AiMAdvances in Microbiology2165-3402Scientific Research Publishing10.4236/aim.2022.123011AiM-116159ArticlesBiomedical&Life Sciences Biodiversity and Distribution of <i>Aspergillus</i> and Their Toxins in Maize from Western and Eastern Regions of South Africa NgumQueenta Nji1AdetunjiModupeade Christianah2*AtebaCollins Njie1MwanzaMulunda3Department of Animal Health, Faculty of Agriculture, Science and Technology, North West University, Mafikeng Campus, Mmabatho, South AfricaDepartment of Biological Sciences, Trinity University, Yaba, Lagos, NigeriaDepartment of Microbiology, Faculty of Natural and Agricultural Sciences, North West University, Mafikeng Campus, Mmabatho, South Africa07032022120312114919, December 202122, March 2022 25, March 2022© Copyright 2014 by authors and Scientific Research Publishing Inc. 2014This work is licensed under the Creative Commons Attribution International License (CC BY). http://creativecommons.org/licenses/by/4.0/

Aspergillus species and aflatoxins production are more prevalent during times of high heat and drought. In South Africa, there is frequent occurrence of drought as a result of climate change. The aim of this study was to investigate the biodiversity and distribution of Aspergillus species with their corresponding toxins in maize from main maize producing regions of South Africa; [Western Regions (WR) and Eastern Regions (ER)]. One hundred and twenty-three (64 from WR and 59 from ER) maize samples from the two agro-climatic regions in South Africa were analyzed using cultural, molecular and analytical methods. Across agro-climatic regions, Aspergillus species contaminated about 62% of the maize samples, while Aspergillus flavus was the most prevalent (47.15%) followed by Aspergillus fumigatus (4.69%) while the least was Aspergillus parasiticus (0.81%). The Western Regions showed a higher distribution of varieties of Aspergillus species compared to the Eastern Regions. Aflatoxins contaminated only 27.64% of the maize samples with a mean total aflatoxin concentration of 2.40 μg/kg which is below the South Africa’s set standard for total aflatoxin in food (5 μg/kg). About 10.57% of the samples produce aflatoxins above the 5 μg/kg permissible limit for total aflatoxin in foods. The ratio of toxigenic to atoxigenic strains of Aspergillus flavus was generally low in all the regions of South Africa. This study could aid policy makers to make informed decisions in developing remediation strategies for Aspergillus mycotoxins.

<i>Aspergillus</i> Species Aflatoxins Maize Aflatoxicity Agro-Climatic South Africa1. Introduction

The filamentous fungus; Aspergillus flavus is a cosmopolitan soil-borne saprophytic organism with opportunistic parasitic behaviors to plants, animals and humans. Aflatoxins are metabolites produced mainly by toxigenic strains of Aspergillus flavus and A. parasiticus, which grow in soil, hay, decaying vegetation and grains [1]. Aflatoxins (AFs) are toxic and carcinogenic to livestock and humans [2] [3] [4]. Contamination by aflatoxin is one of the most serious food and feed safety problems worldwide and causes significant economic losses yearly [5]. Thus, food safety has become a very important issue worldwide and the potential effects of climate change on yields and quality of crops especially for mycotoxins, have received special attention in the past years from a risk analysis perspective [6] [7]. Benkerroum [8] reported past major outbreaks of aflatoxicosis associated with maize-based foods which occurred in West-India (1974), Kenya (1981 and 2004) and Tanzania (2016, 2017). Due to the toxicity and impact of aflatoxins on health, there exist regulatory limits on the quantity of aflatoxins permitted in food and feed in several countries [9] [10].

Climate change plays a major role in the production of AFs in crops by Aspergillus spp. [6] [11] [12] [13] [14]. Climate change affects interactions between different mycotoxigenic spp. and toxins produced in foods and feeds [15] [16]. In particular high temperatures and drought stress directly affect maize and the occurrence of A.flavus favoring fungal growth, conidiation and spore dispersal, and impairing the growth and development of maize [17]. Aspergillus flavus is the dorminant fungal spp. in maize kernels during warm and dry seasons [18] [19] [20] [21] resulting in high levels of aflatoxin contamination of maize in the field [22]. Events related to contamination by aflatoxins are more prevalent during times of high heat and drought which can stress the host plant thereby facilitating infection by A. flavus [23].

Mycotoxin toxicity occurs at very low concentrations, therefore sensitive and reliable methods for their detection are required. Different qualitative and quantitative analytical methods having different sensitivity, specificity and accuracy have been developed such as thin layer chromatography (TLC), high performance liquid chromatography (HPLC) liquid chromatography mass spectrometry tandem (LCMS/MS) and many other techniques.

Literature is depleted in information on the distribution and biodiversity of Aspergillus species and their toxins in silo stored maize cultivated in different climate regions of the country. Hence, this study aimed at filling the information gap by investigating the biodiversity and distribution of Aspergillus species with their corresponding toxins in maize samples from the different agro-climatic areas of SA. Molecular biomarkers to differentiate the ambiguity between the Aspergillus species were employed to identify the different Aspergillus species present in SA maize. The presence of the five most important genes in the aflatoxin biosynthesis pathway among Aspergillus isolates was also evaluated.

This study will aid policy makers to make informed decisions in developing strategies based on monitoring and characterization of risks, prevention, intervention and remediation strategies for Aspergillus mycotoxins, which start from critical points along the production chain such as field, storage, processing and transportation. It will also provide useful information that will enhance global efforts in ensuring production of quality food/feed as well as food security.

2. Materials and Methods2.1. Sampling Site and Collection of Samples2.1.1. Sampling Site

Figure 1 The dotted lines divide the maize production areas in the country into Western (North West and Western part of Free State) and Eastern (Guateng and Eastern part of Free State) regions [24]. Maize samples were collected from main

maize producing areas of the different agro-climatic regions of South Africa; Free State (45%), North West (17%) and Guateng (5%) [25]. The Western regions situated in the drier and warmer areas of the country and the Eastern regions with higher rainfall and cooler areas [24]. Samples from these regions were collected from silos managed by SENWESS.

2.1.2. Samples

One hundred and twenty three (123) maize samples were randomly collected from selected silo sites from different agricultural regions of South Africa (Gauteng, North West and Free State). A structured sampling model was used for sampling where 10 kg of maize was taken from different points of the silos while the maize was routinely moved. The collected samples were mixed and 5 kg subsequently collected. The samples were collected separately, finely milled using a warring blender (IKA, Model M20, Germany) and put into sterile zip lock polythene bags, labeled and stored at 4˚C prior analysis to arrest any formation of mycotoxins before analysis. Fifty nine samples (59) were from the Eastern region (ER) while sixty four (64) were from the Western region (WR).

2.2. Determination of Moisture Content

The moisture content of samples was determined using the AOAC method [26]. Five grams (5 g) of milled maize samples were dried in an oven (Labcon, Model FSOM4 Maraisburg) at 105˚C for 1 hour. Triplicates of each sample were weighed and the initial moisture content (%) calculated as shown in the formula below:

Moisturecontent ( % ) = ( Initialweight − Finalweight ) Initialweight × 100

2.3. Mycobiota Enumeration in Maize Samples

The mycobiota population in the maize samples was enumerated using the dilution plate techniques as described by [27]. Appropriate 10-fold dilutions of milled maize samples in sterile water were spread plated on potato dextrose agar (PDA) and incubated in the dark at 28˚C for 72 h. Thereafter, the mycobiota present in each sample was enumerated and reported as colony forming units per gram (cfu/g) of maize.

cfu = Numberofcolonies × reciprocalofthedilutionfactor Platingvolume   ( 1   ml )

The percentage occurrence of each of the isolates was calculated by comparing the ratio of the number of individual organism to the total number of organism present in each sample.

Percentage of appearance (PA) was calculated according to the following formula:

PA = Numberofsampleswiththesamegeneraorspp . Totalnumberofsamplesofgeneraofspp .

2.4. Isolation and Purification

The previously cultured plates were returned to the incubator for additional 2 - 4 days for isolation of surface sample mycoflora and purification [28] [29]. Purified isolates from various plates were grouped on the basis of their colony morphology (colour and pigmentation of colony) and characteristics of spores (sclerotial production) [30] [31] [32] among others. Actual identification was carried out using the Polymerase Chain Reaction (PCR) method with different primers. A representative from all the various groups was scrapped using a wooden spatula and put in special extraction tubes (lysis tube) prior extraction of DNA.

2.5. Molecular Identification of Aspergillus Isolates

Extraction of the genomic DNA of fungal isolates was done using the Zymo Research kit (Zymo-Research fungal/Bacterial Soil Microbe DNA, D6005, USA), following the manufacture’s protocols. A reaction volume of 25 μl was used containing 7 μl nuclease-free water, 12 μl PCR Master Mix (Biolabs), 1.5 μl each of oligonucleotide forward and reverse primers (3 μm) and 3 μL template DNA mixed in the PCR tubes [33]. Amplification of the Internal Transcribed Spacer Region (ITS1–5.8S-ITS2-ITS; approximately 550 - 600 bp) of fungal isolates from the maize was done through the Polymerase Chain Reaction (PCR), using the universal ITS1 primer (5’-CTTGGTCATTTA GAGGAAGAAGTAA-3’) and ITS 4 (5’-TCC TCC GCT TAT TGA TATGC-3) with intial denaturation at 95˚C for 2 min, 35 cycles of 95˚C for 30 s, 54˚C for 30 s, and 72˚C for 1 min, and an additional 72˚C for 10 min [34]. Species level differentiation of closely related Aspergillus species was done by amplification and sequencing of the β-tubulin (benA) and calmodulin (CaM) genes. Camodulin primer (CMD5-5’-CCGAGTACAAGGARGCCTTC-3’ and CMD6-5’CCGATR GAGGTCATR ACGTGG-3’) with approximately 550 bp amplicon size at 94˚C for 1 min, 35 cycles of 94˚C for 1 min, 55˚C for 30 s, and 72˚C for 1 min, and a final extension of 72˚C for 10 min as the PCR optimization condition [35]. β-tubulin primerben A (ben2f-5-GGTAACCAAATCGGTGCTGCTTTC-3’ and bt2b 5’-ACCCTCAGTGTAGTGACCCTTGGC-3’) with 500 bp amplicon size and PCR optimization condition with initial denaturation of 94˚C for 3 min, 32 cycles of 94˚C for 45 s, 55˚C for 45 s, and 72˚C for 1 min, and a final extension at 72˚C for 5 min [36]. These primers were commercially synthesised by Inqaba biotechnical Industrial (Pty) Ltd (Pretoria, South Africa). The PCR amplicons were analyzed by electrophoresis on 1 % (w/v) agarose gel using 1 × TAE (Tris acrylamide-EDTA) buffer. The gels were run for about 160 min at 120 V on a 20 cm gel to ensure separation of bands, and then detected by ethidium bromide fluorescence on a UV trans illuminator to confirm the expected size of the amplicons and visualised using Chemi Doc Image Analyzer [37].

