【作者】 刘敏；

【导师】 柏连阳；

【作者基本信息】 湖南农业大学 ， 农药学， 2015， 博士

【摘要】 二氯喹啉酸是由德国巴斯夫公司（BASF）开发的新型激素型除草剂,主要用于防除水稻田稗草及部分双子叶杂草。因其具有用量少、持效期长、对稗草特效等优点而被广泛应用于农业。但由于其结构稳定,在田间降解缓慢,残留时间长,给环境安全带来潜在危害,并容易对后茬敏感作物产生残留药害,特别是在稻烟轮作区,由于前茬稻田使用除草剂二氯喹啉酸,其在土壤中的残留会导致后茬烟草产生严重的药害,严重影响了烤烟的产量和质量。本论文在此背景下,对二氯喹啉酸在土壤中的消解动态及其对后茬作物的药害进行了研究；从受二氯喹啉酸药害的烟草根系中分离出一株二氯喹啉酸内生降解菌Q3,通过形态特征、生理生化特性研究及16S rDNA序列同源性分析对其进行了鉴定,并对其降解特性进行了研究；同时,采用高效液相色谱-串联质谱（HPLC-MS/MS）对二氯喹啉酸的微生物降解产物进行了分析鉴定,提出了其可能的降解途径；通过室内盆栽试验及田间小区试验初步研究了菌株Q3对烟草药害的生物修复作用,探讨了二氯喹啉酸胁迫下降解菌对烟草保护酶系活性的影响,为其田间应用提供理论依据；对二氯喹啉酸内生降解菌的全基因组DNA进行了测序,并对其全基因组进行了初步分析,为阐明巨大芽孢杆菌Q3降解二氯喹啉酸的分子机理提供了理论基础。全文主要研究结果如下：采用优化的QuEChERS方法对土壤中的二氯喹啉酸进行前处理,建立了二氯喹啉酸在土壤中残留分析的高效液相色谱（HPLC）方法。当土壤中二氯喹啉酸添加浓度为0.05～1.0mg/kg时,采用优化的QuEChERS方法进行前处理的二氯喹啉酸添加回收率在90.17%-95.27%范围内,相对标准偏差在1.94%3.22%之间,能满足常规残留分析的要求,同时,采用QuEChERS方法可以大量减少有机溶剂的使用,且操作步骤比较简单,因而更适合用作二氯喹啉酸残留检测的常规前处理方法。采用配有紫外检测器（UV Detector）的岛津高效液相色谱仪（LC-20AT）对二氯喹啉酸的残留进行检测,发现低剂量（推荐剂量）下二氯喹啉酸的消解方程为Cx=0.984.e-0.024t,相关系数为0.9719,半衰期28.88 d；高剂量（2倍推荐剂量）下二氯喹啉酸的消解方程为Cx=1.5518.e-0.026t,相关系数为0.9905,半衰期为26.65 d。室内生测结果表明,二氯喹啉酸对烟草药害的主要症状表现为叶片扭曲畸形,在1mg/kg的浓度下烟草的生长完全停止并最终死亡。对其它几种后茬作物药害临界浓度的测定结果表明：二氯喹啉酸对番茄、辣椒的药害临界浓度为104mg/L；对胡萝卜、芹菜的药害临界浓度为10-5mg/L；对黄瓜的药害临界浓度为10-3mg/L。几种作物对二氯喹啉酸的敏感程度从高到低依次为：胡萝卜、芹菜、辣椒、番茄、黄瓜。由此可见,二氯喹啉酸对几种作物的生长均存在不同程度的抑制作用,在进行水旱轮作时,应根据二氯喹啉酸田间实际残留浓度合理选择轮作作物,减少除草剂残留药害的产生。采用无机盐平板划线法,从受二氯喹啉酸药害胁迫的烟草根系分离得到一株对二氯喹啉酸具有较好降解效果的内生降解菌,标记为Q3,Q3为革兰氏阳性菌。通过形态学特性、生理生化特性分析、biolog鉴定以及16S rDNA序列同源性分析,最终将菌株Q3鉴定为巨大芽孢杆菌（Bacillus megaterium）。抗生素敏感性测试表明,该菌对青霉素及氨苄青霉素显示抗性。研究了二氯喹啉酸起始浓度、培养温度、培养基pH、接种量、摇床转速以及降解时间等因素对菌株Q3生长及降解二氯喹啉酸效果的影响。结果表明：适合Q3生长及降解二氯喹啉酸的最佳条件为：二氯喹啉酸起始浓度20mg/L、培养温度30℃C、pH 8.0、接种量6%、摇床转速180 rpm,在最佳培养条件下,菌株Q3在降解第7d对二氯喹啉酸的降解效果可达93%。采用HPLC-MS/MS方法对二氯喹啉酸的微生物降解产物进行了分析鉴定。结果表明：在二氯喹啉酸的微生物降解过程中,共检测到3种降解产物,分别为3,7-二氯-8-甲基喹啉、3-氯-8-羧酸喹啉、8一羧酸喹啉。通过降解产物推测菌株Q3对二氯喹啉酸的微生物降解途径可能是通过羧基还原作用及脱氯作用进行的。采用室内盆栽试验及田间小区试验,研究了二氯喹啉酸胁迫下内生降解菌Q3对烟草药害的修复效果,同时,分析了二氯喹啉酸胁迫下内生降解菌Q3对烟草植株内抗氧化酶等保护酶系（SOD、CAT、POD、APX等）活性以及MDA含量的影响。盆栽试验结果表明：在移栽后30d,菌株Q3生物修复组（Quinclorac+Q3）的叶长、叶宽及株高恢复到了空白对照的89.49%、92.14%及93.64%；移栽后45d,分别恢复到了空白对照的93.40%、91.70%及92.29%。田间小区试验结果表明：在菌株处理后11d,菌株Q3生物修复组（Quinclorac+Q3）的叶长、叶宽及株高恢复到了空白对照的90.49%、90.69%及88.95%；在菌株处理后22d,恢复到了空白对照的96.92%、93.83%及92.58%,盆栽试验和田间小区试验的结果均显示Q3对烟草二氯喹啉酸药害具有良好的恢复效果。对烟草抗氧化酶系活力测定的结果表明,在二氯喹啉酸胁迫下,内生降解菌Q3还可以提高烟草植株内保护酶系SOD、CAT、POD以及APX等酶的活性,并降低烟草植株内MDA的含量,从而减轻二氯喹啉酸对烟草的药害。采用第三代单分子实时测序系统PacBio RS平台对Q3菌株进行了全基因组测序,得到了其全基因组序列。测序原始数据经过过滤后,采用HGAP软件进行拼接,拼接序列进行反复校正,最终得到了Q3菌株的全基因组图谱。Q3的全基因组由一个大小为5,153,539 bp的环状染色体及一个大小为76,260 bp的环状质粒组成。染色体GC含量为38%。经PROKKA软件进行基因组注释后,共预测得到5,264个编码蛋白基因,172个RNA基因（包括130个tRNA基因和42个rRNA基因）。经与其它报道的巨大芽孢杆菌的全基因组比较,发现在巨大芽孢杆菌Q3的全基因组中,共有4694个核心基因,294个泛基因,216个特异基因。Q3菌株中含有1个质粒（Q3_p1）,与菌株WSH-002中的质粒WSH-002_p1 （NC₀17139）同源,与WSH-002_p1质粒相比,Q3_p1只有个基因BACMQ3₀5432在WSH-002_p1中没有相似序列,为其特有基因。质粒Q3_p1中含有一套完整的rRNA基因集,还含有17个tRNA基因,组织结构与chromosome类似。更多还原

【Abstract】 Quinclorac is a new auxin herbicide developed by BASF,and mainly used for controlling barnyard grass and certain dicot grasses in paddy field. Due to the low dosage, long lasting period and special effective control of barnyard grass, quinclorac is widely used in rice field. However, due to high stability of quinclorac, degradation of quinclorac in nature is very slow and its residence time is long,which will bring potential hazard to the envioronment and cause residual phytotoxicity to sensitive crops.Especially in some areas of rice-tobacco rotation field, residues of quinclorac cause serious phytotoxicity to sensitive tobacco and have a serious effect on yield and quality