Journal of Food Safety ISSN 1745-4565
INCIDENCE, PHYLOGENY AND MYCOTOXIGENIC POTENTIALS
OF FUNGI ISOLATED FROM RICE IN NIGER STATE, NIGERIA
jfs_305
334..349
HUSSAINI ANTHONY MAKUN1,2,4, MICHAEL FRANCIS DUTTON2, PATRICK BERKA NJOBEH2,
JUDITH ZANELE PHOKU2 and CLARENCE SUH YAH3
1
Department of Biochemistry, Federal University of Technology, P.M.B 65, Minna 920001, Niger State, Nigeria
Food, Environment and Health Research Group, Faculty of Health Science, University of Johannesburg, Doornfontein Campus, Gauteng, South Africa
3
Toxicology and Biochemistry Department, National Institute for Occupational Health (NIOH), 25 Hospital Road, Constitution Hill, Johannesburg, South
Africa
2
4
Corresponding author. TEL:
+2348035882233; FAX: +23466224482;
EMAIL: hussainimakun@yahoo.com
Accepted for Publication January 10, 2011
doi:10.1111/j.1745-4565.2011.00305.x
ABSTRACT
The study reports on the natural occurrence of fungi in 21 samples of field (10),
stored (6) and marketed (5) rice (Oryza sativa L.) collected from Niger State, Nigeria.
Fungal isolates were primarily identified based on morphological characteristics,
while representative isolates were characterized genetically. An evolutionary tree was
constructed from the resulting sequences of the isolated fungi. The toxigenic potentials of some of the isolated fungi were also determined. A total of 357 fungal isolates
of nine genera including Aspergillus, Fusarium, Sarocladium, Acremonium, Curvularia Botryosphaeria, Penicillium Alternaria and Ascomycota in decreasing order of
predominance were identified. The most frequent fungal contaminants of the rice
samples were A. flavus, A. fumigates, A. niger, A. parasiticus and F. proliferatum. All
strains of A. flavus (aflatoxins B1 and B2), A. parasiticus (aflatoxins B1, B2, G1 and G2),
A. ochraceus (ochratoxin A), F. proliferatum and F. verticillioides (fumonisins B1 and
B2) tested, were excellent producers of their respective mycotoxins. Patulin was produced by A. terreus, whereas deoxynivalenol, zearalenone and T-2 toxin were produced by F. chlamydosporum and other Fusarium spp. The increased prevalence of
toxigenic fungi in rice, a highly consumed food grain in Nigeria, poses serious health
concerns to the general public.
PRACTICAL APPLICATIONS
This study investigated the natural fungal occurrence of rice grown in Nigeria using
polymerase chain reaction-based techniques as well as the toxigenic potentials of
some of the identified fungi. The resultant fungal profile of rice, gene sequences of
the fungi detected in the survey, which were deposited in the GenBank and the constructed evolutionary tree, will serve as reference data for the incidence of fungal
species in rice and will help to evaluate the safety of Nigerian rice. It could be used in
developing and predicting the degree of mycotoxin contamination in rice from
Nigeria for effective mycotoxin control. The toxigenic fungi acquired from the work
will be excellent microbial sources for production of mycotoxin standards namely
aflatoxins BI, B2, G1 and G2, ochratoxin A and fumonisin B1 and B2.
INTRODUCTION
Rice (Oryza sativa) is one of the world’s most extensively cultivated crops and equally a staple food of over half of the
world’s total population (FAO 2002) with its consumption
increasing significantly in Africa within the last few decades
334
(Chang 1987). In Nigeria, this commodity is the sixth most
cultivated crop after Sorghum, millet, cowpea, cassava and
yam. Production of rice has increased over the years in
Nigeria with an estimated 4.7 million tonnes produced
in 2007, making it the second largest producer in Africa
with Egypt as principal producer (FAO 2008). Paradoxically,
Journal of Food Safety 31 (2011) 334–349 © 2011 Wiley Periodicals, Inc.
H.A. MAKUN ET AL.
Nigeria is the third major rice importing country after Iran
and the Philippines in spite of such productivity. Accordingly,
about 2 million tonnes of rice (USDA 2008) is imported and
about 5 million tonnes consumed annually (USDA 2008a).
In Nigeria, rice is produced in all six agroecological zones of
the country with the highest yield coming from the northern
guinea savannah (Ezedinma 2005). Niger State, a generally
hot and humid area with average annual temperature and
relative humidity of 31.7C and 51.6%, respectively, falls
within the guinea savannah region and after Kaduna State, it
is the second largest producer of rice (Erenstein and Lancon
2003) contributing approximately 16% of total rice production (Ezedinma 2005). However, like for other food commodities, it is subjected to microbial contamination.
Toxigenic fungi can attack rice in the field and during
storage resulting in increased mycotoxin levels in this commodity. Aspergillus, Fusarium and Penicillium are the predominant fungal genera associated with food grains during
storage (CAST 2003). The ingestion of grains contaminated
with mycotoxins especially those that are nephrotoxic, immunotoxic, teratogenic and mutagenic can provoke acute and
chronic effects in man and animals ranging from disorders of
central nervous, cardiovascular, pulmonary and intestinal
tract systems to death (Hussein and Brasel 2001; Bhat and
Vasanthi 2003). The involvement of these toxins in human
hepatoma and esophageal cancer (Neal 1995; Richard 2007;
Shephard 2008), increased susceptibility to diseases especially
in children and childhood pre-five mortality with reduced life
expectancy (Sherif et al. 2009), is of major concern with
regards to public health.
Studies conducted to establish the prevalence of fungi
and their toxins in foods and feedstuffs and ensure a healthy
food supply to animals and humans are scanty in Nigeria
unlike the case may be for other countries of the world. Very
limited reports (Opadokun and Ikeorah 1979; Obidoa and
Gugnani 1992; Ikeorah and Okoye 2005; Ayejuyo et al. 2008;
Amadi and Adeniyi 2009) on fungi and mycotoxins contaminations of rice from some parts of Nigeria are available.
Except for Makun et al. (2007), there is no information
on fungi and mycotoxins in rice from Niger State, Nigeria.
The previous survey (Makun et al. 2007) used conventional
fungi identification technique that invariably necessitated
more comprehensive surveys using accurate standardized
methods. The present study was, therefore, conducted to
examine the distribution and phylogeny of fungi in rice
from Niger State as well as their ability to produce mycotoxins using polymerase chain reaction (PCR)-based technique and thin-layer chromatography (TLC), respectively.
Knowledge on the toxigenicity of common fungal contaminants of rice is vital in elucidating animal and human
mycotoxicoses expected from such a commodity and will
equally be helpful in addressing the problem of mycotoxin
control in Nigeria.
Journal of Food Safety 31 (2011) 334–349 © 2011 Wiley Periodicals, Inc.
MYCOTOXIGENIC FUNGI IN RICE FROM NIGERIA
MATERIALS AND METHODS
Materials
All chemicals used were of analar grade unless otherwise
stated.
(1) Antibiotics: streptomycin and chloramphenicol (Sigma,
Aldrich).
(2) All chemicals for PCR analysis including the Fungal/
Bacterial DNA extraction kit were purchased from Zymo
Research Corporation, Irvine, CA.
(3) Strong anion exchange (SAX) cartridge (ANATECH,
Gauteng, South Africa).
(4) Mycotoxins standards: Aflatoxins B1, B2, G1 and G2, ochratoxin A (OTA), zearalenone (ZEA), deoxynivalenol (DON),
T-2 toxin (T2) and patulin (PAT) reference standards were
obtained from Sigma, St. Louis, MO. Fumonisin B1 (FB1), B2
and B3 were purchased from PROMEC, MRC, Tygerberg,
South Africa.
Sampling
Twenty-one representative rice samples from the fields (10),
storage facilities (6) and market outlets (5) were randomly
collected through donations and purchases in December
2008 from 21 villages in the traditional rice growing area of
Niger State, Nigeria. Samples were collected from separate
batches by thorough mixing of the contents of traditional
storage facilities and market containers to obtain homogeneity and representative samples collected from the top, middle
and bottom of the containers. In the case of field samples,
between seven to ten bunches of rice from stalks at the front,
middle and back of the farm were randomly collected and
thoroughly mixed to give a sample. Samples (about 0.5 kg
each) were put in sealed plastic bottles and transported to our
laboratory in South Africa where they were finely milled to
pass through a no. 20 sieve and stored in the deep freezer at
-20C for a week until analyzed.
Isolation and Identification of Fungi
The mycological analytical procedure involving four steps
according to the method of Kaufman et al. (1963) was used
including fungal isolation on potato dextrose agar (PDA) and
Ohio agricultural and experimental station agar (OAESA),
subculturing on PDA, malt extract agar (MEA) and Czapek
yeast extract agar (CYA), macro- and microscopic identification and finally, phylogenetics of fungi. The phylogeny of
fungi was determined following DNA extraction, PCR amplification, purification of PCR product, product quantification
and DNA sequencing for a confirmation of various species of
fungi. The culture media used (PDA, OAESA, MEA and CYA)
were prepared as described by Atlas (2004).
335
H.A. MAKUN ET AL.
MYCOTOXIGENIC FUNGI IN RICE FROM NIGERIA
Primarily, each milled sample was subjected to a six-level
serial dilution technique in which 1 g was diluted in a 9-mL
ringer solution, vortexed and subsequently, 1 mL of the suspension was transferred to a 9-mL ringer solution and vortexed, and so forth. One milliliter of each suspension was
inoculated on solid PDA and OAESA in 90-mm Petri dishes
and incubated at 25C for 7–14 days. Between the 5th and 7th
day of incubation, all colonies were counted using a colony
counter and results presented as number of fungal colonies
per gram of sample calculated and expressed in colonyforming units per gram (cfu/g).
The fungi so screened were subcultured on CYA, MEA and
PDA, incubated at 30C for 7–14 days and identified to species
level where possible. In this case, the hyphae and conidia from
each colony representing each fungal species were transferred
aseptically on three spots diagonally on each Petri dish containing the medium. Identification to species level was done
based on the macroscopic and microscopic characteristics of
the isolates following the identification keys of Klich (2002)
for Aspergillus spp. Pitt and Hocking (1997) for Penicillium
and Nelson et al. (1983) for Fusarium spp. Isolates were
subcultured on PDA slants and stored at 4C until further
analyzed.
Eight of the 48 fungi isolated from the rice samples were
precisely identified by this conventional method. These
include Aspergillus niger, A. parasiticus and A. penicillioides
and A. flavus. Isolates were further identified at the Inqaba
Biotechnological Laboratories, Pretoria, South Africa by
comparison of the nucleic acid profiles of individual fungal
species as described by Samson et al. (2004). To this end, the
mycelia of isolates on PDA slants were subcultured for 7
days as previously described and mycelia harvested, freezedried and then subjected to DNA extraction, PCR amplification, purification and quantification of PCR product, DNA
sequencing and analysis as described by Samson et al.
(2004) and Geiser et al. (2004) with some modifications.
Accordingly, a Fungal/Bacterial DNA extraction kit (Zymo
Research Corporation, Irvine, CA) was used for DNA
extractions in addition to MSB Spin PCRapace (Invitek
GmbH, Berlin, Germany) that was used for ultrafast purification and concentration of PCR-fragments. The freezedried cultures were thawed for 1 h and genomic DNA
extracted. To this effect, 60 mg sample was weighed and
resuspended in 200 mL phosphate-buffered saline (PBS)
contained in a 1.5-mL ZR Bashing Bead™ lysis tube (Zymo
Research Corporation, Irvine, CA). This tube was then
placed in a genie disruptor for 5 min and then centrifuged at
10,000¥ g for 1 min. The supernatant was recovered in a
Zymo-Spin™ IV spin filter placed in a 1.5 mL Eppendorf
tube, again centrifuged at 7,000¥ g for 1 min, filtered into a
collection tube and 1,200 mL of fungal/bacterial DNA
binding buffer added and vortexed. Eight-hundred microliters of the mixture was transferred to a Zymo-Spin™ IIC
336
column placed in a collection tube, centrifuged for 1 min at
10,000¥ g and the supernatant discarded (¥2). A 200-mL
aliquot of DNA pre-wash buffer I was added to the ZymoSpin™ IIC column in a new collection tube, centrifuged at
10,000¥ g for 1 min and filtrate discarded, while retaining
the column, which was then placed into a new tube. Into the
Zymo-Spin™ IIC column, 500 mL fungal/bacterial DNA
wash buffer II was added and again centrifuged at 10,000¥ g
for 1 min. The Zymo-Spin™ column was transferred to a
sterile 1.5 mL Eppendorf tube and 100 mL DNA elution
buffer added directly to the column matrix, centrifuged at
10,000¥ g for 30 s and DNA eluted and preserved for PCR
analysis.
The PCR procedure followed is covered by US Patents
4,683,195 and 4,683,202 (Hoffmann-LaRoche AG, Basel,
Switzerland). The primers (ITS_1 and ITS_4) used were
synthesized at a 0.01-mM scale and purified using reversephase cartridge purification (Inqaba). These primers were
resuspended in 2 mM TE buffer prepared from a stock solution concentration of 100 mM. PCR was performed using
the Fermentas 2 X PCR mix (Fermentas Life Science,
Lithuania). The PCR mixture for each sample consisted of
25 mL of 2 X PCR mix, 1 mL each of 2 mM primers, 1 mL of
DNA (final concentration of 10 mM), and constituted to a
final volume of 50 mL with nuclease free water. A negative
control, containing all of the reagents used except the DNA
was also prepared. PCR was performed using an Eppendorf
96-well Thermocycler (Eppendorf, Westbury, NY). The PCR
cycling conditions were set as follows: Pre-dwelling at 95C
for 3 min, 35 cycles denaturation at 95C for 1 min, annealation at 58C for 45 s, extension at 72C for 1 min 30 s, postdwelling at 72C for 10 min and held at 4C until samples
were retrieved.