Sequencing was conducted on an ABI Prism 3500 XL DNA Sequencer (Applied Biosystems, Foster City, California) at the Inqaba Biotechnical Industrial, Pretoria, South Africa. Finch TV software version 1.4.0 was used to analyse chromatograms, resulting from sequencing reaction for good quality sequence assurance. The resulting chromatographs were blasted in the NCBI database (http://www.ncbi.nlm.nih.gov) with the Basic Local Alignment Search Tool (BLASTn) for homology in order to identify probable organisms in question [38]. The sequences were later deposited in the GenBank for allocation of accession number.

2.6. Aflatoxigenicity Test of Aspergillus Isolates2.6.1. Cultural Method for Detection of Aflatoxigenic Fungi in Maize

Yeast Extract Sucrose (YES) Agar (yeast extract; 20 g, sucrose; 150 g, agar; 20 g and MgSO4; 0.5 g) was used as described by Criseo, Bagnara [39]. Mycelia plugs of Aspergillus isolates were inoculated on petri dishes containing YES medium in duplicates and the isolates incubated unilluminated at 28˚C for 3 - 14 days. The YES agar plates checked on the 7th and 14th day for aflatoxin producing ability by the ammonium hydroxide vapour-induced colour change test as described by Jefremova, Ostrý [40]. 2 ml of concentrated ammonium hydroxide solution (W/V, 25%,) was placed on the inside of the lid of the inverted petri dish containing the isolate on YES agar and left for 5 - 10 minutes. Colour change at the reverse side of the agar plate was then observed for those that tested positive. Plates that tested negative were re-examined on the 14th day for colour change.

2.6.2. Thin Layer Chromatographic Method for Qualitative Detection of Aflatoxins Producing Isolates among Aspergillus Species

Extraction of aflatoxin from the isolates was done in accordance with Midorikawa, Pinheiro [41] and Yin, Yan [42]. The 7-day old Aspergillus isolates on YES agar were divided into two equal halves using sterile surgical blades (one half for TLC analysis while the other half was kept for other analyses). The first half of the agar containing the isolates was scooped into 50 ml centrifuge tubes and 15 - 20 ml of 70% methanol-water (70:30) added to it and kept on a shaker for 30 minutes. Thereafter, it was centrifuged at 5000 rpm for 5 minutes and the extracts decanted into clean tubes. The extracts were then evaporated to dryness by air blowing in the dark evolution chamber. The residues were then reconstituted with 500 μl of 100% methanol. A 20 μl volume of each of the reconstituted extracts were spotted on a 20 × 20 glass backed 250 μm thick silica gel coated TLC plates (Merck KGaA, Darmstadt Germany) and developed in the TLC tank containing the mobile phase; chloroform-ethyl acetate-propane-2-ol (90:5:5, v/v/v). The presence of aflatoxins was determined by viewing the plates under the UV light at 365 nm for the presence of a bright blue or blue green fluorescence at the same migration level with the total aflatoxin standard (Sigma Aldrich) on the silica plate.

2.6.3. Detection of Aflatoxin Producing Gene in Aspergillus Isolates

The DNA of suspected Aspergilla were examined for the presence of five important aflatoxin-producing genes (aflR, aflJ, aflM, aflQ, aflD and omt-A) present in the aflatoxin biosynthesis pathway by PCR method, using previously reported primer sets [43] [44]. Optimization of polymerase chain reaction conditions was done separately for the different target genes. A reaction volume of 25 μl was used containing 7 μl nuclease-free water, 12 μl PCR Master Mix, 1.5 μl each of oligonucleotide forward and reverse primers (3 μm) and 3 μL template DNA mixed in the PCR tubes [33]. The PCR amplified products were resolved on 1% agarose gel and stained with ethidium bromide. After electrophoresis, gel was removed from the gel-casting platform and exposed to UV transillumination.

The following primers were used for the detection of the aflatoxin producing gene: Nor primer (Nor 1-5-ACC GCT ACG CCG GCA CTC TCG GCA C-3 and Nor 2-5-GTT GGC CGC CAG CTT CGA CAC TCC G-3 with 400 bp amplicon with the Nor (AflD) gene); Ver primer (Ver 1-5-GCC GCA GGC CGC GGA GAA AGT GGT-3 and Ver 2-5-GGG GAT ATA CTC CCG CGA CAC AGC C-3 with 537 bp amplicon with Ver (AflM) gene); Omt primer (Omt 1-5-GTG GAC GGA CCT AGT CCG ACA TCA C-3 and Omt 2-5-GTC GGC GCC ACG CAC TGG GTT GGG G-3 with a 797 amplicon size having the gene OmtA); AflR primer (AflR 1-5-TAT CTC CCC CCG GGC ATC TCC CGG-3 and AflR 2-5-CCG TCA GAC AGC CAC TGG ACA CGG-3 with 1032 bp amplicon size having the AflR gene); and the AflJ primer (AflJ F-5-TGA ATC CGT ACC CTT TGA GG-3 and AflJ R-5-GGA ATG GGA TGG AGA TGA GA-3 with 737 bp amplicon size having the gene AflJ gene). The PCR optimization conditions for the primers are presented in Table 1.

2.7. Liquid Chromatographic Analyses for Fungal Isolates2.7.1. Liquid Chromatography Mass Spectrometry Mass Tandem (LCMS/MS) Parameters

An LC-MS/MS method was designed and validated for the analysis of 123 analytes. A Waters Acquity UPLC system coupled to a Xevo TQS mass spectrometer

Polymerase chain reaction conditions for aflatoxin primers
Primer code (gene)Pre-denaturationDenaturationAnnealingElongation
Nor 1&2 (Nor(aflD)94˚C—10 min94˚C—1 min65˚C—1 min72˚C—2 min (33 cycles)72˚C—5 min (1 cycle
Ver 1&2 (Ver(aflM)95˚C—4 min95˚C—1 min58˚C—1 min72˚C—30 sec (30 cycles)72˚C—10 min (1 cycle
Omt 1&2 (omt)94˚C—5min94˚C—1 min72˚C—2 min72˚C—2 min (33 cycles)72˚C—10 min (1 cycle
AflR 1&2 (aflR)95˚C—4 min95˚C—1 min60˚C—1 min72˚C—30 sec (30 cycles)72˚C—10 min (1 cycle
AflJ F&R (aflJ)95˚C—10 min95˚C—50 sec58˚C—50 sec72˚C—2 min (30 cycles)72˚C—10 min (1 cycle

(Waters, Milford, MA, USA) was used to analyse the samples, equipped with Mass LynxR (version 4.1) and Quan Lynx R (version 4.1) software (Waters, Manchester, UK) for data acquisition and processing. A ZORBAX Eclipse XDB C18-column (1.8 μm, 100 × 2.1 mm) was applied (Agilent Technologies, Diegem, Belgium). The mobile phase consisted of water/methanol (55/45, v/v), adjusted with 9mg potassium bromide and 1ml 65% nitric acid at a flow rate of 0.4 mL/min. The gradient elution programme started at 100% mobile phase. Then, the mobile phase increased with a linear increase to 99% in the 20th minute. Duration of each LC run was 30 minutes including re-equilibration. The capillary voltage was 3 kV and nitrogen applied as spray gas. Source and desolvation temperatures were set at 120˚C and 400˚C respectively. The argon collision gas pressure was 9 × 10−6 bars, cone gas flow was 35 L/h and desolvation as flow was 800 L/h. For increased sensitivity and selectivity, the instrument was operated in the selected reaction monitoring (SRM) mode and two SRM transitions monitored for each analyte. Matrix-matched calibration plots were constructed for the determination of AFB1, AFB2, AFG1 and AFG2. The limit of detection (LOD) was calculated as three times the standard error of the intercept divided by the slope of the standard curve; the limit of quantification (LOQ) was computed in a similar way except for the standard error, which was by a factor of six. The calculated LOD and LOQ were verified by the signal-to-noise ratio (s/n), which was more than 3 and 10 respectively in accordance with the IUPAC guidelines [45].

