Mycotoxin contamination in rice is less reported, compared to that in wheat or maize, however, some Fusarium fungi occasionally infect rice in the paddy field. Fumonisins are mycotoxins mainly produced by Fusarium verticillioides, which often ruins maize. Rice adherent fungus Gibberella fujikuroi is taxonomically near to F. verticillioides, and there are sporadic reports of fumonisin contamination in rice from Asia, Europe and the United States. Therefore, there exists the potential risk of fumonisin contamination in rice as well as the need for the validated analytical method for fumonisins in rice. Although both natural and spiked reference materials are available for some Fusarium mycotoxins in matrices of wheat and maize, there are no reference materials for Fusarium mycotoxins in rice. In this study, we have developed a method for the preparation of a reference material containing fumonisins in Thai rice. A ShakeMaster grinding machine was used for the preparation of a mixed material of blank Thai rice and F. verticillioides-infected Thai rice. The homogeneity of the mixed material was confirmed by one-way analysis of variance, which led this material to serve as an in-house reference material. Using this reference material, several procedures to extract fumonisins from Thai rice were compared. Accordingly, we proved the applicability of an effective extraction procedure for the determination of fumonisins in Japanese rice.
Mating studies were conducted on 78 isolates of Fusarium species section Liseola from rice, sugarcane and maize. From the crosses with tester strains of Gibberella fujikuroi species complex, 64.1% (50 out of 78 isolates) were cross-fertile with tester strains of mating populations A to E. The results of the mating studies showed that of the 50 isolates, 19 belonged to mating population A (Gibberella moniliformis), 18 to mating population B (Gibberella sacchari), 4 to mating population E (Gibberella subglutinans), 6 to mating population D (Gibberella intermedia) and 3 to mating population C (G. fujikuroi). Identification of several mating populations from rice, sugarcane and maize could be important biological entities under field conditions.
Mating studies were conducted on 78 isolates of Fusarium species section Liseola from rice, sugarcane and maize. From the crosses with tester strains of Gibberella fujikuroi species complex, 64.1% (50 out of 78 isolates) were cross-fertile with tester strains of mating populations A to E. The results of the mating studies showed that of the 50 isolates, 19 belonged to mating population A (Gibberella moniliformis), 18 to mating population B (Gibberella sacchari), 4 to mating population E (Gibberella subglutinans), 6 to mating population D (Gibberella intermedia) and 3 to mating population C (G. fujikuroi). Identification of several mating populations from rice,sugarcane and maize could be important biological entities under field conditions.
Fumonisin B1 (FB1) is a common mycotoxin produced by Fusarium species particularly F. proliferatum and F. verticillioides. The toxin produced can cause adverse effects on humans and animals. The objectives of this study were to detect the production of FB1 based on the amplification of FUM1 gene, to quantify FB1 produced by the isolates using Ultra-fast Liquid Chromatography (UFLC) analysis, to examine the embryotoxicity effect of FB1 and to determine EC50 toward the larvae of zebrafish (Danio rerio). Fifty isolates of Fusarium species were isolated from different hosts throughout Malaysia. Successful amplification of the FUM1 gene showed the presence of this gene (800 bp) in the genome of 48 out of 50 isolates. The highest level of FB1 produced by F. proliferatum isolate B2433 was 6677.32 ppm meanwhile F. verticillioides isolate J1363 was 954.01 ppm. From the assessment of embryotoxicity test of FB1 on larvae of zebrafish, five concentrations of FB1 (0.43 ppm, 0.58 ppm, 0.72 ppm, 0.87 ppm and 1.00 ppm) were tested. Morphological changes of the FB1 exposed-larvae were observed at 24 to 168 hpf. The mortality rate and abnormality of zebrafish larvae were significantly increased at 144 hpf exposure. Meanwhile, the spontaneous tail coiling showed a significant difference. There were no significant differences in the heartbeat rate. As a conclusion, the presence of FUM1 in every isolate can be detected by FUM1 gene analysis and both of the species produced different concentrations of FB1. This is the first report of FB1 produced by Fusarium species gave a significant effect on zebrafish development.
