Mechanism Analysis Of Self-control Cu-Fe Nanocrystal Film Against Botrytis Cinerea And The Preservation Application

Grey mould caused by Botrytis cinerea（B.cinerea）is one of the top 10 most destructive plant diseases in the world,which can infect more than 1400 fruit and vegetable crops including strawberries,and cause significant economic losses to the fruit and vegetable industry.Although chemical inhibitors are effective in controlling post-harvest fruit and vegetable infestation by B.cinerea,the selective pressure they exert on the fungus has caused the rising resistance of B.cinerea.Therefore,it is urgent to develop a new strategie for the control of B.cinerea with high efficiency and low induced resistance.Nanomaterials,with their advantages of tiny size and functionalizability,provide new ideas for exploring novel B.cinerea control strategies.Compared with traditional chemical inhibitors,the non-specific inhibitory properties of nanomaterials make them less prone to drug resistance while possessing good antifungal activity.In particular,the new intelligent-response chemodynamic therapy（CDT）antifungal strategy,which makes use of the internal microenvironment of the fungus（oxidative stress）to stimulate the production of antimicrobial molecules,is expected to become a new weapon to alleviate the drug resistance of B.cinerea and to achieve precise prevention and treatment,due to its specific inhibition of the fungus and the absence of toxic side effects on normal tissues.Based on this,B.cinerea was took as the research object in the study,and combined with the design concept that nanomaterials can play an inhibitory role in response to the oxidative stress of fungi,a fungal self-control Fenton antifungal system based on Cu-Fe nanocrystals（CuFeNCs）was reasonably constructed to reduce the toxicity and side effects of nanomaterials as well as the drug resistance of fungi.The relationship between the structural properties of CuFeNCs and the rate of hydroxyl radical（·OH）generation was systematically investigated using multidimensional physicochemical characterisation and evaluation methods such as antifungal activity,which elucidated the mechanism of action for the improvement of Fenton efficiency.Cu₈Fe₂NCs（Cu:Fe=8:2）were further preferred,the fungal self-control property and long-lasting antifungal effect（up to 210 days）of Cu₈Fe₂NCs were revealed at multiple levels from the perspectives of antifungal activity,metal homeostasis,and intracellular changes in the fungal cells.The transcriptomics approach was used to comprehensively analyse the potential inhibition mechanism of Cu₈Fe₂NCs against B.cinerea at the genetic level,and further explored the freshness preservation effect of long-lasting antifungal cling film（Cu-Fe/SA/GL）loaded with Cu₈Fe₂NCs in practical applications.This study provides a theoretical basis for the rational design and construction of novel responsive nanomaterials and their application in antifungal freshness preservation and the study of inhibition mechanism.The main research and conclusions of this research are as follows:（1）Preparation and characterisation of CuFeNCs and preliminary investigation of their inhibitory effect on B.cinerea.CuFeNCs were prepared by the one-pot method,and their structures were characterised with multi-dimensional physicochemical characterisation such as transmission electron microscopy（TEM）,high-resolution transmission electron microscopy（HRTEM）,and energy dispersive spectroscopy（EDS）,which indicated that the CuFeNCs were composed of Cu O and Fe₃O₄domains with a size of 100 nm and a uniform distribution of Cu,Fe,and O elements.X-ray photoelectron spectroscopy（XPS）was used to analyse the surface elemental composition as well as the chemical state of CuFeNCs.In addition,the Cu₈Fe₂NCs had the highest ratio of Cu and Fe in the reduced state and the lowest reaction potential,which were conducive to promoting the Fenton reaction.Finally,the antifungal activities of Cu_xFe_yNCs with different Cu-Fe ratios were comprehensively evaluated from the perspectives of·OH production capacity,mycelial growth and spore germination.Cu₈Fe₂NCs with excellent antifungal activity were successfully screened,laying a solid research foundation for the subsequent antifungal research.（2）The inhibitory effect of Cu₈Fe₂NCs on B.cinerea and fungal self-control properties was revealed.With the above screening of Cu₈Fe₂NCs as the research object,using the determination of mycelial growth,spore germination,cell viability and the observation of mycelial morphology and microstructure,results indicated that Cu₈Fe₂NCs had an excellent antifungal effect on B.cinerea,which significantly inhibited the growth of the mycelium and the production of the conidium,and the inhibitory effect showed a concentration-dependent increase.Through the measurement of zeta potential,internal metal homeostasis,cell membrane integrity and ROS,results indicated that that Cu₈Fe₂NCs would immediately release metal ions into the fungui to destroy metal homeostasis and lead to the increase of oxidative stress,which ultimately triggered the occurrence of Fenton reaction in the fungui,and achieved the long-lasting inhibition of B.cinerea（up to 210 days）.In addition,the safety of Cu₈Fe₂NCs was comprehensively evaluated by in vitro toxicity assay and in vivo toxicity assay in mice,which showed that Cu₈Fe₂NCs had no toxic effects on normal cells,and only had damaging effects on B.cinerea.（3）Clarify the inhibitory mechanism of Cu₈Fe₂NCs against B.cinerea.Based on the Illumina hiseq sequencing platform for transcriptomics research,the key signalling pathways involved in regulation after Cu₈Fe₂NCs treatment were further analysed.The study showed that a total of 470 differentially expressed genes（DEGs）were found in B.cinerea after treatment with Cu₈Fe₂NCs,of which,224 were up-regulated genes and 246 were down-regulated genes,which indicated that the treatment of Cu₈Fe₂NCs had a greater impact on the expression of B.cinerea m RNAs and interfered with the expression of some genes.The functional enrichment of gene sets indicated that Cu₈Fe₂NCs further promoted the production of H₂O₂and Fenton reaction within the fungus and finally achieved the inhibitory effect on B.cinerea by regulating oxidative stress-related pathways（mitochondrial autophagy biosynthesis pathway,pentose phosphate pathway biosynthesis pathway and glutathione metabolism pathway）as well as ergosterol biosynthesis-related pathways.（4）Preparation of fungal self-control antifungal cling film and research on freshness preservation application.In order to further make efficient use of the dual functional properties of Cu₈Fe₂NCs for fungal self-control and long-lasting antifungal resistance against B.cinerea,the Cu-Fe/SA/GL film was reasonably constructed by solidifying Cu₈Fe₂NCs into sodium alginate/gelatin film.Using the multidimensional physicochemical characterisation and the determination of film properties,it was shown that the Cu-Fe/SA/GL film as a new type of food packaging not only had good mechanical and long-lasting anti-fungal properties,but also slowed down the evaporation of water during the preservation of fruits and vegetables.Further,a model of strawberry infected by B.cinerea was constructed to investigate the regulatory role of Cu-Fe/SA/GL film in the strawberry preservation,which indicated that the Cu-Fe/SA/GL film prolonged the storage time by inhibiting the growth of B.cinerea in strawberries while delaying the changes of the physicochemical properties and nutritional components of strawberries during the storage period.In summary,this study successfully prepared CuFeNCs with fungal self-control properties and achieved long-lasting antifungal effects against B.cinerea.The inhibition mechanism of CuFeNCs against B.cinerea was elucidated,and the antifungal freshness preservation of Cu-Fe/SA/GL film was explored,which provides a new idea for the safer and more efficient application of nanomaterials in the research of antifungal prevention and control,and is of great significance for the safeguarding of the national healthy life as well as the high-quality development of fruit and vegetable industries.