of tobacco.In this paper,residue detection method of quinclrac in soil and its degradation dynamics were studied, and phytotoxicity caused by quinclorac to sensitive crops was determined;An endophytic quinclorac-degarading bacteria was isolated from root of tobacco grown in quinclorac contaminated soils and strain Q3 was identified based on morphological characteristics, bio log identification and 16S rDNA gene sequence analysis.Then the degradation characteristics of Q3 was studied. The microbial degrading products of quinclorac were analyzed and identified by liquid chromatography-tandem mass spectrometry （HPLC-MS/MS） and possible degrading pathway of quinclorac was proposed.Bioremediation effect of Q3 on tobacco phytotoxicity was studied through pot experiment and field experiment,then effect of Q3 on activity of tobacco protective enzymes was discussed,which could provide theoretical basis for its field application.The whole genome DNA of the quinclorac degrading strain Q3 was sequenced and preliminary analysis was conducted, which would provide theoretical basis for study molecular mechanism of microbial degradation of quinclorac. The results were as follows:With simplified QuEChERS method for pretreatment of quinclorac in soil,HPLC method for detection of quinclorac in soil was established.When the addition concentration of quinclorac was 0.05～1.0mg/kg, recovery of quinclorac with QuEChERS method was between 90.17%～95.27%, and the relative standard deviation（RSD） was between 1.94%～3.22%,which could meet the requirement of regular residue analysis. Meanwhile, the operating procedure of QuEChERS method was simple and less organic solvent would be used,so the QuEChERS method is more suitable for pretreatment of quinclorac in soil.Quinclorac residue in soil was detected by HPLC-20AT equipped with a UV detector. The results showed that the degradation dynamics equation of low concentration（Recommended dose） of quinclorac was Cx=0.984.e-0.024t with a correlation index of 0.9719,and the half life was 28.88d; While the degradation dynamics equation of high concentration（Double recommended dose） of quinclorac was Cx=1.5518.e-0.026t with a correlation index of 0.9905,and corresponding half life of quinclorac was 26.65 d.The results of indoor bioassay indicated that quinclorac caused phytotoxicity symptom to tobacco is mainly leaf twist, and growth of tobacco would stop and die at last under quinclorac residue concentration of 1mg/kg. The results also showed that the critical concentration causing tomato and pepper phytotoxicity was 10-4mg/L, while it was 10-5mg/L for carrot and celery, and 10-3mg/L for cucumber. The sensibility level to quinclorac from high to low were carrot, celery, pepper, tomato and cucumber. The results indicated that quinclorac has a certain inhibiting effect on growth of these crops, so logical choice of rotation crops according to actual quinclorac residue concentration may help reduce phytotoxicity caused by herbicide residue.With mineral salt streak plate method, an endophytic quinclorac-degarading bacteria named Q3 was isolated from root of tobacco grown in quinclorac contaminated soils. Strain Q3 was a gram-positive bacterium. Based on morphologic properties, physiological-biochemical characteristics, biolog identification and 16S rDNA gene sequence analysis,strain Q3 was finally identified as Bacillus megaterium. Antibiotic sensibility test indicated that strain Q3 was resistant to penicillin