Aflatoxins and Fumonisins Contamination of Maize in Bangladesh: An Emerging Threat for Safe Food and Food Security

Maize (Bhutta) is one of the important growing cereal crops in Bangladesh. Toxigenic fungi such as Aspergillus and Fusarium infect stored maize grains. Enzyme-linked immusorbent assay (ELISA) was used to determine total aflatoxins and fumonisins contamination in stored maize grains collected from 15 Bangladeshi maize-producing areas. The highest total concentration of aflatoxins (103.07 µg/kg) and fumonisin (9.18 mg/kg) was found in Chuadanga and Gaibandha, whereas the lowest was detected for aflatoxins (1.07 µg/kg) and (0.11 mg/kg) in Dinajpur and Cumilla, respectively. The findings clearly demonstrated that aflatoxin concentrations in samples from six regions and fumonisin concentrations in samples from 10 regions were beyond the regulatory limit of aflatoxin (10 ppb) and fumonisin (1 ppm), respectively, as set by European Union (EU). However, a positive correlation between aflatoxins with toxigenic A. flavus, and fumonisins with toxigenic Fusarium spp. was observed. The fungi associated with maize grains were identified by sequencing of ITS regions. Moreover, toxigenic A. flavus was confirmed using primers specific to nor, apa2, omtA and primer FUM1 for F. proliferatum and F. oxysporum. Since the Bangladesh Food Safety Authority has not authorized any precise regulation limits for maize mycotoxin contamination, these results will serve as a benchmark for monitoring mycotoxin contamination in maize and also to develop globally practiced biocontrol approach for producing safe food and feed.


Introduction
or Zea mays L. (corn) is one of the supreme vital cereals in the globe which belongs to Poaceae family and it has been ranked as a third position in the last few decades after wheat and rice [1]. A fair number of food and industrial commodities such as maize flour, animal feed, cooking ingredient, corn syrup, grain alcohol and whiskey are processed from maize [2]. Maize has been known as a significant emerging crop in Bangladesh as well as maize production is familiarized destructive and highly abundant fumonisin toxins where FB1 is the most ruinous due to its availability of high concentration on host ranging from 70 % to 80 % of all fumonisins [36][37][38]. Several biotic (temperature, water stress) and abiotic (osmotic stress, pH, and fungicides) factors are resposnsible for Fusarium growth and Fumonisin production [39,40]. At maturity stage damage occurs by insects, during flowering wet warm weather, rain before harvest, humidity, and media composition for both the Fusarium spp., all the activites are related to fumonisins production [41,42]. FUM1 gene can also expressed by ecological conditions reported by [43,44]. As Fusarium is widespread and ubiquitous in all cereal growing regions of the globe and corresponding mycotoxins are produced which has been influenced by storage methods and crop production [45]. In the midst of milling, storage, processing, cooking of food and feed, Fusarium are highly stable due to its structure and humans and animals are exhibited to them to a certain degree [46][47][48]. In Bangladesh, animal feed samples were detected and found fumonisin contamination mainly maize based feed contamination [49].
An investigation came out that in South Asia has been ranking as the utmost prevalent continent in case of exposing aflatoxins contamination (82 %) in the globe as well as 41 % maize samples were detected higher amount of aflatoxins contamination than the permissible limit of lenient EU criteria [49]. The very first outbreak of mycotoxin (Sterigmatocystin) was found in Bangladesh in rice straw [50], later in maize and poultry birds [51]. Liver cancer and hepatitis B infection promotes carcinogenic potency in specific individuals by aflatoxins [52,53]. In Japan, in the year of 1991-2009, violation cases were detected exceeded 1500 in foods which were imported at a level of 10-4918 mg/kg [54]. 62 % children with the age of 3 are at a complete risk of infecting with aflatoxins as aflatoxins biomarkers was detected in plasma of their blood [55]. According to WFP (World Food Program), permissible limit of aflatoxins is 10 ppb (10 μg/kg) and for fumonisins it is 1 ppm (1 mg/kg) [56]. Fumonisins toxin may causes esophageal carcinoma in humans [57], as well as contaminated with folate uptake in cellular level [58] and surging the intensity of neural tube defect [59]. 52 % positive rate of fumonisins was found with an overall level of 936 mg/kg in Asia [60]. Fusarium mycotoxin can cause leukoencephalomalacia, porcine pulmonary edema and rat hepatocarcinoma in human and livestock as well [55,61,62] detected that in Dhaka, Bangladesh 62 % of 3 year old children had aflatoxin biomarkers in their blood plasma revealing chronic aflatoxin exposure as reported earlier that significant amount aflatoxins were found from corn selling in the Bangladeshi market. Probably 1311 cases of liver cancer was detected every year in Bangladesh [63]. In can be deduced from abovementioned fact that determining aflatoxins and fumonisins and all other mycotoxins in food and feed are the prime need for the country like Bangladesh as these mycotoxin substantially subverts our plants yield concurrently human and animal lives as well. Thus, more research needs to be conducted to elicit the specific mycotoxin hampering specific food, feed and plants, besides to find out the plausible management for controlling these mycotoxins. This study highly exhibited the aflatoxins and fumonisins toxin level in Bangladesh from maize samples of different regions as it has been concerned as one of the burning issues for ensuring safety food.

