar effects have been
reported in human population in a few African countries
such as Ghana and it has been recently shown that 99%
of all children weaned from mother’s milk to maize-based
diets in Benin and Togo had aflatoxin in their blood,
indicating ingestion of aflatoxin-contaminated food
(Ranajit et al., 2005).
In developing countries, the contamination of crops with
aflatoxin leads not only to economic losses, but also has
a severe impact on human health. In Africa, a continent
that relies on vulnerable crops such as groundnuts and
maize as dietary staples, aflatoxin contamination causes
major health problems (Shephard, 2003). People in rural
areas may have no option but to consume contaminated
crops on a daily basis. This moderate, chronic intake of
aflatoxin via food can lead to severe pathological
conditions, including liver cancer, immune system
deficiency and impaired development of children
(Williams et al., 2004). Malnutrition, a common condition
in rural Africa, increases disease prevalence and further
reduces the ability of the human body to cope with
aflatoxin exposure. Chronic aflatoxin poisoning reduces
life expectancy.
Acute aflatoxin poisoning is caused by ingestion of high
levels of the toxin. Immediate consequences are severe
liver damage, acute jaundice and hepatitis, which may
subsequently result in death (Bennett and Klich, 2003).
Although on a global basis, deaths from acute aflatoxin
poisoning are rare, Kenya has experienced dramatic
outbreaks of mycotoxin poisoning resulting in loss of
lives. In 2004, an acute aflatoxicosis outbreak occurred in
Machakos, Kenya resulting in 317 cases and 125 deaths,
while cases of liver cancer have been linked to high
levels of aflatoxins in the Lake Victoria Basin (LVB)
(Anonymous, 2004).
MATERIALS AND METHODS
Source of maize and soil samples
A total of 113 maize and 113 soil samples obtained randomly from
maize farms and rural households in Makueni, Nyeri, Bungoma
south, Moiben and Ugunja districts were used in this study. These
districts were selected to represent the following counties
respectively: Makueni, Nyeri, Bungoma, Uasin Gishu and Siaya
(Figure 1). Makueni, Nyeri and Siaya counties were selected due
to the fact that aflatoxin poisoning cases have been previously
reported in those counties, while Bungoma County lies on a transit
route where exchange of maize from other countries such as
Uganda is possible. Uasin Gishu County was selected due to the
fact that there is high maize production in the county every year. In
each district, four villages were selected, that is, towards the east,
west, north and south of the district. In each village, five homes
(200 to 300 m apart) were randomly selected based on information
gathered from local residents as to which homes had renowned
maize farmers and sampling was done in those homes. Most of
these areas are in mid-altitude agro ecological zones with warm
and humid conditions such as Ugunja while Nyeri and Moiben
districts have high relative humidity thus mould invasion is primarily
due to inadequate drying and improper storage (Pitt, 2000). These
factors favour development of moulds and production of mycotoxins
(Kaaya et al., 2006). These areas have unpredictable rainfall
patterns making it difficult for small scale farmers to efficiently dry
their produce. In Nyeri, Bungoma south, Moiben and Ugunja
districts 20 maize samples (500 g each) and 20 soil samples (100 g
each) were collected. However, in Makueni district 33 maize
samples (500 g each) and 33 soil samples (100 g each) were
collected. Soil samples were collected from maize farms. This
higher number of samples collected from Makueni district was
attributed to the fact that the district had several reported cases of
aflatoxin poisoning. Soil samples were collected from floors of
maize stores, maize farms and the bare ground in the homestead
where maize is usually dried. The samples were collected in
properly labeled khaki paper bags to minimize saprophytic fungal
contamination and transported in a cool box to the laboratory for
analysis. The samples were stored at 4°C until further analysis.
Isolation from maize and soil samples
The maize grains were surface sterilized in 2% sodium hypochlorite
and rinsed three times with sterile distilled water. A total of 20
grains were randomly picked per sample and plated (five per plate)
on Czapek Dox Agar medium (CZ) amended with 50 mg of
streptomycin and 50 mg of penicillin. The soil samples were first
serially diluted before plating. One gram of the soil sample was
dissolved in 9 ml sterile distilled water and serially diluted to 10
-4
.
