猕猴桃对软腐病病原菌Botryosphareia dothidea侵染的应答机制研究

kiwifruit rot is one of the most important kiwifruit diseases with high incidence rate and widely happened in different kiwifruit production areas, causing serious economic loss. Botryosphareia dothidea is the one of the main pathogen causing kiwifruit rot, but the infection process and pathogenesis were not yet clear. From the perspective of the stress response of kiwifruit infected with pathogen, artificial inoculation was carried out during the flowering, different development periods and postharvest ripening periods of kiwifruits. Histopathology and quantitative real-time PCR detection technology based on the specific primers of B. dothidea will be performed to study the infection way and process. Dynamic changes of metabolites during the disease development will be analyzed, and transcriptome sequencing will also be used to reveal the response mechanism of kiwifruit infected with B. dothidea. The aim of this project is to elaborate the infection process of kiwifruit rot pathogen, the dynamic variation of the morphology and metabolite in the process of infection, the regulation pattern of defense responses- and signal transduction- related genes, and to explore the formation mechanism of rot and unpleasant odours, ultimately, to lay the foundation for disease control and resistance breeding research.

猕猴桃软腐病在各个猕猴桃产区都有大量发生，严重影响猕猴桃的经济效益。葡萄座腔菌属真菌Botryosphareia dothidea是其主要病原菌之一，但其侵染过程以及致病机理尚不清楚。本项目从猕猴桃接种病原菌后的应激反应角度出发，在花期、果实不同生长发育期以及采后不同后熟期接种B. dothidea，通过组织病理学观察、基于特异性引物的实时荧光定量PCR检测等手段研究猕猴桃软腐病菌的侵染途径和过程；通过对发病过程中的代谢物动态变化分析和转录组测序揭示猕猴桃对软腐病病原菌侵染的应答机制。本项目的开展，旨在阐明猕猴桃软腐病病原菌的侵染过程、发病过程中的组织形态和代谢物的动态变化规律以及猕猴桃防御反应、信号传导等基因的调控模式，揭示软腐与异味形成机制，最终为病害防治与抗病育种等研究奠定基础。

猕猴桃果实软腐病发生广泛，很多猕猴桃园的发病率高于50%%，造成严重的经济损失。本项目从猕猴桃接种病原菌后的应激反应角度出发，在采后接种B. dothidea，通过组织病理学观察手段研究猕猴桃软腐病菌的侵染途径和过程；通过对发病过程中的代谢物动态变化分析和转录组测序揭示猕猴桃对软腐病病原菌侵染的应答机制。研究建立了一套有效的离体接种体系，采后10天接种，环境因子设置为25 °C，相对湿度≥ 90 %。在猕猴桃果皮表面的皮孔位置观察到了B. dothidea的分生孢子器，在接种5 d后，表皮表面与内部细胞内可见菌丝体着生，皮孔可能为自然侵染的重要通道。取接种后12 h、24 h、72 h与120 h的猕猴桃组织进行转录组测序与代谢物检测，四个时间点样品得到8342个差异表达基因与403种代谢物质。组学数据分析表明，接种12 h后，猕猴桃组织启动了基础防御反应，PR1、CHIT1B基因、植物-病原菌互作基因、苯丙素代谢途径基因上调表达。接种72 h后，PAL基因表达受抑制；接种120h后，果胶酯酶（Acc01461）、木葡聚糖基水解酶基因（XTH16）、聚半乳糖醛酸酶（Acc19706）等基因表达受促进，细胞降解途径增强，有利于病斑形成与组织腐烂。代谢物集中在黄酮类、脂类、氨基酸、酚酸与有机酸，其中黄酮类、木酚素、香豆素物质是植物抗病/抗菌主要物质，其中检测到的11种单宁酸属于一类苦涩物质，可能与猕猴桃组织腐烂后的苦涩味有关。本项目通过微观手段观察到猕猴桃果实表面的B. dothidea的分生孢子器，皮孔可能为侵染重要通道。建立离体接种体系观测到发病过程，为后续猕猴桃软腐病病原菌的鉴定提供帮助；接种中后期，细胞壁降解途径受到促进，有利于腐烂的发生，酚酸、单宁酸可能是引起腐烂果肉苦涩味的重要物质。本项目的开展对于认知猕猴桃软腐病的发病过程有重要意义。