Sample collection
Composite stored maize grain samples were collected from 15 maize growing areas of Bangladesh such as Bogura, Kushtia, Meherpur, Chuadanga, Kishoreganj, Manikganj, Cumilla, Rajshahi, Dinajpur, Rangpur, Natore, Thakurgaon, Panchagarh, Nilphamary and Jashore. Maize samples were collected from stores of traders in local markets of different districts. Ten markets were sampled in each district having at least five traders in each market. At least two quarter of kilogram unique samples were coalesced from each trader for laboratory analysis. Samples were collected after thoroughly mixing maize in the bag to increase chances of getting the fungi. The samples were stored at temperatures below 4° C to await analysis.

Procedure of sample preparation
A representative sample was taken and it was grounded with blender so that 75 % of that grounded portion can pass through a 20-mesh sieve, then thoroughly the subsample portion was mixed. 50 g of ground sample was weighed out into a clean conical flask that can be tightly sealed. 250 mL of methanol (70 % methanol diluted in water) extraction solution was added to the ground sample and the flask was sealed. Then the conical flask containing the sample was shaken for 3 min. The sample was allowed to settle down, then the top layer of extract was filtered through a Whatman #1 filter paper and the filtrate sample was collected. The prepared extract was diluted at 1:20 with distilled water. Sample was ready for testing without further preparation.

Assay protocol for aflatoxins
200 μL conjugate solution was pipetted into dilution wells. 100 μL of each standard or sample extract was added into the dilution wells. The mixture was mixed well and 100 μL of the mixture (conjugate and standard or samples) was transferred into antibody-coated wells. The plate was then incubated for 15 min with slow shaking and washed with distilled water for 5 times. The plate was then tap dried. 100 μL of substrate solution was pipetted into antibody coated wells. The plate was incubated with shaking for 5 min. 100 μL of stop solution was pipetted into antibody coated wells. The absorbance of each well was read at 450 nm with a differential filter at 630 nm. As the aflatoxin limit was (0-40) ppb but we found more than that which was diluted by dilution factor in three regions (Bogura, Nilphamari, Rangpur) by four times dilution.

Assay procedure for fumonisins detection
200 μL conjugate solution was pipetted into dilution wells with 100 μL of each standard and sample extract. The mixture was mixed well and 100 μL of the mixture (conjugate and standard or samples) was transferred into antibody-coated wells. The plate was then incubated for 15 min with slow shaking and then washed with distilled water for 5 times. The plate was then tap dried. 100 μL of substrate solution was pipetted into antibody coated wells. The plate was incubated with shaking for 5 min. 100 μL of stop solution was pipetted into antibody coated wells. The absorbance of each well was read at 450 nm with a differential filter at 630 nm.

