AwSclB regulates a network for Aspergillus westerdijkiae asexual sporulation and secondary metabolism independent of the fungal light control.

As a prevalent pathogenic fungus, Aspergillus westerdijkiae poses a threat to both food safety and human health. The fungal growth, conidia production and ochratoxin A (OTA) in A. weterdijkiae are regulated by many factors especially transcription factors. In this study, a transcription factor AwSclB in A. westerdijkiae was identified and its function in asexual sporulation and OTA biosynthesis was investigated. In addition, the effect of light control on AwSclB regulation was also tested. The deletion of AwSclB gene could reduce conidia production by down-regulation of conidia genes and increase OTA biosynthesis by up-regulation of cluster genes, regardless under light or dark conditions. It is worth to note that the inhibitory effect of light on OTA biosynthesis was reversed by the knockout of AwSclB gene. The yeast one-hybrid assay indicated that AwSclB could interact with the promoters of BrlA, ConJ and OtaR1 genes. This result suggests that AwSclB in A. westerdijkiae can directly regulate asexual conidia formation by activating the central developmental pathway BrlA-AbaA-WetA through up-regulating the expression of AwBrlA, and promote the light response of the strain by activating ConJ. However, AwSclB itself is unable to respond to light regulation. This finding will deepen our understanding of the molecular regulation of A. westerdijkiae development and secondary metabolism, and provide potential targets for the development of new fungicides.

First record of Leaf Spot of Ficus religiosa Caused by Alternaria alternata in Pakistan.

Ficus religiosa (L.) belongs to the family Moraceae, native to India and commonly known as 'Peepal'. It has high medicinal value due to its antibacterial, antiviral and antioxidant properties (Singh et al., 2015; Kalpana et al., 2009). In August 2021, leaf spots were observed on F. religiosa trees in Pabbi forest park Kharian (32°50'01.4"N 73°50'17.7"E), District Gujrat, Pakistan. The disease incidence was recorded approximately 30%. The leaf spots were irregular in shape, brown in colour, 3-9 mm in size and encircled by yellowish halo. In severe condition, the spots coalesced and produced necrotic areas on leaf surface (Figure 1). The samples (n=21) were collected based on symptoms and infected leaf segments were excised into small pieces, surface disinfected with 1% NaClO for 20s and rinsed 3 times with sterilized distilled water. The pieces were plated on Potato Dextrose Agar (PDA) medium and incubated at 28°C for 7 days. All the pure cultures were obtained through single spore method on PDA and preserved in 30% glycerol at -80°C. The colonies were olive green to dark brown with white margin and later turned dark olive or black with enormous sporulation. Conidia (n=24) were obclavate, ovoid, brown in colour and measuring 10.2 to 34.1 µm long x 5.9 to 12.3 µm wide with 1 to 6 transverse and 1 to 3 longitudinal septa (Figure 2). Based on these characteristics, the pathogen was identified as Alternaria alternata (Gilardi, G., et al. 2019). For molecular identification, the Internal Transcribed Spacers (ITS) region, endopolygalacturonase (endoPG) gene and major allergen (Alt a1) gene were amplified using ITS1/4 (White et al. 1990), PG3/PG2b (Andrew et al. 2009) and Alt-4for/Alt-4rev (Lawrence et al. 2013) primers respectively. Based on molecular characteristics, all isolates were identified as A. alternata. The sequences of the representative isolate FLB-1 were submitted in the GenBank with the accession numbers OL514181 for ITS, OK315658 for endoPG and OK315659 for Alt al showing 100% similarity with ITS accession KP124298, and endoPG accession AY205020 and 99.7% with Alt al accession KP123847 sequences of CBS106.24 A. alternata after BLASTn queries. The Phylogenetic reconstruction based on maximum likelihood, using Mega X (Kumar et al. 2018) and FLB-1 grouped with A. alternata (Figure 3). Pathogenicity test was performed on nine months old healthy F. religiosa (L.) seedlings (n=12) to fulfil the Koch's postulates. The leaves were pinpricked and sprayed with FLB-1 conidial suspension (107 spores/ml) by using atomizer (Bajwa et al., 2010). The leaves of F. religiosa (L.) seedlings (n=12) sprayed with sterilized distilled water served as control. All the seedlings were incubated at 25 ± 3°C in the glasshouse. The experiment was performed three times under the same conditions. The typical symptoms appeared on inoculated leaves after 7-14 days that were similar to the symptoms observed on original infected F. religiosa (L.) trees. In the control treatment leaves remained asymptomatic (Figure 4). The pathogen from the artificial infected leaves was re-isolated and identified as A. alternata based on morphological and molecular characteristics. To our knowledge, this is the first report of leaf spot of F. religiosa (L.) caused by A. alternata in Pakistan. The leaves of F. religiosa (L.) are commonly used in Asia for different purposes and this leaf spot disease may represent a significant threat to F. religiosa (L.) tree health.