One millilitre of the 10
-3
and 10
-4
dilutions were plated in CZ
amended with 50 mg of streptomycin and 50 mg penicillin. The
plates were then incubated at 28°C for 7 days and the number of
kernels showing growth of Aspergillus species in each Petri dish
was counted (Plate 1A) while for the dilution plates the number of
Aspergillus colonies per plate was counted (Plate 1B). Aspergillus
colonies were sub-cultured on potato dextrose agar (PDA) and
incubated at 28°C for 7 days. Treatments were replicated four times
and the experiment was done in a complete randomized design.
Identification and characterisation of Aspergillus species
The resulting cultures were identified to species level based on
cultural and morphological characteristics like colony diameter,
colony colour on agar and reverse, colony texture and zonation
(Klich, 2002). Morphological features were studied under the
microscope and the major and remarkable microscopic features
that were considered were conidiophores, conidial shape, phialides
and metulae, presence and shape of vesicles (Larone, 1995).
Contemporary diagnosis of the Aspergillus species was based on
the descriptions and keys of Klich (2002). For microscopic
characterisation, slide cultures of the isolates were prepared and
incubated in moist chambers at 28°C for 5 days before observation
under a light microscope.
Data analysis
SAS version 9.0 was used in the data analysis. One way analysis of
variance (ANOVA) was used to test whether the Aspergillus isolates
obtained from maize and soil in the five districts were significantly
different from each other based on their frequency of occurrence.
Odhiambo et al. 4381
Figure 1. Map of the study area. Key: Bungoma (Mean temperature: 20°C, RH: 68 to 76%), Makueni (Mean temperature: 22.5°C,
RH: 48 to 57%) Uasin Gishu (Moiben) (Mean Temp: 17.5°C, RH: 75 to 80%), Nyeri (Mean temperature: 16°C, RH: 64-83%) and
Siaya County (Mean temperature: 23.5°C, RH: 53 to 60%). Source: Cartographer, Department of Environmental Science, Egerton
University (2013).
4382 Afr. J. Microbiol. Res.
Plate 1. (a) Aspergillus spp. growth on maize kernels cultured on CZ media. (b) Aspergillus spp. colonies growth
from soil serial dilutions cultured on CZ media.
Table 1. Frequency of Aspergillus isolates from maize and soil samples and distribution of various Aspergillus species across
all the five districts.
Source
Makueni
Nyeri
Moiben
Ugunja
Bungoma
Total
% Total
Maize isolates
99
17
7
14
7
144
82.3
Soil isolates
2
5
11
4
8
30
17.7
Aspergillus species
A. flavus
96
15
4
10
11
136
78.5
A. versicolor
0
2
8
2
2
14
8.0
A. parasiticus
1
0
1
4
0
6
3.4
A. clavatus
0
0
4
0
0
4
2.3
A. sydowii
1
2
0
1
0
4
2.3
A. fumigatus
1
2
0
0
1
4
2.3
A. glaucus
1
0
1
0
0
2
1.1
A. nidulans
1
1
0
0
0
2
1.1
A. candidus
0
0
0
1
0
1
0.6
A. wentii
0
0
0
0
1
1
0.6
Total No.
101
22
18
18
15
174
100
Total (%)
58.0
12.6
10.4
10.4
8.6
100
One way ANOVA was also used to test whether the distribution of
the various Aspergillus spp. were significantly different in all the five
districts.
RESULTS
Aspergillus isolates from maize and soil and their
incidences across all the districts
The incidence of the maize and soil isolates from all the
districts was recorded as shown in Table 1. The
percentage ratio of maize to soil Aspergillus isolates in
each district was as follows: Makueni 98%: 2%, Nyeri
77.3%: 22.7%, Moiben 38.9%: 61.1%, Ugunja 77.8%:
22.2% and Bungoma South 46.7%: 53.3%. In Makueni,
Nyeri and Ugunja districts, there was a higher number of
maize isolates than soil isolates, while in Moiben and
Bungoma South districts there was slightly more soil
isolates than maize isolates.
