Flavus Growth Research Articles

Inhibitory effect of (E)-2-heptenal on Aspergillus flavus growth revealed by metabolomics and biochemical analyses

The prevention of fungal proliferation in postharvest grains is critical for maintaining grain quality and reducing mycotoxin contamination. Fumigation with natural gaseous fungicides is a promising and sustainable approach to protect grains from fungal spoilage. In this study, the antifungal activities of (E)-2-alkenals (C5-C10) on Aspergillus flavus were tested in the vapor phase, and (E)-2-heptenal showed the highest antifungal activity against A. flavus. (E)-2-Heptenal completely inhibited A. flavus growth at 0.0125 µL/mL and 0.2 µL/mL in the vapor phase and liquid contact, respectively. (E)-2-Heptenal can disrupt the plasma membrane integrity of A. flavus via leakage of intracellular electrolytes. Scanning electron microscopy indicated that the mycelial morphology of A. flavus was remarkably affected by (E)-2-heptenal. Metabolomic analyses indicated that 49 metabolites were significantly differentially expressed in A. flavus mycelia exposed to 0.2 µL/mL (E)-2-heptenal; these metabolites were mainly involved in galactose metabolism, starch and sucrose metabolism, the phosphotransferase system, and ATP-binding cassette transporters. ATP production was reduced in (E)-2-heptenal-treated A. flavus, and Janus Green B staining showed reduced cytochrome c oxidase activity. (E)-2-Heptenal treatment induced oxidative stress in A. flavus mycelia with an accumulation of superoxide anions and hydrogen peroxide and increased activities of superoxide dismutase and catalase. Simulated storage experiments showed that fumigation with 400 µL/L of (E)-2-heptenal vapor could completely inhibit A. flavus growth in wheat grains with 20% moisture; this demonstrates its potential use in preventing grain spoilage. This study provides valuable insights into understanding the antifungal effects of (E)-2-heptenal on A. flavus. KEY POINTS : • (E)-2-Heptenal vapor showed the highest antifungal activity against A. flavus among (C5-C10) (E)-2-alkenals. • The antifungal effects of (E)-2-heptenal against A. flavus were determined. • The antifungal actions of (E)-2-heptenal on A. flavus were revealed by metabolomics and biochemical analyses.

Antifungal Activity of Essential Oil and Plant-Derived Natural Compounds against Aspergillus flavus.

Aspergillus flavus is a facultative parasite that contaminates several important food crops at both the pre- and post-harvest stages. Moreover, it is an opportunistic animal and human pathogen that causes aspergillosis diseases. A. flavus also produces the polyketide-derived carcinogenic and mutagenic secondary metabolite aflatoxin, which negatively impacts global food security and threatens human and livestock health. Recently, plant-derived natural compounds and essential oils (EOs) have shown great potential in combatting A. flavus spoilage and aflatoxin contamination. In this review, the in situ antifungal and antiaflatoxigenic properties of EOs are discussed. The mechanisms through which EOs affect A. flavus growth and aflatoxin biosynthesis are then reviewed. Indeed, several involve physical, chemical, or biochemical changes to the cell wall, cell membrane, mitochondria, and related metabolic enzymes and genes. Finally, the future perspectives towards the application of plant-derived natural compounds and EOs in food protection and novel antifungal agent development are discussed. The present review highlights the great potential of plant-derived natural compounds and EOs to protect agricultural commodities and food items from A. flavus spoilage and aflatoxin contamination, along with reducing the threat of aspergillosis diseases.

Use of MALDI-TOF MS to Discriminate between Aflatoxin B1-Producing and Non-Producing Strains of Aspergillus flavus.

