Interactive effects of temperature and water activity on growth, mycotoxin production, and transcriptomic profiles of Aspergillus flavus, Aspergillus parasiticus, and Fusarium verticillioides.
In vitro stress responses of Aspergillus flavus, Aspergillus parasiticus, and Fusarium verticillioides are investigated across broad gradients of temperature and water activity to provide a theoretical basis for developing targeted mycotoxin mitigation strategies under environmental factors change.
Abstract
Maize is frequently contaminated with aflatoxins (AFs) and fumonisins (FBs), posing a significant global food safety threat. Understanding how environmental factors regulate fungal toxigenesis under global warming is crucial. This study systematically investigated the in vitro stress responses of Aspergillus flavus, Aspergillus parasiticus, and Fusarium verticillioides (strains isolated from contaminated maize) across broad gradients of temperature (15-40 °C) and water activity (0.93-0.995 aw). Phenotypically, primary and secondary metabolism exhibited striking divergence: A. flavus grew optimally at 30 °C and 0.98 aw, yet intriguingly accumulated peak AFB2 specifically under severe drought stress (0.93 aw). A. parasiticus showed broad thermal adaptability for growth, but its AFB1 production was strictly confined to 25 °C. F. verticillioides maximized FB1 at 20 °C and 0.98 aw,. Transcriptomics revealed novel regularity underlying these patterns: under drought stress, A. flavus AFB2 biosynthesis uniquely decoupled from the classical aflM-dependent pathway via metabolic flux redirection; lepI was identified as an unprecedentedly strict temperature-dependent switch for A. parasiticus AFB1; and a FUM13-centric hub network coordinated FB1 synthesis in F. verticillioides. These findings provide a theoretical basis for developing targeted mycotoxin mitigation strategies under environmental factors change.
Verticillium dahliae is a destructive soil-borne fungus with a broad host range, and its persistence in soil complicates control. Current measures, mainly resistant cultivars and chemicals, are limited and environmentally risky, promoting biocontrol as a green alternative. Here, we investigated the biocontrol mechanisms of Bacillus velezensis L33a against V. dahliae JR2 in tomato. In vitro assays on PDA plates at 26°C for 9 d showed that L33a inhibited JR2 by 58.6%, caused hyphal malformation and disruption, and its volatile organic compounds suppressed pathogen growth. In pot experiments, tomato roots dipped in JR2 suspension (1 ×10⁶ CFU/mL) for 30 min at 7 d after transplanting and grown for 21 d achieved 60.9% control efficacy. Physiological assays indicated reduced peroxidase and catalase activities, while qPCR revealed that L33a alone upregulated JA signaling (SlJAZ1, SlMYC2, SlPI II) and antioxidant (SlCAT, SlAPX) genes, with further enhancement upon JR2 co-treatment. To track their interactions, we generated GFP-labeled JR2 and RFP-labeled L33a; dual fluorescence labeling showed that L33a endophytically colonized Arabidopsis thaliana roots and competed with JR2 for the same niche, correlating with reduced pathogen colonization. Integrated metabolomic and transcriptomic analysis further revealed that L33a treatment altered pathways related to ABC transporters, amino acid metabolism, cell wall integrity, and energy metabolism in JR2, with tyrosine metabolism significantly enriched at both levels. Collectively, these findings suggest that L33a is a promising biocontrol strain for green management of tomato Verticillium wilt.
With increased mycotoxin contamination, the search for eco-friendly biocontrol strategies has become increasingly important. This study aimed to evaluate the efficiency of stilbenoid-enriched Merlot (Me) and Tannat (Ta) vine root extracts, and their major active molecule, vitisin B (VIT), against Aspergillus carbonarius growth and its ochratoxin A (OTA) production using in vitro liquid and solid, and ex vivo grape-berry assays. In liquid assay, Me and Ta (125 mg/L) and VIT (36 mg/L) significantly inhibited extracellular OTA. Additionally, VIT temporarily reduced both fungal growth and total OTA. Transcriptomics showed that VIT and Me slightly induced OTA biosynthesis genes. In solid and ex vivo assays, doses above 1 g/L decreased OTA production by up to 48% without impacting fungal growth. This lack of correlation suggested a distinct regulatory response to extract-induced stress. Importantly, the fungicide tebuconazole showed method-dependent effects. On in vitro solid medium, it significantly inhibited the fungal growth without affecting OTA yield, whereas in ex vivo berry assays, it significantly inhibited both. Our findings emphasize the potential of stilbenoid-enriched extracts as biocontrol agents targeting OTA production, highlighting the value of multi-assay evaluations. Future studies should elucidate their modes of action and optimize their efficacy.