Inhibitory effect of allyl and benzyl isothiocyanates on ochratoxin a producing fungi in grape and maize.

The purpose of this study was to evaluate the inhibitory effect of allyl-isothiocyanate (AITC) and benzyl-isothiocyanate (BITC) on fungal growth and Ochratoxin A (OTA) production by Aspergillus ochraceus, A. carbonarius and A. niger. Here, we found that spore germination and fungal growth of the three fungi were significantly inhibited when the concentration of AITC and BITC was higher than 1.25 µg/mL. The inhibitory effect of AITC or BITC on A. carbonaceus and A. ochraceus was significantly stronger than that of A. niger. Scanning electron microscopy showed that the mycelia of all three fungi were changed by AITC and BITC. Compared with A. ochraceus and A. carbonarius, the damage to A. niger was lower. For OTA production, AITC and BITC could significantly down-regulated the expression of all five OTA biosynthesis genes in A. niger and A. carbonarius. In A. ochraceus, although several OTA biosynthesis genes were up-regulated, the key PKS gene was down-regulated by AITC and BITC. Twenty-five µg/mL of AITC or BITC could reduce the infection of the three fungi on grapes with inhibition rates of 28%-36% during 14 days and prolong the shelf life of grapes. In maize, the OTA production of the three fungi was significantly reduced by 25 µg/mL of AITC and BITC with the inhibition rates 68.04%-93.49% and 65.87%-75.45%, respectively. These results suggest that AITC and BITC can be used as natural fungicides to prevent A. niger, A. carbonarius and A. ochraceus from infecting grapes and maize and control OTA contamination.

iTRAQ proteome analysis of the antifungal mechanism of citral on mycelial growth and OTA production in Aspergillus ochraceus.

BACKGROUND: Aspergillus ochraceus causes food spoilage and produces mycotoxin ochratoxin A (OTA) during storage of agricultural commodities. In this study, citral was used to inhibit A. ochraceus growth and OTA accumulation, proteomic analysis was employed to verify the mechanism of citral. RESULTS: Citral was found to significantly inhibit fungal growth and mycotoxin production in A. ochraceus. Specifically, 75, 125, 150 and 200 µL L-1 citral suppressed mycelial growth by 33%, 46%, 50% and 100%, respectively. Additionally, 75 µL L-1 citral inhibited OTA accumulation by 25%. Proteomic analysis was performed to elucidate the inhibitory mechanism of citral on mycelial growth and OTA production at subinhibitory concentrations (75 µL L-1 ). Proteomics analysis identified 2646 proteins in A. ochraceus fc-1, of which 218 were differentially expressed between control and 75 µL L-1 citral treatment samples. Differentially expressed proteins were identified by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of biological process, cellular component and molecular function terms. Potential factors affecting mycelial growth and OTA production were analysed, and OTA production was revealed to be a complex process involving many associated factors related to various processes including nutrient intake, sterol biosynthesis, ribosome biogenesis, energy metabolism, oxidative stress and amino acid metabolism. In addition, citral at 75 µL L-1 down-regulated OTA biosynthetic genes including pks and nrps, but slightly up-regulated the global regulatory factors veA, velB and laeA. CONCLUSION: The findings further demonstrate the potential of citral for the preservation of grains and other agricultural products, and provide new insight into its antifungal mechanisms at subinhibitory concentrations. © 2021 Society of Chemical Industry.

Recent developments and applications of hyperspectral imaging for rapid detection of mycotoxins and mycotoxigenic fungi in food products.

