Molecular Characterization of Fumonisin Mycotoxin Genes of Fusarium sp Isolated from Corn and Rice Grains

Fungi mycotoxins can be a serious risk to health and lead to substantial economic loss. The environmental conditions of Saudi Arabia, with its mostly warm temperatures, are conducive to the growth of toxigenic fungi resulting in mycotoxin production in different food items. The current study elucidates the natural occurrence of toxigenic fungi and mycotoxin production in grains in Saudi Arabia. Samples of white rice and corn (yellow, red) grains were collected from different local markets and houses. Three fungal isolates were obtained from the corn and rice grains and examined using Potato Dextrose Agar (PDA) and Carnation Leaf Agar (CLA) media. Fusarium spp. were the most prominent fungi in yellow corn, red corn and white rice grains. Three isolated F. moniliforme strains were identified using molecular characterization of the trichothecene 3-O acetyltransferase (TRI101) toxin gene. The DNA genome of the three Fusarium moniliforme isolates (namely, F. moniliforme_1, F. moniliforme_2 and F. moniliforme_3, which correspond to isolates from yellow corn, red corn and white rice, respectively) were used as a template for PCR to amplify trichothecene 3-O acetyltransferase (TRI101). Partially sequenced fragments amplified using a specific primer set were used to confirm the identification of, and to evaluate the phylogenetic relationships among the three isolates as well as to identify the corresponding antigenic determinants. The epitope prediction analysis demonstrated that there were four epitopes with scores equal to 1 in F. moniliforme_1, F. moniliforme_2 and F. moniliforme_3, respectively. Interestingly, there were great dissimilarities in the epitope sequences among the three isolates except in NSTPRACASEQEVS, STSSRADSSSLSTD and CTLCPRSLMASSVR. This indicates that the unique antigenic determinants predicted in the trichothecene 3-O acetyltransferase (TRI101) toxin gene could be used for designing a broad spectrum antibody for rapid detection of Fusarium spp. in foods.


