KMS XINJIANG INSTITUTE OF ECOLOGY AND GEOGRAPHY，CAS
| 新疆野苹果腐烂病病原菌鉴定及诱导宿主抗病相关基因表达分析 | |
| 刘晓洁 | |
| Subtype | 硕士 |
| Thesis Advisor | 张道远 |
| 2018-06-01 | |
| Degree Grantor | 中国科学院大学 |
| Place of Conferral | 新疆乌鲁木齐 |
| Degree Discipline | 工程硕士 |
| Keyword | 新疆野苹果 腐烂病 致病性 PR 基因 Malus sieversii canker pathogenicity PR gene |
| Abstract | 近十年来，由于多种原因，新疆野苹果林遭受了毁灭性的生态危害，大面积的野苹果纯林衰败甚至死亡，腐烂病的爆发成为野苹果林大面积死亡的重要原因之一。苹果树腐烂病不仅是天山野果林中新疆野苹果的主要病害，也是我国乃至世界性的栽培苹果生产中的毁灭性病害，是制约我国苹果安全生产和危害世界苹果产业的主要限制因子。已有研究对分离自栽培苹果（Malus domestica）的腐烂病菌进行了分离与鉴定，认为主要是黑腐皮壳菌（Cytospora mali，又名 Valsa mali）对宿主栽培苹果造成了危害，但是，对新疆野苹果腐烂病的研究很少。本研究一方面对新疆野苹果腐烂病病原菌进行分离、鉴定及致病力分析，并建立快速接菌鉴定新疆野苹果腐烂病抗性株筛选方法；另一方面系统研究了新疆野苹果侵染腐烂病不同时间点病程相关基因表达动态，以期为后续新疆野苹果腐烂病发生机制及防控研究作出贡献。本研究以实验室获得的 5 株苹果腐烂病病原菌（EGI1、 EGI2、 EGI3、 EGI4、EGI5）以及黑腐皮壳菌 C. mali 标准菌株（03-8-1 西北农林科技大学馈赠），和C. parasitica 分离株（1342 新疆农业大学馈赠）共 7 个菌株进行研究，取得主要结论如下：1. 通过培养菌落形态观察及 SEM扫描电镜菌株气生菌丝形态观察，发现 EGI1、EGI3 和 03-8-1 形态一致，而 EGI4、 EGI5 和 1342 菌株形态一致；2. 进一步进行分子生物学鉴定，利用 ITS， tef1-α 和 tub2 基因片段，以 Diaporthevaccinia 为外群，分别通过单基因片段和多基因片段联合分析构建系统进化树。结果表明， 结合邻接树（NJ）、 最大似然构树（ML）和最大简约树（MP）联合分析， EGI1， EGI2， EGI3 与 C. mali 03-8-1 菌株聚为一支，而 EGI 4，EGI 5 和 1342 与 C. parasitica 菌株聚为一支。说明从新疆野苹果腐烂病病枝上分离并鉴定出 C. mali 和 C. parasitica 两个主要腐烂病病原菌种；3. 通过致病力鉴定，室内病原菌回接新疆野苹果 3 年生实生苗枝条和叶片并检测各分离株致病性，发现所分离的 5 株菌 EGI1、 EGI2、 EGI3、 EGI4、 EGI5均为致病菌，且三株 C. mali 菌株（EGI 1、 EGI2 和 EGI 3 菌株）为强致病性菌株， C. parasitica（EGI 4、 EGI 5 和 1342 菌株）为弱致病性菌株。进一步在新源野果林苗圃地进行病原菌接菌及致病力鉴定验证，得到一致结论。在此基础上，申报发明专利 1 项：一种苹果树腐烂病病原菌快速接菌及致病力快速鉴定的方法。该方法包括对枝条以及叶片 2 个体系的快速接菌及鉴定，具有快速、高效而准确的特点，尤其适用于田间大样本量操作；4. 研究了新疆野苹果侵染腐烂病不同时间点病程相关基因表达动态，其中包括水杨酸（Salicylic acid, SA）、 茉莉酸（Jasmonic acid, JA） 合成及信号传导关键基因和病程相关（Pathogen Related gene, PR gene）PR 基因。通过菌株 EGI1侵染新疆野苹果枝条，对其进行 qRT-PCR，定量获得抗病相关基因表达模式，发现 SA（MsPAL, MsNPR1）， JA（MsLOX, MsCOI1, MsAOS, MsAOC） 合成及信号传导关键基因经腐烂病菌侵染后有明显上调， MsPR5， MsPR8，MsPR10 基因也有显著上调，从转录水平说明植物激素和 PR 基因积极响应新疆野苹果腐烂病。 |
| Other Abstract | In the past ten years, due to various reasons, wild apple forests in Xinjiang havesuffered devastating ecological damage, and large areas of pure wild apple forestshave declined or even died. The outbreak of canker disease has become one of themost important causes of death in wild apple forest. Apple tree canker disease is notonly the main disease of Xinjiang wild apple forest in Tianshan Mountain， it is also adevastating disease in the production of cultivated apple in China and the world. It isthe main limiting factor that restricts the safe production of apple in China andendangers the world apple industry. Some studies have been carried out on theisolation and identification of canker strains isolated from cultivated apple M.domestica. It is believed that the main reason is that Cytospora mali (also known asValsa mali) is harmful to host cultivated apple. On the one hand, the pathogen of valsacanker on M. sieversii was isolated and identified. The rapidly screening method ofinoculation was established to identify the resistance to Cytospora canker of M.sieversii. On the other hand, the expression dynamics of genes related to the cankerdisease course at different time points in M. sieversii were systematically