Pseudomonas syringae致病变种的分子鉴定技术及其应用
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摘要
植物病原丁香假单胞菌(Pseudomonas syringae)根据其寄主植物以及所致病害的不同症状被分为许多致病变种(pathovar)。致病变种的鉴定是一项重要的基础工作,通常依据一系列复杂的理化特征等细菌学方法来鉴定。随着分子生物学技术的飞速发展,核酸杂交技术、基因组DNA指纹图谱分析技术、保守基因以及特定基因的序列分析技术,为植物病原丁香假单胞菌基因组种团(genomospecies)的划分以及致病变种的鉴定提供了先进方法。
本研究通过分析16S rDNA、16S-23S ITS、hrp基因在丁香假单胞菌致病变种鉴定中的可能作用,并把分子鉴定结果与常规的细菌学鉴定结果相互验证,旨在筛选出适于丁香假单胞菌致病变种快速鉴定的指标分子,建立一整套快速鉴定致病变种的分子鉴定技术体系。
本研究所取得的主要结果如下：
1.以来自8个丁香假单胞菌致病变种P. s. pv. tomato、P. s. pv. angulata、P. s. pv. tabaci、P. s. pv. syringae、P. s. pv. garcae、P.s. pv. glycinea、P. s. pv. phaseolicola、P. s. pv. maculicola以及相关菌株P. viridiflava、P. gingeri的共16个标准菌株的基因组DNA为模板,利用16S rDNA通用引物进行PCR扩增,得到长度约为1560bp的特异性条带,通过T/A克隆获得含有目标片段的重组质粒,经测序得到了各致病变种16S rDNA基因的近全长序列。序列Blast同源性检索以及序列系统进化分析结果显示：植物假单胞病菌的16S rDNA序列高度保守,适于属以及种水平上的鉴定。丁香假单胞菌的16S rDNA序列更为保守,16S rDNA基因的系统发育分析表明,这一高度保守的16S rDNA基因不能满足丁香假单胞菌致病变种分子鉴定的要求。
2.利用16S-23S rDNA的保守序列设计了扩增ITS全长序列的简并引物,以8个丁香假单胞菌致病变种的16个标准菌株的基因组DNA为模板进行PCR扩增,得到长度为750bp的特异性条带。PCR产物分别经限定性内切酶HincⅡ及DdeⅠ酶切片段的RFLP分析表明,丁香假单胞菌的16S-23S ITS同样高度保守,P. syringae 8个致病变种的ITS扩增产物的RFLP图谱没有足够的多态性用来鉴别致病变种,仅有P. gingeri、P. viridiflava与P. syringae各致病变种的ITS-PCR-RFLP图谱明显不同。因此,ITS序列同样不是丁香假单胞菌致病变种分子鉴定的理想指标。
3. hrpZ基因是病原细菌所独有的,其编码产物既与致病性又与诱导非寄主的过敏性有关。利用设计的4对hrpZ基因引物,以丁香假单胞菌致病变种P. s. pv. tomato、P. s. pv. angulata、P. s. pv. tabaci、P. s. pv. syringae、P.s. pv. garcae、P. s. pv. glycinea、P. s. pv. phaseolicola、P. s. pv. maculicola及其它相关供试菌株P. viridiflava、P. gingeri的基因组DNA为PCR扩增模板,分析了hrpZ基因的PCR扩增片段长度多态性。结果表明,引物对HrpABF/R能够有效区分全部8个供试丁香假单胞菌致病变种；引物对HrpZ08F/R1与HrpZ359/1115对于P. s. pv. garcae以及相关菌株无特异性扩增结果或者多态性结果。引物对HrpZ09UP/DW对供试菌株也有特异性或多态性扩增结果,且在P. s. pv. syringae不同菌株之间存在条带亮度差异。除引物对HrpABF/R外,引物对HrpZ08F/R、HrpZ09UP/DW与HrpZ359/1115都不能对P. s. pv. angulala和P. s. pv. tabaci进行有效区分,说明了P. s. pv. angulata和P. s. pv. tabaci二者的hrpZ可能相同。与16S rDNA以及ITS相比,hrpZ基因的PCR扩增产物的多态性在致病变种间非常丰富,而且致病变种内的保守性强,扩增结果清晰易辩。hrpZ基因的系统进化分析也表明了该基因是丁香假单胞菌致病变种快速鉴定的理想分子指标。
4.常规细菌学鉴定实验表明,细菌形态特征、经典LOPAT以及GATTa等一系列理化鉴定结果与基于hrpZ基因的分子鉴定结果相一致,但基于hrpZ的分子鉴定具有快速、准确、易操作、结果客观等独特优势,说明本研究成功建立了基于hrpZ基因的致病变种分子鉴定技术体系,为植物病原丁香假单胞菌及其致病变种的快速鉴定提供了一个有效策略。
5.本研究中多次发现野火致病变种与角斑致病变种在形态学特性、理化特性、PCR扩增特点等十分相似。为了区分二者,利用设计的引物对tabAF/R的特异性扩增区分了P. s. pv. angulata和P. s. pv. tabaci两个致病变种。结果表明野火致病变种P. s. pv. tabaci能够得到片段长度1263bp的特异性条带,而角斑致病变种P. s. pv. angulata则没有该特异性条带。说明能够利用特定基因(即烟毒素编码基因)的扩增策略能够区分开tab+菌株和tab-菌株。
6.利用本研究建立的基于hrpZ基因以及tabA基因的分子鉴定技术体系,成功地对豫西烟草细菌性叶斑病进行了病原菌的快速鉴定,最终确定该细菌性叶斑病的病原为P. s. pv. tabaci的tab-菌株(即P. s. pv. angulata),这一鉴定结果同样得到了常规细菌学鉴定的验证与支持。
Pseudomonas syringae group includes a number of pathovars that vary in their host range and/or disease symptoms. The traditional bacteriological identification is a basic but important work, including a series of physiological and morphyological characteristics tests. With the development of molecular biological technology, DNA-DNA hybridization, genomic DNA fingerprint analysis and DNA sequence analysis of conservative genes and special genes have been used to distinguish the strains or pathovars of P. syringae and to classify P. syringae into several groups-genomospecies.