Introduction
ungi cause major crop diseases during harvest and storage under higher temperature and humidity conditions [1]. While more than 25 different fungi species are known to invade stored grains and legumes [2], certain species such as Aspergillus, Fusarium and Penicillum are responsible for most spoilage and germ damage during storage [3,4]. They cause a reduction in baking quality and nutritive value, produce undesirable odors, color and change the appearance of stored food grade seeds [5]. Mycotoxins are secondary metabolites produced by fungi, which cause health hazards to animals and human beings [6,7]. Moreover, fungal infestation of the seed coat may not only decrease seed viability, but also cause abnormal seedling development [1,7]. A large number of mycotoxin producing fungi which are associated with groundnuts, peanuts, cereals such as maize, rice, sorghum, wheat, barley and oats, and spices such as black pepper, ginger, nutmeg, chilly, etc. are of great significance worldwide [8], but knowledge regarding fungal seed decay and its importance for plant demographic and community processes is quite limited [9,10]. Fungal genera, such as Aspergillus sp; Fusarium sp; Penicillium sp; Alternaria sp; and Epicoccum sp. have been isolated from seeds of beans, cowpea, peas, and cocoa [3,11,12]. Regarding legumes in Saudi Arabia, very little information exists with respect to natural contamination with toxigenic fungi and mycotoxins. Aflatoxin(s) have been detected in some Aspergillus isolates while fumonisin has been found in some Fusarium isolates [13]. Among food contaminants, mycotoxins may cause substantial economic loss due to reducing availability of commodities with acceptable levels of mycotoxins present, and their possibly greater cost [14]. Mycotoxins continue to pose various health risks to consumers depending on the specific mycotoxin consumed and the level of exposure, and the health status of individuals in the population [15]. The majority of mycotoxins of greatest concern for human and animal health are produced by the genera Aspergillus, Penicillium, and Fusarium, the so-called field fungi, which frequently infect various food commodities [10,15], and outbreaks of mycotoxicoses in humans and animals, caused by ingestion of products containing mycotoxins have been reported [4,16]. However, further studies confirm that the toxic effects depend on intake dose, toxin type, duration of exposure, metabolism, mode of action, and defense mechanism [17,18]. Humans are exposed to mycotoxins throughout their lives due to consumption of fungus-contaminated food products, but sufficient quantities of mycotoxins in food and feedstuff can adversely affect human and animal health [14,18]. Many human diseases, especially carcinogenic, teratogenic, hepatic, and gastrointestinal ones, have been found to be linked to the ingestion of mycotoxin-contaminated products [4,19,20].
This study was conducted to determine the bioinformatics characterization of Fumonisins isolated from corn and rice grain in Saudi Arabia. Fumonisins are produced by species of Fusarium genera, principally F. proliferatum, F. verticillioides, and F. nygamai [21]. Fumonisin B 1 (FB 1 ) is the most abundantly occurring and toxicologically most significant derivative [22]. Fumonisins are widely found as contaminants in corn, rice, figs, beer, and other commodities. Temperatures of 15 to 30 °C and 0.9 to 0.995 water activity have been reported as optimum for fumonisin production [23]. FB 1 , due to its cancer-promoting activity, is designated as a possible human carcinogen [24], and fumonisins are probably linked to human esophageal cancer [4,25]. It is also known that they are nephrotoxic and hepatotoxic [26] and cause neural tube defects in experimental animal species, and may also affect humans [27]. Several studies have revealed mycotoxin contamination worldwide in rice, for example, aflatoxins have been found in the United Arab Emirates [28], fumonisins in Iran and Argentina [25,29], OTA in Morocco [30], ZEA in Nigeria [31], DON in Italy [32], nivalenol in Korea [33] and citrinin in Egypt [34]. In this study, we have hypothesized that mycotoxins affect human populations because the storage conditions in local markets and houses are conducive to mycotoxin production. As most of the corn and rice are grown during the wet season, they are susceptible to mycotoxin contamination. Rice is shown to be a good substrate for toxigenic fungi like A. flavus, A. ochraceus, Penicillium citrinum, and F. proliferatum [35,36]. Humidity, temperature, storage conditions, and transport time are the factors that influence mycotoxin production in rice. In the early 20th century, many human diseases occurring in Japan and other Asian countries were attributed to mycotoxins consumed in mold-damaged rice [4,37]. Unfortunately, enactment of stringent rules for mycotoxin control in food is not always the best solution [4,38]. A beneficial effect in Saudi Arabian mycotoxin-contaminated food is left for domestic population and developing grain producing countries [39].
The impact of mycotoxin standards is more drastic for the population of developing countries [1,39]. Therefore, whilst in terms of quantity, availability of food for consumption might not be a problem, the availability of high-quality food which is free from mycotoxins, or at least, having toxin levels in permissible limits, is a matter of great concern in Saudi Arabia and other highly populated areas of the world [1,39]. Thus, the regulatory authorities should aim to facilitate trade without compromising the protection of consumers' health [4,39]. Therefore, the aim of this study was to determine the Fusarium species that are naturally occurring in contaminating corn and rice seeds (as the main crops imported in Saudi Arabia) by the molecular identification of toxigenic mycotoxin profiles of those species and protein structural analysis depicted from the gene(s) responsible for toxin biosynthesis. We have hypothesized that by studying F the molecular properties of Fumonisins, we could in future be able to produce vaccines for those species of Fusarium genera which have a higher prevalence in developing countries [4,10,39].

Grain samples:
150 samples of corn (yellow and red grains) and rice were collected from markets and houses from different areas of Saudi Arabia (Riyadh, Hail, Qasim, Asir,Tabuk, Jizan, Jouf, Jeddah and Dammam). About 0.5 -1 kg of samples was taken randomly and collected in clean dry packaging.

Isolation of mycotoxigenic Fusarium species
Agar plate and blotter tests were used to isolate Fusarium spp. as described by Neergaard [40]. The 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. Half of each group was plated on potato dextrose agar (PDA). All dishes were incubated for 5 to 7 days at 25 °C.

Purification and identification of Fusarium species
Fusarium isolates were identified as species based on the morphological characteristics 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 light microscope [41]. When macroconidia, microconidia and mycelial characteristics from SNA were insufficient for identifying the species, further examination of the samples were done on different agar media. Potato Dextrose Agar (PDA) was used to identify colony pigment characteristics of aerial mycelium on the agar [40,41] Carnation Leaf Agar (CLA) was used to identify macroconidia, chlamydospores and the presentation of aerial mycelium. A single colony was transferred and purified by the hypha tip technique onto a DA medium in the presence of streptomycin (50 mg /ml). Cultured fungi were processed for molecular identification using specific primers for the trichothecene 3-O acetyltransferase (TRI101) toxin gene. All conditions of isolation and purification of mycotoxins were performed under sterilization to prevent any external agent from polluting the seeds.

