Inhibitor Effect And Mechanism Of Enterobacter Cloacae 3J1EC On Aflatoxin Production By Aspergillus Flavus

Peanut,corn and other agricultural products are highly susceptible to aflatoxin contamination.Aflatoxins are a kind of highly toxic and carcinogenic substances that heavily threaten to public health.which are mainly produced by Aspergillus flavus and Aspergillus parasiticus.A.flavus is mainly from soil saprophytic fungus.Accompanied by agricultural products,they contaminate the agricultural products in the whole industrial chain inlcduing harvesting,storage,transportation,and processing stage.Therefore,it is very important to develop the inhibitors of aflatoxin to ensure the quality and safety of agricultural products.To explore an effective and safe biological inhibitor,we evaluated the A.flavus toxin-producing inhibition and biological safety of potential inhibitor of Enterbacter cloacae 3J1EC,and further studied the inhibiting mechanisms of Enterobacter cloacae 3J1EC on A.flavus toxin-producing in the transcriptome level in this thesis.1.Enterobacter cloacae 3J1EC showed a strong inhibitory effect on aflatoxin production.Dynamic changes of alfatoxin contents and spore were investigated to determine the key time points of spore germination and alfatoxin production.Moreover,A.flavus 3.4408 and 6 A.flavus isolated from soils by our lab were co-cultured with Enterobacter cloacae 3J1EC to evaluate its inhibitory effect on aflatoxin production by determining the alftoxin contents.The dynamic anaylsis results showed that A.flavus germinated completely in 8 hours and started to produce aflatoxins in culture medium at 72 hours.Furthermore,we found that the content of aflatoxin produced by A.flavus treated with Enterobacter cloacae 3J1EC decreased by 95.9%?99.5%and the dry weight of A.flavus 3.4408 decreased by 54%.More importantly,Enterobacter cloacae 3J1EC can inhibit not only standard A.flavus 3.4408 but not A.flavus isolated from soils to produce aflatoxins,indicating that Enterobacter cloacae 3J1EC showed potential to be a broad-spectrum inhibitor for aflatoxins.2.The molecular mechanism of Enterobacter cloacae 3J1EC inhibiting aflatoxin production was preliminarily investigated.The transcriptome of A.flavus 3.4408 at 0,8 and 72 hour were analyzed to trace the dynamic changes of transcriptome by using RNA-Seq technique.The results indicated that laeA,aflR and aflS were up-regulated by 3.1,2.34 and 1.26 times higher than those at 0 h,respectively.Their expression level kept increasing up to 3.79,4.02 and 2.91 times at 72 h.When Enterobacter cloacae 3J1EC was used,laeA,aflR and aflS were down-regulated at 72h.The results showed that Enterobacter cloacae 3J1EC inhibited the expression of LaeA,aflR and aflS,thus reduced the toxin production of Aspergillus flavus.The results provide a scientific basis for biocontrol using Enterobacter cloacae 3J1EC in agricultural practices.3.The biological safety of Enterobacter cloacae 3J1EC was evaluated.Pathogenicity of acute intraperitoneal injection,acute oral toxicity and acute percutaneous toxicity of Enterobacter cloacae3J1EC were investiagated.Among them,pathogenicity test of acute intraperitoneal injection was conducted according to General Biosafety Standard for Microbial Fertilizers?NY/T 1109-2017?.Meanwhile,acute oral toxicity and acute percutaneous toxicity tests were conducted according to the toxicological test method of pesticide registration?GB/T 15670.5-2017?.The test results were as follows:?1?the pathogenicity rate of the test was 0,?2?LD₅₀>5000mg/kg·BW for female rats,and?3?LD₅₀>2000mg/kg·BW for female and male rats.Three tests indicated that Enterobacter cloacae3J1EC met the requirements of microbial fertilizer.