Therefore, the aim of this study was to determine the Aspergillus species by the molecular identification of toxigenic mycotoxin profiles of those species that are naturally occurring in contaminating corn and rice seeds (as the main crops imported in Saudi Arabia) and protein structural analysis depicted from the gene(s) responsible for toxin biosynthesis. Which can be used as screening test for cost-effective control of mycotoxin and their products. We have hypothesized that by study the molecular characterizations and bioinformatics properties of Aflatoxins could in future be able to produce vaccine for species of Aspergillus genera which have higher prevalence rate in development countries (Bhatnagar et al. 2003; Pasquali et al. 2016).

One hundred fifty grains corn (yellow and red grains) and rice were collected from different area of Saudi Arabia (Riyadh, Hail, Qasim, Asir,Tabuk, Jizan, Jouf, Jeddah and Dammam), where collected from storage markets and houses. The collected grains were randomly and its weight between 0.5 – 1 kg of each grain in cleans and dries packaging. Agar plate and blotter tests were used to isolate Aspergillus sp. as described by Neergaard (1977). Grains were divided into two groups, the first group was disinfected with sodium hypochlorite 1% for 2 min and the second group was non-disinfected. All grains were washed several times by sterilized water, and then dried between sterilized filter papers. The half of each group was plated on potato dextrose agar (PDA) (Sigma-Aldrich, USA). All dishes were incubated for 5 – 7 days at 25 °C. All isolation process under sterilized conditions to prevent any contamination of grain.

Aspregillus species were initially identified to species based on the morphological characteristics (Leslie, Summerell and Bullock, 2006) of the macroconidia, microconidia and general mycelium presentation from a single spore isolate grown for 7 – 10 days on SNA with an Olympus BH-2 (Olympus America, New York) light microscope. Potato Dextrose Agar (PDA) was used to identify colony pigment characteristics of aerial mycelium on the agar (Leslie, Summerell and Bullock, 2006). Carnation Leaf Agar (CLA) (bio-WORLD, USA) was used to identify macroconidia, chlamydospores and the presentation of aerial mycelium single colony was transferred and purified by hypha tip technique onto PDA medium in the presence of streptomycin (50 mg.ml-1). The developing fungi were prepared for molecular identification using primers specific for the Aspergillus flavus AFLR (aflR) toxin gene. All conditions of isolation and purification of mycotoxins were performed under sterilization for prevent any external agent of seeds pollutions.

Total DNA was extracted from A. flavus _ YC; A. flavus _ RC; A. flavus _ Rice isolates infected grains. Aspergillus flavus AFLR (aflR) toxin gene was amplified from isolated DNA of mycelium using PCR reaction mixture and specific primer sets. PCR amplicons were allowed for sequencing reaction through cycle sequencing method. The DNA amplicons returned as electropherogram files. Electropherogram showed distinct peaks for each base cell as well as high Q values for each cell. Sequences obtained for each primer for each isolate had sufficient overlap between them and used to form one continuous sequence (Coting). The nucleotide partial sequence of Aspergillus flavus AFLR (aflR) toxin gene in the three isolates was compared with published isolates on Gen Bank. The sequence homology revealed that the gene of interest Aspergillus flavus AFLR (aflR) toxin gene and the test fungal isolates was Aspergillus flavus isolates. A multiple sequence alignment was constructed using ClustalW software (GNU Lesser GPL) between the three studied isolates. The alignment showed many conserved regions in all sequences as well as distinguished the heterogeneity positions among the aligned sequences (Figure 1a, 1b, 1c). Phylogenetic analysis was performed by construction of phylogenetic tree using a neighbor joining method to unravel the relationships among all Aspergillus flavus isolates (Figure 2). The phylogenetic tree resulted in two clades in which A. flavus _ Rice (white rice isolate) and A. flavus _ RC (red corn isolate) were in the same cluster whilst A. flavus _ YC (yellow corn isolate) was separate in a different cluster (Figure 2). Thus, the molecular identification based on sequence homology of the Aspergillus flavus AFLR (aflR) toxin gene confirmed the identity and phylogeny of the studied three Aspergillus flavus isolates.

The epitope prediction analysis demonstrated that there were 1, 2, 3 and 4 epitopes whose score were equal 1 in A. flavus _ YC (yellow corn), B: A. flavus _ RC (red corn) and C: A. flavus _ Rice (white rice). Also, there were great variations in the epitope sequences among the three isolates except in RLQEGGDDAAGIPA, SPPPPVETQGLGGD, RPSESLPSARSEQG and PAHNTYSTPHAHTQ were found to be common between all isolates. These residues with high frequencies of occurrences in antigenic determinants were highlighted (yellow) in the antigenicity profile (Figure 3). Figure (3) also show the variability in the positions and types of amino acid residues with high antigenic frequency. Table. 1