2.7.2. High Performance Liquid Chromatography

This is a quantitative determination method for the extraction of aflatoxins. Extraction of aflatoxins from the samples was done using the Easi-Extract Aflatoxin (R-Biopharm Rhone LTD) immuno-affinity columns kits in conjunction with HPLC or LC-MS/MS as described in the product’s extraction kit. The total aflatoxin content of the samples was determined and quantified from maize samples with a high performance liquid chromatography column (HPLC) using the Shimadzu corporation model (Kyoto, Japan). LC-20AB liquid chromatography equipped with CBM-20A communication bus module, LC-20AB degasser, CTO-20A column oven, SIL-20A auto sampler, RF-10AxL fluorescence detector, RID-10A refractive index detector and SPD-M20A photodiode array detector linked to LC solutions version 1.22 software release. The chromatographic separation of analytes and standards was performed by passing through a reverse phase Symmetry column C18 (Waters). The oven temperature was maintained at 30˚C. Peak areas and retention times of mycotoxins were used to determine the amount of specific mycotoxins per sample based on those of standard mycotoxins using a calibration curve. To determine AFs, the fluorescence detector RF 10AXL was coupled with a Coring cell (CoBrA cell) (DR Weber Consulting, Germany) as electrochemical cell for the derivatisation of AFs. The mobile phase consisted of water/methanol (55/45, v/v), adjusted with 9 mg potassium bromide and 1 ml 65% nitric acid. The HPLC method used was validated by determining its linearity, accuracy and sensitivity. Linearity was determined by constructing calibration curves from standards of AFB1, AFB2, AFG2, AFG2, total aflatoxin (AFtot) and from extracts of blank samples of previously analyzed maize samples that did not contain any of the aflatoxins. Linear range was examined at 3 different concentrations (0.025 µg/ml, 0.25 µg/ml and 2,5 µg/ml). The matrix-matched calibration curves were built by spiking blank samples with selected aflatoxin standards after the extraction process. Calibration curves were constructed by plotting peak areas against concentration and linear functions applied to the calibration curves. Matrix effect (ME) was calculated for each analyte by comparing the slope of the standard calibration curve with the matrix-matched calibration curve for the same levels of concentration. Sensitivity of the methodology or system used was evaluated by limit of detection (LOD) and limit of quantification (LOQ), estimated for a signal-to-noise ratio (S/N) ×3 and ×10 respectively from chromatograms of samples spiked at the lowest level validated. Accuracy was evaluated through recovery studies and determined by calculating the ratio of the peak areas for each aflatoxin by analyzing the samples spiked before and after extraction at three additional levels of 25, 50, and 100 µg/kg for all aflatoxins analyzed (AFB1, AFB2, AFG1, AFG2 AFtot). Quantification of the toxins was performed by measuring peak areas, retention time and comparing them with the relevant standard calibration curves.

3. Results3.1. Moisture Content and Mycobiota Count in Maize Sample

The moisture content of the maize samples ranged from 7.38% - 8.84% across both regions, with ranges of 7.38% - 8.81% and 7.64% - 8.84% for ER and WR respectively. The mean moisture content for maize in both regions (8.30% and 8.26%) is not significantly different (p > 0.05) from one another (Table 2).

Mycobiota count in the maize ranged from 0 - 48 cfu/g and 0 - 23 cfu/g for WR and ER respectively, with mean count in WR (9.75 cfu/g) higher than those obtained in the ER (5.93 cfu/g). Sample 045/M/2015 from ER had the highest Aspergillus spp. load (9 cfu/g), with fungal load ranging from 0 - 7.33 cfu/g and 0 - 9 cfu/g for WR and ER respectively (Table 2).

Mean moisture content, fungal and Aspergillus load in maize samples
RegionsRangeMean ± SD
Moisture (%)WR (n = 64)7.64 - 8.848.30 ± 0.27
ER (n = 59)7.38 - 8.818.26 ± 0.26
Fungi (cfu/g)WR (n = 64)0 - 48.009.75 ± 9.55
ER (n = 59)0 - 23.005.93 ± 14.76
Aspergillus (cfu/g)WR (n = 64)0 - 7.330.82 ± 6.05
ER (n = 59)0 - 9.001.09 ± 6.42
SD = Standard deviation
3.2. Mycobiota Distribution in Maize from Different Regions

A total of 123 maize samples were analyzed (WR; 64 and ER; 59). Fungi spp. belonging to eight genera; Fusarium, Aspergillus, Penicillium, Rasamsonia, Talaromyces, Paecilomyces, Byssochlamys and Verticillium were isolated from the maize and four distinct genera Talaromyces, Paecilomyces, Byssochlamys, Verticillium found in maize were from the ER (Table 3). Fusarium spp. had the highest distribution while Penicillium spp. remained the least distributed among other fungal spp. from both regions (Figure 2).

Frequency of Mycobiota distribution in maize
Fungi isolateNo. of infected samples from each RegionFrequency of isolation (%) from each RegionsTotal No. of infected samplesTotal frequency of isolation (%)
Western Region (n = 64)Eastern Region (n = 59)Western Region (64)Eastern Region (59)(n = 123)(n = 123)
Aspergillus spp.472973.4449.157661.79
Fusarium spp.524481.2574.589678.05
Penicillium spp.12318.755.081512.2
Other Fungi311048.4416.954133.33
3.3. Molecular Identification of Aspergillus Isolates

Out of the total 123 samples, 76 (29 from ER and 47 from WR) were morphologically identified as Aspergillus isolates and these represented 11 Operational Taxonomic Unit (OTU) and four different Aspergilli sections; Aspergillus flavi section (A. flavus, A. parasiticus, A. oryzae); Aspergillus nigri section (A. niger, A. awamori, A. brasiliensis, A. luchuensis, A. welwitschiae); Aspergillus fumigate section (A. fumigatus) and Aspergillus usti section (A. insuetus, A. ustus, A. aff. ustus, A. mnutus) by matching the sequences of the isolates with ITS sequences in the database, as well as those of partial benA and caM genes of closely related type strains in the GenBank (Table 4). Accession number were then assigned to each isolates after successful submission to the genbank. Aspergillus spp. contaminated 61.79% of the maize samples with Aspergillus flavus being the most frequent (WR; 73.44%, ER; 49.15%.) in both region.

Molecular identification of Aspergillus isolates
GroupIsolatesDescritionFungal ID (ITS)Fungal ID (CMD)Fungal ID (BT2)
122, G275, 76, 100, 24, 40, 15, 55, 74, 66, 68, 50, 17, 51, 108, 58, 57, 117, 49, 27, 16, 29, 105, 107, 87, 102, 91, 5Greenish colony without scerotiaA. flavus A. oryzaeA. flavusA. flavus
2108, 59, 36, 111, 113, 110, 1, 192, 48, 32, 6, 54, 37, 67, 109, 72, 51, 94, 84, 95, 62, 2,Greenish yellow colony with scerotiaA. oryzaeA. flavusA. flavus
3G208, 47, 88, 26, 13, 35, 31, 21, 118, 46, 119, 114, 53, 99, 40, 107, 10, 70, 6, 101, 45, 16, 64, 80, 82, 104Greenish brown colony with timidly visible scerotiaA. flavusA. flavusA. flavus
438Dark greenA. parasiticusA. parasiticusA. parasiticus
5108, 16Greenish thallus structureA. aff. ustus A. ustus A. insuetusA. insuetus A. mnutusA. insuetus
627, 119Blue greyA. fumigatusA. fumigatusA. fumigatus
7119, 64, 19, 6, 87, G218, 103, 111, 70Dark green to greyA. fumigatusA. fumigatusA. niger A. brasiliensis
8111, 12, 42Dark with yellow tones on the reverse sideA. nigerA. nigerA. niger A. awamori
9108, 1, 70BlackN/AA. luchuensisN/A
10115, 45, 80, 84, 2BlackA. nigerA. welwitschiaeA. niger
1158, 95Dark brown to blackA. brasiliensis Aspergillus sp.A. brasiliensisRasamsonia columbiensis
3.4. Frequency of Isolation of Aspergillus Isolates

Thirteen different Aspergillus spp. were isolated from the 11 OTUs group out of which only six were found in maize from the ER while all the spp. were found in maize from the WR in varying loads, spp. such as A. parasiticus, A. insuetus, A. awamori, A. mnutus, A. ustus as well as A. aff. ustus were not found in Eastern regions (Table 5). Amongst the Aspergillus spp. isolated, A. flavus was the most common spp with the highest frequency of isolation from both regions (47.15%) as compared with other spp. Aspergillus parasiticus had the least frequency of isolation (0.81%) and was found only in the WR.

3.5. Incidence of Aflatoxin Producing Genes in Aspergillus Species

The result of screening for aflatoxin producing genes in the Aspergillus section flavi revealed that none of the isolates from both regions possessed the AflR gene (Figure 3(a)). The hierarchy of distribution of the other genes screened for in isolates from WR and ER is; aflM (33%) > aflJ (19%) > aflD (8%) > Omt-A (3%) and aflM (17%) > aflJ (7%) > aflD (7%) > Omt-A (2%). The aflatoxin producing ability of the isolates was also confirmed with the cultural and TLC methods. Only 14% and 5% of the isolates in WR and ER respectively tested positive using the cultural NRDCA method and 6.25% and 1.7% with TLC method (Figure 3(b)). A 9.09% of the isolates from both region tested positive for aflatoxigenicity using the polyphasic approach (molecular, cultural and TLC), 20.45% were positive for two methods while 70.45% were positive for at least one of the methods.

Percent frequency of isolation of Aspergillus isolates across different regions
Aspergillus isolateTotal number sample (n = 123)Frequency of isolation (%)Eastern RegionWestern Region
Sample (n = 59)Frequency of isolation (%)Sample (n = 64)Frequency of isolation (%)
A. flavus (A. oryzae)5847.152135.593757.81
A. fumigatus86.558.4734.69
A. niger32.440034.69
A. parasiticus10.810011.56
A. brasiliensis21.6311.6911.56
A. luchuensis21.6323.3911.56
A. aff. ustus21.630023.13
A. awamori32.440032.42
A. insuetus A. mnutus21.630021.63
21.630021.63
A. ustus21.630021.63
A. welwitschiae54.0743.2510.81
3.6. Fungal Metabolites and Mycotoxins in Maize Samples3.6.1. Analysis of Liquid Chromatography Mass Spectrometry Mass Tandem (LCMS/MS) for Fungal Metabolites in Maize Samples

Aflatoxin was not detected in any of the maize samples analyzed using the LCM-S/MS. However, other detected metabolitesviz: 3-Nitropropionic acid; Sterigmatocystin; seco-sterigmatocystin; averufin; Kojic acid; and orsellinic acid. The limit of detection (LOD) ranged between 0.04 µg/kg (averufin and seco-sterigmatocystin) and 21 µg/kg (kojic acid), while limit of quantification (LOQ) ranged from 0.13 µg/kg (averufin) to 68 µg/kg (kojic acid). Percentage recovery ranged from 75.5% (seco-sterigmatocystin) to 168.8% (orsellinic acid) (Table 6). At least 36.59% of maize samples had one or more of the metabolites mentioned above while 20.33% of the samples had traceable amounts of kojic acid, but below limit of quantification (data not shown).

