Aspergillus flavus is a major foodborne fungus capable of producing aflatoxins that threaten food quality and safety. Nitrogen metabolism is closely associated with fungal development and secondary metabolism; however, the roles of nitrate-responsive regulators in aflatoxin biosynthesis remain unclear. In this study, the biological functions of AfnirA and AfcrnA, two regulators involved in nitrate utilization, were investigated in A. flavus through gene deletion and overexpression analyses. Transcriptional profiling showed that both genes responded to changes in nitrogen sources and were induced under nitrogen imbalance conditions. Functional analyses demonstrated that AfnirA and AfcrnA differentially affected fungal growth, conidiation, conidial germination, and sclerotial development. Deletion of AfnirA abolished sclerotia formation, whereas disruption of AfcrnA significantly increased sclerotia production. In addition, the ΔAfcrnA mutant exhibited enhanced sensitivity to osmotic, oxidative, and cell wall stresses, accompanied by increased intracellular reactive oxygen species accumulation. Notably, deletion of AfcrnA markedly promoted aflatoxin B1 production, with an approximately 2.3-fold, and upregulated the expression of aflatoxin biosynthetic genes, including aflC, aflD, and aflP. Subcellular localization analysis revealed nuclear localization of AfnirA, whereas AfcrnA displayed vacuolar-associated cytoplasmic localization. These findings indicate that AfnirA and AfcrnA play distinct roles in regulating fungal development, oxidative stress adaptation, and aflatoxin biosynthesis in A. flavus.

Fusarium root rot, caused by Fusarium solani, is one of the major postharvest diseases of sweet potato. Cinnamaldehyde (CA), a natural product from cinnamon bark, has demonstrated promising antifungal efficacy; however, its mechanism of action, especially regarding fungal sugar metabolism, remains poorly understood. CA above 0.375 g L-1 inhibited F. solani mycelial growth, induced glucose deprivation, and triggered the accumulation of reserve carbohydrates (trehalose and glycogen). Moreover, CA promoted the accumulation of reactive oxygen species and hydrogen peroxide (H2O2), protein carbonylation, and DNA fragmentation, indicating that CA caused oxidative damage and led to apoptotic cell death in F. solani. Notably, CA suppressed the function of the metabolic regulator sucrose non-fermenting protein kinase 1 (Snf1) by disrupting its transcription and translation. Genetic knockout of Snf1 increased the inhibition rate of mycelial growth, amplified intracellular glucose scarcity, promoted the accumulation of reserve carbohydrates, accelerated H2O2 accumulation and protein oxidative damage, and ultimately exacerbated the extent of cell apoptosis. Complementarily, activation of Snf1 attenuated the antifungal effect of CA. Moreover, CA reduced rot incidence and better preserved nutritional quality (soluble sugars and starch), particularly in sweet potato roots infected with Snf1-deletion mutants. These findings reveal that CA exerts its antifungal effect by disrupting Snf1-mediated glucose supply and inducing oxidative stress in F. solani to control sweet potato postharvest decay. This study provides novel insights into the antifungal mechanism of plant essential oils and supports the potential application of CA as an eco-friendly preservative for controlling postharvest spoilage in carbohydrate-rich fresh produce.

Aspergillus flavus is a predominant fungal contaminant, producing aflatoxin B1 (AFB1), one of the most potent mycotoxins posing serious risks to global food safety. In this study, we evaluated the antifungal efficacy of Bacillus velezensis ATC-AL and its active antifungal substances (AAS) against A. flavus and other mycotoxinproducing fungi. The AAS treatment displayed broad-spectrum antifungal activity, completely suppressing AFB1 synthesis at a minimum inhibitory concentration of 8 mu L/mL. It also significantly reduced deoxynivalenol (DON) accumulation by Fusarium graminearum, indicating multi-target antitoxin potential. Mechanistic analyses revealed that AAS disrupts fungal membrane integrity and induces oxidative stress, as evidenced by elevated reactive oxygen species (ROS) levels, lipid peroxidation, and structural damage to the cell membrane and cell wall inA. flavus. Metabolomic profiling further revealed perturbations in the TCA cycle, glutathione metabolism, and nucleic acid biosynthesis, consistent with mitochondrial dysfunction and impaired energy metabolism. Correspondingly, AAS treatment caused a collapse in mitochondrial membrane potential and decreased ATP levels, suggesting apoptosis-like cell death. In grain kernel assays, AAS significantly inhibited fungal colonization and eliminated mycotoxin production, effectively protecting grains from infection by A. flavus and F. graminearum. Collectively, these findings demonstrate that B.velezensis ATC-AL functions as a promising biocontrol agent acting through multiple antifungal and anti-mycotoxin pathways, offering a sustainable strategy for mitigating fungal contamination and mycotoxin risk in grain storage systems.