Mycotoxin detection is a major problem in developing countries where contaminated food commodities may readily reach food stores and homes (Bilodeau, 2011; Boutigny et al., 2012; Taha et al., 2017). The risk of contamination by mycotoxins is an important food safety concern for grains and other field crops (Abdulkadar et. al., 2004; Mwanza et al., 2013). Humans are exposed to mycotoxins throughout their life time due to consumption of fungus-contaminated food products and many human diseases, especially carcinogenic, teratogenic, hepatic, and gastrointestinal ones, have been found linked with the ingestion of mycotoxin-contaminated products (Fung and Clark, 2004; Shephard, 2008; Mwanza et al., 2013). Sufficient quantities of mycotoxins in food and feedstuff can adversely affect human and animal health (Qiu and Shi, 2014). Environmental factors and host species have a strong impact on the occurrence of a specific chemotype and the incidence of Aspergillus species (Bhatnagar et al., 2003). The distribution of Aspergillus species in maize is influenced by optimal climatic conditions, pathogenicity and competition between other fungi (Bhatnagar et al., 2006). The type of environmental factor identified in the incidence of Aspergillus species as demonstrated in recent EU maize surveys (Creepy, 2002). In those studies, the prevalence of species varied year-to-year and was believed to be associated with the differences in climatic conditions between years (Caldas et al., 2002; Scauflaire et al., 2011). As was reported by Covarelli et al. (2015) the emergence of toxigenic fungi on small grains has a negative impact on the safety and quality of feed and food. in this study We isolate mycotoxins strains of Aspergillus sp from contaminated corn and rice grains by a genomic library through amplication mycotoxins structure genes (aflR) by polymerase chain reaction (PCR) and sequencing of amplicans this result was agree with (Chen et al., 2002). Data clearly reveal that the PCR technique is efficient in distinguishing mycotoxins (Bilodeau, 2011; de Souza et al., 2005; Taha et al., 2012; Allam et al., 2015) from commonly inhabiting stored grains. We isolate Aspergillus sp. from both zellow and red corn which used in food and feed in human and animal; also isolated from long and short rice (Niessen, 2007; Fox and Howlett, 2008). Aspergillus species being able to grow at moderate to high temperature (Ehrlich et al., 2003) and its responsible for spoilage of food commodities during transport and storage. In addition, reduction in nutritive value, insipidness and discoloration are other problems resulted from contamination of grains by Aspergillus (Dean et al., 2012). Rapid and accurate identification of Aspergillus and/or their metabolites are mandatory for the implementation of preventive measures in the whole food production system as was reported by Bok and Keller, (2004); Bhatnagar et al. (2003) and Dean et al. (2012). The molecular characterization of three Aspergillus sp. isolated from small grains (yellow corn, white rice and red corn) using the mycotoxins gene, aflatoxin AFLR (aflR) allowed for coupled identification and mycotoxins screening in the three Aspergillus isolates (Bhatnagar et al., 2006). Mycotoxins-producing fungi were isolated from sorghum grains from Saudia Arabia before (Mahmoud et al., 2013; Yassin et al., 2010). Following the molecular identification of Aspergillus sp. B-cell epitopes in the aflatoxin AFLR (aflR) gene were predicted. The characterization of B-cell epitopes using computational tools is highly advantageous for the synthesis of specific antibodies for rapid detection of microbial pathogens in their environments. The epitopes prediction saves labor and time for validation experiments. The identification of epitopes plays a crucial role in the vaccine design, immunodiagnostic testing and antibody production (Sette and Fikes, 2003). In this study, BCPREDS server was used to predict epitopes found in the primary amino acids sequence of aflatoxin AFLR (aflR) protein. BCPREDS proved high efficiency to predict linear B-cell epitopes in SARS-CoV S protein (El-Manzalawy et al., 2008a; El-Manzalawy et al., 2008b). There was variability in the sequence and numbers of epitopes among the three toxin proteins analyzed. Here, a fixed length of epitopes (14 residues) was observed. The epitope prediction analysis demonstrated that there were 1, 2, 3 and 4 epitopes whose score were equal 1 in A. flavus _ YC; A. flavus _ RC; A. flavus _ Rice respectively. In this study, there were great dissimilarity in the epitope sequences among the three isolates. But we found that there some epitope sequences were common between all isolates in RLQEGGDDAAGIPA, SPPPPVETQGLGGD, RPSESLPSARSEQG and PAHNTYSTPHAHTQ. This result suggesting its exploitation for design of a specific antibody to be used for rapid detection of different Aspergillus species in small grains (Ehrlich et al., 2003; Degola et al., 2007). The highly frequent residues with high antigenicity profiles such as valine, leucine, isoleucine, aspartic acid, glutamine and glutamic acid are mostly hydrophobic (Duan et al., 2007). The occurrence of hydrophobic residues in epitopes is frequent and do have a hierarchy signature (Zhang, 2002; Aftabuddin and Kundu, 2007; Suga and Galel, 2007; Scauflaire et al., 2011). Epitope prediction has many implications in pathogen detection and differentiation applications. The consideration of occurrence of Aspergillus sp. on small grains is important in risk assessment of mycotoxins and setting up preventive measures proactively. The main problems concentrated in high risk outbreaks of aflatoxicosis have been reported in different countries all over the world; that because the widespread of aflatoxin contamination especially in developing countries (Pitt 2000a; Pitt 2000b; Kovacs, 2004). No doubt, that the safety control in food and feed processing should be by prevention of mycotoxin contamination in agriculture by used as simple and rapid screening tests for cost-effective control of food diseases by production of vaccines (Sette and Fikes, 2003; Probst et al., 2014; Mwanza et al., 2013; Allam et al., 2015).