In all the regions, orsellinic acid had the highest percentage of occurrence (29.27%; concentration range between 235.76 and 829.60 µg/kg), followed by kojic acid (5.69%; concentration range of 65.61 to 587.51 µg/kg) while the rest were found in only one sample (0.81%). Fifty percent (50%) of maize samples from WR contained at least, one of these metabolites while about twenty-two percent (22.03%) of maize samples from ER had these metabolites (Table 7). 3-Nitropropionic acid was not detected in maize samples from ER, metabolites such as sterigmatocystin and seco-sterigmatocystin were also detected in maize from WR. 3-Nitropropionic acid (15.70 µg/kg), Sterigmatocystin (1.69 µg/kg), seco-sterigmatocystin (0.36 µg/kg) and averufin (0.48 µg/kg) were detected in only one sample.

LCMS/MS detected metabolites in maize and their frequency of occurrence
AnalytesLOQ (µg/kg)LOD (µg/kg)Recovery (%)Frequency of occurrence
WR (n = 64)ER (n = 59)
32 (50%)13 (22.03%)
Kojic acid682178.64 (6.25%)3 (5.08%)
3-Nitropropionic acid2.50.7587.81 (1.56%)0
Sterigmatocystin0.250.0891.301 (1.69%)
Seco-sterigmatocystin0.140.0475.501 (1.69%)
Averufin0.130.04104.101 (1.69%)
Orsellinic acid6020168.827 (42.19%)9 (15.25%)
Aflatoxin G1-----
Aflatoxin G2-----
Aflatoxin B1-----
Aflatoxin B2-----
 Mean aflatoxin concentrations and frequency of distribution of toxins in maize from different regions (HPLC method)
Western RegionEastern Region
SampleB2B1Total Aflatoxin (µg/kg)SampleB2B1Total Aflatoxin (µg/kg)
Mean0.453.023.53Mean0.2519.0119.26
Range0 - 170 - 650 - 65Range0 - 12.90 - 1069.80 - 1082.7
Frequency of distribution
G2G1B2B1Total
WR (n = 64)009 (14.06%)16 (25%)22 (34.38%)
ER (n = 59)003 (5.08%)11 (18.64%)12 (20.34%)
SA (n = 123)0012 (9.76%)27 (21.95%)34 (27.64%)
3.6.2. Detection of Aflatoxins in Maize Samples Using High Performance Liquid Chromatography

Standard curves were constructed by calculating the ratio of the peak areas for each aflatoxin standard samples spiked before and after extraction at three levels of 25, 50 and 100 µg/ kg for all aflatoxins analyzed (Suppl. Figure 1). The toxins (G2, G1, B2 and B1) were eluted at 6 - 7, 7 - 8, 8 - 9 and 10 - 11 minutes respectively. The aflatoxin content of the maize samples was measured in µg/kg.

Aflatoxins G1 and G2 were absent in all analyzed samples as revealed by HPLC technique. However, aflatoxins B1 and B2 were found in both regions in varying amounts. The ranges for aflatoxin B1 concentration (0 - 65 and 0 - 10.70 µg/kg) was higher than AFB2 (0 - 17 µg/kg to 0 - 12.9 µg/kg) across WR and ER. The ER had a mean value of aflatoxins B1 of 1.04 µg/kg compared to the WR with a mean value of 3.11 µg/kg. The same trend was observed for AFB2 with WR having a higher mean value of 0.05 µg/kg and ER with a mean value of 0.98 µg/kg. Thus, the mean total aflatoxin was higher in WR (3.59 µg/kg) compared to ER (1.09 µg/kg) (Table 6). There was no significant difference between aflatoxins B2 and B1 in ER and WR. The WR recorded a higher percentage contamination of samples for both aflatoxins B1 (25%) and B2 (14.06%) compare to aflatoxins B1 (18.64%) and B2 (5.08%) from ER (Table 6).

4. Discussion

Moisture content of grain at post-harvest and during storage is critical in the quality of the product after storage. A moisture content of 13% is the maximum moisture content required for grain storage [46]. Analysis of moisture content in maize samples in this study revealed a lower range of mean moisture content (7.38% and 8.84%) in samples from both regions. Previous authors have also reported similar moisture content rangesof 10.23% - 14.12% and 11% - 13% in maize grain from regions in Southern Africa [47] [48]. Contamination by fungi and production of mycotoxin in grain can happen in the field or during storage, hence, fungi are classified as field and/or storage microbes. Aspergillus species are both field and storage fungi [49]. Therefore, with such low moisture, the fungal load observed and contamination by aflatoxin in maize in this study could have been due to invasion in the field and proliferation during storage [50] [51]. For example, Kimanya, De Meulenaer [52] found levels of aflatoxin up to 21,667 μg/kg in freshly harvested maize before sorting and 1758 μg/kg in the same stocks of maize after sorting and stored for 5 months. Moisture content was found to have no significant effect on contamination by aflatoxin in maize. Studies have shown that moisture below 13% has little or no influence on the growth of fungi and mycotoxin production during storage.

The three main genera of fungi known for producing mycotoxins; Aspergillus, Fusarium, and Penicillium [53] were found in maize grown in SA. Although this study focused onAspergillus spp., fungal spp. belonging to eight genera (Fusarium, Aspergillus, Penicillium, Rasamsonia, Talaromyces, Paecilomyces, Byssochlamys and Verticillium) were found in maize from the different agro-climatic regions of SA. Out of the eight genera, four distinct genera (Talaromyces, Paecilomyces, Byssochlamys and Verticillium) were not found in maize from the Eastern Regions. From this study the mycotoxin producing genera were more in the WR than the ER. This result corroborate earlier studies in SA maize where similar genera of microbes were isolated in their maize [54] [55]. The latter author reported incidence of mycotoxin-producing genera of Aspergillus, Penicillium and Fusarium in maize produced in Tunisia and Egypt; Fusarium spp. 95.3%, Aspergillus 87.5% and Penicillium 64.0% [55]. The geographic location of sample could be the primary factor affecting composition of microbiota in grains [56].

Out of the thirteen Aspergillus species isolated in this study, A. flavus (47.15%) was the most predominant followed by A. niger (4.69%) and A. fumigatus (4.69%), which contradicts the reports of Egbuta [57] and Chilaka, De Kock [54] who found higher occurrences of A. fumigatus (45%) than A. flavus (43%) in SA commercial maize (73.4%) and Nigeria (66.7%) respectively. The high incidence of A. flavus in both regions compared to other members of Aspergillus section flavi group (A. parasiticus, A. mnutus etc.) could be due to previous reports that Aspergillus flavus are more prevalent during times of high heat and drought [23]. Also, A. flavus are natural habitant of soil, hay, decaying vegetation, and grains acting as the reservoir of inoculums for infection of kernels in the field. Aspergillus flavus possess a higher adaptability to growth substrates in a range of environments (field and storage fungi) and can produce spores that remain viable even under extremely strict conditions [1] [58] [59]. Fungi that thrive in a particular area are strongly determined by the prevailing climatic conditions [56] [60]. The findings of this study corroborate those of Kankolongo, Hell [61] in Zambian maize in which A. flavus and A. niger were the most prevalent fungal isolates of maize grains.

In this study, 27.64% of the A. flavus isolates were aflatoxins producers where 10.57 of the isolates produced aflatoxins concentration above the South Africa’s set standard (5 µg/kg). Gruber-Dorninger, Jenkins [62] found that only 0.7% of the 282 maize samples collected in South Africa had aflatoxins above the set standard for SA, and concluded that aflatoxins showed a low prevalence in feed samples from South Africa and detected levels were mostly unproblematic. On the contrary, AFB1 were reported in 70% of the 40 maize samples in an assessment conducted in 2010 from two feed mills in KwaZulu-Natal [54]. In addition, the results of this study contradicts those of Meyer, Skhosana [63] who in a multi-mycotoxin long-term monitoring period (2014-2018) study, reported specifically that no aflatoxin was found in SA commercial maize. Meyer and collaborators further explained that total aflatoxin contamination were reported in three white maize samples from areas in the North West province in the 2014-2015 production seasons. In the summers of 2010, 2011, 2012 especially 2015, South Africa suffered severe droughts [62] [64] [65] that led to low crop yields in South Africa, for instance, there was a 30% reduction in maize production in 2015 (from 14,250,000 tons in 2014 to 9,955,000 tons) when SA experienced very low rainfall that amounted to 141 mm from 1991 to 2014 [62]. In already hot climates, more frequent drought may result in higher mycotoxin production [66]. Numerous studies agree on the main role of drought and high temperatures in higher aflatoxin production in maize [23] [67] [68].

Aspergillus flavus were found in 61% of the maize samples from both regions where about 28% did produce aflatoxins. Fungal growth and AFs production in cereals depends on temperature, moisture, etc. [69]. The non-production of aflatoxins could be attributed to: high temperatures and water stress which reduces production of AFB1, despite the growth of A. flavus under these conditions [70], then 2015 been a year of drought and very low rainfall (141 mm). Interactions between water activity and temperature have prominent effects on Aspergillus spp. and production of aflatoxin [71] [72]. These authors studied the effect of interactions of temperature and water activity (aw) on the biosynthetic regulatory gene (aflR) expression and production of AFB1 by A. flavus in maize. They observed the greatest growth of A. flavus at 30˚C/0.99 aw with no growth at 20˚C/0.90 aw [71] [72]. Based on the investigation by Battilani, Toscano [73] on the possible emergence of AFB1 in cereals as a result of climate change, they projected that for every 2˚C increase in temperature, there is an increase in AFs risk. In addition, Shooshtari, Mohammadi [74] found that a mutation including substitution of some bases and many other different physiological conditions affecting biosynthesis of aflatoxin, does affect production of aflatoxin. Furthermore, isolated spp. are genetically different and the nucleotide sequence is not associated with the mentioned primer. For example, the ITS molecular marker identified most of the Aspergillus flavus as Aspergillus oryzea. A. oryzae is a domesticated variant of A. flavus [75], which could be another reason aflatoxins were not produced.