I solation, purification, identification and preservation of mycotoxigenic fungi
Isolation & purification of Aspergillus spp. and Fusarium spp. were collected from stored maize grain samples which was conducted by blotter method [64,65]. In this DOI: http://dx.doi.org /10.5772/intechopen.101647 method, 400 maize grains were tested for the identification of toxigenic Aspergillus spp. and Fusarium spp. for each sample collected from different locations and 40 plastic pestridishes were used for each sample. Then 10 maize grains were placed in the sterile plastic petridish containing three layers of wet blotter papers. The petridish was incubated at 25 ± 1° C under 12/12 h light and darkness cycle for 7 days. Each seed was observed under stereo microscope (Stemi 508, Germany) in order to record the presence of fungal colonies and temporary slides were prepared from the fungal colonies for morphological identification under compound microscope (Primo Star, Germany). Or one of the quarter kilo samples from each trader milled into fine floor using a Laboratory Milling machine. Ten grams of the ground sample was mixed with 100 ml sterile water to make a stock solution and serially diluted up to dilution 10 3 . The suspension was plated in Potato Dextrose Agar Medium (PDA) [66,67] by mixing 1 ml suspension in molten PDA cooled to 40° C. Isolation media was prepared by weighing 39 g of PDA into 1 L of water. The mixture was autoclaved for 15 min at 121° C and 15 PSI pressure. The media was allowed to cool to about 50° C and then amended with 25 ng/L of streptomycin and tetracycline [68,69]. Petri dishes were labeled appropriately and a milliliter of the diluted sample was poured into a sterile petri dish aseptically and then 18 ml of PDA media at 40° C will was poured on the same plate and the mixture swirled gently to mix. The mixture was allowed to cool and solidify in the laminar flow hood and then sealed using parafilm for incubation. The plates were incubated at room temperature for 5-7 days.

DNA extraction
Before DNA extraction each purified Aspergillus spp. and Fusarium spp. was grown on PDA for 7-10 days at 28° C in an incubator. Then a 5 mm culture block was transferred on the conical flask containing PDA broth and the flasks were incubated at 28° C in an incubator for 7-10 days. Mycelium of each isolate was harvested and preserved at −80° C.
Genomic DNA was extracted from the fungal species isolated from maize grains following Wizard Genomic DNA extraction kit (Promega, USA) according to the manufacturer instructions from 100 mg fungal tissue ground with liquid nitrogen. Fungal tissue was processed by freezing with liquid nitrogen and grinding into a fine powder using a microcentrifuge tube pestle or a mortar and it was pestled. 0.04 g of this fungal tissues powder was added to a 1.5 ml microcentrifuge tube. 600 μl of Nuclei Lysis Solution was added and it was vortexed for 1-3 s to wet the tissue. The sample was incubated at 65° C for 15 min. 3 μl of RNase Solution was added to the cell lysate, and the sample was mixed by inverting the tube 2-5 times. The mixture was incubated at 37° C for 15 min. The sample was allowed to cool to room temperature for 5 min before proceeding. 200 μl of Protein Precipitation Solution was added, and it was vortexed vigorously at high speed for 20 s. The sample was centrifuged for 3 min at 13,000-16,000 × g. The precipitated proteins were formed into a tight pellet. The supernatant was carefully removed containing the DNA (leaving the protein pellet behind) and it was transferred to a clean 1.5 ml microcentrifuge tube containing 600 μl of room temperature isopropanol. The solution was gently mixed by inversion until thread-like strands of DNA form a visible mass. Then the sample was centrifuged at 13,000-16,000 × g for 1 min at room temperature. The supernatant carefully decanted. 600 μl of room temperature 70 % ethanol was added and was inverted gently into the tube several times to wash the DNA. It was centrifuged at 13,000-16,000 × g for 1 min at room temperature. The ethanol was aspirated carefully using either a drawn Pasteur pipette or a sequencing pipette tip. The DNA pellet was very loose at this point and care must be used to avoid aspirating the pellet into the pipette. The tube was inverted onto clean absorbent paper and the pellet was air-dried for 15 min. 100 μl of DNA Rehydration Solution was added and the DNA was rehydrated by incubating at 65° C for 1 h. Periodically the solution was mixed by gently tapping the tube. Alternatively, the DNA was rehydrated by incubating the solution overnight at room temperature or at 4° C. The DNA was stored at 2-8° C.

