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Investigation of Diversity and Dominance of Fungal Biota in Stored Wheat

Grains from Governmental Warehouses in West Bengal, India

Article in Journal of the Science of Food and Agriculture · January 2019

DOI: 10.1002/jsfa.9568

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Research Article
Received: 16 August 2018 Revised: 22 November 2018 Accepted article published: 8 January 2019 Published online in Wiley Online Library:

(wileyonlinelibrary.com) DOI 10.1002/jsfa.9568

Investigation of diversity and dominance of

fungal biota in stored wheat grains from
governmental warehouses in West
Bengal, India
Ranjana Kumari,a Lakshmi E Jayachandranb and Ananta K Ghosha*

Abstract
BACKGROUND: Fungal infestation is a leading cause of qualitative and quantitative deterioration of stored wheat grains. Limited
information is available on the spatial distribution of fungal biota associated with stored wheat grains in India. Fungi were
isolated and characterized from nine stored wheat grain samples in three warehouses of the Food Corporation of India, located
in three agro-climatic zones (Paschim Medinipur, Bankura and Purulia) of West Bengal in India.

RESULTS: Maximum density and fungal diversity were observed in dichloran glycerol agar (DG-18) medium and the number
increased with the increase of storage duration. Samples collected from Purulia showed maximum fungal diversity than that
from Bankura and Paschim Medinipur. A total of 284 fungal isolates were obtained, classified into 29 operational taxonomic
units (based on amplified ribosomal DNA restriction analysis of 18S and internal transcribed spacer sequences), and identified
as 24 different fungal species. The majority of fungal isolates belonged to Aspergillus flavus (35%) followed by Rhizopus oryzae
(13%) and Eurotium amstelodami (9%). Aspergillopepsin O (PEPO) gene and aflatoxin biosynthetic pathway gene, nor-1, were
amplified by polymerase chain reaction (PCR) from 91% and 71% of Aspergillus flavus isolates, respectively, indicating their
aflatoxin producing ability. Aflatoxin production was further confirmed by ammonia vapour test, thin layer chromatography
(TLC) and high-performance liquid chromatography (HPLC).

CONCLUSION: The presence of toxigenic fungi in stored wheat grain emphasizes the necessity of quarantine measures of stored
grains before placing them in the public domain to save consumers from health hazards.
© 2019 Society of Chemical Industry

Keywords: stored wheat grain; fungal prevalence; amplified ribosomal DNA restriction analysis; ITS region sequencing; phylogenetic
analysis

INTRODUCTION destruction caused by fungal infestation depends on its diversity

In India, a gradual increase in the productivity of food grains has and density, which in turn is directly related to geographical and
been registered due to advancement in production technology climatic conditions.4,5
as well as availability of good quality seeds, fertilizers, pesticides Due to a lack of proper knowledge and management in develop-
and adequate irrigation.1 Approximately 60–70% of food grain ing countries, more than five billion people are regularly exposed
produced in the country is stored at the domestic level and the bal- to aflatoxins by unknowingly consuming contaminated foods.
ance quantity is supplied to the government procurement agen- Although there are several reports of mycotoxin epidemics in
cies. The Food Corporation of India (FCI) is the single largest grain Indian states like Gujarat, Bihar, Andhra Pradesh, Uttar Pradesh,
storage agency having a capacity of 26.62 million Mt, which is Tamil Nadu, Punjab and Karnataka, mycotoxin contamination
about 30% of total produce. It controls operations such as procure- in the stored wheat grain is rarely assessed and almost never
ment, storage, preservation, interstate movement and distribution controlled. In order to protect public health, most countries and
of the grains.
Grain deterioration during storage can happen due to biotic
(fungi, insects, pests) as well abiotic (temperature, moisture) fac- ∗ Correspondence to: AK Ghosh, Department of Biotechnology, Indian Insti-
tors. The stored grain microbiota, including bacteria and fungi, can tute of Technology Kharagpur, Kharagpur, West Bengal 721302, India.
adversely affect the physical, chemical, and biological properties E-mail: [email protected]
of the grain. The growth of some fungal species such as flavus,
a Department of Biotechnology, Indian Institute of Technology Kharagpur,
parasiticus and nominus can produce toxic secondary metabo-
Kharagpur, India
lites such as mycotoxins that can affect human health and cause
feed refusal.2 A large number of fungal contaminants have been b Department of Agricultual and Food Engineering, Indian Institute of Technol-
reported to be associated with the stored grain.3 The severity of ogy Kharagpur, Kharagpur, India

J Sci Food Agric (2019) www.soci.org © 2019 Society of Chemical Industry

 www.soci.org R Kumari, LE Jayachandran, AK Ghosh

international organizations have already established regulations

150.0 ± 100 (6)

150.0 ± 133 (7)

170.0 ± 144 (8)
350.0 ± 150 (8)
250.0 ± 108 (9)

Values represents mean ± standard deviation. Number of fungal isolates examined by amplified ribosomal DNA restriction analysis (ARDRA) and sequencing from each sample are shown within
0.645 ± 0.019
S9 (3 years)
for the allowed level of aflatoxins in food samples. Screening of
the prevalent fungal species and mycotoxin levels in different

38
grain samples (maize, sorghum and paddy) of southern states of
India have shown presence of ten different Aspergillus species and
samples having aflatoxin B1 (AFB1) and ochratoxin A (OTA) levels
above the allowed limit.6 Gautam et al.7 have also reported con-

150.0 ± 150 (6)

100.0 ± 118 (5)
50.0 ± 126 (5)
0.558 ± 0.020
80.0 ± 78 (5)

40.0 ± 99 (3)
tamination of 25 stored rice samples collected from district Mandi,

S8 (2 years)
Purulia
Himachal Pradesh of India with different fungal genera with high-

24
est frequency of Aspergillus flavus, Aspergillus niger and Rhizopus.
Alternaria has been identified as the most dominant genera along
with highly toxic species such as cryptococcus in stored wheat
grains collected from different states of Australia.8 Species such as

12.0 ± 100 (7)

0.537 ± 0.011

10.0 ± 85 (1)
6.0 ± 38 (2)

4.0 ± 58 (5)
3.0 ± 48 (3)
Penicillium citrinum, Aspergillus candidus and Fusarium proliferatum

