CZECH MYCOL.

63(1): 93107, 2011

Mycobiota and aflatoxins associated with imported

rice grains stored in Uganda
1

HANINGTON K. TALIGOOLA , MADY A. ISMAIL

1,2*

, SAMSON K. CHEBON

1, 3

Department of Botany, Faculty of Science, Makerere University, P.O. Box 7062, Uganda;
[email protected]
2
Department of Botany, Faculty of Science, Assiut University, P.O. Box 71516, Assiut, Egypt;
[email protected]
3
Moi High School-Kabarak, P.O. Box, 7049, Nakuru, Kenya
*
corresponding author

Taligoola H. K., Ismail M. A., Chebon S. K. (2011): Mycobiota and aflatoxins associated with imported rice grains stored in Uganda. Czech Mycol. 63(1): 93107.
Milled rice grains imported into Uganda from Pakistan were investigated for natural contamination
by fungi and aflatoxins. The direct plating method using five isolation media was used to enumerate and
isolate the fungi during a 270-day storage period. Fungi were isolated and identified to species level and
the percentage contamination levels were calculated. A total of 35 species belonging to 16 genera were
recorded. The broadest species spectrum were found in the genera Aspergillus, Penicillium, Eurotium
and Fusarium, which were represented by 11, 7, 4, and 3 species, respectively. Throughout the storage
period, xerophilic fungi including Aspergillus candidus, Eurotium amstelodami and E. chevalieri were
predominantly isolated. Species of the genus Penicillium (particularly P. pinophilum) and its
teleomorph Talaromyces ranked second in predominance, while Aspergillus flavus, Fusarium spp. and
other field fungi occurred only sporadically. Aflatoxins were recorded in rice samples during most storage periods with one sample recording 2050 ppb. The moisture content increased in rice grains attaining
values of over 14 % from the 180th day of storage onwards. A positive correlation was observed between
moisture content and incidence of xerophiles, including A. candidus and E. amstelodami.
Key words: rice grain, xerophilic fungi, nephrotoxigenic penicillia, Fusarium, aflatoxins.

Taligoola H. K., Ismail M. A., Chebon S. K. (2011): Mykobiota a aflatoxiny v ri importovan do Ugandy. Czech Mycol. 63(1): 93107.
Mlet re importovan z Pakistnu do Ugandy byla studovna s ohledem na vskyt hub a aflatoxin. Bylo zaznamenno 35 druh z 16 rod. Druhov nejpoetnj byly rody Aspergillus, Penicillium,
Eurotium a Fusarium (11, 7, 4, 3 druhy). Aflatoxiny byly zaznamenny ve vlhkch obdobch. Byla pozorovna pozitivn korelace mezi vlhkost a vskytem xerofilnch druh, vetn A. candidus a E.
amstelodami.

INTRODUCTION
Developing countries including those in sub-Saharan Africa, which includes
Uganda, have considerable import and export trade in agricultural commodities.
The imports from the Asiatic countries, including Pakistan, are cereal grains, par93