Isolation of genomic DNA
The mycelium mass of Fusarium species isolates grown on a PDA broth medium was harvested by centrifugation at 6000 rpm for 10 min. The pellets were washed twice by PBS buffer and stored at 20 °C. Total DNA of three isolates was isolated using the lysozyme-dodecyl sulfate lysis method as described by Leach et al. [41].

Amplification and purification of trichothecene 3-O acetyltransferase (TRI101) gene
Specific PCR reactions were conducted to assess the presence of TRI101 gene. The primers used were: FAD-U1 (5′-GATCTCGACATGGCCTTTGTCCCC-3′); FAD-D1 (5′-GAACAGGTGGTGAATGACGTGCTTC-3′) [40]. The PCR amplification conditions included initial denaturation at 94 ° C for 5 min, then 35 cycles at 94 °C for 30 s, 55 °C for 60 s followed by extension step at 72 °C for 90 s and a final extension at 72 °C for 7 min. The amplification reaction was performed by thermal cycler (COT Thermocycler model 1105). Purification of PCR product was detected by electrophoresis using agarose 1.5% in 1x TAE buffer and stained with ethidium bromide [21,41]. The trichothecene 3-O acetyltransferase (TRI101) gene fragment was excised from the gel and purified using a QIA quick gel extraction kit (Qiagen, Berlin, Germany).
DNA sequencing by purified PCR products were prepared for Sanger sequencing technology using the DNA sequencer technique (Sigma, central lab, PNU, KSA). DNA sequences of Fusarium isolates were aligned using Bio Edit software version 7 (www. Mbio-NCUs. Edu/bio. Edit) and were compared to the reference sequences accessions of Fusarium spp. available in the nucleotide database at NCBI using BIASTn-algorithm to identify closely related sequences (http/WWW.NCbI.Nih.Gov). Dendrograms were constructed using un-weighted pair group method with Arithmetic (UPGMA) on Genbank.

Epitope prediction and antigenicity
The primary amino acid sequence of the trichothecene 3-O acetyltransferase (TRI101) protein was evaluated from the corresponding nucleotide sequence using MEGA 6.0 software. The linear B-cell epitopes in the primary amino acid sequence of the coat protein was performed using the BCPREDS server with default parameters (http://ailab.cs.iastate.edu/bcpreds/), which implements a support vector machine (SVM) and the subsequence kernel method [42]. Flexible length linear B-cell epitopes were predicted using the FBCP red [43] method with a specificity cut-off, 75%. The antigenicity of each amino acid residue in the primary protein sequence was determined using a semi-empirical method, which makes use of the physicochemical properties of each amino acid and its frequency of occurrence in experimentally known segmental epitopes.

Results
The Fusarium isolates were selected for molecular identification using trichothecene 3-O acetyltransferase (TRI101) gene sequencing. Three Fusarium isolates represented grains from yellow corn, red corn and white rice and were designated as F. moniliforme_1, F. moniliforme_2 and F. moniliforme_3, respectively.

Molecular characters of toxin gene:
Total DNA was extracted from Fusarium isolates [F. moniliforme_1 (yellow corn isolate), F. moniliforme_2 (red corn isolate) and F. moniliforme_3 (white rice isolate)] from infected grains. The trichothecene 3-O acetyltransferase (TRI101) gene of three F. moniliforme isolates was amplified from isolated DNA of mycelium. The nucleotide partial sequence of the trichothecene 3-O acetyltransferase (TRI101) gene in the three isolates was compared with published isolates in the GenBank. The sequence homology revealed that the gene of interest was the trichothecene 3-O acetyltransferase (TRI101) gene and the test fungal isolates were Fusarium moniliforme isolates.
A multiple sequence alignment (MSA) was constructed using Clustal W software between the three studied isolates ( Figure 1A). The alignment showed many conserved regions in all sequences and also distinguished the heterogeneity positions among the aligned sequences. Phylogenetic analysis was performed by construction of a phylogenetic tree using a neighbor-joining method to unravel the relationships among all Fusarium moniliforme isolates ( Figure 1B). The phylogenetic tree resulted in two clades in which Fusarium moniliforme_1 (yellow corn isolate) and Fusarium moniliforme_2 (red corn isolate) were in the same cluster whilst Fusarium moniliforme_3 (white rice isolate) was separate in a different cluster (Figure 2A, B). Thus, the molecular identification based on sequence homology of the trichothecene 3-O acetyltransferase (TRI101) gene confirmed the identity and phylogeny of the studied three Fusarium moniliforme isolates.   .....................................................   The epitope prediction analysis demonstrated that there were 1, 2, 3 and 4 epitopes with a score equal to 1 in F. moniliforme _1, F. moniliforme _2 and F. moniliforme_3, respectively. Also there were great variations in the epitope sequences among the three isolates except for NSTPRACASEQEVS, STSSRADSSSLSTD and CTLCPRSLMASSVR, which where common among all isolates. These residues with high frequencies of occurrence in antigenic determinants are highlighted (yellow) in the antigenicity profile ( Figure 3). Figure 3 also shows the variability in the positions and types of amino acid residues with high antigenic frequency.