S7 (1 year)
have been detected as most prevalent fungi, in Korean polished

18
stored rice.9
Culture based methods, such as serial dilution and plating, is sim-
ple to use, relatively inexpensive, and sensitive for fungal detection

Note: PDA, potato dextrose agar; CZA, czapex dox agar; MEA, malt extract agar; DG-18, dichloran glycerol agar; DRBC, dichloran rose bengal agar.
but cannot identify fungus at the species level. But various molecu-

150.0 ± 142 (5)

80.0 ± 105 (7)

110.0 ± 80 (6)
0.587 ± 0.016

50.0 ± 96 (4)
50.0 ±75 (4)
S6 (3 years)
lar techniques, such as amplified ribosomal DNA (rDNA) restriction
analysis (ARDRA),10 complete or partial sequencing of ribosomal

26
RNA (rRNA) gene,11 temperature and denaturing gradient gel elec-
trophoreses (TGGE and DGGE) of rRNA gene,12 DNA Microarray13
and real time polymerase chain reaction (PCR)14 are used for faster

110.0 ± 113 (6)

and accurate identification of fungal species. Internal transcribed

80.0 ± 155 (4)

0.555 ± 0.007
70.0 ± 84 (2)

60.0 ± 58 (2)
50.0 ± 77 (3)
S5 (2 years)
Bankura
spacer (ITS) region of the fungus has been proposed as the stan-

17
dard barcode for identification of fungal species.15,16 It tends to be
unique among species and remains fairly unchanged among indi-
Table 1. Analysis of fungal load in stored wheat samples collected from different warehouses in West Bengal

viduals of the same species. Besides, ITS region (∼600 bp) is easy to
amplify from small amounts of genomic DNA as it exists in multiple

20.0 ± 102 (1)

copies (∼250 copies) per cell.17 The molecular characterization of 0.524 ± 0.012
15.0 ± 64 (6)

20.0 ± 88 (2)
70.0 ± 92 (4)
50.0 ± 67 (2)
S4 (1 year)

microbiota along with morphological studies help to identify fun-

15
gal biota with higher accuracy at the species level.18
Food safety is a major global health concern, which demands
integrated efforts and practices to guarantee safe food through the
entire supply chain from the farm to the consumer. Therefore, an
270.0 ± 112 (21)

250.0 ± 146 (15)

380.0 ± 148 (13)
320.0 ± 111 (8)
200.0 ± 98 (9)
0.695 ± 0.025

investigation of microbial load and diversity in stored food grains

S3 (3 years)

at an early stage is needed to prevent the spread of toxic fungal 66

outbreaks. The objective of this study is to analyse the diversity
and dominance of fungal biota present in stored wheat grains of
different FCI warehouses of West Bengal, in India.
Paschim Medinipur

34.0 ± 100 (20)

0.622 ± 0.016

2.0 ± 28 (11)
15.0 ± 46 (6)

26.0 ± 74 (6)
2.2 ± 33 (6)
S2 (2 years)

MATERIALS AND METHODS

49

Sample procurement
Wheat grains stored for 1, 2 and 3 years, were collected randomly
from three different FCI Warehouses (Paschim Medinipur, Bankura
and Purulia) of West Bengal (Table 1). During collection the wheat
0.573 ± 0.009

1.7 ± 25 (15)
2.0 ± 40 (7)

0.4 ± 14 (1)
8.0 ± 36 (5)
0.8 ± 28 (3)
S1 (1 year)

samples did not show any visible microbial contamination. The

water activity (aw ) of wheat samples was measured at ambient
31

temperature using a water activity meter (Model: HygroLab C1;

Make: Rotronic AG, Bassersdorf, Switzerland).
Total number of isolates

Isolation of fungal contaminants from stored wheat grains

examined by ARDRA
CFU/g of sample × 103

Pure fungal colonies were isolated from the collected wheat sam-
Water activity (aW )

and sequencing
(Storage duration)

ples by serial dilution and plating method on different media

Sample name

plates as described by Harley and Prescott.19 Briefly, 10 g of wheat

parentheses.
on media
Location of
warehouse

grains was soaked in 90 mL peptone water (0.1%) and shaken

at 120 rpm for 30 min. Thereafter, it was homogenized, serially
DG-18
DRBC
MEA
PDA
CZA

diluted up to 10−5 dilution, and 100 μL of each dilution was spread

on five different media plates such as potato dextrose agar (PDA),

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Fungal biota in stored wheat grains www.soci.org

