【作者】 朱勃；

【导师】 谢关林；

【作者基本信息】 浙江大学 ， 植物病理学， 2010， 博士

【摘要】 2006年初夏在浙江杭州地区的桑树主栽区发生一种严重的桑树新病害—桑细菌性枯萎病,病害逐年加重,对蚕桑生产造成了严重损失。为有效治理该病,本研究对该病原及其检测方法作了深入的研究。以6株桑细菌性枯萎病分离菌株为代表,通过一系列的研究方法证明了该病原是一种肠杆菌(Enterobacter)病原；利用rpoB序列设计了一对特异性引物,对桑细菌性枯萎病病原进行检测；同时还应用生物信息学技术对肠杆菌科细菌由于基因的获得与丢失而造成的多样性进行了分析与评估。取得了以下主要成果：(1)通过6株桑细菌性枯萎病病原细菌代表菌株的常规细菌学分析、生理生化分析、电镜观察、致病性测试及Koch氏假说测定、Biolog鉴定、脂肪酸分析、ERIC-PCR分析、16S rRNA及rpoB序列分析,首次明确了该桑病是国内外未见报道的一种由肠杆菌引起的新病害,最终通过代表菌株R18-2 (ZJUPD3)与9种与植物有关的肠杆菌标准菌株的DNA-DNA杂交,明确了同源性关系,病原归属于Enterobacter cloacae complex (Ecc群)中,并将其定名为Enterobacter morus, R18-2 (ZJUPD3)菌株已作为该新种的模式菌株分别保藏于中国微生物菌种保藏管理中心(CGMCC 1.10322T)和比利时王国菌种保藏管理中心(BCC-LMG 25706)。(2)利用rpoB序列设计并合成了一对桑细菌性枯萎病病原Enterobacter morus的特异性引物,通过与其它9种近缘肠杆菌标准菌株、4株桑树细菌性枯萎病、2株桑青枯病病原细菌、2株桑疫病病原细菌及50株桑腐生(附生)菌株的测试后发现其特异性达到了预期的要求；并利用实时定量PCR技术对枯萎菌的灵敏性进行测试,结果发现在一个20μl反应体系中如果含有一个活菌细胞即能有效的检测出来,体现了其良好的灵敏性；此外还利用桑树根、茎田间采样样本直接进行PCR检测,发现无论是已有还是未见明显症状的样本,只要是后期实验中能分离到桑枯萎病病原的样本,通过本PCR手段都能有效检测。研究结果充分体现了该对引物的灵敏性及特异性。首次建立了具有自主知识产权的桑细菌性枯萎病菌检测体系。并申报了国家发明专利(发明专利申请号：200910157005.4)。(3)本研究通过对肠杆菌科细菌的基因水平转移分析,发现基因的获得与丢失在肠杆菌科的进化历史中占据了非常重要的作用；肠杆菌科的核心基因和泛基因的数量差异非常明显,根据估算,肠杆菌科的核心基因数量约为164个,而泛基因数量可以达到约10万个,我们根据其变化的规律拟合了其趋势公式,分别为根据我们利用基因的存在与丢失所构建的马科夫模型推测,肠杆菌科细菌在进化过程中首先经历了一次大量的基因丢失的过程,产生了基因组非常小的内生菌,随后在进化的不同的时期获得了大量的基因,产生了致病力及寄主各不相同的各类病原菌；此外该模型推测超过1／3的肠杆菌科的基因是通过水平转移获得的,表明了水平转移对于肠杆菌科类细菌的影响是非常巨大的；利用最大似然法对这些水平转移所获得的基因与核心基因进行正向选择的分析,结果表明表明水平转移所获得的基因更容易受到强烈的正向选择的影响。更多还原

【Abstract】 A new mulberry wilt disease was observed in summer of 2006, which caused severe economic lost in mulberry plantations in Hangzhou, Zhejiang, China and the disease severity increased year by year. For effective management of the disease, the exact bacterial pathogen needs to be determined and the scientific name has to be nomenclatured. An accurate, rapid and sensitive molecular detection method for the pathogen has to be established. Based on a series of phytopathological methods for the 6 representative strains, the pathogen of this new mulberry bacterial wilt disease was proved to be a new species in Enterobacter genus; a pair of specific primers based on the rpoB regions was designed to detect the pathogen; the bioinformatics tools was used to analyze and evaluate the diversity of Enterobacteriaceae based on the gene gain and loss results. The major results are listed as follows:(1). The results of traditional bacteriology tests, physiological and biochemical analysis, electron microscope observation, pahogenicity and hypersensitive tests, biolog tests, ERIC-PCR analysis,16S rRNA and rpoB sequences analysis of the 6 representative strains show this pathogen is a new species in Enterobacter genus, which has not been reported before. Based on the results of DNA-DNA hybridization of a representative strain R18-2 (ZJUPD3) with the nine Enterobacter type strains, the pathogen was classified into Enterobacter cloacae complex and was nomenclatured as Enterobacter morus. Strain R18-2(ZJUPD3) has been deposited in culture collection center of China and BCCM/LMG Bacteria Collection of Belgium with the deposite ID CGMCC 1.10322T and LMG 25706T, respectivey.(2). A specific primer of the Enterobacter morus was designed within regions of the RNA polymeraseβ-subunit (rpoB) gene. An accurate, sensitive and rapid method was established and tested by performing on 9 type species of Enterobacter,4 E. morus,2 Ralstonia solanacearum strains,2 Pseudomonas syringae pv. mori strains and 50 unidentified epiphytic bacterial isolates from mulberry plants. The real-time PCR assays reliably detected one single cell in a 20μl reaction system, reflecting its strong sensitivity. Moreover, mulberry roots and stems were directly used for PCR detection and it was found that samples with E. morus (acquired by isolation) can be detected by this method whether it has obvious symptoms or not. The results indicated the sensitivity and specificity of the primer. This is the first report of establishing detection system for E. morus causing mulberry bacterial wilt and we applied a China national invention patent (Patent Application Number:200910157005.4).(3). Based on the horizontal gene transfer (HGT) analysis of Enterobacteriaceae, it can be concluded that gene gain and loss is the major force in Enterobacteriaceae evolution and the divergence is huge between core and pan genome. The number of core genome was only 164 while pan genome was more than 100,000. The size of the species core genome for a large number of sequenced strains was extrapolated by fitting the exponential decaying 2 functions to the amount of conserved genes while the number of strain-specific genes was extrapolated by fitting the Multiple Multiplicative Factor (MMF) model to the amount of strain-specific genes. We may speculate that the small genome endophyte in Enterobacteriaceae was first evolved with a gene loss event in this family. Then, species of pathogens were arised with continous HGT events in different evolutionary scale. The markov models suggested that more than 1/3 of genes in Enterobacteriaceae were horizontally transferred, which indicated that HGT played an important role in evolution. Positive selection analysis also revealed that the transferred genes were much easier to be influenced by positive selection pressure than those of the conserved genes.更多还原