Aflatoxin B1 (AFB1) is one of the most toxic mycotoxins. One of the producers of AFB1 is Aspergillus flavus. Therefore, its rapid identification plays a key role in various sectors of the food and feed industry. MALDI-TOF mass spectrometry is one of the fastest and most accurate methods today. Therefore, the aim of this research was to develop the rapid identification of producing and non-producing strains of A. flavus based on the entire mass spectrum. To accomplish the main goal a different confirmatory MALDI-TOF MS and TLC procedures such as direct AFB1 identification by scraping from TLC plates, A. flavus mycelium, nutrient media around A. flavus growth, and finally direct AFB1 identification from infected wheat and barley grains had to be conducted. In this experiment, MALDI-TOF mass spectrometry with various modifications was the main supporting technology. All confirmatory methods confirmed the presence of AFB1 in the samples of aflatoxin-producing strains of A. flavus and vice versa; AFB1 was not detected in the case of non-producing strains. Entire mass spectra (from 2 to 20 kDa) of aflatoxin-producing and non-producing A. flavus strains were collected, statistically analyzed and clustered. An in-depth analysis of the obtained entire mass spectra showed differences between AFB1-producing and non-producing strains of A. flavus. Statistical and cluster analysis divided AFB1-producing and non-producing strains of A. flavus into two monasteries. The results indicate that it is possible to distinguish between AFB1 producers and non-producers by comparing the entire mass spectra using MALDI-TOF MS. Finally, we demonstrated that if there are established local AFB1-producing and non-producing strains of A. flavus, the entire mass spectrum database identification of aflatoxigenic A. flavus strains can be even faster and cheaper, without the need to identify the toxin itself.

Differential Expression of Genes Related to Growth and Aflatoxin Synthesis in Aspergillus flavus When Inhibited by Bacillus velezensis Strain B2.

Aspergillus flavus is a saprophytic soil fungus that infects and contaminates seed crops with the highly carcinogenic aflatoxin, which brings health hazards to animals and humans. In this study, bacterial strains B1 and B2 isolated from the rhizosphere soil of camellia sinensis had significant antagonistic activities against A. flavus. Based on the phylogenetic analysis of 16SrDNA gene sequence, bacterial strains B1 and B2 were identified as Bacillus tequilensis and Bacillus velezensis, respectively. In addition, the transcriptome analysis showed that some genes related to A. flavus growth and aflatoxin synthesis were differential expressed and 16 genes in the aflatoxin synthesis gene cluster showed down-regulation trends when inhibited by Bacillus velezensis strain B2. We guessed that the Bacillus velezensis strain B2 may secrete some secondary metabolites, which regulate the related gene transcription of A. flavus to inhibit growth and aflatoxin production. In summary, this work provided the foundation for the more effective biocontrol of A. flavus infection and aflatoxin contamination by the determination of differential expression of genes related to growth and aflatoxin synthesis in A. flavus when inhibited by B. velezensis strain B2.

The Inhibitory Effect of Pseudomonas stutzeri YM6 on Aspergillus flavus Growth and Aflatoxins Production by the Production of Volatile Dimethyl Trisulfide.

Aspergillus flavus and the produced aflatoxins cause great hazards to food security and human health across all countries. The control of A. flavus and aflatoxins in grains during storage is of great significance to humans. In the current study, bacteria strain YM6 isolated from sea sediment was demonstrated effective in controlling A. flavus by the production of anti-fungal volatiles. According to morphological characteristics and phylogenetic analysis, strain YM6 was identified as Pseudomonas stutzeri. YM6 can produce abundant volatile compounds which could inhibit mycelial growth and conidial germination of A. flavus. Moreover, it greatly prevented fungal infection and aflatoxin production on maize and peanuts during storage. The inhibition rate was 100%. Scanning electron microscopy further supported that the volatiles could destroy the cell structure of A. flavus and prevent conidia germination on the grain surface. Gas chromatography/mass spectrometry revealed that dimethyl trisulfide (DMTS) with a relative abundance of 13% is the most abundant fraction in the volatiles from strain YM6. The minimal inhibitory concentration of DMTS to A. flavus conidia is 200 µL/L (compound volume/airspace volume). Thus, we concluded that Pseudomonas stutzeri YM6 and the produced DMTS showed great inhibition to A. flavus, which could be considered as effective biocontrol agents in further application.

