两种植原体病害的分子检测与鉴定
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摘要
植原体是一类能引起多种植物病害的植物病原原核生物。本研究利用分子生物学技术对我国部分地区的枣疯病植原体以及河南地区杨树黄叶病进行了检测和鉴定,主要结果如下：
1)利用植原体通用引物对R16F2n/R16R2、fTufu/rTufu和rp(v)F1A/rp(v)R1A对北京和河北地区枣疯病植原体的16S rDNA基因,延伸因子tuf基因,核糖体蛋白基因(rp)进行PCR扩增,分别得到1.2 kb,0.8 kb以及1.2 kb的特异片段。经序列测定和同源性比较表明：北京地区枣疯病植原体16S rDNA基因与其它地区枣疯病植原体株系差异不大；tuf基因与16SrV组的葡萄黄叶病(FD)同源性最高,为92.84%,而与已经公布的其它地区(陕西杨凌)的枣疯病植原体tuf基因同源性较低,为57.29%；北京地区枣疯病植原体rp基因与16SrV组的枣疯病泰山株系(JWB-Taishan)以及大麻丛枝病植原体(HFWB)同源性最高,均为99.83%,与16SrV组的成员同源性均在96%以上。
2)利用植原体16S rDNA基因保守序列通用引物对P1/P7与R16F2n/R16R2对河南地区表现叶片黄化症状的杨树总DNA进行巢式PCR扩增,结果得到约1.2 kb的特异性DNA片段；利用植原体rp基因检测引物对rpF1/rpR1对河南地区表现叶片黄化症状的杨树总DNA进行直接PCR扩增,再运用rp(Ⅰ)F1/rp(Ⅰ)R1引物对进行巢式PCR扩增,得到大小约1.2 kb的特异片段。经序列测定和同源性比较表明此植原体归属于16SrI组。
通过对北京及河北地区枣疯病植原体PCR扩增,发现其tuf基因与其它地区枣疯病tuf基因存在较大的差异,从而为枣疯病在亚组水平上的分类提供了新的参考依据；用植原体检测引物对从河南地区杨树黄叶病症的样品中扩增到特异性条带,表明河南杨树黄叶病株中存在植原体,这是在国内首次报道植原体侵染杨树。
Phytoplasma is a plant pathogenic prokaryotes that can cause many kinds of plant diseases. By use of molecular biology techniques, the JWB phytoplasma in some parts of China and poplar yellows pathogen in henan province were detected and identified.The main results read as follows:
1) By use of PCR, the 16S rDNA,elongation factor Tu (tuf gene) and ribosomal protein (rp) gene of phytoplasma associated with JWB in Beijing and Hebei districts were amplified separately with universal primer pairs R16F2n/R16R2, fTufu/rTufu and rp(v)F1A/rp(v)R1A. We obtained 16S rDNA gene of 1.2 kb, tuf gene of 0.8 kb and rp gene of 1.2 kb from the diseased samples. The results of sequencing and homologous comparison with other phytoplasmas showed that JWB in Beijing shared a high identity with other JWB phytoplasmas in 16S rDNA gene; JWB in Beijing shared 92.84% similarity with Flavescence doree (FD) phytoplasma (Candidatus Phytoplasma vitis) in tuf gene. The tuf gene of JWB in Beijing shared a low identity of 57.29% with JWB tuf gene in Shaanxi district which had been already reported.The homology analysis for sequences of rp gene showed that a high identity with members of the 16SrV group phytoplasmas, with homology rate higher than 96 %. It shared mostly identity of 99.83% to the 16SrV group of JWB strain Taishan and Hemp fiber witches'-broom phytoplasma (HFWB).
2) By use of universal primers P1/P7 and R16F2n/R16R2 for 16S rDNA gene, a specific fragment of 1.2 kb in length was amplified with nested PCR from poplar showing typical phytoplasma symptom of yellowing in henan province. By use of universal primers pairs rpF1/rpR1 and rp(Ⅰ)F1/rp(Ⅰ)R1 for rp genes, a specific fragment of 1.2 kb was amplified with nested PCR from poplar diseased sample.The results of sequencing and homologous comparison with other phytoplasmas showed that the phytoplasma strain is one of the members of 16Sr I group.
According to the PCR with JWB phytoplasmas in Beijing and Hebei districts, we discovered the JWB tuf gene in Beijing and Hebei had a high difference between with other districts. The results provided the basis for the classification in subgroup of the 16SrV group phytoplasmas. By use of universal primers for dectection of phytoplasma, DNA fragments were obtained from the total DNA of poplar yellows sample.The results indicated existence of phytoplasm aassociated with poplar in Henan province. This is the first report of phytoplasma associated with poplar yellows disease in China.