Mycotoxins are the foremost naturally occurring contaminants of food products such as corn, peanuts, tree nuts, and wheat. As the secondary metabolites, mycotoxins are mainly synthesized by many species of the genera Aspergillus, Fusarium and Penicillium, and are considered highly toxic and carcinogenic to humans and animals. Most mycotoxins are detected and quantified by analytical chemistry-based methods. While mycotoxigenic fungi are usually identified and quantified by biological methods. However, these methods are time-consuming, laborious, costly, and inconsistent because of the variability of the grain-sampling process. It is desirable to develop rapid, non-destructive and efficient methods that objectively measure and evaluate mycotoxins and mycotoxigenic fungi in food. In recent years, some spectroscopy-based technologies such as hyperspectral imaging (HSI), Raman spectroscopy, and Fourier transform infrared spectroscopy have been extensively investigated for their potential use as tools for the detection, classification, and sorting of mycotoxins and toxigenic fungal contaminants in food. HSI integrates both spatial and spectral information for every pixel in an image, making it suitable for rapid detection of large quantities of samples and more heterogeneous samples and for in-line sorting in the food industry. In order to track the latest research developments in HSI, this paper gives a brief overview of the theories and fundamentals behind the technology and discusses its applications in the field of rapid detection and sorting of mycotoxins and toxigenic fungi in food products. Additionally, advantages and disadvantages of HSI are compared, and its potential use in commercial applications is reported.

Biodetoxification of fungal mycotoxins zearalenone by engineered probiotic bacterium Lactobacillus reuteri with surface-displayed lactonohydrolase.

Zearalenone (ZEN) is one of the common mycotoxins with quite high occurrence rate and is harmful to animal and human health. Lactobacillus reuteri is known as a probiotic bacterium with active immune stimulating and high inhibitory activity against pathogenic microorganisms. In this study, we expressed the lactonohydrolase from Rhinocladiella mackenziei CBS 650.93 (RmZHD) in L. reuteri via secretion and surface-display patterns, respectively. Endogenous signal peptides from L. reuteri were first screened to achieve high expression for efficient ZEN hydrolysis. For secretion expression, signal peptide from collagen-binding protein showed the best performance, while the one from fructose-2,6-bisphosphatase worked best for surface-display expression. Both of the engineered strains could completely hydrolyze 5.0 mg/L ZEN in 8 h without detrimental effects on bacterial growth. The acid and bile tolerance assay and anchoring experiment on Caco-2 cells indicated both of the abovementioned engineered strains could survive during digestion and colonize on intestinal tract, in which the surface-displayed strain had a better performance on ZEN hydrolysis. Biodetoxification of model ZEN-contaminated maize kernels showed the surface-displayed L. reuteri strain could completely hydrolyze 2.5 mg/kg ZEN within 4 h under low water condition. The strain could also efficiently detoxify natural ZEN-contaminated corn flour in the in vitro digestion model system. The colonized property, survival capacity, and the efficient hydrolysis performance as well as probiotic functionality make L. reuteri strain an ideal host for detoxifying residual ZEN in vivo, which shows a great potential for application in feed industry.

The bZIP transcription factor Afap1 mediates the oxidative stress response and aflatoxin biosynthesis in Aspergillus flavus.

Aflatoxin is a carcinogenic secondary metabolite produced mainly by Aspergillus flavus and Aspergillus parasiticus, which can seriously endanger the health of humans and animals. Oxidative stress is a common defense response, and it is known that reactive oxygen species (ROS) can induce the synthesis of a series of secondary metabolites, including aflatoxin. By using mutants lacking the afap 1 gene, the role of afap1 gene in oxidative stress and aflatoxin synthesis was assessed. The growth of the mutant strains was significantly inhibited by the increase in the concentration of H2O2, inhibition was complete at 40mmol/l. However, in the quantitative analysis by HPLC, the concentration of AFB1 increased with the increased H2O2 until 10mmol/l. Following an analysis based on the information provided by the NCBI BLAST analysis, it was assumed that Afap1, a basic leucine zipper (bZIP) transcription factor, was associated with the oxidative stress in this fungus. Treatment with 5mmol/l H2O2 completely inhibited the growth of the mutant strains in afap 1 but did not affect the growth of the CA14PTs strain (non-mutant strain). In addition, the concentration of AFB1 in the mutant strains was approximately » of that observed in the CA14PTs strain. These results suggested that Afap1 plays a key role in the regulation of oxidative stress and aflatoxin production in A. flavus.