CZECH MYCOL. 63(1): 93107, 2011

ticularly rice (Oryza sativa). These agricultural products are transported from
farm to collection centre, from inland to port, from port of the exporting country
to that of the importing one, and also from port to areas of consumption in the
country of import. In each of these stages, the commodities have to be stored, the
period of storage depending on such factors as distance involved, mode and speed
of transport, customs clearance, weather conditions like monsoons or the ElNio, and conditions of storage at the various transit points as well as during
transportation (Bhat 1988, Milton & Pawsey 1988).
The time lag involved during transportation particularly includes long journeys
overseas lasting several weeks to months during which time the moisture content
of the otherwise sufficiently dried grain increases considerably to levels ideal for
growth of xerophilic fungi. Further, the metabolic moisture and heat resulting
from respiration of both the fungi and rice grain may become ideal for the growth
of less xerotolerant fungi. This triggers a chain of reaction that ultimately results
in massive colonisation of the grain bulk by various types of fungi (Magan et al.
1984, Bhat 1988, Makun et al. 2007, Reddy et al. 2009, Taligoola et al. 2010).
The study of fungi in cereal grains which have undergone transport is significant due to the presence of mycotoxins and the relationship of these toxins to
mycotoxicoses of domestic animals as well as humans, when they consume contaminated grains as food. Several studies on fungal contamination of cereals in
storage and those having undergone prolonged transportation have been made
worldwide (Wallace & Sinha 1975, Hesseltine 1982, Sidik & Pedersen 1986, Bhat
1988, Milton & Pawsey 1988, Mossel 1988, Wareing 1997, Makun et al. 2007, Reddy
et al. 2009). In Uganda, several studies on fungi and mycotoxins, particularly aflatoxin contamination, have been focused on local staple foods including rice, corn,
soybean and peanuts (Lopez & Crawford 1967; Apert et al. 1971; Sebunya &
Yourtee 1990; Ismail et al. 2003; Taligoola et al. 2004, 2010). However, studies on
fungal and aflatoxin contamination of imported foods which are a significant part
of diets in Uganda, particularly cereals including rice grain, are yet undocumented.
In the present study, milled rice grains imported into Uganda from Pakistan
were analysed for fungal contamination under storage conditions. Aflatoxin levels
in the rice grain were also monitored.

MATERIALS AND METHODS

R i c e s a m p l e s. A set of two nylon bags of milled rice grains (each weighing
50 kg) imported from Pakistan into Uganda were bought from markets in the capital city Kampala. The date of packaging as indicated on the bags was 22nd April
1998. The rice was stored in a room at a temperature of 25 2 C for a period of
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TALIGOOLA H. K., ISMAIL M. A., CHEBON S. K.: MYCOBIOTA AND AFLATOXINS IN RICE GRAINS

270 days between Nov. 1998 and Aug. 1999. During this time the rice grains were
periodically sampled 8 times: on the 1st day upon arrival from the market, and then
on the 45th, 90th, 135th, 180th, 210th, 240th and 270th day, to analyse fungal and aflatoxin contamination, as well as the moisture content of the rice grains.
S a m p l i n g a n d s u r f a c e s t e r i l i s a t i o n. After every storage period,
a representative sub-sample of 2 kg, was withdrawn for analysis from the top,
middle and bottom of each of the two bags using a nobble trier into a clean conical
flask and carefully mixed. Two further working sub-samples of 500 g each from
these sub-samples, to be used for determination of fungal contamination, were
obtained and first surface-sterilised using 70 % ethanol pre-rinse prior to a 0.8 %
chlorine treatment for 2 minutes (Andrews 1996). Excess disinfectant was
drained off from the grains, followed by rinsing the grains three times with sterilised tap water. Excess water on the grains was mopped using sterile filter paper.
The grains were then plated on suitable isolation media at a plating rate of 10 rice
grains per plate in all the media used.
I s o l a t i o n o f f u n g i. The direct plating method was used to determine seedborne fungi. A general purpose enumeration medium and four selective agar media were used to detect and isolate the following groups of fungal species: (i) fungi
in general, using dichloran rose-bengal chloramphenicol agar, DRBC (King et al.
1979), modified by Pitt & Hocking (1985); (ii) xerophilic fungi, using dichloran
18 % glycerol agar, DG18 (Pitt & Hocking 1980); (iii) aflatoxigenic Aspergillus
spp., using Aspergillus flavus/parasiticus agar, AFPA (Pitt et al. 1983); (iv)
nephrotoxigenic Penicillium spp., using pentachloronitrobenzene rose-bengal
yeast extract sucrose agar, PRYES (Frisvad 1983); and (v) Fusarium spp., using
pentachloronitrobenzene-potato sucrose agar, PCNB-PSA (Nash & Synder 1962,
Booth 1971). Fifty grains per sub-sample were plated on DRBC and PCNB-PSA,
while 100 grains were plated for the other remaining selective media. All plates
were incubated under natural conditions of light and darkness at 25 2 C for 78
days except for those containing AFPA, which were incubated at 30 C for 4248
hrs, DG18, which was incubated for 1420 days, and PCNB-PSA, which was incubated under continuous light from an ordinary 40-watt, fluorescent tube for 78
days. The occurrence of fungi for each sub-sample of the two bags was calculated
per total number of rice grains plated on each medium and the resulted incidences
were used to calculate the mean occurrence of fungi (as a percentage) on grains
from the two bags, since the second bag was a duplicate.
I d e n t i f i c a t i o n o f f u n g i. Fungi were identified on the basis of their macroscopic and microscopic features using the keys of Raper & Fennell (1965),
Booth (1971), Ellis (1971), Pitt (1979), Moubasher (1993), Pitt & Hocking (1997),
Leslie & Summerell (2006). Six identification media were used including Czapek
yeast extract agar supplemented with 20 % sucrose (CY20S Thom & Raper 1941)
for the identification of Eurotium spp., Czapek yeast extract agar (CYA Pitt
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CZECH MYCOL. 63(1): 93107, 2011

