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

Background and Methods Assessing 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 Conclusion Treatment 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.研究背景与方法 当前评估农药危害对土壤微生物过程的影响，多采用仅能有限揭示原位发生过程的分析方法。本研究提出一种更为全面的研究策略。农药的副作用可表现为特定微生物基因的表达变化，或群落多样性的改变。为评估农药对基因表达的影响，我们聚焦于参与氨氧化过程的amoA基因（amoA）。我们构建了土壤微宇宙体系，分别施加棉隆（dazomet）、代森锰锌（mancozeb）或不施加农药作为空白对照。我们提出假说：农药施用后amoA基因的转录本数量会下降，并通过逆转录定量PCR（reverse-transcription qPCR）验证该假说。我们同时提出另一假说：农药会影响细菌群落多样性。针对该假说，我们分别针对16S核糖体RNA（16S rRNA）与16S rRNA基因开展测序及多样性分析，二者分别代表活性土壤细菌群落与总土壤细菌群落。 研究结果与结论 棉隆处理可使细菌与古菌的amoA基因转录本数量均降低两个数量级以上，且效应持续时长超过28天。代森锰锌同样可抑制amoA基因转录本数量，但仅产生短暂效应。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物。此外，在添加氮源的土壤微宇宙体系中，细菌amoA基因转录本数量与硝酸盐生成量呈显著相关。棉隆可使总细菌数量降低一个数量级，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在12天后似乎得以重建。然而，在第12天，基于16S核糖体RNA基因序列反映的总细菌群落多样性仍以厚壁菌门（Firmicutes）和变形菌门（Proteobacteria）为主，这大概率反映了早期机会主义菌群的生长停滞，以及更为多样的群落得以重建。我们未观察到代森锰锌对群落多样性产生任何影响。