聚合酶链反应-变性梯度凝胶电泳分析在毒死蜱胁迫下土壤真菌群落结构演替

Analyses of soil fungal community succession under chlorpyrifos stress using PCR-DGGE

为评价毒死蜱施用后对土壤真菌群落结构的影响,将农田土壤用终质量分数5mg/kg毒死蜱处理,同时以不施用毒死蜱为对照,定期采集土样进行毒死蜱残留测定并提取土壤DNA,采用基于染色体内转录间隔区(internaltranscribed spacers,ITS)ITS1f-GC/ITS2和28SrDNA U1-GC/U2引物对进行聚合酶链反应-变性梯度凝胶电泳,获得的指纹图谱利用Quantity One软件进行数字化分析,计算样品的多样性指数(H),并用Matlab软件进行主成分分析.ITS1f-GC/ITS2和U1-GC/U2扩增区域分析结果表明,毒死蜱施用后第7～60天内土壤真菌群落结构与对照相比发生很大改变,且毒死蜱处理样品真菌群落结构保持基本稳定,多样性指数明显提高;主成分分析显示,毒死蜱处理样品与对照明显分在2个不同的区域,充分表明毒死蜱对土壤真菌群落结构影响迅速且持久.

Chlorpyrifos（CP）is a broad-spectrum,moderately toxic organophosphorus insecticide.It is widely used throughout the world,especially in recent years with the restrictions or eliminations of highly toxic organophosphorus compounds.The fate of CP after a single application in soil and its effects on soil microbial population,microbial functional diversity,microbial respiration,and enzyme activities have been well investigated.However,the influence of CP application on the soil fungal community succession remains unknown.Denaturing gradient gel electrophoresis（DGGE） is currently used in the characterization of soil bacterial and fungal community.DGGE analysis can provide a picture composed of an array of bands with different intensities,and the banding patterns of DGGE are analyzed using Quantity One V4.62software（Bio-Rad）.An Excel worksheet was used to calculate the Shannon-Wiener index（H）,and Matlab software（MathWorks） was performed to do principal component analysis（PCA） of DGGE data.The aim of this study was to assess the effect of CP application on soil fungal community using PCR-DGGE based on internal transcribed spacer（ITS） region and 28SrDNA amplification products.After preincubation in the dark for one week at 25℃,CP was applied to soils with a predetermined volume of their commercial formulation,following proper dilution with distilled water,to give a final concentration of 5 mg / kg corresponding to the recommended dose.Soil samples received the same amount of sterilized water without CP were used as controls.Each treatment was conducted thrice,and a total of six microcosms were prepared.At 1,7,15,30,45,and 60dafter CP treatment,soil samples were collected to detect CP residue in the soil and to extract soil DNA for polymerase chain reaction（PCR）-DGGE analysis.The degradation curve of CP in soil showed that the CP residues in the soil samples were significantly decreased to 2.086,0.355,and 0.008mg / kg respectively after 7,30and 60dof CP treatment.The degradation of CP in soil was interpreted with the first-order kinetics,y=5.357e-0.09x,R 2 =0.905.The half-life of CP was 7.99dcalculated by firstorder function.ITS1f-GC / ITS2 and U1-GC / U2 primer pairs were used to amplify ITS region and 28SrDNA respectively,and thereafter DGGE was used to further detect fungal community.DGGE fingerprint showed that the composition of fungal community was obviously changed by CP treatment,and the Shannon-Wiener diversity index（H） was increased and kept in higher level since 7dafter CP treatment.PCA analysis showed that the samples of CP treatment were significantly separated from those of controls.In conclusion,our data and research demonstrate that the CP application can affect the soil fungal community in a quick and persistent way.