SQU Journal for Science, 2019, 24(2), 78-87 DOI: http://dx.doi.org/10.24200/squjs.vol24iss2pp78-87
Sultan Qaboos University
78
Molecular Characterization of Fumonisin
Mycotoxin Genes of Fusarium sp Isolated
from Corn and Rice Grains
Latifa A. Al-Husnan
1
, Muneera D.F. Al-Kahtani
1
and Randa M.A. Farag
2
*
1
Biology department, Faculty of Science, Princess Noruah bint Abdulrahman University (PNU),
Riyadh, Kingdom of Saudi Arabia;
2
Virology-Molecular biology, Health Sciences Research Center
(HSCR), Princess Noruah bint Abdulrahman University (PNU), Riyadh, Kingdom of Saudi Arabia;
*Email: rmfaraj@pnu.edu.sa.
ABSTRACT: 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.
Keywords: Fumonisin; PCR; Sequences; Phylogenetic tree; Antigenic determinants.
الذرة و االرز حبوب من المعزول لفطرالفيوزاريوم فيومونيسين سموم لجينات الجزيئى الوصف
فرج أحمد رانده محمد و القحطانى منيرة ،لطيفة الحسينان
ظروف مما الشك فيه أن االصابات الفطرية للمواد الغذائية تمثل عواقب وخيمة بسبب المخاطر على الصحة العامة والخسائر االقتصادية.كما ان ال :صلخمال
في المواد الغذائية البيئية و ارتفاع درجات الحرارة بالمملكة العربية السعودية توفر البيئة المناسبة لنمو الفطريات مما يؤدي إلى إنتاج السموم الفطرية
عينات من األرز المختلفة. توضح الدراسة الحالية التواجد الطبيعي للفطريات السمية والسموم الفطرية في الحبوب في المملكة العربية السعودية. تم جمع
ن الحبوب المطحونة وحبوب األرز األبيض والذرة )الصفراء والحمراء( من مختلف األسواق المحلية والمنازل ,حيث تم عزل ثالث عزالت فطرية م
الفيوزاريوم كان األكثرانتشار في الذرة الصفراء .,PDA Dextro ) ) Carnation Leaf Agar (CLA)باستخدام اوساط غذائية مناسبه لنمو الفطريات
O-3م توصيف جزيئي لجين باستخدا M. moniliforme والذرة الحمراء وحبوب األرز األبيض. تم تحديد ثالث سالالت معزولة من الفطر
acetyltransferase (TRI101). تم استخدام جينوم الحمض النووي لعزالت الفيوزاريوم الثالثة وهي moniliforme (F. moniliforme_1 و F.
moniliforme_2 و F. moniliforme_3 تم استخدام أجزاء متسلسلة جزئياً ، والتي تقابل العزالت من الذرة الصفراء والذرة الحمراء واألرز األبيض ،
لتحليل الجزيئى تم تضخيمها باستخدام مجموعة برايمر محددة لتأكيد التحديد ، لتقييم العالقات البينيه لالحماض االمينيه بين العزالت الثالثة. أظهر ا
على F. moniliforme_3 و F. moniliforme_2 و F. moniliforme_1 في 1لألحماض االمينية أن هناك أربع بروتينات ذات درجات تساوي
MOLECULAR CHARACTERIZATION OF FUMONISIN MYCOTOXIN GENES
79
، NSTPRACASEQEVS التوالي. من المثير لالهتمام ، كان هناك اختالف كبير في تسلسل االحماض االمينية من بين ثالث عزالت إال في
STSSRADSSSLSTD و CTLCPRSLMASSVR. 3المتنبأ بها في جينة هذا يشير إلى أن المحددات الفريدة لمولدات االحماض االمينية-O
acetyltransferase (TRI101) يمكن استخدامها لتصميم جسم مضاد واسع النطاق للكشف السريع عن Fusarium ،sp. األطعمة ألغراض في
.مراقبة الجودة
.تعين التغيرات الجينية لالحماض االمينيةو شجرة العالقات الجينية ،تتابع القواعد النيتروجينيه ،تفاعل البلمرة المتسلسل ،الفيوموسين :مفتاحيةال كلماتال
1. 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 B1 (FB1) 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]. FB1, 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
AL-HUSNAN, L.A. ET AL
80
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].
2. Materials and Methods
2.1 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.
2.2 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.
2.3 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.
2.4 Molecular identification of trichothecene 3-O acetyltransferase (TRI101) toxin gene
2.4.1 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].
2.4.2 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.
2.5 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.
