青蒜挥发物调控黄瓜霜霉病抗性的化感作用机理

Biological measure is a promising technique direction to overcome plant diseases which is the bottleneck of facility vegetable production industry in China. Our previous research has proved that cucumber/green garlic intercropping system can effectively alleviate the continuous obstacle and disease in facility fruit vegetable production. Especially, the system can significantly reduce the incidence of cucumber downy mildew at early growth stage, but the mechanism of the green garlic volatiles function is still not clear. This project aims to isolate and identify the main allelochemicals in volatiles of green garlic by GC/MS and bioassay method; to study the inhibition effect of green garlic volatiles and its main effect of allelochemical on pathogen of cucumber downy mildew. By inoculate the pathogen of cucumber downy mildew in cucumber/green garlic co-culture system with root-zone isolated in a semi-closed circumstance to study the leaf tissue structural and physiological changes during infection process to explore the inducible resistance in cucumber and investigate genes expression profiles by RNA-seq, analyse the sensitive metabolic pathways and functions of genes that response to garlic allelochemical and confirm the temporal and spatial expression characteristics and transcription level of major genes by qRT-PCR. Finally, the physiological and molecular mechanism of cucumber resistance to downy mildew induced by green garlic volatiles are to be elucidated. This research can provide the theoretical principle for alleviating continuous cropping obstacle in fruit vegetable intercropped with green garlic system and lay foundations for theoretical and technological basis for the practical utilization of green garlic allelopathic volatiles in alleviating airborne disease of vegetable.

探索应用生物措施解决设施蔬菜病害问题是现代蔬菜产业技术的发展方向。本课题组前期研究表明，套作青蒜可有效消减黄瓜连作障碍和病害，尤其是套作青蒜可显著降低黄瓜早期霜霉病的发生，但青蒜活体挥发物对黄瓜霜霉病的作用机制尚未明确。本项目采用GC/MS和生物测试法鉴定青蒜挥发物主效化感物质，以黄瓜及其霜霉病菌为对象，①研究青蒜活体挥发物及其主效化感物质对黄瓜霜霉病菌的抑制作用；②在青蒜与黄瓜半密闭环境根区隔离共培养体系接种霜霉病菌以及主效化感物质处理后接种，分析病菌侵染过程中黄瓜叶片组织结构和生理生化变化，探讨黄瓜诱导抗性反应；③进行转录组测序，分析代谢途径和筛选差异表达基因，对其进行时空表达分析和基因功能分析，揭示青蒜活体挥发物诱导黄瓜霜霉病抗性的生理和分子机制。该研究将为套作青蒜消减设施果菜连作障碍的栽培模式提供进一步的理论依据，为直接利用青蒜挥发性化感物质减轻设施蔬菜气传病害奠定理论和技术基础。

黄瓜是我国设施栽培的第一大蔬菜作物，在高湿的设施环境中黄瓜霜霉病发病严重，造成重大的经济损失。本课题组前期研究发现，套作青蒜可显著降低黄瓜早期霜霉病的发生。本研究采用GC-MS和生物测试法鉴定青蒜挥发物主效化感物质，通过在青蒜与黄瓜半密闭环境根区隔离共培养体系接种霜霉病菌探讨黄瓜诱导抗性反应，利用转录组测序探究青蒜挥发物诱导黄瓜霜霉病抗性的生理和分子机制。主要研究结果如下：.1. 明确了青蒜挥发物的主效化感物质及其含量。.GC-MS鉴定和生物测试结果表明二烯丙基二硫（DADS）是青蒜挥发物中的主效化感物质，每克切段青蒜大约可释放0.08 mg的DADS，且切段后释放的青蒜挥发物中DADS的含量约是自然生长释放的5倍。.2. 揭示了不同浓度的青蒜挥发物和1 mmol L-1 DADS对黄瓜活性氧清除的化感作用。.通过青蒜挥发物和DADS处理不同品种黄瓜幼苗，发现青蒜挥发物和DADS对不同品种的作用有所不同，但都能增加黄瓜叶片H2O2的积累，调控抗氧化酶活性、丙二醛含量和防御酶基因的表达。.3. 揭示了DADS对黄瓜霜霉菌的抑制效应和诱导黄瓜抗性的生理机制。.接种霜霉菌后用DADS处理，可以抑制黄瓜霜霉菌休止孢的萌发和孢囊梗的数量，从而抑制霜霉病菌的侵入和扩展。青蒜与黄瓜共培养、切段青蒜及DADS处理都能显著降低黄瓜霜霉病的病情指数。用DADS处理黄瓜幼苗10 d后接种霜霉菌，发现DADS处理显著诱导黄瓜对霜霉病的抗性，这一过程伴随着木质素和H2O2含量显著提高，POD活性显著增强，PAL活性显著降低，IAA和SA含量增加。.4. 初步揭示了青蒜挥发物诱导黄瓜霜霉病抗性的相关基因和代谢通路。.转录组分析表明，大量差异基因参与了防御反应的生物学过程，特别是DADS处理时会诱导“苯丙烷生物合成”、“苯丙氨酸代谢”、“植物-激素信号转导”和“MAPK信号转导”途径的DEG显著上调表达，说明DADS处理的黄瓜幼苗产生了诱导抗性以抵抗病原菌的入侵。WGCNA分析筛选到9个关键基因。.该研究为直接利用青蒜挥发性化感物质减轻设施蔬菜气传病害奠定了理论和技术依据。