The PCR products were further analyzed on an ABI
PRISM 3700 Genetic analyzer (AB, Applied Biosystems,
Nieuwerkerk a/d Yssel, the Netherlands). The forward and
reverse sequences of the PCR products were assembled with a
DYEamic ET Terminator Cycle Sequencing Kit (Amersham,
Bioscience, Roosendaal, the Netherlands) using the programs
SeqMan and EditSeq from the LaserGene package (DNAStar,
Inc., Madison, WI). DNA sequences and identities of fungi
were obtained from Inqaba Finch server.
Phylogenetic Analysis
Sequences of the rice fungal isolates in FASTA format
obtained from Inqaba Finch server were further subjected to
identification on the PCR amplification of the 16S and internal transcribed spacer (ITS_4) regions using the GenBank
Blast. The evolutionary tree was constructed using PHYLIP
package (version. 3.6) from http://evolution.genetics.
washington.edu/phylip.html and the evolutionary distances
matrix generated.
Journal of Food Safety 31 (2011) 334–349 © 2011 Wiley Periodicals, Inc.
H.A. MAKUN ET AL.
Determination of Mycotoxin-Producing
Potentials of Fungi Isolated
Each strain representing the fungal species of Aspergillus,
Penicillium and other genera with the exception of Fusarium
were further tested for toxigenicity to determine their ability
to produce the following mycotoxins: aflatoxin B1 (AFB1);
aflatoxin B2 (AFB2); aflatoxin G1 (AFG1); aflatoxin G2
(AFG2); OTA; and PAT. These isolates were cultured individually on solid yeast extract sucrose agar (YES) agar in a
90-mm Petri dish and incubated at 25C for 28 days according to the method of Singh et al. (1991). Mycotoxins synthesized by each fungus were extracted by dissolving 5 g of
isolate including the medium in 10 mL of dichloromethane
(DCM). The crude extract obtained was filtered through a
Whatman no. 2V filter paper and the filtrate put in a screwcap vial, dried under a stream of N2 gas and stored at 4C
until analyzed.
The mycotoxins in the crude extracts were detected by a
two-dimensional TLC technique devised by Patterson and
Roberts (1979). To this end, extracts were reconstituted with
200 mL DCM, vortexed and 20 mL of the extract solution
spotted about 10 mm from the edge of a silica gel TLC plate. A
similar procedure was followed for mycotoxin standards as a
reference for detecting mycotoxins of interest. The developing
solvents for the first and second runs were DCM/ethyl
acetate/propan-2-ol (90:5:5, v/v/v) and toluene/ethyl acetate/
formic acid (6:3:1, v/v/v), respectively. After the second run,
plates were dried and visualized under ultraviolet radiation at
wavelength of 365 nm. For PAT detection, plates were sprayed
with 0.5% 3-methyl-2-benzothiazolinone hydrazone hydrochloride solution and heated at 120C for 3 min. PAT appears
as a yellow spot with an orange fluorescence. Visual comparison of retention factor (RF) values and fluorescing color of
spots of extracts to that of standards was the basis for identification of toxins.
The toxigenic potentials of the isolated Fusarium spp. in
producing ZEA, DON, T-2, FB1 and FB2 were evaluated. Representative isolates of each Fusarium spp. were further plated
on solid YES agar and incubated at 25C for 28 days. Fusarium
mycotoxins were extracted according to the method of
Hinojo et al. (2005) with some modifications following
clean-up procedures depending on the type of mycotoxin to
be determined. Fusarium toxins in 10 g of agar-containing
mycelia was extracted into 25 mL CH3OH/H2O (60/40, v/v)
and shaken on a mechanical shaker for 1 h. The entire content
was filtered through a Whatman no. 2V filter paper to obtain
the crude extract for each fungus. For FB analysis, the extract
was stored at 4C until used. ZEA, DON and T-2 toxins were
extracted three times from the crude extracts with 25 mL of
DCM. The bottom layer was passed through a bed of Na2SO4
anhydrous into a 500 mL round bottom flask and dried by
rotary evaporation. The content was then reconstituted with
Journal of Food Safety 31 (2011) 334–349 © 2011 Wiley Periodicals, Inc.
MYCOTOXIGENIC FUNGI IN RICE FROM NIGERIA
200 mL DCM into a 0.5-mL screw-cap vial, dried by passing
through a stream of N2 gas and then stored at 4C until
analyzed.
Methanol was used for fumonisin extraction. The clean-up
method of Sydenham et al. (1992) was adopted for FB analysis. The stored filtrate was passed through a SAX column
cartridge (ANATECH) previously conditioned with 5 mL
methanol followed by 5 mL methanol : water (3:1, v/v). The
column was washed with 8 mL methanol : water (3:1, v/v)
and then 3 mL methanol. The absorbed FB was then eluded
with 10 mL 1% acetic acid in methanol. The eluent was
evaporated to dryness and the residue stored in a screw-cap
vial at 4C until analyzed.
A two-dimensional TLC technique (Patterson and Roberts
1979) as previously described was used for the detection of
ZEA, DON and T-2 toxin. Here, dried extracts were reconstituted with 200 mL DCM and 20 mL was applied to the origin
of a two-dimensional TLC plates as previously described but
this time, those for ZEA analysis were run twice in DCM : acetone (9:1, v/v) and derivatized with cold dianisidine reagent
prepared according to Malaiyandi et al. (1976). The mobile
solvents for trichothecenes were DCM/ethyl acetate/propan2-ol (90:5:5, v/v/v) and toluene/ethyl acetate/formic acid
(6:3:1, v/v/v), respectively. Plates were derivatized with chromotropic acid (Baxter et al. 1983), while those for fumonisin
analysis were developed in dicholoromethane/methanol/
acetic acid (80:20:2 v/v/v) and butanol/water/acetic acid
(12:5:3 v/v/v), respectively. Fumonisins were visualized as
purple spots on dried plates that were sprayed with
p-anisaldehyde reagent, followed by heating for 3 min at
120C. The retardation factors (RF1 and RF2) and color of the
individual spots on TLC were calculated and compared with
those of standard mycotoxins to aid in the identification of
mycotoxins present.
Statistical Analysis
Data on the cfu were subjected to statistically analysis. An
analysis of variance was used to derive mean values and standard deviation using SigmaStat 3.5 for Windows (Systat Inc.,
San Jose, CA, 2006), which were compared by least significant
difference. Mean values were deemed to significantly differ if
P ⱕ 0.5.
RESULTS
PCR Analysis
The results of the evolutionary history and identities of
the rice fungi based on 18S ribosomal RNA gene, partial
sequence; internal transcribed spacer 1, 5.8S ribosomal RNA
gene and internal transcribed spacer 2, complete sequence;
and 28S ribosomal RNA gene, partial sequence are presented
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MYCOTOXIGENIC FUNGI IN RICE FROM NIGERIA
in Fig. 1 and Table 1. PCR was successfully performed and
bands (550 bp) were obtained. Each sequence was compared
with those of the same species deposited at GenBank. Similarity distances ranged from 0.010 to 0.452. Total nucleotides
amplified were 470–533 bp and corresponded to the ITS_4
complete regions; the 3, portion of the 18S gene, 5.8S complete sequence, and the 5, end of the 28S gene. The sequences
and names of fungi processed by Inqaba Finch server were in
complete conformity with our GenBank blasting results
except in two instances. The sequence of HM 23, which was
identified by Inqaba as Curvularia sp., was identified in the
GenBank as Curvularia affinis (AF071335.1) with a query
coverage of 51% and maximum identity of 99%. Similarly,
the sequence data of HM 34 that was blasted as only Fusarium
sp. by Inqaba was identified in the GenBank as Pseudofusarium purpureum strain MUCC 248 (EU301058.1) with
an 84% query coverage and maximum identity of 99%. The
identities obtained for these isolates from the GenBank were
therefore adopted forthwith in this study. HM 22 failed to
grow enough and HM 25 was a mixed culture, which did not
allow for PCR analysis of the isolates so their identities as
Alternaria and Curvularia spp. obtained via conventional
methods based on morphological characterization were
adopted.
Fungal Contamination
The survey generated data on the fungal contamination of
rice samples that are presented in Table 2. A total of 357 fungi
belonging to nine different fungal genera including Aspergillus, Fusarium, Penicillium, Acremonium, Alternaria, Ascomycota, Botryosphaeria, Curvularia and Sarocladium, were
isolated and identified in the survey. In overall, Aspergillus
(62.75%) was the most predominant fungal genera identified
followed by Fusarium (21.85%) in addition to the less frequent members of Sarocladium (3.92%), Acremonium
(3.64%), Curvularia (3.08%), Botryosphaeria (1.68%), Penicillium (1.12%), the Alternaria (0.56%) and Ascomycota
(0.56%) that were also isolated. The calculated mean
⫾ standard deviation values of the cfu for the different
types of samples, though not significantly different
(P ⱕ 0.05), showed lower fungal contamination in field
(2.6 ¥ 102 ⫾ 5.0 ¥ 102) than stored (5.0 ¥ 102 ⫾ 1.6 ¥ 102)
and marketed (5.0 ¥ 103 ⫾ 2.0 ¥ 102) samples, an indication
of increasing fungal contamination from field to storage and
subsequently market.
Although, there was no clear distinction between field and
storage fungi, some species were strictly field or storage fungi.
All isolates each of A. aculeatus, A. niveus, two of A. terreus, A.
tubingensis, Alternaria sp. and Ascomycota sp. were considered
field fungi, whereas all isolates of A. oryzae (6), A. sclerotiorum
(3), Penicillium oxalicum (4), F. chlamydosporum (2), F.
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H.A. MAKUN ET AL.
pseudonygamai (2) and F. verticillioides (4) were found only in
the stored samples (stored and marketed samples) as seen in
Table 2.
Aspergillus members accounted for 227 of the total 357
fungi isolated with A. flavus being the most dominant. Fourteen species of Aspergillus were found among which A. flavus
was recovered in a total of 19 samples analyzed, recording an
incidence of 90.5%. The next sets of dominant members of
the Aspergillus were A. fumigatus and A. niger, each isolated
in 81% as well as A. parasiticus in 71% of samples. Other
members belonging to this genus had low incidence rates and
contamination by these species in decreasing order of predominance was A. ochraceus, A. unguis, A. candidus, A. oryzae,
A. pencillioides and A. terreus. For those isolated in only one
sample were A. aculeatus, A. niveus and A. sclerotiorum. The
three strains of Eurotium found in this study were all belonging to E. amstelodami being recovered from two rice samples.
The genus Fusarium was also frequently isolated, accounting for 78 of the 357 fungal isolates from rice reported herein
with F. proliferatum being the most dominant, occurring in 11
(52.4%) of the samples. In all, six members of this genus
were isolated alongside others including Fusarium sp.
(33.3%), Pseudofusarium purpureum (23.8%), F. verticillioides (14.3%), F. chlamydosporum (9.5%) and F. pseudonygamai (9.5%).
The third most dominant fungal genera found in the study
after Aspergillus and Fusarium is the Sarocladium occurring in
10/21 samples analyzed. The two species found namely S.
attenuatum and S. oryzae contaminated three and eight
samples, respectively. Other genera were of low incidence.
Acremonium sp. was found in 38% of the samples. Eleven isolates of two species of the Curvularia family contaminated 8
of the 21 rice samples. The species namely Curvularia sp. and
Curvularia affinis had incidence rates of 38 and 14.3%,
respectively. Six Botryosphaeria dothidea were isolated in four
samples, while the four isolates of the only species of Penicillium, P. oxalicum were found in two samples. The two
least predominant genera, Ascomycota (two isolates) and
Alternaria (two isolates) contaminated two and one samples,
respectively.
This mycological survey also showed that none of the
samples analyzed was free of fungal contamination with
samples having very high frequency of co-occurrences
of between 5 and 14 fungal species. The commonest of
such multioccurrences were the simultaneously contamination by A. flavus, A. fumigatus, A. parasiticus, A. niger and F.
proliferatum.
Mycotoxigenic Potentials of Fungal Isolates
Randomly selected representative isolates from each strain of
the species were screened for their toxigenic potentials. Data
on the toxigenicity of fungi tested in the present study are
Journal of Food Safety 31 (2011) 334–349 © 2011 Wiley Periodicals, Inc.
H.A. MAKUN ET AL.
MYCOTOXIGENIC FUNGI IN RICE FROM NIGERIA
FIG. 1. PHYLOGENETIC TREE INFERRED FROM ITS-4 GENE SEQUENCE OF FUNGAL ISOLATES FROM RICE IN NIGER STATE, NIGERIA IN RELATION TO
REFERENCE CULTURES IN THE LITERATURE
Journal of Food Safety 31 (2011) 334–349 © 2011 Wiley Periodicals, Inc.
339
H.A. MAKUN ET AL.