Effects of Four Isothiocyanates in Dissolved and Gaseous States on the Growth and Aflatoxin Production of Aspergillus flavus In Vitro.

Aflatoxins (AFs), a class of toxins produced by certain species of the genus Aspergillus, occasionally contaminate food and cause serious damage to human health and the economy. AFs contamination is a global problem, and there is a need to develop effective strategies to control aflatoxigenic fungi. In this study, we focused on isothiocyanates (ITCs) as potential chemical agents for the control of aflatoxigenic fungi. We quantitatively evaluated the effects of four ITCs (allyl ITC (AITC), benzyl ITC (BITC), and methyl and phenylethyl ITCs) in dissolved and gaseous states on the growth and aflatoxin B1 production of Aspergillus flavus. In experiments using dissolved ITCs, BITC was found to be the strongest inhibitor of growth and aflatoxin B1 production by A. flavus. Meanwhile, in the gaseous state, AITC strongly inhibited the A. flavus growth. When the concentration of ITCs in the liquid medium was quantified over time, AITC levels decreased to below the detection limit within 24 h, whereas BITC levels remained stable even after 48 h. These results suggested that when ITCs are utilized to control aflatoxigenic fungi, it is necessary to use them in a dissolved or gaseous state, depending on their volatility.

Assessment of Erythrophleum suaveolens (Brenan) leaves and stem bark water extracts antifungal potency on Aspergillus flavus incidence from maize (Zea mays L.) seeds

Aspergillus flavus has been associated with fungi attacks on stored maize seeds. Erythrophleum suaveolens had been reported to have antifungal properties which could protect some crops against fungi attack. This study aimed at evaluating the antifungal protection potency of both water extract of E. suaveolens leaves and stem bark on maize seeds varieties from A. flavus attack post six months of its application. Water extract of E. suaveolens leaves and stem barks (1, 10 and 100% concentration) were independently applied on four different varieties of maize seeds (OBA 98, OBA Super 6, SAMAZ 52 and SAMAZ 47) and stored alongside maize seeds without the extract application for 6 months. The fungal growth determination was carried out on Potato Dextrose Agar (PDA) media plates by placing maize seeds from both the group to which the extract was applied and those without the extract and incubating at 28 °C for 48 hours. The results indicated that all the maize seeds that the E. suaveolens leaves and stem bark water extract was applied at a varying concentration (1, 10 and 100%), recorded no growth of A. flavus unlike the maize seeds without the water extracts application which shows the incidence of A. flavus growth. In conclusion, E. suaveolens leaves and stem bark water extracts under varying concentration of 1, 10 and 100% had demonstrated to be an effective antifungal agent against A. flavus even after 6 months post application.

Evaluation of Natural Antifungal Compounds from Artemisia afra and its Effectiveness on Prevention of Aflatoxins

Artemisia afra is a medicinal plant with antimicrobial activities, known to treat tuberculosis, measles, cough, and influenza. In consequence, its applications have increasingly become pivotal to control various microbes which cause different diseases. This study therefore, evaluated the chemical composition and the antifungal activity of A. afra essential oil. The extraction of essential oil was done using steam distillation. The essential oil chemical composition was analysed by GC-MS. The analysis revealed 19 compounds from A. afra. The main compounds were borneol (71.529%), terpinen-4-ol (8.582%), β-thujone (3.283%), β-terpineol (2.590%), α-terpineol (1.791%), chrysanthenone (1.507%), myrtenal (1.393%), eucalyptol (1,8-cineole) (1.263%), cis-sabinene hydrate (1.172%) and trans-carane, 4,5-epoxy (1.088%). The growth and aflatoxin production inhibitions against A. flavus were determined via antifungal and antiaflatoxigenic bioassays of the A. afra essential oil at the concentration range of 10–50 µL/mL using a poisoned-food method. The quantification of the aflatoxins production was carried out using high-performance liquid chromatography. The antifungal activity of A. afra essential oil was effective against A. flavus with growth inhibition of 99.97–99.99% and controlled aflatoxins production by 99.25–99.63%. The results indicated that A. afra essential oil can become an alternative natural fungicide for the control of A. flavus growth and aflatoxins production.
 Keywords: Artemisia afra; essential oil; A. flavus; aflatoxins; GC-MS

Effect of Plant Extracts on Suppression of Aspergillus flavus Growth and Aflatoxins Production in Peanuts

Effect of Plant Extracts on Suppression of Aspergillus flavus Growth and Aflatoxins Production in Peanuts

Mechanisms underlying the inhibitory effects of linalool on Aspergillus flavus spore germination.