Microfungi isolated from Malay traditional vegetables such as Centella asiatica, Cosmos caudatus, Oenanthe javanica, Persicaria odorata and Psophocarpus tetragonolobus are well diverse. A total of 40 isolates of the fungi were identified and classified into four genera such as Aspergillus, Fusarium, Penicillium and Trichoderma. Five species of Fusarium were morphologically identified as F. oxysporum, F. semitectum, F. proliferatum, F. solani and F. konzum. Three species of Aspergillus were identified as A. niger, A. fumigatus and A. flavus. The highest number of microfungi was isolated from Cosmos caudatus (12 isolates), followed by Persicaria odorata (9 isolates), Oenanthe javanica (8 isolates), Centella asiatica (6 isolates) and Psophocarpus tetragonolobus (5 isolates). Four isolates of Fusarium species were able to produce moniliformin (MON) and five isolates were able to produce fumonisin B1 (FB1). This is the first report on diversity of microfungi associated with some Malay traditional vegetables.
This study was carried out to examine the efficacy of two biocontrol agents (Clonostachys rosea 016, BCA1; Gram-negative bacterium, BCA5) for control of FUM1 gene expression and fumonisin B1 (FB1) production by F. verticillioides FV1 on maize cobs of different ripening stages: R3, Milk (0.985 aw); R4, Dough (0.976 aw); R5, Dent (0.958 aw). Initially, temporal studies on FUM1 gene expression and FB1 production were performed on maize kernels for up to 14days. This revealed that day 10 was optimum for both parameters, and was used in the biocontrol studies. Maize cobs were inoculated with 50:50 mixtures of the pathogen:antagonist inoculum and incubated in environmental chambers to maintain the natural aw conditions for ten days at 25 and 30°C. The growth rates of F. verticillioides FV1, the relative expression of the FUM1 gene and FB1 production were quantified. It was found that, aw×temp had significant impacts on growth, FUM1 gene expression and FB1 production by F. verticillioides FV1 on maize cobs of different maturities. The fungal antagonist (BCA1) significantly reduced FB1 contamination on maize cobs by >70% at 25°C, and almost 60% at 30°C regardless of maize ripening stage. For the bacterial antagonist (BCA5) however, FB1 levels on maize cobs were significantly decreased only in some treatments. These results suggest that efficacy of antagonists to control mycotoxin production in ripening maize cobs needs to take account of the ecophysiology of the pathogen and the antagonists, as well as the physiological status of the maize during silking to ensure effective control.
The Fusarium species are notoriously known for causing various plants and animal diseases and producing a number of harmful mycotoxins. The mycotoxins production by species recovered from non-agricultural hosts such as wild grasses have hitherto never been given attention. We examined 30 strains representing 12 Fusarium species i.e. F. oxysporum, F. solani, F. semitectum, F. nelsonii, F. compactum, F. equiseti, F. chlamydosporum, F. proliferatum, F. subglutinans, F. sacchari, F. lateritium and F. incarnatum-equiseti species complex isolated from wild grasses in Peninsular Malaysia for the production of four major mycotoxins i.e. moniliformin (MON), fumonisin BI (FB1), zearalenone (ZEN) and beauvericin (BEA) using TLC and HPLC techniques. BEA was the highest frequency of mycotoxin detected, followed by MON, ZEN and FB1. This study also presented the first report of BEA production by F. solani, F. compactum and F. chlamydosporum. All mycotoxins were not produced by F. nelsonii and F. lateritium. All Fusarium species were isolated from asymptomatic grasses, hence they are likely to exist as endophytes or latent pathogens.
