两种植原体病害的分子检测与鉴定
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摘要
本研究采用分子生物学方法,对引起桃树黄化和玉兰黄化的植原体进行了鉴定,主要结果如下：
桃树是我国重要的果树作物,桃树黄化病表现为发病的叶片除叶脉仍然保持绿色外,叶片的其他部位均表现出均匀的黄化现象,在部分发病严重的枝梢上,出现叶片卷曲,皱缩的情况。病害危害1-2年后,病株整株出现黄化,部分枝梢落叶情况非常严重。桃树黄化病害发生的地点是一种点状的发生为主,病害有明显的扩散趋势。但是这种扩散趋势不是一种爆发式,而是通过一种年度积累扩散。
本研究用分子生物学方法对我国北京地区利用针对植原体的通用引物,通过巢式PCR从自然表现黄化的桃树植株中扩增到了植原体16S rRNA。克隆、测序、进行序列分析并运用新的分类方法对其进行了分类,利用国际基因数据库GenBank中的BLAST程序对测序结果进行同源性检索发现,与其他16SrⅠ翠菊黄化组(Aster yellows group)组中的各植原体亲缘关系均在98%以上,充分肯定了其作为16SrⅠ组成员的分类地位。在荧光显微镜下,桃树黄化病枝韧皮部成熟筛管中产生健康植株中没有的特异性黄绿色荧光,嫁接实验证明植原体是桃树黄化的病原。
由于在自然发病的桃树黄化病叶中植原体的含量很少,本研究对DNA提取方法和PCR的条件进行了一系列的摸索和改进,建立了优化的实验条件,包括在DNA提取过程中去掉叶肉、选用组培病苗等,进行PCR时对模板DNA稀释、选择最佳退火温度等。
通过巢式PCR证明表现黄化症状的玉兰树中含有植原体。16SrRNA序列的结果表明与玉兰黄化相关的植原体属于翠菊黄化组16SrI亚组。
本实验中的桃树黄化病、玉兰黄化病是国内外首次报道。本研究对桃树黄化植原体和玉兰黄化植原体进行了分子鉴定,初步研究了它与其它植原体的系统进化关系和分子差异；建立了桃树黄化植原体快速准确的检测、鉴定与鉴别的技术体系；为这些植原体的科学分类和命名、病原基因组学和功能基因组学研究、亚组及变异分析、病原检测、病害检疫及病害的治理等都奠定了良好基础。
In this dissertation, molecular biological methods were applied to identify peach yellws and magnolia yellows phytoplasma isolate collected from different regions in China, the main results read as follows.
Peach (Purnus persica) is a very important fruit tree in China. Purnus persica yellows (PY) disease, whose typical symptom is leaves show yellows besides nervation,.In some of branches with serious disease, the leaves show curly and crimple symptom.
The peach tree that the pathogen infect, after 1-2 years later, leaves of the whole tree are yellow, a few branches fall leaves serious. The spot adherence is the main way of the place where Peach yellows happened. The disease has obviously disffused tendency. This tendency is accumulate by years but not explosive. In this research, using phytoplasma universal primers for 16SrRNA gene to detect phytoplasma associated with peach leaf showing yellows symptom. A 1.4kb DNA fragment was amplified by nested-PCR from the total DNA of diseased peach samples. After cloning and nucleotide sequencing of the amplified fragment, and the new classification standard were adopted to identify PY phytoplasma isolates collected from different regions in China. Using the BLAST procedures of international gene database Gene Bank, the results demonstrated that the strain shared identities of more than 98% with other members in 16SrI group, but is obviously under 97.0% with other groups, So the results made it clear that this phytoplasma strain is one of the members of Aster yellows phytoplasma group. The phloem of Peach yellws have differential fluorescence that isn't observed in healthy peach. The phytoplasma particles were observed under fluorescence microscope. Graft experiment also conformed that air-plant phyllody is caused by phytoplasma.
Because the phytoplasma of Peach yellws in nature is very few, the methods of DNA extraction and the conditions of PCR were studied and modified. the optimized experimental conditions were built up, such as removal of leaf mesophyll and use of in vitro cultured plantlets when extracting DNA, serial dilution of template DNA and optimizing of annealing temperature during PCR amplification.
Using phytoplasma universal primers for 16SrRNA gene to detect phytoplasma asso-ciated with magnolia leaf showing yellows symptom. the results of 16S rRNA made it clear that this phytoplasma strain is one of the members of Aster yellows phytopla-sma group.