MOLECULAR CHARACTERIZATION OF FUMONISIN MYCOTOXIN GENES
81
3. 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.
3.1 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.
Figure 1. A and B are MSA for Phylogeny of the three studied Fusarium sp. isolates (F. moniliforme_1 (red corn
isolate) and F. moniliforme_2 (white rice) were in the same cluster whilst Fusarium F. moniliforme_3 (yellow corn
isolate) was separate in a different cluster.
AL-HUSNAN, L.A. ET AL
82
A
1 11 21 31 41 51 60
| | | | | | |
KWLSRYSSTPSASYQASFRSTPKSVYSTPSLIPLNIPLLSVPSSKVLSASPNPSHGSQAR 60
......EEEEEEEEEEEEEE...........................EEEEEEEEEEEEE
SKPRALARETQELPLSSLSRTFLVLKTSAMILQRPRSRAERRHTLWRCLTRTSSRQGRRY 120
E...............................EEEEEEEEEEEEEE..............
LLDLVLVPTTQSLFYCSSTSSRADSSSLSTDITVLWIWAKMRSVYSPRRAVTTHSPKRKR 180
..EEEEEEEEEEEEEE.EEEEEEEEEEEEEE.....EEEEEEEEEEEEEE..........
PTSIARRFLTLKTTRLAPRIIRLSNLMLVVTLFSRRSVQAGRSSHSALRPCQSSRMLLPR 240
............................................................
LLTHQQSSCRLTMLFRRSSGSRPLACVSKESMALHLPSSAVLLMLDRQWVSRTTTQAFFK 300
....................EEEEEEEEEEEEEE.........................E
TPTTTRPSAKSPTSHSAQQHHAFVQNSTPRACASEQEVSRRICTTTPTSPTYPRLMRTHL 360
EEEEEEEEEEEEE............EEEEEEEEEEEEEE.EEEEEEEEEEEEEE......
PASCVLGPRWDSGITTLDSDWLSPRLDGQSLSLLRACTLCPRSLMASSVRRLLGTRIWTD 420
.....EEEEEEEEEEEEEE.................EEEEEEEEEEEEEE..........
RRIRSGP 427
.......
B
1 11 21 31 41 51 60
| | | | | | |
KWLSRYSSTPSASYQASFRSTPKSVSSTPSLIPLNIPLLSVPSSKVLSASPKPSHGSQAR 60
.EEEEEEEEEEEEEE.EEEEEEEEEEEEEE.................EEEEEEEEEEEEE
SKPRSLARETQELPSSLLRTFLVLKTSAMILQRPRSRAERRDTLWRCLTRTSSRQGRRYL 120
E.EEEEEEEEEEEEEE...............EEEEEEEEEEEEEE...............
LDLVLVPTTQSLFYCSSTSSRADSSSLSTDITVLWIWAKMRSVYSPRRAVTTHSPKRRPT 180
.EEEEEEEEEEEEEE.EEEEEEEEEEEEEE.....EEEEEEEEEEEEEE...........
SIARRFLTLKTTRLAPRIIRLSNLMLVVTLFSRRSVQAGRSSHSAARPCQSSRMLLPRLL 240
............................................................
THQQSSCRLTMLFRRSSGNRPLACVSKESMALHLPSSAVLLMLDRQWVSRTTTQAFFKSP 300
..................EEEEEEEEEEEEEE.........................EEE
TTTRPSAKSPKSHSAQQHHAFVQNSTPRACASEQEVSRRICTTTPTSPTYPRLMRTHLPA 360
EEEEEEEEEEE............EEEEEEEEEEEEEE.EEEEEEEEEEEEEE........
SCVLGPRWDSGIKTLGSDWVSPRLDGQSLSLLRACTLCPRSLMASSVRRFLGTRIWTDRR 420
...EEEEEEEEEEEEEE.................EEEEEEEEEEEEEE............
IRSGP 425
.....
C
1 11 21 31 41 51 60
| | | | | | |
KWLSRYSSTPSASYQASFRSTPKSVSSTPSLIPLNIPLLSVPSSKVLSASPKPSHGSQAR 60
.EEEEEEEEEEEEEE.EEEEEEEEEEEEEE.................EEEEEEEEEEEEE
SRPRALAKETQELPLSSLLRTFLVLKTSAMILQRPRSRAERRDTLWRCLTRTSSRQGRRY 120
E.EEEEEEEEEEEEEE................EEEEEEEEEEEEEE..............
