基于微透析的土壤酚酸动态及其对水稻秸秆还田对油菜幼苗化感作用的意义

Negative effects limited the extension of crop straw returning techniques in recent years, among them allelopathic effect can not be ignored. Historically, simple phenolic acids have been the most frequently identified allelopathic agents in decomposing organic materials. However, the difficulties of monitoring allelochemical concentrations in soil and their dynamics over time have been a major barrier to testing hypotheses of allelopathic effects. Recently, a novel sampling technique based on the microdialysis principle shows the potential for making break through progress in the research due to its advantage of in vivo analysis and continuous monitoring without withdrawal of soil solution. The objective of this research is to evaluate the allelopathic activity of rice straw on rapeseed with emphasize on dynamics of phenolic acids in rhizosphere monitored by microdialysis sampling method coupled to high performance liquid chromatography (HPLC). First, the method is to be established by constructing the concentric microdialysers and selecting proper thickness of dialyzing membrane, composition and rate of perfusate, factors that influence the extract efficiency, et al. Second, concentration and composition of phenolic acids in bulk and rhizosphere environment under control and rice straw returning treatment are to be monitored in vivo by microdialysis.We will then analyze the role of phenolic acids in allelopathic effect of rice straw returning by analyzing the correlativity between their concentration in rhizosphrere environment and the response of the rapeseed seedlings including leaf expansion rate, root activity and lignification, membrane perturbation of root cells. Flux and dynamic of phenolic acids in bulk and rhizospheric environment and their effect on rapeseed seedlings will also be verified utilizing model systems. The direct evidence presented in this research will make it clear whether phenolic acids are the main cause of the allelopathic effect of rice straw on rapeseed seedling. This knowledge can also contribute to improve the crop straw returning techniques in the future.

近些年负效应成为制约秸秆还田技术推广的主要瓶颈，化感作用为其中不容回避的一个方面。虽有研究表明秸秆腐解过程中常释出以酚酸类物质为主的化感物质，但因其在土壤中的真实动态难以测得，它们在秸秆还田后起的作用尚不明了。原位、实时的微透析取样技术有望突破此瓶颈。本项目拟采用微透析取样技术，以油菜幼苗为受体，对水稻秸秆还田处理土壤中酚酸的释放、迁移规律及其化感作用进行基础科学研究。具体内容包括：自制微透析探针，筛选微透析加HPLC法测定土壤中有效酚酸浓度的条件；原位测定水稻秸秆还田处理下非根际和根际土壤中酚酸浓度与组成的动态；根据根际酚酸动态与幼苗生长速率的相关关系，以及根系活力、膜透性、木质化程度分析酚酸的化感作用。同时，以酚酸添加模拟研究进一步验证其结果。本研究从酚酸释放、迁移及其生物影响的角度，阐释水稻秸秆还田对油菜幼苗的化感作用；利于推动植物化感作用的理论发展，为完善秸秆还田技术提供理论依据。

在长江中下游地区最普遍的轮作模式——稻-油轮作系统中，水稻秸秆直接还田常导致油菜出苗不齐、幼苗生长受抑制的问题尚未得到妥善解决。本项目以长江中下游主栽油菜品种为受体，以腐解液-培养皿滤纸法及土壤混合培养法对水稻秸秆的化感效应进行生物测试，并探讨了酚酸在其中所起的作用。结果表明：水稻秸秆对长江中下游主栽油菜品种普遍表现出了明显的化感抑制效应。水稻秸秆的腐解速率和化感抑制效应在腐解早期强于后期，化感效应强度与水稻秸秆腐解导致水溶态酚酸总量的变化有关，但也不排除其他化感物质的影响。水稻秸秆对油菜的化感抑制作用在厌氧腐解条件下表现为降低种子发芽势和强烈抑制胚根生长，在好氧腐解条件下则仅表现为对地上部生长的抑制。两者之间的差异应该归因于腐解环境不同造成的化感物质不同，对其进一步研究分析将助于全面揭示水稻秸秆对油菜的化感作用机制。本项目还引入了基于扩散原理实现实时、原位和对环境微扰动的微透析技术，探索并优化了测量土壤环境中游离酚酸含量的条件，建立了微透析-HPLC平台，并进一步研究了水稻秸秆处理对土壤中游离态酚酸含量动态的影响，上述方法测出的土壤游离态酚酸包括阔马酸、阿魏酸、对羟基苯甲酸、二甲氧基苯甲酸、水杨酸；其中含量最高的是对羟基苯甲酸，含量为0.00~0.93μg/g soil，与常规方法测定的土壤水溶态单酚酸含量同数量级。水稻秸秆的主效应正在处理、分析中。总的看来，此方法在植物化感作用机理的研究中具有较好的实用价值。但也存在取样耗时长、效率低；提取液微量、目标物质浓度低、杂质多，对测定技术要求高；回收率受提取介质温度、均匀度影响大；微透析半透膜微小脆弱且重复利用率低，试验成本高等问题。