竹小叶病植原体的分子鉴定

 2008年在杨凌采集到具有典型植原体侵染症状的菲白竹,应用植原体16S rRNA基因的通用引物对R16mF2／R16mR1和R16F2n／R16R2对其进行检测,巢式PCR得到约1.2 kb的特异性片段。对扩增片段进行测序并进行系统进化树分析,结果表明,该病原属于翠菊黄化组(Candidatus Phytoplasma asteris),与该组成员同源性均在98%以上。随后用16Sr Ⅰ组和Ⅴ组特异引物对R16（Ⅰ）F1／R16（Ⅰ）R1和R16（Ⅴ）F1／R16（Ⅴ）R1也证明其属于翠菊黄化组,RFLP 分析表明该植原体属于16SrⅠ-B亚组。植原体侵染菲白竹在中国属首次报道。     

天津滨海新区泡桐丛枝病植原体16S rDNA基因序列分析

利用分子生物学技术对天津滨海新区泡桐丛枝病病原进行分类鉴定。采用植原体16S rDNA通用引物R16mF2/R16mR1对患病植株总DNA进行PCR扩增,得到约1.4 kb特异性片段。克隆测序、Blast比对和iPhyClassifier分析结果表明,天津滨海新区泡桐丛枝植原体16S rDNA基因片段长1 432 bp,与国内泡桐丛枝植原体PY株系相似性最高,达99.86%,归属于16SrI组(aster yellows group,翠菊黄化组)D亚组。系统树构建与分析显示,泡桐丛枝病天津滨海株PaWB-TJBH与16SrI其他亚组亲缘关系较近,同在16SrI组进化枝上,与16Sr I-D组亲缘关系最近;16S rDNA序列RFLP电子酶切图谱表明,PaWB-TJBH属于16SrI-D组一个成员,与同源性比较和系统进化分析结果一致。     

滇朴丛芽病植原体的分子鉴定

滇朴Celtis kunmingensis Cheng et Hong是云南的乡土树种,适宜全国大部分地区种植,极具观赏价值,是近年来最热门的绿化首选树种―绿化行道树,云南部分地区滇朴近年常表现丛芽的症状。本研究采用形态学与分子生物学结合的方法,对染病的幼嫩枝条进行扫描电镜(SEM)观察;利用16S rDNA植原体通用引物P1/P7和R16F2/R16R2进行常规PCR和巢式PCR,分别获得1.8 kb和1.2 kb的特异性基因片段,将该特异性片段与其他已知分类地位的植原体16S rDNA片段进行同源性比对分析,同时利用邻接法(NJ)构建系统发育树。结果表明在染病的滇朴韧皮部组织中可见植原体存在,滇朴丛芽病植原体与芝麻叶状植原体同源性高达99.40%,通过系统发育树可进一步推测滇朴丛芽病植原体是属于16SrⅠ-B亚组成员,本研究结果为该病害的诊断与防治提供了理论依据。     

桃红叶植原体检测及鉴定

对表现红叶的桃植株进行植原体16SrRNA基因PCR扩增,得到1.2kb的特异片段.将此片段与pGEM T Easy载体连接并转化到大肠杆茵DH5α感受态细胞中.通过酶切、PCR鉴定,对筛选得到的重组阳性克隆进行序列测定及同源性比较分析,确定该株系属于翠菊黄化植原体组(Aster yellows group,16SrI).在国内首次报道了翠菊黄化组中的植原体侵染桃树.     