A Consensus Ochratoxin A Biosynthetic Pathway: Insights from the Genome Sequence of Aspergillus ochraceus and a Comparative Genomic Analysis.

Ochratoxin A (OTA) is a toxic secondary metabolite produced by Aspergillus and Penicillium species that widely contaminates food and feed. We sequenced and assembled the complete ∼37-Mb genome of Aspergillusochraceus fc-1, a well-known producer of OTA. Key genes of the OTA biosynthetic pathway were identified by comparative genomic analyses with five other sequenced OTA-producing fungi: A. carbonarius, A. niger, A. steynii, A. westerdijkiae, and Penicillium nordicum OTA production was completely inhibited in the deletion mutants (ΔotaA, ΔotaB, ΔotaC, ΔotaD, and ΔotaR1), and OTA biosynthesis was restored by feeding a postblock substrate to the corresponding mutant. The OTA biosynthetic pathway was unblocked in the ΔotaD mutant by the addition of heterologously expressed halogenase. OTA biosynthesis begins with a polyketide synthase (PKS), OtaA, utilizing acetyl coenzyme A (acetyl-CoA) and malonyl-CoA to synthesize 7-methylmellein, which is oxidized to OTß by cytochrome P450 monooxygenase (OtaC). OTß and l-ß-phenylalanine are combined by a nonribosomal peptide synthetase (NRPS), OtaB, to form an amide bond to synthesize OTB. Finally, OTB is chlorinated by a halogenase (OtaD) to OTA. The otaABCD genes were expressed at low levels in the ΔotaR1 mutant. A second regulator, otaR2, which is adjacent to the biosynthetic gene, could modulate only the expression of otaA, otaB, and otaD Thus, we have identified a consensus OTA biosynthetic pathway that can be used to prevent and control OTA synthesis and will help us understand the variation and production of the intermediate components in the biosynthetic pathway.IMPORTANCE Ochratoxin A (OTA) is a significant mycotoxin that contaminates cereal products, coffee, grapes, wine, cheese, and meat. OTA is nephrotoxic, carcinogenic, teratogenic, and immunotoxic. OTA contamination is a serious threat to food safety, endangers human health, and can cause huge economic losses. At present, >20 species of the genera Aspergillus and Penicillium are known to produce OTA. Here we demonstrate that a consensus OTA biosynthetic pathway exists in all OTA-producing fungi and is encoded by a gene cluster containing four highly conserved biosynthetic genes and a bZIP transcription factor.

Characterization and toxicological potential of Alternaria alternata associated with post-harvest fruit rot of Prunus avium in China.

Post-harvest fruit rot caused by Alternaria species is one of the most important threats to the fruit industry. Post-harvest rot on sweet cherry (Prunus avium) fruit was observed in the fruit markets of the Haidian district of Beijing, China. The fungal isolates obtained from the infected sweet cherry fruits matched the descriptions of Alternaria alternata based on the morphology and multi-gene (ITS, endo-PG, and Alta1) sequence analysis. Pathogenicity tests indicated that ACT-3 was the most virulent isolate, exhibiting typical post-harvest fruit rot symptoms. Physiological studies revealed that the optimal conditions for the growth of ACT-3 were temperature of 28°C, water activity of 0.999, and pH of 8 with 87, 85, and 86 mm radial growth of ACT-3 on a potato dextrose agar (PDA) medium, respectively, at 12 days post-inoculation (dpi). Moreover, the fungus showed the highest growth on a Martin agar medium (MAM) modified (85 mm) and a PDA medium (84 mm) at 12 dpi. The proliferation of the fungus was visualized inside the fruit tissues by confocal and scanning electron microscope (SEM), revealing the invasion and destruction of fruit tissues. Alternaria mycotoxins, tenuazonic acid (TeA), and alternariol (AOH) were detected in five representative isolates by HPLC analysis. The highest concentrations of TeA (313 µg/mL) and AOH (8.9 µg/mL) were observed in ACT-6 and ACT-3 isolates, respectively. This study is the first to present a detailed report on the characteristics and proliferation of A. alternata associated with sweet cherry fruit rot and the detection of toxic metabolites.

Fus3 Interacts with Gal83, Revealing the MAPK Crosstalk to Snf1/AMPK to Regulate Secondary Metabolic Substrates in Aspergillus flavus.