This study revealed the molecular specialty of PY,MY phytoplasma and its phylogenetic relationship and molecular differentiation with other phytoplasmas. And the protocol for the rapid and accurate detection, identification and diferentiation of PY,MY phytoplasma is outlined. These approaches lay the foundation for the scientific classification and nomenclature,phytoplasmal genomics, pathogen detection, disease quarantine, epidemiological researches and disease management
桃树是我国重要的果树作物,桃树黄化病表现为发病的叶片除叶脉仍然保持绿色外,叶片的其他部位均表现出均匀的黄化现象,在部分发病严重的枝梢上,出现叶片卷曲,皱缩的情况。病害危害1-2年后,病株整株出现黄化,部分枝梢落叶情况非常严重。桃树黄化病害发生的地点是一种点状的发生为主,病害有明显的扩散趋势。但是这种扩散趋势不是一种爆发式,而是通过一种年度积累扩散。
本研究用分子生物学方法对我国北京地区利用针对植原体的通用引物,通过巢式PCR从自然表现黄化的桃树植株中扩增到了植原体16S rRNA。克隆、测序、进行序列分析并运用新的分类方法对其进行了分类,利用国际基因数据库GenBank中的BLAST程序对测序结果进行同源性检索发现,与其他16SrⅠ翠菊黄化组(Aster yellows group)组中的各植原体亲缘关系均在98%以上,充分肯定了其作为16SrⅠ组成员的分类地位。在荧光显微镜下,桃树黄化病枝韧皮部成熟筛管中产生健康植株中没有的特异性黄绿色荧光,嫁接实验证明植原体是桃树黄化的病原。
由于在自然发病的桃树黄化病叶中植原体的含量很少,本研究对DNA提取方法和PCR的条件进行了一系列的摸索和改进,建立了优化的实验条件,包括在DNA提取过程中去掉叶肉、选用组培病苗等,进行PCR时对模板DNA稀释、选择最佳退火温度等。
通过巢式PCR证明表现黄化症状的玉兰树中含有植原体。16SrRNA序列的结果表明与玉兰黄化相关的植原体属于翠菊黄化组16SrI亚组。
本实验中的桃树黄化病、玉兰黄化病是国内外首次报道。本研究对桃树黄化植原体和玉兰黄化植原体进行了分子鉴定,初步研究了它与其它植原体的系统进化关系和分子差异；建立了桃树黄化植原体快速准确的检测、鉴定与鉴别的技术体系；为这些植原体的科学分类和命名、病原基因组学和功能基因组学研究、亚组及变异分析、病原检测、病害检疫及病害的治理等都奠定了良好基础。
In this dissertation, molecular biological methods were applied to identify peach yellws and magnolia yellows phytoplasma isolate collected from different regions in China, the main results read as follows.
Peach (Purnus persica) is a very important fruit tree in China. Purnus persica yellows (PY) disease, whose typical symptom is leaves show yellows besides nervation,.In some of branches with serious disease, the leaves show curly and crimple symptom.
The peach tree that the pathogen infect, after 1-2 years later, leaves of the whole tree are yellow, a few branches fall leaves serious. The spot adherence is the main way of the place where Peach yellows happened. The disease has obviously disffused tendency. This tendency is accumulate by years but not explosive. In this research, using phytoplasma universal primers for 16SrRNA gene to detect phytoplasma associated with peach leaf showing yellows symptom. A 1.4kb DNA fragment was amplified by nested-PCR from the total DNA of diseased peach samples. After cloning and nucleotide sequencing of the amplified fragment, and the new classification standard were adopted to identify PY phytoplasma isolates collected from different regions in China. Using the BLAST procedures of international gene database Gene Bank, the results demonstrated that the strain shared identities of more than 98% with other members in 16SrI group, but is obviously under 97.0% with other groups, So the results made it clear that this phytoplasma strain is one of the members of Aster yellows phytoplasma group. The phloem of Peach yellws have differential fluorescence that isn't observed in healthy peach. The phytoplasma particles were observed under fluorescence microscope. Graft experiment also conformed that air-plant phyllody is caused by phytoplasma.
Because the phytoplasma of Peach yellws in nature is very few, the methods of DNA extraction and the conditions of PCR were studied and modified. the optimized experimental conditions were built up, such as removal of leaf mesophyll and use of in vitro cultured plantlets when extracting DNA, serial dilution of template DNA and optimizing of annealing temperature during PCR amplification.
Using phytoplasma universal primers for 16SrRNA gene to detect phytoplasma asso-ciated with magnolia leaf showing yellows symptom. the results of 16S rRNA made it clear that this phytoplasma strain is one of the members of Aster yellows phytopla-sma group.
This study revealed the molecular specialty of PY,MY phytoplasma and its phylogenetic relationship and molecular differentiation with other phytoplasmas. And the protocol for the rapid and accurate detection, identification and diferentiation of PY,MY phytoplasma is outlined. These approaches lay the foundation for the scientific classification and nomenclature,phytoplasmal genomics, pathogen detection, disease quarantine, epidemiological researches and disease management
引文
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