In order to screen some useful molecular identification marker and build up a new available strategy,8 pathovars and their 16 identified strains were used. Their 16S rDNA genes were sequenced, the 16S-23S rDNA internal transcriptional spacer sequences were analysed by PCR-RFLP, and one important hypersensitive-response and pathogenesis related gene, hrpZ gene was cloned and analysied. At the same time, the traditional bacteriological identification were performed for the validity of the established molecular identification strategy.
The main results of this study were followed:
1. Sixteen standard strains belonging to eight Pseudomonas syringae pathvars including P. s. pv. tomato, P. s. pv. angulata, P. s. pv. tabaci, P. s. pv. syringae, P. s. pv. garcae, P. s. pv. glycinea, P. s. pv. phaseolicola, P. s. pv. maculicola and two strains from P. viridiflava and P. gingeri were used in the experiments. The full sequences of 16S rDNA gene were cloned with the universal 16S rDNA primers. The unique, band of about 1560bp in size were recovered, cloned and sequenced. These sequences were blasted in Genebank database for researching their homologies. According to the results of phylogenetic analysis,16S rDNA sequences were so highly conservative that they only were an available molecular marker for genus and species identification of Pseudomonas syringae. However, the 16S rDNA sequence was not a useful molecular marker for pathvar determination.
2. The full-length ITS of the tested strains were amplified by PCR with the degenerate primers. Unique product of about 750bp in size were recovered and degested with HincⅡand DdeⅠ, respectively, in order to analyse the PCR-RFLP. The results showed that there just were some differences at inter-species level, while revealing highly consistent at inter-pathovar and intra-pathovar level. Therefore, the ITS sequence was neither an ideal molecular marker.
3. The hrp genes in plant pathogenic bacteria play important roles in causing disease on host plants and eliciting hypersensitive response on non-host plants. The sequences of hrpZ gene of the tested strains were amplified by PCR with the degenerate primers. The sequence polymorphism of hrpZ gene were analyzed and the NJ-tree was constructed. The target fragments coule be amplified with HrpABF/HrpABR, HrpZ09UP/HrpZ09DW primers, and also distinguished by HrpZ08F/HrpZ08Rl and HrpZ359/HrpZl 115 but for P. s. pv. garcae and related strain P. viridiflava and P. gingeri. We also found that there were some differences between the tested strains of P. s. pv. syringae. Comparing with the 16S rDNA and ITS molecular marker, as a new molecular identification marker hrpZ gene was very conservative in different strains of a certain Pseudomonas syringae pathvar and highly homologous in different pathvars. The differences between the tested pathvars could be detected clearly. In conclusion, hrpZ gene was a perfect molecular marker for the rapid identification of Pseudomonas syringae pathvars.
4. Consistent with the trantional identification results, which were based on characteristics of physical and chemical, hrpZ-based molecular identification with clear and objective result was much more rapid, accurate and easy to carry out. It revealed us that as the special gene hrpZ was a new perfect molecular identification marker and the strategy based on this gene was viable.
5. During the study, usually, P. s. pv. angulata and P. s. pv. tabaci were not only familiar with most of the characteristics of physical and chemical, but also with the same band amplified by PCR. To differentiate P. s. pv. angulata from P. s. pv. tabaci, tabAF/tabAR primers were designed for amplification by PCR. P. s. pv. angulata lacked this special band as a important feature and was distinguished from P. s. pv. tabaci. Thus the tab A gene was another useful molecular marker, which was used to distinguish the two pathvars.
