【作者】 张晶；

【导师】 杨清香；

【作者基本信息】 河南师范大学 ， 微生物学， 2009， 硕士

【摘要】 本研究以小麦作为研究对象,选择应用最广泛的兽药抗生素-四环素和土霉素,研究了兽药抗生素暴露情况下小麦根际微生物群落结构的变化和小麦苗期生长所受到的影响,并对抗生素污染状况下小麦抗性内生细菌的种类及种属分布进行了初步探索,为抗生素的合理使用提供了科学依据。研究所取得的主要结论有以下三方面:(一)表征无抗生素暴露条件下小麦根际微生物群落的结构特征及优势细菌鉴定:小麦根际土壤中微生物总数达到5×108cfu g-1,约为非根际土壤的二倍。细菌比例最高,放线菌次之,真菌最低;从中分离出10株数量优势细菌,经16S rRNA鉴定后与芽孢杆菌属(Bacillus)有最大相似性;优势菌的生长在低浓度四环素和土霉素存在时就出现抑制,对土霉素的敏感性高于四环素。(二)小麦抗性内生细菌的数量及在植株中的分布研究:1)小麦内生菌数目在1×104-1.95×106 cfu g-1之间,抗性内生菌数目介于6.9×103和5.67×105,从中分离出22株土霉素抗性内生菌,包括15株G+和7株G-,经16s rRNA基因序列分析,它们与Bacillus,Alphaproteobacteria和Gammaproteobacteria三大类微生物聚在一起,其中Bacillus类细菌最多,占59.1%。2)对这22株抗性内生菌进行土霉素敏感性试验,其最低抑菌浓度(MIC)值介于16-256ug ml-1之间;在小麦不同部位分离的抗性内生菌中,芽孢杆菌属内生菌明显占优势,其中地衣芽孢杆菌(Bacillus licheniformis)在小麦根际土壤、根、茎等器官中均分离得到,另外在植株的同一器官均分离到不同种属的抗土霉素抗性内生菌。在小麦种子中没有分离到任何土霉素抗性的内生菌。(三)水培试验系统下四环素类抗生素污染对小麦苗期生长发育的影响:1)在有效抗生素剂量暴露下(0、0.8、4、20、60、100mg l-1),小麦种子发芽率、根伸长和芽伸长对四环素和土霉素的生态毒性敏感顺序依次为:根伸长>发芽率>芽伸长,土霉素处理产生的生态毒性明显高于四环素;小麦的根伸长抑制率(或芽伸长抑制率)与抗生素浓度间均存在明显的剂量效应,20 mg l-1以上浓度抑制作用尤为显著;水培液中微生物群落在两种抗生素的选择压力下,优势类群由细菌向真菌方向转变。2)水培液细菌类群的PCR-DGGE图谱可分为两种模式:0.8和4 mg l-1水培液中微生物群落图谱与对照组相似,而高浓度(20 mg l-1、60 mg l-1、100 mg l-1)处理下DGGE图谱结果比较一致。通过对高强度条带的切胶、测序、比对,发现它们与Acinetobacter,Sphingobacterium multivorum,Chloroflexi和Bacillus等种属具有比较近的亲缘关系。更多还原

【Abstract】 Under the pressure of veterinary antibiotics, including tetracycline(OTC) and oxytetracycline(TC), The microbial community dynamic change in wheat rhiosphere and wheat growth status were investigated, Primary research also focused on the entophyte varieties and distribution in wheat, which guided the proper use of antibiotics, then to reduce the production of drug resistance stains and low their eco-toxicity. The results showed in the following:(1) The microbial community structures in wheat rhizosphere and dominant bacteria identify without antibiotic pollution: The population of total microorganisms in rhizosphere soil was 5.251×108 cfu g-1, which was almost 2 times of it in non-rhizosphere soil; bacteria, actinomyces and fungi were cultivated and the proportion was 99.04%, 0.90% and 0.06%, respectively; 10 dominant bacteria were isolated from wheat rhizosphere. All of them were grouped into genus Bacillus according to phylogenetic analysis based on their full length 16S rRNA gene sequences. Sensitivity of the 10 isolates was observed under low concentrations of oxytetracycline and tetracycline, and higher sensitivity appeared in oxytetracycline.(2) Isolation and distribution of oxytetracycline-resistance entophytic bacteria in plant of wheat:1) The CFU number and species distribution of wheat entophytic bacteria in rizosphere, root, stem, leaves and seeds were researched. The results indicated that the total amount of entophytic bacteria in wheat was between 1×104cfu g-1 and 1.95×106cfu g-1, in which the antibiotic resistance entophytic bacteria were from 6.9×103 to 5.67×105. 22 strains of oxytetracycline resistant entophytic bacteria were isolated, including 15G+ and 7G.-. The 22 isolates were clustered into Bacillus, Alphaproteobacteria and Gammaproteobacteria based on their full length 16S rRNA gene sequences, in which strains of Bacillus occupied 59.1%.2) The minimum inhibition concentration (MIC) values of the 22 entophytic bacteria to oxytetracycline were between 16 ug ml-1 and 256 ug ml-1. Comparing the entophytic bacteria in different organs of wheat; strains belonging to Genus Bacillus were obviously predominant. Especially Bacillus licheniformis, it was isolated from wheat rizosphere, root and stem. Different species of bacteria could be isolated from the same organ in wheat. However, no oxytetracycline resistant bacterium could be detected from wheat seeds. (3) Influence of tetracycline antibiotics on the growth of wheat and microbe dynamic change under the condition of water solution culture:1) Oxytetracycline showed higher eco-toxicity than tetracycline with the designed concentrations(0, 0.8, 4, 20, 60, 100 mg l-1)according to root elongation and shoot elongation variance analysis results; Sequence orders was root elongation>shoot elongation>germinating rate. The critical value of wheat growth inhibition could be defined on 20 mg l-1 tetracycline primarily. The relation between the wheat root elongation control rate (or shoot elongation control rate) and antibiotics dosages all existed, significant inhibition appeared in the concentrations higher than 20 mg l-1. OTC and TC exerted a selective pressure on soil microbial community in form of a shift from soil bacteria to soil fungi.2) The nested PCR-DGGE was also used to evaluate community structure difference happened in DNA levels among water samples subjected to antibiotic treatment, The DGGE profile could be divided into two patterns, the 0, 0.8 and 4 mg l-1 fell into the first one, which had similar DGGE profile with the negative control, while the other three concentrations (20, 60, 100 mg l-1)made up the second group and kept consistent bacterial community. Eight DGGE bands were excised, re-amplified, sequenced and aligned with Blast. The phylogenetic analysis results indicated that they had high affinity with Acinetobacter, Sphingobacterium multivorum, Chloroflexi and Bacillusand respectively.更多还原