Biogenic volatile organic compounds hold remarkable potential for controlling fungal decay in agro- and food products. Recently, we reported that linalool, the major volatile component of the Zanthoxylum schinifolium pericarp, showed great potential as a biofumigant to control Aspergillus flavus growth in postharvest grains. In this study, the inhibitory effects of linalool on A. flavus growth in stored grains and its underlying mechanism were investigated through transcriptomic and biochemical analyses. Linalool vapor at 800 μL/L can effectively prevent A. flavus growth in 22% moisture wheat grains. Linalool at 2 μL/mL completely inhibited the germination of A. flavus spores, and 10 μL/mL caused spore death. Scanning electron microscopy revealed that linalool treatment caused wrinkling and spore breakage. Transcriptomics showed that 3806 genes were significantly differentially expressed in A. flavus spores exposed to 2 μL/mL linalool, predominantly showing enrichment regarding the ribosome, DNA replication, glutathione metabolism, peroxisome, and MAPK signaling pathways. Flow cytometry showed that linalool treatment caused hyperpolarization of mitochondrial membrane potential. 4,6-Diamidino-2-phenylindole staining indicated that linalool caused DNA fragmentation in A. flavus spores, and monodansylcadaverine staining confirmed that linalool induced autophagy in A. flavus spores. We thus propose that linalool can damage the plasma membrane, cause mitochondrial dysfunction and DNA damage, and induce autophagy in A. flavus spores. These findings considerably improve our understanding of the mechanisms underlying the inhibitory effects of linalool on A. flavus, which is crucial regarding the development of applications to prevent postharvest grain spoilage due to A. flavus infestations. KEY POINTS: • The inhibitory potency of linalool on A. flavus spore germination was determined. • Transcriptomic analyses were performed to identify differentially expressed genes of A. flavus exposed to linalool. • A functional mechanism underlying the inhibitory effects of linalool on A. flavus spore germination is proposed.

Preparation and characterization of a novel nanoemulsion consisting of chitosan and Cinnamomum tamala essential oil and its effect on shelf-life lengthening of stored millets

This study aimed to improve the stability of Cinnamomum tamala essential oil (CTEO) via encapsulating into chitosan nanoemulsion (CsNe) through an ionic-gelation technique and explore its food preservative efficacy against aflatoxigenic strain of Aspergillus flavus (AFLHPSi-1, isolated from stored millet), aflatoxin B1 (AFB1) contamination, and lipid peroxidation, causing qualitative deterioration of stored millets. The CTEO was characterized through gas chromatography–mass spectrometry (GC–MS) analysis that confirmed the presence of linalool as a major component occupying approximately 82.64% of the total oil. The synthesized nanoparticles were characterized through scanning electron microscopy (SEM), fourier transform infrared (FTIR) spectroscopy, and X-ray diffraction (XRD) analysis. The encapsulation efficiency (EE) and loading capacity (LC) of CTEO-CsNe were found to be 97.71% and 3.33%, respectively. In vitro release study showed a biphasic release pattern: with an initial burst release followed by a controlled release of CTEO. During investigation of efficacy, the CTEO-CsNe caused complete inhibition of A. flavus growth, and AFB1 biosynthesis at 1.0 and 0.8 μL/mL, respectively. The CTEO-CsNe exhibited its antifungal mode of action by altering fungal plasma membrane integrity (ergosterol inhibition) and permeability (leakage of important cellular constituents), and antiaflatoxigenic mode of action by inhibiting cellular methylglyoxal biosynthesis. CTEO-CsNe showed high free radical scavenging capacity (IC50 = 5.08 and 2.56 μL/mL) against DPPH•+ and ABTS•+ radicals, respectively. In addition, CTEO-CsNe presented remarkable preservative efficacy, inhibiting AFB1 and lipid peroxidation in model food system (Setaria italica) without altering their organoleptic properties. Based on overall results, CTEO-CsNe can be recommended as a novel shelf-life enhancer of stored millet samples.

