基于化感自毒作用理论的半夏连作障碍机理研究

Pinelliae ternata is an important medicinal plant. At the present time, this herb largely relies on artificial cultivation. However, continuous cropping obstacle is always the key problem which severely inhibited the sustainable development of cultivation of Pinellia ternata. In the present study, based on the theory of allelopathic autotoxicity, we fistly research the physical and chemical properties, and microbial community diversity of soil. Then isolation and identify allelopathic autotoxicity compounds by the technology of extraction separation → column chromatography →GC-MS, combine with bioassay method to track the active part. And sifting major allelopathic autotoxicity compound of continuous cropping obstacle of Pinellia ternata through bioassay method to validate its activtiy and the content of allelopathic autotoxicity compounds in the soil mensurated by HPLC. Finally, treat the seedling of Pinellia ternata and soil with autotoxicity compound, to research the influences of autotoxicity compound on the activity of several protective enzyme of plant cell and the activity of root system, and parameter of photosynthesis and chlorophyll fluorescence of Pinellia ternata seedling, as well as the influences of autotoxicity compound on the physical and chemical properties and microbial community diversity of soil. We expect to reveal the allelopathic autotoxicity mechanism of continuous cropping obstacle of Pinellia ternata.

半夏Pinellia ternata是一种重要药用植物。目前，药用半夏主要来源于人工栽培，但是连作障碍一直是困扰半夏生产发展的重大技术难题。本项目立足于化感自毒作用，以化感自毒物质为切入点。首先对土壤理化特性、微生物多样性和土壤自毒潜力进行研究，探讨半夏连作障碍与土壤微生态特性的关系。然后通过生物测试活性追踪的方法，利用有机溶剂萃取→硅胶柱色谱分离→气相色谱质谱连用技术，分离鉴定自毒物质；并通过生物测试活性验证和土壤中自毒物质含量定量分析的方法，筛选主要自毒物质。再用自毒物质处理半夏幼苗和土壤，通过对幼苗根系活力、细胞保护酶活性、叶片光合参数、叶绿素荧光参数，以及土壤理化性质和微生物多样性的分析，明确自毒物质对幼苗生理和土壤微生态特性的影响。从而从植物生理和土壤微生态两个方面，阐明连作障碍发生与自毒物质的关系，揭示半夏连作障碍发生的化感自毒作用机理，为其连作障碍解除措施的研究提供参考。

本项目立足于化感自毒作用，从土壤自毒潜力评价、自毒物质来源、自毒物质分离与鉴定、自毒物质验证、连作与间作的土壤微生态等方面开展了研究。（1）构建了半夏无菌苗培育体系，叶片和叶柄一步成苗培养基分别为“MS+1.5mg/L NAA+1.0mg/L 6-BA +0.1%活性炭”和“MS+1.0mg/L NAA+1.0mg/L 6-BA +0.1%活性炭”；（2）研究了自毒物质来源，发现化感自毒物质主要存在于半夏植株腐解液的Fr2部位，通过萃取分离、生物测试活性追踪及气质联用技术，分离鉴定出Fr2组分的30种化合物，包括2,6-二叔丁基对甲酚、芥酸酰胺、邻苯二甲酸二异辛酯、邻苯二甲酸二异丁酯、2,2′‐亚甲基双（4甲基-6-叔丁基苯酚）等五种化感物质；（3）验证了芥酸酰胺等5种物质对半夏的化感作用，初步确定2,6-二叔丁基对甲酚、芥酸酰胺、2,2′-亚甲基双（4甲基-6-叔丁基苯酚）为半夏化感自毒物质，它们对半夏珠芽萌发、幼苗生长、植株生理都具有化感抑制作用；（4）评价了土壤自毒潜力，半夏根际土壤浸提液对其珠芽萌发、幼苗和根系生长具有化感作用，但均不强，因此，半夏根际土壤对半夏的自毒作用较弱；（5）半夏连作会显著减少土壤细菌和放线菌数量，增加真菌数量，使土壤B/F值呈显著的逐渐减小趋势，并对土壤养分和理化性质有一定影响，因此，种植或连作半夏会导致土壤由“细菌型”土壤向“真菌型”土壤转变，推测土壤微生物区系的改变可能是半夏连作障碍的原因之一；（6）间作对土壤性质和微生物都有一定影响，合理间作可以减缓土壤向“真菌型”土壤转变，提高土壤酶活性，改善肥力状况。因此，间作是减缓半夏连作障碍的可行途径，尤以与决明或大豆间作为首选。