czapek dox agar (CZA), malt extract agar (MEA), dichloran glyc- been deposited in the GenBank under the accession numbers:
erol agar (DG-18) and dichloran rose bengal agar (DRBA) contain- KY065343-KY065371, and KY260656-KY260682. A phylogenetic
ing 75 μg/mL of chloramphenicol to prevent bacterial growth. The tree was constructed by a neighbour joining (NJ) method and evo-
experiment was done in triplicate and the plates were incubated lutionary distances were computed using the Kimura 2-parameter,
at 30 ∘ C for 5 days. The colony forming unit (CFU) was calculated with complete deletion (i.e. no positions containing gaps were
by using Eqn ((1): considered in the phylogeny analysis). All reconstructions were
validated by bootstrapping with 1000 replicates.
Total number of colonies × Dilution factor
CFU∕g = (1)
Plating volume (mL) × Wheat extract (g∕mL)
Morphological characterization
For morphological characterization, pure fungal isolate from each
Amplification of 18S with complete ITS ARDRA group was inoculated separately on PDA media plate
Genomic DNA of all the isolates was extracted using the modi- and incubated at 25 ∘ C in the dark for 7 days. The full grown
fied mini prep method.20 The rRNA gene segment comprising colonies were observed for size, appearances, mycelial textures
of 18S-ITS1-5.8S-ITS2 was amplified by PCR using univer- and pigmentations on both obverse and reverse side of the
sal primers NS1F (5′ -GTAGTCATATGCTTGTCTC-3′ ) and ITS4R colonies. The structure of conidial heads and the shapes of conidia
(5′ -TCCTCCGCTTATTGATATGC-3′ ). The amplicon (approximately were also observed under the microscope.24
1.6 kb) was size fractionated through 1% agarose gel and visual-
ized under ultraviolet (UV) light.
Molecular characterization
Species specific PCR assay
Amplified ribosomal DNA restriction analysis (ARDRA)
The most prevalent fungal isolates were further charac-
ARDRA of all the amplicons (18S-ITS1-5.8S -ITS2) of different fungal terized by PCR using species-specific primer set PEPO1
isolates was performed.21 In brief, each amplicon (1 μg) was dou- (5′ -CGACGTCTACAAGCCTTCTGGAAA-3′ ) and PEPO2 (5′ CAGCAG
ble digested with 2.5 U of restriction enzymes (HhaI and HaeIII) in ACCGTCATTGTTCTTGTC-3′ ). The amplicon (200 bp)
reaction volume of 20 μL for 3 h at 37 ∘ C and then analysed on 3% of aspergillopepsin (PEOP) biosynthesis gene segment was
agarose gel. The gel was stained with ethidium bromide, visual- analysed on 1.5% agarose gel and then sequenced.
ized and photographed using a gel documentation system (Model
BioView Transilluminator UST-20M-8E; Make: BioView® Transillu-
minator, Biostep, Burkhardtsdorf, Germany). Only the bands more Aflatoxigenic potential analysis
than 100 bp were considered for analysis and isolates showing sim- Molecular detection of different fungal strains in food products
ilar restriction digestion pattern were grouped together as a par- based on amplification of toxin genes by PCR using species spe-
ticular operational taxonomic unit (OTU). cific primers have been used by several authors.25–27 To determine
the toxin biosynthesis potential of the isolated Aspergillus, its
nor-1 gene, which codes for norsolorinic acid, an intermediate in
Cloning and sequencing of ITS region including 5.8S rRNA
gene the aflatoxin biosynthetic pathway28 was amplified by PCR using
NOR1F (5′ -ACCGCTACGCCGGCACTCTCGGCAC-3′ ) and NOR1R
The ITS region (ITS1-5.8S-ITS2) of rRNA gene was ampli-
(5′ - GTTGGCCGCCAGCTTCGACACTCCG-3′ ) primers.29 The ampli-
fied from fungal genomic DNA of each OTU by universal
con (400 bp) was visualized under UV light after electrophoresis
primer set ITS1F (5′ TCCGTAGGTGAACCTGCGG-3′ ) and ITS4R
on 1.5% agarose gel and stained with ethidium bromide. The
(5′ -TCCTCCGCTTATTGATATGC-3′ ). For all isolates, amplification
amplicon was then sequenced for further confirmation.
of ITS region yielded a single band of an approximately 600 bp.
The PCR amplicon was cloned into T/A cloning vector, pTZ57R/T
(Thermo Fisher Scientific, Waltham, Massachusetts, USA), trans- Chemical analysis of aflatoxin production
formed into chemically competent Top10 Escherichia coli cells All the Aspergillus flavus isolates showing amplification of nor-1
and sequenced in an automated DNA sequencer (Model Genetic gene were further analysed for the production of aflatoxin by
analyzer 3500; Make: Applied Biosystem, California, USA). culturing on yeast extract sucrose agar media. The fungal cul-
ture plates were examined by ammonium vapour test as well
Sequence assembly and analysis as visualized in UV trans-illuminator at 365 nm.30 Aflatoxins
were extracted from culture media by the solvent extraction
The obtained nucleotide sequences were analysed using
method (acetonitrile–methanol (85:15, v/v) and extracts were
‘Sequencher Version 5.3’ software (Gene Codes Corporation,
analysed in thin layer chromatography (TLC) using toluene–ethyl
Ann Arbor, MI, USA). Homology search was carried out using
acetate–formic acid (6:3:1) as mobile phase. Aflatoxin purification
local alignment search tool (BLAST) against the International
and quantification was performed by high-pressure liquid chro-
Nucleotide Sequence Collaboration databases (GeneBank, EMBL
matography (HPLC) as described by Nollet and Toldrá.31 HPLC was
and DDBJ) to determine their identities. The fungal isolate was
performed using C18 reversed-phase column (250 mm × 4.6 mm,
named as a particular species by considering coverage and iden-
5 μm particle size), using methanol– acetonitrile–water (20:20:60)
tity of sequences (identity > 95%, for the same genus and identity
as the mobile phase at a flow rate of 1 mL/min, and monitored in
> 98%, for the same species with taxonomically characterized
diode array detector (DAD) at 360 nm (Model: Agilent 1260 Infinity
organisms). Multiple sequence alignment was then carried out
Quaternary LC; Make: Agilent Technologies, California, USA).
using ClustalW22 at the European Bioinformatics Institute Website
(http://www. ebi.ac.uk/clustalw/). Phylogenetic analyses were
performed using MEGA6 software using reference sequences Statistical analysis
from GenBank databases.23 Nucleotide sequences of more than Incidence of fungal diversity in different stored wheat sample was
500 bp of all the fungal isolates reported in the present study have quantitatively analysed for species richness and abundance. The

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 www.soci.org R Kumari, LE Jayachandran, AK Ghosh

Figure 1. Amplified ribosomal DNA restriction analysis (ARDRA) pattern produced by double digestion of 18S with internal transcribed spacer (ITS) region
of ribosomal DNA using HaeIII and HahI restriction enzyme. (A) L, 100 bp DNA ladder; 1, OTU11; 2, OTU9; 3, OTU1; 4, OTU21; 5, OTU4; 6, OTU16; 7, OTU5;
8, OTU6; 9, OTU7. (B) L, 100 bp DNA ladder; 1, OTU28; 2, OTU22; 3, OTU29; 4, OTU26; 5, OTU12. (C) L, 100 bp DNA ladder; 1, OTU2; 2, OTU3; 3, OTU10; 4,
OTU15; 5, OTU18; 6, OTU19; 7, OTU20; 8, OTU17. (D) L, 100 bp DNA ladder; 1, OTU23; 2, OTU13; 3, OTU24; 4, OTU25; 5, OTU14; 6, OTU8; 7, OTU27.

