柑橘黄龙病菌及与其混合发生病毒的分子特性及超低温脱除研究
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摘要
柑橘黄龙病(Citrus Huanglongbing，HLB)是世界性柑橘生产上的毁灭性病害。易与其混合发生的几种柑橘病毒病及类似病害主要包括衰退病、裂皮病和碎叶病；病原分别为Citrus tristeza virus(CTV)，Citrus exocortis viroid(CEVd)，Citrus tatter leaf
virus(CTLV)。本研究主要以HLB病菌、CTV、CEVd、CTLV为对象，从生物学角度及分子水平上对其部分特性进行研究；目的是建立上述4种病原的高灵敏度的分子检测技术体系；在分子水平上明确其部分分子特性。另外初步探讨运用超低温技术脱除HLB病菌、CTV、CEVd的可能性及机理。主要获得结果如下：
1．HLB病菌生物学鉴定及PCR检测体系优化。以长春花和椪柑为指示植物，部分待检材料表现了明显HLB症状。通过对影响PCR技术的各个主要因素的调整，建立了HLB病菌PCR、Semi-Nested PCR及Nested-PCR检测技术；并对三种方法的检测灵敏度进行比较，结果发现Semi-Nested PCR及Nested-PCR检测灵敏度远高于常规PCR，是常规PCR的10~4倍。运用Nested-PCR技术在黄皮上检测出HLB病菌(Candidatus Liberobacter asiaticus)，在国内外属于首次报道。
2．不同引物检测HLB病菌灵敏度比较。对基于rplA／rplK，β-operon，16SrDNA和16S／23SrDNA不同基因组序列设计的5对引物依次为fP400／rP400，fP535／rP535，fA2／rJ5，fOI1／rOI2c，fOI2／r23S1检测HLB病菌灵敏度进行了比较，结果发现不同引物检测灵敏度不一样，其检测灵敏度由高到低依次为：fP400／rP400＞fP535／rP535＞fA2／rJ5＞fOI1／rOI2c＞fOI2／r23S1。
3．HLB菌亚洲株系分子鉴定。HLB病菌16SrDNA片段RFLP-SSCP分析及基于16SrDNA序列中国柑橘黄龙病菌属分类地位的确立，分析结果显示来自中国7省区9个代表性的分离物16SrDNA高度保守，全部属于亚洲菌系(Candidatus
Liberobacter asiaticus)。HLB病菌16S／23SrDNA间区序列分析及系统进化树分析结果显示：来自中国7省区分布在不同的寄主体内、导致寄主表现不同症状的18个分离物在该区段没有发生显著变异，各分离物之间的同源性达99％以上，全部与亚洲菌系(Candidatus Liberobacter asiaticus)高度同源，而与非洲菌系(Candidatus Liberobacter africanum)差异较大。
4．HLB病菌Southern blotting检测。利用α-~(32)P-dCTP标记16SrDNA探针进行HLB病菌Dot-blotting检测，结果发现Dot-blotting检测灵敏度高于常规PCR而低于Semi-Nested PCR及Nested-PCR，最低检测下限为30fg总核酸。
5．CTV生物学鉴定、RT-PCR检测技术的建立及来自不同寄主的21个分离物3'端、5'端分子特性分析。以墨西哥莱檬和甜橙为指示植物，待检样品表现典型的CTV致病症状。核苷酸序列多重比对分析结果显示：21个CTV分离物的5'端和3'端共4个变异区(A区，F区，CP，P20)存在明显的差异。A区的核苷酸序列
Citrus huanglongbing globally has been regarded as one of the most destructive disease to citrus production. Pothogens easily mix infect with it are Citrus tristeza virus(CTV), Citrus exocortis viroid (CEVd) and Citrus tatter leaf virus (CTLV). Biological and molecular characteristic of these four pathogens were studied. The objective is to establish highly sensitive molecular detection method for their rapid detection; and to study part of their molecular character; another objective is to make sure the possibility and the mechanism of eliminating the above 3 pathogen (HLB, CTV, CEVd) by cryopreservation protocol. The main results are described as follows:
1. Biological identification and optimization of PCR system for the identification of HLB pathogen. Several HLB samples were inoculated to Catharanthus roseas and Citrus reticulata Blanco by mechanical method and grafting-inoculation respectively. These isolates caused typical symptoms on indicator plants. PCR, Semi-Nested PCR and Nested-PCR were established by adjusting the main factors in PCR system. Results showed the sensibility of Semi-Nested PCR and Nested-PCR was 10~4 times higher than single PCR. HIB pathogen (Candidatus Liberobacter asiaticus) was firstly detected out on wampee by Nested-PCR. It is the first report that HLB pathogen can infect wampee throughout the world.
2. Comparison of sensitivity of different primer sets for the detection of HLB pathogen. Based on the sequence of rplA/rplK, β-operon, 16SrDNA and 16S/23S rDNA, 5 different primer sets were designed. The detection results showed sensitivity of different primer sets were not identical, from the highest to the lowest were fP400/r P400> fP535/r P535>fA2/rJ5> fOIl/rOI2c> fOI2/r23S1.
3. Identification of Candidatus Liberobacter asiaticus, amplification of 16SrDNA, the consequent analysis of its products by RFLP, SSCP and phylogenetic tree analysis for 16SrDNA. Results showed that 9 different representative isolates coming from 7 provinces were all belong to Candidatus Liberobacter asiaticus. Sequence analysis of 16S/23SrDNA intergenic region and phylogenetic tree constructed showed that 18 representative isolates coming from different hosts, showing different symptoms (7 provinces) had no variation; the homology among isolates was above 99%. They were all highly homologous with Candidatus Liberobacter asiaticus, and distinct from Candidatus Liberobacter africanum.