Discussion
Fungal infections not only cause considerable economic loss, there is no doubt that contamination of grains and foodstuffs with mycotoxins has become a danger that can't be ignored [40,43]. Many species are well known mycotoxin producers with various toxicological properties which pose high risk to human and animal health [44,45].
Environmental factors and host species have a strong impact on the occurrence of a specific chemotype and the incidence of Fusarium species [46]. The distribution of Fusarium species in maize is influenced by climatic conditions, pathogenicity and competition between other fungi [47]. The type of environmental factor identified in the incidence of Fusarium species was demonstrated in recent EU maize surveys [48]. In these studies, the prevalence of species varied year-to-year and is believed to be associated with the differences in climatic conditions between years [49,50].
As was reported by [51], the presence of toxigenic fungi on small grains has a negative impact on the safety and quality of animal feed and human food [51]. The genus Fusarium includes cosmopolitan and ubiquitous mold fungi in which saprobes and plant pathogens are many. Fusarium species causes yield losses in processing and production [51,52]. Being able to grow at low temperature, Fusarium spp. are responsible for spoilage of food through contamination during transport and storage [9,17,52]. In addition, reduction in nutritive value, insipidness and discoloration are other problems resulting from contamination of grains by Fusarium [53].
The advance of rapid and accurate identification of Fusarium and/or their metabolites are mandatory for the implementation of preventive measures in the whole food production system, as was reported by [54]. The molecular characterization of three Fusarium spp. isolated from small grains (yellow corn, white rice and red corn) using the mycotoxin gene, trichothecene 3-O acetyltransferase (TRI101), allowed for coupled identification and mycotoxin screening in the three Fusarium isolates [54,55]. Following the molecular identification of Fusarium spp., B-cell epitopes in the trichothecene 3-O acetyltransferase (TRI101) 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 [56]. Epitope prediction saves labor and time for validation experiments. The identification of epitopes plays a crucial role in vaccine design, immunodiagnostic testing and antibody production [56]. In other study BCPREDS serves were used to predict epitopes found in the primary amino acids sequence of trichothecene 3-O acetyltransferase (TRI101) protein, where BCPREDS has proved highly efficient for predicting linear B-cell epitopes in SARS-CoV S protein [56,57]. There was variability in the sequence and numbers of epitopes among the three toxin proteins analyzed [57].
In the present study, a fixed length of epitopes (14 residues) was observed. The epitope prediction analysis demonstrated that there were 1, 2, 3 and 4 epitopes with scores equal to 1 in F. moniliforme_1, F. moniliforme_2 and F. moniliforme_3, respectively. Interestingly, there were great dissimilarities in the epitope sequences among the three isolates except for NSTPRACASEQEVS, STSSRADSSSLSTD and CTLCPRSLMASSVR, which were common among all isolates. This result suggests its exploitation for the design of a specific antibody to be used for rapid detection of different Fusarium species in small grains. Epitope prediction has many implications in pathogen detection and differentiation applications. Consideration of the occurrence of Fusarium spp. on small grains is important in the risk assessment of mycotoxins and in proactively setting up preventive measures [57].

Conclusion
The identification of immunodiagnostic testing and antibody production of a fixed length of epitopes (14 residues) observed in the present study plays a crucial role in the vaccine design. This helps to control the incidence of mycotoxins in small grains (rice and corn). We also need to design bagged information about Epitope prediction for identification of mycotoxins in crops with economic value and high consumption rate.