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African Journal of Biotechnology Vol. 8 (14), pp. 3303-3308, 20 July, 2009
Available online at http://www.academicjournals.org/AJB
ISSN 1684–5315 © 2009 Academic Journals
Full Length Research Paper
Aflatoxin B1 producing potential of Aspergillus flavus
strains isolated from stored rice grains
K. R. N. Reddy1*, P. Saritha2, C. S. Reddy2 and K. Muralidharan2
1Agroinnova, Centre of Competence for Innovation in the Agro-Environmental Sector, University of Torino, Torino, 10095
Grugliasco, Italy.
2Department of Plant Pathology, Directorate of Rice Research, Rajendranagar, Hyderabad- 500 030, Andhra Pradesh,
India.
Accepted 10 June, 2009
Aflatoxin B1 (AFB1) producing potential of different strains of Aspergillus flavus, isolated from 1,200
stored rice grains collected from 43 locations in 20 rice growing states in India was investigated.
Eighty-five strains of A. flavus were isolated from the discolored rice grains and tested for their AFB1
producing potential on different agar media. Among these, 43 strains were identified as AFB1
producers (ranging 0.2 – 40 µg/g agar). All the 43 strains of A. flavus produced AFB1 on yeast extract
sucrose agar media (YES). However, 65% of the strains produced AFB1 on Czapek’s agar, 53% of the
strains on potato dextrose agar (PDA) and none of the strains on Aspergillus flavus and parasiticus
agar media (AFPA). The strain, DRAf 009 produced maximum AFB1 (4.0 – 40 µg/g agar) on all the agar
media tested. Five strains of A. flavus producing high amounts of AFB1 identified in agar media were
evaluated for their AFB1 production on milled rice cultivars. The five strains produced AFB1 on all the
rice cultivars. Out of 5 strains, the DRAf 009 produced maximum AFB1 (386 – 415 µg/g grain) on all the
rice cultivars tested.
Key words: Rice, Aspergillus flavus, AFB1.
INTRODUCTION
Rice (Oryza sativa L.) is the most important staple food
crop in India and the bulk of rice is grown in kharif or wet
season. Frequent and heavy rainfall and floods particu-
larly near to harvest in coastal areas in eastern, southern
and western regions of the country, wet the crop and
make panicles more prone to invasion by Aspergillus sp.
(Reddy et al., 2004). In a preliminary study A. flavus iso-
lates from rice grains were shown to possess the ability
to produce AFB1 (Reddy et al., 2005). However, myco-
toxin producing fungi is less commonly reported for rice
than for many other cereal crops (Tanaka et al., 2007)
but rice represents a very good substrate for fungal grow-
th and toxinogenesis since it is used as an ideal culture
medium to test the toxigenic potential of isolated strains
(Bars and Bars, 1992). Among the aflatoxins, AFB1 is the
most toxic form for mammals and presents hepatotoxic,
∗Corresponding author. E-mail: drkrnreddy@gmail.com. Tel.:
0039-011-6706383.
teratogenic and mutagenic properties, causing damage
such as toxic hepatitis, hemorrhage, edema, immunosup-
pression and hepatic carcinoma (Speijers and Speijers,
2004). It has been classified as a class 1 human carcino-
gen by the International Agency for Research on Cancer
(IARC, 1993). Several disease outbreaks of aflatoxicosis
in humans and animals have been reported due to the
consumption of aflatoxin contaminated food and feed
(Reddy and Raghavender, 2007). Fouzia and Samajpati
(2000) isolated mycotoxin-producing fungi from contami-
nated grains of rice sold in the local markets of Calcutta,
India. Waghray et al. (1988) reported Aspergillus sp. as
the most predominant fungi in the grain samples of flood-
affected paddy variety NLR 9672 collected from standing
crop, threshing floors and storage sites in the Nellore
district of Andhra Pradesh, India. Almeida et al. (1991)
identified aflatoxigenic strains of Aspergillus in milled rice
from different regions of Brazil. Jayaraman and
Kalyanasundaram (1990) explored the rice bran for
toxigenic mycoflora and reported that A. flavus as the
major contaminant. Trung et al. (2001) had reported that
3304 Afr. J. Biotechnol.
Table 1. States and locations from where the seed was collected.