Though there are regulatory limits for aflatoxins in only 15 African countries, the regulations vary widely among these countries. Exposure to aflatoxin in food is a significant risk factor [76], as they are teratogenic and mutagenic and chronic exposure to aflatoxins have resulted in reduced immune activities [77], malnutrition [78] and growth impairment [79]. All South Africans consume maize in one form or another due to its use as an ingredient in different food products (maize meal, grits, corn flakes and snacks) [80] [81]. Maizeconsumed by livestock and poultry, also end up in the human food chain, through meat products, dairy products, cheeses and eggs. Therefore, SA maize quality has a direct impact on the health of humans and animals who consume the products. To find the problem attributable to aflatoxin contamination of food, both detected concentrations and consumption habits mustn’t be ignored [82]. The mean value of total aflatoxins concentration in this study ranged between 1.09 and 3.59 μg/kg for the regions, which is below the recommended daily standards for human consumption in South Africa and USA [62] [83].

Maize from both regions in SA contained AFs and traceable amounts of other metabolites using HPLC and LCMS/MS techniques. HPLC detected AFs while LCMS/MS did not detect any aflatoxins in the same sample rather detected a number of other metabolites, some of which are precursors of aflatoxins. The reason might be that, while HPLC is vastly used for chromatography fingerprinting of the constituents, where standards (AFB1, AFB2, AFG1 and AFG2) solvent calibration thereby reprogramming the HPLC for the targeted aflatoxins. Unfortunately, these standards did not exist for the co-metabolites, therefore could not be picked up by the HPLC method. On the other hand, LC-MS/MS is the most successful interface and a powerful approach for identification of the unknown constituents [84]. The reason might be that LC-MS/MS is effective in quantifying trace level contaminants in food and feed as well as a range of parent compounds and their metabolites [85], many of which are easy to ionise and give good sensitivity often with crude sample preparation. This is because most of these metabolites (averufin, sterigmatocystin and seco-sterigmatocystin) are active precursors of AFB1 formation [86]. Using AF standards enhanced the HPLC method specificity. The results of this study corroborate those of Meyer, Skhosana [63] who detected no aflatoxins in maize samples using the LCMS/MS technique. In this study, we detected 6 metabolites in some maize samples using LCMS/MS compared to the study conducted in Nigeria by [44] where they detected about 65 metabolites in maize samples grown in Nigeria using the same method. Thirteen mycotoxin regulated metabolites in South Africa [63]; maize samples from SA used in this study contained about 23% of the regulated metabolites (aflatoxins B1 and B2 and 3-Nitropropionic acid). Apart from the AFB1 and AFB2 detected by the HPLC technique, the LCMS/MS detected metabolites such as: averufin, sterigmatocystin, seco-sterigmatocystin, 3-Nitropropionic acid (3NPA), Kojic acid and Orsellinic acid.

Among the metabolites found in the maize samples, averufin, sterigmatocystin and seco-sterigmatocystin were found only in one sample from the ER. Averufin, sterigmatocystin and seco-sterigmatocystin metabolites are active precursors for AFB1 formation [86]. Averufin (AVF) is a non-toxic early precursor of aflatoxin by a blocked mutant of Aspergillus parasiticus. Early steps in the synthesis of AFB1 involves the conversion of norsolorinic acid (NOR) to averantin (AVN), which is oxidized to 59-hydroxyaverantin (HAVN) and then to averufin (AVF). Yabe, Ando [87] found that HAVN is an intermediate in the enzymatic conversion of AVN to AVF. Yu, Chang [88] found that the avnA gene encodes a cytochrome P-450-type monooxygenase, necessary for converting AVN to HAVN. However, the enzyme involved in the oxidation of HAVN was not identified.

Sterigmatocystin is a metabolite produced mainly by Aspergillus fungi, and is an intermediate in the biosynthesis of aflatoxin B1. Sterigmatocystin is a potentially health hazardous mycotoxin, produced mainly by Aspergillus species and is usually detected in food and feed as a natural contaminant [89] [90], recent data indicates that exposure to sterigmatocystin can also occur through direct skin contact or inhalation. As a precursor of aflatoxin biosynthesis with hepatotoxic action, sterigmatocystin is classified as group 2B carcinogen by IARC [91] [92] [93] due to its association with immunotoxic, immune modulatory activities as well as mutagenic effects. The structure and bioactivity of sterigmatocystin are similar to those of aflatoxin; however, its toxicity is weaker compared to aflatoxin and is observed in the liver and the lung [94]. Suggestions are that sterigmatocystin is 1/10 as potent as aflatoxin B1 with a 10 day LD50 of 120 mg/kg in rats [95] [96]. The IARC-classification of sterigmatocystin is group 2B, which means it is carcinogenic in other species and possibly carcinogenic to humans, however, a definitive link between human exposure and cancer has not been proved.

The 3-Nitropropionic acid, one of the regulated metabolites detected by the LCMS/MS method, was found only in one sample (074/M/2015) from the WR contaminated with A. flavus. 3NPA is a natural potent environmental toxin and mitochondrial inhibitor [96], toxic to both humans and animals. 3NPA, a metabolite produced by a number of fungi, including Aspergillus species and widely found in food such as cereals and sugar cane, among others [97] and caused mostly by extreme weather, stressed crop growth and storage conditions [98], which can further rise under global warming conditions [99]. This might be the reason 3NPA was found only in WR, where weather conditions are extreme. Acute encephalopathy can develop to dystonia in humans exposed to low doses of 3-NPA [100]. 3NPA induces neurodegeneration in the caudate putamen in humans and experimental animals, resembling Huntington’s disease so, 3NPA poisoning primarily used as an animal model of selective neurodegeneration [101] [102] where toxin-treated experimental animals showed decreased motor performance [103]. Also, 3-NPA inhibits succinate dehydrogenase, a key enzyme for oxidative energy production [104], and causing ATP levels in the brain to fall. This effect develops fast and is not limited to the sites of morphological damage [101]. Since the nervous system requires lots of energy to work, mitochondrial damage is probably reflected in the electrical activity of the brain.

Kojic acid (5-hydroxy-2-(hydrxymethyl)-4-pyrone; KA), a non-regulated metabolite, is an organic acid secreted by several species of Aspergillus, especially A. oryzae [105]. It is multifunctional and has weak acidic property, non-hazardous biodegradation, making it an attractive and profitable skeleton for development of biologically active compounds by its derivatives [106]. Kojic acid (KA) is use as food derivative, antibiotic, antioxidant [105], a skin whitening agent, treatment of chloasma [107], antitumor agent [108] and radio protective agent [109]. This metabolite was the second highest detected in samples from both regions, while other samples had it but in unquantifiable amounts.

Orsellinic acid is a common salicylic acid unit in the biosynthesis of secondary metabolites in actinomycetes, fungi and lichens, formally isolated from chaetomium cochliodes in 1959 [110]. Orsellinic acid is a key biosynthetic intermediate of many depside metabolites in lichen and fungi. Orsellinic acid is also an important polar co-metabolite present in many fungi; however, its contribution to overall bioactivity is not well understood. Orsellinic acid is a useful standard for bioassay and analytical techniques for dereplication of common co-metabolites. This non-regulated metabolite was the most frequent metabolite detected in maize samples from both regions. Most of the metabolites detected (KA, orsellinic acid and seco-sterigmatocystin), usually colonise plant tissues and offer significant benefits to their host plants, by producing growth regulators, antimicrobials and antiviral, which are survival advantages [111].

Among the 58 Aspergillus flavus isolates tested if any of the five aflatoxigenic genes were present (aflD, aflM, omtA, aflJ and aflR), no Aspergillus flavus isolate had the regulatory aflR gene, three isolates possessed the omt-A gene (two isolates from WR and one from ER) eleven isolates possessed the aflD gene (seven from WR and four from ER) while sixteen isolates possessed the aflJ gene (twelve from WR and four from ER). Majority of the isolates that tested positive, possessed the aflM gene (twenty-one from WR and ten from ER). One isolate (from ER) possessed up to three of the five genes tested, fifteen isolates possessed two out of the five genes tested (thirteen from WR and two from ER) while twenty-eight isolates possessed, at least, one of the genes tested. 50% and 25.86% of tested isolates from WR and ER respectively tested positive for aflatoxigenicity. This study has shown that there is a clear similarity between A. flavus and aflatoxicity in both regions, which, agrees to earlier studies conducted using RT-PCR targeting aflD, aflQ, aflO, aflP, aflR and aflS genes [112] [113] [114] [115] [116]. Mahmoud [117] also reported a correlation between PCR amplification of four aflatoxin biosynthetic pathway genes (aflD, aflM, aflP, and aflQ), with the aflatoxins production ability of A. flavus isolates from stored peanuts in Egypt. Aflatoxins-producing genes present in an Aspergillus isolate does not necessarily confirm the isolate to be aflatoxigenic [44] [114] [118] [119]. PCR-based techniques are important to detect aflatoxigenic potential of Aspergillus isolates [120] [121] [122], since these genes encode the key enzymes and regulatory factors in the pathway of biosynthesis of aflatoxins.

5. Conclusion

Climate change is clear and has been increasing over the past few years and this could join a threat to food security and safety. Even though the findings of this study show that irrespective of the climatic variations of the different regions in South Africa, Aspergillus species and aflatoxin production is not yet a threat to South Africa’s commercial food/feed industry, the frequent occurrence of drought in South Africa as experienced in 2015 is a clear sign that climatic zones that appear safe now might later lead to risk of disease and/or loss in crop production as climatic conditions change. There is, therefore, a need for long-term and continuous monitoring of Aspergillus species and their toxins in maize across different agro-climatic areas of South Africa (4 to 5 years) as well as other suppliers of agricultural maize and maize grown and stored by small-scale farmers. This will help simulate and model a trend that can clearly predict the long-term effects of climate change on aflatoxins in South African maize. Extensive studies will be carried out on strategies such as competitive exclusion and touch inhibition and real application be done on South African fields comparatively in the different agro-climatic regions.

Funding

This research was funded by North West University, Mafikeng and The APC was funded by North West University, Mafikeng.

