【作者】 李智；

【导师】 李国怀；

【作者基本信息】 华中农业大学 ， 果树学， 2014， 博士

【摘要】 桃流胶病(Peach gummosis)是桃树生产上的主要病害之一,分布广泛,尤其在我国长江流域以及以南地区发病更为严重。桃流胶病危害严重,导致树势衰弱、产量降低、品质下降、甚至树体死亡,已经成为制约桃产业健康、可持续发展的重要因素之一。研究报道,葡萄座腔菌属(Botryosphaeria spp.)真菌是引起桃流胶病的病原菌,主要包括三个种,分别为Lasiodiplodia theobromae, Diplodia seriata和Botryosphaeria dothidea。本文从组织病理、生理生化、糖代谢以及基因表达的角度对桃流胶病发病过程进行研究,也对病原菌真菌毒素、生长类物质对桃流胶病的影响和硫酸锌的防治作用进行了探讨。主要结果如下：1.通过致病条件研究表明空气相对湿度和伤口是桃流胶病发病的主要影响因子。在桃与病原真菌L. theobromae的互作中,桃枝条在遭受病原菌侵染后的应激反应包括生理生化的改变以及防御相关基因的表达,具体表现为花青素和过氧化氢含量增加,PAL活性、以及防御相关基因的表达增强。另一方面,病原菌诱导了细胞降解和胶体形成,具体表现为MDA含量增加,叶绿素含量显著降低,细胞壁降解,涉及到细胞壁降解的相关基因表达显著增加。2.在桃病原性流胶病发病过程中,糖代谢发生了明显的变化。接种病原菌后桃枝条内的可溶性糖,甘露糖,阿拉伯糖和木糖含量具有不同程度的增加；蔗糖、山梨醇和果糖含量总体呈下降趋势；通过qRT-PCR分析表明,L-galactose和GDP-L-galactose的合成受到抑制,UDP-D-glucuronate、UDP-D-galactose、UDP-D-xylose和UDP-D-arabinose的合成增强,而这些物质是多糖合成的直接前体；通过切面观察和PAS染色表明接种病原菌后桃一年生枝条中多糖有明显积累；糖转运相关蛋白表达增强。3.接种病原菌前用乙烯利处理可促进桃流胶病的发生,而接种病原菌后用乙烯利处理可抑制病害发生,乙烯利在调控桃流胶形成过程中扮演着双重作用。接种后处理抑制胶体形成,表现为较低的流胶率、流胶量和较小的病斑直径；1-MCP(乙烯信号抑制剂)处理可以缓减乙烯利的作用而促进胶体的形成；乙烯利抑制中性转化酶的表达,而1-MCP诱导其表达；另外,乙烯利具有抑制病原真菌生长的效果。接种前处理促进胶体形成,比对照提前1d形成胶体,而且导致较高的流胶率、流胶量和较大的病斑直径。两种处理均诱导了糖含量的增加,促进了衰老相关基因的表达。但是,与接种后处理相比,乙烯利接种前处理导致更低的蔗糖(24hpi)、葡萄糖(48和72hpi)和果糖(12、48和72hpi)含量。4.建立了葡萄座腔菌JMB-122真菌粗毒素的制备体系,通过GC-MS鉴定了9种真菌次生代谢物。9种真菌次生代谢物分别为油酸酰胺、芥酸酰胺、2,4-叔丁基苯酚、3-(3,5-二叔丁基-4-羟基苯基)丙酸甲酯、硬脂酸、棕榈酸、单硬脂酸甘油酯和甘油脂肪酸酯；通过分析7种无机化合物对真菌毒素致病性的影响,发现高锰酸钾和硫酸锌可以有效抑制由JMB-122毒素引起的桃流胶病；外源高锰酸钾处理可减少胶体的形成。5.硫酸锌不仅能缓解流胶病的发生,还可以抑制真菌的生长发育和致病性。桃流胶病发病过程中,锌含量减少；在接种病原菌后的桃一年生枝条上喷施硫酸锌能缓解流胶病的发生,硫酸锌可显著减小病斑形成和降低流胶量；硫酸锌抑制了JMB-122真菌毒素引起的流胶病,通过qRT-PCR分析,硫酸锌诱导了5个防御相关基因的表达。硫酸锌可以有效抑制病原真菌菌丝的生长。随硫酸锌浓度的增加,菌落颜色变深；形态观察表明25mM硫酸锌下,菌丝表面变皱,畸形；50mM硫酸锌下,菌丝顶端膨大,伸长受到抑制。硫酸锌处理JMB-122真菌20min后接种桃一年生枝条,25mM有效降低了流胶量,减小了病斑直径；50mM的硫酸锌完全抑制了流胶病的发生。更多还原