The high number of isolates in maize than in soil
especially in Makueni may be due to on-farm maize grain
processing as this has been reported to be a common
practice among the farmers, especially of eastern Kenya
(Makueni) (Strosnider et al., 2006). Results obtained by
Muthomi et al. (2012) showed that maize and maize
products sampled at farm level had a higher risk of
contamination by Aspergillus spp. and aflatoxins. These
results show that maize and soil across all the five
districts are highly contaminated with fungi of the genus
Aspergillus. The frequency of the Aspergillus isolates
A B
across all the five districts analyzed through ANOVA, was
not significantly different (P = 0.5489). This was expected
because the ubiquitous nature of these Aspergillus spp.
enables them to grow on dead organic matter
everywhere in nature, their presence is only visible to the
unaided eye when mould colonies form and they derive
energy from the organic matter in which they live (Ryan
and Ray, 2004).
Morphological, cultural and microscopic
characterisation of the Aspergillus species
Morphological, cultural and microscopic features of the
isolates were studied and recorded (Table 2). A total of
10 Aspergillus species were identified and characterized.
Most isolates of the same species, despite originating
from different districts, showed similarities in their
morphological and cultural characteristics in PDA media.
A. flavus strains EM324, EM244, EM182 and EM1112
from Makueni; NM091, NM083 and NM084 from Nyeri;
BM071 and BS116 from Bungoma UM127 from Ugunja
and RS013, RM023-2 from Moiben, all had similar
surface colour of olive green with whitish margins and
reverse colour of creamish to yellow on PDA. Similarly,
there were only slight variations in their colony diameters
which ranged between 37 to 42 mm. For A. glaucus,
strains RS024 from Moiben and EM211 from Makueni
also had less variable colony colour of green with yellow
areas and reverse colour of creamish yellow, and the
colony diameter ranged between 26 and 29 mm. A.
parasiticus strain UM082 (Plate 2b) showed
characteristics similar to A. flavus strains (Plate 2b) apart
from the colony colour which was conifer green. A.
versicolor strains BS203 from Bungoma had a colony
diameter of 17.0 mm which differed with other A.
versicolor strains RS016 and RS162 from Moiben which
had colony diameters ranging from 32 to 34 mm.
Aspergillus clavatus strains were from Moiben district and
were all similar in their morphological characteristics.
Aspergillus sydowii strains from Makueni, Nyeri and
Ugunja were not different from each other in their
morphological characteristics. Aspergillus fumigatus from
Makueni, Nyeri and Bungoma districts also did not show
major differences in their morphological characteristics.
Aspergillus nidulans strains from Makueni and Nyeri were
not different from each other morphologically. Aspergillus
wentii and A. candidus occurred singly from Bungoma
and Ugunja districts, respectively, and had no strains to
be compared with. The cultural, morphological and
microscopic characteristics of the various Aspergillus
spp. were recorded as shown in Table 2. The 10 different
Aspergillus spp. identified and characterized are shown in
Plate 2a - j. Plate 3a and b show microscopic
characteristics of A. flavus and A. parasiticus.
Identification of A. flavus is not an easy task due to its
similarities with A. parasiticus and A. nomius. However,
Odhiambo et al. 4383
the other Aspergillus spp. are distinctly different from
each other, and with the help of the descriptions and keys
by Klich (2002), it was possible to achieve a reliable
identification and discrimination of the various isolates of
Aspergillus species.
Distribution of the Aspergillus species and their
incidences in the five districts
The Aspergillus species, having been identified and
characterized to species level as shown in Table 2, were
grouped according to their species and district of origin
and data recorded as shown in Table 1. Aspergillus
flavus species was the most frequently occurring species
in almost all the districts apart from Moiben district which
had more A. versicolor than A. flavus. These results
(Table 1) are evidence that A. flavus is the major
contaminant of maize and soil across all the five districts
under this study. Statistically, these results further
showed that there was no significant difference (P =
0.0699) in the frequency of occurrence among the
various Aspergillus species found in all the five districts at
a 95% confidence limit. SAS version 9.0 was used in the
data analysis.