1973), malt extract agar (MEA Blakeslee 1915) and 25 % glycerol nitrate agar
(G25N Pitt 1973) for identification of Penicillium spp., potato dextrose agar
(PDA Booth 1971, Leslie & Summerell 2006) for identification of Fusarium spp.,
and malt extract agar (MEA Pitt & Hocking 1997) for identification of yeasts.
E x t r a c t i o n a n d e s t i m a t i o n o f a f l a t o x i n s. Aflatoxin was determined between the 135th and 270th day of storage such that 10 samples of rice were
screened. A semi-quantitative test for the determination of total aflatoxins in the
rice grains was carried out whereby a commercial immunological test kit,
aflascan (from Rhne Diagnostics and Technologies Ltd., Glasgow, U.K.) was
used. A comparator card, a component of the aflascan, was used in the determination of the levels of aflatoxins in g/kg (ppb). The total aflatoxin level (aflatoxin
B1, B2, G1 and G2) in the grain was determined according to the procedure outlined
in the aflascan. Samples to be analysed were at least 1 kg of rice grain, aseptically
and thoroughly ground to fine powder, from which a 50 g sub-sample was withdrawn for the aflatoxin assay. A filtered 50100 ml extract consisting of a blend of
4 g of sodium chloride and 250 ml of 60 % high perfomance liquid chromatography
(HPLC) analytical methanol was collected, from which 10 ml was pumped
through an antibody-containing immunoaffinity column at 23 ml/minute, a component of the aflascan, using a glass syringe, also a component of the aflascan.
Residues in the column were washed off by pumping 10 ml of distilled water three
times at 5 ml/minute. Any aflatoxins bound onto the antibodies in the
immunoaffinity column were extracted during elution, a process that involved
pumping HPLC analytical grade methanol (eluant) through the column at a maximum flow rate of 1 drop per second. The eluant, containing aflatoxins, was collected in a glass tube put below the column, into which 1.0 ml of distilled water
and chloroform was later added. Upon shaking the liquid mixture, two separate
layers resulted, chloroform being at the bottom. A Florisil tip, a component of the
aflascan, was attached to the bottom of the glass syringe and a carefully pipetted
chloroform layer was pumped slowly through it. To estimate the aflatoxin level,
the Florisil tip was placed under an ultra-violet light box at 360 nm. Comparison of
the intensity of any blue and/or green fluorescence on the Florisil tip with the fluorescent comparator card provided a semi-quantitative estimation of the total aflatoxin in ppb of the original sample. For 10 ml filtrate, the comparator card was
viewed on a scale of 0 ppb, 10 ppb, 20 ppb, 50 ppb and 100 ppb.
D e t e r m i n a t i o n o f m o i s t u r e c o n t e n t. The moisture content of each
rice grain sample was periodically detemined during the 270-day storage period
by finding the loss in weight of the rice grain upon heating for a 24-hour period in
an oven at 110 C and expressing it as a percentage of the fresh weight (Gariboldi
1973). Triplicate sub-samples of 50 g each per each rice sample from each bag
were used. The mean moisture content value of rice from the two bags at a certain
period of storage was then calculated.
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TALIGOOLA H. K., ISMAIL M. A., CHEBON S. K.: MYCOBIOTA AND AFLATOXINS IN RICE GRAINS

