| Aspergillus flavus is a ubiquitous filamentous fungus in agricultural and sideline products,can produce highly toxic and carcinogenic aflatoxin through secondary metabolism,which poses a severe threat to the life of human and animal.Essential oils（EOs）and their main components have attracted the attention of many scholars due to their high safety and broad-spectrum antimicrobial activity,and their antimicrobial mechanism in both liquid and gaseous phase have also been widely studied.At present,many studies related to the antimicrobial mechanism of EOs are based on the their continuous treatment on microorganisms.However,EOs are highly volatile,whose concentration decrease over time.These existing studies can not accurately guide the practical application of EOs in food preservation and the persistent effect on the state of microorganisms after the removal of EO is also worthy of further study.In this study,the effects of transient CA vapor treatment on the growth and aflatoxin synthesis of A.flavus were explored,and the persistent effects on the physiological characteristics of A.flavus were explored.At the same time,the molecular regulation mechanism of the persistent effects of transient CA vapor treatment on the growth and aflatoxin synthesis of A.flavus was analyzed by transcriptome sequencing.The main results are as follows:（1）The effect of transient CA vapor treatment on the growth of A.flavus was related to the concentration of gaseous CA.After being treated with gaseous CA at a concentration not higher than 0.4μL/m L,A.flavus could completely recover its growth by removing CA.Under the same transient treatment,when the concentrations of gaseous CA were 0.6μL/m L and 0.8μL/m L,A.flavus partially resumed growth.When the concentration of gaseous CA was 1.6μL/m L,A.flavus could not grow.The sensitivities of different A.flavus to gaseous CA were different,and A.flavus JX-3,A.flavus JX-4 and A.flavus HN-1 were relatively sensitive to gaseous CA.After transient CA vapor treatment,the synthesis of AFB1 in A.flavus was significantly promoted.A.flavus regulated the damage repair and AFB1 synthesis by up-regulating the expression of drug efflux transporter related genes and down-regulating the expression of velvet complex protein related genes.（2）The transient treatment of gaseous CA destroyed the integrity of A.flavus cell membrane,led to the leakage of protein,resulted in the accumulation of ROS and lipid peroxidation,and improved the activities of antioxidant enzymes.After gaseous CA removal,ergosterol and protein synthesis returned to normal,ROS were removed,MDA content and the antioxidant enzymes activities decreased.The results of SEM and TEM showed that the surface and internal structure of cells were destroyed by the transient treatment of gaseous CA.After removal,the structure of A.flavus cells gradually returned to normal,but there was cavitation related to apoptosis in the cells,and the cells could not return to normal state.Meantime,the disordered mitochondrial membrane potential and inhibited ATPase activity could not be restored to the normal state,and the transient gaseous CA treatment caused irreversible damage to mitochondria.（3）Transcriptome sequencing analysis showed that the genes related to drug efflux transporters in A.flavus were significantly up-regulated and amino acid and relevant functional protein metabolism were active after the removal of gaseous CA to offset the adverse effects on cells.However,the normal function of A.flavus mitochondria has not recovered,and the related enzymes and energy metabolism pathways in mitochondria have been inhibited.A.flavus promotes the synthesis of AFB1 by up-regulating the structural genes of aflatoxin synthesis instead of the regulatory genes.In addition,A.flavus can promote secondary metabolism by up-regulating the key gene flu G of velvet complex protein,up-regulating the glycolytic pathway and down-regulating the pentose phosphate pathway.To sum up,this study preliminarily explored the molecular regulation mechanism of the persistent effect of transient CA vapor treatment on growth and aflatoxin synthesis on A.flavus from different perspectives such as cell structure,energy metabolism and gene expression,and provided a new understanding for the response mechanism of A.flavus to EOs and the rational application. |
