【摘要】：乙草胺、芐嘧磺隆以及乙·芐復配制劑是目前最廣泛使用的稻田新型除草劑。但大量施用對后茬敏感作物造成明顯藥害,對農田生態(tài)環(huán)境帶來嚴重污染。除草劑在土壤中的殘留一般通過物理、化學與生物學過程而消失,其中微生物降解是主要途徑。因此,利用降解微生物消除環(huán)境污染具有重要意義。通過富集培養(yǎng)篩選出一個能以乙草胺為唯一碳源、并耐受芐嘧磺隆的復合菌系66C,從66C復合菌系中分離得到4個細菌菌株,分別命名為66C-1、66C-2、66C-3、66C-4。通過形態(tài)特征、分子系統(tǒng)發(fā)育分析和生理生化3方面對菌株進行鑒定,將66C-1、66C-2、66C-3、66C-4分別鑒定為粘質沙雷氏菌、Psedomona shunanensi s、惡臭假單胞菌、硝化還原假單胞菌。單一的菌株不能降解乙草胺,只有當這4株菌以1:1:1:1混合培養(yǎng)時才會生長,達到共同降解乙草胺的目的,將4株菌等比例構成的混合菌重新命名為HXDW。在乙草胺初始濃度為100mg/L、pH9.0、NaCl濃度為2%的無機鹽培養(yǎng)基中25℃培養(yǎng)時,混合菌HXDW生長最好。10mg/L P b2+明顯促進復合菌的生長,而10mg/L的Cd2+和Mn2+抑制復合菌的生長。降解菌系只能利用乙草胺作為唯一碳源,而不能利用丁草胺、異丙甲草胺和吡氟酰草胺。利用LC-MS測定混合菌HXDW培養(yǎng)液中乙草胺的殘留量,發(fā)現(xiàn)乙草胺的7d降解率為14%。利用UHPLC-QTOF-MS測定混合菌HXDW對乙草胺的代謝產物,初步斷定乙草胺經羥基化、氧化、脫氯等過程形成中間產物3-羥基-2-氨基苯甲酸。SDS-PAGE電泳分析顯示,經100mg/L乙草胺或100m g/L乙草胺與30mg/L芐嘧磺隆的復配劑誘導后,混合菌HXDW的全蛋白圖譜出現(xiàn)3條約58.0、50.0和43.0kDa大小的特異性蛋白條帶;而30mg/L芐嘧磺隆誘導的HXDW培養(yǎng)液全蛋白圖譜呈現(xiàn)出80.0、64.0、58.0、50.0、43.0、38和25kDa等7條特異性的蛋白條帶。通過滲透休克法提取混合菌HXDW胞內、周質空間及胞外粗酶液進行乙草胺的降解研究,結果表明24h的乙草胺降解率分別為17.43%、18.34%、44.95%,乙草胺降解酶主要來自復合菌的胞外酶,而胞內也存在少量的降解酶。當用乙·芐復配劑(20mg/L芐嘧磺隆和60mg/L乙草胺)脅迫敏感菌-大腸桿菌和枯草芽孢桿菌后,SOD酶活迅速下降,至處理后2h達到最低值;之后回升至處理后12h到達最高值,SOD酶活分別為處理前1.63倍和1.08倍。而大腸桿菌的C AT酶活曲線顯示出兩個峰值,最大值為處理前的1.23倍;枯草芽孢桿菌的CAT酶活在處理初期變化緩慢,處理18h時達到最高值,此時酶活為處理前的4.20倍�？傮w上,G-菌-大腸桿菌、G+菌-枯草芽孢桿菌經低濃度乙·芐復配劑脅迫后,可緩慢通過SOD酶和CAT酶的提高對抗氧化脅迫。而超過該濃度后,兩菌株的CAT酶活顯著降低,SOD酶活不變,表明高于60mg/L乙草胺及20mg/L芐嘧磺隆對敏感菌株造成損害。
[Abstract]:Acetochlor, bensulfuron-methyl and acetylbensulfuron-methyl are the most widely used herbicides in paddy fields. However, a large amount of application caused obvious pesticide damage to the next crop and caused serious pollution to the ecological environment of farmland. The residues of herbicides in soil are generally disappeared through physical, chemical and biological processes, in which microbial degradation is the main pathway. Therefore, the use of degradation microorganisms to eliminate environmental pollution is of great significance. Through enrichment culture, a compound strain 66C, which can use Acetochlor as the sole carbon source and tolerance to bensulfuron-methyl, was selected. Four bacterial strains were isolated from 66C complex strain and named 66C-1O66C-2O66C-3O66C-4 respectively. The strains were identified by morphological characteristics, molecular phylogenetic analysis and physiological and biochemical analysis. 66C-1A66C-2O66C-3O66C-4 was identified as Pseudomonas, Psedomona shunanensi s, and Pseudomonas pseudomonas was nitrified. A single strain could not degrade Acetochlor, and only when the four strains were mixed with 1: 1: 1: 1: 1, could they grow, so that the co-degradation of Acetochlor could be achieved. The four strains with the same proportion were renamed HXDW.. When the initial concentration of Acetochlor was 100 mg 路L ~ (-1), pH 9.0% NaCl concentration was 2%, the mixed bacteria HXDW grew best at 25 鈩，

本文編號：2360008