Acknowledgements

The authors wish to acknowledge the North-west University for the privilege to carry out this study and support to publish the findings.

Conflicts of Interest

The authors declare no conflict of interest.

Cite this paper

Nji, N.Q., Christianah, A.M., Njie, A.C. and Mulunda, M. (2022) Biodiversity and Distribution of Aspergillus and Their Toxins in Maize from Western and Eastern Regions of South Africa. Advances in Microbiology, 12, 121-149. https://doi.org/10.4236/aim.2022.123011

ReferencesDhakal, A. and Sbar, E. (2020) Aflatoxin Toxicity. StatPearls [Internet].IARC. International Agency for Research on Cancer (2012) Review of Human Carcinogens: Chemical Agents and Related Occupations. World Health Organization, Geneva.Rushing, B.R. and Selim, M.I. (2019) Aflatoxin B1: A Review on Metabolism, Toxicity, Occurrence in Food, Occupational Exposure, and Detoxification Methods. Food and Chemical Toxicology, 124, 81-100. https://doi.org/10.1016/j.fct.2018.11.047Udovicki, B., et al. (2018) Overview on the Mycotoxins Incidence in Serbia in the Period 2004-2016. Toxins, 10, Article 279. https://doi.org/10.3390/toxins10070279Pankaj, S.K., Shi, H. and Keener, K.M. (2018) A Review of Novel Physical and Chemical Decontamination Technologies for Aflatoxin in Food. Trends in Food Science & Technology, 71, 73-83. https://doi.org/10.1016/j.tifs.2017.11.007Magan, N., Medina, A. and Aldred, D. (2011) Possible Climate-Change Effects on Mycotoxin Contamination of Food Crops Pre-And Postharvest. Plant Pathology, 60, 150-163. https://doi.org/10.1111/j.1365-3059.2010.02412.xMiraglia, M., et al. (2009) Climate Change and Food Safety: An Emerging Issue with Special Focus on Europe. Food and Chemical Toxicology, 47, 1009-1021.https://doi.org/10.1016/j.fct.2009.02.005Benkerroum, N. (2019) Retrospective and Prospective Look at Aflatoxin Research and Development from a Practical Standpoint. International Journal of Environmental Research and Public Health, 16, Article 3633. https://doi.org/10.3390/ijerph16193633Ojuri, O.T., et al. (2018) Assessing the Mycotoxicological Risk from Consumption of Complementary Foods by Infants and Young Children in Nigeria. Food and Chemical Toxicology, 121, 37-50. https://doi.org/10.1016/j.fct.2018.08.025Onyeke, C.C., et al. (2017) Aflatoxins Composition of Maize (Zea mays L.), Guinea Corn (Sorghum bicolor L.) Cold Paps and Peanut (Arachis hypogea L.) Butter in Nsukka, Nigeria. Journal of Basic Pharmacology and Toxicology, 1, 18-22.Paterson, R.R.M. and Lima, N. (2011) Further Mycotoxin Effects from Climate Change. Food Research International, 44, 2555-2566. https://doi.org/10.1016/j.foodres.2011.05.038Paterson, R.R.M. and Lima, N. (2010) How Will Climate Change Affect Mycotoxins in Food? Food Research International, 43, 1902-1914. https://doi.org/10.1016/j.foodres.2009.07.010Wu, F., et al. (2011) Climate Change Impacts on Mycotoxin Risks in US Maize. World Mycotoxin Journal, 4, 79-93. https://doi.org/10.3920/WMJ2010.1246Wu, S., et al. (2011) Magnetic Nanobead-Based Immunoassay for the Simultaneous Detection of Aflatoxin B1 and Ochratoxin A Using Upconversion Nanoparticles as Multicolor Labels. Biosensors and Bioelectronics, 30, 35-42.https://doi.org/10.1016/j.bios.2011.08.023Magan, N., et al. (2010) Environmental Factors and Interactions with Mycobiota of Grain and Grapes: Effects on Growth, Deoxynivalenol and Ochratoxin Production by Fusarium culmorum and Aspergillus carbonarius. Toxins, 2, 353-366.https://doi.org/10.3390/toxins2030353Paterson, R. and Lima, N. (2012) Climate Change, Fumonisins and Animal Feed.Ojiambo, P.S., et al. (2018) Cultural and Genetic Approaches to Manage Aflatoxin Contamination: Recent Insights Provide Opportunities for Improved Control. Phytopathology, 108, 1024-1037. https://doi.org/10.1094/PHYTO-04-18-0134-RVWBattilani, P., Barbano, C. and Piva, G. (2008) Aflatoxin B1 Contamination in Maize Related to the Aridity Index in North Italy. World Mycotoxin Journal, 1, 449-456.https://doi.org/10.3920/WMJ2008.x043Giorni, P., et al. (2009) Control of the Development of Aspergillus flavus in Maize during Post-Harvest. Tecnica Molitoria, 60, 261-267.Lahouar, A., et al. (2016) Effects of Temperature, Water Activity and Incubation Time on Fungal Growth and Aflatoxin B1 Production by Toxinogenic Aspergillus flavus Isolates on Sorghum Seeds. Revista Argentina de Microbiologia, 48, 78-85.https://doi.org/10.1016/j.ram.2015.10.001Piva, G., Battilani, P. and Pietri, A. (2006) Emerging Issues in Southern Europe: Aflatoxins in Italy. Wageningen Academic Publishers, Wageningen.Scheidegger, K. and Payne, G. (2003) Unlocking the Secrets behind Secondary Metabolism: A Review of Aspergillus flavus from Pathogenicity to Functional Genomics. Journal of Toxicology: Toxin Reviews, 22, 423-459. https://doi.org/10.1081/TXR-120024100Kachapulula, P., et al. (2017) Aflatoxin Contamination of Groundnut and Maize in Zambia: Observed and Potential Concentrations. Journal of Applied Microbiology, 122, 1471-1482. https://doi.org/10.1111/jam.13448Boutigny, A.L., et al. (2012) Quantitative Detection of Fusarium Pathogens and Their Mycotoxins in South African Maize. Plant Pathology, 61, 522-531.https://doi.org/10.1111/j.1365-3059.2011.02544.xSaifaddin, G. (2021) South Africa: Maize Production, by Province 2019/2020.AOAC (2007) Official Methods of Analysis. 18th Edition, Association of Official Analytical Chemists.Samson, R.A. (1995) Introduction to Food-Borne Fungi. Centraalbureau voor Schimmelcultures, Utrecht.Aziz, N.H, et al. (1998) Comparative Antibacterial and Antifungal Effects of Some Phenolic Compounds. Microbios, 93, 43-54.Suleiman, M.N. and Taiga, A. (2009) Efficacy of Aquenous Extracts of Neem and Sharf from the Control of Fungi Associated with Milled and Unmilled Store Rice Grain. 5th Proceeding of the Humboldt Kellog Annual Agriculture Conference, 71-73.Pitt, J.I. and Hocking, A.D. (2009) Fungi and Food Spoilage. Vol. 519, Springer Science & Business Media, Berlin. https://doi.org/10.1007/978-0-387-92207-2R&oslash;nhede, S., et al. (2005) Hydroxylation of the Herbicide Isoproturon by Fungi Isolated from Agricultural Soil. Applied and Environmental Microbiology, 71, 7927-7932. https://doi.org/10.1128/AEM.71.12.7927-7932.2005Tambekar, P. and Wate, S. (2007) Study of Phosphate Solubilization Efficiencies of Fungi and Bacteria Isolated from Saline Belt of Puma River Basin. Research Journal of Agriculture and Biological Sciences, 3, 701-703.Montso, K.P. and Ateba, C.N. (2014) Molecular Detection of Clostridium Species in Beef Obtained from Retail Shops in North West Province, South Africa. Journal of Food and Nutrition Research, 2, 236-243. https://doi.org/10.12691/jfnr-2-5-5Pinto, A.J. and Raskin, L. (2012) PCR Biases Distort Bacterial and Archaeal Community Structure in Pyrosequencing Datasets. PLoS ONE, 7, e43093.https://doi.org/10.1371/journal.pone.0043093Sabino, R., et al. (2014) Molecular Screening of 246 Portuguese Aspergillus Isolates among Different Clinical and Environmental Sources. Medical Mycology, 52, 519-529. https://doi.org/10.1093/mmy/myu006Glass, N.L. and Donaldson, G.C. (1995) Development of Primer Sets Designed for Use with the PCR to Amplify Conserved Genes from Filamentous Ascomycetes. Applied and Environmental Microbiology, 61, 1323-1330.https://doi.org/10.1128/aem.61.4.1323-1330.1995Green, M.R. AND Sambrook, J. (2012) Molecular Cloning. A Laboratory Manual 4th.Vol. 1, Cold Spring Harbor Laboratory Press, New York.Altschul, S.F. and Koonin, E.V. (1998) Iterated Profile Searches with PSI-BLAST—A tool for Discovery in Protein Databases. Trends in Biochemical Sciences, 23, 444-447. https://doi.org/10.1016/S0968-0004(98)01298-5Criseo, G., Bagnara, A. and Bisignano, G. (2001) Differentiation of Aflatoxin-Producing and Non-Producing Strains of Aspergillus flavus Group. Letters in Applied Microbiology, 33, 291-295. https://doi.org/10.1046/j.1472-765X.2001.00998.xJefremova, M., et al. (2016) Rapid Diagnostic Testing of Toxigenic Microfungi Isolated from Foodstuffs. Acta Veterinaria Brno, 85, 151-156. https://doi.org/10.2754/avb201685020151Midorikawa, G., et al. (2008) Characterization of Aspergillus flavus Strains from Brazilian Brazil Nuts and Cashew by RAPD and Ribosomal DNA Analysis. Letters in Applied Microbiology, 47, 12-18.https://doi.org/10.1111/j.1472-765X.2008.02377.xYin, Y.