Black rot, caused by Ceratocystis fimbriata, is a destructive disease of sweetpotato, resulting in significant global losses. This study aimed to identify an effective biocontrol strain and elucidate its antifungal mechanisms against C. fimbriata. Here, Bacillus velezensis ATC-AL was isolated and identified, and its active antifungal substances (AAS) were extracted from the culture supernatant. AAS exhibited strong inhibitory effects on the growth and spore germination of C. fimbriata. In addition, genome analysis predicted nine secondary metabolites biosynthetic gene clusters, and LC-MS/MS analysis further confirmed the presence of several major antifungal compounds. Mechanistic investigations revealed that AAS disrupted cell wall and membrane integrity, induced oxidative stress, and triggered mitochondrial dysfunction and apoptosis in C. fimbriata. Collectively, these findings suggest that B. velezensis ATC-AL exhibits multi-target antifungal activity and represents a promising natural biocontrol agent for the management of postharvest fungal diseases in sweetpotato.

Sweetpotato black rot, caused by Ceratocystis fimbriata, is a major postharvest disease that leads to substantial storage losses worldwide. In this study, a salt-tolerant rhizobacterial strain, Bacillus albus SSR3, was isolated from the rhizosphere of sweetpotato grown in saline-alkali soil, with broad-spectrum antagonistic activity against postharvest fungal pathogens. LC-MS/MS analysis revealed diverse bioactive metabolites associated with its antifungal activity. Integrated transcriptomic and metabolomic analyses showed that SSR3 bioactive metabolites extensively reprogrammed fungal metabolism, particularly pathways involved in carbohydrate and amino acid metabolism, antioxidant defense, and energy production. These alterations were accompanied by disruption of cell wall and membrane integrity, excessive reactive oxygen species accumulation, and mitochondrial dysfunction, ultimately inhibiting fungal growth. Here, we also found that SSR3 bioactive metabolites effectively inhibited aflatoxin B1 production by Aspergillus flavus and deoxynivalenol accumulation in Fusarium graminearum. In vivo assays further demonstrated that SSR3 bioactive metabolites significantly reduced sweetpotato black rot severity and effectively limited fungal colonization and mycotoxin contamination in stored agricultural commodities. Collectively, our findings demonstrate that B. albus SSR3 suppresses postharvest fungal pathogens through coordinated metabolic reprogramming, oxidative stress induction, and cellular integrity disruption, highlighting its potential as a sustainable biocontrol agent for postharvest disease management.

The genomic signatures of microbial domestication remain poorly understood within the context of natural population variation. Here, we demonstrate that Aspergillus oryzae, the filamentous fungus used in soy sauce production, shares more recent ancestry with a predominantly northern, largely non-aflatoxigenic population of Aspergillus flavus (population C). Strikingly, A. oryzae isolates also overlap with the recently described, clinically enriched A. flavus population D, suggesting the possibility of multiple domestication events. Although A. oryzae exhibits reduced virulence compared to A. flavus, all isolates tested retained pathogenicity in a zebrafish infection model. At the transcriptomic level, Aspergillus populations are significantly differentiated, with distinct responses to population density, indicating that population-specific transcriptomes adapt differently to ecological conditions. These differences extend beyond gene content and are not always explained by phylogenetic relationships, suggesting that phenotypic diversification occurs through the rapid reorganization of transcriptomic architectures. For example, A. oryzae displays significantly elevated expression of a module enriched for carbohydrate metabolism. Population-specific variation is also evident among secondary metabolite (SM) gene clusters. While A. oryzae shows markedly reduced expression of specific SM genes, particularly those involved in aflatoxin biosynthesis, this trend does not extend across the entire secondary metabolome. Using machine-learning-based gene regulatory network inference, we identified population-specific transcriptomic differences linked to distinct transcription factors, with evidence for both cis- and trans-acting regulatory divergence, but no changes in global regulators such as laeA. Together, these findings provide new insights into the domestication of A. oryzae, its global significance, and the microevolution of fungal secondary metabolic pathways.

