Risk Assessment Of Fusarium Toxin In Rice And Preliminary Exploration Of Biological Control Techniques

In order to realize the safety of fusarium toxins from genetically modified（GM）rice,it is necessary to investigate the contamination of Fusarium and fusarium toxins in rice.The biological control technology against Fusarium infection and toxin contamination can provide guarantee for human health.To ensure food security,this study which based on metabonomics evaluated the food safety of GM rice from the aspects of fusarium toxins content and the diversity of endophytic fungi.The detoxification effect of biological control technology on zearalenone in rice was further explored.The details are as follows:（1）Evaluation of untargeted metabolome variation and mycotoxin risk warning in transgenic riceHigh performance liquid chromatography-mass spectrometry（HPLC-MS）was used to analyzed Japonica rice varieties Nipponbare（FP1）and PJ574（FP2）and their corresponding transgenic line（FT16、FT23）.Multivariate statistical methods such as principal component analysis and partial least squares discriminant analysis were used to detect 448 metabolites from rice samples.Different metabolites were screened and it was found that the concentration variation of zearalenone（ZEN）,L-lactate,10E,12Z-octadecanodienoic acid and spermine were significantly affected by gene modification or variety differences.There were9,23,and 13 unique differential metabolites in groups FP1-FP2,FP1-FT16 and FP2-FT23,respectively,with the highest variation in group FP1-FT16.The unique differential metabolites of the FP1-FT16 group mainly included deoxyguanolate,coffee salt and gluconic acid.The unique differential metabolites of the FP2-FT23 group mainly contained prostaglandin B2,L-cystine,and 16-hydroxypalmitic acid.The unique differential metabolites of FP1-FP2 group mainly include inositol,and stearate amides.The effects of transgenic breeding and variety differences are similar,both of which affect arginine biosynthesis and multiple amino acid metabolic pathways to a certain extent,but the variation in metabolic pathways in different varieties of transgenic rice is different.Although the variation of metabolic components and metabolic pathways induced by transgenic breeding may affect the nutritional quality of rice,the extent of variation within the safe range is not greater than that of rice varieties.Enzyme-linked immunoassay（ELISA）showed that the zearalenone content in rice samples ranged from 4.2 to 6μg/kg,with a significant difference between the two parent varieties（P<0.01）,but the content of zearalenone in transgenic rice was substantially equivalent to that in the parents.The ZEN content synthesized by rice endophytic Fusarium in rice is significantly lower than the national standard（60μg/kg）but there are risks of fungal outbreak and mycotoxin accumulation under certain conditions.（2）Safety assessment of fusarium toxin and genetic analysis of endophytic fungi in riceBy the ELISA technique,low doses of fusarium toxins were detected in the four rice samples,with ZEN contents ranging from 3.7 to 6.1μg/kg,Deoxynivalenol（DON）contents ranging from 13.3 to 25.8μg/kg,and T-2 toxin contents ranging from 0.4 to 1.2μg/kg,but there were no significant differences among the four rice samples（P<0.05）.The substance of transgenic rice was identical to that of parent rice.Fumonisin（FB₁）were detected only in FT16 rice samples,whose content was 8.5μg/kg.The content of fusarium toxin detected in rice samples from 4 shelf periods is far lower than the national standard and does not pose a food safety risk.No DON or FB₁were detected in 16 Fusarium strains,and the ZEN content was at 5.5～21.5μg/kg.T-2 toxin was detected in 9 Fusarium strains,and its content was between 0.1 and 0.5μg/kg.In addition,a total of 9 endophytic phyla and 68 fungal genera were detected by ITS high-throughput sequencing of four rice species,among which the dominant phyla were ascomycetes,and the dominant fungal genera were mainly Fusarium and Curvularia.Among the endophytic fungalαdiversity of the four rice samples,only the Shannon index of FP1 and FP2 had significant differences（P<0.05）.The Fusarium richness of GM rice FT16 was 59.61 times higher than that of its parent FP1,identified as the dominant strain,but the difference was not significant（P<0.05）.The endophytic fungal groups of GM rice were essentially equivalent to those of their parent rice.The safety risk caused by transgenic mutation is low,but the prevalence of low dose toxins and the presence of endophytic fungi are more worthy of caution.（3）Antifungal activity of endophytic Bacillus BR-1 in rice and its detoxification effect on zearalenoneIn our previous study,endophytic Bacillus BR-1 was isolated from rice.The strain BR-1was sequenced by whole genome sequencing,and the antifungal activity and zearalenone degrading properties of the strain BR-1 were evaluated.The strain BR-1 was identified as Bacillus velezensis.There are four biosynthetic gene clusters of Bacillus velezensis BR-1including surfactin,fengycin,bacillaene and bacillibactin,which are responsible for the biosynthesis of lipopeptides.Four candidate genes with homology of 67.91%～100%were identified by BLASTP comparison between the genome sequence of strain BR-1 and the known genes related to ZEN degrading enzymes.In addition,strain BR-1 had a strong antagonistic activity based on the colony diameter,spore germination and mycelium growth of Fusarium.The supernatant of strain BR-1 exhibited the highest detoxification effect against on ZEN,with a degrading rate of approximately 80%.M9 medium and 24 h culture time were selected as the optimal conditions for degradation of ZEN.The optimal degrading reaction conditions were as follows:concentration of ZEN substrate 100 ng/m L,time 2 h,p H=7,and reaction temperature 37℃.The chemical nature component of biodegrading ZEN substance by strain BR-1 was investigated.It was found that the biodegradable ZEN substance was a heat-resistant protein.In the prevention and control application of rice,strain BR-1 showed significant antagonistic effects against Fusarium and ZEN degradation on rice infected by Fusarium graminearum,and compared with the control group,whose toxin degradation rate reached 54.77～63.31%.In the ZEN degradation,the degradation rate of ZEN was 60.36%after 24 h of treatment with the strain BR-1,and the degradation rate was 76.77%after 72 h of treatment.Overall,Bacillus velezensis BR-1 showed good potential for mildew control in grain storage.In summary,safety assessment of GM rice in terms of metabolomics revealed the detection of ZEN in rice samples during the shelf-life,the investigation of its causes and preliminary risk assessment,and the investigation of the ability of endophytic Fusarium to produce toxins in rice.In general,GM rice showed substantial equivalence with the parental species,but the detection of fusarium toxin in the shelf-life rice and the wide distribution of the endophytic Fusarium fungus are alarming phenomena.By identifying and evaluating the effective degradation strains of ZEN,and understanding the antagonistic activity of endophytic Bacillus velezensis BR-1 against Fusarium and the degradation characteristics of ZEN,we can help to improve the safety of mycotoxin in rice.