MYCOTOXIGENIC FUNGI IN RICE FROM NIGERIA
TABLE 1. LIST OF FUNGAL ISOLATES IN RICE FROM NIGERIA IN RELATION TO REFERENCE CULTURES
Species name
Aspergillus
A. aculeatus
A. aculeatus
A. candidus
A. candidus
A. candidus
A. candidus
A. flavus
A. flavus
A. flavus
A. flavus
A. fumigatus
A. fumigatus
A. fumigatus
A. fumigatus
A. niveus
Fennellia nivea
A. ochraeus
A. ochraceus
A. oryzae
A. oryzae
A. oryzae
A. oryzae
A. sclerotiorum
A. sclerotiorum
A. terreus
A. terreus
A. tubingensis
A. awamori
A. unguis
A. unguis
E. amstelodami
E. amstelodami
Fusarium
F. chlamydosporum
Fusarium sp
F. proliferatum
F. proliferatum
F. pseudonygamai
F. pseudonygamai
Fusarium sp
Pseudofusarium purpureum
Fusarium sp
Fusarium sp
F. verticillioides
F. verticillioides
F. verticillioides
Gibberella moniliformis
Penicillium
P. oxalicum
Penicillium sp
Acremonium
Acremonium sp
Acremonium sp
Acremonium sp
Acremonium sp
340
Accession No
Geographic origin
Host plant/code
HM 6
EU645715.1
HM 15
HM 36
FJ441640.1
AY373843.1
HM 7
HM 20
HM 21
FJ011545.1
HM 17
HM 24
HM 28
FJ844610.1
HM 19
FJ155814.1
HM 48
FJ878632.1
HM 44
HM 46
FJ654485.1
FJ654483.1
HM 35
AY373866.1
HM 14
GQ461901.1
HM 13
EF151436.1
HM 27
FJ878626.1
HM 16
GQ120984.1
Tasaba, Nigeria
Oryzae sativa F2
Seafan Aspergillosis
Oryzae sativa, F8
Oryzae sativa M 20
Dama, Nigeria
Lafiyagi, Nigeria
Xiamen, China
USA
Maitumbi, Nigeria
Tawi, Nigeria
Bida, Nigeria
Southern China
Pigi, Nigeria
Kwarkwata, Nigeria
Innagi, Nigeria
China
Dama, Nigeria
Mexico
Gbadadan, Nigeria
Greece
Danzariya, Nigeria
Ekosa, Nigeria
Indian Western Ghats
Indian Western Ghats
Lafiyagi, Nigeria.
USA
Maitumbi, Nigeria
Greece
Dama, Nigeria
China
Shata, Nigeria
Greece
Gbadadan, Nigeria
Mediterranean Sea
Kwarkwata, Nigeria
Malaysia
Rimi, Nigeria
China
Danzariya
China
Kodoko, Nigeria
Dust samples
Oryzae sativa F1
Oryzae sativa S 13
Oryzae sativa M 19
Reference
Zuluaga et al. (unpublished)
Zhang and Huang, 2008
Haugland et al. 2004
Xi et al. (unpublished)
Oryzae sativa F3
Oryzae sativa S15
Oryzae sativa M18
Zhang and Shi, 2009
Oryzae sativa F8
Orange peel
Oryzae sativa S14
Solis et al. 2008
Arabatzis and Velegraki (unpublished)
Oryzae sativa S16
Oryzae sativa M17
Megamai forest
Megamai forest
Oryzae sativa M20
Dust samples
Oryzae sativa F1
Venkatesan and Muthuchelian (unpublished)
Venkatesan and Muthuchelian (unpublished)
Haugland et al. 2004
Arabatzis and Velegraki (unpublished)
Oryzae sativa F8
Xie and Jian (unpublished)
Oryzae sativa F6
Arabatzis and Velegraki (unpublished)
Oryzae sativa F8
Marine algae
Larriba et al. (unpublished)
HM 18
GQ352485.1
HM 33
FJ040179.1
HM 47
FJ154075.1
HM 34
EU301058.1
HM 41
GU257906.1
HM 29
HM 40
HM 42
GU257904.1
Kwarkwata, Nigeria
Ekosa, Nigeria
Bida, Nigeria
India
HM 8
EU301633.1
Innagi, Nigeria
China
Oryzae sativa M18
Forest soil
Zhou et al. (unpublished)
HM 30
HM 31
HM 38
GU055562.1
Kodoko, Nigeria
Tawi, Nigeria
Bida, Nigeria
Austria
Oryzae sativa F7
Oryzae sativa S13
Oryzae sativa M19
Agricultural soil
Klaubauf et al. (unpublished)
Isheli, Nigeria
Oryzae sativa F15
Sim et al. 2009
Oryzae sativa. F4
Oryzae sativa
Oryzar sativa S16
Soil
Oryzae sativa F7
Oryzae sativa M21
India
Oryzae sativa S 15
Oryzae sativa M17
Oryzae sativa M19
Wang et al. (unpublished)
Zhao and Gao (unpublished)
Chandra et al. (unpublished)
Chandra et al. (unpublished)
Journal of Food Safety 31 (2011) 334–349 © 2011 Wiley Periodicals, Inc.
H.A. MAKUN ET AL.
MYCOTOXIGENIC FUNGI IN RICE FROM NIGERIA
TABLE 1. CONTINUED
Species name
Alternaria
A. azukiae
A. azukiae
Ascomycota
Ascomycota sp
Ascomycota sp
Botryosphaeria
B. dothidea
B. dothidea
B. dothidea
Curvularia
Curvularia sp
Curvularia affinis
Curvularia sp
Sarocladium
S. attenuatum
S. attenuatum
S. oryzae
S. oryzae
S. oryzae
Accession No
Geographic origin
Host plant/code
HM 10
FJ467366.1
Maitumbi, Nigeria
China
Oryzae sativa F1
HM 45
EU682958.1
Chanchaga, Nigeria
China
Oryzae sativa F10
HM 39
HM 43
GQ855797.1
Rimi, Nigeria
Isheli, Nigeria
East China
Oryzae sativa F4
Oryzae sativa M21
Apple
HM 23
AF071335.1
GQ184733.1
Gsada, Nigeria
Oryzae sativa F8
HM 37
AY566997.1
HM 26
HM 32
AY566996.1
Yikangbe, Nigeria
Oryzae sativa F5
Pigi, Nigeria
Tawi, Nigeria
Oryzae sativa F3
Oryzae sativa s13
Reference
Wu and Li (unpublished)
Sun et al. (unpublished)
China
Liu et al. (unpublished)
Zhang and Pan, 2009
Bills et al. 2004
Bill et al. 2004
The references and their sequences in this table were all obtained from the National Center for Biotechnology Information (NCBI) web site http://
blast.ncbi.nlm.nih.gov/Blast.cgi.
presented in Table 3. As found, all 10 strains of A. flavus tested
were excellent producers of AFB1 and AFB2. A similar observation was followed for A. parasiticus with all tested strains
producing both the B and G types of aflatoxins (AFB1, AFB2,
AFG1 and AFG2). OTA was recovered from cultures of A.
ochraceus tested. Other ochratoxigenic fungi isolated were
A. niger and A. sclerotiorum (HM 35). Two of the strains of A.
terreus both originally from field rice samples were shown to
produce PAT. Results of the toxicity screening of the Fusarium
spp. indicate that all the strains of F. proliferatum (HM 33)
and F.verticillioides (HM 29, 40, 40a and 42) produced FB1
and B2 (FB2). The two strains of F. chlamydosporum (HM 18)
with other Fusarium spp. (HM 41) were excellent producers
of DON and ZEA, while only one strain of Fusarium spp.
(HM 41) produced T-2 toxin. None of the species of Penicillium, Acremonium, Sarocladium, Curvularia Alternaria, Eurotium, Ascomycota and Botryosphaeria produced the toxins
tested. However, many of them produced metabolites that
could not be confirmed because of the lack of reference data.
DISCUSSION
This study provides the first comprehensive documentation
of the distribution and toxigenicity of fungal species contaminating rice from a major traditional rice producing
region of Niger State in Nigeria. Fungal species belonging to
nine genera viz: Asperillus, Fusarium, Penicillium, Acremonium, Ascomycota, Alternaria, Botryosphaeria, Curvularia and
Sarocladium were found to be major fungal contaminants of
Journal of Food Safety 31 (2011) 334–349 © 2011 Wiley Periodicals, Inc.
rice from the 21 villages under study. It was also shown in the
survey that fungal contamination increased from field to
storage with co-occurrence of toxigenic fungi being highly
frequent. Data from the toxigenic screening of the isolated
fungi indicate that there are many toxigenic strains recovered
from the rice samples that were producers of AFB1, AFB2,
AFG1 and AFG2, OTA, ZEA, DON, T-2, FB1 and FB2 under
laboratory conditions. The observed increase in fungal contamination from field to storage is expected as grains are
infected by the ubiquitous fungi in the field and under favorable conditions, the pre-harvest fungi proliferate during
storage (Ominski et al. 1994) with a resultant increase in
fungal concentration and subsequent mycotoxin formation.
The lack of distinction between field and storage with regards
to the fungal strains recorded in the rice samples is also
expected in tropical regions such as Niger State, where fungi
belonging to especially the genus Aspergillus occur in a
natural environment and colonize grains before harvest or
during period of drought stress and insect damage (Moss
1987). Therefore, fungi present on grains in the field could
easily persist during storage under water stress and insect
damage conditions (Mclean and Berjack 1987) such that
there is no definite demarcation between field and storage
fungi. Grain infection in the tropics therefore, is a continuous
process with available predominant species of fungi at any
time depending upon prevailing micro-environmental
conditions of the grain (Mycock and Berjack 1999). This
explains the noted incidence of typical storage fungi A.
aculeatus, A. niveus, A. terreus and A. tubingensis in the field
341
1
1
1
4
1
1
7
3
3
4
8
6
3
4
1
2
2
1
2
74
1
3
3
2
1
1
2
2
3
1
1
1
41
1
1
1
2
1
1
3
2
2
2
2
37
1
2
2
2
1
1
1
1
8
6
1
1
1
2
1
1
2
2
1
1
1
3
7
7
1
1
2
1
3
2
2
9
3
4
2
38
2
2
2
1
1
1
5
2
1
2
2
1
2
2
3
1
1
1
1
3
1
1
1
1
1
1
2
2
4
8
3
2
1
26
1
2
1
1
6
1
1
1
13
1
1
2
2
6
3
1
8
3
1
2
11
Total
1
Sarocladium oryzae
Curvularia sp.
1
Sarocladium attenuatum
Curvularia affinis
Botryosphaeria.dothidea
Alternaria sp
Ascomycota. sp
Alternaria azukiae
Fusaruim spp.
Pseudofusarium purpureum
1
2
2
3
3
30
1
1
2
1
1
2
1
3
1
2
3
4
2
3
1
1
1
8
1
3
2
6
2
5
Acremonium sp
1
11
2
2
1
2
1
1
F.verticillioides
2
F. pseudonygamai
1
F. proliferatum
1
Fusarium .chlamydosporum
1
Penicillium .oxalicum
Eurotium amstelodami
1
A. unguis
A. sclerotiorum
A. penicillioides
A. parasiticus
A. oryzae
1
1
1
1
A. tubingensis
6
6
2
3
3
A. terreus
10
1
2
4
5
7
6
A. ochraceus
4.2 ¥ 102
2.6 ¥ 102
8.0 ¥ 102
1.3 ¥ 104
1.7 ¥ 104
7.0 ¥ 103
1.7 ¥ 104
1.3 ¥ 104
5.0 ¥ 104
1.1 ¥ 104
1.1 ¥ 103
5.0 ¥ 102
1.1 ¥ 103
1.3 ¥ 104
1.2 ¥ 104
1.6 ¥ 104
8.0 ¥ 103
8.9 ¥ 103
2.0 ¥ 104
1.3 ¥ 104
5.0 ¥ 103
A. niveus
Field
Field
Field
Field
Field
Field
Field
Field
Field
Field
Store
Store
Store
Store
Store
Store
Market
Market
Market
Market
Market
A. niger
Maitumbi
Tasaba
Pigi
Rimi
Yikangbe
Shata
Kodoko
Dama
Gsada
Chanchage
Kodoko
Bupbe
Tawi
Gbadadan
Kwarkwata
Danzariya
Ekosa
Innagi
Bida
Lafiyagi
Isheli
A .fumigatus
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
Total
A. flavus
cfu g/mL
A. candidus
Type of sample
Aspergillus. aculeatus
Location of samples
29
21
12
19
14
14
11
13
17
19
11
16
23
19
18
22
16
17
15
16
15
357
H.A. MAKUN ET AL.
Journal of Food Safety 31 (2011) 334–349 © 2011 Wiley Periodicals, Inc.
S/no
MYCOTOXIGENIC FUNGI IN RICE FROM NIGERIA
342
TABLE 2. ABSOLUTE FREQUENCIES OF THE DIFFERENT FUNGAL SPECIES IN RICE FROM NIGERIA
H.A. MAKUN ET AL.
MYCOTOXIGENIC FUNGI IN RICE FROM NIGERIA
TABLE 3. MYCOTOXIGENIC POTENTIALS OF FUNGI ISOLATED FROM RICE IN NIGERIA
Fungi
No. of strains isolated
No. of strains tested
No. of positive strains
Type of mycotoxins produced
A. aculeatus
A. candidus
A. flavus
A. fumigatus
A. niger
A. niveus
A. ochraceus
A. oryzae
A. parasiticus
A. penicillioides
A. sclerotiorum
A. terreus
A. tubingensis
A. unguis
P. oxalicum
F. chlamydosporum
F. proliferatum
F. pseudonygamai
F. verticillioides
Fusaruim spp.
1
7
74
41
37
1
8
6
30
3
3
2
2
9
4
2
38
2
4
28
1
5
10
10
10
1
8
5
10
3
3
2
2
9
4
2
10
2
4
15
–
–
10
–
1
–
8
–
10
–
1
1
–
–
–
2
6
–
4
5
Pseudofusarium purpureum
Acremonium sp
Alternaria azukiae
Alternaria sp
Ascomycota. sp
Botryosphaeria.dothidea
Curvularia affinis
Curvularia sp.
