基于毛细作用-微萃取联用的化感物质原位采样分析技术及化感作用的研究

Based on the theory of capillarity, an in situ sampling-microextraction apparatus for studying allelochemicals without disturbance to soil will be developed by imitating the design of capillarity micropore ceramic filter with combination of microextraction theory and technique.Using the apparatus and modern analytical technique, the identification and concentration of allelochemicals secreted by conventional allelopathic and autotoxicity plants will be studied, and the new methods for extraction and analysis of allelochemicals as well as the allelopathy evaluation method will be established.The sampling technique and methods developed can eliminate the drawbacks from extracting allelochemicals by acid, basic or organic solvent, overcome the limitations of collecting allelochemicals by tissue culture and resin adsorption in water,in which the effect of microorganism to allelochemicals is ignored, and also avoid the defects of continuous vacuum extraction sampling method, in which gravity water is more easily collected and the sampling site is not accurate and the disturbance to soil is heavy. By using the technique developed, allelochemicals can be sampled, separated, concentrated and analysed in true state of nature. It has important practical significance for overcoming the bottleneck in allelopathy study, in order to get objective and accurate results. In the mean time, the practicability of solid-phase microextraction and liquid-phase microextraction for complicated matrix will be further improved and broadened.

基于毛细作用理论，模仿毛细微孔陶瓷过滤机的设计原理，并结合微萃取理论和技术，研制土壤化感物质非扰动原位采样-微萃取装置。用上述装置结合现代分析技术研究经典化感植物、自毒植物产生的化感物质种类、浓度，建立化感物质萃取分析及化感活性评价、化感物质判定的新方法。该采样技术和建立的方法应用到化感研究领域，可回避酸碱液、有机溶剂提取土壤化感物质方法的缺点；同时可避免无菌组织培养、水培树脂吸附等化感物质收集方法的局限性（没考虑微生物转化化感物质的可能）；避免负压连续抽提采样法易采到重力水、采样幅度大从而难以精准定点采样、采样对土壤扰动大等缺陷，使化感物质在自然真实的状态下得以采样、分离、富集及分析，对突破化感作用研究领域的瓶颈，获得客观准确的研究结果，具有重要的现实意义。同时改进和拓宽固相微萃取和液相微萃取技术在复杂基质样品中的实用性。

采用基于毛细作用的原位采样方法收集模式作物辣椒根系分泌的化感物质；利用微萃取分析技术，检测潜在的化感物质；经分析得到38种潜在化感物质，包括酯类，烯烃类，酚类，烷烃类，醇类，酮类和芳香烃类。利用原位-微萃取装置实现了多种化感物质的重复检测，如2,4-二叔丁基苯酚（2,4-DTBP）；对原位采样装置的吸附能力做了评价，初步实现了对模式作物土壤中化感物质的定性和定量分析。对可重复测定到的潜在化感物质进行化感作用研究，发现浓度大于500 mg/L的2,4-DTBP、己二酸二异丁酯、柠檬烯对莴苣发芽生长有明显抑制作用；对2,4-DTBP和辣椒连作土壤对黄瓜苗的他感作用研究发现，2,4-DTBP和辣椒连作土壤使黄瓜抗氧化酶活性提高， MDA和ROS含量增加，并对黄瓜叶片的光合速率、蒸腾速率、气孔导度有着显著抑制作用，对根系活力也有着明显的抑制作用，从而影响了黄瓜植株的正常生长。对模式植物连作培养时，随着连作年限增加，辣椒自毒抑制作用影响逐年增强。连作土壤中的潜在化感物质对辣椒种子的萌发，幼苗组织的酶活性也有很强的自毒作用。该装置和技术应用到化感作用研究领域，可使化感物质在自然真实的状态下得以采样、分离、浓缩及分析，克服了传统取样方法的不利因素干扰，能够获得客观准确的研究结果，对化感作用的研究具有重要的现实意义。项目资助发表了期刊论文12篇，已申请专利2项。