species diversity within the sample (𝛼 diversity) was measured (aw < 0.95), mesophiles (aw = 0.95), and hygrophiles (aw > 0.95).34
using Simpson’s index (Dominance index) and Shannon–Weaver The aw of the analysed samples are less than 0.95 and hence
index (Information statistic index).32 infested mainly with xerophilic fungi.
Different fungal media had different nutrient composition and
∑
s
pH to maximize the chance of growth of all possible fungi present
Simpson′ s index (D) = p2i (2)
in wheat grains. Maximum number (in CFU/g) and diversity of
i=1
fungi were obtained for the samples grown on the DG-18 media.
∑
s This suggests that the DG-18 media is most suitable for the growth
Shannon index (H) = − pi . log2 pi (3) and enumeration of fungi in stored wheat grains with respect to
i=1 other media used in the current study. Hocking35 recommended
this media for the enumeration of xerophilic yeasts and moulds
where P is proportion (n/N); n is the number of a particular from dried and semi-dried foods.
species found; N is the total number of individuals; Σ is sum
of the calculations; s is number of species. Value of D ranges ARDRA based grouping of fungal isolates
from 0 (infinite diversity) to 1 (no diversity), and higher values of ARDRA method is an effective tool to study the genetic changes,
H indicates greater diversity. The species diversity between the enabling quick comparison of fungal communities over a period
samples (𝛽 diversity) was measured using principle component of time, in different environmental conditions. But it gives only
analysis (PCA).33 limited information about the type of microorganisms.36 ARDRA of
284 fungal isolates showed 29 different types of unique restriction
patterns and therefore grouped into 29 OTUs (Fig. 1). The isolates
RESULTS AND DISCUSSION of sample S3 were classified into 15 different OTUs, indicating
Determination of fungal load the presence of a high level of fungal diversity. Similarly, the
A total of 284 different fungi were isolated from all the wheat isolates of sample S2 were grouped in to 11 different OTUs,
grain samples. The fungal density (in CFU/g) increased with the followed by S1 and S9, which were grouped in to 10 different OTUs,
increase in storage time and water activity of stored wheat grain suggesting moderate diversity. Fungal isolates from other wheat
samples (Table 1). For instance, sample S3 stored for 3 years samples were less diverse and grouped into small number of
(aw = 0.695) contained higher fungal diversity and density. The OTUs (Fig. 2). A maximum number of isolates were found in OTU4
moisture of stored grains increases during storage due to the (35%), followed by OTU1 (13%) and OTU16 (9%) (Fig. 3). ARDRA
metabolic activity of seeds, insects and fungi, which further leads has been successfully employed for differentiating the microbial
to the proliferation of more fungal species. Based on optimum population in normal and bulking sludge samples for a duration
aw for growth, fungal groups can be categorized as xerophiles of 19 months.37

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Fungal biota in stored wheat grains www.soci.org

Figure 2. Stacked bar diagram showing relative abundance of different operational taxonomic units (OTUs) of fungal isolates according to amplified
ribosomal DNA restriction analysis (ARDRA).

Figure 3. Prevalence of different fungal species in stored wheat grains.

Fungal identification and phylogenetic study isolates of different OTUs identified them in the same species, i.e.
Sequencing and analysis of ITS region of rRNA genes can help Gordonia sp.
in the identification of microorganisms even at the species level. Phylogenetic analysis using the NJ method clearly divided the
Based on ITS sequence analysis, 24 different fungal species were identified stored grain fungal communities in to three broad
identified from 29 OTUs. Fungal isolates from two or more dif- fungal phyla namely Ascomycota, Basidiomycota, and Zygomy-
ferent OTUs were found to contain same nucleotide sequences cota, which are separated from each other with high levels of
of ITS region, indicating the presence of same fungal species. bootstrap support. The formed tree also included all the reference
As an example OTU2, OTU3 and OTU22 were found to contain taxa correctly along with the identified fungal species. Maximum
fungi were observed in the Ascomycota phylum and the main
Lichtheimia ramose species. Similarly, Rhizopus oryzae was found
dominating species were Aspergillus flavus, followed by Eurotium
in OTU1, OTU11, OTU14 and Aspergillus flavus in OTU4 and OTU15.
amstelodami, Aspergillus candidus, Aspergillus niger, Alternaria
The appearance of different ARDRA patterns of the same fungal
alternata, Aspergillus sydowii along with nine other different
species may be due to mutation in restriction site of HaeIII and
fungal species (Fig. 4).
HhaI present in rRNA genes, and consequently showing changes in
the RFLP pattern. A similar type of study conducted by Waturangi
et al.38 grouped 21 bacterial isolates of different morphological Diversity analysis of stored grain fungal biota
characteristics from mouth microflora into nine OTUs based The study showed that maximum fungal isolates belonged
on ARDRA of 16S rRNA gene but DNA sequence analysis of six to Aspergillus flavus (35%) followed by Rhizopus oryzae (13%) and

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 www.soci.org R Kumari, LE Jayachandran, AK Ghosh

SW81 |KY065357|
SW30 |KY260663|
SW4 |KY260658|
86 SWB3 |KY260677|
SW165 |KY065346|
SW18 |KY260662|
gi|969812107|dbj|LC105688.1| Aspergillus flavus
SW97 |KY065359|
70
SW140 |KY260674|
SW84 |KY260665|
gi|363903384|gb|JN252102.1| Aspergillus candidus
gi|969812101|dbj|LC105682.1| Aspergillus niger
79
SW14 |KY065351|
SW83 |KY260664|
SWB9 |KY260678|
SWB19 |KY260679|

99 SW95 |KY065362|
gi|1002734513|gb|KU687806.1| Aspergillus sydowii

97 SW13 |KY065350|
gi|662009307|ref|NR 121327.1| Penicillium cinnamopurpureum
80
99 SW142 |KY065366|
gi|665387924|gb|KJ775519.1| Aspergillus penicillioides
SW82 |KY065358|
100
100 SW116 |KY260669|
SW173 |KY260676|
gi|21666896|gb|AF455464.1| Eurotium amstelodami
86
100 SW101 |KY065363|
gi|511801492|ref|NR 103664.1| Talaromyces islandicus