6. Using the molecular identification technology strategy, it was successful to identify the pathogen causing bacterial leaf spot on tobacco in the west of Henan province. The pathogen was tab- strain of P. s. pv. tabaci. At the same time the identified result was validated by the characteristics of physical and chemical.
本研究通过分析16S rDNA、16S-23S ITS、hrp基因在丁香假单胞菌致病变种鉴定中的可能作用,并把分子鉴定结果与常规的细菌学鉴定结果相互验证,旨在筛选出适于丁香假单胞菌致病变种快速鉴定的指标分子,建立一整套快速鉴定致病变种的分子鉴定技术体系。
本研究所取得的主要结果如下：
1.以来自8个丁香假单胞菌致病变种P. s. pv. tomato、P. s. pv. angulata、P. s. pv. tabaci、P. s. pv. syringae、P. s. pv. garcae、P.s. pv. glycinea、P. s. pv. phaseolicola、P. s. pv. maculicola以及相关菌株P. viridiflava、P. gingeri的共16个标准菌株的基因组DNA为模板,利用16S rDNA通用引物进行PCR扩增,得到长度约为1560bp的特异性条带,通过T/A克隆获得含有目标片段的重组质粒,经测序得到了各致病变种16S rDNA基因的近全长序列。序列Blast同源性检索以及序列系统进化分析结果显示：植物假单胞病菌的16S rDNA序列高度保守,适于属以及种水平上的鉴定。丁香假单胞菌的16S rDNA序列更为保守,16S rDNA基因的系统发育分析表明,这一高度保守的16S rDNA基因不能满足丁香假单胞菌致病变种分子鉴定的要求。
2.利用16S-23S rDNA的保守序列设计了扩增ITS全长序列的简并引物,以8个丁香假单胞菌致病变种的16个标准菌株的基因组DNA为模板进行PCR扩增,得到长度为750bp的特异性条带。PCR产物分别经限定性内切酶HincⅡ及DdeⅠ酶切片段的RFLP分析表明,丁香假单胞菌的16S-23S ITS同样高度保守,P. syringae 8个致病变种的ITS扩增产物的RFLP图谱没有足够的多态性用来鉴别致病变种,仅有P. gingeri、P. viridiflava与P. syringae各致病变种的ITS-PCR-RFLP图谱明显不同。因此,ITS序列同样不是丁香假单胞菌致病变种分子鉴定的理想指标。
3. hrpZ基因是病原细菌所独有的,其编码产物既与致病性又与诱导非寄主的过敏性有关。利用设计的4对hrpZ基因引物,以丁香假单胞菌致病变种P. s. pv. tomato、P. s. pv. angulata、P. s. pv. tabaci、P. s. pv. syringae、P.s. pv. garcae、P. s. pv. glycinea、P. s. pv. phaseolicola、P. s. pv. maculicola及其它相关供试菌株P. viridiflava、P. gingeri的基因组DNA为PCR扩增模板,分析了hrpZ基因的PCR扩增片段长度多态性。结果表明,引物对HrpABF/R能够有效区分全部8个供试丁香假单胞菌致病变种；引物对HrpZ08F/R1与HrpZ359/1115对于P. s. pv. garcae以及相关菌株无特异性扩增结果或者多态性结果。引物对HrpZ09UP/DW对供试菌株也有特异性或多态性扩增结果,且在P. s. pv. syringae不同菌株之间存在条带亮度差异。除引物对HrpABF/R外,引物对HrpZ08F/R、HrpZ09UP/DW与HrpZ359/1115都不能对P. s. pv. angulala和P. s. pv. tabaci进行有效区分,说明了P. s. pv. angulata和P. s. pv. tabaci二者的hrpZ可能相同。与16S rDNA以及ITS相比,hrpZ基因的PCR扩增产物的多态性在致病变种间非常丰富,而且致病变种内的保守性强,扩增结果清晰易辩。hrpZ基因的系统进化分析也表明了该基因是丁香假单胞菌致病变种快速鉴定的理想分子指标。
4.常规细菌学鉴定实验表明,细菌形态特征、经典LOPAT以及GATTa等一系列理化鉴定结果与基于hrpZ基因的分子鉴定结果相一致,但基于hrpZ的分子鉴定具有快速、准确、易操作、结果客观等独特优势,说明本研究成功建立了基于hrpZ基因的致病变种分子鉴定技术体系,为植物病原丁香假单胞菌及其致病变种的快速鉴定提供了一个有效策略。
5.本研究中多次发现野火致病变种与角斑致病变种在形态学特性、理化特性、PCR扩增特点等十分相似。为了区分二者,利用设计的引物对tabAF/R的特异性扩增区分了P. s. pv. angulata和P. s. pv. tabaci两个致病变种。结果表明野火致病变种P. s. pv. tabaci能够得到片段长度1263bp的特异性条带,而角斑致病变种P. s. pv. angulata则没有该特异性条带。说明能够利用特定基因(即烟毒素编码基因)的扩增策略能够区分开tab+菌株和tab-菌株。
6.利用本研究建立的基于hrpZ基因以及tabA基因的分子鉴定技术体系,成功地对豫西烟草细菌性叶斑病进行了病原菌的快速鉴定,最终确定该细菌性叶斑病的病原为P. s. pv. tabaci的tab-菌株(即P. s. pv. angulata),这一鉴定结果同样得到了常规细菌学鉴定的验证与支持。
Pseudomonas syringae group includes a number of pathovars that vary in their host range and/or disease symptoms. The traditional bacteriological identification is a basic but important work, including a series of physiological and morphyological characteristics tests. With the development of molecular biological technology, DNA-DNA hybridization, genomic DNA fingerprint analysis and DNA sequence analysis of conservative genes and special genes have been used to distinguish the strains or pathovars of P. syringae and to classify P. syringae into several groups-genomospecies.
