Development of a real-time PCR and multiplex PCR assay for the detection and identification of mycotoxigenic fungi in stored maize grains.

This study aimed to identify important mycotoxigenic fungi and accurate detection of mycotoxin in stored maize grains using molecular methods. The current study also optimised the real-time PCR (RT-PCR) assay. The melting curve was established to identify isolated fungal species of Aspergillus (4), Fusarium (3), Penicillium (3), and Alternaria (one). A multiplex polymerase chain reaction (mPCR) technique was developed for the detection and characterisation of mycotoxin producing fungi, mycotoxin metabolic pathway genes, and the determination of eleven mycotoxins in stored maize grains using high-performance liquid chromatography (HPLC). The mPCR results indicated positive signals for potentially mycotoxigenic fungal species tested of Aspergillus, Fusarium, Penicillium, and Alternaria. A protocol for multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was tested to distinguish between free and contaminated, stored maize with aflatoxin B1 (AFB1). The expression pattern of four aflatoxin biosynthetic pathway genes, AFB1 (aflQ, aflP, aflO, and aflD), was a good marker for contaminated, stored maize grains. HPLC analysis showed that maize grain samples were contaminated with mycotoxins, and the concentration was above the detection level. The results indicate that the polyphasic approach might provide a sensitive, rapid, and accurate method for detecting and identifying mycotoxigenic fungal species and mycotoxins in stored maize grains.

Efficacy of the antifungal metabolites of Streptomyces philanthi RL-1-178 on aflatoxin degradation with its application to prevent aflatoxigenic fungi in stored maize grains and identification of the bioactive compound.

Aflatoxin B1 is a potent carcinogen produced by Aspergillus flavus (A. flavus) and Aspergillus. parasiticus (A. parasiticus), mainly during grain storage. The efficacy of the freeze-dried culture filtrate of Streptomyces philanthi (S. philanthi) strain RL-1-178 (DCF) on degradation of aflatoxin B1 (AFB1) were evaluated and its bioactive compounds were identified. The DCF at a concentration of 9.0% (w/v) completely inhibited growth and AFB1 production of A. parasiticus TISTR 3276 and A. flavus PSRDC-4 after 7 days tested in yeast-extract sucrose (YES) medium and on stored maize grains after 28 and 14 days incubation, respectively. This indicated the more tolerance of A. parasiticus over A. flavus. The DCF and bacterial cells of S. philanthi were capable to degrade AFB1 by 85.0% and 100% for 72 h and 8 days, respectively. This confirmed the higher efficacy of the DCF over the cells. After separation of the DCF on thin-layer chromatography (TLC) plate by bioautography bioassay, each active band was identified by liquid chromatography-quadrupole time of flight mass spectrometer (LC-Q-TOF MS/MS). The results revealed two compounds which were identified as azithromycin and an unknown based on mass ions of both ESI+ and ESI- modes. The antifungal metabolites in the culture filtrate of S. philanthi were proved to degrade aflatoxin B1. It could be concluded that the DCF may be applied to prevent the growth of the two aflatoxin-producing fungi as well as the occurrence of aflatoxin in the stored maize grains.

Predictive model for cadmium uptake by maize and rice grains on the basis of bioconcentration factor and the diffusive gradients in thin-films technique.

It is possible for heavy metals in soils to be adsorbed by crop roots and then accumulated in crops, which eventually causes great health risk when the crops are ingested by humans. Thus, it is valuable to understand the enrichment model of heavy metals in crops. Diffusive gradients in thin-films (DGT) technique, as an in-situ passive sampling method, can be used to evaluate the bioavailable heavy metals contents in soils. In this study, data of the bioavailable cadmium (Cd) in soils determined by DGT and Cd contents uptake in rice and maize grains in Tianjin, Zhejiang and Guangxi provinces of China were collected from previous references in Web of Science. By comparing bioconcentration factors, it was found that the heavy metal concentrations accumulated in rice and maize followed a general order roots > stems or leaves > grains. An accurate and robust model for the prediction of Cd content in maize and rice grains was established based on bioconcentration factor (BCF) and the bioavailable Cd content determined by DGT method, with R2 0.986 and root mean square error (RMSE) 0.128. This result suggests that the DGT method can be good tool for predicting heavy metals uptake in crops.

