The effect of cold atmospheric pressure plasma on Aspergillus ochraceus and ochratoxin A production.

Aspergillus ochraceus is a soil fungus known to produce ochratoxin A, a harmful secondary metabolite. Prevention and control of fungal pathogens mostly rely on chemical fungicides, which is one of the contributing factors in the emergence of the fungal resistance, hence novel methods for fungal eradication have been extensively researched. The cold atmospheric pressure (CAP) plasma generated in ambient air has been recently applied in microbial decontamination. Here we used the diffuse coplanar surface barrier discharge in inactivation of a toxigenic strain A. ochraceus. The plasma-treated conidia and mycelium exhibited morphological changes such as ruptures and desiccation. Mycelium dehydration and changes in the chemical composition of hyphal surface accompanied plasma treatment. The growth of 26 h old mycelia were significantly restricted after 30 s of plasma treatment. The conidial vitality declined 4 logs after 180 s of plasma exposure leading to almost complete decontamination. After shorter plasma treatment of conidia, the ochratoxin A (OTA) production increased at the early stage of cultivation, but the overall level was significantly reduced compared to untreated samples after longer cultivation. Our results indicated that the fungal growth and the OTA production were significantly changed by plasma treatment and underscored CAP plasma as a promising method in the decontamination of A. ochraceus without a risk to generate strains with increased OTA production.

Antifungal Activity of Essential Oil Compounds (Geraniol and Citral) and Inhibitory Mechanisms on Grain Pathogens (Aspergillus flavus and Aspergillus ochraceus).

The grain contamination by Aspergillus spp. has been a serious issue. This study exhibited the excellent antifungal effects of the essential oil compounds (EOCs) geraniol and citral against common grain pathogens (A. flavus and A. ochraceus) in vitro and in situ. The inhibitory mechanisms were also evaluated from the perspective of cell membrane permeability, reactive oxygen species (ROS) generation, and Aspergillus spp. growth-related gene expression. Meanwhile, the combined effects of EOCs in the vapor phase and modified atmosphere packaging (MAP) were examined to find an alternative preservation method for controlling Aspergillus spp. The results indicated that citral exhibited the antifungal activity mainly by downregulating the sporulation- and growth-related genes for both pathogens. Geraniol displayed inhibitory effectiveness against A. flavus predominantly by inducing the intracellular ROS accumulation and showed toxicity against A. ochraceus principally by changing cell membrane permeability. Furthermore, the synthetic effects of EOCs and MAP (75% CO2 and 25% N2) induced better grain quality than the current commercial fumigant AlP. These findings reveal that EOCs have potential to be a novel grain preservative for further application.

Effect of Cinnamaldehyde on Morphological Alterations of Aspergillus ochraceus and Expression of Key Genes Involved in Ochratoxin A Biosynthesis.

Ochratoxin A (OTA) is a potent nephrotoxic, hepatotoxic, and teratogenic compound which is a significant mycotoxin contaminates cereals during storage. Aspergillus ochraceus is the most common producer of OTA in cereals and cereal-derived products. Cinnamaldehyde is a natural substance derived from plant cinnamon playing an important role in the reduction of OTA contamination. In this study, the antifungal and antitoxigenic effect of cinnamaldehyde was investigated with its mechanisms of inhibition of fungal growth at the morphological and ultrastructural levels, and inhibition of OTA biosynthesis at the transcriptional level. Significant A. ochraceus growth was inhibited at 0.4⁻1.6 mmol/L with fumigation. A. ochraceus exposed to 0.4 mmol/L of cinnamaldehyde indicated irreversible harmful morphological and ultrastructural modifications such as the folding of the cell, the loss of integrity of the cell wall, the disruption of plasma membrane, the destruction of the mitochondria, and the absence of intracellular organelles. These alterations may be attributed to its inhibition of enzymatic reactions that regulate cell wall synthesis, thus disturbing the morphogenesis and growth of A. ochraceus. In the presence of cinnamaldehyde, the tested biosynthetic and regulatory genes like pks, nrps, veA, laeA and velB were highly downregulated. Moreover, the downregulation effect of cinnamaldehyde increased proportionally with the concentrations. These results suggest that the decrease of OTA production by cinnamaldehyde is attributed to the downregulation of the transcriptional levels of OTA biosynthetic and regulatory genes besides the inhibition of fungal growth. The study reveals the mechanisms of the antifungal and antitoxigenic activities of cinnamaldehyde against A. ochraceus, and further emphasizes that cinnamaldehyde could be a safe and effective natural agents against OTA contamination during cereals storage.

