Screening And Identification Of Quinclorac Degrading Bacteria And Research On Its Degradation Characteristics

Quinclorac(QNC)is a highly selective,hormonal,and low-toxic herbicide with a long duration.It is mainly used to control barnyard grass in paddy.The growth and development of subsequent crops are easily affected by QNC-accumulated in the soil.In addition,the growth and development of animals will be affected by the residual QNC in the environment,and the community structure and the abundance of microorganisms will also be affected.In order to solve the problem of QNC-polluted rice fields,the isolation,identification and application of QNC degrading bacteria were carried out to explore the possibility of remediation of QNC contamination by QNC-degrading strains.The main research results are as follows:(1)A method was established for the determination of QNC in paddy soil,paddy water and rice by high liquid chromatography tandem triple quadrupole mass spectrometry(LC-MS/MS).Combined with the Qu EChers pre-treatment method,samples were extracted with 2%formic acid acetonitrile.,and purified by two dispersed solid phase extraction adsorbents of C₁₈and PSA.The method had good linearity in the linear range of 10.0-1000.0?g/L(R²?0.999);the limit of quantification in paddy water was 12.5?g/L,and the limit of quantification in paddy soil and rice plants was all 50?g/kg.The average recoveries of QNC at three spiked levels was 84.7%-111.5%;the relative standard deviation(RSD)was 1.1%-9.3%(n=6).This method established met the requirements of modern pesticide residues detection.Compared with the previous QNC detection method,this method simplified the operation steps of pre-treatment to extract QNC,saved the economic costs,and was suitable for the detection of QNC in paddy soil,paddy water and rice plants.(2)Using the method of enrichment culture,eight QNC-degrading strains were isolated and screened from the paddy soil.Two strains,strain 15^#and strain D,with the best degradation effect were selected.Through morphological observation,physiological and biochemical tests,phospholipid fatty acid(PLFA)identification and 16S r DNA homology comparison analysis,strain 15^#and strain D were identified as Achromobacter sp.and Cellulosimicrobium cellulans strain sp.,respectively.(3)Exploring the QNC degradation characteristics of strains through single factor experiments.Strain 15^#had the strongest degradation ability of 43.0%for 21d when the temperature is 40?,p H=6,QNC concentration is 100 mg/L,inoculation amount is 7%,yeast extract is 0.1%,and N source is peptone Strain D had the strongest degradation ability of 45.8%for 21d at a temperature of 30?,p H=6,a QNC concentration of 50mg/L,an inoculum amount of 7%,an added amount of yeast extract powder of 0.1%,and a N source of(NH₄)₂SO₄The degradation effect of strain D was better than strain 15^#.So,strain D was selected for follow-up research.(4)In order to investigate the effect of degrading bacteria D on the QNC-phytotoxicity treatment of paddy fields,the degrading bacteria D was aadded to unsterilized soil(ordinary paddy soil)and sterilized soil,and potted experiments were carried out to further verify its degradation effects and provided a theoretical basis for the practical application of QNC-degrading strain D.The results showed that in the paddy field polluted by higher concentration of QNC(750 g/hm²),the 75.45%of QNC could be degraded by bacteria D within 21 days;what's more,it could reduce the phytotoxicity of QNC to rice and promote the growth of rice.(5)Based on high-throughput sequencing technology,the effects of QNC and QNC-degrading bacteria D on the structure and function of microbial communities in the paddy field were studied by analyzing the composition of the microbial sequence of soil samples.The results showed that the relative abundance of Actinomycete bacteria decreased,and the relative abundance of Proteus and Bacteroidetes increased significantly in the treatment group with QNC.QNC had an inhibitory effect on ascomycetes,and this effect was positively correlated with the concentration of QNC,and QNC-degrading strain D could effectively reduce the inhibitory of QNC.The results of bacterial community structure diversity showed that QNC would increase the diversity of soil bacterial communities,and the addition of QNC-degrading bacteria would reduce the diversity of soil bacteria.The results of the diversity of fungal community structure showed that QNC would also increase the diversity of soil fungi.The addition of QNC and QNC-degrading bacteria had little effect on the abundance of soil fungi.By predicting the functions of soil bacteria and fungi,it could be found that QNC could inhibit some functions of bacteria and fungi,while QNC-degrading strain D could alleviate the inhibition of QNC on soil microbial communities.