自然环境中T-2毒素降解菌的筛选与鉴定

摘要

[目的]在自然环境中筛选和鉴定能够降解T-2毒素的菌株.[方法]取对虾养殖池水样、养殖池沉泥样品和对虾混合饲料中分离到的镰孢菌,于GYM产毒培养基中培养14 d后引入自然条件下气载细菌,继续培养至28 d,采用LC-MS/MS检测其中T-2毒素含量.然后结合稀释涂布、平板划线、革兰氏染色、镜检,从镰孢菌产毒培养液中毒素含量明显降低的菌悬液中筛选出T-2毒素降解菌,用16S rRNA分析方法对其进行系统发育分析及菌种鉴定,并验证两菌株的降毒能力与不同基质中二者的联合降毒能力.[结果]以从对虾养殖环境中分离到的5株镰孢菌作为试验菌种,在其产毒培养过程中分离到2株T-2毒素降解菌,16S rRNA鉴定结果分别为弯曲假单胞菌和尼泊尔葡萄球菌,对T-2毒素的降解率分别为90.9％和85.5％,但两者降毒能力并无显著差异(P＞0.05)；其联合作用也有较好降毒效果,与两菌株单独作用无显著差异(P＞0.05),不同基质对其联合降毒作用影响不大(P＞0.05).[结论]新的T-2毒素降解菌的发现为进一步探明T-2毒素降解基因和开发T-2毒素生物降解酶提供了研究基础.%[Objective] This study was to find the T-2 toxin-degrading strains,which were isolated and identified from natural environment.[Methods] Fusarium producing T-2 toxin were isolated from the samples of shrimp culture pond water,pond sediment and shrimp mixed feed,respectively.When it was cultured in GYM medium for 14 days,the airborne bacteria in nature environment were inoculated.The content of T-2 toxin was detected by LC-MS/MS at 28 day in its cultivation.T-2 toxin-degrading strains were screened from the bacteria suspension in which the content of T-2 toxin has obviously decreased with dilution spread method,plat streaking method,Gram stain method and microscopic examination.And then the identification and phylogenetic analysis of the screened strains were performed by 16S rRNA method.The method was also used to validate the toxin-degrading capacities of the screened strains respectively and the combined effect of them in different matrixes.[Results] Two T-2 toxin-degrading strains were screened out during the cultivation of five Fusarium strains which were isolated as indicator bacteria from shrimp culture environment.The results of 16S rRNA analysis showed that they were Pseudomonas geniculate and Staphylococcus nepalensis,and their degradation rates to T-2 toxin were 90.9％ and 85.5％ respectively.But there was no significant difference between the degradation effects of the two strains (P＞0.05).In addition,the combined effect was good but had no significant difference with the single strain's degradation effect (P＞0.05).And the combined degradation effects were not much affected by different matrixes (P＞0.05).[Conclusion] This new discovery established a basis for further ascertaining T-2 toxin degrading genes and developing T-2 toxin degrading enzymes.