Inhibitory effect of essential oils on Aspergillus ochraceus growth and ochratoxin A production.

Ochratoxin A (OTA) is a mycotoxin which is a common contaminant in grains during storage. Aspergillus ochraceus is the most common producer of OTA. Essential oils play a crucial role as a biocontrol in the reduction of fungal contamination. Essential oils namely natural cinnamaldehyde, cinnamon oil, synthetic cinnamaldehyde, Litsea citrate oil, citral, eugenol, peppermint, eucalyptus, anise and camphor oils, were tested for their efficacy against A. ochraceus growth and OTA production by fumigation and contact assays. Natural cinnamaldehyde proved to be the most effective against A. ochraceus when compared to other oils. Complete fungal growth inhibition was obtained at 150-250 µL/L with fumigation and 250-500 µL/L with contact assays for cinnamon oil, natural and synthetic cinnamaldehyde, L. citrate oil and citral. Essential oils had an impact on the ergosterol biosynthesis and OTA production. Complete inhibition of ergosterol biosynthesis was observed at ≥ 100 µg/mL of natural cinnamaldehyde and at 200 µg/mL of citral, but total inhibition was not observed at 200 µg/mL of eugenol. But, citral and eugenol could inhibit the OTA production at ≥ 75 µg/mL and ≥ 150 µg/mL respectively, while natural cinnamaldehyde couldn't fully inhibit OTA production at ≤ 200 µg/mL. The inhibition of OTA by natural cinnamaldehyde is mainly due to the reduction in fungal biomass. However, citral and eugenol could significant inhibit the OTA biosynthetic pathway. Also, we observed that cinnamaldehyde was converted to cinnamic alcohol by A. ochraceus, suggesting that the antimicrobial activity of cinnamaldehyde was mainly attributed to its carbonyl aldehyde group. The study concludes that natural cinnamaldehyde, citral and eugenol could be potential biocontrol agents against OTA contamination in storage grains.

SEM study of water activity and temperature effects on the initial growth of Aspergillus ochraceus, Alternaria alternata and Fusarium verticillioides on maize grain. Scanning electron microscopy.

A scanning electron microscopy (SEM) study of the effect of water activity (a(w)), temperature and fungal interactions on the very early phases of Aspergillus ochraceus, Alternaria alternata and Fusarium verticillioides development on maize grains was carried out. Germination and growth of individual fungal strains were assayed at 0.92, 0.95 and 0.98 a(w), and 20 and 30 degrees C. Hyphal lengths were measured on micrographs taken by SEM at different periods of incubation. A. alternata had the highest linear growth at 0.98 a(w), and was the only species able to grow under all conditions tested, whereas A. ochraceus was not able to germinate at 0.92 a(w) at any temperature assayed. F. verticillioides demonstrated a different behaviour depending on growth temperature. Fungal interactions were studied at 0.95a(w) and 30 degrees C. A. ochraceus germination was inhibited when it was co-inoculated with one or two of the other species. A. alternata showed an increased growth rate when growing together with the other fungi, whereas growth of F. verticillioides was significantly inhibited when paired with A. ochraceus.

Variants of Aspergillus alutaceus var. alutaceus (formerly Aspergillus ochraceus) with altered ochratoxin A production.

The present studies, using Aspergillus alutaceus var. alutaceus Berkeley et Curtis (formerly A. ochraceus Wilhelm) NRRL 3174 along with three other wild-type strains, were undertaken in an attempt to understand the effects of irradiation and other treatments on mycotoxin production in grain. Bedford barley was inoculated with spores of NRRL 3174, gamma irradiated, and incubated at 28 degrees C and 25% moisture. After 10 days of incubation, two colony types, ochre (parental) and yellow (variant), were isolated from the grain. Further culturing of the yellow variant resulted in the spontaneous appearance of a white variant that exhibited greatly enhanced fluorescence under UV light. In subsequent work, we have also isolated variants producing a soluble red pigment. In addition, in model experiments involving irradiation (1 kGy) of pure cultures, induction frequencies ranging between 2 and 4% (survival basis) were observed for the yellow and red variants. Inoculation of these variants into wheat and incubation for 14 days at 28 degrees C and 32% moisture resulted in ochratoxin A production in the relative amounts of 0.09:1:4.6:9.3 for the red, ochre (parental), yellow, and white variants, respectively. Additional characteristics of these isolates are described. Confirmation that the white high-ochratoxin-A-producing variants were derived from the parental strain was demonstrated by obtaining revertant sectors in monoclonal cultures of the variants.