Influence of a glyphosate-based herbicide on growth parameters and aflatoxin B1 production by Aspergillus section Flavi on maize grains.

Glyphosate-based herbicides (GBH) are the main pesticides applied worldwide on maize production. Glyphosate-resistant weeds led to the repeated application of high doses of the pesticide. In addition to environmental conditions, the presence of GBH affects the development of Aspergillus species and aflatoxin B1 (AFB1) production under in vitro conditions. The aim of this work was to evaluate the influence of a commercial GBH on growth and AFB1 production by Aspergillus flavus and Aspergillus parasiticus strains under different water activity (aW) conditions. The following concentrations of active ingredient glyphosate were evaluated: 20, 50, 200 and 500mM. The lag phase prior to growth and growth rate did not change at 20 and 50mM (that is, at field recommended doses) at 0.98 and 0.95 aW; however, at increasing GBH concentrations, between 200 and 500mM, the growth rate decreased at all aW conditions. In general, as the GBH concentration increased, AFB1 production decreased. However, a significant increase in toxin accumulation was found only at one of the aW conditions (0.95) at 21 days with 50mM of GBH in A. flavus and 20 and 50mM of GBH in A. parasiticus. These results show that, even though Aspergillus section Flavi growth did not increase, AFB1 production increased on maize grains at GBH concentrations similar to those of field recommended doses under favorable water availability and temperature conditions.

The Efficacy of Composite Essential Oils against Aflatoxigenic Fungus Aspergillus flavus in Maize.

The efficacy of eleven essential oils (EOs) against Aspergillus flavus NRRL 3357 was investigated. The highest antifungal activity against this aflatoxigenic fungus was exhibited by cinnamon, oregano and lemongrass, which showed low minimum inhibitory concentration (MIC) values under vapor conditions. Interactions of the three EOs were evaluated by the fractional inhibition concentration index (FICI), and the composite essential oils (CEO) showed synergistic inhibitory activities. Chemical analysis of the composite essential oils of cinnamon, oregano, and lemongrass (COL-CEO) revealed that (Z)-citral (33.44%), (E)-citral (32.88%) and carvacrol (19.84%) were the dominant components, followed by limonene (4.29%) and cinnamaldehyde (3.76%). COL-CEO not only inhibited fungal growth but also decreased aflatoxin B1 production by A. flavus. Downregulation of the relative expression of aflatoxin genes in the aflatoxin biosynthetic pathway by COL-CEO revealed its anti-aflatoxigenic mechanism. COL-CEO could also affect the colonization of A. flavus on maize grains. Therefore, COL-CEO may be considered as a potential natural antifungal agent, which could be used for the storage of maize and other grains.

Inhibitory effect of C. zeylanicum, C. longa, O. basilicum, Z. officinale, and C. martini essential oils on growth and ochratoxin A content of A. ochraceous and P. verrucosum in maize grains.

In the study, antifungal and ochratoxin A (OTA) production inhibitory activities of essential oils (EOs) of Cinnamomum zeylanicum, Curcuma longa, Ocimum basilicum, Zingiber officinale, and Cymbopogon martini were reported on Aspergillus ochraceus and Penicillium verrucosum. EOs were obtained by hydrodistillation and GC-MS technique was chosen to deduce their chemical profile. Major chemical compounds in EOs of C. zeylanicum, C. longa, O. basilicum, Z. officinale, and C. martini were (E)-cinnamaldehyde (35.81 %), ar-turmerone (46.13 %), eugenol (36.58 %), geranyl proprionate (18.93 %), and geranyl acetate (14.88 %), respectively. The EOs shown potent antioxidant activity by DPPH and ABTS assays. The EOs presented superlative antifungal activity against P. verrucosum related to A. ochraceus. The C. zeylanicum and C. martini EOs shown superlative antifungal activity related to other EOs. The C. zeylanicum and C. martini EOs completely inhibited the growth and OTA production of P. verrucosum and A. ochraceous at 1500 and 2500 µg/g in maize grains, respectively.

Risk assessment of human exposure to lead and cadmium in maize grains cultivated in soils irrigated either with low-quality water or freshwater.