Palm kernel cake (PKC) is the solid residue following oil extraction of palm kernels and useful to fatten animals either as a single feed with only minerals and vitamins supplementation, or mixed with other feedstuffs such as corn kernels or soy beans. The occurrence of mycotoxins (aflatoxins, ochratoxins, zearalenone, and fumonisins) in feed samples affects the animal's health and also serves as a secondary contamination to humans via consumption of eggs, milk and meats. Of these, aflatoxin B₁ (AFB₁) is the most toxically potent and a confirmed carcinogen to both humans and animals. Methods such as High Performance Liquid Chromatography (HPLC) and Liquid Chromatography-Mass Spectrometry (LC-MS/MS) are common in the determination of mycotoxins. However, these methods usually require sample pre-treatment, extensive cleanup and skilled operator. Therefore, in the present work, a rapid method of electrochemical immunosensor for the detection of AFB₁ was developed based on an indirect competitive enzyme-linked immunosorbent assay (ELISA). Multi-walled carbon nanotubes (MWCNT) and chitosan (CS) were used as the electrode modifier for signal enhancement.N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide (EDC) andN-hydroxysuccinimide (NHS) activated the carboxyl groups at the surface of nanocomposite for the attachment of AFB₁-BSA antigen by covalent bonding. An indirect competitive reaction occurred between AFB₁-BSA and free AFB₁ for the binding site of a fixed amount of anti-AFB₁ antibody. A catalytic signal based on horseradish peroxidase (HRP) in the presence of hydrogen peroxide (H₂O₂) and 3,3',5,5'-tetramethylbenzidine (TMB) mediator was observed as a result of attachment of the secondary antibody to the immunoassay system. As a result, the reduction peak of TMB(Ox)was measured by using differential pulse voltammetry (DPV) analysis. Based on the results, the electrochemical surface area was increased from 0.396 cm² to 1.298 cm² due to the electrode modification with MWCNT/CS. At the optimal conditions, the working range of the electrochemical immunosensor was from 0.0001 to 10 ng/mL with limit of detection of 0.1 pg/mL. Good recoveries were obtained for the detection of spiked feed samples (PKC, corn kernels, soy beans). The developed method could be used for the screening of AFB₁ in real samples.
The secondary metabolites produced by fungi known as mycotoxins, are capable of causing mycotoxicosis (diseases and death) in human and animals. Contamination of feedstuffs as well as food commodities by fungi occurs frequently in a natural manner and is accompanied by the presence of mycotoxins. The occurrence of mycotoxins' contamination is further stimulated by the on-going global warming as reflected in some findings. This review comprehensively discussed the role of mycotoxins (trichothecenes, zearalenone, fumonisins, ochratoxins, and aflatoxins) toward gut health and gut microbiota. Certainly, mycotoxins cause perturbation in the gut, particularly in the intestinal epithelial. Recent insights have generated an entirely new perspective where there is a bi-directional relationship exists between mycotoxins and gut microbiota, thus suggesting that our gut microbiota might be involved in the development of mycotoxicosis. The bacteria-xenobiotic interplay for the host is highlighted in this review article. It is now well established that a healthy gut microbiota is largely responsible for the overall health of the host. Findings revealed that the gut microbiota is capable of eliminating mycotoxin from the host naturally, provided that the host is healthy with a balance gut microbiota. Moreover, mycotoxins have been demonstrated for modulation of gut microbiota composition, and such alteration in gut microbiota can be observed up to species level in some of the studies. Most, if not all, of the reported effects of mycotoxins, are negative in terms of intestinal health, where beneficial bacteria are eliminated accompanied by an increase of the gut pathogen. The interactions between gut microbiota and mycotoxins have a significant role in the development of mycotoxicosis, particularly hepatocellular carcinoma. Such knowledge potentially drives the development of novel and innovative strategies for the prevention and therapy of mycotoxin contamination and mycotoxicosis.