studied tocontribute to the further study on the mechanism and prevention and control ofCanker Disease of M. sieversii in Xinjiang.In this study, five strains of apple canker pathogen (EGI1, EGI2, EGI3, EGI4,EGI5) were obtained. The standard strains of C. mali 03-8-1 and C. parasitica 1342were donated by Northwest University of Agriculture and Forestry Science andTechnology and Xinjiang Agricultural University. A total of 7 strains were studied andthe main conclusions were as follows:1. The morphology of cultured colony and the morphology of aerial hyphae ofSEM scanning electron microscope strain were observed. The morphology ofstrains EGI1, EGI2, EGI3 were consistent with that of strain C. mali 03-8-1,while the morphology of strain EGI 5, EGI 4 and 1342 were consistent with thatof C. parasitica.2. Through the pathogenicity identification, the indoor pathogenic strains wereinoculated with the twigs and leaves of the 3-year-old seedlings of Xinjiangwild apple (M. sieversii), and the pathogenicity of the isolates were detected.Six strains of EGI1, EGI2, EGI3, EGI4, EGI5 and 1342 were classified. Three strains EGI1, EGI2 and EGI3(C. mali) were found to be strong pathogenicity;and three strains EGI4, EGI5 and 1342(C. parasitica) were weak pathogenicity.The pathogenicity identification on leaves was carried out in the nursery ofXinyuan wild fruit forest and the results were consistent with that pathogenicityidentification on twigs. On this basis, the invention patent one item: a method ofrapid inoculation and pathogenicity identification of pathogen of apple treecanker disease, which includes the rapid inoculation and identification of twosystems of branches and leaves. The characteristics of high efficiency andaccuracy are especially suitable for the operation of large sample size in thefield.3. The expression dynamics of phytohormone-related and pathogenesis-relatedgenes at different time points in inoculated with canker pathogen of wild applein Xinjiang were studied, including the key genes of synthesis and signaltransduction of SA, JA and PR genes. The strain EGI 1 was used to infect thetwigs of wild apple in Xinjiang, and the expression pattern of disease resistancerelated genes was obtained by qRT-PCR. The key genes of SA (MsPAL,MsNPR1) and JA (MsLOX, MsCOI1, MsAOS, MsAOC) synthesis and signaltransduction were up-regulated by Cytospora pathogen, and so was the gene ofMsPR5, MsPR8 and MsPR10, which indicated that plant hormones and PRgenes responded positively to the canker disease of wild apple in Xinjiang fromthe level of transcriptional translation. |
| Subject Area | 生物工程 |
| Language | 中文 |
| Document Type | 学位论文 |
| Identifier | http://ir.xjlas.org/handle/365004/14976 |
| Collection | 研究系统_荒漠环境研究室 |
| Affiliation | 中国科学院新疆生态与地理研究所 |
| First Author Affilication | 中国科学院新疆生态与地理研究所 |
| Recommended Citation GB/T 7714 | 刘晓洁. 新疆野苹果腐烂病病原菌鉴定及诱导宿主抗病相关基因表达分析[D]. 新疆乌鲁木齐. 中国科学院大学,2018. |
| Files in This Item: | There are no files associated with this item. | |||||
Items in the repository are protected by copyright, with all rights reserved, unless otherwise indicated.
Edit Comment