Aflatoxins (AFs), highly carcinogenic natural products, are produced by the secondary metabolism of fungi such as Aspergillus flavus. Essential for the fungi to respond to environmental changes and aflatoxin synthesis, the pheromone mitogen-activated protein kinase (MAPK) is a potential regulator of aflatoxin biosynthesis. However, the mechanism by which pheromone MAPK regulates aflatoxin biosynthesis is not clear. Here, we showed Gal83, a new target of Fus3, and identified the pheromone Fus3-MAPK signaling pathway as a regulator of the Snf1/AMPK energy-sensing pathway modulating aflatoxins synthesis substrates. The screening for Fus3 target proteins identified the ß subunit of Snf1/AMPK complexes using tandem affinity purification and multiomics. This subunit physically interacted with Fus3 both in vivo and in vitro and received phosphorylation from Fus3. Although the transcript levels of aflatoxin synthesis genes were not noticeably downregulated in both gal83 and fus3 deletion mutant strains, the levels of aflatoxin B1 and its synthesis substrates and gene expression levels of primary metabolizing enzymes were significantly reduced. This suggests that both the Fus3-MAPK and Snf1/AMPK pathways respond to energy signals. In conclusion, all the evidence unlocks a novel pathway of Fus3-MAPK to regulate AFs synthesis substrates by cross-talking with the Snf1/AMPK complexes.

Analysis of the impact of drying on common wheat quality and safety.

Mycotoxin contamination in grain has been an ongoing concern in the world. Wheat, as a staple crop in China, is particularly notable for its mycotoxin contamination. The main mycotoxins in wheat include deoxynivalenol (DON) and its derivates, zearalenone (ZEN) and aflatoxin B1 (AFB1). After harvest, drying process is an effective technique and a necessary step to ensure the long-term safe storage of wheat. In this study, the moisture content, the concentrations of total fungi and main mycotoxins in post-harvest wheat of three wheat growing areas in the North China Plain were examined, and the effect of different drying methods on wheat quality was evaluated. The results showed that 87.5% of wheat samples were simultaneously contaminated with two or more mycotoxins. Due to the pre-harvest heavy rainfall, the moisture content, the levels of total fungi and mycotoxins in wheat samples of Liaocheng city were significantly higher compared to other regions. Moreover, the effects of different drying methods on the starch gelatinization and viscosity properties of wheat were investigated. The results showed that both natural air drying and dryer drying altered the crystal structure within starch particles and affected the gelatinization and viscosity properties of wheat starch. However, there is no significant difference between the wheat samples treated with two drying methods.

Bacillus amyloliquefaciens A-1 inhibiting fungal spoilage in agricultural products is improved by metabolic engineering of enhancing surfactin yield.

Fungi and subsequent mycotoxins contamination in agricultural products have caused enormous losses and great harm to human and animal health. Biological control has attracted the attention of researchers due to its advantages, including mild conditions, low cost, high efficiency and low nutrient loss. In this study, a newly isolated strain Bacillus amyloliquefaciens A-1 (A-1), was screened for its ability to inhibit the growth and Aflatoxin B1 (AFB1) production of Aspergillus flavus NRRL 3357. Electron microscopy results revealed that mycelium and conidia of A. flavus were destroyed by A-1, affecting hyphae, cell walls, cell membranes and organelles. RNA-seq analysis indicated disturbance in gene expression profiles of A. flavus, including amino acid degradation and starch and sucrose metabolism pathways. Importantly, the biosynthesis of AFB1 was significantly inhibited by the down-regulation of key regulatory genes, aflR and aflS, and the simultaneous down-regulation of most structural genes. Genome analysis predicted six secondary metabolites biosynthetic gene clusters. Then, four surfactin synthesized by cluster C were identified as the main active substance of A-1 using HPLC-Q-TOF-MS. The addition of alanine, threonine, Fe2+ increased surfactin production. Notably, the overexpression of comX also improved surfactin production. The vivo test results indicated that A-1 could significantly inhibit the decay of pear by Aspergillus westerdijkiae, and the mildew of maize and peanuts. Especially, the overexpression of comX in A-1 could enhance the inhibitory activity. In conclusion, the inhibition mechanism of A-1 was revealed, and comX was found can improve the production of surfactin and subsequent activities, which provides the scientific basis for the development of biocontrol agents to reduce spoilage in agricultural products.