The development of a reversed phase high performance liquid chromatography fluorescence method for the determination of the mycotoxins fumonisin B(1) and fumonisin B(2) by using silica-based monolithic column is described. The samples were first extracted using acetonitrile:water (50:50, v/v) and purified by using a C(18) solid phase extraction-based clean-up column. Then, pre-column derivatization for the analyte using ortho-phthaldialdehyde in the presence of 2-mercaptoethanol was carried out. The developed method involved optimization of mobile phase composition using methanol and phosphate buffer, injection volume, temperature and flow rate. The liquid chromatographic separation was performed using a reversed phase Chromolith(®) RP-18e column (100 mm × 4.6 mm) at 30 °C and eluted with a mobile phase of a mixture of methanol and phosphate buffer pH 3.35 (78:22, v/v) at a flow rate of 1.0 mL min(-1). The fumonisins separation was achieved in about 4 min, compared to approximately 20 min by using a C(18) particle-packed column. The fluorescence excitation and emission were at 335 nm and 440 nm, respectively. The limits of detections were 0.01-0.04 μg g(-1) fumonisin B(1) and fumonisin B(2), respectively. Good recoveries were found for spiked samples (0.1, 0.5, 1.5 μg g(-1) fumonisins B(1) and B(2)), ranging from 84.0 to 106.0% for fumonisin B(1) and from 81.0 to 103.0% for fumonisin B(2). Fifty-three samples were analyzed including 39 food and feeds and 14 inoculated corn and rice. Results show that 12.8% of the food and feed samples were contaminated with fumonisin B(1) (range, 0.01-0.51 μg g(-1)) and fumonisin B(2) (0.05 μg g(-1)). The total fumonisins in these samples however, do not exceed the legal limits established by the European Union of 0.8 μg g(-1). Of the 14 inoculated samples, 57.1% contained fumonisin B(1) (0.16-41.0 μg g(-1)) and fumonisin B(2) (range, 0.22-50.0 μg g(-1)). Positive confirmation of selected samples was carried out using liquid chromatography-tandem mass spectrometry, using triple quadrupole analyzer and operated in the multiple reaction monitoring mode.
Adsorption plays an important role in the removal of mycotoxins from feedstuffs. The main objective of this study was to investigate the efficacy of using magnetic graphene oxide nanocomposites (MGO) as an adsorbent for the reduction of Fusarium mycotoxins in naturally contaminated palm kernel cake (PKC). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to assess the mycotoxins in animal feed. Target mycotoxins included the zearalenone (ZEA), the fumonisins (FB1 and FB2) and trichothecenes (deoxynivalenol (DON), HT-2 and T-2 toxin). Response surface methodology (RSM) was applied to investigate the effects of time (3-7 h), temperature (30-50 °C) and pH (3-7) on the reduction. The response surface models with (R2 = 0.94-0.99) were significantly fitted to predict mycotoxins in contaminated PKC. Furthermore, the method ensured a satisfactory adjustment of the polynomial regression models with the experimental data except for fumonisin B1 and B2, which decrease the adsorption of magnetic graphene oxide (MGO). The optimum reduction was performed at pH 6.2 for 5.2 h at of 40.6 °C. Under these optimum conditions, reduced levels of 69.57, 67.28, 57.40 and 37.17%, were achieved for DON, ZEA, HT-2, and T-2, respectively.
Fungi are distributed worldwide and can be found in various foods and feedstuffs from almost every part of the world. Mycotoxins are secondary metabolites produced by some fungal species and may impose food safety risks to human health. Among all mycotoxins, aflatoxins (AFs), ochratoxin A (OTA), trichothecenes, deoxynivalenol (DON and T-2 toxin), zearalenone (ZEN), and fumonisins (FMN) have received much attention due to high frequency and severe health effects in humans and animals. Malaysia has heavy rainfall throughout the year, high temperatures (28 to 31 °C), and high relative humidity (70% to 80% during wet seasons). Stored crops under such conditions can easily be contaminated by mycotoxin-producing fungi. The most important mycotoxins in Malaysian foods are AFs, OTA, DON, ZEN, and FMN that can be found in peanuts, cereal grains, cocoa beans, and spices. AFs have been reported to occur in several cereal grains, feeds, nuts, and nut products consumed in Malaysia. Spices, oilseeds, milk, eggs, and herbal medicines have been reported to be contaminated with AFs (lower than the Malaysian acceptable level of 35 ng/g for total AFs). OTA, a possible human carcinogen, was reported in cereal grains, nuts, and spices in Malaysian market. ZEN was detected in Malaysian rice, oat, barley, maize meal, and wheat at different levels. DON contamination, although at low levels, was reported in rice, maize, barley, oat, wheat, and wheat-based products in Malaysia. FMN was reported in feed and some cereal grains consumed in Malaysia. Since some food commodities are more susceptible than others to fungal growth and mycotoxin contamination, more stringent prevention and control methods are required.