100 SW89 |KY065360|

72 gi|930715694|gb|KR105942.1| Myceliophthora verrucosa

75 SWC5 |KY065369|
100 SWC6 |KY260681|

98 gi|62948152|gb|AY943046.1| Westerdykella globosa

gi|1024292790|gb|KU179665.1| Alternaria alternata
100
SW90 |KY065361|
99 70
SW93 |KY260667|
SW141 |KY260673|

74 SW10 |KY065348|
100 gi|928505210|gb|KR085964.1| Meyerozyma guilliermondi
76 SW11 |KY260660|

100 SW17 |KY065354|

97 gi|1012636559|gb|KU950724.1| Candida tropicalis
gi|731446285gb|KP132795.1|Trichomonascus ciferrii
100
gi|747040110|ref|NR 130678.1| Candida mucifera
99 85
SW115 |KY260668|
SW143 |KY065349|
SW114 |KY065365|

75 SW137 |KY065355|
100 SW119 |KY260670|

70 gi|731445368|gb|KP131878.1| Cryptococcus albidus

gi|338221869|emb|FR870473.1| Cryptococcus rajasthanensis
100
75 SWB40 |KY260680|
SWB41 |KY065368|
97
SW79 |KY065352|
96
100 gi|326468402|gb|HQ670680.1| Sporidiobolus ruineniae
SW15 |KY260661|
100
SW133 |KY260671|

99 SW156 |KY260675|
SW8 |KY260659|

70 SW41 |KY065347|
gi|326468412|gb|HQ670690.1|Rhodosporidium toruloides

100 SWC9 |KY065371|

gi|148578148|emb|AM745433.1| Mucor circinelloides

76 SWC13 |KY065370|
81
100 gi|746818086|gb|KM527221.1| Rhizopus microsporus
SWC8 |KY260682|
100 SW1 |KY260656|
gi|974500224|gb|KT899481.1| Rhizopus oryzae
100
SW136 |KY065343|
SW27 |KY065353|
SW33 |KY065356|
SW135 |KY260672|

100 SWC18 |KY065367|

gi|953764018|dbj|LC097194.1| Syncephalastrum racemosum
96 71 SW104 |KY065364|
SW86 |KY260666|
100
SW63 |KY065344|
gi|329458495|gb|HQ285700.1| Lichtheimia ramosa
SW112 |KY065345|
SW2 |KY260657|

0.05

Figure 4. Phylogenetic analysis of wheat grain fungi, compared with closest matched reference sequences in BLAST analysis, belonging to Ascomycota
( ), Zygomycota ( ) and Basidiomycota ( ) phylum by ‘neighbour-joining’ grouping method, using ‘Kimura-2 parameter’, based on the sequences of
ITS1-5.8S-ITS4 of rDNA. Bootstraps values > 50% (1000 replicates) are shown at the nodes.

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Fungal biota in stored wheat grains www.soci.org

Table 2. Analysis for fungal species richness and abundance by measuring diversity indices in stored wheat samples

Model name S1 S2 S3 S4 S5 S6 S7 S8 S9

Simpson’s index 0.20 0.15 0.27 0.26 0.36 0.24 0.20 0.44 0.18
Shannon–Weaver index 2.75 3.00 2.80 2.10 1.82 2.40 2.41 1.16 2.9

Figure 5. Principle component analysis (PCA) of and fungal species community structure present in stored wheat grain samples collected from different
Food Corporation of India (FCI) warehouses. (a) Score plot and (b) loading plot. The axis explains 64.5% and 14.2% of total variability.

Eurotium amstelodami (9%) and the rest 43% belonged to 21 racemosum was exclusively present in S8 making them the host
other fungal species (Fig. 3). Prevalence of fungal species of genus of different fungal population. This could be attributed to the
Aspergillus in stored wheat grain has been reported earlier.3,39 subtropical (dry and hot) climate of the region. These samples
Aspergillus flavus mainly produces aflatoxin B1 and B2,40 and were an epitome of the xerophilic fungal species such as Eurotium
aflatoxin B1 is accounted as a category 1A natural carcinogen Amstelodami, Aspergillus niger, Alternaria alternata and Aspergillus
by the International Agency for Research on Cancer.41 The Rhi- candidus. In the loading plot of PCA, Aspergillus flavus has been
zopus oryzae can produce extracellular amylase, hence has the placed apart from other species owing to its prevalence in all the
ability to breakdown starch, which can affect seeds nutritional wheat samples studied. Moreover, it lies in the same direction
value.42 Eurotium amstelodami, a xerophilic fungus, infests stored of the cluster of samples formed in the loading plot, illustrating
grains and nuts at low moisture content and causes oxidative its dominance in these samples. Similarly, S5 is dominated with
rancidity.43 At the phylum level most of the fungi were found Rhizopus oryzae, while S7 is dominated with Aspergillus candidus.
to be in Ascomycota (64%), followed by Zygomycota (26%), and PCA plot suggests that the ascomycotes are the most prevalent
Basidiomycota (10%) (Fig. 4). The results of the statistical analysis storage fungi in the stored wheat samples from FCI warehouses
provided further insight into the earlier observations (Table 2). The while the Basidiomycetes have a very limited occurrence.
measurement of diversity within community using corresponding The effect of geographical location on the fungal contaminants
D- and H-indices showed highest diversity in S2 sample (0.15, 3.00) of wheat grains from across four states in Australia have been pre-
and least diversity in S8 (0.36, 1.16). viously reported by Barkat et al.8 A difference in the patterns of
The PCA of fungal species community between samples fungal communities is observed between different states, possi-
explained 64.5% and 14.2% of the variance in data through prin- bly due to variation in the climatic conditions and subsequent
cipal component 1 (PC1) and principal component 2 (PC2) (Fig. 5). variations in the local mycoflora. Similarly, studies on mycoflora
The score plot (Fig. 5(a)) and loading plot (Fig. 5(b)) explained associated with stored wheat grains have shown spatial varia-
the similarities and differences of fungal contamination between tion in the occurrence of prominent storage fungi. Aureobasid-
the stored wheat samples collected from different locations. For ium, Alternaria and Fusarium sp. have been found to be dominant
instance, samples collected from Paschim Medinipur (S1, S2, S3) in Australian wheat,44 Aspergillus sp. in Algerian varieties,45 Fusar-
and Bankura (S4, S5, S6) district were positively correlated and clus- ium and Alternaria in Tunisia46 and Cladosporium and Pencillium, in
tered together in the score plot, which showed that these samples Iranian.47
were infested with similar types of fungal species like Aspergillus
flavus, Rhizopus oryzae, Eurotium amstelodami, Aspergillus niger,
Rhodosporidium toruloides, Alternaria Alternata and Aspergillus Morphological analysis of fungal species
candidus. However, samples S7, S8 and S9 obtained from Puru- Accurate identification of fungi up to the species level was done
lia, were widely dispersed, which indicated negative correlation earlier using both morphological and molecular methods.48 The
and showed the presence of diverse fungal species. Species key morphological features of all the fungi such as size, appearance
such as Westerdykella globosa, Rhizopus microsporus and Mucor of colony, texture, colour matched perfectly with the reported
circinelloids were present in S7 and S9, while Syncephalastrum details available in the literature49 (Fig. 6). The fungi belonging