1)利用植原体通用引物对R16F2n/R16R2、fTufu/rTufu和rp(v)F1A/rp(v)R1A对北京和河北地区枣疯病植原体的16S rDNA基因,延伸因子tuf基因,核糖体蛋白基因(rp)进行PCR扩增,分别得到1.2 kb,0.8 kb以及1.2 kb的特异片段。经序列测定和同源性比较表明：北京地区枣疯病植原体16S rDNA基因与其它地区枣疯病植原体株系差异不大；tuf基因与16SrV组的葡萄黄叶病(FD)同源性最高,为92.84%,而与已经公布的其它地区(陕西杨凌)的枣疯病植原体tuf基因同源性较低,为57.29%；北京地区枣疯病植原体rp基因与16SrV组的枣疯病泰山株系(JWB-Taishan)以及大麻丛枝病植原体(HFWB)同源性最高,均为99.83%,与16SrV组的成员同源性均在96%以上。
2)利用植原体16S rDNA基因保守序列通用引物对P1/P7与R16F2n/R16R2对河南地区表现叶片黄化症状的杨树总DNA进行巢式PCR扩增,结果得到约1.2 kb的特异性DNA片段；利用植原体rp基因检测引物对rpF1/rpR1对河南地区表现叶片黄化症状的杨树总DNA进行直接PCR扩增,再运用rp(Ⅰ)F1/rp(Ⅰ)R1引物对进行巢式PCR扩增,得到大小约1.2 kb的特异片段。经序列测定和同源性比较表明此植原体归属于16SrI组。
通过对北京及河北地区枣疯病植原体PCR扩增,发现其tuf基因与其它地区枣疯病tuf基因存在较大的差异,从而为枣疯病在亚组水平上的分类提供了新的参考依据；用植原体检测引物对从河南地区杨树黄叶病症的样品中扩增到特异性条带,表明河南杨树黄叶病株中存在植原体,这是在国内首次报道植原体侵染杨树。
Phytoplasma is a plant pathogenic prokaryotes that can cause many kinds of plant diseases. By use of molecular biology techniques, the JWB phytoplasma in some parts of China and poplar yellows pathogen in henan province were detected and identified.The main results read as follows:
1) By use of PCR, the 16S rDNA,elongation factor Tu (tuf gene) and ribosomal protein (rp) gene of phytoplasma associated with JWB in Beijing and Hebei districts were amplified separately with universal primer pairs R16F2n/R16R2, fTufu/rTufu and rp(v)F1A/rp(v)R1A. We obtained 16S rDNA gene of 1.2 kb, tuf gene of 0.8 kb and rp gene of 1.2 kb from the diseased samples. The results of sequencing and homologous comparison with other phytoplasmas showed that JWB in Beijing shared a high identity with other JWB phytoplasmas in 16S rDNA gene; JWB in Beijing shared 92.84% similarity with Flavescence doree (FD) phytoplasma (Candidatus Phytoplasma vitis) in tuf gene. The tuf gene of JWB in Beijing shared a low identity of 57.29% with JWB tuf gene in Shaanxi district which had been already reported.The homology analysis for sequences of rp gene showed that a high identity with members of the 16SrV group phytoplasmas, with homology rate higher than 96 %. It shared mostly identity of 99.83% to the 16SrV group of JWB strain Taishan and Hemp fiber witches'-broom phytoplasma (HFWB).
2) By use of universal primers P1/P7 and R16F2n/R16R2 for 16S rDNA gene, a specific fragment of 1.2 kb in length was amplified with nested PCR from poplar showing typical phytoplasma symptom of yellowing in henan province. By use of universal primers pairs rpF1/rpR1 and rp(Ⅰ)F1/rp(Ⅰ)R1 for rp genes, a specific fragment of 1.2 kb was amplified with nested PCR from poplar diseased sample.The results of sequencing and homologous comparison with other phytoplasmas showed that the phytoplasma strain is one of the members of 16Sr I group.
According to the PCR with JWB phytoplasmas in Beijing and Hebei districts, we discovered the JWB tuf gene in Beijing and Hebei had a high difference between with other districts. The results provided the basis for the classification in subgroup of the 16SrV group phytoplasmas. By use of universal primers for dectection of phytoplasma, DNA fragments were obtained from the total DNA of poplar yellows sample.The results indicated existence of phytoplasm aassociated with poplar in Henan province. This is the first report of phytoplasma associated with poplar yellows disease in China.
引文
[1]安凤秋,吴云锋,孙秀芹,等.小麦蓝矮病植原体延伸因子(EF-Tu)tuf基因序列的同源性分析[J].中国农业科学,2006,39(1)：74-80.
[2]车海彦,罗大全.植原体病害的检测方法研究进展[J].华南热带农业大学学报,2006,12(3)：69-73.
[3]车海彦,罗大全,符瑞益,等.海南长春花黄化病植原体的16S rDNA序列分析研究[J].植物病理学报,2009,39(2)：212-216.
[4]陈胜红,张草根,王敏.香樟黄化病综合防治研究初报[J].现代园艺,2007,(9)：28-29.
[5]段彦丹,张玉星,张殿生,等.梨树的铁营养与黄叶病矫治的研究进展[C].全国第四届梨科研,生产与产业化学术研讨会,梨科研与生产进展(三),2005：285-290.
[6]葛泉卿,M.MAIXN ER,温孚江.嵌套引物P1/P7-U3/U5检测葡萄黄化(sto-lbur)植原体时扩增出的额外片段分析[J].植物病理学报,2005,35(2)：97-103.
[7]葛泉卿.葡萄植原体黄化病及其检疫技术[D].2006.山东农业大学博士论文.
[8]顾沛雯,安凤秋,吴云锋,等.小麦蓝矮病植原体16S rDNA基因片段的比较分析[J].植物病理学报,2005,35(5)：403-411.
[9]顾沛雯,吴云锋,安凤秋.小麦蓝矮植原体寄主范围的鉴定及RFLP分析[J].植物病理学报,2007,37(4)：390-397.
[10]郭晓军,田国忠,赵少波,等.抗枣疯病枣树品种的DAPI荧光染色检测[J].河北林业科技,2003,04：1-3.
[11]黄云,王靖,赵艳琴,等.桃黄化病病原植原体的鉴定及16S rDNA序列分析[J].园艺学报,2009,36(3)：341-346.
[12]淮稳霞,赵文霞,田国忠,等.河南杨树黄叶症病因的初步诊断[C].中国植物病理学会2007年学术年会论文集.2007.