DISCUSSION
The high occurrence of Aspergillus spp. moulds in the
maize and soil samples in the various districts can be
attributed to factors such as warmth and humidity in the
LVB region (Ugunja), high relative humidity with low
temperatures in Nyeri, Bungoma and Moiben districts
leading to improper drying of the maize and high
temperatures with drier conditions in Makueni district
which predisposes maize to the moulds at pre-harvest
stage in the field and post-harvest stage in storage
(Okoth et al., 2012). The fungus forms sclerotia that
allows for saprophytic survival for extended periods in the
soil, maize residue and maize-cobs (Wagacha and
Muthomi, 2008). The propagules in the soil and crop
debris act as the primary source of contamination,
infecting maturing maize crops (Atehnkeng et al.,
2008b).
These results were also anticipated in the Lake Basin
Region (Ugunja), because of the relatively high
temperature and relative humidity which provided
optimum growth conditions for the Aspergillus spp.
(Anonymous, 2004). Higher number of Aspergillus spp.
was recorded in grain samples from the semi-arid
Makueni district than those from the humid regions in
Moiben, Nyeri, Bungoma and Ugunja. These results are
in agreement with the findings of Muthomi et al. (2012)
where higher Aspergillus spp. isolation frequencies were
recorded in grain samples from the semi-arid eastern
region than those from the humid North Rift regions.
4384 Afr. J. Microbiol. Res.
Table 2. Cultural, morphological and microscopic characterization of Aspergillus isolates.
Group
no.
Isolate
Colony colour on PDA
Colony
size (mm)
Conidiophore
Conidial
head
Shape of
Vesicles
Seriation
Conidial
shape
Isolate
name
Conidia
Reverse
G3
EM324
Olive green with whitish
margin
Yellowish with grey
margin
41.3±3.1
Short, finely roughened
wall and colourless
Radiate
Subclavate
Uniseriate
Spherical
A. flavus
G7
NM091
Olive green with dirty
white margin
Cream centre with
alternating grey and
cream periphery
35.3±5.1
Short, smooth walled and
colourless
Radiate
Subclavate
Uniseriate
Spherical
A. flavus
G8
NM083
Olive green with white
margin
Creamish
34.7±0.6
Slightly long, rough walled
and colourless
Radiate
Subclavate
Biseriate
Spherical
A. flavus
G9
EM244
Olive green with white
margin
Cream centre with
alternating grey and
cream rings
34.0±4.0
Short, finely roughed
walled and colourless
Radiate
Subclavate
Uniseriate
Spherical
A. flavus
G13
NM084
Olive green with cream
margin
Cream center with
alternative grey and
cream concentric rings
37.7±2.5
Short, smooth walled and
colourless
Radiate
Subclavate
Uniseriate
Spherical
A. flavus
G16
EM182
Olive green with whitish
margin
Creamish
32.3±3.2
Short, smooth walled and
colourless
Columnar
Subclavate
Uniseriate
Spherical
A. flavus
G17
BM071
Olive green with cream
margin
Cream center with
alternative grey and
cream concentric rings
36.7±2.3
Short, rough walled and
colourless
Columnar
Subclavate
Uniseriate
Spherical
A. flavus
G18
EM111
2
Olive green with whitish
margin
Cream with grey
margin
35.0±1.0
Short, smooth walled and
colourless
Radiate
Globose
Uniseriate
Spherical
A. flavus
G19
UM127
Olive green with whitish
margin
Cream with grey
margin
28.3±1.5
Slightly long, smooth
walled and colourless
Radiate
Subclavate
Uniseriate
Spherical
A. flavus
G21
BS203
Grey with a yellow at the
middle
Greenish centre with
creamish margin
17.0±2.7
Short, finely roughened
wall and colourless
Columnar
Subclavate
Biseriate
Spherical
A.
versicolor
G22
RS016
Creamish centre with
green to sulphur yellow
margins
Sulphur yellow
34.0±1.0
Short, finely roughened
wall and colourless
Radiate
Globose
Biseriate
Spherical
A.
versicolor
G31
RS024
Green with sulphur margin
Yellow
26.7±2.1
Long, smooth walled and
colourless
Radiate
Subclavate
Uniseriate
Spherical
A. glaucus
G32
BS023
Sulphur yellow centre with
alternating grey and
sulphur yellow concentric
rings
Sulphur yellow
26.0±2.0
Short, smooth walled and
colourless
Columnar
Subclavate
Biseriate
Spherical
A. wentii
Odhiambo et al. 4385
Table 2. Contd
G34
RS162
Grey centre with
yellowish margin
Yellow
32.7±1.2
Short, smooth walled and
colourless
Radiate
Globose
Biseriate
Spherical
A.