【Abstract】 Peach gummosis is one of the most important and damaging diseases of peach in the south peach production area of the Yangtze River of China. Peach gummosis causes significant growth stunting, yield losses, and tree death. It has been become a restriction factor for healthy and sustainable development of peach production. The three species of Botryosphaeria, L. theobromae, B. dothidea, and Diplodia seriata were the predominant causes of the disease. The objective of this study was to investigate the histopathology, biochemical changes, carbohydrate metabolism, and expression patterns during peach gummosis progression caused by L. theobromae. In addition, we also investigate the mycotoxin of L. theobromae, effect of ethephon on gum formation, and the disease control of zinc sulfate. The main results are as follows:1. The biochemical changes associated with the interaction between peach shoots and L. theobromae suggest that peach plants respond to the fungus by increasing anthocyanin levels, PAL activity, H2O2concentrations, and the expression of defence-related genes. However, the fungal infection triggered cell death and gum formation, as indicated by the increased MDA content, the expression of cell wall degrading-related genes, and reduced chlorophyll a and b content.2. These changes in carbohydrate metabolism were directly associated with the symptom of peach gummosis. Soluble sugars, glucose, mannose, arabinose, and xylose significantly increased in inoculated tissues of peach shoots compared with control tissues at different times after inoculation. Analysis using qRT-PCR revealed that the abundance of key transcripts on the synthesis pathway of UDP-D-glucuronate, UDP-D-galactose, and UDP-D-arabinose increased, but the synthesis of L-galactose and GDP-L-galactose were inhibited. Accumulation of polysaccharides was also observed by section observation and periodic acid Schiff’s reagent staining during infection. After inoculation, the transcript levels of sugar transport-related genes, namely, SUT, SOT, GMT, and UGT, were induced.3. In this study, we investigated the effect of chemical treatments on peach gummosis after inoculation with Lasiodiplodia theobromae. The results showed that ethephon significantly decreased the gum formation rate. Gum formation was promoted by ethephon treatment prior to pathogen inoculation, but inhibited by ethephon applied after the pathogen. Our results show that ethephon has a dual function in regulating gum formation by affecting the peach shoots and the pathogen. For the ehtephon post-treatment, the gum formation was inhibited by ethephon with lower gum weight, gum formation percent, and lesion diameter. The relative expression of neutral invertase was induced by1-MCP, but inhibited by ethephon. The inhibitory effect was counteracted by1-methylcyclopropane, which is an ethylene signal inhibitor.1-Methylcyclopropane also promoted gum formation. Exposure of the three stains of Botryosphaeria to ethephon inhibited mycelial growth. For the ethephon pre-treatment, the gum formation was promoted by ethephon with more gum weight, gum formation percent, and lesion diameter. Both treatment methods increased the sugar content at12and24hpi. However, Lower levels of sucrose, glucose, and fructose were detected in the ethephon-pretreated shoots than those in the post-treated shoots, with significant differences at24hpi for sucrose,48and72hpi for glucose, and24,48, and72hpi for fructose.4. An experiment system was established to obtain the mycotoxins of L. theobromae strain of JMB-122.9kinds of metabolite compounds of L. theobromae strain of JMB-122, namely,9-octadecenamide,13-docosenamide,2,4-bis(1,1-dimethylethyl)-Phenol, butylated hydroxytoluene,3,5-bis(1,1-dimethylethyl)-4-hydroxybenzenepropanoic acid methyl ester, octadecanoic acid, hexadecanoic acid, octadecanoic acid,2,3-dihydroxypropyl ester and hexadecanoic acid2,3-dihydroxypropyl ester, were identified by GC-MS analysis. The bioassay of those compounds will be determined in the next step. By investigating the effect of inorganic compounds on phytotoxin of L. theobromae, zinc sulfate and KMnO4inhibited the pathogenicity casuing by L. theobromae phytotoxin. The gum wight was significantly decreased after application with KMnO4on inoculated peach shoots.5. Zinc sulfate not only alleviated the development of peach gummosis, but also affected the growth, development, and pathogenicity of L. theobromae. Zinc content was reduced during the development of peach gummosis. For the application of zinc sulfate on inoculated peach current shoots, the lesion diameter, gum weight and both was siginificantly decreased by zinc sulfate. Zinc sulfate treatments significantly reduced disease severity and gum formation and effectively controlled peach gummosis under phytotoxin stress of L. theobromae. Zinc sulfate also induced the expression of defense-related genes of CHI, PR4, GNS3, PGIP, and PAL by qRT-PCR analysis. Those results might be responsible for the resistance increase. Results showed that mycelial growth was significantly inhibited by zinc sulfate. With the increase of concentration of zinc sulfate, the black colony exhibits an increase trend at PDA. Zinc sulfate caused abnormal hyphae at25mM and swelling hyphal tips at50mM by morphologic observation. In contrast to the control the black septate hypha increased with an increase in zinc sulfate concentration.更多还原