梨果实表皮角质层结构及化学组分对Alternaria alternata侵染的调控机理研究

Cuticular layer of fruit is the first line of defence againt infection of postharvest pathogen. The physical structure and chemical composition of this layer may influence infection structure formation or pathogenetic factors production by pathogen. Our previous results found that the percentage latent infection of Alternaria alternata significantly increased with the increase of fruit skin thickness and integration, but infection mechanism involved is not fully elucidated. On the basis of our previous studies, changes in physical structure (thickness, integration, surface morphology) and chemical composition (wax and cutin monomers) of cuiticular layer of Pingguoli pear will be observed or analysized systematically by scanning electron microscope (SEM), transmission electron microscopy (TEM) or gas chromatography-mass spectrometry (GC-MS). In addition, effect and regulation mechanism of changes in physical structure and chemical composition of cuiticular layer on mycelium growth, spore germination, infection structure formation, infection process and penetration-related enzymes activity including esterase and cutinase of A. alternata, the causal agent of Alternaria rot on Pingguoli pear, will also be studied in vivo and in vitro. Expected results of the project will elucidate change pattern of physical structure and chemical composition of cuiticular layer during development and storge period and their regulatory role on pathogen-host interaction,and also provide theoretical basis for screening resistant varieties and effectively controlling postharvest diseases of fruit and vegetables.

角质层是病原物侵染果实的第一道屏障，其结构和化学组分的变化对病原物侵染结构的形成和致病因子的产生具有重要的影响。我们前期研究发现随苹果梨角质层厚度和完整度的提高Alternatia alternata的侵染率明显增加，但其侵染机制尚不清楚。本研究以苹果梨为试材，采用扫描电镜、透射电镜和气相色谱-质谱联用技术（GC/MS），系统观察和分析苹果梨果实发育期及贮藏期角质层结构（厚度、完整度及表皮形态）和化学组分（蜡质及角质单体）的变化，同时采用体内（in vivo）体外（in vitro）相结合的方法研究角质层结构和化学组分变化对苹果梨采后主要致腐病原物A. alternata菌丝生长、孢子萌发、侵染结构形成、侵入过程和侵入相关酶（酯酶、角质酶）活性的影响及调控机理。以阐明果实角质层结构及化学组分的变化规律及其在寄主-病原物互作中调控作用,以期为采后病害的有效控制及抗性品种的选育提供理论依据。

本项目以苹果梨为试材，通过显微观察、GC/MS分析及病理学方法系统的观察和分析了发育贮藏期及采后处理果皮角质层显微结构及其化学组分变化，并通过in vivo和in vitro试验研究揭示了表皮蜡质化学组分、疏水性变化对Alternaria alternata侵染结构形成、角质酶活性及基因表达的调控机理，同时探讨了角质组分变化对果实失水的影响。结果表明：. 1. 苹果梨花后30d表皮蜡膜开始形成，花后60d蜡膜层进一步增厚，局部区域形成微裂纹，发育后期表面覆有大量薄片状晶体。贮藏期间蜡膜厚度没有明显变化，贮藏后期表面覆有光滑块状晶体。采后1-MCP处理后表皮蜡质较为粗糙，形成颗粒状蜡质晶体，而乙烯利处理后表皮形成颗粒或棒状晶体。 .2. 透射电镜观察发现果实花后30d角质层较薄且分布不均匀，花后60d角质膜逐渐完整，并在部分表皮细胞的垂周壁上形成了可见的角质栓，随后果皮角质层进一步沉积变厚，形成了大量的垂周壁；贮藏期间表皮清晰的垂周壁角质栓大量形成。.3. 苹果梨发育贮藏期角质提取物中脂肪酸含量占绝对优势，其含量随发育贮藏期的延长而增加，且呈偶数碳优势的脂肪酸链长、饱和度均因发育和贮藏时期而不同。采后1-MCP处理显著增加了蜡质组分中烷烃和醛类含量，而抑制了脂肪酸和萜类含量的增加；而乙烯利处理后烯烃和萜类含量显著增加。.4. A. alternata孢子在梨表皮上首先萌发形成芽管，芽管伸长、顶端膨大形成附着胞，部分附着胞进一步分化形成侵染菌丝，进而通过皮孔或表皮侵入。体内和体外试验表明完整果皮及涂有蜡质提取物的洋葱膜、Formvar膜、硅烷化载玻片均能有效的促进A. alternata附着胞和侵染菌丝的形成，且促进作用因发育和贮藏期而不同。相关性分析表明蜡质疏水性与A. alternata附着胞及侵染菌丝形成呈正相关。果皮蜡质对A. alternata角质酶基因表达及酶活性也具有诱导作用。.5. 果实失水率与角质组分22:02呈显著正相关，而与C18 Epoxy ω-OH、16:0 Di-OH、18:0 Tri-OH和16:0 ω-OH呈负相关。.综上所述，苹果梨表皮角质层结构和角质组分随果实的发育贮藏及采后处理而变化，其蜡质化学组分、疏水性以及表皮结构变化对A. alternata侵入过程、侵染结构形成及致病性均具有一定调控作用。