丛枝菌根真菌介导氧化亚氮减排的作用机制

The diversity of arbuscular mycorrhizal fungi (AMF) is remarkably reduced in chinese intensive agriculture due to the high input of external chemical resources. Consequently, the delivery of ecosystem functions by AMF is greatly suppressed. Roots of major crops are well colonized by AM fungi. The extensive extraradical mycelia of AMF have the potential to reduce the nitrous oxide emissions. However, the mechanism and the key affecting factors are not fully elucidated. We propose a new conceptual strategy of “reducing N2O emissions by AMF, and improving nitrogen use efficiency by enhance the ecological functions of indigenous AMF”. The current research project is aimed to understand the contribution of mycorrhiza and external hyphae to the reduction of nitrous oxide emissions by integrating the knowledge from both the laboratory and field experiments. The techniques, including microbial molecular ecology, isotopic labelling, metagenomics and meta-transcriptomics, will be employed. The role of mycorrhiza and external hyphae in reducing N2O emissions in response to inorganic nitrogen supply, rhizobia and plant residues will be investigated. The regulation of nitrification and denitrification processes by AMF, and the underlying mechanisms, including the mycosphere and hyphosphere effects, e.g. the alteration of physical, chemical and especially microbial properties will be unraveled. The key affecting factors will be elucidated. The regulation of AMF in the decomposition of degraded nodules will be evaluated to investigate the potential interactions between AMF and rhizobia in the capture of available nitrogen and N2O emissions. The coupling of microbial C-N-P in the decomposition of plant residues will be studied by means of metagenomics and meta-transcriptomics. Field experiments will be conducted to assess the reduction of N2O emissions by the indigenous AM fungi. Within the framework of agriculture green development, this project provides theoretical and scientific evidence for the potential to stimulate rhizosphere beneficial microbiomes to reduce N2O emissions while simultaneously improve nutrient use efficiency.

我国高投入集约化农业生产降低了土壤中有益微生物丛枝菌根真菌的多样性，严重影响其生态系统功能的发挥。绝大多数作物根系能被丛枝菌根真菌侵染，菌根庞大的地下菌丝网络可减少N2O排放，但其减排的作用机制及关键影响因子尚不明确。本项目提出“以菌减排、强菌增效“的新思路，针对无机氮供应、根瘤衰老和豆科残体释放N2O的问题，结合室内和大田试验，采用微生物分子生态学、同位素标记、宏基因组和宏转录组方法，明确菌根和菌丝际通过硝化和反硝化过程影响N2O排放的作用，理解相关物理、化学尤其是生物学机制，摸清主要影响因子；探究菌根降低根瘤衰老过程N2O排放的作用及根瘤菌与菌根菌的互作机制；利用宏基因组和宏转录组解析根际/菌丝际微生物C-N-P代谢的耦合作用；通过田间原位试验，综合评价土著菌根真菌减排的作用潜力。在农业绿色发展新命题下，项目为强化根际有益微生物互作提高资源效率进行绿色减排提供重要的理论和科学依据。

作物残体还田可以提高土壤养分，增加土壤有机碳等，因此是一项重要的农业生产措施。然而作物残体尤其是豆科作物收获后，在根茬区出现一个N2O的排放高峰，这将部分抵消豆科固氮带来的益处。因此如何降低豆科根茬区N2O排放量，对缓解气候变化，促进农业绿色发展具有重要指导意义。丛枝菌根真菌（Arbuscular mycorrhizal fungi，简称AMF）可以与陆地上约80%的植物形成共生体，菌根纤细的根外菌丝在土壤中大量延伸生长，帮助植物吸收磷和氮等养分。最新的研究发现，菌丝能从作物残茬和衰老根瘤中吸收氮素，还能通过影响硝化和反硝化过程调控N2O排放，但其作用机制还不清楚。为系统解析AMF介导菌丝际微生物减少残体热区N2O排放的机制，本研究首先采用盆栽试验，利用功能基因高通量测序和培养组学技术，通过动态监测根茬区N2O排放以及该区域反硝化微生物丰度和群落结构对AMF根外菌丝的响应，发现AMF根外菌丝通过招募和富集nosZ型反硝化细菌尤其是假单胞菌，形成区别于土体的细菌群落结构，且上调了nosZ基因表达，促进了N2O还原过程，从而减少了蚕豆根茬区N2O排放（减排量最高达63%）。为进一步解析AMF和菌丝际微生物互作调控N2O排放的机制，开展了系列的纯培养和回接菌试验。通过分离纯化蚕豆根茬斑块中AMF富集的反硝化菌、收集AMF菌丝分泌物，在纯培养条件下研究菌丝分泌物对反硝化优势菌的趋化和N2O排放的影响，发现菌丝分泌物中的羧酸盐是触发所分离荧光假单胞菌菌株JL1 nosZ基因上调并减缓净N2O排放的主要物质。进而将JL1重新接种至灭菌后的残留斑块中进行验证，发现添加柠檬酸后，JL1的N2O减排效果与原位菌丝处理相当。最后开展了大田效应试验，在11年的玉米/蚕豆间作以及蚕豆间作试验田进行了大田取样分析，进一步验证了菌丝密度与nosZ基因丰度之间存在显著正相关性。研究结果表明加强AMF与菌丝际微生物之间的协同互作能够缓解豆科根茬区的N2O排放。该研究为挖掘和利用土壤中有益微生物资源，开发合成菌群调控反硝化微生物降低温室气体排放提供了一条有效的途径。