Eurotium amstelodami
Sarocladium attenuatum
Sarocladium oryzae
6
13
1
1
2
6
3
8
3
3
11
6
5
1
1
2
5
3
5
3
3
3
–
–
–
–
–
–
–
–
–
–
–
Not detected
Not detected
AFB1 and AFB2
Not detected
OTA
Not detected
OTA
Not detected
AFB1, AFB2, AFG1 and AFG2
Not detected
OTA
Patulin
Not detected
Not detected
Not detected
ZEA and DON
FB1 and FB2
Not detected
FB1 and FB2
4 (ZEA + DON) and 1 (T-2
toxin)
Not detected
Not detected
Not detected
Not detected
Not detected
Not detected
Not detected
Not detected
Not detected
Not detected
Not detected
and occurrence of acclaimed field fungi; F. chlamydosporum,
F. pseudonygamai and F. verticillioides in stored samples.
However, there were some few exceptions to such observations in this study. Alternaria spp. being strictly field fungi and
A. oryzae (6), A. sclerotiorum (3), P. oxalicum found only in
storage samples in the present work is in conformity with the
classical grouping of fungi based on colonization period and
moisture requirement by earlier scientists (Javis 1971; Lillehoj 1973).
Aspergillus spp. being the most dominant fungal contaminants of Nigerian food commodities as proven in this survey
is well documented (Bankole and Adebanjo 2003; Bankole
et al. 2003; Atehnkeng et al. 2008). Two reports of Makun
et al. (2007) and Amadi and Adeniyi (2009) on fungi in Nigerian rice are also in agreement with the observations made
herein. Although the distribution of members of the Aspergillus family varied with sample type, i.e., field, store and market
samples, A. flavus was the most dominant species contaminating all rice samples. Similarly, high occurrence frequencies
of this fungus have previously been reported in Nigerian
Journal of Food Safety 31 (2011) 334–349 © 2011 Wiley Periodicals, Inc.
foods including rice by the aforementioned authors. The very
high incidence of A. flavus seen when compared with other
members of Aspergillus can be explained by the corresponding high levels of Aspergillus section Flavi in the soil, plant
debris and insects (Horn and Dorner 1999; Nesci and Etcheverry 2002; Jaime-Garcia and Cotty 2004), which serves as the
reservoir of inoculum for infection of grains in the field. As
reported, A. flavus is the most predominant member of
Aspergillus section Flavi in soils in West Africa (Cardwell and
Cotty 2002; Donner et al. 2006).
All the isolates of A. flavus screened for toxigenicity were
found to produce their attendant mycotoxins and this is in
agreement with the findings of Atehnkeng et al. (2008) who
found significantly higher incidence of toxigenic strains of
the species in Bida and Mokwa than in other parts of
Nigeria. These are the same regions that were also sampled in
the present study. The inherent high temperatures and drier
conditions of Niger State favor grain infection by A. flavus
with subsequent production of AF (Jones et al. 1981; Diener
et al. 1987), which could explain the very high toxigenic
343
MYCOTOXIGENIC FUNGI IN RICE FROM NIGERIA
potentials of the isolated A. flavus in this study. It could also
be that the agricultural management practices in the study
area have created unique ecological niches that promote
the toxigenic potential of strains of this species (Bilgrami
et al. 1981).
Other members of the Aspergillus reported in this study
have all been shown as common fungal contaminants of rice
from Nigeria (Amadi and Adeniyi 2009) and other parts of
the world (Udagawa 1976; Park et al. 2005; Reddy et al. 2006).
The A. parasiticus screened for toxigenicity were producers of
both the B and G types of AF. This assessment distinguished it
from other Aspergilli and served as a confirmation of its identity that there was no reason for it to be sent for confirmation
via PCR-based method. Apart from the AF producers, three
ochratoxigenic species belonging to the Aspergilli were also
isolated. In this case, A. niger, A. ochraceus and A. sclerotiorum
were shown in this work to be OTA producers as reported
elsewhere in literature (Pitt and Hocking 1997; Klich 2002),
whereas strains of A. ochraceus were profuse producers of
OTA; 50% of A. sclerotiorum tested were equally toxigenic
with only 1 of the 10 strains of A. niger screened found to be
ochratoxigenic. A. niger is usually regarded as a benign fungus
and toxin production does not seem to be common (Pitt and
Hocking 1997), so the recorded very low toxigenic potential
of the species is not surprising. Since the metabolic profile of
fungi is subject to the growth medium (Kokkonen et al.
2005), it is also possible that the YES medium used is not very
suitable for OTA production by A. niger. Contamination of
Nigerian foods commodities by high levels of hepatocarcinogenic AF and nephrotoxic OTA (Bankole and Adebanjo 2003)
is related to the abundant presence of aflatoxigenic and ochratoxigenic fungi as seen in this study, and is likely to be associated with increased incidences of human primary liver
cancer (Olubuyide and Solanke 1990) and chronic renal failures (NAN 2008) experienced in the country.
Fusarium contamination of rice is documented in Nigeria
(Ngala 1983; Makun et al. 2007; Amadi and Adeniyi 2009)
and other parts of the world (Reddy et al. 2006). Ngala (1983)
found F. verticillioides as the second major contaminant of
Nigerian rice. Of the five species of Fusarium isolated in this
study, four, namely F. proliferatum, F. verticillioides, F. chlamydosporum and Fusarium sp., were shown in this mycological
study to be mycotoxigenic producing FB, ZEA, DON and T-2
on YES. F. proliferatum and F. verticillioides, which were
shown herein as producers of FB1 and FB2, are not only
important toxin-producing fungi associated with maize
worldwide (Dutton 1996; Kpodo et al. 2000; Marasas 2001),
but have been isolated from other food commodities (Reddy
et al. 2006) including rice (Pitt and Hocking 1997; Pacin et al.
2002; Makun et al. 2007; Maheshwar et al. 2009). In fact, Park
et al. (2005) found F. proliferatum as the most frequent
Fusarium spp. in rice. The potentials of these fungi to synthesize FB1, which is linked to increased incidence of human
344
H.A. MAKUN ET AL.
esophageal cancer in South Africa (Marasas et al. 1988;
Sydenham et al. 1990) and China (Chu and Li 1994; Wang
et al. 1995) and equine leukoencephalomalacia and porcine
pulmonary edema (PPE) (Marasas 2001), should be of public
health concern to the population of Nigeria. ZEA and the trichothecenes (TH)-DON and T-2 are also toxic metabolite
products of the Fusarium spp. of the Nigerian rice samples.
Though they are not acutely toxic per se, their presence in
cereals has been associated with certain animal and human
diseases. For example, ZEA, an estrogenic toxin that causes
infertility in animals, is associated with outbreaks of precocious pubertal changes in children in Puerto Rico and has
been suggested to have a possible involvement in human cervical cancer (Zinedine et al. 2007), while the TH, which are
protein inhibitors, immunosuppressants, cause death due to
internal hemorrhage in animals and man (Sudakin 2003).
The genus Penicillium was a rare group in the studied rice
samples. And even the only species of this family, Penicillium
oxalicum that was found in 2 of the 21 rice samples is the more
ubiquitous member of the genus Penicillium and a normal
representative of the mycobiota of the soil (Pitt and Hocking
1999). It can be reasonably inferred that its notable occurrence here and its widespread incidence in tropical commodities including rice (Pitt and Hocking 1997) is expected and
normal. Though the metabolites of this fungus was not identified in this study, secalonic acid D is reported as the major
metabolite of P. oxalicum and despite the fact that there has
been contradictory experimental reports on its toxicity, its
role in causing certain human and animal diseases is yet to be
ascertained (Pitt and Hocking 1997). The practical rarity of
Penicilli in this mycological study is supported by the findings
of Amadi and Adeniyi (2009) who did not find any Penicillium spp. and their toxins in Nigerian rice from the same
agroecological region studied herein. It can therefore be reasonably supposed that OTA in rice from Nigeria is produced
by A. ochraceus. Penicillium spp. primarily produce OTA in
temperate climates, whereas A. ochraceus are more commonly
associated with warmer climates (Sweeney and Dobson
1998).
The near absence of Penicilli from Nigerian rice is the fundamental difference between the fungal profile of Nigerian
rice and that from other major rice exporting countries of
south east of Asia such as Thailand, the Philippines, Vietnam,
Taiwan, Nepal, Sri Lanka, Bangladesh and Indonesia and
Nepal. While all the fungal species isolated in this survey have
been found in Asian rice, the Penicillium species especially P.
citreonigrum, P. islandicum and P. citrinum alongside their
toxins; luteoskyrin, cyclochlorotine and citreoviridin, which
are common contaminants of Asian rice and linked to yellow
rice disease (Uraguchi and Yamazaki 1978; Gangopayay and
Chakraberti 1982; Garajapathy and Indira 1986; Rama Devi
et al. 1988; Waghray et al. 1988; Jayaraman and Kalyanasundaram 1990; Misra et al. 1995; Desjardins et al. 1999;
Journal of Food Safety 31 (2011) 334–349 © 2011 Wiley Periodicals, Inc.
H.A. MAKUN ET AL.
Udagawa and Tatsuno 2004; Park et al. 2005; Tanaka
et al. 2007; Reddy et al. 2008), may not likely be present in
Nigerian rice.
The genus Sarocladium are known rice pathogens as demonstrated in this investigation. Sarocladium oryzae (Sawada)
is a plant pathogen causing sheath rot disease in rice and also
produces such phytoxins as helvolic acid and cerulin, which
induce chlorosis and reduce seed viability and seedling health
in the infected grains (Sakthivel et al. 2002). Sarocladium
attenuatum has been shown to cause of rice grain spotting
(dirty panicles) in Nigeria (Ngala 1983). Although their
metabolites were not determined in this work, there is no
association of this group of fungi with mycotoxin production
or mycotoxicoses (Pitt and Hocking 1997). Acremonium spp.,
commonly referred to as Cephalosporium, are a ubiquitous,
cosmopolitan fungi with wide distribution among cereals,
maize being the most susceptible, have been shown not only
in this study as contaminants of rice, but in other studies (Pitt
and Hocking 1997). No mycotoxin has yet been ascribed to
this family of fungi in literature. Frequently low to moderate
infection of rice by Curvularia spp. as recorded in 7 of the 21
rice samples, is common in Nigeria (Ngala 1983; Makun et al.
2007) and elsewhere (Misra et al. 1995; Reddy et al. 2008).
The mycotoxins synthesized by this species of fungi are not
reported in this analysis but other reports indicate that they
indeed produce curvularin, a phytotoxin that inhibits cell
division by disrupting mitotic spindle formation (Kobayashi
et al. 1988). Cytochalasins are another class of mycotoxins
ascribed to Curvularia spp. that inhibit cytokinesis, protein
synthesis and cause pulmonary hemorrhage and brain edema
in mice (Visconti and Sibilia 1994).
Botryosphaeria diothidea is one important common tree
pathogen associated with die back and canker diseases of
woody plants, reducing the production of fruit crops such as
apricot, peach and pistachio (Li et al. 1995; Smith et al. 1996;
Ma et al. 2002; Slipper et al. 2004). While its ubiquitous
nature particularly in forest areas could explain its interesting
and unexpected presence in the present work, its low incidence could therefore, be related to the fact that cereals, rice
inclusive, are not its natural habitats but orchard trees. The
demonstration of Alternaria spp. as field rice pathogens
reported in this study is in conformity with the reports of
Makun et al. (2007) in the region under study and Manabe
and Tsuruta (1975) in Japan, who also found them as field
contaminants of rice. Members of this fungal family though
not demonstrated here, are known to produce a wide spectrum of mycotoxins with only the mutagenic altertoxins,
tenuazonic acid and cytochalasins providing adverse effects
on animal and human health (Visconti and Sibilia 1994).
Ascomycota or sac fungi, account for about 75% of all
described fungi including those found in this study.
Finally, it is worth mentioning here that the much higher
frequency of co-contamination of fungi within the same food
Journal of Food Safety 31 (2011) 334–349 © 2011 Wiley Periodicals, Inc.
MYCOTOXIGENIC FUNGI IN RICE FROM NIGERIA
matrix particularly those that produce AF, OTA and FB (A.
flavus, A. fumigatus, A. parasiticus, A. niger and F. proliferatum
and other Fusarium) as found in this study is of concern. With
such simultaneous occurrences therefore, it is highly likely to
find natural co-occurrences of unrelated mycotoxins (Rizzo
et al. 2004) in similar samples such as those already mentioned above. This is most likely the case especially when such
fungi are toxigenic as has been observed herein. This will certainly increase the severity of health-related problems generated from consumption of such contaminated food products
as consumption of multiple mycotoxin in foods may exert
both synergistic and additive effects (Placinta et al. 1999;
Casado et al. 2001; Creppy et al. 2004; Speijers and Speijers
2004; Luongo et al. 2008) in both animal and man. Rice is the
main stable food consumed in Nigeria particularly in the
urban settlements and data provided in this study show this
commodity is of low quality with respect to fungal contamination. However, more number of samples from Niger State,
Nigeria needs to be studied with regards to assessing fungal
contamination both in the field and during storage, which
may provide data that is critical in developing and predicting
the degree of mycotoxin contamination in rice from the
region for effective mycotoxin control. It is equally imperative
to study similar samples based on their mycotoxin profiles as
several of these samples were found to contain several toxigenic strains of fungi.