Antifungal potential of lipopeptides produced by the Bacillus siamensis Sh420 strain against Fusarium graminearum.

Biological control is a more sustainable and environmentally friendly alternative to chemical fungicides for controlling Fusarium spp. infestations. In this work, Bacillus siamensis Sh420 isolated from wheat rhizosphere showed a high antifungal activity against Fusarium graminearum as a secure substitute for fungicides. Sh420 was identified as B. siamensis using phenotypic evaluation and 16S rDNA gene sequence analysis. An in vitro antagonistic study showed that Sh420's lipopeptide (LP) extract exhibited strong antifungal properties and effectively combated F. graminearum. Meanwhile, lipopeptides have the ability to decrease ergosterol content, which has an impact on the overall structure and stability of the plasma membrane. The PCR-based screening revealed the presence of antifungal LP biosynthetic genes in this strain's genomic DNA. In the crude LP extract of Sh420, we were able to discover several LPs such as bacillomycin, iturins, fengycin, and surfactins using ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry. Microscopic investigations (fluorescent/transmission electron microscopy) revealed deformities and alterations in the morphology of the phytopathogen upon interaction with LPs. Sh420 LPs have been shown in grape tests to be effective against F. graminearum infection and to stimulate antioxidant activity in fruits by avoiding rust and gray lesions. The overall findings of this study highlight the potential of Sh420 lipopeptides as an effective biological control agent against F. graminearum infestations.IMPORTANCEThis study addresses the potential of lipopeptide (LP) extracts obtained from the strain identified as Bacillus siamensis Sh420. This Sh420 isolate acts as a crucial player in providing a sustainable and environmentally friendly alternative to chemical fungicides for suppressing Fusarium graminearum phytopathogen. Moreover, these LPs can reduce ergosterol content in the phytopathogen influencing the overall structure and stability of its plasma membrane. PCR screening provided confirmation regarding the existence of genes responsible for biosynthesizing antifungal LPs in the genomic DNA of Sh420. Several antibiotic lipopeptide compounds were identified from this bacterial crude extract using ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry. Microscopic investigations revealed deformities and alterations in the morphology of F. graminearum upon interaction with LPs. Furthermore, studies on fruit demonstrated the efficacy of Sh420 LPs in mitigating F. graminearum infection and stimulating antioxidant activity in fruits, preventing rust and gray lesions.

A Novel and Label-Free Chemiluminescence Detection of Zearalenone Based on a Truncated Aptamer Conjugated with a G-Quadruplex DNAzyme.

Zearalenone (ZEN), one of the most frequently occurring mycotoxin contaminants in foods and feeds, poses considerable threat to human and animal health, owing to its acute and chronic toxicities. Thus, rapid and accurate detection of ZEN has attracted broad research interest. In this work, a novel and label-free chemiluminescence aptasensor based on a ZEN aptamer and a G-quadruplex DNAzyme was constructed. It was established on a competitive assay between ZEN and an auxiliary DNA for the aptamer, leading to activation of the G-quadruplex/hemin DNAzyme and subsequent signal amplification by chemiluminescence generation after substrate addition. To maximize the detection sensitivity, numerous key parameters including truncated aptamers were optimized with molecular docking analysis. Upon optimization, our aptasensor exhibited a perfect linear relationship (R2 = 0.9996) for ZEN detection in a concentration range of 1-100 ng/mL (3.14-314.10 nM) within 40 min, achieving a detection limit of 2.85 ng/mL (8.95 nM), which was a 6.7-fold improvement over that before optimization. Most importantly, the aptasensor obtained a satisfactory recovery rate of 92.84-137.27% and 84.90-124.24% for ZEN-spiked wheat and maize samples, respectively. Overall, our label-free chemiluminescence aptasensor displayed simplicity, sensitivity, specificity and practicality in real samples, indicating high application prospects in the food supply chain for rapid detection of ZEN.

Metagenome Analysis Identifies Microbial Shifts upon Deoxynivalenol Exposure and Post-Exposure Recovery in the Mouse Gut.