Sweetpotato, a vital global crop, cash, and fodder crop, faces significant threats from black rot disease caused by Ceratocystis fimbriata (C. fimbriata). Benzyl isothiocyanate (BITC) is a biologically active essential oil derived from cruciferous plants, widely used for food preservation and exhibiting antimicrobial and antitumor properties. In this study, BITC significantly inhibited C. fimbriata growth and induced cellular damage. Furthermore, through proteomics and bioinformatics analyses, mitochondrial peroxiredoxin PRX1 (PRX1) was identified as a potential target protein of BITC in C. fimbriata, confirming its role in regulating the response of BITC-treated C. fimbriata to oxidative damage. Finally, sweetpotato storage simulation experiments demonstrated that BITC effectively prevents and controls the growth of C. fimbriata during sweetpotato storage. These results provide the basis for using BITC to control sweetpotato black rot, offering insights into developing highly selective and low-negative-impact antisweetpotato black rot disease compounds, and presenting a new strategy for controlling C. fimbriata contamination in sweetpotatoes.

Corn and peanuts are prone to contamination by Aspergillus flavus and subsequent aflatoxin production during storage. Autophagy, a conserved degradation pathway, is essential for normal cell growth and development. In this study, we used homologous recombination to construct Afatg3 gene deletion (zAfatg3) and complementary (zAfatg3:Afatg3) strains to investigate the role of the autophagy-related gene Afatg3 in A. flavus growth, development, and aflatoxin biosynthesis. Quantitative Real-time polymerase chain reaction revealed significant downregulation of autophagy-related genes in the zAfatg3 strain, while flow cytometry indicated disrupted lysosome content, confirming impaired autophagy. Compared to wild-type (WT) and zAfatg3:Afatg3 strains, zAfatg3 exhibited significantly reduced colony diameters and sparse, atrophied hyphae and conidia on all media except low-glucose media. Delayed germination and reduced sporulation in zAfatg3 were linked to the downregulation of abaA (abacus A), brlA (bristle A), and wetA (wettable A) transcription factors. Infection experiments showed that the autophagy-deficient zAfatg3 strain had reduced spore production and parasitism. Aflatoxin production was reduced to 20 % of levels observed in WT and zAfatg3:Afatg3 strains, primarily due to a significant decreased in the expression of aflD (aflatoxin biosynthesis gene D) and aflR (aflatoxin biosynthesis gene R) genes within the aflatoxin synthesis gene cluster. Additionally, zAfatg3 showed increased sensitivity to various environmental and pharmacological stresses. These findings highlight Afatg3's crucial role in regulating sporulation, aflatoxin biosynthesis, and drugs stress response, suggesting its potential as a target to mitigate fungal pathogenicity and enhance food safety.

The growing demand for sustainable food additives has spurred interest in pectin extracted from agricultural byproducts. This study investigated the structural, gelling, and antimicrobial properties of pectin extracted from sweetpotato residue (SPP) using a green hydrothermal method. Optimal extraction conditions (122.4 °C, 19.1 min, liquid-solid ratio of 20.8 mL/g) yielded a maximum pectin extraction rate of 3.05 %. The extracted SPP was characterized as a highly methoxylated pectin with a main peak molecular weight of 57.87 ± 4.6 kDa. Structural analysis revealed abundant galacturonic acid and rhamnogalacturonic acid I domains in both SPP and alkali-modified sweetpotato pectin (MSPP), as confirmed by FT-IR and 1H NMR. SPP and MSPP exhibited smaller particle sizes and higher negative charges compared to citrus pectin (CP) and modified citrus pectin (MCP). Modified pectin exhibited reduced antioxidant activity versus CP. MSPP formed Ca2+-induced gels across pH 4-11.5 without sugar, showing superior water retention and stable networks. Additionally, 3 % SPP effectively inhibited the growth of E. coli and S. aureus. As a coating, SPP extended beef shelf life by 3 days by maintaining color stability and suppressing microbial growth. These findings position sweetpotato residue pectin as a promising, Eco-preservative, with tailored functionality achievable through structural modification.

