| Microbial volatile organic compounds(VOCs)refer to active volatile substances produced by bacteria and fungi during metabolism.In recent years,VOCs have been increasingly used to assess fungal contamination in cereal grains.Fusarium graminearum,a common mycotoxinproducing fungus,can cause fusarium head blight and also produce zearalenone,which causes reproductive abnormalities in mammals.Microbial VOCs can be used as a monitoring indicator for Fusarium graminearum in addition to a green inhibition agent against Fusarium graminearum to reduce the pollution of the environment by chemical inhibition agents.In this study,solid phase microextraction combined with two-dimensional gas chromatography-timeof-flight mass spectrometry(SPME-GC×GC-TOFMS)was utilized to identify and compare the VOCs produced by Fusarium graminearum PH-1 in different growth substrates.Multivariate data analysis was employed to determine the characteristic VOCs that were highly positively correlated with three factors: the growth time of the PH-1 strain,the infection rate of PH-1 in wheat,and ZEN production.Subsequently,classification models were used to investigate the predictive capabilities of non-characteristic VOCs and characteristic VOCs for the level of the PH-1 infestation.Finally,the inhibitory effects of pyrazine VOCs on the growth and mycotoxin production of PH-1 were examined,and the potential mechanisms of growth and mycotoxin inhibition were explored by integrating metabolomics,lipidomics techniques.The main research content of this paper can be summarized into the following three points:1.Identification of characteristics VOCs with growth-related of Fusarium graminearum.The VOCs produced by Fusarium graminearum PH-1 were identified and classified,and partial least squares discriminant analysis revealed changes in VOCs at different growth stages of PH-1.Characteristic VOCs such as 1,4-dichlorobenzene,1,2,4-trimethylbenzene,and 6-chloro-2-phenylhexanenitrile were found to be highly positively correlated with colony diameter,indicating their potential as predictors for PH-1 growth.By comparing five classification models,the neural network model based on characteristic VOCs performed the best in predicting PH-1 growth time with an area under the ROC curve(AUC)of 0.798.Furthermore,the predictive capabilities of different classification models based on characteristic VOCs were generally stronger than those based on non-characteristic VOCs.A comparison of the effects of different substrates revealed that PH-1 produced fewer types of VOCs in wheat,suggesting that its metabolic pathways may change in different environments.2.Study on the relationship between VOCs and Fusarium graminearum pollution in wheat.The SPME-GC×GC-TOFMS method was applied to analyze VOCs in wheat samples with varying degrees of PH-1 infection.Principal component analysis revealed that VOCs changed with increasing infection rates.Characteristic VOCs highly positively correlated with PH-1 infection rates were identified,such as 5-pentylcyclohex-1,3-diene,3-hexanone,and 1,3-octadiene.Comparing five classification models,it was found that the model performance based on non-characteristic VOCs and characteristic VOCs did not differ significantly.Subsequently,the fungal communities in PH-1 infected wheat samples were analyzed in-depth,revealing that fungal VOCs possess certain species specificity.The relationships between mycotoxins and VOCs in wheat samples were investigated,identifying potential indicators related to ZEN content,including 6-butyl-1,4-cycloheptadiene,hexahydro-3-methylenebenzofuran-2(3H)-one and(E,E)-3,5-octadien-2-one.It was found that using characteristic VOCs improved the model’s predictive capabilities,with the highest AUC(0.906)for the neural network model based on characteristic VOCs.Finally,the predictive ability of the classification model combined with VOCs for ZEN content was verified in different batches of samples measured by GC-MS,finding that the AUC of the neural network model based on characteristic VOCs remained high(0.951).3.Investigation of the mechanism of exogenous pyrazine VOCs inhibiting the growth and mycotoxin production of Fusarium graminearum PH-1.Based on the group’s preliminary research and pre-experiments on VOCs,and combined with literature research,the exogenous microbially produced pyrazine VOCs were selected to inhibit PH-1.Growth inhibition was found to be up to 23.4% and ZEN production inhibition was up to 85.4%.Metabolomics and lipidomics techniques were employed,revealing significant changes in compounds related to mycotoxin synthesis,such as endogenous purine nucleoside 9-β-Dfuranoribofuranosyladenine and intermediate 3-methyl-2-oxovaleric acid in the branched-chain amino acid metabolic pathway.Lipids related to cell membrane stability,such as PC O-35:8,SM 38:9;2O and PC 32:1,also exhibited significant changes.It is speculated that pyrazine VOCs will reduce the expression level of valine,leucine and isoleucine biosynthesis,thereby affecting the front-end synthesis of mycotoxins.Through quantitative real-time polymerase chain reaction technology,changes in mycotoxin-producing genes were measured,revealing that after treatment with pyrazine VOCs,the expression of DON-related genes(TRI4,TRI5,TRI6,and TRI10)and ZEN-related genes(ZEB1,ZEB2,PKS3,and PKS14)decreased.The cytotoxicity of pyrazine VOCs was investigated using the CCK-8 assay,which showed that pyrazine VOCs exhibited cytotoxicity only at very high concentrations(1000 μg/L).Finally,it was confirmed that pyrazine VOCs also have the ability to inhibit PH-1 in wheat ears.In summary,firstly,characteristic VOCs associated with PH-1 growth were identified,and the neural network model based on characteristic VOCs was able to predict growth time.Secondly,the neural network model based on the characteristic VOCs was found to be able to predict the infestation rate and ZEN production of PH-1.Finally,pyrazine VOCs were found to inhibit the growth production of PH-1 and could be used at the wheat planting stage. |
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