ACKNOWLEDGMENTS
This study was funded by the University of Johannesburg and
National Research Foundation of South Africa. The authors
wish to acknowledge the rural farmers of Niger State, Nigeria
that donated some of the rice samples in this study.
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HUSSAINI ANTHONY MAKUN1,2,4, MICHAEL FRANCIS DUTTON2, PATRICK BERKA NJOBEH2,
JUDITH ZANELE PHOKU2 and CLARENCE SUH YAH3
1
Department of Biochemistry, Federal University of Technology, P.M.B 65, Minna 920001, Niger State, Nigeria
Food, Environment and Health Research Group, Faculty of Health Science, University of Johannesburg, Doornfontein Campus, Gauteng, South Africa
3
Toxicology and Biochemistry Department, National Institute for Occupational Health (NIOH), 25 Hospital Road, Constitution Hill, Johannesburg, South
Africa
2
4
Corresponding author. TEL:
+2348035882233; FAX: +23466224482;
EMAIL: hussainimakun@yahoo.com
Accepted for Publication January 10, 2011
doi:10.1111/j.1745-4565.2011.00305.x
ABSTRACT
The study reports on the natural occurrence of fungi in 21 samples of field (10),
stored (6) and marketed (5) rice (Oryza sativa L.) collected from Niger State, Nigeria.
Fungal isolates were primarily identified based on morphological characteristics,
while representative isolates were characterized genetically. An evolutionary tree was
constructed from the resulting sequences of the isolated fungi. The toxigenic potentials of some of the isolated fungi were also determined. A total of 357 fungal isolates
of nine genera including Aspergillus, Fusarium, Sarocladium, Acremonium, Curvularia Botryosphaeria, Penicillium Alternaria and Ascomycota in decreasing order of
predominance were identified. The most frequent fungal contaminants of the rice
samples were A. flavus, A. fumigates, A. niger, A. parasiticus and F. proliferatum. All
strains of A. flavus (aflatoxins B1 and B2), A. parasiticus (aflatoxins B1, B2, G1 and G2),
A. ochraceus (ochratoxin A), F. proliferatum and F. verticillioides (fumonisins B1 and
B2) tested, were excellent producers of their respective mycotoxins. Patulin was produced by A. terreus, whereas deoxynivalenol, zearalenone and T-2 toxin were produced by F. chlamydosporum and other Fusarium spp. The increased prevalence of
toxigenic fungi in rice, a highly consumed food grain in Nigeria, poses serious health
concerns to the general public.
PRACTICAL APPLICATIONS
This study investigated the natural fungal occurrence of rice grown in Nigeria using
polymerase chain reaction-based techniques as well as the toxigenic potentials of
some of the identified fungi. The resultant fungal profile of rice, gene sequences of
the fungi detected in the survey, which were deposited in the GenBank and the constructed evolutionary tree, will serve as reference data for the incidence of fungal
species in rice and will help to evaluate the safety of Nigerian rice. It could be used in
developing and predicting the degree of mycotoxin contamination in rice from
Nigeria for effective mycotoxin control. The toxigenic fungi acquired from the work
will be excellent microbial sources for production of mycotoxin standards namely
aflatoxins BI, B2, G1 and G2, ochratoxin A and fumonisin B1 and B2.
INTRODUCTION
Rice (Oryza sativa) is one of the world’s most extensively cultivated crops and equally a staple food of over half of the
world’s total population (FAO 2002) with its consumption
increasing significantly in Africa within the last few decades
334
(Chang 1987). In Nigeria, this commodity is the sixth most
cultivated crop after Sorghum, millet, cowpea, cassava and
yam. Production of rice has increased over the years in
Nigeria with an estimated 4.7 million tonnes produced
in 2007, making it the second largest producer in Africa
with Egypt as principal producer (FAO 2008). Paradoxically,
Journal of Food Safety 31 (2011) 334–349 © 2011 Wiley Periodicals, Inc.
H.A. MAKUN ET AL.
Nigeria is the third major rice importing country after Iran
and the Philippines in spite of such productivity. Accordingly,
about 2 million tonnes of rice (USDA 2008) is imported and
about 5 million tonnes consumed annually (USDA 2008a).
In Nigeria, rice is produced in all six agroecological zones of
the country with the highest yield coming from the northern
guinea savannah (Ezedinma 2005). Niger State, a generally
hot and humid area with average annual temperature and
relative humidity of 31.7C and 51.6%, respectively, falls
within the guinea savannah region and after Kaduna State, it
is the second largest producer of rice (Erenstein and Lancon
2003) contributing approximately 16% of total rice production (Ezedinma 2005). However, like for other food commodities, it is subjected to microbial contamination.
Toxigenic fungi can attack rice in the field and during
storage resulting in increased mycotoxin levels in this commodity. Aspergillus, Fusarium and Penicillium are the predominant fungal genera associated with food grains during
storage (CAST 2003). The ingestion of grains contaminated
with mycotoxins especially those that are nephrotoxic, immunotoxic, teratogenic and mutagenic can provoke acute and
chronic effects in man and animals ranging from disorders of
central nervous, cardiovascular, pulmonary and intestinal
tract systems to death (Hussein and Brasel 2001; Bhat and
Vasanthi 2003). The involvement of these toxins in human
hepatoma and esophageal cancer (Neal 1995; Richard 2007;
Shephard 2008), increased susceptibility to diseases especially
in children and childhood pre-five mortality with reduced life
expectancy (Sherif et al. 2009), is of major concern with
regards to public health.
Studies conducted to establish the prevalence of fungi
and their toxins in foods and feedstuffs and ensure a healthy
food supply to animals and humans are scanty in Nigeria
unlike the case may be for other countries of the world. Very
limited reports (Opadokun and Ikeorah 1979; Obidoa and
Gugnani 1992; Ikeorah and Okoye 2005; Ayejuyo et al. 2008;
Amadi and Adeniyi 2009) on fungi and mycotoxins contaminations of rice from some parts of Nigeria are available.
Except for Makun et al. (2007), there is no information
on fungi and mycotoxins in rice from Niger State, Nigeria.
The previous survey (Makun et al. 2007) used conventional
fungi identification technique that invariably necessitated
more comprehensive surveys using accurate standardized
methods. The present study was, therefore, conducted to
examine the distribution and phylogeny of fungi in rice
from Niger State as well as their ability to produce mycotoxins using polymerase chain reaction (PCR)-based technique and thin-layer chromatography (TLC), respectively.
Knowledge on the toxigenicity of common fungal contaminants of rice is vital in elucidating animal and human
mycotoxicoses expected from such a commodity and will
equally be helpful in addressing the problem of mycotoxin
control in Nigeria.
Journal of Food Safety 31 (2011) 334–349 © 2011 Wiley Periodicals, Inc.
MYCOTOXIGENIC FUNGI IN RICE FROM NIGERIA
MATERIALS AND METHODS
Materials
All chemicals used were of analar grade unless otherwise
stated.
(1) Antibiotics: streptomycin and chloramphenicol (Sigma,
Aldrich).
(2) All chemicals for PCR analysis including the Fungal/
Bacterial DNA extraction kit were purchased from Zymo
Research Corporation, Irvine, CA.
(3) Strong anion exchange (SAX) cartridge (ANATECH,
Gauteng, South Africa).
(4) Mycotoxins standards: Aflatoxins B1, B2, G1 and G2, ochratoxin A (OTA), zearalenone (ZEA), deoxynivalenol (DON),
T-2 toxin (T2) and patulin (PAT) reference standards were
obtained from Sigma, St. Louis, MO. Fumonisin B1 (FB1), B2
and B3 were purchased from PROMEC, MRC, Tygerberg,
South Africa.
Sampling
Twenty-one representative rice samples from the fields (10),
storage facilities (6) and market outlets (5) were randomly
collected through donations and purchases in December
2008 from 21 villages in the traditional rice growing area of
Niger State, Nigeria. Samples were collected from separate
batches by thorough mixing of the contents of traditional
storage facilities and market containers to obtain homogeneity and representative samples collected from the top, middle
and bottom of the containers. In the case of field samples,
between seven to ten bunches of rice from stalks at the front,
middle and back of the farm were randomly collected and
thoroughly mixed to give a sample. Samples (about 0.5 kg
each) were put in sealed plastic bottles and transported to our
laboratory in South Africa where they were finely milled to
pass through a no. 20 sieve and stored in the deep freezer at
-20C for a week until analyzed.
Isolation and Identification of Fungi
The mycological analytical procedure involving four steps
according to the method of Kaufman et al. (1963) was used
including fungal isolation on potato dextrose agar (PDA) and
Ohio agricultural and experimental station agar (OAESA),
subculturing on PDA, malt extract agar (MEA) and Czapek
yeast extract agar (CYA), macro- and microscopic identification and finally, phylogenetics of fungi. The phylogeny of
fungi was determined following DNA extraction, PCR amplification, purification of PCR product, product quantification
and DNA sequencing for a confirmation of various species of
fungi. The culture media used (PDA, OAESA, MEA and CYA)
were prepared as described by Atlas (2004).
335
H.A. MAKUN ET AL.
MYCOTOXIGENIC FUNGI IN RICE FROM NIGERIA
Primarily, each milled sample was subjected to a six-level
serial dilution technique in which 1 g was diluted in a 9-mL
ringer solution, vortexed and subsequently, 1 mL of the suspension was transferred to a 9-mL ringer solution and vortexed, and so forth. One milliliter of each suspension was
inoculated on solid PDA and OAESA in 90-mm Petri dishes
and incubated at 25C for 7–14 days. Between the 5th and 7th
day of incubation, all colonies were counted using a colony
counter and results presented as number of fungal colonies
per gram of sample calculated and expressed in colonyforming units per gram (cfu/g).
The fungi so screened were subcultured on CYA, MEA and
PDA, incubated at 30C for 7–14 days and identified to species
level where possible. In this case, the hyphae and conidia from
each colony representing each fungal species were transferred
aseptically on three spots diagonally on each Petri dish containing the medium. Identification to species level was done
based on the macroscopic and microscopic characteristics of
the isolates following the identification keys of Klich (2002)
for Aspergillus spp. Pitt and Hocking (1997) for Penicillium
and Nelson et al. (1983) for Fusarium spp. Isolates were
subcultured on PDA slants and stored at 4C until further
analyzed.
Eight of the 48 fungi isolated from the rice samples were
precisely identified by this conventional method. These
include Aspergillus niger, A. parasiticus and A. penicillioides
and A. flavus. Isolates were further identified at the Inqaba
Biotechnological Laboratories, Pretoria, South Africa by
comparison of the nucleic acid profiles of individual fungal
species as described by Samson et al. (2004). To this end, the
mycelia of isolates on PDA slants were subcultured for 7
days as previously described and mycelia harvested, freezedried and then subjected to DNA extraction, PCR amplification, purification and quantification of PCR product, DNA
sequencing and analysis as described by Samson et al.
(2004) and Geiser et al. (2004) with some modifications.
Accordingly, a Fungal/Bacterial DNA extraction kit (Zymo
Research Corporation, Irvine, CA) was used for DNA
extractions in addition to MSB Spin PCRapace (Invitek
GmbH, Berlin, Germany) that was used for ultrafast purification and concentration of PCR-fragments. The freezedried cultures were thawed for 1 h and genomic DNA
extracted. To this effect, 60 mg sample was weighed and
resuspended in 200 mL phosphate-buffered saline (PBS)
contained in a 1.5-mL ZR Bashing Bead™ lysis tube (Zymo
Research Corporation, Irvine, CA). This tube was then
placed in a genie disruptor for 5 min and then centrifuged at
10,000¥ g for 1 min. The supernatant was recovered in a
Zymo-Spin™ IV spin filter placed in a 1.5 mL Eppendorf
tube, again centrifuged at 7,000¥ g for 1 min, filtered into a
collection tube and 1,200 mL of fungal/bacterial DNA
binding buffer added and vortexed. Eight-hundred microliters of the mixture was transferred to a Zymo-Spin™ IIC
336
column placed in a collection tube, centrifuged for 1 min at
10,000¥ g and the supernatant discarded (¥2). A 200-mL
aliquot of DNA pre-wash buffer I was added to the ZymoSpin™ IIC column in a new collection tube, centrifuged at
10,000¥ g for 1 min and filtrate discarded, while retaining
the column, which was then placed into a new tube. Into the
Zymo-Spin™ IIC column, 500 mL fungal/bacterial DNA
wash buffer II was added and again centrifuged at 10,000¥ g
for 1 min. The Zymo-Spin™ column was transferred to a
sterile 1.5 mL Eppendorf tube and 100 mL DNA elution
buffer added directly to the column matrix, centrifuged at
10,000¥ g for 30 s and DNA eluted and preserved for PCR
analysis.
The PCR procedure followed is covered by US Patents
4,683,195 and 4,683,202 (Hoffmann-LaRoche AG, Basel,
Switzerland). The primers (ITS_1 and ITS_4) used were
synthesized at a 0.01-mM scale and purified using reversephase cartridge purification (Inqaba). These primers were
resuspended in 2 mM TE buffer prepared from a stock solution concentration of 100 mM. PCR was performed using
the Fermentas 2 X PCR mix (Fermentas Life Science,
Lithuania). The PCR mixture for each sample consisted of
25 mL of 2 X PCR mix, 1 mL each of 2 mM primers, 1 mL of
DNA (final concentration of 10 mM), and constituted to a
final volume of 50 mL with nuclease free water. A negative
control, containing all of the reagents used except the DNA
was also prepared. PCR was performed using an Eppendorf
96-well Thermocycler (Eppendorf, Westbury, NY). The PCR
cycling conditions were set as follows: Pre-dwelling at 95C
for 3 min, 35 cycles denaturation at 95C for 1 min, annealation at 58C for 45 s, extension at 72C for 1 min 30 s, postdwelling at 72C for 10 min and held at 4C until samples
were retrieved.