S t a t i s t i c a l a n a l y s e s. Data were subjected to the analysis of variance

(ANOVA), t-test, and F-test. Statements of significance are based on P 0.05
(Erricker 1979). Correlation was used to determine the relationship between the
various variables.

RESULTS
Occurrence of various types of fungi on DRBC
A total of 28 species belonging to 15 genera were isolated from Pakistani rice
grains in storage as determined on DRBC medium. These fungi contaminated
72.5 % of the grains. The dominant fungi causing contamination were species of
Eurotium, Aspergillus and Penicillium (Tab. 1). Eurotium species including
E. amstelodami and E. chevalieri were consistently isolated at relatively higher
incidence levels than E. repens and E. rubrum. E. amstelodami and E. chevalieri
occurred on the rice grains throughout the storage period, but E. repens and
E. rubrum occurred sporadically.
Among aspergilli, A. candidus was consistently isolated during the storage period except at day 135 of storage. A. fumigatus recorded the highest incidence
level (18 %) among aspergilli at the end of storage period (Tab. 1). Among
penicillia, P. pinophilum had the highest number of isolates while P. islandicum,
P. variabile and P. oxalicum contaminated only very few rice grains. Talaromyces
occurred sporadically, occurring in 36 % of the grains on day 180. Other species
coming after the former ones in incidence were A. niger, Chaetomium globosum
and Rhodotorula mucilaginosa. The remaining species had low incidence levels.
These included Aspergillus tamarii, A. terreus, Paecilomyces variotii, Phoma
spp., Pseudogliomastix protea (teleomorph: Wallrothiella subiculosa), Scopulariopsis brumptii, S. candida, and Penicillium spp., apart from those mentioned
in Tab. 1.
Occurrence of various xerophilic fungi on DG18
Milled imported rice grains in storage contained 10 species from 6 genera of
xerophilic fungi as determined on dichloran 18 % glycerol agar. Several
xerotolerant fungi were also isolated. Among the xerophiles, Eurotium
amstelodami, E. chevalieri, E. rubrum and Aspergillus candidus were isolated
in high incidences (Tab. 2). Other species such as A. wentii, Chrysosporium
farinicola and Polypaecilum pisce occurred occasionally and at comparatively
lower incidence levels than the Eurotium spp. Eurotium amstelodami and
E. chevalieri were encountered throughout the storage period, occurring at incidence levels ranging from 30.052.5 % and 8.027.5 %, respectively.
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CZECH MYCOL. 63(1): 93107, 2011

Tab. 1. Percentage occurrence of fungi (mean number of colonies calculated per 100 grains tested
from the two bags) on milled Pakistani rice during a 270-day storage on dichloran rose bengal
chloramphenicol agar medium (DRBC).
Storage period (days)
Fungi*

45

90

135

180

210

240

Acremonium strictum

Aspergillus candidus

14

11

A. flavus

A. fumigatus

18

A. niger

A. oryzae

A. sydowii

Chaetomium globosum

Cladosporium sphaerospermum

Cochliobolus bicolor

Eurotium amstelodami

40

26

22

32

44

38

33

35

E. chevalieri

270

24

10

14

15

14

17

12

E. repens

11

E. rubrum

11

Fusarium graminearum

Monascus ruber

Penicillium islandicum

P. oxalicum

P. pinophilum

19

P. variabile

Polypaecilum pisce

Talaromyces spp.

19

36

Sterile mycelia (white)

Rhodotorula mucilaginosa

Other yeasts

*Fungi which occurred at very low incidence levels are not included in the table.