Maize is the third important cereal crop after wheat and rice, especially in Egyptian villages. It is used in baking as a substitution component in wheat products and a main component in snacks for children. The target of this study was to estimate the risk assessment of lead (Pb) and cadmium (Cd) in maize grains cultivated in the agricultural soil irrigated by the contaminated water in comparison with that irrigated by freshwater. Lead (Pb) and cadmium (Cd) levels in irrigation water, soils and maize grains collected from different sites in Egypt were determined using ICP-OES. The studied samples were collected from 5 agricultural sites irrigated with freshwater (Nile River water and groundwater) as well as 4 agricultural sites irrigated with low-quality water (contaminated by sewage and industrial wastewater). Results exhibited that the levels of Pb and Cd in soil and maize grains were significantly affected by their levels in irrigation water; where, the levels of Pb and Cd in soil and maize grains irrigated by low-quality water possessed the multiple concentrations in comparison with those irrigated by freshwater. Specific water sources such as Kafr-Dokhmais and Al-Nasiria sites, Kafr El-Sheikh governorate had the highest levels of metals in the samples of irrigation water, soil and maize grains (p < 0.05). Metals levels in water and soil samples were within the permissible limits except Cd in low-quality water samples. Levels of Pb in maize grains irrigated by low-quality water were above the permissible limits (0.20 mg kg-1), while Cd levels were within the permissible limits (0.1 mg kg-1) except Al-Nasiria samples. Levels of Pb and Cd in maize grains irrigated by low-quality water were 19-30 folds those of maize grains irrigated by freshwater. The risk assessment of Pb and Cd levels in maize grains was estimated by daily intake of metals (DIM) and health risk index (HRI). All determined HRI was <1 indicating a non potential health risk for both adults and children.

Risk management of ochratoxigenic fungi and ochratoxin A in maize grains by bioactive EVOH films containing individual components of some essential oils.

Aspergillus steynii and Aspergillus tubingensis are possibly the main ochratoxin A (OTA) producing species in Aspergillus section Circumdati and section Nigri, respectively. OTA is a potent nephrotoxic, teratogenic, embryotoxic, genotoxic, neurotoxic, carcinogenic and immunosuppressive compound being cereals the first source of OTA in the diet. In this study bioactive ethylene-vinyl alcohol copolymer (EVOH) films containing cinnamaldehyde (CINHO), linalool (LIN), isoeugenol (IEG) or citral (CIT) which are major components of some plant essential oils (EOs) were produced and tested against A. steynii and A. tubingensis growth and OTA production in partly milled maize grains. Due to the favourable safety profile, these bioactive compounds are considered in the category "GRAS". The study was carried out under different water activity (0.96 and 0.99 aw), and temperature (24 and 32 °C) conditions. ANOVA showed that class of film, fungal species, aw and temperature and their interactions significantly affected growth rates (GR), ED50 and ED90 and the doses for total fungal growth inhibition and OTA production. The most effective EVOH films against both species were those containing CINHO. ED50, ED90 and doses for total growth and OTA inhibition were 165-405, 297-614, 333-666 µg of EVOH-CINHO/plate (25 g of maize grains), respectively, depending on environmental conditions. The least efficient were EVOH-LIN films. ED50, ED90 and doses for total growth and OTA inhibition were 2800->3330, >3330 and >3330 µg of EVOH-LIN/plate (25 g of maize grains), respectively. The effectiveness of the bioactive films increased with increasing doses. Overall, A. tubingensis was less sensitive to treatments than A. steynii. Depending on the species, aw and temperature affected GR and OTA production in a different way. In A. steynii cultures, optimal growth occurred at 0.96 aw and 32 °C while optimal OTA production happened at 0.99 aw and 32 °C. In A. tubingensis cultures optimal growth happened at 0.99 aw and 32 °C, although the best conditions for OTA production were 0.99 aw and 24 °C. Thus, these species can be very competitive in warm climates and storage conditions. The EVOH-CINHO films followed by EVOH-IEG and EVOH-CIT films, designed in this study and applied in vapour phase, can be potent antifungal agents against A. steynii and A. tubingensis and strong inhibitors of OTA biosynthesis in maize grains at very low doses. This is the first study on the impact that interacting environmental conditions and bioactive films containing individual components of EOs have on the growth of these ochratoxigenic fungi and on OTA production in maize grains.