Risk Assessment And Control Mechanism Of Mixed Contamination Of Common Fungicides And Ochratoxin A In Grapes

Grape fruits are popular among the public,because they taste delicious and are rich in nutrition.They can be eaten freshly and processed into grape wine,raisins,grape juice,etc.In recent years,the cultivation areas and yield have also increased significantly.China is a major producer and consumer of grapes and wine.Because grape has high content of glucose and is hard to clean,and it is suitable for microorganism coherence and growth.That will lead to the occurrence of fungal diseases during plant growth,storage,transportation,etc.Some pathogenic fungi can not only cause fruit rot,but also produce harmful mycotoxins.In order to control the occurrence of fungal diseases and obtain high-quality grapes,the use of fungicides is essential during the cultivation and storage of grapes.This leads to mixed contamination of biological hazards（pathogens and mycotoxins）and chemical hazards（fungicides）.In this paper,grape was selected as the research object to evaluate the risk assessment of grapes during the harvest period in the main producing areas of northern China.The change of carbendazim,hymexazol and Ochratoxin A during wine processing was investigated,the mechanism of inhibition of Mycotoxins production by typical pesticides was explored at the transcriptome level.The study will provide reference for risk assessment of factors of biological harm and chemical hazards in grapes,the prevention and control of Ochratoxin A in processing,and provides new ideas for prevention and control of Ochratoxin A production of Aspergillus carbonarius ultimately.The main conclusions are as follows:First,the analytical method was established to determine Ochratoxin A and 13 kinds of commonly used fungicides（thiophanate-methyl,myclobutanil,metalaxyl,pyrimethanil,tebuconazole,imazalil,triazolinone,pyraclostrobin,prochloraz,propiconazole,difenoconazole,azoxystrobin,dimethomorph）in grapes,wine,raisins and grape juice.Samples were extracted using acetonitrile,using C18 for purification and detected by ultra performance liquid chromatography-tandem mass spectrometry.The external standard method was used for quantification.The average recovery was 73.7%–117.9%with the relative standard deviation between 1.1%and 20.1%.The limit of quantification of this method for Ochratoxin A was 0.1μg kg^-1,for 13 kinds of commonly used fungicides was 1μg kg^-1.The method is simple,can save time and has high efficiency,has a wide range for concentration determination and high sensitivity.The method is also accurate in quantitative analysis and can be used for detection of Ochratoxin A in food of animal origin and simultaneous detection of residue of Ochratoxin A and 13 kinds of commonly used fungicides.That can lay a foundation for subsequent sample detection and risk assessment.Second,residue levels of 13 kinds of commonly used fungicides were tested for 829samples collected from main producing areas in northern China.The risk of dietary intake for the main fungicides in domestic grapes was evaluated in accordance with the average human dietary data,the average weight of the population,and based on the Monte Carlo mathematical probability model.The results showed that 13 fungicides were detected at different degrees.In the fresh grape samples,the phenomenon of multiple residues of pesticides was more serious,but the residual levels were low,most of which were in trace levels.The dietary intake risk assessment results showed that 13 kinds of commonly used fungicides had low risk of chronic dietary intake and the risk was acceptable.The risk of acute dietary intake was far higher than that of chronic dietary intake,but they were all at acceptable levels.Among them,tebuconazole has a high risk of acute dietary intake,which has reached 91.37%,that should arouse widespread concern in the use of pesticides and the risk of daily dietary intake.This study can provide reference for the supervision of grape quality and safety in China and the systematic risk assessment of agricultural product quality and safety in the future.Third,evaluation of the effect of commonly used fungicides on the growth of Aspergillus carbonarius colonies and the production of Ochratoxin A in grapes.The inhibitory effect of each fungicide on the colony growth of Aspergillus carbonarius was determined by the inhibition mycelial growth rate method.The Ochratoxin A produced by which was extracted and determined.And the effects of different fungicides on Ochratoxin A roduction were compared.The results showed that carbendazim,imazalil,fludioxonil,and prochloraz can completely inhibit the growth of Aspergillus carbonarius and the production of Ochratoxin A,they are ideal pesticides for controlling the disease.Fourth,the RNA was extracted and analyzed by different genes in the above three treatment groups and the blank control group.The results of transcriptomics analysis show that,Hymexazol,azoxystrobin and procymidone have a significant effect on the expression of multiple genes of Aspergillus carbonarius,resulting in several changes of GO term,and then influence the related pathways.There are a lot of differences in genes of the three treatments,such as those associated with the function of the binding of metal ions for molecular function,transmembrane transport for biochemical process,and the cell membrane for subcellular fractions,which could contribute to make differences in the pathways of metabolic pathways,biosynthesis of secondary metabolites and antibiotic biosynthesis.Whether the differences in these genes and pathways affect the production of Ochratoxin A,the mechanism of its action needs to be further studied.Fifth,the influence on growth and Ochratoxin A production of Aspergillus carbonarius result from was carbendazim or hymexazol was investigated during the process of wine-making,when exploring the effect of mixed contamination of biological harm（carbendazim and hymexazol）and chemical hazards（Aspergillus carbonarius）.At the same time,tracking detection of Ochratoxin A content and residues of carbendazim and hymexazol during the entire processing period.The results showed that mixed contamination of carbendazim or hymexazol and Aspergillus carbonarius,the growth of Aspergillus carbonarius was influenced and the content of Ochratoxin A was decreased by the two fungicides during fermentation.However,there is no significant difference in the content of Ochratoxin A in the product wine after the whole process;for the three target compounds,the processing factor was less than 1 for each step,indicating that the residual levels of the two fungicides and Ochratoxin A are reduced during the processing of the wine,and the main processing step for the reduction of the concentrations of the three compounds was fermentation;in the clarification step,the addition of bentonite significantly reduced the residual level of carbendazim and had no significant effect on hymexazol and Ochratoxin A.