[13]何放亭,武红巾,陈子文.用DAPI荧光染色诊断植物类菌原体病害[J].植物保护,1994,5：37.
[14]侯晓杰,李正楠,冉隆贤,等.越冬病枝水培与PCR结合检测枣疯病植原体的存活动态[J].中国森林病虫,2007,4.
[15]金开璇,梁成杰,邓丹荔.泡桐丛枝病传毒昆虫研究[J].林业实用技术,1981,(6)：23-24.
[16]金开璇,田国忠,汪跃.组织化学技术快速检测泡桐丛枝病研究[J].植物病理学报,1989,19(3)：185-188.
[17]金开璇,汪跃.紫穗槐带化病中发现类菌原体(MLO)[J].植物病理学报,1992,24：96.
[18]赖帆,李永,徐启聪,等.植原体的最新分类研究动态[J].微生物学通报,2008,35(2)：291-295.
[19]赖帆.重阳木丛枝病植原体的分子鉴定及与其他几种植原体的相关性研究[D].2007.中国林业科学研究院硕士论文.
[20]梁振普,张小霞,乔冠华,等.河南地区杨树“黄叶病”研究初报[J].植物保护科学,2008,24(1)384-388.
[21]李永,田国忠,朴春根,等.我国几种植物上植原体的快速分子鉴别与鉴定的研究[J].植物病理学报,2005,35(4)：293-299.
[22]李永.我国几种木本植物植原体的分子检测与鉴定[D].中国林业科学研究院硕士论文,2004.
[23]李永,田国忠,徐启聪,等.臭矢菜丛枝病植原体的分子鉴定研究[J].植物病理学报,2009,39(4)：377-384.
[24]廖晓兰,朱建裕,朱水芳.实时荧光PCR检测椰子致死黄花病原的建立研究初报[J].福建农业大学学报(增刊),2001,30：196-198.
[25]廖晓兰,朱水芳,陈红运,等.植原体TaqMan探针实时荧光PCR检测鉴定方法的建立[J].植物病理学报,2002,32(4)：361-367.
[26]林木兰,杨继红,陈捷,等.泡桐丛枝病类菌原体单克隆抗体的研制及初步应用[J].植物学报,1993,35(9)710-715.
[27]林木兰,张春立,杨继红,等.用核酸杂交技术检测泡桐丛枝病类菌原体[J].科学通报,1994,36(4)：376-379.
[28]林彩丽.泡桐丛枝病植原体质粒分子特征及编码蛋白功能研究[D].2008.中国农业大学博士学位论文,
[29]罗飞,朱水芳,张成良.泡桐丛枝病媒介昆虫体中植原体的检测和鉴定[M].见：刘仪主编.植物病害研究与防治.北京：中国农业科学出版,1998：280-283.
[30]刘仲健,罗焕亮,张景宁.植原体病理学[M].北京：中国农业出版社,1999.
[31]刘永光.山东省桑萎缩病、枣疯病、竹枝病及三种新植原体病害分子检测与鉴 定[D].2009.山东农业大学硕士论文.
[32]史春霖,张凤舞,陈子文.冰冻断裂的枣疯病病树韧皮组织中类支原体的扫描电镜观察[J].微生物学报,1984,24：139-141.
[33]史英姿.泡桐丛枝植原体中国株系的分子鉴定及免疫膜蛋白基因表达研究[D].西北农林科技大学硕士论文,2007.
[34]邵平绪,洪瑞芬,彭春华,等.泡桐丛枝病传病昆虫研究Ⅰ.茶翅蝽传病试验及电镜观察[J].山东林业科技,1982,1：42.
[35]田国忠.DAPI染色技术及其在树木类菌质体病害研究中的应用[J].河南林业科技,1990b,3：25-27.
[36]田国忠.泡桐丛枝病植原体与泡桐的生化和分子互作研究[D].中国农业大学博士论文,1999.
[37]魏婷,吴云锋,侯伟,等.柳树黄化病植原体的分子分类[J].微生物学报,2009,49(3)：389-394.
[38]马俊青,宋宏伟,翟小巧,等.叶片活性铁与杨树黄叶病的关系研究[J].安徽农业科学,2009,37(27)：13120-13121.
[39]漆艳香,谢艺贤,张辉强,等.植原体DNA提取方法的改良[J].生物技术通报,2004,4：44-46.
[40]王海妮,吴云锋,安凤秋,等.枣疯病和酸枣丛枝病植原体16S rDNA和tuf基因的序列同源性分析[J].中国农业科学,2007,40(10)：2200-2205.
[41]王海妮.枣疯病和酸枣丛枝病植原体的分子鉴定及相关基因的克隆和生物信息学分析[D].2008.西北农林科技大学硕士论文.
[42]王洁.泡桐丛枝病植原体延伸因子tuf基因分析及其他寄主植物的检测[D].2008.山东农业大学硕士论文.
[43]王祈楷,徐绍华,陈子文,等.枣疯病的研究[J].植物病理学报,1981,11(1)：15-18.
[44]徐启聪,田国忠,王振亮,等.中国各地不同枣树品种上枣疯病植原体的PCR检测及分子变异分析[J].微生物学报,2009,49(11)：1510-1519.
[45]张荣,崔晓艳,孙广宇,等.小麦蓝矮病植原体转运蛋白secY基因片段序列分析[J].植物病理学报,2007,37(4)：446-448.
[46]赵锦,刘孟军,周俊义,等.枣疯植原体的分布特点及周年消长规律[J].林业科学,2006,42,(8)：144-147.