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 www.soci.org R Kumari, LE Jayachandran, AK Ghosh

Figure 6. Morphological characteristics of different fungal species grown on potato dextrose agar (PDA) media. OTU1, Rhizopus oryzae; OTU2, Lichtheimia
ramose; OTU3, Lichtheimia ramose; OTU4, Aspergillus flavus; OTU5, Rhodosporidium toruloides; OTU6, Meyerozyma guilliermondii; OTU7, Trichomonascus
sp.; OTU8, Penicillium cinnamopurpureum; OTU9, Aspergillus niger; OTU10, Sporidiobolus ruineniae; OTU11, Rhizopus oryzae; OTU12, Candida tropicalis;
OTU13, Cryptococcus albidus; OTU14, Rhizopus oryzae; OTU15, Aspergillus flavus; OTU16, Eurotium amstelodami; OTU17, Aspergillus candidus; OTU18,
Myceliophthora verrucosa; OTU19, Alternaria alternate; OTU20, Aspergillus sydowii; OTU21, Talaromyces islandicus; OTU22, Lichtheimia ramose; OTU23,
Candida sp.; OTU24, Aspergillus penicillioides; OTU25, Syncephalastrum racemosum; OTU26, Cryptococcus rajasthanensis; OTU27, Westerdykella globose;
OTU28, Rhizopus microspores; OTU29, Mucor circinelloides.

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Fungal biota in stored wheat grains www.soci.org

Figure 7. Representative agarose gel image (1.5%) showing amplification of (A) PEPO and (B) afltoxigenic (nor1) gene in Aspergillus flavus isolates. Lane L,
100 bp ladder; lanes 1–10, samples; lane B, negative control.

Figure 8. (A) Reverse side view of Aspergillus flavus colony on YESA media: (a) untreated with ammonia vapour, (b) treated with ammonia vapour, (c)
exposed in normal light, (d) exposed with ultraviolet light (365 nm). (B) Analysis of extracted aflatoxin by thin-layer chromatography (TLC). Lane1, aflatoxin
standard mix; lanes 2 and 3, aflatoxin extracted from two different Aspergillus flavus isolates. (C) Analysis of extracted aflatoxins by high-performance liquid
chromatography (HPLC).

to Ascomycota phylum contains both unicellular yeast as well as side of plates. This colour change is associated with the aflatoxin
multicellular mould whereas Zygomycota phylum contains only production.30 Isolates showing dark red colour indicated its abil-
mouldy fungi and Basidiomycota phylum contains only unicellular ity to produce higher quantities of aflatoxins than the isolates that
yeast. have shown bright red or pink colour (Fig. 8(A)). The TLC anal-
ysis showed that 84% of the nor-1 positive isolates were able
to produce aflatoxin chemo-types (B1, B2, G1 and G2) on YESA
Molecular characterization of fungal species
media (Fig. 8(B)). HPLC elution profile of extracted aflatoxin cor-
Analysis by PCR using species specific primers showed amplifica-
related with the that of standard and confirmed the production
tion of 200 bp PEPO gene segment in 91% of the Aspergillus iso-
of four different chemo-types with maximum production of AFB1
lates confirming them as Aspergillus flavus (Fig. 7(A)). Logotheti,
chemo-type (Fig. 8(C)).
Kotsovili-Tseleni, Arsenis and Legakis50 have developed species
The aflatoxigenic isolates produced AFB1 in the range of 6 to
specific PEPO primer set targeting first exon of aspergillopepsin
40 μg/g media, which was higher than the permissible limit of
genes for identification of Aspergillus and shown amplification of
0.03 μg/kg in all food commodities in India.52 This emphasizes the
PEPO gene (confirmed by sequencing) in 30 out of 40 Aspergillus
high toxin producing potential of the fungal isolates collected
flavus. Further, amplification of 400 bp nor-1 gene showed that
from the stored wheat grains. Consumption of significant amounts
77% Aspergillus flavus species contained this gene and have the
of these contaminants could lead to severe health issues among
potential for aflatoxin production (Fig. 7(B)). Molecular identifica-
humans and animals.
tion of aflatoxigenic potential of Aspergillus flavus species through
the amplification of nor-1 gene (which encodes norsoloronic acid;
an intermediate in the aflatoxin B1 synthetic pathway) indicates
CONCLUSION
a high percentage of Aspergillus flavus in stored grain mycobiota
Fungal communities present in stored wheat grains were iso-
can produce aflatoxin. Amplification of this gene in 60 out of 89
lated and molecularly characterized by a combination of ARDRA
Aspergillus flavus isolates, using the same primer set has been
and sequencing of ITS region of rRNA gene. The wheat grains were
reported by Priyanka et al.29 A similar study conducted by Erami
found to be infested with 24 diverse species of fungi belonging
et al.51 has shown amplification of nor-1 gene in 7 of 14 isolates
to three broad phylum of fungus namely Ascomycota, Zygomycota
capable of producing aflatoxins.
and Basidiomycota. The diversity and fungal load varied with stor-
age duration, aw level and geographical location of the storage
Chemical analysis of aflatoxin warehouse. The grains were mainly infected with xerophilic fungi
The ammonium vapour test showed moderate to dark pink colour which include very high frequency of toxigenic Aspergillus flavus
change and UV-induced greenish blue fluorescence on reverse species (confirmed by the amplification of nor gene through PCR,