S/N State Locations No. of samples
1 Andhra Pradesh 6 257
2 Andaman and Nicobar 1 34
3 Assam 3 40
4 Bihar 2 24
5 Chattishgarh 1 11
6 Delhi 1 122
7 Gujarat 2 12
8 Jarkhand 2 21
9 Jammu & Kashmir 2 27
10 Karnataka 4 92
11 Kerala 2 294
12 Madhya Pradesh 1 19
13 Maharashtra 1 7
14 Meghalaya 1 9
15 Puducherry 2 47
16 Rajasthan 1 10
17 Tamil Nadu 4 85
18 Tripura 2 11
19 Uttaranchal 1 28
20 West Bengal 4 50
Total 43 1,200
A. flavus is the most predominant fungi in rice grains from
South Vietnam.
To date comprehensive studies on aflatoxin production
by the strains of A. flavus on different agar media and on
native solid substrates (rice grains) are limited. Therefore,
in our investigation, we have explored the production of
AFB1 by different strains of A. flavus isolated from disco-
lored rice grains in India.
MATERIALS AND METHODS
Rice seed sample collection
A total of 1200 discolored rice samples were collected from 43 loca-
tions in 20 rice growing states in India (Table1). The seeds collec-
ted were either from areas exposed to different weather conditions
or stored at various storage conditions:
(1) Seeds from the crop exposed to heavy rains and floods.
(2) Seeds from the submerged or damp conditions.
(3) Seeds stored in the godown for 1 - 4 years.
(4) Seeds from the grain market.
To avoid the sampling error due to highly heterogeneous nature of
fungal distribution, each 2 kg composite sample collected from one
storehouse was a mixture of 10 sub-samples (200 g each). Such
sub-samples were collected from five diagonal on each of the up-
per, middle and lower layers of each storehouse (Liu et al., 2006).
Isolation of aflatoxin producing A. flavus from seed samples
Using agar plate method (ISTA, 1966), A. flavus was isolated from
all the seed samples. Four hundred seeds from each sample were
plated on one-half strength potato dextrose agar (PDA) medium
containing Rose Bengal at 50 ppm (Cotty, 1994). The plates were
incubated at room temperature and the presence of A. flavus was
observed after 6 days. The A. flavus isolated from samples were
further purified individually by sub culturing PDA slants. They were
then identified according to Raper and Fennell (1965) and Klich
(2002).
Extraction of AFB1 from A. flavus strains grown on agar media
Eighty five strains of A. flavus isolated from rice grain samples were
grown on sterilized different agar media (AFPA, Czapeks agar, PDA
and YES agar) for 5 days at 25 ± 2°C. Three replications were
maintained for each isolate for each media. AFB1 were extracted
by grinding the moldy agar (20 g) in waring blender for 5 min with
methanol (100 ml) containing 0.5% potassium chloride (KCl) (Lee,
1965). The mixture was filtered through Whatman No.1 filter paper
and the clarified filtrate was concentrated in vacuum on a rotary
evaporator to 1 ml and identified on thin layer silica gel chromato-
graphy (TLC) for presence and absence of AFB1. Then the dried
residue was dissolved in minimum quantity of methanol and esti-
mated by indirect competitive ELISA.
Preparation of spore suspension
Five strains of high AFB1 producing A. flavus on agar media (DRAf
002, DRAf 006, DRAf 009, DRAf 012 and DRAf 018) isolated from
rice grains and maintained on potato dextrose agar slants at 4°C
were harvested by adding 10 ml of sterile distilled water to give a fi-
nal concentration of approximately 2 x 1012 spores/ml.