4. Detection of HLB pathogen by Southern blotting. The α-~(32)P-dCTP labeled probe of
virus(CTLV)。本研究主要以HLB病菌、CTV、CEVd、CTLV为对象，从生物学角度及分子水平上对其部分特性进行研究；目的是建立上述4种病原的高灵敏度的分子检测技术体系；在分子水平上明确其部分分子特性。另外初步探讨运用超低温技术脱除HLB病菌、CTV、CEVd的可能性及机理。主要获得结果如下：
1．HLB病菌生物学鉴定及PCR检测体系优化。以长春花和椪柑为指示植物，部分待检材料表现了明显HLB症状。通过对影响PCR技术的各个主要因素的调整，建立了HLB病菌PCR、Semi-Nested PCR及Nested-PCR检测技术；并对三种方法的检测灵敏度进行比较，结果发现Semi-Nested PCR及Nested-PCR检测灵敏度远高于常规PCR，是常规PCR的10~4倍。运用Nested-PCR技术在黄皮上检测出HLB病菌(Candidatus Liberobacter asiaticus)，在国内外属于首次报道。
2．不同引物检测HLB病菌灵敏度比较。对基于rplA／rplK，β-operon，16SrDNA和16S／23SrDNA不同基因组序列设计的5对引物依次为fP400／rP400，fP535／rP535，fA2／rJ5，fOI1／rOI2c，fOI2／r23S1检测HLB病菌灵敏度进行了比较，结果发现不同引物检测灵敏度不一样，其检测灵敏度由高到低依次为：fP400／rP400＞fP535／rP535＞fA2／rJ5＞fOI1／rOI2c＞fOI2／r23S1。
3．HLB菌亚洲株系分子鉴定。HLB病菌16SrDNA片段RFLP-SSCP分析及基于16SrDNA序列中国柑橘黄龙病菌属分类地位的确立，分析结果显示来自中国7省区9个代表性的分离物16SrDNA高度保守，全部属于亚洲菌系(Candidatus
Liberobacter asiaticus)。HLB病菌16S／23SrDNA间区序列分析及系统进化树分析结果显示：来自中国7省区分布在不同的寄主体内、导致寄主表现不同症状的18个分离物在该区段没有发生显著变异，各分离物之间的同源性达99％以上，全部与亚洲菌系(Candidatus Liberobacter asiaticus)高度同源，而与非洲菌系(Candidatus Liberobacter africanum)差异较大。
4．HLB病菌Southern blotting检测。利用α-~(32)P-dCTP标记16SrDNA探针进行HLB病菌Dot-blotting检测，结果发现Dot-blotting检测灵敏度高于常规PCR而低于Semi-Nested PCR及Nested-PCR，最低检测下限为30fg总核酸。
5．CTV生物学鉴定、RT-PCR检测技术的建立及来自不同寄主的21个分离物3'端、5'端分子特性分析。以墨西哥莱檬和甜橙为指示植物，待检样品表现典型的CTV致病症状。核苷酸序列多重比对分析结果显示：21个CTV分离物的5'端和3'端共4个变异区(A区，F区，CP，P20)存在明显的差异。A区的核苷酸序列
Citrus huanglongbing globally has been regarded as one of the most destructive disease to citrus production. Pothogens easily mix infect with it are Citrus tristeza virus(CTV), Citrus exocortis viroid (CEVd) and Citrus tatter leaf virus (CTLV). Biological and molecular characteristic of these four pathogens were studied. The objective is to establish highly sensitive molecular detection method for their rapid detection; and to study part of their molecular character; another objective is to make sure the possibility and the mechanism of eliminating the above 3 pathogen (HLB, CTV, CEVd) by cryopreservation protocol. The main results are described as follows:
1. Biological identification and optimization of PCR system for the identification of HLB pathogen. Several HLB samples were inoculated to Catharanthus roseas and Citrus reticulata Blanco by mechanical method and grafting-inoculation respectively. These isolates caused typical symptoms on indicator plants. PCR, Semi-Nested PCR and Nested-PCR were established by adjusting the main factors in PCR system. Results showed the sensibility of Semi-Nested PCR and Nested-PCR was 10~4 times higher than single PCR. HIB pathogen (Candidatus Liberobacter asiaticus) was firstly detected out on wampee by Nested-PCR. It is the first report that HLB pathogen can infect wampee throughout the world.
2. Comparison of sensitivity of different primer sets for the detection of HLB pathogen. Based on the sequence of rplA/rplK, β-operon, 16SrDNA and 16S/23S rDNA, 5 different primer sets were designed. The detection results showed sensitivity of different primer sets were not identical, from the highest to the lowest were fP400/r P400> fP535/r P535>fA2/rJ5> fOIl/rOI2c> fOI2/r23S1.
3. Identification of Candidatus Liberobacter asiaticus, amplification of 16SrDNA, the consequent analysis of its products by RFLP, SSCP and phylogenetic tree analysis for 16SrDNA. Results showed that 9 different representative isolates coming from 7 provinces were all belong to Candidatus Liberobacter asiaticus. Sequence analysis of 16S/23SrDNA intergenic region and phylogenetic tree constructed showed that 18 representative isolates coming from different hosts, showing different symptoms (7 provinces) had no variation; the homology among isolates was above 99%. They were all highly homologous with Candidatus Liberobacter asiaticus, and distinct from Candidatus Liberobacter africanum.
4. Detection of HLB pathogen by Southern blotting. The α-~(32)P-dCTP labeled probe of
引文
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