Aspergillus flavus, one of the most widely distributed and abundant genus of Aspergillus worldwide, poses an evident threat as a source of food contamination in grains and cereals. Perillaldehyde (PAE), a volatile essential oil extracted from the stem and leaves of Perilla frutescens L., exhibits potent antifungal activity. In our study, we investigated the role of Cox10, a key enzyme in the heme A synthesis pathway crucial for maintaining mitochondrial function. We found that cox10 is a sensitive gene of A. flavus in response to PAE by gene expression assay and GFP fluorescent localization tagging, and then we found that the deletion of this gene affects the growth and development of A. flavus, but the drug resistance is elevated. Through transcriptome sequencing and its experimental validation, the molecular mechanisms of stress triggered by the deletion of cox10 were further clarified, such as the decrease in intracellular drug content due to the increase in the expression of drug efflux proteins, and the increase in the thickness of cell wall due to the increase in the content of cell wall chitin. Clearly, cox10 plays a critical role in regulating various cellular processes of A. flavus, including growth, reproduction, development, as well as pathogenicity and drug resistance. These significant findings establish a solid theoretical foundation for the development of environmentally friendly, safe, and effective antifungal agents to combat A. flavus contamination.

Autophagy, a conserved cellular recycling process, plays a crucial role in maintaining homeostasis under stress conditions. It also regulates the development and virulence of numerous filamentous fungi. In this study, we investigated the specific function of ATG8, a reliable autophagic marker, in the opportunistic pathogen Aspergillus flavus. To investigate the role of atg8 in A. flavus, the deletion and complemented mutants of atg8 were generated according to the homologous recombination principle. Deletion of atg8 showed a significant decrease in conidiation, spore germination, and sclerotia formation compared to the WT and atg8C strains. Additionally, aflatoxin production was found severely impaired in the ∆atg8 mutant. The stress assays demonstrated that ATG8 was important for A. flavus response to oxidative stress. The fluorescence microscopy showed increased levels of reactive oxygen species in the ∆atg8 mutant cells, and the transcriptional result also indicated that genes related to the antioxidant system were significantly reduced in the ∆atg8 mutant. We further found that ATG8 participated in regulating the pathogenicity of A. flavus on crop seeds. These results revealed the biological role of ATG8 in A. flavus, which might provide a potential target for the control of A. flavus and AFB1 biosynthesis.

Abstract Aflatoxin, mainly produced by Aspergillus flavus, is one of the most notorious mycotoxin for its toxicity and carcinogenicity. Despite extensive efforts, effective strategies to control A. flavus and AFB1 contamination remain elusive. Here, we investigate the potential of p‐anisaldehyde (AS), an aldehyde derived from plant essential oils, as a natural antifungal agent against A. flavus and its ability to modulate AFB1 biosynthesis. We found that AS exhibited broad‐spectrum antifungal activities against Aspergillus spp. and effectively inhibited A. flavus asexual development, AFB1 production, and pathogenicity. AS treatment disrupted the cell surface structure and membrane integrity, as observed by scanning electron microscopy and PI staining. RNA‐sequencing analysis revealed that AS significantly altered the expression of genes involved in redox homeostasis, plasma membrane function, and cell cycle progression. Further investigation demonstrated that AS induced a reduction in mitochondrial membrane potential (Δψm) and accumulation of reactive oxygen species (ROS), leading to cell cycle arrest at the G2/M phase. The AS‐induced ROS accumulation was found to be mitigated by the superoxide dismutase‐mediated antioxidant system, which is regulated by transcriptional factor Ap1. Notably, the Ap1‐regulatory ROS detoxification system was also found to be involved in A. flavus pathogenicity and AFB1 production. Overall, these findings provide valuable insights into the inhibitory mechanism of AS against A. flavus, paving the way for its potential application as a natural strategy to mitigate AFB1 contamination in both food and agriculture crops.