Extraction of AFB1 from A. flavus strains grown on rice grains
The high aflatoxin producing strains in agar medium was used for
this study. Polished milled rice grains from cultivars Koshihikari (ja-
ponica), Mugad Suganda (aromatic and scented indica), Pusa
Basmati and Rasi (non-scented indica) were soaked 2 h in tap
water (25 ml/50 g grain) and were sterilized by autoclaving. Fer-
mentation was carried out in 500 ml Erlenmeyer flasks containing
50 g of rice inoculated with 1 ml of spore suspension (2 x 1012 cfu/
ml) of five isolates of A. flavus for five days at 25 ± 2°C. Five iso-
lates of A. flavus (DRAf 002, DRAf 006, DRAf 009, DRAf 012 and
DRAf 018) from different samples collected across the country were
used and for each isolate six replications were maintained. Grains
with fungal growth (20 g) from individual cultures were blended in
waring blender for 5 min with methanol (100 ml) containing 0.5%
KCl (Reddy et al., 2009). The mixture was filtered through Whatman
No.1 filter paper and the clarified filtrate was concentrated in va-
cuum on a rotary evaporator to 1 ml and identified on thin layer sili-
ca gel chromatography (TLC) for presence and absence of AFB1.
Then the dried residue was dissolved in a minimum quantity of
methanol and estimated by indirect competitive ELISA.
Identification of AFB1 on TLC
Aflatoxin production was evaluated by standard TLC to confirm the
presence or absence of AFB1. Two replicates were analyzed by
spotting crude extract of aflatoxins. The TLC plates used were
coated with silica gel 60 F254 on aluminum sheet, 20 x 20 cm and
developed in chloroform/methanol (97:3) (Reddy et al., 2004). The
plates were then observed under UV light at 365 nm and compared
with standard aflatoxin spotted on the same plate.
Materials for ELISA
AFB1, AFB1-bovine serum albumin (BSA) conjugate, goat anti-rab-
bit IgG-ALP conjugate, -nitrophenyl phosphate and BSA used
were from Sigma, St Louis, USA, and microtiter plates (Maxi-sorp
F96) were from Nunc (Nalge Nunc International, Denmark). All
other chemicals were of reagent grade or chemically pure. Highly
specific polyclonal antibodies for AFB1 were purchased from Inter-
national Crop Research Institute for the Semi Arid Tropics, Patan-
cheru, India (Devi et al., 1999).
Estimation of AFB1 by ELISA
The AFB1 was estimated by the method of Reddy et al. (2009).
Microtiter plates were coated with 100 ng/ml of AFB1-BSA in
sodium carbonate buffer, pH 9.6 (150 µl/well) and left overnight at
4°C. They were then washed in PBST, added with BSA (0.2%) and
allowed to stand at 37°C for 1 h. ELISA plates were again washed
with PBST and added with 100 µl AFB1 standards ranging from 25
ng to 10 pg/ml. Pre-incubation was carried out with 50 µl antiserum
diluted in PBST-BSA (1:6000) and held for 45 min at 37°C. Filtrate
samples extracted from rice grains and agar media with aqueous
methanol-KCl as described earlier were added to wells at 1:10
dilution in PBST-BSA, that is, 100 µl/well. Goat antirabbit immuno-
globulins conjugate to alkaline phosphatase were used at a 1:4000
dilution to detect rabbit antibodies attached to AFB1-BSA.
ρ
-nitro-
phenyl phosphate was used as a substrate at 0.5 mg/ml. Absor-
Reddy et al. 3305
bance was recorded at 405 nm with an ELISA plate reader (Bio-
Rad-680) after incubation at 28°C for 45 - 60 min. Standard curves
were obtained by plotting log10 values of AFB1 dilutions at A405.
The AFB1 (ng/ml) in samples was determined from the standard
curves as: AFB1 µg/kg of agar or milled rice = [aflatoxin (ng/ml) in
sample x buffer (ml) x extraction solvent (ml)]/sample weight (g). In
order to test the recovery of AFB1, 20 g healthy rice grain was
mixed with pure AFB1 (Sigma, St Louis, USA) to give concentra-
tions ranging from 5 to 100 µg/kg. Rice samples were extracted and
assayed as for unknown samples.
RESULTS AND DISCUSSION
AFB1 production by A. flavus isolates on agar media
Out of 85 strains of A. flavus tested, 43 strains produced
AFB1 on TLC compared with standard at Rf 0.47. Other
strains of A. flavus did not produce any toxin on any me-
dia tested. The results on production of AFB1 by the
strains of A. flavus through ELISA revealed that the capa-
city to produce was in the range of 0.2 - 40 µg/g of agar.