versicolor
G38
US098
Greyish blue with
white margin
Yellow
29.0±1.0
Slightly long, smooth
walled and colourless
Radiate
Globose
Biseriate
Spherical
A. sydowii
G41
RS013
Olive green with
cream margin
Yellow centre with
cream margin
36.0±2.0
Long, smooth walled and
colourless
Radiate
Globose
Uniseriate
Spherical
A. flavus
G49
UM082
Conifer green with
cream margin
Yellow centre with
cream margin
26.7±0.6
Slightly long, smooth
walled and colourless
Radiate
clavate
Uniseriate
Spherical
A.
parasiticu
s
G50
BM092
Grey centre with
green margin
Cream centre with grey
margin
41.3±3.1
Slightly long, smooth
walled and colourless
Radiate
Globose
Uniseriate
Spherical
A.
fumigatus
G58
US184
White
Deep yellow
15.7±1.5
Short, finely roughened
wall and colourless
Radiate
Globose
Uniseriate
Spherical
A.
candidus
G64
RM143
Bluish green with
white margin
Brown center with
alternating cream and
brown concentric rings
31.7±1.5
Short, finely roughened
wall and brownish
Radiate
Subclavate
Uniseriate
Spherical
A.
clavatus
G68
ES042
Green
Deep red
23.7±2.1
Short, smooth walled and
colourless
Columnar
Subclavate
Biseriate
Spherical
A.
nidulans
G71
BS116
RM023-
2
Olive green
Creamish yellow
36.0±2.0
Short, smooth walled and
colourless
Columnar
Subclavate
Uniseriate
Spherical
A. flavus
G72
EM211
Green centre with
alternating yellow
and green concentric
rings
Cream
29.0±1.7
Slightly long, smooth
walled and colourless
Columnar
Subclavate
Uniseriate
Spherical
A.
glaucus
E, Eastern (Makueni); R, Rift valley (Moiben); B, Bungoma; N, Nyeri; U, Ugunja; M, maize; S, soil; (01-32), sample number in each district; (1 -12), isolate number from each sample; (-2), purification
number.
Mechanical damage during and after harvest,
facilitates entry of the fungal spores either in
maize cobs or grains (Pitt, 2000). This could
explain why very high quantities of the Aspergillus
spp. were isolated from the maize samples
because some of them had damaged grains that
might have predisposed the grains to the fungus
infection.
These results resonates with the study of Muthomi
et al. (2012) in which the specific Aspergillus spp.
isolated from whole and unprocessed maize grain
and soil from North Rift and Eastern regions were:
A. flavus, A. niger, A. fumigatus, A. versicolor, A.
terreus, A. clavatus and A. ochraceus. The most
frequently isolated were A. flavus and A. niger,
while A. clavatus was the least frequently isolated
Aspergillus species and was mainly isolated in
samples from the humid North Rift region.
Similarly, in this study, A. flavus had the highest
incidences in all the districts apart from Moiben.
Moiben (North Rift) had the highest incidence of
A. clavatus and this support the findings of
Muthomi et al. (2012) where A. clavatus was
predominantly isolated from the humid North rift
4386 Afr. J. Microbiol. Res.
Plate 2. Morphological and cultural characteristics of
the 10 Aspergillus species growing on PDA media
after seven days of growth at 28°C. a (i) A. flavus
NM091 (surface); a (ii) A. flavus NM091 (reverse); b
(i) A. parasiticus UM082 (surface); b (ii) A. parasiticus
UM082 (reverse).
Plate 2 Contd. Morphological and cultural
characteristics of the 10 Aspergillus species
growing on PDA media after seven days of growth
at 28°C. c (i) A.versicolor BS203 (surface) ; c
(ii) A. versicolor BS203 (reverse); d (i) A. clavatus
RM143 (surface) d (ii) A. clavatus RM143
(reverse); e (i) A. sydowii US098 (surface) 2e (ii) A.
sydowii US098 (reverse).