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 www.soci.org R Kumari, LE Jayachandran, AK Ghosh

ammonia vapour test, UV imaging, TLC and HPLC). The presence of isolated from tropical bed bugs in Northern Peninsular Malaysia,
high levels of toxigenic fungal species in stored wheat grains of the Cimex hemipterus (Hemiptera: Cimicidae). Asian Pacific J Trop Biomed
5:707–713 (2015).
FCI warehouse alerts the necessity for quarantine measures before 19 Harley J and Prescott L, Basic laboratory and culture techniques, in
distribution in to the public domain. Laboratory Exercises in Microbiology, 2nd edn. W.C. Brown, Dubuque,
IA, pp. 14–46 (1993).
20 Lee S, Milgroom M and Taylor J, A rapid, high yield mini-prep method
for isolation of total genomic DNA from fungi. Fungal Genetics
ACKNOWLEDGEMENTS Reports 35:23–24 (1988).
The authors would like to acknowledge financial support from 21 Alves A, Phillips AJ, Henriques I and Correia A, Evaluation of amplified
the Ministry of Human Resource Development and Depart- ribosomal DNA restriction analysis as a method for the identification
ment of Biotechnology, Government of India for conducting this of Botryosphaeria species. FEMS Microbiol Lett 245:221–229 (2005).
22 Thompson JD, Higgins DG and Gibson TJ, CLUSTAL W: improving
research work.
the sensitivity of progressive multiple sequence alignment through
sequence weighting, position-specific gap penalties and weight
matrix choice. Nucleic Acids Res 22:4673–4680 (1994).
REFERENCES 23 Tamura K, Dudley J, Nei M and Kumar S, MEGA4: molecular evolu-
1 Ali J, Sharma A and Rani P, Overview of grain drying and storage tionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol
problems in India. Int J Eng Res Gen Sci 3:674–678 (2015). 24:1596–1599 (2007).
2 Davari E, Mohsenzadeh M, Mohammadi G and Rezaeian-Doloei R, 24 Pitt JI and Hocking AD, The ecology of fungal food spoilage, in Fungi
Characterization of aflatoxigenic Aspergillus flavus and A. parasiticus and food spoilage. Springer, Berlin (2009).
strain isolates from animal feedstuffs in northeastern Iran. Iran J Vet 25 Nirmaladevi D, Venkataramana M, Srivastava RK, Uppalapati S, Gupta
Res 16:150–155 (2015). VK, Yli-Mattila T et al., Molecular phylogeny, pathogenicity and tox-
3 Tournas VH and Niazi NS, Potentially toxigenic fungi from selected igenicity of Fusarium oxysporum f. sp. lycopersici. Sci Rep 6:21367
grains and grain products. J Food Saf 38:12422 (2018). (2016).
4 Lal S and Srivastava B, Insect pests of stored wheat in Madhya Paradesh 26 Mudili V, Siddaih CN, Nagesh M, Garapati P, Naveen Kumar K, Murali
(India). J Entomol Res 9:141–148 (1985). HS et al., Mould incidence and mycotoxin contamination in freshly
5 Getachew A, Chala A, Hofgaard IS, Brurberg MB, Sulyok M and Tron- harvested maize kernels originated from India. J Sci Food Agric
smo A-M, Multimycotoxin and fungal analysis of maize grains from 94:2674–2683 (2014).
south and southwestern Ethiopia. Food Addit Contam Part B Surveill 27 Venkataramana M, Shilpa P, Balakrishna K, Murali H and Batra H, Inci-
11:64–74 (2018). dence and multiplex PCR based detection of trichothecene chemo-
6 Priyanka SR, Venkataramana M, Kumar GP, Rao VK, Murali HCS types of Fusarium culmorum isolates collected from freshly har-
and Batra HV, Occurrence and molecular detection of toxigenic vested maize kernels in southern India. Braz J Microbiol 44:401–406
Aspergillus species in food grain samples from India. J Sci Food Agric (2013).
94:537–543 (2014). 28 Rodrigues P, Venâncio A, Kozakiewicz Z and Lima N, A polyphasic
7 Gautam AK, Gupta H and Soni Y, Screening of fungi and mycotoxins approach to the identification of aflatoxigenic and non-aflatoxigenic
associated with stored rice grains in Himachal Pradesh. Int J Theor strains of Aspergillus section Flavi isolated from Portuguese
Appl Sci 4:128–133 (2012). almonds. Int J Food Microbiol 129:187–193 (2009).
8 Barkat E, Hardy GSJ, Ren Y, Calver M and Bayliss K, Fungal contaminants 29 Priyanka S, Ramana MV, Balakrishna K, Murali H and Batra H, A novel
of stored wheat vary between Australian states. Australas Plant non radioactive PCR-DNA probe for the detection of aflatoxin pro-
Pathol 45:621–628 (2016). ducing Aspergillus species from major food crops grown in India.
9 Park JW, Choi S-Y, Hwang H-J and Kim Y-B, Fungal mycoflora and Adv Microbiol 2:577 (2012).
mycotoxins in Korean polished rice destined for humans. Int J Food 30 Saito M and Machida S, A rapid identification method for
Microbiol 103:305–314 (2005). aflatoxin-producing strains of Aspergillus flavus and A. parasiticus by
10 Smit E, Leeflang P and Wernars K, Detection of shifts in microbial com- ammonia vapor. Mycoscience 40:205–208 (1999).
munity structure and diversity in soil caused by copper contamina- 31 Nollet LM and Toldrá F, Food Analysis by HPLC, 3rd edn. CRC Press, Taylor
tion using amplified ribosomal DNA restriction analysis. FEMS Micro- and Francis Group, New York, NY (2012).
biol Ecol 23:249–261 (1997). 32 Liu J, Deng JC, Yang CQ, Huang N, Chang XL, Zhang J et al., Fungal
11 Bellemain E, Carlsen T, Brochmann C, Coissac E, Taberlet P and diversity in field mold-damaged soybean fruits and pathogenicity
Kauserud H, ITS as an environmental DNA barcode for fungi: an identification based on high-throughput rDNA sequencing. Front
in silico approach reveals potential PCR biases. BMC Microbiol Microbiol 8:779 (2017).
10:189–197 (2010). 33 Pulido RP, Burgos MJG, Gálvez A and Lucas R, Changes in bacterial
12 Zhou J, Liu X, Jiang H and Dong M, Analysis of the microflora in diversity of refrigerated mango pulp before and after treatment
Tibetan kefir grains using denaturing gradient gel electrophoresis. by high hydrostatic pressure. LWT – Food Sci Technol 78:289–295
Food Microbiol 26:770–775 (2009). (2017).
13 Leinberger DM, Schumacher U, Autenrieth IB and Bachmann TT, Devel- 34 Mannaa M and Kim KD, Influence of temperature and water activity on
opment of a DNA microarray for detection and identification of deleterious fungi and mycotoxin production during grain storage.
fungal pathogens involved in invasive mycoses. J Clin Microbiol Mycobiology 45:240–254 (2017).
43:4943–4953 (2005). 35 Hocking AD, Isolation and identification of xerophilic fungi in stored
14 Schabereiter-Gurtner C, Nehr M, Apfalter P, Makristathis A, Rotter M products, in Fungi and Mycotoxins in Stored Products. Proceedings of
and Hirschl A, Evaluation of a protocol for molecular broad-range an International Conference Held at Bangkok, Thailand, 23-26 April.,
diagnosis of culture-negative bacterial infections in clinical routine p. 65 (1991).
diagnosis. J Appl Microbiol 104:1228–1237 (2008). 36 Gich FB, Amer E, Figueras JB, Charles A, Abella CAA, Balaguer MD et al.,
15 Schoch CL, Seifert KA, Huhndorf S, Robert V, Spouge JL, Levesque CA Assessment of microbial community structure changes by amplified
et al., Nuclear ribosomal internal transcribed spacer (ITS) region as ribosomal DNA restriction analysis (ARDRA). Int Microbiol 3:103–106
a universal DNA barcode marker for fungi. Proc Natl Acad Sci USA (2000).
109:6241–6246 (2012). 37 Błaszczyk D, Bednarek I, Machnik G, Sypniewski D, Sołtysik D, Loch T
16 Ramana MV, Balakrishna K, Murali HCS and Batra HV, Multiplex et al., Amplified ribosomal DNA restriction analysis (ARDRA) as a
PCR-based strategy to detect contamination with mycotoxigenic screening method for normal and bulking activated sludge sample
Fusarium species in rice and fingermillet collected from southern differentiation. Pol J Environ Stud 20:29–36 (2011).
India. J Sci Food Agric 91:1666–1673 (2011). 38 Waturangi DE, Francisca I and Susanto CO, Genetic diversity of methy-
17 Vilgalys R and Hester M, Rapid genetic identification and mapping of lotrophic bacteria from human mouth based on amplified ribosomal
enzymatically amplified ribosomal DNA from several Cryptococcus DNA restriction analysis (ARDRA). Hayati J Biosci 18:77–81 (2011).
species. J Bacteriol 172:4238–4246 (1990). 39 Chelladurai V, Jayas D and White N, Thermal imaging for detecting
18 Ab Majid AH, Zahran Z, Rahim AH, Ismail NA, Rahman WA, Zubairi fungal infection in stored wheat. J Stored Prod Res 46:174–179
KS et al., Morphological and molecular characterization of fungus (2010).