All the strains of A. flavus (43) produced AFB1 on YES
media and none of the strains on AFPA. Isolates from
Tamil Nadu and Maharashtra produced high AFB1 on
agar media. One isolate of A. flavus (DRAf 009) from
Tamil Nadu produced maximum amount of AFB1 (40
µg/g) at 25°C, on YES agar media (Table 2). Sixty-five
percent of the isolates produced AFB1 on Czapeks agar
and 53% of the isolates on PDA agar media.
Fente et al. (2001) evaluated different agar (Czapeks,
YES, coconut agar, aflatoxin producing ability (APA) me-
dia, coconut extract agar and coconut cream agar) media
for aflatoxin production by Aspergillus sp. They reported
more aflatoxin production in YES media compared to
others. In our investigation we also found that YES agar
media is the best media for AFB1 production by A. flavus.
Manisha and Sandip (2003) isolated A. flavus strains
from rice mill surrounding air and tested aflatoxin pro-
ducing capability in Czapeks agar, APA media and CAM
agar. They found high aflatoxin production in Czapeks
media produced by A. flavus compared to other media. In
this study 65% of the A. flavus strains produced AFB1 on
Czapeks agar. Carballo and Miguel (1987) collected 133
samples (mixed feeds and cereal grains) were examined
for the incidence of strains of the A. flavus group. They
tested the ability to produce aflatoxin in all strains isolated
on cracked rice, APA medium and glucose-yeast extract
agar (GYA) medium. Ten of the 67 isolates of A. flavus
were aflatoxin-producing strains in rice and GYA medium;
only three were aflatoxin-positive on the APA test.
AFB1 production by A. flavus isolates on rice grains
Five strains of highest AFB1 producing A. flavus strains,
identified on agar media were evaluated for their potential
of AFB1 production on different rice cultivars. All the 5
strains produced AFB1 ranging from 9-415 µg/g of grains
on all the rice cultivars tested (Table 3). Among the five
3306 Afr. J. Biotechnol.
Table 2. Production of AFB1 (µg/g agar) by A. flavus strains isolated from stored rice grains on
agar media at 25±2°C.
Different agar media and AFB1 (µg/g agar) Isolates Place of collection
Czapeks YES PDA AFPA
DRAf 002 Tamil Nadu 0.5 6.4 - -
DRAf 006 Tamil Nadu 2.0 4.5 - -
DRAf 007 Andhra Pradesh 0.5 1.0 0.5 -
DRAf 009 Tamil Nadu 4.0 40.0 4.0 -
DRAf 010 Jammu & Kashmir 0.5 2.0 1.0 -
DRAf 011 Bihar 1.0 1.0 - -
DRAf 012 Maharashtra - 5.8 - -
DRAf 013 Delhi 1.0 0.5 1.0 -
DRAf 018 Maharashtra 1.0 5.0 2.0 -
DRAf 019 Pondicherry 2.0 0.5 2.0 -
DRAf 022 Assam - 1.5 - -
DRAf 026 Karnataka - 2.0 1.5 -
DRAf 027 Andhra Pradesh 0.5 0.2 - -
DRAf 029 Tamil Nadu - 1.5 0.5 -
DRAf 030 Maharashtra 1.0 3.0 2.0 -
DRAf 031 Chattishgarh - 1.5 1.5 -
DRAf 032 Pondicherry 0.5 0.5 3.0 -
DRAf 033 Jarkhand 1.0 0.5 - -
DRAf 038 Tamil Nadu - 2.0 - -
DRAf 039 Tripura 0.5 2.5 1.0 -
DRAf 040 Andhra Pradesh 2.0 0.5 - -
DRAf 042 West Bengal - 1.0 1.5 -
DRAf 043 Chattishgarh 0.2 1.0 1.0 -
DRAf 044 Tamil Nadu 1.5 0.5 - -
DRAf 046 Rajasthan 2.0 0.5 - -
DRAf 047 Madhya Pradesh - 2.0 1.5 -
DRAf 048 Kerala - 1.0 1.0 -
DRAf 049 Jarkhand - 2.5 - -
DRAf 050 Uttaranchal 0.5 0.2 1.5 -
DRAf 051 Andhra Pradesh 2.0 1.5 - -
DRAf 052 West Bengal - 1.0 0.5 -
DRAf 055 Tamil Nadu 1.0 0.5 0.5 -
DRAf 056 Gujarat 1.0 0.5 - -
DRAf 057 Kerala - 1.0 - -
DRAf 059 Maharashtra 2.0 1.0 1.0 -
DRAf 062 Pondicherry 1.0 1.5 - -
DRAf 065 Tamil Nadu 0.5 2.5 0.5 -
DRAf 068 Andhra Pradesh - 3.0 - -
DRAf 070 Rajasthan 1.0 1.0 0.5 -
DRAf 072 Bihar 0.5 0.2 - -
DRAf 078 Tripura - 1.0 0.5 -
DRAf 080 Tamil Nadu 1.0 0.5 - -
DRAf 081 Meghalaya - 1.5 - -
CD (P=0.05) 0.9 7.4 0.6 -
CV (%) 18.5 26.8 15.8 -
Czapeks, Czapeks agar; YES, yeast extract sucrose agar; PDA, potato dextrose agar; AFPA,
Aspergillus flavus and parasiticus agar; - = no toxin.