[47]朱天生,潘一展,崔廷涛,等.榆树黄化病植原体的分子检测与鉴定[J].植物病理学报,2008,38(4)：401-406.
[48]中国科学院上海生化化学研究所病毒组,山东省果树科学研究所枣疯病研究组.枣疯病病原体的电子显微镜研究.中国科学,1974(6)：622-626.
[49]Arocha Y,Lo'pez M,Almeida R,et al.'Candidatus Phytoplasma graminis' and'Candidatus Phytoplasma caricae',two novel phytoplasmas associated with diseases of sugarcane,weeds and papaya in Cuba [J]. Int J Syst Evol Microbiol, 2005,55,2451-2463.
[50]Bai X,Zhang J,Ewing A,et al. Living with genome instability:the adaptation of phytoplasmas to diverse environments of their insect and plant hosts [J]. Journal of Bacteriology,2006,188:3682-3696.
[51]Bertaccini. Phytoplasmas:diversity,taxonomy,and epidemiology[C]. Frontiers in Bioscience,2007,12,673-689.
[52]Bertolini E,Tomes E,Olmos A. Co-operational PCR couple with dotblot hybridization for detection and 16SrX grouping of phytoplasmas [J].Plant Pathology,2007,1:1-6.
[53]Davies D L,Clark M F. Production and characterization of polyclonal and monoclonal antibodies against peach yellow leafroll MLO-associated antigens [J]. Acta Hortic.1992,309:275-284. International Journal of systematic bacteriology, 1997,47:262-269.
[54]Denes A S and Sinha R C. Extrachromosomal DNA elements of plant pathogenic mycoplasmalike organisms [J]. Canadian Journal of Plant Pathology,1991,13: 26-32.
[55]Deng S J and Hiruki C. Genetic relatedness between two nonculturable mycoplasmalike organisms revealed by nucleic acid hybridization and polymerase chain reaction[J]. Phytopathology,1991,81(12):1475-1479.
[56]Doi,Y.M.,M.Teranaka,et al. Mycoplasma or PLT-group-like microorganisms found in the phloem elements of plants infected with mulberry dwarf,potato witches'-broom,aster yellows or paulownia witches'-broom[J]. Ann Phytopathol Soc Jpn,1967,33:259-266.
[57]Firrao G, Smart C D,Kirkpatrick B C. Physical map of the Western X-disease phytoplasma chromosome [J]. Bacteriol,1996,178(13):3985-3988.
[58]G. R. Prabu, P.G. Kawar,D. Theertha Prasad. Differential filtration approach for isolation and enrichment of Sugarcane Grassy Shoot phytoplasma [J]. Sugar Tech,2008,10(3):274-277.
[59]Hsu H T,Lee I-M,Davis R E. Immunization for generation of hybridoma antibodies specifically reaction with plants infected with a mycoplasmalike origanism(MLO) and their use in detection of MLO antigens [J]. Phytopathology,1990,80:946-950.
[60]International Committee on Systematic Bacteriology,Subcommittee on the Taxonomy of Mollicutes. Revised minimum standards for description of new species of the class Mollicutes (Division Tenericutes) [C]. Int J Syst Bacteriol,1995,45(3):605-612.
[61]International Committee on Systematic Bacteriology, Subcommittee on the Taxonomy of Mollicutes Minutes of the meetings,21 and 28 July,1998,Sydney,Australia[C]. Int J Syst Evol Microbiol,2000,50(3):1393-1397.
[62]Jennifer Berger, Josef Dalla Via,Sanja Baric. Development of a TaqMan allelic discrimination assay for the distinction of two major subtypes of the grapevine yellows phytoplasma Bois noir [J]. Eur J Plant Pathol,2009,124:521-526.
[63]Jiang Y P,Che T A,Chiykowski L N. Production of monoclonal antibodies to peach eastern X-disease agent and their use in disease detection [J]. Can J Plant pathol,1989,11:325-331.
[64]Jung H Y,Toshimi Sawayanagi,Shigeyuki Kakizawa,et al.'Candidatus Phytoplasma ziziphi',a novelphytoplasma taxon associated with jujube witches'-broom disease [J]. International Journal of Systematic and Evolutionary Microbiology,2003,53,1037-1041.
[65]Kirkpatrick B C,Stenger D C,Morris T J,et al. Cloning and detection of DNA from a nonculturable plant pathogenic mycoplasma-like organism[J]. Science, 1987,238:197-2001.
[66]Lauer U and Seemuller E. Physical map of the chromosome of the apple proliferation phytoplasma [J]. J Bacteriol,2000,182(5):1415-1418.
[67]Liefting LW,Shaw ME,Kirkpatrick BC. Sequence analysis of two plasmids from the phytoplasma beet leafhopper-transmitted virescene agent [J]. Microbiology, 2004,150:1809-1817.
[68]Lee I-M,Davis R E,Chen T-A.,et al. A genotype-based system for identification and classification of coplasmalike organisms (MLOs) in the aster yellows MLO strain cluster [J]. Phytopathology,1992,82:977-986.
[69]Lee I-M,Davis R E. Sinclair Genetic relatedness of mycoplasmalike organisms detected in Ulmus spp. in the United States and Italy by means of DNA probes and polymerase chain reactions [J]. Phytopathology,1993a,83 (6):829-833.
[70]Lee I-M. Use of mycoplasmalike orgonismas (MLO) group specific oligonucleotide primers for nested-PCR assays to detect mixed-MLO infections in a single host plant [J]. Phytopathology,1993b,84 (6):559-566.