Nitric oxide (NO) is a signaling molecule with diverse roles in various organisms. However, its role in the opportunistic pathogen Aspergillus flavus remains unclear. This study investigates the potential of NO, mediated by metabolites from A. oryzae (AO), as an antifungal strategy against A. flavus. We demonstrated that AO metabolites effectively suppressed A. flavus asexual development, a critical stage in its lifecycle. Transcriptomic analysis revealed that AO metabolites induced NO synthesis genes, leading to increased intracellular NO levels. Reducing intracellular NO content rescued A. flavus spores from germination inhibition caused by AO metabolites. Furthermore, exogenous NO treatment and dysfunction of flavohemoglobin Fhb1, a key NO detoxification enzyme, significantly impaired A. flavus asexual development. RNA-sequencing and metabolomic analyses revealed significant metabolic disruptions within tricarboxylic acid (TCA) cycle upon AO treatment. NO treatment significantly reduced mitochondrial membrane potential (Δψm) and ATP generation. Additionally, aberrant metabolic flux within the TCA cycle was observed upon NO treatment. Further analysis revealed that NO induced S-nitrosylation of five key TCA cycle enzymes. Genetic analysis demonstrated that the S-nitrosylated Aconitase Acon and one subunit of succinate dehydrogenase Sdh2 played crucial roles in A. flavus development by regulating ATP production. This study highlights the potential of NO as a novel antifungal strategy to control A. flavus by compromising its mitochondrial function and energy metabolism.

Contamination of crop seeds and feed with Aspergillus flavus and its associated aflatoxins presents a significant threat to human and animal health due to their hepatotoxic and carcinogenic properties. To address this challenge, researchers have screened for potential biological control agents in peanut soil and pods. This study identified a promising candidate, a strain of the nonpigmented bacterium, Achromobacter xylosoxidans ZJS2-1, isolated from the peanut rhizosphere in Zhejiang Province, China, exhibiting notable antifungal and antiaflatoxin activities. Further investigations demonstrated that ZJS2-1 active substances (ZAS) effectively inhibited growth at a MIC of 60 μL/mL and nearly suppressed AFB1 production by 99%. Metabolomic analysis revealed that ZAS significantly affected metabolites involved in cell wall and membrane biosynthesis, leading to compromised cellular integrity and induced apoptosis in A. flavus through the release of cytochrome c. Notably, ZAS targeted SrbA, a key transcription factor involved in ergosterol biosynthesis and cell membrane integrity, highlighting its crucial role in ZJS2-1's biocontrol mechanism. Moreover, infection of crop seeds and plant wilt caused by A. flavus can be efficiently alleviated by ZAS. Additionally, ZJS2-1 and ZAS demonstrated significant inhibitory effects on various Aspergillus species, with inhibition rates ranging from 80 to 99%. These findings highlight the potential of ZJS2-1 as a biocontrol agent against Aspergillus species, offering a promising solution to enhance food safety and protect human health.

Abstract Striatin‐interacting phosphatases and kinases (STRIPAKs) are evolutionarily conserved supramolecular complexes that control various important cellular processes such as signal transduction and development. However, the role of the STRIPAK complex in pathogenic fungi remains elusive. In this study, the components and function of the STRIPAK complex were investigated in Fusarium graminearum, an important plant‐pathogenic fungus. The results obtained from bioinformatic analyses and the protein–protein interactome suggested that the fungal STRIPAK complex consisted of six proteins: Ham2, Ham3, Ham4, PP2Aa, Ppg1, and Mob3. Deletion mutations of individual components of the STRIPAK complex were created, and observed to cause a significant reduction in fungal vegetative growth and sexual development, and dramatically attenuae virulence, excluding the essential gene PP2Aa. Further results revealed that the STRIPAK complex interacted with the mitogen‐activated protein kinase Mgv1, a key component in the cell wall integrity pathway, subsequently regulating the phosphorylation level and nuclear accumulation of Mgv1 to control the fungal stress response and virulence. Our results also suggested that the STRIPAK complex was interconnected with the target of rapamycin pathway through Tap42‐PP2A cascade. Taken together, our findings revealed that the STRIPAK complex orchestrates cell wall integrity signalling to govern the fungal development and virulence of F. graminearum and highlighted the importance of the STRIPAK complex in fungal virulence.