Deoxynivalenol (DON) is one of the most prevalent food-associated mycotoxins, and is known to cause a variety of adverse health effects on human and animals. Upon oral exposure, the intestine is the main target organ of DON. The current study unraveled that DON exposure (2 mg/kg bw/day or 5 mg/kg bw/day) can significantly reshape the gut microbiota in a mouse model. The study characterized the specific gut microbial strains and genes changed after DON exposure and also investigated the recovery of the microbiota upon either 2 weeks daily prebiotic inulin administration or 2 weeks recovery without intervention after termination of DON exposure (spontaneous recovery). The results obtained reveal that DON exposure causes a shift in gut microorganisms, increasing the relative abundance of Akkermansia muciniphila, Bacteroides vulgatus, Hungatella hathewayi, and Lachnospiraceae bacterium 28-4, while the relative abundance of Mucispirillum schaedleri, Pseudoflavonifractor sp. An85, Faecalibacterium prausnitzii, Firmicutes bacterium ASF500, Flavonifractor plautii, Oscillibacter sp. 1-3, and uncultured Flavonifractor sp. decreased. Notably, DON exposure enhanced the prevalence of A. muciniphila, a species considered as a potential prebiotic in previous studies. Most of the gut microbiome altered by DON in the low- and high-dose exposure groups recovered after 2 weeks of spontaneous recovery. Inulin administration appeared to promote the recovery of the gut microbiome and functional genes after low-dose DON exposure, but not after high-dose exposure, at which changes were exacerbated by inulin-supplemented recovery. The results obtained help to better understand the effect of DON on the gut microbiome, and the gut microbiota's recovery upon termination of DON exposure.

Combined exposure to deoxynivalenol facilitates lipid metabolism disorder in high-fat-diet-induced obesity mice.

Deoxynivalenol (DON) is a trichothecene toxin that mainly produced by strains of Fusarium spp. DON contamination is widely distributed and is a global food safety threat. Existing studies have expounded its harmful effects on growth inhibition, endocrine disruption, immune function impairment, and reproductive toxicity. In energy metabolism, DON suppresses appetite, reduces body weight, triggers lipid oxidation, and negatively affects cholesterol and fatty acid homeostasis. In this study, high-fat diet (HFD) induced obese C57BL/6J mice were orally treated with 0.1 mg/kg bw/d and 1.0 mg/kg bw/d DON for 4 weeks. The lipid metabolism of mice and the molecular mechanisms were explored. The data showed that although DON reduced body weight and fat mass in HFD mice, it significantly increased their serum triglyceride concentrations, disturbance of serum lipid metabolites, impaired glucose, and resulted in insulin intolerance in mice. In addition, the transcriptional and expression changes of lipid metabolism genes in the liver and epididymis (EP) adipose indicate that the DON-mediated increase in serum triglycerides is caused by lipoprotein lipase (LPL) inhibition in EP adipose. Furthermore, DON down-regulates the expression of LPL through the PPARγ signaling pathway in EP adipose. These results are further confirmed by the serum lipidomics analysis. In conclusion, DON acts on the PPARγ pathway of white adipose to inhibit the expression of LPL, mediate the increase of serum triglyceride in obese mice, disturb the homeostasis of lipid metabolism, and increase the risk of cardiovascular disease. This study reveals the interference mechanism of DON on lipid metabolism in obese mice and provides a theoretical basis for its toxic effect in obese individuals.

Accurate and non-destructive monitoring of mold contamination in foodstuffs based on whole-cell biosensor array coupling with machine-learning prediction models.

Mold contamination in foodstuffs causes huge economic losses, quality deterioration and mycotoxin production. Thus, non-destructive and accurate monitoring of mold occurrence in foodstuffs is highly required. We proposed a novel whole-cell biosensor array to monitor pre-mold events in foodstuffs. Firstly, 3 volatile markers ethyl propionate, 1-methyl-1 H-pyrrole and 2,3-butanediol were identified from pre-mold peanuts using gas chromatography-mass spectrometry. Together with other 3 frequently-reported volatiles from Aspergillus flavus infection, the volatiles at subinhibitory concentrations induced significant but differential response patterns from 14 stress-responsive Escherichia coli promoters. Subsequently, a whole-cell biosensor array based on the 14 promoters was constructed after whole-cell immobilization in calcium alginate. To discriminate the response patterns of the whole-cell biosensor array to mold-contaminated foodstuffs, optimal classifiers were determined by comparing 6 machine-learning algorithms. 100 % accuracy was achieved to discriminate healthy from moldy peanuts and maize, and 95 % and 98 % accuracy in discriminating pre-mold stages for infected peanuts and maize, based on random forest classifiers. 83 % accuracy was obtained to separate moldy peanuts from moldy maize by sparse partial least square determination analysis. The results demonstrated high accuracy and practicality of our method based on a whole-cell biosensor array coupling with machine-learning classifiers for mold monitoring in foodstuffs.