The PCR products were further analyzed on an ABI
PRISM 3700 Genetic analyzer (AB, Applied Biosystems,
Nieuwerkerk a/d Yssel, the Netherlands). The forward and
reverse sequences of the PCR products were assembled with a
DYEamic ET Terminator Cycle Sequencing Kit (Amersham,
Bioscience, Roosendaal, the Netherlands) using the programs
SeqMan and EditSeq from the LaserGene package (DNAStar,
Inc., Madison, WI). DNA sequences and identities of fungi
were obtained from Inqaba Finch server.
Phylogenetic Analysis
Sequences of the rice fungal isolates in FASTA format
obtained from Inqaba Finch server were further subjected to
identification on the PCR amplification of the 16S and internal transcribed spacer (ITS_4) regions using the GenBank
Blast. The evolutionary tree was constructed using PHYLIP
package (version. 3.6) from http://evolution.genetics.
washington.edu/phylip.html and the evolutionary distances
matrix generated.
Journal of Food Safety 31 (2011) 334–349 © 2011 Wiley Periodicals, Inc.
H.A. MAKUN ET AL.
Determination of Mycotoxin-Producing
Potentials of Fungi Isolated
Each strain representing the fungal species of Aspergillus,
Penicillium and other genera with the exception of Fusarium
were further tested for toxigenicity to determine their ability
to produce the following mycotoxins: aflatoxin B1 (AFB1);
aflatoxin B2 (AFB2); aflatoxin G1 (AFG1); aflatoxin G2
(AFG2); OTA; and PAT. These isolates were cultured individually on solid yeast extract sucrose agar (YES) agar in a
90-mm Petri dish and incubated at 25C for 28 days according to the method of Singh et al. (1991). Mycotoxins synthesized by each fungus were extracted by dissolving 5 g of
isolate including the medium in 10 mL of dichloromethane
(DCM). The crude extract obtained was filtered through a
Whatman no. 2V filter paper and the filtrate put in a screwcap vial, dried under a stream of N2 gas and stored at 4C
until analyzed.
The mycotoxins in the crude extracts were detected by a
two-dimensional TLC technique devised by Patterson and
Roberts (1979). To this end, extracts were reconstituted with
200 mL DCM, vortexed and 20 mL of the extract solution
spotted about 10 mm from the edge of a silica gel TLC plate. A
similar procedure was followed for mycotoxin standards as a
reference for detecting mycotoxins of interest. The developing
solvents for the first and second runs were DCM/ethyl
acetate/propan-2-ol (90:5:5, v/v/v) and toluene/ethyl acetate/
formic acid (6:3:1, v/v/v), respectively. After the second run,
plates were dried and visualized under ultraviolet radiation at
wavelength of 365 nm. For PAT detection, plates were sprayed
with 0.5% 3-methyl-2-benzothiazolinone hydrazone hydrochloride solution and heated at 120C for 3 min. PAT appears
as a yellow spot with an orange fluorescence. Visual comparison of retention factor (RF) values and fluorescing color of
spots of extracts to that of standards was the basis for identification of toxins.
The toxigenic potentials of the isolated Fusarium spp. in
producing ZEA, DON, T-2, FB1 and FB2 were evaluated. Representative isolates of each Fusarium spp. were further plated
on solid YES agar and incubated at 25C for 28 days. Fusarium
mycotoxins were extracted according to the method of
Hinojo et al. (2005) with some modifications following
clean-up procedures depending on the type of mycotoxin to
be determined. Fusarium toxins in 10 g of agar-containing
mycelia was extracted into 25 mL CH3OH/H2O (60/40, v/v)
and shaken on a mechanical shaker for 1 h. The entire content
was filtered through a Whatman no. 2V filter paper to obtain
the crude extract for each fungus. For FB analysis, the extract
was stored at 4C until used. ZEA, DON and T-2 toxins were
extracted three times from the crude extracts with 25 mL of
DCM. The bottom layer was passed through a bed of Na2SO4
anhydrous into a 500 mL round bottom flask and dried by
rotary evaporation. The content was then reconstituted with
Journal of Food Safety 31 (2011) 334–349 © 2011 Wiley Periodicals, Inc.
MYCOTOXIGENIC FUNGI IN RICE FROM NIGERIA
200 mL DCM into a 0.5-mL screw-cap vial, dried by passing
through a stream of N2 gas and then stored at 4C until
analyzed.
Methanol was used for fumonisin extraction. The clean-up
method of Sydenham et al. (1992) was adopted for FB analysis. The stored filtrate was passed through a SAX column
cartridge (ANATECH) previously conditioned with 5 mL
methanol followed by 5 mL methanol : water (3:1, v/v). The
column was washed with 8 mL methanol : water (3:1, v/v)
and then 3 mL methanol. The absorbed FB was then eluded
with 10 mL 1% acetic acid in methanol. The eluent was
evaporated to dryness and the residue stored in a screw-cap
vial at 4C until analyzed.
A two-dimensional TLC technique (Patterson and Roberts
1979) as previously described was used for the detection of
ZEA, DON and T-2 toxin. Here, dried extracts were reconstituted with 200 mL DCM and 20 mL was applied to the origin
of a two-dimensional TLC plates as previously described but
this time, those for ZEA analysis were run twice in DCM : acetone (9:1, v/v) and derivatized with cold dianisidine reagent
prepared according to Malaiyandi et al. (1976). The mobile
solvents for trichothecenes were DCM/ethyl acetate/propan2-ol (90:5:5, v/v/v) and toluene/ethyl acetate/formic acid
(6:3:1, v/v/v), respectively. Plates were derivatized with chromotropic acid (Baxter et al. 1983), while those for fumonisin
analysis were developed in dicholoromethane/methanol/
acetic acid (80:20:2 v/v/v) and butanol/water/acetic acid
(12:5:3 v/v/v), respectively. Fumonisins were visualized as
purple spots on dried plates that were sprayed with
p-anisaldehyde reagent, followed by heating for 3 min at
120C. The retardation factors (RF1 and RF2) and color of the
individual spots on TLC were calculated and compared with
those of standard mycotoxins to aid in the identification of
mycotoxins present.
Statistical Analysis
Data on the cfu were subjected to statistically analysis. An
analysis of variance was used to derive mean values and standard deviation using SigmaStat 3.5 for Windows (Systat Inc.,
San Jose, CA, 2006), which were compared by least significant
difference. Mean values were deemed to significantly differ if
P ⱕ 0.5.
RESULTS
PCR Analysis
The results of the evolutionary history and identities of
the rice fungi based on 18S ribosomal RNA gene, partial
sequence; internal transcribed spacer 1, 5.8S ribosomal RNA
gene and internal transcribed spacer 2, complete sequence;
and 28S ribosomal RNA gene, partial sequence are presented
337
MYCOTOXIGENIC FUNGI IN RICE FROM NIGERIA
in Fig. 1 and Table 1. PCR was successfully performed and
bands (550 bp) were obtained. Each sequence was compared
with those of the same species deposited at GenBank. Similarity distances ranged from 0.010 to 0.452. Total nucleotides
amplified were 470–533 bp and corresponded to the ITS_4
complete regions; the 3, portion of the 18S gene, 5.8S complete sequence, and the 5, end of the 28S gene. The sequences
and names of fungi processed by Inqaba Finch server were in
complete conformity with our GenBank blasting results
except in two instances. The sequence of HM 23, which was
identified by Inqaba as Curvularia sp., was identified in the
GenBank as Curvularia affinis (AF071335.1) with a query
coverage of 51% and maximum identity of 99%. Similarly,
the sequence data of HM 34 that was blasted as only Fusarium
sp. by Inqaba was identified in the GenBank as Pseudofusarium purpureum strain MUCC 248 (EU301058.1) with
an 84% query coverage and maximum identity of 99%. The
identities obtained for these isolates from the GenBank were
therefore adopted forthwith in this study. HM 22 failed to
grow enough and HM 25 was a mixed culture, which did not
allow for PCR analysis of the isolates so their identities as
Alternaria and Curvularia spp. obtained via conventional
methods based on morphological characterization were
adopted.
Fungal Contamination
The survey generated data on the fungal contamination of
rice samples that are presented in Table 2. A total of 357 fungi
belonging to nine different fungal genera including Aspergillus, Fusarium, Penicillium, Acremonium, Alternaria, Ascomycota, Botryosphaeria, Curvularia and Sarocladium, were
isolated and identified in the survey. In overall, Aspergillus
(62.75%) was the most predominant fungal genera identified
followed by Fusarium (21.85%) in addition to the less frequent members of Sarocladium (3.92%), Acremonium
(3.64%), Curvularia (3.08%), Botryosphaeria (1.68%), Penicillium (1.12%), the Alternaria (0.56%) and Ascomycota
(0.56%) that were also isolated. The calculated mean
⫾ standard deviation values of the cfu for the different
types of samples, though not significantly different
(P ⱕ 0.05), showed lower fungal contamination in field
(2.6 ¥ 102 ⫾ 5.0 ¥ 102) than stored (5.0 ¥ 102 ⫾ 1.6 ¥ 102)
and marketed (5.0 ¥ 103 ⫾ 2.0 ¥ 102) samples, an indication
of increasing fungal contamination from field to storage and
subsequently market.
Although, there was no clear distinction between field and
storage fungi, some species were strictly field or storage fungi.
All isolates each of A. aculeatus, A. niveus, two of A. terreus, A.
tubingensis, Alternaria sp. and Ascomycota sp. were considered
field fungi, whereas all isolates of A. oryzae (6), A. sclerotiorum
(3), Penicillium oxalicum (4), F. chlamydosporum (2), F.
338
H.A. MAKUN ET AL.
pseudonygamai (2) and F. verticillioides (4) were found only in
the stored samples (stored and marketed samples) as seen in
Table 2.
Aspergillus members accounted for 227 of the total 357
fungi isolated with A. flavus being the most dominant. Fourteen species of Aspergillus were found among which A. flavus
was recovered in a total of 19 samples analyzed, recording an
incidence of 90.5%. The next sets of dominant members of
the Aspergillus were A. fumigatus and A. niger, each isolated
in 81% as well as A. parasiticus in 71% of samples. Other
members belonging to this genus had low incidence rates and
contamination by these species in decreasing order of predominance was A. ochraceus, A. unguis, A. candidus, A. oryzae,
A. pencillioides and A. terreus. For those isolated in only one
sample were A. aculeatus, A. niveus and A. sclerotiorum. The
three strains of Eurotium found in this study were all belonging to E. amstelodami being recovered from two rice samples.
The genus Fusarium was also frequently isolated, accounting for 78 of the 357 fungal isolates from rice reported herein
with F. proliferatum being the most dominant, occurring in 11
(52.4%) of the samples. In all, six members of this genus
were isolated alongside others including Fusarium sp.
(33.3%), Pseudofusarium purpureum (23.8%), F. verticillioides (14.3%), F. chlamydosporum (9.5%) and F. pseudonygamai (9.5%).
The third most dominant fungal genera found in the study
after Aspergillus and Fusarium is the Sarocladium occurring in
10/21 samples analyzed. The two species found namely S.
attenuatum and S. oryzae contaminated three and eight
samples, respectively. Other genera were of low incidence.
Acremonium sp. was found in 38% of the samples. Eleven isolates of two species of the Curvularia family contaminated 8
of the 21 rice samples. The species namely Curvularia sp. and
Curvularia affinis had incidence rates of 38 and 14.3%,
respectively. Six Botryosphaeria dothidea were isolated in four
samples, while the four isolates of the only species of Penicillium, P. oxalicum were found in two samples. The two
least predominant genera, Ascomycota (two isolates) and
Alternaria (two isolates) contaminated two and one samples,
respectively.
This mycological survey also showed that none of the
samples analyzed was free of fungal contamination with
samples having very high frequency of co-occurrences
of between 5 and 14 fungal species. The commonest of
such multioccurrences were the simultaneously contamination by A. flavus, A. fumigatus, A. parasiticus, A. niger and F.
proliferatum.
Mycotoxigenic Potentials of Fungal Isolates
Randomly selected representative isolates from each strain of
the species were screened for their toxigenic potentials. Data
on the toxigenicity of fungi tested in the present study are
Journal of Food Safety 31 (2011) 334–349 © 2011 Wiley Periodicals, Inc.
H.A. MAKUN ET AL.
MYCOTOXIGENIC FUNGI IN RICE FROM NIGERIA
FIG. 1. PHYLOGENETIC TREE INFERRED FROM ITS-4 GENE SEQUENCE OF FUNGAL ISOLATES FROM RICE IN NIGER STATE, NIGERIA IN RELATION TO
REFERENCE CULTURES IN THE LITERATURE
Journal of Food Safety 31 (2011) 334–349 © 2011 Wiley Periodicals, Inc.
339
H.A. MAKUN ET AL.