During storage, a change in species diversity among the xerophilic fungi was
recorded. Six species were encountered during the initial 135 days of storage, but
all the 10 species occurred between 210 and 270 days of storage on the grains
(Tab. 2). Similarly, marginal xerophiles including Aspergillus fumigatus and
A. penicillioides also occurred during the second half of the storage period. The
incidence of xerophiles, particularly A. candidus and E. amstelodami, increased
during storage so that the highest incidence levels were recorded during the second half of the storage period (Tab. 2).
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TALIGOOLA H. K., ISMAIL M. A., CHEBON S. K.: MYCOBIOTA AND AFLATOXINS IN RICE GRAINS
Tab. 2. Percentage occurrence of fungi (mean number of colonies; calculated per 200 grains tested
from the two bags) on milled Pakistani rice during a 270-day storage on dichloran 18 % glycerol agar
medium (DG 18).
Storage period (days)
Fungi*

45

90

135

180

210

240

270
22.5

Xerophilic fungi
Aspergillus candidus

4.5

3.5

2.5

A. wentii

Chrysosporium farinicola

0.5

3.5

Eurotium amstelodami

35

30

50.5

42

45

47.5

41

E. chevalieri

25

16.5

15

15

14.5

27.5

16

6.5

E. repens

52.5
8
0.5

E. rubrum

4.5

4.5

4.5

Monascus ruber

0.5

Polypaecilum pisce

1.5

2.5

Wallemia sebi

Aspergillus flavus

1.5

A. fumigatus

A. niger

0.5

0.5

0.5

2.5

A. penicillioides

1.5

Cladosporium sphaerospermum

0.5

Penicillium pinophilum

0.5

11.5

2.5

0.5

0.5

Penicillium spp.

0.5

Rhodotorula mucilaginosa

7.5

Other yeasts

0.5

0.5

0.5

Xerotolerant fungi

36.5

*Fungi which occurred at very low incidence levels are not included in the table.

The increasing incidence levels of xerophiles including A. candidus and

E. amstelodami on the imported Pakistani rice were positively correlated with increasing moisture content during storage (Tabs. 1, 2).
Occurrence of aflatoxigenic Aspergillus species on AFPA
Rice grains in storage were scarcely contaminated by aflatoxigenic Aspergillus
spp. Only A. flavus was recorded on the rice grain and it occurred sporadically,
only twice out of the 8 occasions of plating. Among the other seven Aspergillus
species found to contaminate rice, A. candidus and A. niger were the most frequent, occurring on 7 and 4 out of 8 occasions, respectively. The remaining species each occurred on only 2 or 1 occasion out of 8. However, all species of
Aspergillus except A. candidus had incidence levels of less than 10 %. The highest
incidence of A. candidus was 32 % (Tab. 3).
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CZECH MYCOL. 63(1): 93107, 2011

Tab. 3. Percentage occurrence of fungi on milled Pakistani rice during a 270-day storage period (calculated as mean number of colonies per total number of grains tested from two bags) on Aspergillus
flavus/parasiticus agar (AFPA, 200 grains), pentachloronitrobenzene yeast extract sucrose agar
(PRYES, 200 grains) and pentachloronitrobenzene potato sucrose agar (PCNB PSA, 100 grains).
Storage period (days)
0

45

90

135

180

210

240

270

2.5

A. candidus

0.5

12.5

32

11

2.5

A. fumigatus

A. niger

0.5

A. ochraceus

0.5

A. oryzae

0.5

1.5

A. penicillioides

0.5

A. sydowii

1.5

6.5

Fungi*
On AFPA medium
A. flavus
Other Aspergillus spp.

On PRYES medium
Penicillium chrysogenum

0.5

0.5

P. fellutanum

0.5

P. jensenii

0.5

P. oxalicum

0.5

0.5

P. pinophilum

1.5

0.5

1.5

2.5

Penicillium sp.

0.5

Talaromyces spp.