[71]Lee I-M,Gundersen-Rindal D E,Bertaccini A. Phytoplasma:ecology and genomic diversity [J]. Phytopathology,1998a,88:1359-1366.
[72]Lee I-M,Gundersen D E,Davis R E,et al. Revised classification scheme of phytoplasmas based on RFLP analyses of 16S rRNA and ribosomal protein gene sequences [J]. Int J Syst Evol Microbiol,1998b,48:1153-1169.
[73]Lee I-M,Davis R E,Gundersen-Rindal D E. Phytoplasma:phytopathogenic mollicutes [J]. Annu Rev Microbiol,2000,54:221-255.
[74]Lee I-M,Gundersen D E,Davis R E,et al.'Candidatus Phytoplasma asteris',a novel phytoplasma taxon associated with aster yellows and related diseases[J]. International Journal of Systematic and Evolutionary Microbiology,2004a,54: 1037-1048.
[75]Lee I-M,Martini M,Marcone C,et al. Classification of phytoplasma strains in the elm yellows group (16SrV) and proposal of'Candidatus Phytoplasma ulmi'for the phytoplasma associated with elm yellows [J]. International Journal of Systematic and Evolutionary Microbiology,2004b,54:337-347.
[76]Lee I-M,Zhao Y,Bottner K D. SecY gene sequence analysis for finer differentiation of diverse strains in the aster yellows phytoplasma group [J]. Molecular and Cellular Probes,2006,20:87-91.
[77]Lim,P-O,Sears,B. B. Evolutionary relationships of a plant-pathogenic mycoplasmalike organism and Acholeplasma laidlawii deduced from two ribosomal protein gene sequences [J]. J Bacteriol,1989,174,2606-2611.
[78]Lin C L,Li H F,Fan Z F. Two novel plasmids from paulownia witches'-broom phytoplasma and its relatednss to other phytoplasmas [C]. Proceedings of the Third Asian Conference on Plant Pathology,Yogyakarta,Indonesia August, 20-24,2007:149-150.
[79]Lin C P,Chen T A. Monoclonal antibodies against the aster yellows agent [J]. Science,1985,227:1233-1235.
[80]Marcone C, Lee I M,Davis R E,et al. Classification of aster yellows-group phytoplasmas based on combined analyses of rRNA and tuf gene sequences [J]. International Journal of Systematic and Evolutionary Microbiology,2000,50: 1703-1713.
[81]Marcone C, Schneider B and Seemuller E.'Candidatus Phytoplasma cynodontis', the phytoplasma associated with Bermuda grasswhite leaf disease [J]. International Journal of Systematic and Evolutionary Microbiology,2004,54:1077-1082.
[82]Oshima K and Kakizawa S. Reductive evolution suggested from the complete genome sequence of a plant-pathogenic phytoplasma [J]. Nat Genet,2004,36(1): 27-29.
[83]Padovan A C and Firrao G. Chromosome mapping of the sweet potato little leaf phytoplasma reveals genome heterogeneity within the phytoplasmas [J]. Microbiology,2000,146 (4):893-902.
[84]Schneider B,Gibb K S,Seemuuller E. Sequence and RFLP analysis of the elongation factor Tu gene used in differentiation and classification of phytoplasmas [J]. Microbiology,1997,143:3381-3389.
[85]Seruga M,Skoric D,Botti S,et al. Molecular characterization of a phytoplasma from the asteryellows (16SrI) group naturally infecting Populus nigra L. 'Italica'trees in Croatia [J]. For. Path.2003,:33:113-125.
[86]Tran-Nguyen,L.T.,Kube,M. et al. Comparative genome analysis of'Candidatus Phytoplasma australiense'(subgroup tuf-Australia I; rp-A) and'Ca. Phytoplasma asteris'Strains OY-M and AY-WB [J]. J. Bacteriol,2008,190 (11):3979-3991.
[87]Woese C R.Bacterial evolution [J].Microbiol Rev,1987,51(2):221-271.
[88]Wei W,Robert E,Davis I-M,et al. Computer-simulated RFLP analysis of 16s rRNA genes:identification of ten new phytoplasma groups [J]. Int J Syst Evol Microbiol,2007,57:1855-1867.
[89]Yi C K,La YJ. Mycoplasma-like bodies found in the phloem elements of jujube trees infected with witches'-broom disese [J].Research Reports of the Forest Research Institute of Korea,1973,20:111-114.
[90]Zreik S,Carle L,Bove P,et al. Characterization of the mycoplasmalike organism associated with witches-broom disease of lime and proposition of a Candidatus taxon for the organism," Candidatus Phytoplasma aurantifolia." [J]. Int J Syst Bacteroil,1995,45:449-453.
[2]车海彦,罗大全.植原体病害的检测方法研究进展[J].华南热带农业大学学报,2006,12(3)：69-73.
[3]车海彦,罗大全,符瑞益,等.海南长春花黄化病植原体的16S rDNA序列分析研究[J].植物病理学报,2009,39(2)：212-216.
[4]陈胜红,张草根,王敏.香樟黄化病综合防治研究初报[J].现代园艺,2007,(9)：28-29.
[5]段彦丹,张玉星,张殿生,等.梨树的铁营养与黄叶病矫治的研究进展[C].全国第四届梨科研,生产与产业化学术研讨会,梨科研与生产进展(三),2005：285-290.
[6]葛泉卿,M.MAIXN ER,温孚江.嵌套引物P1/P7-U3/U5检测葡萄黄化(sto-lbur)植原体时扩增出的额外片段分析[J].植物病理学报,2005,35(2)：97-103.