Chitosan encompassed Aniba rosaeodora essential oil as innovative green candidate for antifungal and antiaflatoxigenic activity in millets with emphasis on cellular and its mode of action.

The present study demonstrates first time investigation on encapsulation of Aniba rosaeodora essential oil into chitosan nanoemulsion (AREO-CsNe) with the aim of improvement of its antifungal, and aflatoxin B1 (AFB1) inhibitory performance in real food system. The GC–MS analysis of AREO revealed the presence of linalool (81.46%) as a major component. The successful encapsulation of EO into CsNe was confirmed through SEM, FTIR, and XRD analysis. The in-vitro release study showed the controlled release of AREO. AREO-CsNe caused complete inhibition of Aspergillus flavus (AFLHPSi-1) growth and AFB1 production at 0.8 and 0.6 μl/ml, respectively, which was far better than AREO (1.4 and 1.2 μl/ml, respectively). Impairment of ergosterol biosynthesis coupled with enhancement of cellular materials leakage confirmed plasma membrane as the possible antifungal target of both AREO and AREO-CsNe. Significant inhibition of methylglyoxal (AFB1 inducer) synthesis in AFLHPSi-1 cells by AREO and AREO-CsNe confirmed their novel antiaflatoxigenic mode of action. In-silico molecular docking studies revealed effective interaction of linalool with Ver-1 and Omt-A proteins, leading to inhibition of AFB1 biosynthesis. Further, AREO-CsNe showed enhanced antioxidant activity with IC50 values 3.792 and 1.706 μl/ml against DPPH• and ABTS•+ radicals, respectively. In addition, AREO-CsNe caused 100% protection of stored millets (Setaria italica seeds) from AFB1 contamination and lipid peroxidation over a period of 1 year without compromising its sensory properties and exhibited high safety profile with LD50 value 9538.742 μl/kg body weight. Based on enhanced performance of AREO-CsNe over AREO, it can be recommended as a novel substitute of synthetic preservative for preservation of stored millets.

Efficacy and mechanism of action of food isolated yeasts in the control of Aspergillus flavus growth on pistachio nuts

Due to its nutritional characteristics, dried fruit and in particular pistachio is considered an important component in the daily diet. Unfortunately, pistachio nuts can be contaminated during storage with a wide range of pathogenic fungi, including Aspergillus flavus. The present work has evaluated how the use of two yeast strains belonging to the species Wickerhamomyces anomalus and Metschnikowia pulcherrima can inhibit the in vitro and in vivo growth of A. flavus. Both yeast strains demonstrated a good in vitro antifungal activity attributable to a specific mechanism of action, although higher efficacy was evidenced by W. anomalus strain. The production of volatile organic compounds (VOCs) and lytic enzymes was hypothesized as the main mechanisms of action exerted by W. anomalus, while the nutritional competition for iron was assumed as the main biocontrol mechanism for M. pulcherrima. Moreover, from the results of the in vivo test carried out on artificially infected pistachio seeds, it was clear as M. pulcherrima strain showed the same efficacy of W. anomalus in inhibiting the growth and sporulation of A. flavus mold, despite W. anomalus was the most effective strain during in vitro assay. Altogether, these results indicate that both strains could be considered as potential biocontrol agents against A. flavus fungal growth, notwithstanding it is always important considering the tritrophic interaction (yeast-mold- host), which could play a crucial role in determining the final results.