4.5

4.5

2.5

0.5

Fusarium graminearum

F. oxysporum

F. solani

13.2
0.1

13.6
0.09

13.53
0.03

13.97
0.12

14.03
0.13

14.23
0.12

14.27
0.06

14.48
0.12

On PCNBPSA medium

Moisture content (%)

*Fungi which occurred at very low incidence levels are not included in the table.

Occurrence of Penicillium species on PRYES

Rice grains were found to be contaminated by 5 Penicillium species and one
unidentified fungus on the nephrotoxigenic Penicillium agar medium, pentachloronitrobenzene rose-bengal yeast extract sucrose (PRYES). Several Talaromyces species were also isolated. Well-known nephrotoxigenic Penicillium spp. including P. aurantiogriseum and P. viridicatum were not among the isolated species. However, the incidence levels of Penicillium spp. on the imported rice were
low with only 4.0 % being the highest level recorded in one sample (Tab. 3). Among
the Penicillium species occurring on Pakistani rice grain in storage, P. pinophilum
was predominant. Penicillium chrysogenum and P. oxalicum were less frequent,
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TALIGOOLA H. K., ISMAIL M. A., CHEBON S. K.: MYCOBIOTA AND AFLATOXINS IN RICE GRAINS

while P. fellutanum and P. jensenii were rare (Tab. 3). Talaromyces spp.
(teleomorphs of penicillia) scored 4.5 % as the highest incidence level.
Occurrence of Fusarium species on PCNB-PSA
Milled rice grains in storage were found contaminated with only 3 species of
Fusarium on pentachloronitrobenzene potato sucrose agar (PCNB-PSA). These
Fusarium spp. were each found only in 1 out of 8 occasions (Tab. 3). The percentage of rice grains contaminated by these Fusarium species was equally low;
F. solani had the highest occurrence level of only 8 %, while F. oxysporum and
F. graminearum contaminated 4 % and 2 % of the grains, respectively. However,
despite the increase in moisture content of the rice grains from 13.2 to 14.48 %, the
incidence levels of fusaria did not increase during storage because this range of
moisture contents is not ideal for the growth of Fusarium spp. on rice.
Occurrence of aflatoxins and their relationship between incidence of aflatoxigenic aspergilli, and the moisture content of rice grains.
Eight samples out of the 10 milled Pakistani rice samples screened for
aflatoxins were found to be contaminated. Four levels of contamination were recorded: 0 ppb, 010 ppb, 1020 ppb and 2050 ppb (Tab. 4). The aflatoxin level of
2050 ppb recorded is above the maximum level internationally allowed in foods
(20 ppb). It was noted that all the positive samples for aflatoxins had moisture
contents above the recommended level of 14.0 % for safe storage of milled rice
grains. Of the important toxigenic fungi only A. flavus was found to contaminate
the rice grains. It occurred sporadically, only at the start of the study and on the
240th day on each of the two samples of rice studied (Tab. 4).
Tab. 4. Occurrence of aflatoxins, aflatoxigenic aspergilli on Aspergillus flavus/parasiticus agar (AFPA)
and moisture content of milled Pakistani rice during a storage period of 270 days.
Milled Paki- Analysis
stani rice
Sample 1

Aflatoxin level (ppb)*

Aflatoxigenic aspergilla**

Sample 2

Storage period ( days)

135

180

210

240

270

010

010

010

Moisture content (%)

13.90.1

13.90.1

14.20.2

140.5

14.30.0

Aflatoxin level (ppb)*

1020

2050

010

010

1020

14.10.1

14.150.15

14.30.1

14.350.14

14.60.2

Aflatoxigenic aspergilli**
Moisture content (%)

*ppb: Parts per billion (micrograms per kilogram)