Primers, PCR conditions and sequencing of ITS region
The extracted DNA samples were amplified with PCR reaction for ITS regions. The forward primer: ITS1-5.8S (5′-GGAAGTAAAAGTCGTAACAAGG-3′) and the reverse primer rDNA-ITS4 (TCCTCCGCTTATTGATATGC) were used [70]. PCRs were performed in 25 μl reaction volume containing 12.5 μl master mix, 1 μl ITS1, 1 μl ITS4, 9.5 μl Nuclease free water and 1 μl templet DNA (100 ng/μl). PCR products were visualized in 2 % agarose gel, dyed with ethidium bromide and the photograph was taken using a Gel documentation system (Dynamica, GelView Master). The conditions for PCR reaction was: initial denaturation for 5 min at 95° C, followed by 34 cycles at 95° C for 30s, at 55° C for 1 min and at 72° C for 1 min and then final elongation at 72° C for 6 min. The amplified products were stored at −20° C. PCR products were sequenced using ITS1 primer via commercial outsourcing at Macrogen, Korea via Biotech concern. Finaly, Sequence data were imported by Chromas Software version 2. Sequence data were analyzed by BLAST program (Basic Local Alignment Search Tool) and GenBank (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

Primers for PCR amplification
Primers specific for fumonisins producing Fusarium spp. (FUM1 Forward-CCATCAC AGTGGGACACAGT, FUM1 Reverse-CGTATC GTCAGCATGATGTAGC) were used previously [72]. PCR were performed in mixture 15 μl volume containing 1 μl of DNA sample, 7.5 μl of master mix, 1 μl FUM1 forward primer, 1 μl FUM1 reverse primer, 4.5 μl nuclease free water. PCR was performed using T100 Thermocycler (BioRad, Hercules, USA). The PCR condition for FUM1 regions include 94° C for 4 min for initial denaturation, followed by 35 cycles of denaturation at 94° C for 1 min, primer annealing at 58° C for 1 min, primer extension at 72° C for 1 min. The final extension was set at 72° C for 10 min. 4 μl of the PCR product was electrophoresed on 1.5 % agarose gel, stained with ethidium bromide, illuminated and documented using Gel documentation system (Dynamica, GelView Master).

Statistical analyses
The collected data were analyzed statistically by using Minitab software version 17 (www.minitab.com). The mean of all the treatments were compared by critical difference value at 5 % level of significance.

Determination of total Aflatoxins contamination in stored maize grain samples collected from some selected growing areas of Bangladesh
The study was performed at the Laboratory of Department of Plant Pathology, Bangladesh Agricultural University, Mymensingh. Composite stored maize grain samples were collected from 15 maize growing areas of Bangladesh including Panchagarh, Thakurgaon, Dinajpur, Nilphamari, Rangpur, Lalmonirhat, Gaibandha, Bogura, Natore, Kushtia, Jashore, Chuadanga, Kishoreganj, Manikganj and Cumilla.
In terms of total aflatoxins concentration in μg/kg, the highest and lowest amount of aflatoxins concentration was recorded in Chuadanga (101.57 μg/kg) and Dinajpur (1.08 μg/kg) which exposed no significant relationship to each other. The moderate amount of afalatoxin level was detected in Gaibandha (68.73 μg/kg), Kushtia (31.48 μg/ kg), Kishoreganj (30.86 μg/kg), Rangpur (20.56 μg/kg) and Cumilla (11.91 μg/kg) revealing more aflatoxins contamination than the regulatory limit (10 μg/kg) in which only aflatoxins concentration from Kushtia and Kishoreganj revealed statistically significant data, besides, rest of the location exhibited below level of aflatoxins contamination of regulatory limit showing more or less statistically significant data.
Total aflatoxins associated with maize grains were detected in 2020, with the supreme value was detected in Chuadanga (30.5 %) followed by Kushtia (29.5 %), Nilphamari (22.5 %), Panchagarh (19.25 %) and the minimal aflatoxins was detected in Manikganj (3.2 %), rest of the samples from other districts revealed lower to moderate level of aflatoxins, moreover, data from Chuadanga and Kushtia, Cumilla, Jashore and Natore, Thakurgaon and Rangpur, Lalmonirhat and Kishoreganj regions revealed ststistically similar data while data from other regions exhibited statistically dissimilar data.