LLDLLLVPTTQCLFYCSSTSSRADSSSLSTDITVLWIWAKMRPVYSPRRAVTTHSPKRKR 180
..EEEEEEEEEEEEEE.EEEEEEEEEEEEEE.....EEEEEEEEEEEEEE..........
PTSIARRFLTLKTTRLAPRIIRLSNLMLVVTLFSRRSVQAGRSSHSAPRPCQSSRMLLPR 240
............................................................
LLTHQQSSCRLTMLFRRSSGIRPLACVSKESMALHLPSSAVLLMLDRQWVSRTTTQAFFK 300
................EEEEEEEEEEEEEE.............................E
TPTTTRPSAKSPTSHSAQQHHAFVQNSTPRACASEQEVSRRICTTTRTSPTYPRLMRTHL 360
EEEEEEEEEEEEE............EEEEEEEEEEEEEE..EEEEEEEEEEEEEE.....
PASCVLGPRWHSGITTLGSDWVSPRLDGQSLSLLRACTLCPRSLMASSVRRFLGTRIWTD 420
.....EEEEEEEEEEEEEE.................EEEEEEEEEEEEEE...RRIRSGP 427
Figure 2. Amino acid residues of trichothecene 3-O acetyltransferase (TRI101) protein in A: Fusarium spp._1 (yellow
corn), B: Fusarium spp._2 (red corn) and C: Fusarium spp._3 (white rice) showing predicted epitopes (Red) that are
highlighted.
MOLECULAR CHARACTERIZATION OF FUMONISIN MYCOTOXIN GENES
83
Table 1. Flexible length predictions of epitopes in the amino acids sequence of trichothecene 3-O acetyltransferase
(TRI101) protein of the three studied Fusarium isolates
No. Epitope/Fusarium sp._1
(yellow corn)
Score/ Epitope/ Fusarium
sp._2 (red corn)
Score/ Epitope/ Fusarium
sp._3(white rice)
Score/
1 RICTTTPTSPTYPR 1 RICTTTPTSPTYPR 1 ICTTTRTSPTYPRL 1
2 KTPTTTRPSAKSPT 1 KSPTTTRPSAKSPK 1 KTPTTTRPSAKSPT 1
3 NSTPRACASEQEVS 1 NSTPRACASEQEVS 1 NSTPRACASEQEVS 1
4 SSTPSASYQASFRS 0.998 SFRSTPKSVSSTPS 1 SFRSTPKSVSSTPS 1
5 SASPNPSHGSQARS 0.996 SASPKPSHGSQARS 0.998 SASPKPSHGSQARS 0.998
6 STSSRADSSSLSTD 0.993 STSSRADSSSLSTD 0.993 LGPRWHSGITTLGS 0.997
7 SRPLACVSKESMAL 0.974 QRPRSRAERRDTLW 0.984 STSSRADSSSLSTD 0.993
8 LGPRWDSGITTLDS 0.967 WLSRYSSTPSASYQ 0.974 QRPRSRAERRDTLW 0.984
9 QRPRSRAERRHTLW 0.94 NRPLACVSKESMAL 0.949 WLSRYSSTPSASYQ 0.974
10 CTLCPRSLMASSVR 0.892 LGPRWDSGIKTLGS 0.896 RSSGIRPLACVSKE 0.967
11 IWAKMRSVYSPRRA 0.871 CTLCPRSLMASSVR 0.892 PRALAKETQELPLS 0.953
12 DLVLVPTTQSLFYC 0.794 IWAKMRSVYSPRRA 0.871 CTLCPRSLMASSVR 0.892
13 - DLVLVPTTQSLFYC 0.794 IWAKMRPVYSPRRA 0.883
14 - PRSLARETQELPSS 0.704 DLLLVPTTQCLFYC 0.708
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.
4. 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
AL-HUSNAN, L.A. ET AL
84
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].
A
B
C
Figure 3. Kolaskar and Tongaonkar antigenicity scale for prediction of antigenic determinants in trichothecene 3-O
acetyltransferase (TRI101) in A: Fusarium spp._1 (yellow corn), B: Fusarium spp._2 (red corn) and C: Fusarium
spp._3 (white rice) Amino acid residues of high frequencies in epitopes are distinguished (yellow).
MOLECULAR CHARACTERIZATION OF FUMONISIN MYCOTOXIN GENES
85
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.
Conflict of Interest
The authors declare no conflict of interest.
Acknowledgement
The authors are grateful to the Deanship of Scientific Research, PNU for financial support.
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Received 9 October 2018
Accepted 7 February 2019