-N., et al. (2008) Biological Control of Aflatoxin Contamination of Crops. Journal of Zhejiang University Science B, 9, 787-792. https://doi.org/10.1631/jzus.B0860003Rashid, M., et al. (2008) Categorization of Aspergillus flavus and Aspergillus parasiticus Isolates of Stored Wheat Grains in to Aflatoxinogenics and Non-Aflatoxinogenics. Pakistan Journal of Botany, 40, 2177-2192.Adetunji, M., et al. (2014) Fungal and Bacterial Metabolites of Stored Maize (Zea mays, L.) from Five Agro-Ecological Zones of Nigeria. Mycotoxin Research, 30, 89-102. https://doi.org/10.1007/s12550-014-0194-2Currie, L.A. (1995) Nomenclature in Evaluation of Analytical Methods including Detection and Quantification Capabilities (IUPAC Recommendations 1995). Pure and Applied Chemistry, 67, 1699-1723. https://doi.org/10.1351/pac199567101699Laca, A., et al. (2006) Distribution of Microbial Contamination within Cereal Grains. Journal of Food Engineering, 72, 332-338. https://doi.org/10.1016/j.jfoodeng.2004.12.012Mohale, S., et al. (2013) Mycotoxigenic Fungi and Mycotoxins Associated with Stored Maize from Different Regions of Lesotho. Mycotoxin Research, 29, 209-219.https://doi.org/10.1007/s12550-013-0176-9Muga, F.C., Marenya, M. and Workneh, T.S. (2019) Effect of Temperature, Relative Humidity and Moisture on Aflatoxin Contamination of Stored Maize Kernels. Bulgarian Journal of Agricultural Science, 25, 271-277.Cleveland, T.E., et al. (2003) United States Department of Agriculture—Agricultural Research Service Research on Pre-Harvest Prevention of Mycotoxins and Mycotoxigenic Fungi in US Crops. Pest Management Science: Formerly Pesticide Science, 59, 629-642. https://doi.org/10.1002/ps.724Maina, A.W., et al. (2017) Assessment of Farmers Maize Production Practices and Effect of Triple-Layer Hermetic Storage on the Population of Fusarium Spp. and Fumonisin Contamination. World Journal of Agricultural Research, 5, 21-30.Maina, A.W., Wagacha, J.M., Mwaura, F.B., Muthomi, J.W. and Woloshuk, C.P. (2016) Postharvest Practices of Maize Farmers in Kaiti District, Kenya and the Impact of Hermetic Storage on Populations of Aspergillus spp. and Aflatoxin. http://erepository.uonbi.ac.ke/handle/11295/103442contaminationKimanya, M., et al. (2008) Human Exposure to Fumonisins from Home Grown Maize in Tanzania. World Mycotoxin Journal, 1, 307-313.https://doi.org/10.3920/WMJ2008.x032Greeff-Laubscher, M.R., et al. (2020) Mycotoxin Production by Three Different Toxigenic Fungi Genera on Formulated Abalone Feed and the Effect of an Aquatic Environment on Fumonisins. Mycology, 11, 105-117.https://doi.org/10.1080/21501203.2019.1604575Chilaka, C.A., et al. (2012) Fungal and Mycotoxin Contamination of South African Commercial Maize. Journal of Food, Agriculture and Environment, 10, 296-303.Lahouar, A., et al. (2015) Toxigenic Molds in Tunisian and Egyptian Sorghum for Human Consumption. Journal of Stored Products Research, 63, 57-62.https://doi.org/10.1016/j.jspr.2015.07.001Chen, T., et al. (2016) Assessment of Drought Impact on Main Cereal Crops Using a Standardized Precipitation Evapotranspiration Index in Liaoning Province, China. Sustainability, 8, Article 1069. https://doi.org/10.3390/su8101069Egbuta, M.A. (2011) Occurrence of Mycotoxins in Nigerian Food Commodities and Health Risk Assessment. Master’s Thesis, University of Johannesburg, South Africa.Magan, N. and Aldred, D. (2007) Post-Harvest Control Strategies: Minimizing Mycotoxins in the Food Chain. International Journal of Food Microbiology, 119, 131-139. https://doi.org/10.1016/j.ijfoodmicro.2007.07.034Nesci, A. and Etcheverry, M. (2002) Aspergillus Section Flavi Populations from Field Maize in Argentina. Letters in Applied Microbiology, 34, 343-348.https://doi.org/10.1046/j.1472-765X.2002.01094.xBryden W.L. ,et al. (2007)Mycotoxins in the Food Chain: Human Health Implications Asia Pacific Journal of Clinical Nutrition 16, 95-101.Kankolongo, M.A., Hell, K. and Nawa, I.N. (2009) Assessment for Fungal, Mycotoxin and Insect Spoilage in Maize Stored for Human Consumption in Zambia. Journal of the Science of Food and Agriculture, 89, 1366-1375.https://doi.org/10.1002/jsfa.3596Gruber-Dorninger, C., Jenkins, T. and Schatzmayr, G. (2018) Multi-Mycotoxin Screening of Feed and Feed Raw Materials from Africa. World Mycotoxin Journal, 11, 369-383. https://doi.org/10.3920/WMJ2017.2292Meyer, H., et al. (2019) Long Term Monitoring (2014-2018) of Multi-Mycotoxins in South African Commercial Maize and Wheat with a Locally Developed and Validated LC-MS/MS Method. Toxins, 11, Article 271. https://doi.org/10.3390/toxins11050271Botai, C.M., et al. (2017) Drought Characteristics over the Western Cape Province, South Africa. Water, 9, Article 876. https://doi.org/10.3390/w9110876Botai, C.M., Botai, J.O. and Adeola, A.M. (2018) Spatial Distribution of Temporal Precipitation Contrasts in South Africa. South African Journal of Science, 114, 70-78. https://doi.org/10.17159/sajs.2018/20170391D&ouml;vényi-Nagy, T., et al. (2020) Pre-Harvest Modelling and Mitigation of Aflatoxins in Maize in a Changing Climatic Environment—A Review. Toxins, 12, Article 768.https://doi.org/10.3390/toxins12120768Jani&cacute; Hajnal, E., et al. (2017) Aflatoxins Contamination of Maize in Serbia: The Impact of Weather Conditions in 2015. Food Additives & Contaminants: Part A, 34, 1999-2010. https://doi.org/10.1080/19440049.2017.1331047Pleadin, J., et al. (2014) Ochratoxin A Reduction in Meat Sausages Using Processing Methods Practiced in Households. Food Additives & Contaminants: Part B, 7, 239-246. https://doi.org/10.1080/19393210.2014.900119Achaglinkame, A.M., Opoku, N. and Amagloh, F.K. (2017) Aflatoxin Contamination in Cereals and Legumes to Reconsider Usage as Complementary Food Ingredients for Ghanaian Infants: A Review. Journal of Nutrition & Intermediary Metabolism, 10, 1-7. https://doi.org/10.1016/j.jnim.2017.09.001Gallo, A., et al. (2016) Effect of Temperature and Water Activity on Gene Expression and Aflatoxin Biosynthesis in Aspergillus flavus on Almond Medium. International Journal of Food Microbiology, 217, 162-169.https://doi.org/10.1016/j.ijfoodmicro.2015.10.026Gizachew, D., et al. (2019) Aflatoxin B1 (AFB1) Production by Aspergillus flavus and Aspergillus parasiticus on Ground Nyjer Seeds: The Effect of Water Activity and Temperature. International Journal of Food Microbiology, 296, 8-13.https://doi.org/10.1016/j.ijfoodmicro.2019.02.017Lv, C., et al. (2019) Interaction of Water Activity and Temperature on the Growth, Gene Expression and Aflatoxin Production by Aspergillus flavus on Paddy and Polished Rice. Food Chemistry, 293, 472-478.https://doi.org/10.1016/j.foodchem.2019.05.009Battilani, P., et al. (2016) Aflatoxin B1 Contamination in Maize in Europe Increases Due to Climate Change. Scientific Reports, 6, Article No. 24328.https://doi.org/10.1038/srep24328Shooshtari, A., et al. (2007) Application of PCR on Detection of Aflatoxinogenic Fungi. Archives of Razi Institute, 62, 95-100.Kurtzman, C., et al. (1986) DNA Relatedness among Wild and Domesticated Species in the Aspergillus flavus Group. Mycologia, 78, 955-959.https://doi.org/10.2307/3807436Wild, C.P. and Gong, Y.Y. (2010) Mycotoxins and Human Disease: A Largely Ignored Global Health Issue. Carcinogenesis, 31, 71-82.https://doi.org/10.1093/carcin/bgp264Turner, P.C., et al. (2003) Modification of Immune Function through Exposure to Dietary Aflatoxin in Gambian Children. Environmental Health Perspectives, 111, 217-220. https://doi.org/10.1289/ehp.5753Tchana, A.N., Moundipa, P.F. and Tchouanguep, F.M. (2010) Aflatoxin Contamination in Food and Body Fluids in Relation to Malnutrition and Cancer Status in Cameroon. International Journal of Environmental Research and Public Health, 7, 178-188. https://doi.org/10.3390/ijerph7010178Gong, Y., et al. (2003) Determinants of Aflatoxin Exposure in Young Children from Benin and Togo, West Africa: the Critical Role of Weaning. International Journal of Epidemiology, 32, 556-562. https://doi.org/10.1093/ije/dyg109Yearbook, F.S. (2013) World Food and Agriculture. Food and Agriculture Organization of the United Nations, Rome, 15.Sydenham, E.W., et al. (1991) Fumonisin Contamination of Commercial Corn-Based Human Foodstuffs. Journal of Agricultural and Food Chemistry, 39, 2014-2018. https://doi.org/10.1021/jf00011a028Marasas, W. (1997) Risk Assessment of Fumonisins Produced by Fusarium Moniliforme in Corn. Cereal Research Communications, 25, 399-406. https://doi.org/10.1007/BF03543742Asghar, M.A., et al. (2014) Occurrence of Aflatoxins Contamination in Brown Rice from Pakistan. Iranian Journal of Public