In order to screen some useful molecular identification marker and build up a new available strategy,8 pathovars and their 16 identified strains were used. Their 16S rDNA genes were sequenced, the 16S-23S rDNA internal transcriptional spacer sequences were analysed by PCR-RFLP, and one important hypersensitive-response and pathogenesis related gene, hrpZ gene was cloned and analysied. At the same time, the traditional bacteriological identification were performed for the validity of the established molecular identification strategy.
The main results of this study were followed:
1. Sixteen standard strains belonging to eight Pseudomonas syringae pathvars including P. s. pv. tomato, P. s. pv. angulata, P. s. pv. tabaci, P. s. pv. syringae, P. s. pv. garcae, P. s. pv. glycinea, P. s. pv. phaseolicola, P. s. pv. maculicola and two strains from P. viridiflava and P. gingeri were used in the experiments. The full sequences of 16S rDNA gene were cloned with the universal 16S rDNA primers. The unique, band of about 1560bp in size were recovered, cloned and sequenced. These sequences were blasted in Genebank database for researching their homologies. According to the results of phylogenetic analysis,16S rDNA sequences were so highly conservative that they only were an available molecular marker for genus and species identification of Pseudomonas syringae. However, the 16S rDNA sequence was not a useful molecular marker for pathvar determination.
2. The full-length ITS of the tested strains were amplified by PCR with the degenerate primers. Unique product of about 750bp in size were recovered and degested with HincⅡand DdeⅠ, respectively, in order to analyse the PCR-RFLP. The results showed that there just were some differences at inter-species level, while revealing highly consistent at inter-pathovar and intra-pathovar level. Therefore, the ITS sequence was neither an ideal molecular marker.
3. The hrp genes in plant pathogenic bacteria play important roles in causing disease on host plants and eliciting hypersensitive response on non-host plants. The sequences of hrpZ gene of the tested strains were amplified by PCR with the degenerate primers. The sequence polymorphism of hrpZ gene were analyzed and the NJ-tree was constructed. The target fragments coule be amplified with HrpABF/HrpABR, HrpZ09UP/HrpZ09DW primers, and also distinguished by HrpZ08F/HrpZ08Rl and HrpZ359/HrpZl 115 but for P. s. pv. garcae and related strain P. viridiflava and P. gingeri. We also found that there were some differences between the tested strains of P. s. pv. syringae. Comparing with the 16S rDNA and ITS molecular marker, as a new molecular identification marker hrpZ gene was very conservative in different strains of a certain Pseudomonas syringae pathvar and highly homologous in different pathvars. The differences between the tested pathvars could be detected clearly. In conclusion, hrpZ gene was a perfect molecular marker for the rapid identification of Pseudomonas syringae pathvars.
4. Consistent with the trantional identification results, which were based on characteristics of physical and chemical, hrpZ-based molecular identification with clear and objective result was much more rapid, accurate and easy to carry out. It revealed us that as the special gene hrpZ was a new perfect molecular identification marker and the strategy based on this gene was viable.
5. During the study, usually, P. s. pv. angulata and P. s. pv. tabaci were not only familiar with most of the characteristics of physical and chemical, but also with the same band amplified by PCR. To differentiate P. s. pv. angulata from P. s. pv. tabaci, tabAF/tabAR primers were designed for amplification by PCR. P. s. pv. angulata lacked this special band as a important feature and was distinguished from P. s. pv. tabaci. Thus the tab A gene was another useful molecular marker, which was used to distinguish the two pathvars.
6. Using the molecular identification technology strategy, it was successful to identify the pathogen causing bacterial leaf spot on tobacco in the west of Henan province. The pathogen was tab- strain of P. s. pv. tabaci. At the same time the identified result was validated by the characteristics of physical and chemical.
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