Volatiles of antagonistic soil yeasts inhibit growth and aflatoxin production of Aspergillus flavus

Minimizing Aspergillus flavus growth is an effective strategy to mitigate aflatoxin contamination in food and agricultural products. In the present investigation, we attempted to utilize soil-associated yeasts from the Western and Eastern Ghats of India against A. flavus to reduce aflatoxin contamination. Forty-five yeast isolates were screened against A. flavus using overlay and dual plate assays. Among them, 12 isolates effectively inhibited the growth of A. flavus. The 18S rDNA gene sequence analysis identified the twelve antagonistic isolates as belonging to Saccharomyces cerevisiae, Suhomyces xylopsoci, Pichia kudriavzevii, and Candida tropicalis. From the isolated yeasts, S. cerevisiae strains were selected for further evaluation based on the potential antagonistic activity. Volatiles of S. cerevisiae effectively suppressed the mycelial growth of A. flavus (P < 0.05) up to 92.1 % at 7 DAI. Scanning electron microscopic images of the fungus exposed to volatiles showed hyphal deformity and mycelial damage. Aflatoxin B1 (AFB1) production was drastically reduced up to 99.0 % in the volatile-exposed fungus compared to the control. The yeast strain YKK1 showed consistent Aspergillus flavus growth inhibition (80.7 %) and AFB1 production (98.1 %) for 14 days. Gas chromatography-mass spectrophotometry analysis of the yeast volatiles revealed the presence of antimicrobial compounds, including 1-pentanol, 1-propanol, ethyl hexanol, ethanol, 2-methyl-1-butanol, ethyl acetate, dimethyl trisulfide, p-xylene, styrene, and 1,4-pentadiene. The evaluated compounds of yeast volatiles, including ethyl acetate, hexanal, 1-propanol, 1-heptanol, 1-butanol, and benzothiazole, inhibited the fungal growth and AFB1 production of Aspergillus flavus when applied as pure chemicals. Benzothiazole at 5 mM was responsible for a high level of growth inhibition (23.6 %) and reduction of AFB1 synthesis (93.5 %). Hence, volatile compounds produced by soil yeast strains could be a potential biocontrol mechanism against aflatoxin contamination.

Characterization of Bacillus velezensis E2 with abilities to degrade ochratoxin A and biocontrol against Aspergillus westerdijkiae fc-1

Ochratoxin A (OTA), primarily produced by the fungi belonging to the species of Aspergillus and Penicillium, is one of the most common mycotoxins found in cereals and fruits. In addition to resulting in huge economic losses, OTA contamination also poses considerable threat to human and livestock health. Microbial degradation of mycotoxins has been considered with great potential in mycotoxins decontamination. In a previous study, Bacillus velezensis E2 was isolated by our laboratory and showed appreciable inhibitory effect on Aspergillus flavus growth and aflatoxin production in rice grains. In this study, B. velezensis E2 was investigated for its ability to remove OTA and biocontrol against the ochratoxigenic Aspergillus westerdijkiae fc-1. The results revealed that B. velezensis E2 has considerable inhibitory effect on A. westerdijkiae fc-1 both on PDA medium and pear fruits, with inhibitory rate of 51.7% and 73.9%, respectively. In addition, its ability to remove OTA was evaluated in liquid medium and the results showed that more than 96.1% of OTA with an initial concentration of 2.5 μg/mL could be removed by B. velezensis E2 in 48 h. Further experiments revealed that enzymatic transformation and alkaline hydrolysis might be the main mechanisms related to OTA degradation by B. velezensis E2, with ring open ochratoxin α (OP-OTα) as a possible degradation product. Our study indicated that the B. velezensis E2 strain could be a potential bacterial candidate in biodegradation of OTA and biocontrol against A. westerdijkiae fc-1.

The antifungal mechanisms of plant volatile compound 1-octanol against Aspergillus flavus growth.