**calculated as percentage occurrence of isolates (out of 100 grains tested at the end of each storage
period).

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DISCUSSION
Pakistani rice, being an imported commodity from Pakistan, is subjected to diverse environmental conditions during its transport oversea whereby the grains
absorb moisture during its long distance transportation to Uganda. Even before
its transportation, the rice may have been subjected to repeated handling between
its harvesting and its packaging for export. The date of packaging as indicated on
the bags was 22nd April 1998; thus its harvesting and transportation to Uganda was
probably around the 1997/98 El-Nio wet weather phenomena. Rice grains imported into the country from Pakistan, Vietnam and India around this period were
reported to have been unfit for human consumption (Mukanga 1999).
Milled Pakistani rice was predominantly contaminated by storage fungi including species of Eurotium, Aspergillus and Penicillium as determined on DRBC.
Cereal grains which have undergone transport have been shown to be commonly
contaminated with these fungi (Wallace & Sinha 1975, Milton & Pawsey 1988,
Taligoola et al. 2010).
The heavy contamination of the imported Pakistani rice with xerophiles right
from the start of the study in November 1998 suggests that the rice was already
contaminated upon its arrival in Uganda. The period of only 7 months which
elapsed between its packaging in Pakistan (April 1998 as was indicated on the
bags) and the start of the study in November 1998 strongly supports this suggestion. It has been demonstrated that it takes 12 months before extreme xerophilic
fungi including Eurotium spp. appear in cereal grains including rice and corn
with an original moisture contents of 13.5 % (Sauer & Christensen 1968, Sidik &
Pedersen 1986). Respiration by the extreme xerophiles on the grains and by the
grain itself produces metabolic water which increases the grains moisture. This
subsequently enhances growth of various fungi on the grains (Harris & Lindblad
1978, Bhat 1988, Reddy et al. 2009).
Under storage, in absence of ventilation of the vessels (containers), cereal
grains which have undergone shipment have been found to create conditions
ideal for growth of extreme xerophiles including A. restrictus and Eurotium spp.
(Bhat 1988). An ecological succession occurs to result in subsequent contamination of the grains by less xerophilic and then xerotolerant fungi including yeasts,
A. candidus, other Aspergillus spp. and Penicillium spp. Most of these fungi are
involved in grain heating during storage (Christensen 1987, Sauer 1988). Shipment
of food-aid grain and from large bag stacks of maize stored in sub-Saharan Africa
have been found to be predominantly contaminated by fungi including
Aspergillus candidus, A. fumigatus, A. flavus, and Thermoascus aurantiacus
(Wareing 1997).
On AFPA, the aflatoxigenic species A. flavus was recorded at incidence levels
of only 2.5 % at the start of the storage period, and 2 % on the 240th day. Therefore,
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Pakistani rice grains had few grains and samples contaminated by A. flavus. This
sparse contamination may be attributed to the moisture contents of the rice
grains, which were found unsuitable for the growth of A. flavus. The highest moisture content of the various samples of Pakistani rice grains during the entire period of storage was 14.48 0.12 %. However, A. flavus has been found to require
a lower limit of moisture content of 18.0 % for its growth (Christensen 1987; Sauer
1988). Aspergillus flavus as the main aflatoxigenic Aspergillus species has similarly been found in various cereal grains including rice from Uganda (Taligoola et
al. 2004, 2010), Nigeria (Makun et al. 2007), Thailand (Lapmak et al. 2009), and India (Reddy et al. 2009), corn from Uganda (Ismail et al. 2003) and Burundi
(Munimbazi & Bullerman 1996), sorghum and barley from Ethiopia (Abate &
Gashe 1985), and in cereal grains, particularly maize, which have undergone
transport in tropical countries including sub-Saharan Africa (Milton and Pawsey
1988, Wareing 1997).
The general scarcity of Penicillum spp. isolated on PRYES from Pakistani rice
grains may also be attributed to their comparatively low moisture contents during
storage, whereby the highest moisture contents recorded was 14.48 0.12 %. Most
Penicillium spp., however, require a minimum moisture of 16.519.0 % for optimal growth in cereal grains (Christensen 1987).
Among Penicillium species isolated, P. pinophilum and P. chrysogenum have
commonly been reported to occur in food grains which have undergone transport
including milled rice (Tonon et al. 1997, Taligoola et al. 2010) and soybean
(Bothast et al. 1979). Penicillium chrysogenum has also been found to occur in
barley (Frisvad 1983). Penicillium pinophilum has been reported as an active
agent of biodeterioration (Pitt 1979) and has been isolated from maize and peanuts (Pitt et al. 1994, Pitt & Hocking 1997).
Fusarium species, being field fungi, have been found to grow at high grain
moisture contents of 2025 % (Bullerman 1979). However, since 14.48 0.12 % was
the highest moisture content recorded in Pakistani rice grains under storage,
growth of Fusarium spp. was thus inhibited and only three species were recorded on PCNB-PSA agar. However, increase in moisture content and storage period were found to cause an increase in incidence of Fusarium spp. on paddy
grains from Egypt, whereby moisture contents of 11.5 %, 17 %, 22.5 % and 28 %
were found in two studies (Abdel-Hafez et al. 1992, Mazen et al. 1993), and on sorghum from Nigeria (Elegbede et al. 1982).
The presence of Fusarium spp. on rice grains suggests a potential for
mycotoxin contamination. Trichothecenes, moniliformin and zearalenone have
been found to be produced by F. oxysporum. F. graminearum is also known to
produce trichothecenes, whose presence in foods is of human health concern
(Bullerman 1979, Abbas et al. 1989).

