| Chlorpyrifos,a highly effective organophosphate insecticide,is widely used in agricultural production.However,its excessive and irrational application may cause the occurrence of agricultural residues in the soil.The residual chlorpyrifos in the soil is taken up and bioaccumulated in plants,which may be harmful in the safety of farm produce and human health.Soil bacterial community is an important part of soil micro-ecology;studies have shown that its diversity can respond to chlorpyrifos pollution.Previous investigations have revealed that some bacterial populations can promote the degradation of chlorpyrifos in the soil,however,the bacterial community diversity and structure in different soils types are unique,and their differences in response to chlorpyrifos contamination remains to be studied.At present,PCR-DGGE has been commonly used to analyze the soil bacterial community.However,it is still difficult to fully and objectively reveal the diversity and dynamic changes of bacterial communities in the soil due to the limitations of PCR-DGGE.In recent years,given the biotechnological advances in the next-generation sequencing(NGS)technologies,such as the high-throughput sequencing Illumina Miseq platform,it has been used to analyze the dynamic changes in bacterial community structure and diversity in some contaminated soils.However,whether the advantages of Illumina Miseq technology can be used in different types of chlorpyrifos contaminated sites are yet to be understood.In this study,chlorpyrifos was used as the model pollutant.Based on the optimized analysis of chlorpyrifos residue in the soil,pot experiments were carried out to study the residual dynamics of chlorpyrifos in red and purple soils.With the help of modern molecular biology methods such as PCR-DGGE and high-throughput sequencing(Illumina Miseq),the dynamic changes in the soil bacterial community structure and diversity after chlorpyrifos contamination were primarily investigated,and the response of related dominant microflora to chlorpyrifos pollution was analyzed.It attempts to provide a basis for revealing the effects of conventional pesticides on soil bacterial communities,rational application of pesticides,maintenance of soil environmental quality,and safety of agricultural products.The main research results are as follows:(1)The analytical method for chlorpyrifos residues in soil was optimized.The petroleum ether-acetone(V:V=1:1)was used as the extractant.After ultrasonic extraction,the extract was purified by chromatography column(anhydrous sodium sulfate and silica gel),and the collected filtrate was concentrated by rotary evaporation and re-dissolved in n-hexane before being analyzed by high performance liquid chromatography(HPLC).The optimized liquid phase conditions were as follows:gradient elution with acetonitrile:water(V:V=9:1)as mobile phase,a flow rate of 0.6 mL/min,an ultraviolet(UV)detection wavelength at 292 nm,and a column temperature of 40℃;The detection limit of chlorpyrifos in HPLC is 0.01 mg/L.Additionally,the recovery rate was used to test the for the feasibility of the chlorpyrifos analysis method in the soil:at the three levels of 0.02,0.2 and 2 mg/kg,the average recoveries of chlorpyrifos in the soil were 113.10%,117.42%,and 89.41%,respectively.The variable coefficient were 8.75%,3.32%,and 2.93%,respectively.The results indicate that the optimized analytical method for this parper can be used in the residue detection of chlorpyrifos in contaminated soil samples.(2)The pot experiment method was used to reveal the residual dynamics of chlorpyrifos in different types of soil.The red paddy soil of Yingtan City,Jiangxi Province and the purple paddy soil of Yanting County of Sichuan Province were used as potting soils with different physicochemical properties.In rice pot experiments from May to October 2017,the initial application concentrations of chlorpyrifos were 300,600,1500,2400 and 3000 g/ha.On the first day of sampling,the residues of chlorpyrifos in the red paddy soil were 1.41,2.70,2.44,4.55 and 2.80 mg/kg,respectively,while the residues of chlorpyrifos in the purple paddy soil were 1.63,0.96,0.85,1.63 and 1.92 mg/kg,respectively.With the increase in the sampling time,the chlorpyrifos residues in both red and purple paddy soils gradually decreased.On the 28th day,chlorpyrifos residues were not detected in the red and purple paddy soil treated with 300,600 and 1500 g/ha,and the residues were 0.10 and 0.30 mg/kg in the red soil potted plants treated with concentrations of chlorpyrifos at 2400 and 3000 g/ha,and the residual concentrations in the purple paddy soil pots were 0.24 and 0.46 mg/kg.On the 56th day,no residues of chlorpyrifos were detected in all potted soils treated.(3)The effects of chlorpyrifos pollution on the dynamic changes of bacterial community structure and diversity in paddy soil were studied by PCR-DGGE and high-throughput sequencing techniques.The PCR-DGGE map can intuitively reflect the changes of soil bacterial communities after contamination with different chlorpyrifos concentrations.The results showed that the number of bands in the purple paddy soil was higher than that in the red soil when no pesticide was applied,i.e.,the richness of bacterial community in the purple paddy soil was higher than that in the red soil.After application,the diversity of bacterial communities in both soils showed a pre-existing period,whereby a trend of decline was observed before a gradual recovery.High-throughput sequencing results further confirmed that the purple soil species richness(OTU=2000)was higher than that of red soil(OTU=1500).Proteobacteria was the main genus in both types of paddy soils,with their proportion in the red and purple paddy soils prevailing at 26.9%and 42.3%,respectively;in addition,High-throughput sequencing also revealed that the genera of chlorpyrifos metabolizing bacteria in paddy potted soils mainly includes Sphingomonas,Bacillus and Flavobacterium.In the red paddy potted soil,when no pesticide is applied,the relative abundances of Sphingomonas,Bacillus and Flavobacterium strains were 0.27%,0.68%and 0.38%,respectively.At application concentrations of 600,1500 and 2400 g/ha,with increasing application concentrations,the relative abundance of Sphingomonas(0.21%,0.25%and 0.18%)and Bacillus(0.85%,0.50%and 0.56%)were reduced;when the application concentration were 600 and 1500 g/ha,the relative abundance of Flavobacterium in the soil had increased up to 1.19%and 1.14%,respectively;when the application concentration was 2400 g/ha,the relative abundance of Flavobacterium reduced significantly to 0.12%.In purple paddy soil,when no pesticide is applied,the relative abundances of Sphingomonas,Bacillus and Flavobacterium were 0.52%,1.0%and 0.19%,respectively.The increased application concentrations of 600,1500 and 2400 g/ha led to an increase in the relative abundance of Sphingomonas(0.96%,0.80%and 0.58%)and Bacillus(0.57%,0.97%and 1.63%),while reducing the relative abundance of Flavobacterium(0.17%,0.10%,and 0.10%)in purple soil. |
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