The exploitation of active ingredients from plant volatile organic compounds as natural gaseous fungicides shows remarkable potential for controlling fungal decay in postharvest agroproducts. Although 1-octanol is a common component of cereal volatiles, its antifungal potency against spoilage fungi in postharvest grains remains unclear. In this study, we studied the effectiveness of 1-octanol against Aspergillus flavus growth in postharvest grains and its mechanisms of action. 1-Octanol vapor and liquid contact dose-dependently inhibited A. flavus spore germination and mycelial growth at a low concentration. The simulated storage experiment demonstrated that 300 μL/L of 1-octanol vapor completely controlled A. flavus growth in wheat, corn, and paddy grains with 20% moisture content. 1-Octanol treatment irreversibly damaged the conidial and mycelial morphology of A. flavus and caused electrolyte leakage due to reduced plasma membrane integrity. It induced apoptosis along with morphological abnormalities, phosphatidylserine externalization, mitochondrial membrane potential depolarization, intracellular reactive oxygen species accumulation, and DNA fragmentation in A. flavus cells. Metabolomic analysis revealed that 1-octanol treatment disrupted the biosynthesis of unsaturated fatty acids, ATP-binding cassette transporters, amino acid metabolism, and glycerophospholipid metabolism. This study demonstrated the promising application potential of 1-octanol as a biofumigant for preventing fungal spoilage of postharvest cereal grains. KEY POINTS: • (1) 1-Octanol inhibits Aspergillus flavus growth in the vapor phase and liquid contact; • (2) 1-Octanol damages membrane integrity and induces apoptosis of A. flavus; • (3) Metabolomic changes in A. flavus mycelia were analyzed after 1-octanol treatment.

Effect of Aspergillus flavus on Seed Germination and Seedlings Growth of Barley and Some of Associated Weeds

The research was conducted during the 2021 growing season which included a series of experiments to investigate the effect the fungus Aspergillus flavus presence on germination and growth of barley and some barley weeds including annual ryegrass Lolium rigidum, darnel Lolium temulentum and yellow sweet clover Melilotus officinalis. Results of isolation and identification of fungi associated with seedlings roots of barley and the weeds showed that A. flavus was the most prevalent fungus associated with roots and surrounding soil recording the highest frequency among all the found fungi. In plastic pots, Aspergillus flavus was added to the potting soil and tested for its the effect on seed germination and seedlings growth of barley and weeds under study. The results showed after 20 days of planting that the fungus led to an increase in the percentage of germination and fresh weight and lengths of barley seedlings while the presence of the fungus decreased germination and seedlings growth of studied weeds with the highest negative impact on the darnel. Incubation at different temperatures were tested for their effects on A. flavus growth, and results showed that the fungus grows higher when incubated at relatively high temperatures (25-35°C), while the lowest growth was at 15 °C and the fungus did not grow at 10 °C.

Antifungal activity of puroindoline protein from soft wheat against grain molds and its potential as a biocontrol agent

Mold growth reduces the quality of stored grains, besides producing toxins that pose a potential threat to human health. Therefore, prevention of grain mold growth during storage is important to ensure a safe and high-quality product, preferably using an eco-friendly antifungal agent. The puroindoline (PIN) protein was extracted by Triton X-114 and identified by QE mass spectrometry. Aspergillus flavus has attracted much attention because of its toxic secondary metabolites, and PIN protein showed a significant inhibition on A. flavus growth. Scanning electron microscopy revealed altered spore morphology of A. flavus following PIN protein treatment, and propidium iodide staining showed incomplete spore cell membranes. The disruption and deformation of A. flavus spores suggest that the cell walls and cell membranes were compromised. Decreased mitochondrial membrane potential and increased levels of intracellular reactive oxygen specieswere detected using JC-1 and 2,7-dichlorodihydrofluorescein diacetate staining, respectively. PIN protein could effectively inhibit the growth and aflatoxins B1 production of A. flavus in stored grains, such as wheat and rice. PIN proteins can inhibit the growth of many common grain storage molds, including Penicillium, Aspergillus spp. (A. flavus, A. glaucus, A. kawachii, A. ochraceus and A. niger), Alternaria and Fusarium graminearum, in a dose-dependent manner. PIN protein has a significant inhibitory effect on the growth of grain molds, with a stronger inhibitory effect noted in wheat and rice. Our study provides a novel and simple theoretical basis for the selection and storage of mold resistance in grains and food during storage.