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Data on the relationship between incidence of aflatoxigenic Aspergillus spp.

(A. flavus), aflatoxins on the various samples of Pakistani rice grains and their respective moisture contents show that while an increase in moisture content was
recorded, a consistent increase in the incidence of A. flavus and aflatoxins was
not observed. The highest moisture content recorded for the two samples of rice
grains during the entire period of storage was 14.6 %. However, this was considerably below 18.0 %, which is the minimum moisture content for A. flavus, the main
aflatoxigenic species, to grow in cereal grains (Christensen 1987, Sauer 1988).
However, it was noted that all positive samples for aflatoxins had moisture contents above the recommended level for safe storage (14 %) (Christensen &
Kaufmann 1965, Bencini & Walston 1991, Taligoola et al. 2010).

CONCLUSION
The current study revealed that milled rice grain imported from Pakistan into
Uganda recorded fungal contamination predominantly by xerophilic fungi including
Eurotium spp. and Aspergillus candidus throughout a 270-day storage period,
while field fungi including Fusarium spp. occurred scarcely. Other storage fungi
including aflatoxigenic Aspergillus flavus, Penicillium spp. and Talaromyces
spp. were isolated only sporadically. The presence of these fungi, some of which
are toxigenic, might enabe mycotoxin contamination of the rice. Aflatoxin incidence on the rice grains did not record any consistent increase, but the majority
of the samples (80 %) were found contaminated with one sample recording 2050
ppb, which is above the maximum limit of 20 ppb internationally allowed in foods.
Uganda abides by this limit. From the current data, it is apparent that the presence
of fungi and aflatoxins are not due to storage and were most likely present before
the start of the study, i.e. at the source. Moisture contents of the rice grains increased significantly during storage attaining values of over 14 %, which is the recommended safe storage level for milled rice, as from the 180th day of storage onwards. A positive correlation was established between increase of moisture content of the rice grains and their contamination level by xerophilic fungi, particularly A. candidus and E. amstelodami, during storage.

ACKNOWLEDGEMENTS
The authors are deeply indebted to Prof. BukenyaZiraba, Head of the Botany
Department, Makerere University, Kampala for the facilities he provided during
this research. Our gratitude also goes to Managing Director, Uganda Bureau of
Standards for the aflatoxin screening facilities he provided. Grateful acknowledg104

TALIGOOLA H. K., ISMAIL M. A., CHEBON S. K.: MYCOBIOTA AND AFLATOXINS IN RICE GRAINS

ment is due to the Egyptian Fund for Technical Cooperation with Africa for sponsoring Prof. Ismail at Makerere University, giving him the opportunity to act as
a supervisor of Mr. S. K. Chebon.

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