2 Relationship between aflatoxins producing A. flavus and mean aflatoxins concentrations
The regression analysis between toxigenic A. flavus percentage and mean aflatoxin concentrations which was positively correlated by observing regression equation where the slope was = 0.55 and y-intercept was = 50.14, coefficient of determination, R 2 = 0.198 and coefficient of correlation, r = 0.44 which depicted that 1 % surges of toxigenic A. flavus in maize grains ultimately rised 50.137 μg/ kg aflatoxin concentration. In terms of 5 % surges of toxigenic A. flavus in maize grains, the aflatoxin concentration was increased up to 2.75 μg/kg and when toxigenic A. flavus increased 20 % in maize grains, the aflatoxin concentration was escalated up to 11.0 μg/kg (Figure 1).

Identification of A. flavus from the stored maize grain samples collected from some selected growing areas of Bangladesh
Morphological identification of A.flavus was detected by using petridish and culture plate method as well as observing microscopic figures under compound and stereo microscope (Figure 2A(a)-(d)). Thirty five fungal isolates were identified using primers specific to ITS 1 and ITS 4 regions. PCR assays of A. flavus DNA with ITS 1 and ITS 4 primers amplified a single fragment of about 600 bp which revealed that all the isolates obtained were fungi. Sequence analysis of ITS region by BLAST program revealed that all the isolates obtained from maize were belong to A. flavus ( Figure 3A).

PCR based identification and confirmation of aflatoxin producing
Aspergillus flavus species obtained from maize grain samples AF02_Ran, AF01_Lal, AF01_Bog, AF02_Bog, AF03_ Jas, AF04_ Jas, AF01_Chu, AF03_Kis, AF04_Kis, AF01_Man were identified by PCR amplification of ITS region using ITS1 and ITS4 primers and the results of PCR showed an amplification size 600 bp confirmed the fungal isolates ( Figure 3A)      When the isolates of Aspergillus Spp. were analyzed by PCR for aflatoxin producing ability using nor, omtA, apa-2 genes based primers from fifteen maize growing areas. The result showed the amplified DNA fragment was 400 bp, 1024 bp, 1032 bp confirmed that the A. flavus isolates had the ability to produce aflatoxin that encode nor, omtA, apa-2 genes (Figure 3B). Only six species showed a positive result with nor, omtA, apa-2 genes set of primers. The result indicated A. flavus strains were aflatoxins producers as those were an evident from our investigation (Figure 3B).
PCR products were sequenced using ITS-1 primer and sequence data were analyzed by homology search using BLAST Nucleotide program. Isolates were identified as different A. flavus based on the homology percentage with their closest relatives available in the NCBI database.

Determination of total fumonisins contamination in stored maize grain samples collected from some selected growing areas of Bangladesh
The study was conducted at the Laboratory of Department of Plant Pathology, Bangladesh Agricultural University, Mymensingh. Composite stored maize grains samples were collected from 15 maize growing areas of Bangladesh such as Panchagarh, Thakurgaon, Dinajpur, Nilphamari, Rangpur, Lalmonirhat, Gaibandha, Bogura, Natore, Kushtia, Jashore, Chuadanga, Kishoreganj, Manikganj, Cumilla.

Relationship between fumonisins producing Fusarium spp. and mean fumonisin concentrations
The regression analysis between Fusarium spp. infected maize grains and mean fumonisin concentrations which was positively correlated by observing regression equation where the slope was = 0.038 and y-intercept was = 0.882, coefficient of determination, R 2 = 0.198 and coefficient of correlation, r = 0.45 which depicted that 1 percent surges of Fusarium in maize grains ultimately rised 0.038 mg/kg fumonisins concentration. In terms of 5 % surges of Fusarium in maize grains, the fumonisins concentration was increased up to 0.19 mg/kg and when Fusarium increased 20 % in maize grains, the fumonisins concentration was escalated up to 0.76 mg/kg (Figure 4).