Trang Tran-Minh, M. Corio-Costet, Marie Laurens et al.· Toxins· 0 citations
The phytopathogenic fungus Fusarium verticillioides produces fumonisins, mycotoxins that contaminate grains of Zea mays L. (maize) and pose a risk to human and animal health. The aim of this study was to evaluate the efficiency of bacteria and filamentous fungi in controlling toxigenic F. verticillioides in maize. Microorganisms associated with maize grains were isolated and identified, and their ability to inhibit pathogen growth and reduce total fumonisin production was assessed. The microorganism showing the highest efficiency was subsequently identified by molecular methods. Fungi belonging to the genera Fusarium (85.71%), Aspergillus (57.14%), Penicillium (21.43%), and Trichoderma (9.52%) were recovered in maize grain samples, based on the total analyzed. 71.1% of the F. verticillioides isolates were toxigenic, producing fumonisin concentrations ranging from 30 to 4,010 ppb, with strain H08 showing particularly high levels. In antagonism assays, 90.7% of the bacterial isolates and 42.7% of the filamentous fungi exhibited inhibitory activity against the pathogen. Trichoderma atroviride strain 3 showed the highest efficiency, reducing mycelial growth by 84.94% and fumonisin concentration by 99.77%, demonstrating strong potential to reduce maize contamination by toxigenic F. verticillioides.
Cinthya Llanos-Olivera, Marilín Sánchez-Purihuamán, Ada Barturen-Quispe et al.· Journal of Applied Biology &...· 0 citations
Fusarium graminearum causes cereal spoilage and mycotoxin production. Currently, biocontrol microorganisms have emerged as a safe and promising strategy to manage the contamination of toxin-producing fungi in cereal grains. The primary objective was to elucidate the antifungal mechanism of an isolated strain of Lactiplantibacillus pentosus Q11 against F. graminearum and to evaluate the preservative effects of its cell-free supernatant (CFS) in fresh sweet corn. In vitro antifungal assays of L. pentosus Q11 showed strong antagonistic activity against F. graminearum, Aspergillus flavus, and Penicillium expansum, as well as the ability to reduce zearalenone. Moreover, whole-genome analysis revealed no genes related to virulence and antimicrobial resistance, and no hemolytic activity was observed, which suggested the potential safety of strain Q11. Moreover, the CFS of L. pentosus Q11 effectively inhibited the mycelial growth of F. graminearum, disrupted hyphal integrity, and induced fungal cell death. Moreover, 3 organic acids and 38 volatile compounds in the CFS of Q11 strain were identified by SPME-GC-MS and HPLC, in which 2-nonanone, 2-methylpyrazine, lactic acid, acetic acid were predominant compounds. The CFS and lactic acid combined with 2-nonanone severely damaged hyphae of F. graminearum, by disrupting cell membrane and wall, leading to leakage of intracellular substances, showing a synergistic antifungal effect of the two vital compounds. Subsequently, the treatments of CFS and combined agents significantly decreased 88.05% - 89.52% of fungal populations, and AFB1 and DON mycotoxin accumulation in fresh sweet corn after 12 days of storage compared to the control. The malondialdehyde content in the treated corn was decreased by 29.25% - 31.50%, consistent with the higher activities of POD and APX, thereby preserving their sensory attributes and nutritional quality. Metabolomic analysis revealed that L-glutamate, γ-glutamylcysteine, and oxidized glutathione were significantly downregulated in the glutathione metabolism pathway in the treated corn, indicating that CFS containing lactic acid and 2-nonanone could repress fungal growth and alleviate oxidative stress in fresh sweet corn. Overall, these findings demonstrate that L. pentosus Q11 with synergistic volatile organic compounds and lactic acid is a promising biocontrol agent in preserving fresh sweet corn.