Smartphone-based chemiluminescence detection of aflatoxin B1 via labelled and label-free dual sensing systems.

To develop a sensing platform for onsite determination of AFB1 in foodstuffs, we developed smartphone-based chemiluminescence detection of AFB1 via labelled and label-free dual modes. The labelled mode was characteristic of double streptavidin-biotin mediated signal amplification, obtaining limit of detection (LOD) of 0.04 ng/mL in the linear range of 1-100 ng/mL. To reduce the complexity in the labelled system, a label-free mode based on both split aptamer and split DNAzyme was fabricated. A satisfactory LOD of 0.33 ng/mL was generated in the linear range of 1-100 ng/mL. Both labelled and label-free sensing systems achieved outstanding recovery rate in AFB1-spiked maize and peanut kernel samples. Finally, two systems were successfully integrated into smartphone-based portable device based on custom-made components and android application, achieving comparable AFB1 detection ability to a commercial microplate reader. Our systems hold huge potential for AFB1 onsite detection in food supply chain.

Biocontrol potential of lipopeptides produced by the novel Bacillus subtilis strain Y17B against postharvest Alternaria fruit rot of cherry.

The use of synthetic fungicides against postharvest Alternaria rot adversely affects human health and the environment. In this study, as a safe alternative to fungicides, Bacillus subtilis strain Y17B isolated from soil exhibited significant antifungal activity against Alternaria alternata. Y17B was identified as B. subtilis based on phenotypic identification and 16S rRNA sequence analysis. To reveal the antimicrobial activity of this strain, a PCR-based study detected the presence of antifungal lipopeptide (LP) biosynthetic genes from genomic DNA. UPLC Q TOF mass spectrometry analysis detected the LPs surfactin (m/z 994.64, 1022.68, and 1026.62), iturin (m/z 1043.56), and fengycin (m/z 1491.85) in the extracted LP crude of B. subtilis Y17B. In vitro antagonistic study demonstrated the efficiency of LPs in inhibiting A. alternata growth. Microscopy (SEM and TEM) studies showed the alteration of the morphology of A. alternata in the interaction with LPs. In vivo test results revealed the efficiency of LPs in reducing the growth of the A. alternata pathogen. The overall results highlight the biocontrol potential of LPs produced by B. subtilis Y17B as an effective biological control agent against A. alternata fruit rot of cherry.

SakA Regulates Morphological Development, Ochratoxin A Biosynthesis and Pathogenicity of Aspergillus westerdijkiae and the Response to Different Environmental Stresses.

Ochratoxin A (OTA), as a common mycotoxin, has seriously harmful effects on agricultural products, livestock and humans. There are reports on the regulation of SakA in the MAPK pathway, which regulates the production of mycotoxins. However, the role of SakA in the regulation of Aspergillus westerdijkiae and OTA production is not clear. In this study, a SakA deletion mutant (ΔAwSakA) was constructed. The effects of different concentrations of D-sorbitol, NaCl, Congo red and H2O2 on the mycelia growth, conidia production and biosynthesis of OTA were investigated in A. westerdijkiae WT and ΔAwSakA. The results showed that 100 g/L NaCl and 3.6 M D-sorbitol significantly inhibited mycelium growth and that a concentration of 0.1% Congo red was sufficient to inhibit the mycelium growth. A reduction in mycelium development was observed in ΔAwSakA, especially in high concentrations of osmotic stress. A lack of AwSakA dramatically reduced OTA production by downregulating the expression of the biosynthetic genes otaA, otaY, otaB and otaD. However, otaC and the transcription factor otaR1 were slightly upregulated by 80 g/L NaCl and 2.4 M D-sorbitol, whereas they were downregulated by 0.1% Congo red and 2 mM H2O2. Furthermore, ΔAwSakA showed degenerative infection ability toward pears and grapes. These results suggest that AwSakA is involved in the regulation of fungal growth, OTA biosynthesis and the pathogenicity of A. westerdijkiae and could be influenced by specific environmental stresses.