Inhibition of Aspergillus flavus Growth and Aflatoxin Production in Zea mays L. Using Endophytic Aspergillus fumigatus.

Aspergillus flavus infection of vegetative tissues can affect the development and integrity of the plant and poses dangerous risks on human and animal health. Thus, safe and easily applied approaches are employed to inhibit A. flavus growth. To this end, the fungal endophyte, i.e., Aspergillus fumigatus, was used as a safe biocontrol agent to reduce the growth of A. flavus and its infection in maize seedlings. Interestingly, the safe endophytic A. fumigatus exhibited antifungal activity (e.g., 77% of growth inhibition) against A. flavus. It also reduced the creation of aflatoxins, particularly aflatoxin B1 (AFB1, 90.9%). At plant level, maize seedling growth, leaves and root anatomy and the changes in redox status were estimated. Infected seeds treated with A. fumigatus significantly improved the germination rate by 88.53%. The ultrastructure of the infected leaves showed severe disturbances in the internal structures, such as lack of differentiation in cells, cracking, and lysis in the cell wall and destruction in the nucleus semi-lysis of chloroplasts. Ultrastructure observations indicated that A. fumigatus treatment increased maize (leaf and root) cell wall thickness that consequentially reduced the invasion of the pathogenic A. flavus. It was also interesting that the infected seedlings recovered after being treated with A. fumigatus, as it was observed in growth characteristics and photosynthetic pigments. Moreover, infected maize plants showed increased oxidative stress (lipid peroxidation and H2O2), which was significantly mitigated by A. fumigatus treatment. This mitigation was at least partially explained by inducing the antioxidant defense system, i.e., increased phenols and proline levels (23.3 and 31.17%, respectively) and POD, PPO, SOD and CAT enzymes activity (29.50, 57.58, 32.14 and 29.52%, respectively). Overall, our study suggests that endophytic A. fumigatus treatment could be commercially used for the safe control of aflatoxins production and for inducing biotic stress tolerance of A. flavus-infected maize plants.

Linalool, the main volatile constituent from Zanthoxylum schinifolium pericarp, prevents growth of Aspergillus flavus in post-harvest grains

Plant volatile organic compounds (PVOCs) have shown great potential as alternatives to synthetic insecticides or fungicides for stored grain management. Zanthoxylum schinifolium pericarp, a traditional Chinese spice, is conventionally buried in grain piles to prevent fungal spoilage of stored grains in China. However, the chemical basis and antifungal mechanism of PVOCs from Z. schinifolium pericarp by which they inhibit spoilage remain unclear. In this study, the effectiveness of PVOCs from Z. schinifolium pericarp against Aspergillus flavus growth in high-moisture wheat grains was studied under simulated storage conditions. The growth of A. flavus in the grains was inhibited in a dose-dependent manner. The chemical composition of PVOCs from Z. schinifolium pericarp was determined using gas chromatography-mass spectrometry; linalool (50.31%) and d-limonene (20.92%) were the two main components. An antifungal experiment showed that 0.571 and 1.2 μL/mL concentrations of linalool completely inhibited A. flavus growth upon vapor and liquid contact, respectively. d-limonene showed much lower antifungal activity, indicating that linalool is responsible for the antifungal activity of PVOCs from Z. schinifolium pericarp. Linalool treatment disrupted the cell membrane of A. flavus, resulting in increased electrolyte leakage and A260nm in the culture supernatant. After 6 h of exposure to 1.2 μL/mL linalool, metabolomic analysis revealed 90 differentially expressed metabolites in A. flavus mycelia, including 69 upregulated and 21 downregulated metabolites. It was speculated that linalool inhibited A. flavus by disrupting the permeability and integrity of cell membranes, tricarboxylic acid cycle, and ATP binding cassette transport and by inducing mitochondrial dysfunction and oxidative stress. This study shows the potential of PVOCs from Z. schinifolium pericarp as biofumigants for stored grain management and provides a new perspective on their antifungal mechanism against A. flavus growth.