Reddy et al. 3307
Table 3. Production of AFB1 (µg/g rice grain) by A. flavus strains isolated from stored rice grains on rice
kernels.
Rice kernels and AFB1 (µg/g rice grain)
Isolate Place of collection
Koshihikari Mugad Suganda Pusa Basmati 1 Rasi
DRAf 002 Tamil Nadu 54 64 62 60
DRAf 006 Tamil Nadu 11 11 13 9
DRAf 009 Tamil Nadu 386 405 415 392
DRAf 012 Maharashtra 12 11 15 9
DRAf 018 Maharashtra 31 33 36 28
CD (P=0.05) 8.6 6.4 10.2 4.8
CV (%) 1.8 3.2 1.4 1.2
strains, DRAf 009 produced maximum AFB1 on four rice
cultivars (range 386-415 µg/g of grains). The aflatoxin
production capacity of 13 aflatoxigenic moulds isolated
from natural black olive samples were reported to vary
from 0.11 to 5.9 µg/g on rice substrate (Eltem, 1996).
Begum et al. (1994) had reported the production of
aflatoxin by A. flavus at 10.4 mg/kg rice substrate. Demet
et al. (1995) studied the production of AFB1 at different
intervals by artificial inoculation of A. parasiticus on rice
grains. They reported a more or less constant le-vel of 30
mg AFB1/kg of rice grains. Aflatoxin production was
estimated in 11 cultivars of rice and 6 cultivars of wheat
following inoculation of seed with A. parasiticus. A
marked variation was found in the amounts of AFB1 and
AFG1 produced by the different cultivars. In general
wheat grain was a poor source of the toxin compared
with rice grain (Sinha et al., 1991). Refai et al. (1993) had
reported that AFB1 production by A. flavus on rice, maize
and YES media at 52, 40 and 40 µg/50 g substrate, res-
pectively. Nandi and Haggblom (1984) reported aflatoxin
production on rough rice grains (2430 - 10,643 µg/kg) by
inoculating the grains with A. flavus. The total yield of
aflatoxin production in culture by A. flavus isolate from
peanut was 1511 g/g of polished rice substrate in 5 days
in shake cultures at 28°C (Shotwell et al., 1966). But the
maximum amount of aflatoxin production estimated in this
investigation was only 415 µg/g of substrate used at 25°C
in still culture.
Recovery of AFB1 from rice samples
The effectiveness of the extraction procedure was confir-
med by adding pure AFB1 to ground rice and extracted in
methanol-KCl. Recoveries from rice samples estimated
by ELISA were greater than 95%.
Conclusion
In this study, neither AFB2 nor AFG1 was detected and
the bulk of aflatoxins produced by A. flavus isolates from
stored rice grains belonged to AFB1 only on different
agar media and solid substrates. It is therefore, apparent
that A. flavus in tropical rice mostly produce AFB1. Bulk
production of AFB1 on rice grains by high aflatoxin
producing A. flavus strains will help to generate pure
standards at an economical price and to produce anti-
bodies for ELISA detection and estimation of AFB1 con-
tamination in rice intended for domestic and export
markets.
ACKNOWLEDGEMENT
The authors are thankful to Indian Council of Agricultural
Research (ICAR), New Delhi for providing funds through
Aflatoxin net work project.
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