柑橘黄龙病两种植物寄主内生细菌
日你妈
的分离及分析
重庆大学硕士学位论文
（学术学位）
学生姓名：王*
指导教师：王中康教授
专业：生物学
学科门类：理学
重庆大学生命科学学院
二O一四年四月
Separation and Analysis of Endophytic Bacteria from Two Kinds of Huanglongbing’s Host Plant-Citrus and Clauna lansium (Lour.) Skeels
A Thesis Submitted to Chongqing University
in Partial Fulfillment of the Requirement for the
Master‟s Degree of Science
By
Fang Wang
edg什么意思Supervid by Prof. Zhongkang Wang
Specialty: Biology
School of Life Sciences, Chongqing University,
Chongqing, China
April, 2014
摘要
dyp是啥
gloomy什么意思
摇滚青春歌曲柑橘黄龙病因危害严重、分布广泛、传播速度快且尚无有效根治措施，而成为研究柑橘病害的热门课题。该病害的病原菌是一种寄生在寄主韧皮部的不可培养细菌。现阶段，有报道表明，自然界中不同的细菌菌群之间存在着拮抗或共生关系。本研究以此为基础，选取柑橘黄龙病两种芸香科寄主（柑橘与黄皮），研究黄龙病病原自身生活环境中的菌群结构，对比分析同株罹病柑橘植株症状差异组织和病健株黄皮的内生菌群结构差异，从不同角度研究黄龙病菌与内生细菌菌群产生的相互影响。从黄皮韧皮结构中分离并鉴定出具有多种生理功能的细菌，可为黄龙病发病机理研究和病害生物防控提供理论基础。
1、利用基于16S rDNA 的变性梯度凝胶电泳（DGGE）分析同一柑橘黄龙病罹病植株的显症和未显症组织内生细菌多样性，寻找与黄龙病菌(…Candidatus Liberibacter asiaticus‟ (Ca.Las))相互作用的优势菌株，并用定量PCR 方法，对果、枝、叶等不同组织黄龙病菌、优势菌株及细菌总数进行检测。结果显示柑橘黄龙病病株中，显症和无症组织所带黄龙病菌差异很大，且显症部位病菌量明显高于无症部位。对显症和无症组织内生细菌的DGGE结果进行分析得，同一组织内生菌群结构基本相同；对图谱中17条明显条带回收克隆测序，发现其中8个条带均属于沙雷氏菌属（Serratia），占总条带数的47.06%。序列分析显示这8条序列为Serratia marcescens不同的菌株（序列相似性为99.63%）。定量分析各差异显症部位单位组织内的粘质沙雷氏菌和细菌总数，发现相同部位的总细菌量差异不显著，但粘质沙雷氏菌与黄龙病菌的量呈负相关。
蓝鸥xix
guardian angel
2、利用可培养技术及核糖体DNA扩增片段限制性内切酶分析（ARDRA）对健、病黄皮内生细菌菌群多样性进行研究。在兼性厌氧条件下，从黄皮韧皮部中共分离出41种可培养内生细菌，分属于18个属。其中Pudomonas sp.、Acinetobacter sp.为健康黄皮内生菌的优势菌属；Chryobacterium sp.、Bacillus sp.和Sphingomonas sp.为感病黄皮内生菌的优势菌属。健、病黄皮韧皮结构中的内生细菌种类相差很大，有8个属在两者中都存在，包括Acinetobacter sp.、Pudomonas sp.、Bacillus sp.、Chryobacterium sp.、Sphingomonas sp.、Serratia sp.、Stenotrophomonas sp.、Brevundimonas sp.，但其分离频率差异很大。
采用ARDRA指纹图谱分析法，从健株和病株中分别得到27、17个操作分类单元(OTU)，其中Pudomonas sp.在两者中均为优势菌属。
两种分离方法都表明γ-变形菌是黄皮内生细菌的主要菌群。
对可培养内生细菌进行7种功能鉴定（蛋白酶活力、淀粉水解能力、IAA产
生能力、Fe血细胞合成能力、抗生素合成能力、自身固氮能力、ACC脱氢酶合成能力），筛选出具有至少5种功能的菌株有Escherichia hermannii（Cl-04）、Serratia proteamaculans（Cl-06）、Pantoea agglomerans（Cl-17）、Pantoea sp.（Cl-18）、Arthrobacter sp.（Cl-19）、Enterobacter ludwigii（Cl-24），分属于4个属。只有Serratia proteamaculans在健、病植株中都存在，且分离频率
都很低。其余菌株均只从健株中分离到。
本研究第一部分内容表明柑橘黄龙病病株中，各部位所带病菌量不均匀，是否显症与组织内柑橘黄龙病菌的量呈正相关，内生菌群总量与显症无相关性，但粘质沙雷氏菌与黄龙病菌的量呈负相关。粘质沙雷氏菌与黄龙病菌在韧皮部细胞内增殖过程中的相互作用值得深入研究。第二部分研究结果表明，感病与健康黄皮植株中均含有丰富的内生细菌，但它们的菌群结构不同。黄龙病菌侵入植物体后，内生细菌种类变少，可见其对黄皮韧皮部内的菌群产生了一定的影响。从黄皮韧皮部中筛选出的多种功能性细菌，为进一步了解它们与黄龙病菌的相互关系奠定基础。
关键词：黄龙病，内生细菌，多样性，粘质沙雷氏菌，功能分析
ABSTRACT
cousin是什么意思Due to heavy damage, wide distribution area, spread fast and no effective measures to control, citrus Huanglongbing (HLB) became a hot topic in citrus dia rearch. HLB is caud by a kind of nonculturable bacterium belongs to the genus “Candidatus Liberibacter” and belongs to Alphaproteobacteria, which survived in the phloem of hosts only. Now, there are many reports about the mutual antagonism and mutualism between bacteria.
Bad on the previous studies, We lected two kinds of citrus HLB host rutaceae (citrus and Clauna lansium （Lour.）Skeels) , understanding microflora from the “Candidatus Liberibacter” living environment. Comparative analysis of the endophytic bacteria in symptoms and symptomless tissues of single HLB-affected citrus plan and of the diad and healthy wampee, that is the foundation to recognize the effects between Candidatus Liberibacter and the endophytic bacteria. The endophytic bacteria isolated from wampee may antagonize the HLB pathogen, and also may be mutualism, which is the foundation of pathogenesis and biological control.
accounting
1. The population structure and bacterial diversity of endophytic bacteria in symptoms and symptomless tissues of single HLB-affectedcitrus plant were investigated bad on 16S rDNA quences analyzed by PCR-DGGE (PCR-denaturing gradient gel electrophoresis). Quantitative analysis of Ca.Las, dominant bacteria and total amount of bacterial population in symptoms and symptomless tissues of branches, leaves and fruits was carried out by quantitative PCR.
The results showed that the amount of Ca.Las is different significantly in symptoms and symptomless tissues; bacterial population in symptoms citrus tissues was significantly higher than in symptomless tissues. The PCR-DGGE analysis result showed that endophytic bacterial structures are basically same in symptoms and symptomless tissues. Extraction, cloning and quencing of 17
obvious bands in DGGE electrophoretogram revealed that 8 of 17 bands belonged to the genus Serratia, which accounted for 47.06%. Sequence alignment showed that they belonged to different strains of Serratia marcescens with the 99.63% similarity. Quantitative analysis of the total bacteria and Serratia marcescens showed that there is no significant difference in total bacterial count between symptoms and symptomless tissues while the amount of Serratia marcescens showed negatively correlation with