Yuhuan Zhang, Jiang Li, Ye Qiu et al.· Food microbiology· 0 citations
Durum wheat is highly susceptible to Fusarium head blight (FHB), a severe fungal disease caused primarily by Fusarium graminearum. FHB affects durum wheat production by causing significant yield losses and grain contamination with mycotoxins such as deoxynivalenol (DON) and zearalenone (ZEA). Given the limitations of chemical fungicides, finding sustainable biological control agents is essential. This study evaluated the antagonistic and biocontrol capabilities of Trichoderma harzianum ITEM 3636 against F. graminearum through in vitro and greenhouse experiments. In vitro dual and sandwich culture assays demonstrated that T. harzianum significantly inhibits pathogen mycelial growth through direct interaction and the emission of volatile compounds. In a competition test on rice kernels, co-inoculation with ITEM 3636 significantly reduced pathogen biomass, leading to maximum reductions of 96.5% for DON and 98% for ZEA. Furthermore, greenhouse trials on a commercial durum wheat cultivar revealed that T. harzianum ITEM 3636 significantly decreased FHB severity by 40% and reduced DON contamination by up to 32% only when a combined seed-coating and spike-spraying application was performed. Additionally, ITEM 3636 exhibited biostimulant-like effects on yield parameters, causing an increase in kernel weight of 33% in the greenhouse assay. These findings highlight T. harzianum ITEM 3636 as a promising and ecological alternative to synthetic fungicides for managing FHB, safeguarding crop production, and ensuring food safety.
J. Erazo, Paula Vanella, J. Palazzini et al.· Agronomy· 0 citations
Global climate change and the escalating drought cycles have severely compromised agricultural productivity in marginalized agroecosystems worldwide. This has necessitated a deeper understanding of the role of the phytobiome in the host plant's resilience. Although seed biopriming with plant growth-promoting bacteria (PGPB) is widely acknowledged for alleviating abiotic stress, the role of PGPB biofilms in enhancing the efficiency of biopriming remains a critical knowledge gap. In this study, the biofilm-forming capacity of nine thermohalotolerant, ACC-deaminase-producing PGPB strains previously isolated from the rhizosphere of Cyamopsis tetragonoloba grown in arid regions of India was evaluated. Strains were classified into biofilm-forming and biofilm-deficient groups based on pellicle formation, wrinkle formation by macrocolony, safranin staining quantification (OD492 > 2.5 v/s OD492 < 0.5), FTIR spectroscopy, and FESEM analyses. Selected representatives of both groups, Bacillus altitudinis C-17 and Bacillus subtilis J-35 (biofilm-forming), and Enterobacter cloacae C-35 (biofilm-deficient), were subjected to comparative seed biopriming assays, carried out on a drought-susceptible variety of C. tetragonoloba under simulated drought (-1.5 MPa). Results revealed that simulated drought completely arrested seed germination in the unprimed control group, whereas biopriming with the biofilm-forming strain J-35 restored germination by >70% (p < 0.005). The biofilm-deficient strain, C-35, showed a non-significant recovery (p = 0.476). In pot assay conducted under water deficit, biopriming with biofilm-forming PGPBs significantly improved seedling vigor, as evidenced by improved root length, shoot length, leaf area, and wilting index. Statistical analyses revealed that biofilm formation strongly correlated with enhanced seed colonization efficiency (R2 = 0.953, p < 0.005). Furthermore, biofilm-forming strains induced robust modulation of host oxidative homeostasis, as evident from ∼15.8-fold increase in proline content, a 3- to 5-fold increase in superoxide dismutase activity, and a negative correlation with harmful H2O2 accumulation (R = -0.816). These observations clearly demonstrate that enhanced biopriming by biofilm-forming PGPBs stems from the underlying mechanism in which biofilms act as biological anchors, enhancing seed colonization and triggering robust modulation of the host plant's antioxidant machinery. These findings provide a fundamental understanding necessary to exploit the social microbial behavior in developing next-generation bioinoculants for crops grown in climate-vulnerable agroecosystems, including drought-prone arid and semi-arid regions. The core hypothesis tested in this study is that biofilm formation by PGPB serves as more than a microbial survival strategy; it is a critical functional determinant of phytobiome resilience that enhances host physiological plasticity and abiotic stress resilience through persistent host-microbe interaction and the systemic modulation of oxidative stress.
Ragini Dolhey, Kamalpreet Kaur, Mamta Bajiya et al.· Plant physiology and biochem...· 0 citations
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