13C-标记秸秆添加对DNA稳定性同位素探针试验结果的影响

Effect of 13C-labled straw on the results of DNA stable isotope probing experiments

摘要:

[目的]稳定性同位素探针技术（stable isotope probing，SIP）是采用稳定性同位素示踪复杂环境中具有代谢活性微生物的有力工具。然而，在近期利用SIP技术的研究当中，我们发现¹³C-标记物对试验本身有一定程度影响。例如研究土壤秸秆降解微生物，需将¹³C-标记作物秸秆添加到土壤，利用微域培养实验和DNA-SIP技术解析主导降解微生物物种。但是¹³C秸秆的添加以及不同土壤肥力水平是否会影响土壤微生物群落有待商榷。[方法]本研究采集江西鹰潭红壤试验站3种施肥处理（Control、NPK、OM）水稻土壤，分别添加自然丰度（¹²C）和¹³C-标记的高丰度水稻秸秆，进行微域培养试验，研究两种秸秆添加下的响应物种以及不同丰度C对生物质气体的累积排放、细菌α-多样性以及群落结构的影响。[结果]研究发现，3种施肥土壤下，2种丰度秸秆处理间C累计排放无差异。但是，寡营养条件（Control）下，¹³C-标记秸秆处理的细菌α-多样性高，¹²C秸秆处理群落异质性高，稳定性较差，无差异性物种；与¹²C秸秆处理相比，富营养条件（NPK和OM）下，¹³C-标记秸秆处理的细菌α-多样性和群落结构无差异，但存在差异物种，主要集中于变形菌门和稀有物种。[结论]本研究的结果表明¹³C标记秸秆对微生物群落有一定影响，因此在后续的SIP试验中，高丰度秸秆虽可被用来作为标记底物，但需慎用。

Abstract:

[Objective] DNA-based stable isotope probing (DNA-SIP) is a powerful tool for tracing substrate assimilated microorganisms in complex environments. However, in recent studies involving SIP, we found that ¹³C utilization may interfere with experiment results. For example, when microcosm and DNA-SIP are applied to study straw degradation microorganism, ¹³C-labled straw is bound to be amended, but whether the amendment as well as the fertilization of soil will affect the microbial communities is still unknown.[Methods] We sampled three fertilized (Control, NPK, OM) paddy soil from Yingtan red soil ecological experimental station to study the difference of biogas emission, microbial diversity and responding species among three fertilized soils and two kinds of straw. Microcosms were conducted with ¹²C and ¹³C-labled straw amendments afterwards.[Results] We found that no difference of accumulative C emission was detected among three soils with ¹²C and ¹³C-labled straw amendments. Under oligotrophic (Control) conditions, soil bacterial communities of ¹³C-labeled treatment represented a higher diversity while microbial heterogenicity was higher in ¹²C-labeled treatment, and discrepant species were hardly detected between these two treatments. Under copiotrophic (NPK and OM) conditions, bacterial communities of ¹³C-labeled straw treatment performed no difference on both their diversity and structure, but discrepant species were detected, mainly belong to Proteobacteria and rare species. [Conclusion] Our results showed that ¹³C-labeled straw did affect bacterial communities to some extent. Hence, as labeling substrate, high abundance straw can be applied, but it need to be cautious in the following SIP experiments.