MYCOTOXIGENIC FUNGI IN RICE FROM NIGERIA
TABLE 1. LIST OF FUNGAL ISOLATES IN RICE FROM NIGERIA IN RELATION TO REFERENCE CULTURES
Species name
Aspergillus
A. aculeatus
A. aculeatus
A. candidus
A. candidus
A. candidus
A. candidus
A. flavus
A. flavus
A. flavus
A. flavus
A. fumigatus
A. fumigatus
A. fumigatus
A. fumigatus
A. niveus
Fennellia nivea
A. ochraeus
A. ochraceus
A. oryzae
A. oryzae
A. oryzae
A. oryzae
A. sclerotiorum
A. sclerotiorum
A. terreus
A. terreus
A. tubingensis
A. awamori
A. unguis
A. unguis
E. amstelodami
E. amstelodami
Fusarium
F. chlamydosporum
Fusarium sp
F. proliferatum
F. proliferatum
F. pseudonygamai
F. pseudonygamai
Fusarium sp
Pseudofusarium purpureum
Fusarium sp
Fusarium sp
F. verticillioides
F. verticillioides
F. verticillioides
Gibberella moniliformis
Penicillium
P. oxalicum
Penicillium sp
Acremonium
Acremonium sp
Acremonium sp
Acremonium sp
Acremonium sp
340
Accession No
Geographic origin
Host plant/code
HM 6
EU645715.1
HM 15
HM 36
FJ441640.1
AY373843.1
HM 7
HM 20
HM 21
FJ011545.1
HM 17
HM 24
HM 28
FJ844610.1
HM 19
FJ155814.1
HM 48
FJ878632.1
HM 44
HM 46
FJ654485.1
FJ654483.1
HM 35
AY373866.1
HM 14
GQ461901.1
HM 13
EF151436.1
HM 27
FJ878626.1
HM 16
GQ120984.1
Tasaba, Nigeria
Oryzae sativa F2
Seafan Aspergillosis
Oryzae sativa, F8
Oryzae sativa M 20
Dama, Nigeria
Lafiyagi, Nigeria
Xiamen, China
USA
Maitumbi, Nigeria
Tawi, Nigeria
Bida, Nigeria
Southern China
Pigi, Nigeria
Kwarkwata, Nigeria
Innagi, Nigeria
China
Dama, Nigeria
Mexico
Gbadadan, Nigeria
Greece
Danzariya, Nigeria
Ekosa, Nigeria
Indian Western Ghats
Indian Western Ghats
Lafiyagi, Nigeria.
USA
Maitumbi, Nigeria
Greece
Dama, Nigeria
China
Shata, Nigeria
Greece
Gbadadan, Nigeria
Mediterranean Sea
Kwarkwata, Nigeria
Malaysia
Rimi, Nigeria
China
Danzariya
China
Kodoko, Nigeria
Dust samples
Oryzae sativa F1
Oryzae sativa S 13
Oryzae sativa M 19
Reference
Zuluaga et al. (unpublished)
Zhang and Huang, 2008
Haugland et al. 2004
Xi et al. (unpublished)
Oryzae sativa F3
Oryzae sativa S15
Oryzae sativa M18
Zhang and Shi, 2009
Oryzae sativa F8
Orange peel
Oryzae sativa S14
Solis et al. 2008
Arabatzis and Velegraki (unpublished)
Oryzae sativa S16
Oryzae sativa M17
Megamai forest
Megamai forest
Oryzae sativa M20
Dust samples
Oryzae sativa F1
Venkatesan and Muthuchelian (unpublished)
Venkatesan and Muthuchelian (unpublished)
Haugland et al. 2004
Arabatzis and Velegraki (unpublished)
Oryzae sativa F8
Xie and Jian (unpublished)
Oryzae sativa F6
Arabatzis and Velegraki (unpublished)
Oryzae sativa F8
Marine algae
Larriba et al. (unpublished)
HM 18
GQ352485.1
HM 33
FJ040179.1
HM 47
FJ154075.1
HM 34
EU301058.1
HM 41
GU257906.1
HM 29
HM 40
HM 42
GU257904.1
Kwarkwata, Nigeria
Ekosa, Nigeria
Bida, Nigeria
India
HM 8
EU301633.1
Innagi, Nigeria
China
Oryzae sativa M18
Forest soil
Zhou et al. (unpublished)
HM 30
HM 31
HM 38
GU055562.1
Kodoko, Nigeria
Tawi, Nigeria
Bida, Nigeria
Austria
Oryzae sativa F7
Oryzae sativa S13
Oryzae sativa M19
Agricultural soil
Klaubauf et al. (unpublished)
Isheli, Nigeria
Oryzae sativa F15
Sim et al. 2009
Oryzae sativa. F4
Oryzae sativa
Oryzar sativa S16
Soil
Oryzae sativa F7
Oryzae sativa M21
India
Oryzae sativa S 15
Oryzae sativa M17
Oryzae sativa M19
Wang et al. (unpublished)
Zhao and Gao (unpublished)
Chandra et al. (unpublished)
Chandra et al. (unpublished)
Journal of Food Safety 31 (2011) 334–349 © 2011 Wiley Periodicals, Inc.
H.A. MAKUN ET AL.
MYCOTOXIGENIC FUNGI IN RICE FROM NIGERIA
TABLE 1. CONTINUED
Species name
Alternaria
A. azukiae
A. azukiae
Ascomycota
Ascomycota sp
Ascomycota sp
Botryosphaeria
B. dothidea
B. dothidea
B. dothidea
Curvularia
Curvularia sp
Curvularia affinis
Curvularia sp
Sarocladium
S. attenuatum
S. attenuatum
S. oryzae
S. oryzae
S. oryzae
Accession No
Geographic origin
Host plant/code
HM 10
FJ467366.1
Maitumbi, Nigeria
China
Oryzae sativa F1
HM 45
EU682958.1
Chanchaga, Nigeria
China
Oryzae sativa F10
HM 39
HM 43
GQ855797.1
Rimi, Nigeria
Isheli, Nigeria
East China
Oryzae sativa F4
Oryzae sativa M21
Apple
HM 23
AF071335.1
GQ184733.1
Gsada, Nigeria
Oryzae sativa F8
HM 37
AY566997.1
HM 26
HM 32
AY566996.1
Yikangbe, Nigeria
Oryzae sativa F5
Pigi, Nigeria
Tawi, Nigeria
Oryzae sativa F3
Oryzae sativa s13
Reference
Wu and Li (unpublished)
Sun et al. (unpublished)
China
Liu et al. (unpublished)
Zhang and Pan, 2009
Bills et al. 2004
Bill et al. 2004
The references and their sequences in this table were all obtained from the National Center for Biotechnology Information (NCBI) web site http://
blast.ncbi.nlm.nih.gov/Blast.cgi.
presented in Table 3. As found, all 10 strains of A. flavus tested
were excellent producers of AFB1 and AFB2. A similar observation was followed for A. parasiticus with all tested strains
producing both the B and G types of aflatoxins (AFB1, AFB2,
AFG1 and AFG2). OTA was recovered from cultures of A.
ochraceus tested. Other ochratoxigenic fungi isolated were
A. niger and A. sclerotiorum (HM 35). Two of the strains of A.
terreus both originally from field rice samples were shown to
produce PAT. Results of the toxicity screening of the Fusarium
spp. indicate that all the strains of F. proliferatum (HM 33)
and F.verticillioides (HM 29, 40, 40a and 42) produced FB1
and B2 (FB2). The two strains of F. chlamydosporum (HM 18)
with other Fusarium spp. (HM 41) were excellent producers
of DON and ZEA, while only one strain of Fusarium spp.
(HM 41) produced T-2 toxin. None of the species of Penicillium, Acremonium, Sarocladium, Curvularia Alternaria, Eurotium, Ascomycota and Botryosphaeria produced the toxins
tested. However, many of them produced metabolites that
could not be confirmed because of the lack of reference data.
DISCUSSION
This study provides the first comprehensive documentation
of the distribution and toxigenicity of fungal species contaminating rice from a major traditional rice producing
region of Niger State in Nigeria. Fungal species belonging to
nine genera viz: Asperillus, Fusarium, Penicillium, Acremonium, Ascomycota, Alternaria, Botryosphaeria, Curvularia and
Sarocladium were found to be major fungal contaminants of
Journal of Food Safety 31 (2011) 334–349 © 2011 Wiley Periodicals, Inc.
rice from the 21 villages under study. It was also shown in the
survey that fungal contamination increased from field to
storage with co-occurrence of toxigenic fungi being highly
frequent. Data from the toxigenic screening of the isolated
fungi indicate that there are many toxigenic strains recovered
from the rice samples that were producers of AFB1, AFB2,
AFG1 and AFG2, OTA, ZEA, DON, T-2, FB1 and FB2 under
laboratory conditions. The observed increase in fungal contamination from field to storage is expected as grains are
infected by the ubiquitous fungi in the field and under favorable conditions, the pre-harvest fungi proliferate during
storage (Ominski et al. 1994) with a resultant increase in
fungal concentration and subsequent mycotoxin formation.
The lack of distinction between field and storage with regards
to the fungal strains recorded in the rice samples is also
expected in tropical regions such as Niger State, where fungi
belonging to especially the genus Aspergillus occur in a
natural environment and colonize grains before harvest or
during period of drought stress and insect damage (Moss
1987). Therefore, fungi present on grains in the field could
easily persist during storage under water stress and insect
damage conditions (Mclean and Berjack 1987) such that
there is no definite demarcation between field and storage
fungi. Grain infection in the tropics therefore, is a continuous
process with available predominant species of fungi at any
time depending upon prevailing micro-environmental
conditions of the grain (Mycock and Berjack 1999). This
explains the noted incidence of typical storage fungi A.
aculeatus, A. niveus, A. terreus and A. tubingensis in the field
341
1
1
1
4
1
1
7
3
3
4
8
6
3
4
1
2
2
1
2
74
1
3
3
2
1
1
2
2
3
1
1
1
41
1
1
1
2
1
1
3
2
2
2
2
37
1
2
2
2
1
1
1
1
8
6
1
1
1
2
1
1
2
2
1
1
1
3
7
7
1
1
2
1
3
2
2
9
3
4
2
38
2
2
2
1
1
1
5
2
1
2
2
1
2
2
3
1
1
1
1
3
1
1
1
1
1
1
2
2
4
8
3
2
1
26
1
2
1
1
6
1
1
1
13
1
1
2
2
6
3
1
8
3
1
2
11
Total
1
Sarocladium oryzae
Curvularia sp.
1
Sarocladium attenuatum
Curvularia affinis
Botryosphaeria.dothidea
Alternaria sp
Ascomycota. sp
Alternaria azukiae
Fusaruim spp.
Pseudofusarium purpureum
1
2
2
3
3
30
1
1
2
1
1
2
1
3
1
2
3
4
2
3
1
1
1
8
1
3
2
6
2
5
Acremonium sp
1
11
2
2
1
2
1
1
F.verticillioides
2
F. pseudonygamai
1
F. proliferatum
1
Fusarium .chlamydosporum
1
Penicillium .oxalicum
Eurotium amstelodami
1
A. unguis
A. sclerotiorum
A. penicillioides
A. parasiticus
A. oryzae
1
1
1
1
A. tubingensis
6
6
2
3
3
A. terreus
10
1
2
4
5
7
6
A. ochraceus
4.2 ¥ 102
2.6 ¥ 102
8.0 ¥ 102
1.3 ¥ 104
1.7 ¥ 104
7.0 ¥ 103
1.7 ¥ 104
1.3 ¥ 104
5.0 ¥ 104
1.1 ¥ 104
1.1 ¥ 103
5.0 ¥ 102
1.1 ¥ 103
1.3 ¥ 104
1.2 ¥ 104
1.6 ¥ 104
8.0 ¥ 103
8.9 ¥ 103
2.0 ¥ 104
1.3 ¥ 104
5.0 ¥ 103
A. niveus
Field
Field
Field
Field
Field
Field
Field
Field
Field
Field
Store
Store
Store
Store
Store
Store
Market
Market
Market
Market
Market
A. niger
Maitumbi
Tasaba
Pigi
Rimi
Yikangbe
Shata
Kodoko
Dama
Gsada
Chanchage
Kodoko
Bupbe
Tawi
Gbadadan
Kwarkwata
Danzariya
Ekosa
Innagi
Bida
Lafiyagi
Isheli
A .fumigatus
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
Total
A. flavus
cfu g/mL
A. candidus
Type of sample
Aspergillus. aculeatus
Location of samples
29
21
12
19
14
14
11
13
17
19
11
16
23
19
18
22
16
17
15
16
15
357
H.A. MAKUN ET AL.
Journal of Food Safety 31 (2011) 334–349 © 2011 Wiley Periodicals, Inc.
S/no
MYCOTOXIGENIC FUNGI IN RICE FROM NIGERIA
342
TABLE 2. ABSOLUTE FREQUENCIES OF THE DIFFERENT FUNGAL SPECIES IN RICE FROM NIGERIA
H.A. MAKUN ET AL.
MYCOTOXIGENIC FUNGI IN RICE FROM NIGERIA
TABLE 3. MYCOTOXIGENIC POTENTIALS OF FUNGI ISOLATED FROM RICE IN NIGERIA
Fungi
No. of strains isolated
No. of strains tested
No. of positive strains
Type of mycotoxins produced
A. aculeatus
A. candidus
A. flavus
A. fumigatus
A. niger
A. niveus
A. ochraceus
A. oryzae
A. parasiticus
A. penicillioides
A. sclerotiorum
A. terreus
A. tubingensis
A. unguis
P. oxalicum
F. chlamydosporum
F. proliferatum
F. pseudonygamai
F. verticillioides
Fusaruim spp.
1
7
74
41
37
1
8
6
30
3
3
2
2
9
4
2
38
2
4
28
1
5
10
10
10
1
8
5
10
3
3
2
2
9
4
2
10
2
4
15
–
–
10
–
1
–
8
–
10
–
1
1
–
–
–
2
6
–
4
5
Pseudofusarium purpureum
Acremonium sp
Alternaria azukiae
Alternaria sp
Ascomycota. sp
Botryosphaeria.dothidea
Curvularia affinis
Curvularia sp.