[7]葛泉卿.葡萄植原体黄化病及其检疫技术[D].2006.山东农业大学博士论文.
[8]顾沛雯,安凤秋,吴云锋,等.小麦蓝矮病植原体16S rDNA基因片段的比较分析[J].植物病理学报,2005,35(5)：403-411.
[9]顾沛雯,吴云锋,安凤秋.小麦蓝矮植原体寄主范围的鉴定及RFLP分析[J].植物病理学报,2007,37(4)：390-397.
[10]郭晓军,田国忠,赵少波,等.抗枣疯病枣树品种的DAPI荧光染色检测[J].河北林业科技,2003,04：1-3.
[11]黄云,王靖,赵艳琴,等.桃黄化病病原植原体的鉴定及16S rDNA序列分析[J].园艺学报,2009,36(3)：341-346.
[12]淮稳霞,赵文霞,田国忠,等.河南杨树黄叶症病因的初步诊断[C].中国植物病理学会2007年学术年会论文集.2007.
[13]何放亭,武红巾,陈子文.用DAPI荧光染色诊断植物类菌原体病害[J].植物保护,1994,5：37.
[14]侯晓杰,李正楠,冉隆贤,等.越冬病枝水培与PCR结合检测枣疯病植原体的存活动态[J].中国森林病虫,2007,4.
[15]金开璇,梁成杰,邓丹荔.泡桐丛枝病传毒昆虫研究[J].林业实用技术,1981,(6)：23-24.
[16]金开璇,田国忠,汪跃.组织化学技术快速检测泡桐丛枝病研究[J].植物病理学报,1989,19(3)：185-188.
[17]金开璇,汪跃.紫穗槐带化病中发现类菌原体(MLO)[J].植物病理学报,1992,24：96.
[18]赖帆,李永,徐启聪,等.植原体的最新分类研究动态[J].微生物学通报,2008,35(2)：291-295.
[19]赖帆.重阳木丛枝病植原体的分子鉴定及与其他几种植原体的相关性研究[D].2007.中国林业科学研究院硕士论文.
[20]梁振普,张小霞,乔冠华,等.河南地区杨树“黄叶病”研究初报[J].植物保护科学,2008,24(1)384-388.
[21]李永,田国忠,朴春根,等.我国几种植物上植原体的快速分子鉴别与鉴定的研究[J].植物病理学报,2005,35(4)：293-299.
[22]李永.我国几种木本植物植原体的分子检测与鉴定[D].中国林业科学研究院硕士论文,2004.
[23]李永,田国忠,徐启聪,等.臭矢菜丛枝病植原体的分子鉴定研究[J].植物病理学报,2009,39(4)：377-384.
[24]廖晓兰,朱建裕,朱水芳.实时荧光PCR检测椰子致死黄花病原的建立研究初报[J].福建农业大学学报(增刊),2001,30：196-198.
[25]廖晓兰,朱水芳,陈红运,等.植原体TaqMan探针实时荧光PCR检测鉴定方法的建立[J].植物病理学报,2002,32(4)：361-367.
[26]林木兰,杨继红,陈捷,等.泡桐丛枝病类菌原体单克隆抗体的研制及初步应用[J].植物学报,1993,35(9)710-715.
[27]林木兰,张春立,杨继红,等.用核酸杂交技术检测泡桐丛枝病类菌原体[J].科学通报,1994,36(4)：376-379.
[28]林彩丽.泡桐丛枝病植原体质粒分子特征及编码蛋白功能研究[D].2008.中国农业大学博士学位论文,
[29]罗飞,朱水芳,张成良.泡桐丛枝病媒介昆虫体中植原体的检测和鉴定[M].见：刘仪主编.植物病害研究与防治.北京：中国农业科学出版,1998：280-283.
[30]刘仲健,罗焕亮,张景宁.植原体病理学[M].北京：中国农业出版社,1999.
[31]刘永光.山东省桑萎缩病、枣疯病、竹枝病及三种新植原体病害分子检测与鉴 定[D].2009.山东农业大学硕士论文.
[32]史春霖,张凤舞,陈子文.冰冻断裂的枣疯病病树韧皮组织中类支原体的扫描电镜观察[J].微生物学报,1984,24：139-141.
[33]史英姿.泡桐丛枝植原体中国株系的分子鉴定及免疫膜蛋白基因表达研究[D].西北农林科技大学硕士论文,2007.
[34]邵平绪,洪瑞芬,彭春华,等.泡桐丛枝病传病昆虫研究Ⅰ.茶翅蝽传病试验及电镜观察[J].山东林业科技,1982,1：42.
[35]田国忠.DAPI染色技术及其在树木类菌质体病害研究中的应用[J].河南林业科技,1990b,3：25-27.
[36]田国忠.泡桐丛枝病植原体与泡桐的生化和分子互作研究[D].中国农业大学博士论文,1999.
[37]魏婷,吴云锋,侯伟,等.柳树黄化病植原体的分子分类[J].微生物学报,2009,49(3)：389-394.
[38]马俊青,宋宏伟,翟小巧,等.叶片活性铁与杨树黄叶病的关系研究[J].安徽农业科学,2009,37(27)：13120-13121.
[39]漆艳香,谢艺贤,张辉强,等.植原体DNA提取方法的改良[J].生物技术通报,2004,4：44-46.
[40]王海妮,吴云锋,安凤秋,等.枣疯病和酸枣丛枝病植原体16S rDNA和tuf基因的序列同源性分析[J].中国农业科学,2007,40(10)：2200-2205.