and penbritin.Effect of initial quinclorac concentration, culture temperature, culture pH, inoculation size, rotate speed of the shaker and degradation time on Q3 growth and its degrading efficience were studied. The results showed that the optimum condition for Q3 growth and quinclorac degradation were as follows:initial quinclorac concentration of 20 mg/L, culture temperature of 30℃, culture pH of 8.0, inoculation size of 6%（Volume ratio）, shaker rotate speed of 180 rpm. At optimum conditions, more than 93% of quinclorac could be degraded by Q3 in 7 days.Microbial degrading products of quinclorac were analyzed and identified by HPLC-MS/MS. The results showed that three degradation products in all were detected in the microbial degradation process of quinclorac, which were respectively 3, 7-dichloro-8-methyl-quinoline,3-chlorin-8-quinoline-carboxylic and 8-quinoline-carboxylic. Based on the results of mass spectrum, we hypothesized that strain Q3 may employ multiple pathways to degrade quinclorac. One possible pathway is through the reduction of carboxyl and the other pathway is through dechlorination of quinclorac.Bioremediation effect of strain Q3 on tobacco phytotoxicity caused by quinclorac was studied through pot experiment and field plot experiment. Meanwhile, effect of Q3 on activity of tobacco protective enzyme（including SOD, CAT, POD and APX） and MDA content was analyzed. The results of pot experiment showed that leaf length, leaf width and plant height of tobacco with Q3 application were recovered to 89.49%,92.14% and 93.64% of those of control 30 days after transplanting; and 93.40%,91.70% and 92.29% 45 days after transplanting. The results of field plot experiment showed that leaf length, leaf width and plant height of tobacco with Q3 application were recovered to 90.49%,90.69% and 88.95% of those of control 11 days after Q3 application;and 96.92%、93.83% and 92.58% 22 days after Q3 application. Both pot experiment and field plot experiment showed that strain Q3 had perfect bioremediation effect against tobacco phytotoxicity caused by quinclorac. Meanwhile, strain Q3 could also improve the activity of tobacco protective enzymes such as SOD, CAT, POD and APX, and decrease the content of MDA in tobacco, thus alleviated the tobacco phytotoxicity caused by quinclorac.The whole genome of Bacillus megaterium Q3 was sequenced by PacBio RS system and whole genome sequence was obtained. After filtering, the original data of sequencing was jointed by HGAP software,then jointed sequence was adjusted repeatedly and the whole genome draft of Q3 was obtained. The Bacillus megaterium Q3 genome consists of a circular chromosome（5,153,539 bp） and a circular plasmid（76,260 bp）. GC content of the Q3 chromosome was 38%. After genome annotation by PROKKA software, the Bacillus megaterium Q3 genome encodes 5264 protein genes,172 RNA genes（including 130 tRNA genes and 42 rRNA genes）. Compared with genomes of other reported Bacillus megaterium strains, there are 4694 core genes,294 pan genes and 216 special genes in the genome of Bacillus megaterium Q3. There is a plasmid Q3_pl in the genome of Bacillus megaterium Q3,which is homologous with the WSH-002_p1（NC₀17139） plasmid in the genome of strain WSH-002. Compared with the WSH-002_p1 plasmid, there is only one gene BACMQ3₀5432 that is the special gene of strain Q3. Plasmid Q3_p1 has a complete rRNA gene set and 17 tRNA genes,and its structure is similar to the chromosome.更多还原