wileyonlinelibrary.com/jsfa © 2019 Society of Chemical Industry J Sci Food Agric (2019)

Fungal biota in stored wheat grains www.soci.org

40 Reis T, Oliveira T, Baquião A, Gonçalves S, Zorzete P and Corrêa B, 46 Bensassi F, Mahdi C, Bacha H and Hajlaoui MR, Survey of the mycobiota
Mycobiota and mycotoxins in Brazil nut samples from different of freshly harvested wheat grains in the main production areas of
states of the Brazilian Amazon region. Int J Food Microbiol 159:61–68 Tunisia. Afr J Food Sci 5:292–298 (2011).
(2012). 47 Mostafa A, Kazem S, Mohammad S and Rokouei M, Determination of
41 Kumar V, Basu M and Rajendran T, Mycotoxin research and mycoflora wheat grain mycoflora in store-pits Golestan Province. Aust J Basic
in some commercially important agricultural commodities. Crop Prot Appl Sci 5:1070–1076 (2011).
27:891–905 (2008). 48 Jayasiri SC, Hyde KD, Ariyawansa HA, Bhat J, Buyck B, Cai L et al., The
42 Ghosh B and Ray RR, Current commercial perspective of Rhizopus faces of fungi database: fungal names linked with morphology,
oryzae: a review. J Appl Sci 11:2470–2486 (2011). phylogeny and human impacts. Fungal Divers 74:3–18 (2015).
43 Abellana M, Benedi J, Sanchis V and Ramos A, Water activity and tem- 49 Petrini LE and Petrini O, Identifying Moulds: A Practical Guide. J Cramer
perature effects on germination and growth of Eurotium amstelo- in Borntrager Science Publishers, Breganzona, Switzerland, (2013).
dami, E. chevalieri and E. herbariorum isolates from bakery products. 50 Logotheti M, Kotsovili-Tseleni A, Arsenis G and Legakis N, Multiplex PCR
J Appl Microbiol 87:371–380 (1999). for the discrimination of A. fumigatus, A. flavus, A. niger and A. terreus.
44 Berghofer LK, Hocking AD, Miskelly D and Jansson E, Microbiology of J Microbiol Methods 76:209–211 (2009).
wheat and flour milling in Australia. Int J Food Microbiol 85:137–149 51 Erami M, Hashemi S, Pourbakhsh S, Shahsavandi S, Mohammadi S,
(2003). Shooshtari AH et al., Application of PCR on detection of aflatoxino-
45 Riba A, Mokrane S, Mathieu F, Lebrihi A and Sabaou N, Mycoflora and genic fungi. Arch Razi Inst 62:95–100 (2007).
ochratoxin A producing strains of Aspergillus in Algerian wheat. Int 52 Rajarajan P, Rajasekaran K and Asha Devi N, Aflatoxin contamination in
J Food Microbiol 122:85–92 (2008). agricultural commodities. Ind J Pharm Biol Res 1:148–151 (2013).

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