[41]王海妮.枣疯病和酸枣丛枝病植原体的分子鉴定及相关基因的克隆和生物信息学分析[D].2008.西北农林科技大学硕士论文.
[42]王洁.泡桐丛枝病植原体延伸因子tuf基因分析及其他寄主植物的检测[D].2008.山东农业大学硕士论文.
[43]王祈楷,徐绍华,陈子文,等.枣疯病的研究[J].植物病理学报,1981,11(1)：15-18.
[44]徐启聪,田国忠,王振亮,等.中国各地不同枣树品种上枣疯病植原体的PCR检测及分子变异分析[J].微生物学报,2009,49(11)：1510-1519.
[45]张荣,崔晓艳,孙广宇,等.小麦蓝矮病植原体转运蛋白secY基因片段序列分析[J].植物病理学报,2007,37(4)：446-448.
[46]赵锦,刘孟军,周俊义,等.枣疯植原体的分布特点及周年消长规律[J].林业科学,2006,42,(8)：144-147.
[47]朱天生,潘一展,崔廷涛,等.榆树黄化病植原体的分子检测与鉴定[J].植物病理学报,2008,38(4)：401-406.
[48]中国科学院上海生化化学研究所病毒组,山东省果树科学研究所枣疯病研究组.枣疯病病原体的电子显微镜研究.中国科学,1974(6)：622-626.
[49]Arocha Y,Lo'pez M,Almeida R,et al.'Candidatus Phytoplasma graminis' and'Candidatus Phytoplasma caricae',two novel phytoplasmas associated with diseases of sugarcane,weeds and papaya in Cuba [J]. Int J Syst Evol Microbiol, 2005,55,2451-2463.
[50]Bai X,Zhang J,Ewing A,et al. Living with genome instability:the adaptation of phytoplasmas to diverse environments of their insect and plant hosts [J]. Journal of Bacteriology,2006,188:3682-3696.
[51]Bertaccini. Phytoplasmas:diversity,taxonomy,and epidemiology[C]. Frontiers in Bioscience,2007,12,673-689.
[52]Bertolini E,Tomes E,Olmos A. Co-operational PCR couple with dotblot hybridization for detection and 16SrX grouping of phytoplasmas [J].Plant Pathology,2007,1:1-6.
[53]Davies D L,Clark M F. Production and characterization of polyclonal and monoclonal antibodies against peach yellow leafroll MLO-associated antigens [J]. Acta Hortic.1992,309:275-284. International Journal of systematic bacteriology, 1997,47:262-269.
[54]Denes A S and Sinha R C. Extrachromosomal DNA elements of plant pathogenic mycoplasmalike organisms [J]. Canadian Journal of Plant Pathology,1991,13: 26-32.
[55]Deng S J and Hiruki C. Genetic relatedness between two nonculturable mycoplasmalike organisms revealed by nucleic acid hybridization and polymerase chain reaction[J]. Phytopathology,1991,81(12):1475-1479.
[56]Doi,Y.M.,M.Teranaka,et al. Mycoplasma or PLT-group-like microorganisms found in the phloem elements of plants infected with mulberry dwarf,potato witches'-broom,aster yellows or paulownia witches'-broom[J]. Ann Phytopathol Soc Jpn,1967,33:259-266.
[57]Firrao G, Smart C D,Kirkpatrick B C. Physical map of the Western X-disease phytoplasma chromosome [J]. Bacteriol,1996,178(13):3985-3988.
[58]G. R. Prabu, P.G. Kawar,D. Theertha Prasad. Differential filtration approach for isolation and enrichment of Sugarcane Grassy Shoot phytoplasma [J]. Sugar Tech,2008,10(3):274-277.
[59]Hsu H T,Lee I-M,Davis R E. Immunization for generation of hybridoma antibodies specifically reaction with plants infected with a mycoplasmalike origanism(MLO) and their use in detection of MLO antigens [J]. Phytopathology,1990,80:946-950.
[60]International Committee on Systematic Bacteriology,Subcommittee on the Taxonomy of Mollicutes. Revised minimum standards for description of new species of the class Mollicutes (Division Tenericutes) [C]. Int J Syst Bacteriol,1995,45(3):605-612.
[61]International Committee on Systematic Bacteriology, Subcommittee on the Taxonomy of Mollicutes Minutes of the meetings,21 and 28 July,1998,Sydney,Australia[C]. Int J Syst Evol Microbiol,2000,50(3):1393-1397.
[62]Jennifer Berger, Josef Dalla Via,Sanja Baric. Development of a TaqMan allelic discrimination assay for the distinction of two major subtypes of the grapevine yellows phytoplasma Bois noir [J]. Eur J Plant Pathol,2009,124:521-526.
[63]Jiang Y P,Che T A,Chiykowski L N. Production of monoclonal antibodies to peach eastern X-disease agent and their use in disease detection [J]. Can J Plant pathol,1989,11:325-331.
[64]Jung H Y,Toshimi Sawayanagi,Shigeyuki Kakizawa,et al.'Candidatus Phytoplasma ziziphi',a novelphytoplasma taxon associated with jujube witches'-broom disease [J]. International Journal of Systematic and Evolutionary Microbiology,2003,53,1037-1041.
[65]Kirkpatrick B C,Stenger D C,Morris T J,et al. Cloning and detection of DNA from a nonculturable plant pathogenic mycoplasma-like organism[J]. Science, 1987,238:197-2001.
[66]Lauer U and Seemuller E. Physical map of the chromosome of the apple proliferation phytoplasma [J]. J Bacteriol,2000,182(5):1415-1418.
