| 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. |
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