Identification of Fusarium species from the stored maize grain samples collected from some selected growing areas of Bangladesh
Morphological identification of F. oxysporum and F. proliferatum were detected by using petridish and culture plate method as well as observing microscopic figures under compound and stereo microscope (Figure 2B(a)-(f )). Fifteen fungal isolates were identified using primers specific to ITS 1 and ITS 4 region. PCR assays of F. oxysporum DNA with ITS 1 and ITS 4 primers amplified a single fragment of about 600 bp which revealed that all the isolates obtained were fungi (Figure 5A). Sequence analysis of ITS region by BLAST program revealed that all the isolates obtained from maize were belong to F. oxysporum and F. proliferatum.
When the isolates of Fusarium species were analyzed by PCR for fumonisins producing ability using FUM1 gene based primers from fifteen maize growing areas. The result showed the amplified DNA fragment was 183 bp confirmed that the Fusarium had the ability to produce fumonisin that encode FUM1 gene (Figure 5B). Only two Fusarium species showed a positive result with FUM1 gene set of primers. The result was contrary as F. proliferatum and F. oxysporum ( Table 4) were fumonisin-producers as it was evident from our investigation.

Discussion
The experiment was conducted at Plant Bacteriology and Biotechnology Laboratory of Department of Plant Pathology, Bangladesh Agricultural University, Mymensingh during the period of 2019-2020. The purpose of the experiment were to detect the levels of fumonisins and aflatoxins and to identify the aflatoxin and fumonisins producing Aspergillus and Fusarium in maize associated with maize by PCR using nor, omtA, apa-2 and FUM1. Genes involving afl R, ver-1, omt-1 and apa-2 associated with biosynthetic pathway regarding aflatoxins production [73][74][75][76]. Apa-1, Nor-1, Omt-1 and Ver-1 gens belong to four primers were applied to detect aflatoxins contamination [77,78]. A. flavus was quantified by nor-1 gene in several contaminated food samples and cereals using PCR assay [77]. Besides, [56] mentioned that FUM1 gene with an expected amplicon size of 183 bp can easily detect the fumonisin and non-fumonisin producing Fusarium, moreover other researchers also identified the fumonisin by using FUM1 gene which is in accordance with our study [79][80][81]. We gathered samples from 15 maize growing areas to measure the aflatoxins and fumonisins level but not all the Aspergillus strains are capable of engendering mycotoxins, thus screening is crucial and we detected by Agra Quant Total Aflatoxin and Fumonisin Test Kit following ELISA approach for detection and this method also used by [82][83][84][85][86][87] for detecting aflatoxins and fumonisin. In our experiment, we detected the aflatoxins contamination Agra Quant Total Aflatoxins 96 well microtiter plate ELISA test kit produced in Romer Labs, Packers and Stockyards Administration (GIPSA) in US Department of Agriculture (USDA) which ability to detect individual aflatoxins very precisely and accurately with a range of 0-320 ppb in accrodance with an experiment conducted by [82]. A number of approaches have been widely used to detect mycotxin naming high-performance liquid chromatography (HPLC), enzyme-linked immunosorbent assay (ELISA), and thin layer chromatography (TLC) [83,84] and served as a reliable method for detecting aflatoxins and fumonisins [85,88,89]. In Gaibandha and Cumilla region fumonisin contamination were highest and lowest compared to other areas revealing moderate amount of fumonisins. In this study, all of the 15 samples were found positive with fumonisins producing Fusarium and aflatoxin producing fungi Aspergillus which in accordance with the findings of [90,91]. We found positive correlation for both aflatoxins and fumonisins contamination between their toxin percentages which were matched with the findings of [92] who found apositive correlation has been identified between the proportion of FUM1 transcripts and the proportion of fumonisins biosynthesized by the F. verticillioides and F. proliferatum species.
As we observed that both aflatoxin and fumonisin concentration were fluctuate one region to another region which have been also monitored that due to association of several significant factors like temperature, water activity, storage conditions, drought, humidity, insect damage, flowering stage, plant characteristics [94][95][96][97][98]. Ref. [48] revealed that aflatoxin production comprised of several factor including existence of certain genes and in intact that means deletions or insertions within the gene regions, crop stress [99] and in fumonisins two factors temperatures and water potential are fundamental to produce fumonisins [99] along with rainfall patterns, longer durations of drought which has been prominent in Mediterranean regions [100][101][102][103]. These all conditions significantly impact on the variation of the population of mycotoxin producing fungi both Fusarium and Aspergillus [103]. In our experiment, we recorded over all three regions (Chuadanga, Kishoreganj, Gaibandha) were engendering higher amount of aflatoxins and fumonisins production respectively, thus we speculated in Chuadanga, temperature fluctuation influences the mycotoxin production, in Kishoreganj which exposed with flood and severe water stress and the region Gaibandha with drought problems, these might have the feasible factor for Aspergillus and Fumonisins to produce gigantic amount of mycotoxins compared to other areas. Aflatoxin levels rise as a result of drought, insect damage, and heat during fungal growth [25]. Marasas [104] found that, the presence of fumonisins is linked to weather conditions, with larger instances occurring during hot and dry conditions. Abbas et al. [105] revealed that A. flavus grows supreme around 28-37° C with a humidity level of at least 80 %.
Post-harvest factors are also exacerbate mycotoxin production and generate a favorable condition for fungus related to their growth and mycotoxin production and those include storage fungus, insect infestation, contaminant mold respiration, insects and mites, water availability and temperature ultimately deteriorate grain quality [106][107][108]. As [109] also observed that interaction between these factors triggered the mycotoxigenic species growth, mycotoxin production, niche occupation and competitiveness, [110] also revealed the moisture and surrounding air conditions also influenced mycotoxin production by initiating biological and biochemical activity. Maize is a hygroscopic crop which easily absorbs or release moisture and humidity in the surrounding ambience until getting the adjustment with equilibrium conditions which led to swift degradation in storage. Fusarium species can damage stored grain by causing seedling illnesses, root rots, stalk rots, and ear rots in maize which ultimately hazardous to plants and animal [111][112][113][114][115][116]. Due to all correlating factors with aflatoxin production, high amount of aflatoxins were found in Bangladeshi markets [23] and 82 % contamination in South Asia [49]. Decomposing potentiality of AFs are very slow several approaches including DOI: http://dx.doi.org/10.5772/intechopen.101647 physical, chemical have been investigated [19] and monitored changing in sensory property and nutrient diminishment which led to mount food safety problems ultimately. A number of microorganisms have been identified fruitfully working as a biocontrol agents to control mycotoxins such as Bacillus subtilis, Pseudomonas, Trichoderma, atoxigenic strains of A. flavus and A. parasiticus [117][118][119]. Thus, suppressing mycotoxins by biocontrol agent would be a fruitful approach though several experiments need to be conducted precisely in future.