Eurotium amstelodami
Sarocladium attenuatum
Sarocladium oryzae
6
13
1
1
2
6
3
8
3
3
11
6
5
1
1
2
5
3
5
3
3
3
–
–
–
–
–
–
–
–
–
–
–
Not detected
Not detected
AFB1 and AFB2
Not detected
OTA
Not detected
OTA
Not detected
AFB1, AFB2, AFG1 and AFG2
Not detected
OTA
Patulin
Not detected
Not detected
Not detected
ZEA and DON
FB1 and FB2
Not detected
FB1 and FB2
4 (ZEA + DON) and 1 (T-2
toxin)
Not detected
Not detected
Not detected
Not detected
Not detected
Not detected
Not detected
Not detected
Not detected
Not detected
Not detected
and occurrence of acclaimed field fungi; F. chlamydosporum,
F. pseudonygamai and F. verticillioides in stored samples.
However, there were some few exceptions to such observations in this study. Alternaria spp. being strictly field fungi and
A. oryzae (6), A. sclerotiorum (3), P. oxalicum found only in
storage samples in the present work is in conformity with the
classical grouping of fungi based on colonization period and
moisture requirement by earlier scientists (Javis 1971; Lillehoj 1973).
Aspergillus spp. being the most dominant fungal contaminants of Nigerian food commodities as proven in this survey
is well documented (Bankole and Adebanjo 2003; Bankole
et al. 2003; Atehnkeng et al. 2008). Two reports of Makun
et al. (2007) and Amadi and Adeniyi (2009) on fungi in Nigerian rice are also in agreement with the observations made
herein. Although the distribution of members of the Aspergillus family varied with sample type, i.e., field, store and market
samples, A. flavus was the most dominant species contaminating all rice samples. Similarly, high occurrence frequencies
of this fungus have previously been reported in Nigerian
Journal of Food Safety 31 (2011) 334–349 © 2011 Wiley Periodicals, Inc.
foods including rice by the aforementioned authors. The very
high incidence of A. flavus seen when compared with other
members of Aspergillus can be explained by the corresponding high levels of Aspergillus section Flavi in the soil, plant
debris and insects (Horn and Dorner 1999; Nesci and Etcheverry 2002; Jaime-Garcia and Cotty 2004), which serves as the
reservoir of inoculum for infection of grains in the field. As
reported, A. flavus is the most predominant member of
Aspergillus section Flavi in soils in West Africa (Cardwell and
Cotty 2002; Donner et al. 2006).
All the isolates of A. flavus screened for toxigenicity were
found to produce their attendant mycotoxins and this is in
agreement with the findings of Atehnkeng et al. (2008) who
found significantly higher incidence of toxigenic strains of
the species in Bida and Mokwa than in other parts of
Nigeria. These are the same regions that were also sampled in
the present study. The inherent high temperatures and drier
conditions of Niger State favor grain infection by A. flavus
with subsequent production of AF (Jones et al. 1981; Diener
et al. 1987), which could explain the very high toxigenic
343
MYCOTOXIGENIC FUNGI IN RICE FROM NIGERIA
potentials of the isolated A. flavus in this study. It could also
be that the agricultural management practices in the study
area have created unique ecological niches that promote
the toxigenic potential of strains of this species (Bilgrami
et al. 1981).
Other members of the Aspergillus reported in this study
have all been shown as common fungal contaminants of rice
from Nigeria (Amadi and Adeniyi 2009) and other parts of
the world (Udagawa 1976; Park et al. 2005; Reddy et al. 2006).
The A. parasiticus screened for toxigenicity were producers of
both the B and G types of AF. This assessment distinguished it
from other Aspergilli and served as a confirmation of its identity that there was no reason for it to be sent for confirmation
via PCR-based method. Apart from the AF producers, three
ochratoxigenic species belonging to the Aspergilli were also
isolated. In this case, A. niger, A. ochraceus and A. sclerotiorum
were shown in this work to be OTA producers as reported
elsewhere in literature (Pitt and Hocking 1997; Klich 2002),
whereas strains of A. ochraceus were profuse producers of
OTA; 50% of A. sclerotiorum tested were equally toxigenic
with only 1 of the 10 strains of A. niger screened found to be
ochratoxigenic. A. niger is usually regarded as a benign fungus
and toxin production does not seem to be common (Pitt and
Hocking 1997), so the recorded very low toxigenic potential
of the species is not surprising. Since the metabolic profile of
fungi is subject to the growth medium (Kokkonen et al.
2005), it is also possible that the YES medium used is not very
suitable for OTA production by A. niger. Contamination of
Nigerian foods commodities by high levels of hepatocarcinogenic AF and nephrotoxic OTA (Bankole and Adebanjo 2003)
is related to the abundant presence of aflatoxigenic and ochratoxigenic fungi as seen in this study, and is likely to be associated with increased incidences of human primary liver
cancer (Olubuyide and Solanke 1990) and chronic renal failures (NAN 2008) experienced in the country.
Fusarium contamination of rice is documented in Nigeria
(Ngala 1983; Makun et al. 2007; Amadi and Adeniyi 2009)
and other parts of the world (Reddy et al. 2006). Ngala (1983)
found F. verticillioides as the second major contaminant of
Nigerian rice. Of the five species of Fusarium isolated in this
study, four, namely F. proliferatum, F. verticillioides, F. chlamydosporum and Fusarium sp., were shown in this mycological
study to be mycotoxigenic producing FB, ZEA, DON and T-2
on YES. F. proliferatum and F. verticillioides, which were
shown herein as producers of FB1 and FB2, are not only
important toxin-producing fungi associated with maize
worldwide (Dutton 1996; Kpodo et al. 2000; Marasas 2001),
but have been isolated from other food commodities (Reddy
et al. 2006) including rice (Pitt and Hocking 1997; Pacin et al.
2002; Makun et al. 2007; Maheshwar et al. 2009). In fact, Park
et al. (2005) found F. proliferatum as the most frequent
Fusarium spp. in rice. The potentials of these fungi to synthesize FB1, which is linked to increased incidence of human
344
H.A. MAKUN ET AL.
esophageal cancer in South Africa (Marasas et al. 1988;
Sydenham et al. 1990) and China (Chu and Li 1994; Wang
et al. 1995) and equine leukoencephalomalacia and porcine
pulmonary edema (PPE) (Marasas 2001), should be of public
health concern to the population of Nigeria. ZEA and the trichothecenes (TH)-DON and T-2 are also toxic metabolite
products of the Fusarium spp. of the Nigerian rice samples.
Though they are not acutely toxic per se, their presence in
cereals has been associated with certain animal and human
diseases. For example, ZEA, an estrogenic toxin that causes
infertility in animals, is associated with outbreaks of precocious pubertal changes in children in Puerto Rico and has
been suggested to have a possible involvement in human cervical cancer (Zinedine et al. 2007), while the TH, which are
protein inhibitors, immunosuppressants, cause death due to
internal hemorrhage in animals and man (Sudakin 2003).
The genus Penicillium was a rare group in the studied rice
samples. And even the only species of this family, Penicillium
oxalicum that was found in 2 of the 21 rice samples is the more
ubiquitous member of the genus Penicillium and a normal
representative of the mycobiota of the soil (Pitt and Hocking
1999). It can be reasonably inferred that its notable occurrence here and its widespread incidence in tropical commodities including rice (Pitt and Hocking 1997) is expected and
normal. Though the metabolites of this fungus was not identified in this study, secalonic acid D is reported as the major
metabolite of P. oxalicum and despite the fact that there has
been contradictory experimental reports on its toxicity, its
role in causing certain human and animal diseases is yet to be
ascertained (Pitt and Hocking 1997). The practical rarity of
Penicilli in this mycological study is supported by the findings
of Amadi and Adeniyi (2009) who did not find any Penicillium spp. and their toxins in Nigerian rice from the same
agroecological region studied herein. It can therefore be reasonably supposed that OTA in rice from Nigeria is produced
by A. ochraceus. Penicillium spp. primarily produce OTA in
temperate climates, whereas A. ochraceus are more commonly
associated with warmer climates (Sweeney and Dobson
1998).
The near absence of Penicilli from Nigerian rice is the fundamental difference between the fungal profile of Nigerian
rice and that from other major rice exporting countries of
south east of Asia such as Thailand, the Philippines, Vietnam,
Taiwan, Nepal, Sri Lanka, Bangladesh and Indonesia and
Nepal. While all the fungal species isolated in this survey have
been found in Asian rice, the Penicillium species especially P.
citreonigrum, P. islandicum and P. citrinum alongside their
toxins; luteoskyrin, cyclochlorotine and citreoviridin, which
are common contaminants of Asian rice and linked to yellow
rice disease (Uraguchi and Yamazaki 1978; Gangopayay and
Chakraberti 1982; Garajapathy and Indira 1986; Rama Devi
et al. 1988; Waghray et al. 1988; Jayaraman and Kalyanasundaram 1990; Misra et al. 1995; Desjardins et al. 1999;
Journal of Food Safety 31 (2011) 334–349 © 2011 Wiley Periodicals, Inc.
H.A. MAKUN ET AL.
Udagawa and Tatsuno 2004; Park et al. 2005; Tanaka
et al. 2007; Reddy et al. 2008), may not likely be present in
Nigerian rice.
The genus Sarocladium are known rice pathogens as demonstrated in this investigation. Sarocladium oryzae (Sawada)
is a plant pathogen causing sheath rot disease in rice and also
produces such phytoxins as helvolic acid and cerulin, which
induce chlorosis and reduce seed viability and seedling health
in the infected grains (Sakthivel et al. 2002). Sarocladium
attenuatum has been shown to cause of rice grain spotting
(dirty panicles) in Nigeria (Ngala 1983). Although their
metabolites were not determined in this work, there is no
association of this group of fungi with mycotoxin production
or mycotoxicoses (Pitt and Hocking 1997). Acremonium spp.,
commonly referred to as Cephalosporium, are a ubiquitous,
cosmopolitan fungi with wide distribution among cereals,
maize being the most susceptible, have been shown not only
in this study as contaminants of rice, but in other studies (Pitt
and Hocking 1997). No mycotoxin has yet been ascribed to
this family of fungi in literature. Frequently low to moderate
infection of rice by Curvularia spp. as recorded in 7 of the 21
rice samples, is common in Nigeria (Ngala 1983; Makun et al.
2007) and elsewhere (Misra et al. 1995; Reddy et al. 2008).
The mycotoxins synthesized by this species of fungi are not
reported in this analysis but other reports indicate that they
indeed produce curvularin, a phytotoxin that inhibits cell
division by disrupting mitotic spindle formation (Kobayashi
et al. 1988). Cytochalasins are another class of mycotoxins
ascribed to Curvularia spp. that inhibit cytokinesis, protein
synthesis and cause pulmonary hemorrhage and brain edema
in mice (Visconti and Sibilia 1994).
Botryosphaeria diothidea is one important common tree
pathogen associated with die back and canker diseases of
woody plants, reducing the production of fruit crops such as
apricot, peach and pistachio (Li et al. 1995; Smith et al. 1996;
Ma et al. 2002; Slipper et al. 2004). While its ubiquitous
nature particularly in forest areas could explain its interesting
and unexpected presence in the present work, its low incidence could therefore, be related to the fact that cereals, rice
inclusive, are not its natural habitats but orchard trees. The
demonstration of Alternaria spp. as field rice pathogens
reported in this study is in conformity with the reports of
Makun et al. (2007) in the region under study and Manabe
and Tsuruta (1975) in Japan, who also found them as field
contaminants of rice. Members of this fungal family though
not demonstrated here, are known to produce a wide spectrum of mycotoxins with only the mutagenic altertoxins,
tenuazonic acid and cytochalasins providing adverse effects
on animal and human health (Visconti and Sibilia 1994).
Ascomycota or sac fungi, account for about 75% of all
described fungi including those found in this study.
Finally, it is worth mentioning here that the much higher
frequency of co-contamination of fungi within the same food
Journal of Food Safety 31 (2011) 334–349 © 2011 Wiley Periodicals, Inc.
MYCOTOXIGENIC FUNGI IN RICE FROM NIGERIA
matrix particularly those that produce AF, OTA and FB (A.
flavus, A. fumigatus, A. parasiticus, A. niger and F. proliferatum
and other Fusarium) as found in this study is of concern. With
such simultaneous occurrences therefore, it is highly likely to
find natural co-occurrences of unrelated mycotoxins (Rizzo
et al. 2004) in similar samples such as those already mentioned above. This is most likely the case especially when such
fungi are toxigenic as has been observed herein. This will certainly increase the severity of health-related problems generated from consumption of such contaminated food products
as consumption of multiple mycotoxin in foods may exert
both synergistic and additive effects (Placinta et al. 1999;
Casado et al. 2001; Creppy et al. 2004; Speijers and Speijers
2004; Luongo et al. 2008) in both animal and man. Rice is the
main stable food consumed in Nigeria particularly in the
urban settlements and data provided in this study show this
commodity is of low quality with respect to fungal contamination. However, more number of samples from Niger State,
Nigeria needs to be studied with regards to assessing fungal
contamination both in the field and during storage, which
may provide data that is critical in developing and predicting
the degree of mycotoxin contamination in rice from the
region for effective mycotoxin control. It is equally imperative
to study similar samples based on their mycotoxin profiles as
several of these samples were found to contain several toxigenic strains of fungi.
ACKNOWLEDGMENTS
This study was funded by the University of Johannesburg and
National Research Foundation of South Africa. The authors
wish to acknowledge the rural farmers of Niger State, Nigeria
that donated some of the rice samples in this study.
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