Health, 43, 291-299.Yang, T., et al. (2013) Comparative Pharmacokinetic Studies of Andrographolide and Its Metabolite of 14-Deoxy-12-Hydroxy-Andrographolide in Rat by Ultra-Performance Liquid Chromatography-Mass Spectrometry. Biomedical Chromatography, 27, 931-937. https://doi.org/10.1002/bmc.2884Owen, L.J., et al. (2019) Oestradiol Measurement during Fulvestrant Treatment for Breast Cancer. British Journal of Cancer, 120, 404-406. https://doi.org/10.1038/s41416-019-0378-9Rodricks, J.V. (1969) Fungal Metabolites Which Contain Substituted 7,8-Dihydrofuro[2,3-b] Furans (DHFF) and 2,3,7,8-Tetrahydrofuro[2,3-b] Furans (THFF). Journal of Agricultural and Food Chemistry, 17, 457-461.https://doi.org/10.1021/jf60163a007Yabe, K., Ando, Y. and Hamasaki, T. (1991) A Metabolic Grid among Versiconal Hemiacetal Acetate, Versiconol Acetate, Versiconol and Versiconal during Aflatoxin Biosynthesis. Microbiology, 137, 2469-2475.https://doi.org/10.1099/00221287-137-10-2469Yu, J., et al. (1997) avnA, a Gene Encoding a Cytochrome P-450 Monooxygenase, Is Involved in the Conversion of Averantin to Averufin in Aflatoxin Biosynthesis in Aspergillus parasiticus. Applied and Environmental Microbiology, 63, 1349-1356.https://doi.org/10.1128/aem.63.4.1349-1356.1997Ver&scaron;ilovskis, A. and De Saeger, S. (2010) Sterigmatocystin: Occurrence in Foodstuffs and Analytical Methods—An Overview. Molecular Nutrition & Food Research, 54, 136-147. https://doi.org/10.1002/mnfr.200900345Wang, J.-S., et al. (1999) Protective Alterations in Phase 1 and 2 Metabolism of Aflatoxin B 1 by Oltipraz in Residents of Qidong, People's Republic of China. Journal of the National Cancer Institute, 91, 347-354. https://doi.org/10.1093/jnci/91.4.347IARC (1976) Monographs on the Evaluation of Cancinogenic Risks to Humans, in World Health Organization International Agency for Research on Cancer. Lyon, France.IARC (2008) Monographs on the Evaluation of Carcinogenic Risks to Humans, 1,3-Butadiene, Ethylene Oxide and Vinyl Halides (Vinyl Fluoride, Vinyl Chloride and Vinyl Bromide), Vol. 97, pp. 1-510.Yabe, K. and Nakajima, H. (2004) Enzyme Reactions and Genes in Aflatoxin Biosynthesis. Applied Microbiology and Biotechnology, 64, 745-755.https://doi.org/10.1007/s00253-004-1566-xPurchase, I.F.H. and Van der Watt, J. (1970) Carcinogenicity of Sterigmatocystin. Food and Cosmetics Toxicology, 8, 289-290.https://doi.org/10.1016/S0015-6264(70)80004-9IARC (2002) Monograph on the Evaluation of Carcinogenic Risks to Humans: Some Traditional Herbal Medicines, Some Mycotoxins, Naphthalene and Styrene, Iarc Press, Lyon, Vol 82.Roberts, T.J. (2005) 3-Nitropropionic Acid Model of Metabolic Stress. In Stroke Genomics, Humana Press, pp. 203-220.Wang, X. and Yang, R. (2003) Movement Disorders Possibly Induced by Traditional Chinese Herbs. European Neurology, 50, 153-159.https://doi.org/10.1159/000073056Jensen, A.N. and Baggesen, D.L. (2005) Salmonella Infection Risk Associated with Outdoor Organic Pig Production. System Development: Quality and Safety of Organic Livestock Products, pp. 87-93.Bhat, R., Rai, R.V. and Karim, A.A. (2010) Mycotoxins in Food and Feed: Present Status and Future Concerns. Comprehensive Reviews in Food Science and Food Safety, 9, 57-81. https://doi.org/10.1111/j.1541-4337.2009.00094.xLiu, X., Luo, X. and Hu, W. (1992) Studies on the Epidemiology and Etiology of Moldy Sugarcane Poisoning in China. Biomedical and Environmental Sciences, 5, 161-177.Brouillet, E., et al. (1999) Replicating Huntington’s Disease Phenotype in Experimental Animals. Progress in Neurobiology, 59, 427-468.https://doi.org/10.1016/S0301-0082(99)00005-2Maya-López, M., et al. (2017) Neuroprotective Effect of WIN55, 212-2 against 3-Nitropropionic Acid-Induced Toxicity in the Rat Brain: Involvement of CB1 and NMDA Receptors. American Journal of Translational Research, 9, 261-274.Teunissen, C., et al. (2001) Behavioural Correlates of Striatal Glial Fibrillary Acidic Protein in the 3-Nitropropionic Acid Rat Model: Disturbed Walking Pattern and Spatial Orientation. Neuroscience, 105, 153-167. https://doi.org/10.1016/S0306-4522(01)00164-6Coles, C.J., Edmondson, D.E. and Singer, T.P. (1979) Inactivation of Succinate Dehydrogenase by 3-Nitropropionate. Journal of Biological Chemistry, 254, 5161-5167. https://doi.org/10.1016/S0021-9258(18)50574-3Bentley, R. (2006) From Miso, Sake and Shoyu to Cosmetics: A Century of Science for Kojic Acid. Natural Product Reports, 23, 1046-1062. https://doi.org/10.1039/b603758pChaudhary, J., Pathak, A. and Lakhawat, S. (2014) Production Technology and Applications of Kojic Acid. Annual Research & Review in Biology, 2014, 3165-3196.https://doi.org/10.9734/ARRB/2014/10643Terabayashi, Y., et al. (2010) Identification and Characterization of Genes Responsible for Biosynthesis of Kojic Acid, an Industrially Important Compound from Aspergillus oryzae. Fungal Genetics and Biology, 47, 953-961.https://doi.org/10.1016/j.fgb.2010.08.014Tamura, T., et al. (2001) Dose-Threshold for Thyroid Tumor-Promoting Effects of Orally Administered Kojic Acid in Rats after Initiation with N-Bis(2-Hydroxypropyl) Nitrosamine. The Journal of Toxicological Sciences, 26, 85-94. https://doi.org/10.2131/jts.26.85Emami, S., et al. (2007) Kojic Acid and Its Manganese and Zinc Complexes as Potential Radioprotective Agents. Bioorganic & Medicinal Chemistry Letters, 17, 45-48. https://doi.org/10.1016/j.bmcl.2006.09.097Lackner, G., et al. (2013) Assembly of Melleolide Antibiotics Involves a Polyketide Synthase with Cross-Coupling Activity. Chemistry & Biology, 20, 1101-1106.https://doi.org/10.1016/j.chembiol.2013.07.009Stone, J.K., Polishook, J.D. and White, J.F. (2004) Endophytic Fungi. In: Foster, M. and Bills, G., Eds., Biodiversity of Fungi, Elsevier Academic Press, Burlington, 241-270. https://doi.org/10.1016/B978-012509551-8/50015-5Baquiao, A.C., et al. (2016) Expression of Genes by Aflatoxigenic and Nonaflatoxigenic Strains of Aspergillus flavus Isolated from Brazil Nuts. Foodborne Pathogens and Disease, 13, 434-440. https://doi.org/10.1089/fpd.2015.2111Davari, E., et al. (2015) Characterization of Aflatoxigenic Aspergillus flavus and A. parasiticus Strain Isolates from Animal Feedstuffs in Northeastern Iran. Iranian Journal of Veterinary Research, 16, 150-155.Fakruddin, M., et al. (2015) Characterization of Aflatoxin Producing Aspergillus flavus from Food and Feed Samples. SpringerPlus, 4, Article No. 159.https://doi.org/10.1186/s40064-015-0947-1Jamali, M., et al. (2013) Expression of Aflatoxin Genes aflO (omtB) and aflQ (ordA) Differentiates Levels of Aflatoxin Production by Aspergillus flavus Strains from Soils of Pistachio Orchards. Research in Microbiology, 164, 293-299.https://doi.org/10.1016/j.resmic.2012.12.008Sweeney, M.J., Pàmies, P. and Dobson, A.D. (2000) The Use of Reverse Transcription-Polymerase Chain Reaction (RT-PCR) for Monitoring Aflatoxin Production in Aspergillus parasiticus 439. International Journal of Food Microbiology, 56, 97-103.https://doi.org/10.1016/S0168-1605(00)00277-4Mahmoud, M.A. (2015) Detection of Aspergillus flavus in Stored Peanuts Using Real-Time PCR and the Expression of Aflatoxin Genes in Toxigenic and Atoxigenic A. flavus Isolates. Foodborne Pathogens and Disease, 12, 289-296. https://doi.org/10.1089/fpd.2014.1854Accinelli, C., et al. (2008) Aspergillus flavus Aflatoxin Occurrence and Expression of Aflatoxin Biosynthesis Genes in Soil. Canadian Journal of Microbiology, 54, 371-379. https://doi.org/10.1139/W08-018Loureno, A., et al. (2007) Genetic Diversity of Environmental Aspergillus flavus Strains in the State of S&atilde;o Paulo, Brazil by Random Amplified Polymorphic DNA. Memorias do Instituto Oswaldo Cruz, 102, 687-692.https://doi.org/10.1590/S0074-02762007000600005Chen, R.-S., et al. (2002) Polymerase Chain Reaction-Mediated Characterization of Molds Belonging to the Aspergillus flavus Group and Detection of Aspergillus parasiticus in Peanut Kernels by a Multiplex Polymerase Chain Reaction. Journal of Food Protection, 65, 840-844. https://doi.org/10.4315/0362-028X-65.5.840Mayer, Z., Farber, P. and Geisen, R. (2003) Monitoring the Production of Aflatoxin B1 in Wheat by Measuring the Concentration of Nor-1 mRNA. Applied and Environmental Microbiology, 69, 1154-1158. https://doi.org/10.1128/AEM.69.2.1154-1158.2003Shapira, R., et al. (1996) Detection of Aflatoxigenic Molds in Grains by PCR. Applied and Environmental Microbiology, 62, 3270-3273. https://doi.org/10.1128/aem.62.9.3270-3273.1996