[67]Liefting LW,Shaw ME,Kirkpatrick BC. Sequence analysis of two plasmids from the phytoplasma beet leafhopper-transmitted virescene agent [J]. Microbiology, 2004,150:1809-1817.
[68]Lee I-M,Davis R E,Chen T-A.,et al. A genotype-based system for identification and classification of coplasmalike organisms (MLOs) in the aster yellows MLO strain cluster [J]. Phytopathology,1992,82:977-986.
[69]Lee I-M,Davis R E. Sinclair Genetic relatedness of mycoplasmalike organisms detected in Ulmus spp. in the United States and Italy by means of DNA probes and polymerase chain reactions [J]. Phytopathology,1993a,83 (6):829-833.
[70]Lee I-M. Use of mycoplasmalike orgonismas (MLO) group specific oligonucleotide primers for nested-PCR assays to detect mixed-MLO infections in a single host plant [J]. Phytopathology,1993b,84 (6):559-566.
[71]Lee I-M,Gundersen-Rindal D E,Bertaccini A. Phytoplasma:ecology and genomic diversity [J]. Phytopathology,1998a,88:1359-1366.
[72]Lee I-M,Gundersen D E,Davis R E,et al. Revised classification scheme of phytoplasmas based on RFLP analyses of 16S rRNA and ribosomal protein gene sequences [J]. Int J Syst Evol Microbiol,1998b,48:1153-1169.
[73]Lee I-M,Davis R E,Gundersen-Rindal D E. Phytoplasma:phytopathogenic mollicutes [J]. Annu Rev Microbiol,2000,54:221-255.
[74]Lee I-M,Gundersen D E,Davis R E,et al.'Candidatus Phytoplasma asteris',a novel phytoplasma taxon associated with aster yellows and related diseases[J]. International Journal of Systematic and Evolutionary Microbiology,2004a,54: 1037-1048.
[75]Lee I-M,Martini M,Marcone C,et al. Classification of phytoplasma strains in the elm yellows group (16SrV) and proposal of'Candidatus Phytoplasma ulmi'for the phytoplasma associated with elm yellows [J]. International Journal of Systematic and Evolutionary Microbiology,2004b,54:337-347.
[76]Lee I-M,Zhao Y,Bottner K D. SecY gene sequence analysis for finer differentiation of diverse strains in the aster yellows phytoplasma group [J]. Molecular and Cellular Probes,2006,20:87-91.
[77]Lim,P-O,Sears,B. B. Evolutionary relationships of a plant-pathogenic mycoplasmalike organism and Acholeplasma laidlawii deduced from two ribosomal protein gene sequences [J]. J Bacteriol,1989,174,2606-2611.
[78]Lin C L,Li H F,Fan Z F. Two novel plasmids from paulownia witches'-broom phytoplasma and its relatednss to other phytoplasmas [C]. Proceedings of the Third Asian Conference on Plant Pathology,Yogyakarta,Indonesia August, 20-24,2007:149-150.
[79]Lin C P,Chen T A. Monoclonal antibodies against the aster yellows agent [J]. Science,1985,227:1233-1235.
[80]Marcone C, Lee I M,Davis R E,et al. Classification of aster yellows-group phytoplasmas based on combined analyses of rRNA and tuf gene sequences [J]. International Journal of Systematic and Evolutionary Microbiology,2000,50: 1703-1713.
[81]Marcone C, Schneider B and Seemuller E.'Candidatus Phytoplasma cynodontis', the phytoplasma associated with Bermuda grasswhite leaf disease [J]. International Journal of Systematic and Evolutionary Microbiology,2004,54:1077-1082.
[82]Oshima K and Kakizawa S. Reductive evolution suggested from the complete genome sequence of a plant-pathogenic phytoplasma [J]. Nat Genet,2004,36(1): 27-29.
[83]Padovan A C and Firrao G. Chromosome mapping of the sweet potato little leaf phytoplasma reveals genome heterogeneity within the phytoplasmas [J]. Microbiology,2000,146 (4):893-902.
[84]Schneider B,Gibb K S,Seemuuller E. Sequence and RFLP analysis of the elongation factor Tu gene used in differentiation and classification of phytoplasmas [J]. Microbiology,1997,143:3381-3389.
[85]Seruga M,Skoric D,Botti S,et al. Molecular characterization of a phytoplasma from the asteryellows (16SrI) group naturally infecting Populus nigra L. 'Italica'trees in Croatia [J]. For. Path.2003,:33:113-125.
[86]Tran-Nguyen,L.T.,Kube,M. et al. Comparative genome analysis of'Candidatus Phytoplasma australiense'(subgroup tuf-Australia I; rp-A) and'Ca. Phytoplasma asteris'Strains OY-M and AY-WB [J]. J. Bacteriol,2008,190 (11):3979-3991.
[87]Woese C R.Bacterial evolution [J].Microbiol Rev,1987,51(2):221-271.
[88]Wei W,Robert E,Davis I-M,et al. Computer-simulated RFLP analysis of 16s rRNA genes:identification of ten new phytoplasma groups [J]. Int J Syst Evol Microbiol,2007,57:1855-1867.
[89]Yi C K,La YJ. Mycoplasma-like bodies found in the phloem elements of jujube trees infected with witches'-broom disese [J].Research Reports of the Forest Research Institute of Korea,1973,20:111-114.
[90]Zreik S,Carle L,Bove P,et al. Characterization of the mycoplasmalike organism associated with witches-broom disease of lime and proposition of a Candidatus taxon for the organism," Candidatus Phytoplasma aurantifolia." [J]. Int J Syst Bacteroil,1995,45:449-453.