Ceratocystis fimbriata Ellis & Halsted is a ubiquitous and devastating phytopathogen causing significant losses in harvested sweet potatoes. Furanoterpenoid toxins elicited by the C. fimbriata can cause hepatotoxicity, pneumonia and lung edema, and seriously threaten human health. Therefore, controlling C. fimbriata and limiting the storage losses of sweet potatoes are of critical importance to the economic value of sweet potatoes and human health. In this review, the mechanisms of phytopathogen infection and dispersal are summarized. Furanoterpenoid toxins incidence in the tuberous root and their toxicology are reviewed. Furthermore, the control methods of C. fimbriata and postharvest sweet potato spoilage were emphasized. Equipped with these understandings, this review aims to provide an important theoretical basis for the efficient prevention and control of C. fimbriata and the maintaining of the storage quality of sweet potatoes. Additionally, the key problems in sweet potato postharvest storage and areas of research that warrant further studies are analyzed and identified. Per our knowledge, the current manuscript is the first review to provide integrated and up-to-date information for scientists working with C. fimbriata.

RGS proteins, as crucial regulators of the G protein signaling pathway, are widely distributed in fungi, while little is known about their roles in Aspergillus flavus development and aflatoxin. In this study, we identified six RGS proteins in A. flavus and revealed that these proteins have important functions in the regulation of conidia, sclerotia, and aflatoxin formation.

Root rot caused by Fusarium solani is one of the major postharvest diseases limiting sweet potato production. Here, antifungal activity and the action mode of perillaldehyde (PAE) against F. solani were investigated. A PAE concentration of 0.15 mL/L in air (mL/L air) markedly inhibited the mycelial growth, spore reproduction and spore viability of F. solani. A PAE vapor of 0.25 mL/L in air could control the F. solani development in sweet potatoes during storage for 9 days at 28 °C. Moreover, the results of a flow cytometer demonstrated that PAE drove an increase in cell membrane permeability, reduction of mitochondrial membrane potential (MMP) and accumulation of reactive oxygen species (ROS) in F. solani spores. Subsequently, a fluorescence microscopy assay demonstrated that PAE caused serious damage to the cell nuclei in F. solani by inducing chromatin condensation. Further, the spread plate method showed that the spore survival rate was negatively correlated with the level of ROS and nuclear damage, of which the results indicated that PAE-driven ROS accumulation plays a critical role in contributing to cell death in F. solani. In all, the results revealed a specific antifungal mechanism of PAE against F. solani, and suggest that PAE could be a useful fumigant for controlling the postharvest diseases of sweet potatoes.

Root rot caused by Fusarium solani is one of major postharvest diseases limiting sweet potato production. Antifungal effect and possible mode of action of cinnamaldehyde (CA) against F. solani were investigated. CA concentration of 0.075 g/L inhibited conidial viability of F. solani. CA vapor of 0.3 g/L in air completely controlled the F. solani development in sweet potatoes during storage for 10 days at 28 degrees C, and protected soluble sugar and starch in the flesh from depletion by the fungus. Further results demonstrated that CA induced reduction in mitochondrial membrane potential (Delta psi m), ROS accumulation, and cell apoptosis characterized by DNA fragmentation in F. solani. Moreover, CA facilitated decomposition of mitochondria-specific cardiolipin (CL) into its catabolites by the catalytic action of phospholipases. Altogether, the results revealed a specific antifungal mechanism of CA against F. solani, and suggest that CA holds promise as a preservative for postharvest pres-ervation of sweet potato.