Plate 2 Contd. Morphological and cultural
characteristics of the 10 A. species growing on PDA
media after seven days of growth at 28°C. f (i) A.
fumigatus BM092 (surface) ; f (ii) A. fumigatus
BM092 (reverse); g (i) A. glaucus RS024 (surface); g
(ii) A. glaucus RS024 (reverse); h (i) A. nidulans
ES042 (surface); h (ii) A. nidulans ES042 (reverse).
Plate 2 Contd. Morphological and cultural
characteristics of the 10 Aspergillus species
growing on PDA media after seven days of growth
at 28
o
C. i (i) A. candindus US184 (surface) ; i
(ii) A. candindus US184 (reverse); j (i) A. wentii
BM023 (surface); j (ii) A. wentii BM023 (reverse).
a (i)
a (ii)
b
(i)
b
(
i
i)
c (i) c (ii)
d (i) d (ii)
e (i) e (ii)
f (i)
f (ii)
g (i)
g (ii)
h (i)
h (ii)
i (i) i (ii)
j (i) j (ii)
Odhiambo et al. 4387
Plate 3. Microscopic characteristics of A. flavus (EM244) and A. parasiticus (UM082). a: Uniseriate conidial
heads with subclavate vesicle of A. flavus strain EM244. b: Radiate conidial head shape attached to a long
stipe of A. parasiticus strain UM082.
region.
The pervasive nature of Aspergillus spp., their high
ability to colonize diverse substrates and lack of effective
control measures (Souza et al., 2005) could have
contributed to their high occurrences in maize and soil
from the five districts. Aspergillus spp. are more
commonly associated with cereals during drying and
storage. A. flavus and A. parasiticus have a particular
affinity for cereals and can be recognized by yellow-green
or grey green colour on maize kernels in the field and in
storage (Varga et al., 2011). This study found out that A.
flavus was the most prevalent Aspergillus spp. in all the
districts except for Moiben district where A. versicolor
was the predominant species.
These results are in line with the findings of Okoth et al.
(2012) and Muthomi et al. (2012) who reported that A.
flavus was the most dominant Aspergillus spp. in
Makueni and Nandi counties and also in Eastern region
and North Rift region, respectively. Grain samples
collected from farmers in the semi-arid eastern region
(Makueni) during the short rainy seasons had higher
incidences of A. flavus, of up to 14% as compared to
grain harvested during the long rainy seasons (Muthomi
et al., 2012). This statement supports the high levels of A.
flavus in Makueni district since sampling in this study was
done immediately after the short rainy season maize
harvest in all the districts. Additionally, variations in fields
cropping history, cultivation practices, sowing dates, seed
varieties planted and/or soil types can differ greatly in
aflatoxigenic fungi and aflatoxin contamination (Munkvold
et al., 2009). These dynamics may clarify the variances in
the incidences and type of the Aspergillus spp. isolated
from the five districts. Similarly, damaged maize kernels
also favours the growth of A. flavus as compared to any
other Aspergillus species (Pitt, 2000). This could be the
reason why the most frequently isolated Aspergillus
species in the five districts was A. flavus.
Identification of A. flavus is not an easy task due to its
similarities with A. parasiticus and A. nomius. However,
the other Aspergillus spp. are distinctly different from
each other, and with the help of the descriptions and keys
by Klich (2002), it was possible to achieve a reliable
identification and discrimination of the various Aspergillus
spp. isolates. The results of this study showed that even
“good maize perceived to be safe for human consum-
ption is highly contaminated with moulds of the genus
Aspergillus with A. flavus being the major contaminant.
All the “good” maize samples from Makueni, Nyeri,
Moiben, Ugunja and Bungoma South districts were found
to have high levels of A. flavus contamination as
compared to their respective soil samples. It is evident
that residents of these districts consume the “good”
maize oblivious of the health risks that they and their
animals are exposed to.
ACKNOWLEDGEMENTS
This work was supported by the National Council of
Science and Technology, Kenya. The authors would like
to thank the Department of Horticulture of Jomo Kenyatta
University of Agriculture and Technology for providing the
materials necessary for accomplishing this work, and also
Biological Sciences Department of Egerton University for
the cooperation accorded.
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