浮萍根系分泌物芥酸酰胺的释放留存特征及影响反硝化的作用机理研究

Denitrification is an important pathway for agricultural nitrogen loss. The plant-microbe interaction mechanism responsible for affecting nitrogen transformation and utilization is a hot topic in the field of plant nutrition. Plant can stimulate the denitrification activity of microorganisms through root exudates. However, there is a lack of intensive study of its mechanism, and release and accumulation characteristics. With the application of gas chromatography, gas chromatography-mass spectrometry techniques and rhizosphere interactions methods, this project focuses on the plant duckweed widely distributed in agricultural area and the denitrifying bacterium Pseudomonas fluorescens. Based on the N-removal stimulants in duckweed root exudates erucamide, the exudation characteristic of erucamide, the effect of different factors, and the accumulation and biodegradation of erucamide were also investigated to clarify the release and accumulation characteristics of erucamide and the key regulation factor. The effect of erucamide on nitrogen removal pathway, denitrification products, denitrification reductase activity, and factors (NO3- and O2) involved in the stimulation was discussed to reveal the mechanism underlying duckweed root exudate erucamide on bacterial nitrogen removal. This study can provide the scientific basis and applied value for effectively regulating nitrogen loss by denitrification in agro-ecosystems and purifying nitrogen from eutrophic water body by duckweed-microorganism interactions in the rhizosphere.

反硝化是农田氮素损失的重要途径。影响氮素反硝化过程的植物－微生物互作机制是植物营养领域的研究热点。植物可通过根系分泌物来促进微生物的反硝化活性，然而对其释放留存特征和作用机理缺乏深入研究。本项目以农田中广泛存在的浮萍和反硝化细菌Pseudomonas fluorescens为研究对象，综合运用气相色谱、色谱－质谱联用技术和根际互作研究方法，以浮萍根系分泌物脱氮促进物质芥酸酰胺为切入点，研究其在根际环境中的释放规律及不同环境因子的影响，考察芥酸酰胺的积累规律和自身降解情况，以揭示其在浮萍根际环境中的释放留存特征；探讨芥酸酰胺对P. fluorescens脱氮途径、反硝化产物和反硝化还原酶活性的影响以及NO3-、O2 对反硝化促进过程的影响，以期阐明芥酸酰胺促进反硝化脱氮的作用机理。研究成果可为浮萍－微生物根际互作体系调控农田生态系统氮素反硝化损失和净化富营养水体氮提供科学依据和应用基础。

反硝化过程是农田氮素损失的重要途径，理解影响反硝化过程的植物－微生物互作机制对于提高农田氮素利用率和减少农业面源的NO3- 污染具有十分重要的意义。本项目以农田中广泛存在的浮萍和反硝化细菌Pseudomonas fluorescens为研究对象，综合运用气相色谱、色谱-质谱联用技术和根际互作研究方法，以浮萍分泌的非营养性脱氮促进物质芥酸酰胺为切入点，研究其在根际环境中的释放留存特征和作用机理。研究结果表明，气相色谱比液相色谱测定的芥酸酰胺检测限低，而连续收集法收集的浮萍根系分泌物中芥酸酰胺含量显著高于（5倍）静置收集法收集较。芥酸酰胺分泌量在高氮水平下(>300 mg L-1) 显著提高，并与浮萍生长率负相关。芥酸酰胺分泌量同时受两株反硝化荧光假单胞菌诱导，在浮萍根际环境中有效作用时间长。进一步的机理研究表明，芥酸酰胺对P. fluorescens脱氮的促进作用主要是通过反硝化途径实现，关键起作用的化学结构可能是二十二个碳链长度以及13位的双键，还能与其他浮萍源代谢物油酸酰胺和亚麻酸等协同促进微生物反硝化。芥酸酰胺显著促进了P. fluorescens 的NO3-、NO2-、NO还原速率以及N2O产生速率，同时促进了关键反硝化还原酶NAR与NIR的活性。本项目揭示了反硝化调控物质芥酸酰胺的分泌受到高氮和特定反硝化细菌两个因子的诱导，明确了促进反硝化的关键作用位点是反硝化还原酶NAR与NIR。研究结果深化了人们对浮萍植物-反硝化微生物互作体系调控水体富营养化和土壤氮素循环的科学认知，推进了传统营养性反硝化促进剂向非营养性信号调控物质的发展，为今后开发绿色、经济和高效的农业氮素污染水体生物修复技术和产品提供技术支撑。