Conclusion
Aflatoxins and fumonisins are the major source of disease outbreaks due to a lack of knowledge and consumption of contaminated food and feed in Bangladesh. Excessive levels of aflatoxins and fumonisins in food in Bangladesh is a major concern because still majority of the people have not any idea that they are consuming food and feed which crossed the permissible limit set by EU. Another significant factor is no sign of regulating any acceptable limit for this country and that's why people are easily contaminated with several mycotoxins without properly knowing any acceptable limit as well as industries are also not ensuring any precise step to diminish mycotoxins concentration in terms of engendering several products. As our study clearly conceded that most of the regions (Rangpur, Gaibandha, Kushtia, Chuadanga, Kishoreganj, Manikganj, Cumilla) were at higher risk for aflatoxin as well as the regions (Panchagarh, Thakurgoan, Nilphamari, Rangpur, Lalmonirhat, Gaibandha, Bogura, Kushtia, Kishoreganj, Manikganj) were exposed with fumonisins contamination more than that of acceptable limit of fumonisins which ultimately effects animal and mankind by entering our food chain. Thus, several effective approaches (physical, chemical, biological, and genetic engineering techniques) need to be employed as early as possible to suppress the ruinous consequences of mycotoxin contamination of Bangladesh. exploited to reduce aflatoxin exposure and improve health. Annals of the New York Academy of Sciences. 2010;1273(1):7-17 occurrence in feed: A ten-year survey. Toxins (Basel). 2019;11 (7):375