Pesticide Side Effects in an Agricultural Soil Ecosystem as Measured by amoA Expression Quantification and Bacterial Diversity Changes

Background and MethodsAssessing the effects of pesticide hazards on microbiological processes in the soil is currently based on analyses that provide limited insight into the ongoing processes. This study proposes a more comprehensive approach. The side effects of pesticides may appear as changes in the expression of specific microbial genes or as changes in diversity. To assess the impact of pesticides on gene expression, we focused on the amoA gene, which is involved in ammonia oxidation. We prepared soil microcosms and exposed them to dazomet, mancozeb or no pesticide. We hypothesized that the amount of amoA transcript decreases upon pesticide application, and to test this hypothesis, we used reverse-transcription qPCR. We also hypothesized that bacterial diversity is affected by pesticides. This hypothesis was investigated via 454 sequencing and diversity analysis of the 16S ribosomal RNA and RNA genes, representing the active and total soil bacterial communities, respectively.Results and ConclusionTreatment with dazomet reduced both the bacterial and archaeal amoA transcript numbers by more than two log units and produced long-term effects for more than 28 days. Mancozeb also inhibited the numbers of amoA transcripts, but only transiently. The bacterial and archaeal amoA transcripts were both sensitive bioindicators of pesticide side effects. Additionally, the numbers of bacterial amoA transcripts correlated with nitrate production in N-amended microcosms. Dazomet reduced the total bacterial numbers by one log unit, but the population size was restored after twelve days. The diversity of the active soil bacteria also seemed to be re-established after twelve days. However, the total bacterial diversity as reflected in the 16S ribosomal RNA gene sequences was largely dominated by Firmicutes and Proteobacteria at day twelve, likely reflecting a halt in the growth of early opportunists and the re-establishment of a more diverse population. We observed no effects of mancozeb on diversity.背景与方法 当前针对农药危害对土壤微生物过程影响的评估，多依赖仅能对土壤中正在进行的过程提供有限认知的分析手段。本研究提出一种更为全面的研究方法。农药的副作用可能表现为特定微生物基因的表达变化，或是群落多样性的改变。为评估农药对基因表达的影响，本研究聚焦于参与氨氧化过程的amoA基因（amoA gene）。我们构建了土壤微宇宙（soil microcosms）体系，分别施加威百亩（dazomet）、代森锰锌（mancozeb），并设置未施加农药的空白对照组。我们提出假说：农药施用后amoA基因的转录本数量会出现下降，并通过反转录定量PCR（reverse-transcription qPCR）对该假说进行验证。此外，我们还提出另一假说：农药会对细菌群落多样性造成影响。该假说通过对16S核糖体RNA（16S rRNA）及核糖体RNA基因的454焦磷酸测序（454 sequencing）与多样性分析得以验证，二者分别代表土壤中的活性细菌群落与总细菌群落。 结果与结论 威百亩处理组的细菌与古菌amoA基因转录本数量均下降超过2个对数单位，且其长效影响可持续28天以上。代森锰锌同样会抑制amoA基因的转录本数量，但仅表现出短暂的抑制效果。细菌与古菌的amoA基因转录本均可作为农药副作用的灵敏生物指示剂。此外，在添加氮源的土壤微宇宙体系中，细菌amoA基因转录本的数量与硝酸盐生成量呈显著相关。威百亩使总细菌的数量下降1个对数单位，但菌群规模在12天后得以恢复。活性土壤细菌的群落多样性也似乎在12天后得到重建。然而，通过16S核糖体RNA基因序列反映的总细菌群落多样性，在第12天时主要由厚壁菌门（Firmicutes）与变形菌门（Proteobacteria）主导，这大概率反映了早期机会性微生物生长的停滞，以及更为多样的菌群群落的重建。我们未观察到代森锰锌对群落多样性产生任何影响。