云南泡桐丛枝病植原体核糖体蛋白基因片段序列分析

 应用植原体核糖体蛋白基因通用引物对rpF1/rpR1,对采自云南省曲靖市的泡桐丛枝病植原体DNA (PaWB-QJ)进行PCR扩增,得到1.3 kb的特异片段,证明此病株中存在植原体。将此片段与pGEM-T Easy载体连接并转化大肠杆菌JM109感受态细胞,进行PCR鉴定、核糖体蛋白基因部分核苷酸序列测定及分析。结果表明,该株系(PaWB-QJ)核糖体蛋白基因片段长1 244 bp,包含rps19、rpl22和rps3基因。对PaWB-QJ株系的核糖体蛋白基因序列的同源性比较结果显示与16S rI-B亚组的翠菊黄化(Aster yellows,AY)、长春花黄化(Periwinkle yellows,PY)和泡桐丛枝德国株系(Paulownia witches'-broom,PaWB-German)的亲缘关系最近,达到99.0%以上,而与其它组中的株系明显低于97.0%,所以认为该植原体株系属于翠菊黄化组B亚组(16SrI-B)。     

小麦蓝矮植原体寄主范围的鉴定及RFLP分析

 小麦蓝矮是我国首次报道的小麦植原体病害。采用介体接种植物,症状观察和应用植原体16S rDNA基因通用引物对R16mF2/R16mR1进行PCR扩增,在接种小麦和传毒介体中均扩增出1.4kb的特异片段,鉴定出小麦蓝矮植原体新寄主7种。用巢式PCR方法对小麦蓝矮病田自然发病杂草进行分子检测,从表现症状的10种杂草中均扩增出1.2kb的特异片段。利用6种植原体特异性限制性内切酶对10种杂草的扩增片段进行RFLP(restriction fragment length polymor-phism)分析表明:扩增片段的RFLP图谱与目前已知的16Sr I组翠菊黄化植原体的RFLP图谱相近。鉴定出小麦蓝矮植原体田间自然新寄主10种。     

榆树黄化病植原体的分子检测与鉴定

 利用植原体16SrRNA基因的通用引物R16rrLF2/R16mR1和R16F2n/R16R2对山东泰山上发生的榆树(Ulmus parvifolia)黄化病感病植株总DNA进行巢式PCR扩增,得到了约1.2kb的特异性片段,从分子水平证实了榆树黄化病的病原(EY-China)为植原体。将扩增到的片段测序,并进行一致性和系统进化树分析。结果表明,该分离物属于植原体榆树黄化组(Candidatus Phytoplasma ulmi),与该组成员16SrRNA序列的一致性均在98.2%以上,其中与16SrV-B亚组中的纸桑丛枝(Paper mulberry wiches'-broom)和枣疯病(Jujube witches'-broom)植原体一致性最高,达到99.4%,在系统进化树中与该亚组成员聚类到同一个分支,说明该分离物属于植原体16SrV-B亚组。本研究首次对在中国引致榆树黄化病的植原体进行了分子检测,并通过核酸序列分析将其鉴定到亚组水平。     

竹丛枝植原体16SrDNA片段克隆与序列分析

利用植原体16SrRNA基因序列设计合成的引物,对表现丛枝的竹子植株总DNA进行直接PCR及巢式PCR扩增,得到长1.2kb的目的片段。将此片段与pGEMTEasy载体连接并转化到大肠杆菌DH5α感受态细胞中。通过酶切、PCR鉴定,对筛选得到的重组阳性克隆进行核酸序列测定及同源性比较分析,结果表明其与植原体16SrⅠ组中的西方翠菊黄化植原体(SAY)同源率为99%。依据16SrDNA序列建立了竹子丛枝病植原体株系的系统进化树。对云南竹子丛枝病植原体株系分类鉴定与已报道的结果相似。     

海南省木豆丛枝病植原体的分子检测及鉴定

 利用植原体通用引物R16mF2/R16mR1和rp (Ⅱ) F1/rp (Ⅱ) R1对海南木豆丛枝病植原体16S rDNA和部分核糖体蛋白(ribosomal protein,rp)基因序列进行PCR扩增、克隆和测序。获得海南木豆丛枝病植原体16S rDNA基因片段为1430bp,rp基因片段为1170bp。核苷酸同源性比较和系统进化树构建表明,引起海南木豆丛枝病的植原体应属于16SrⅡ组中的亚组ⅲ。本研究首次从分子水平确定了引起我国海南木豆丛枝病的病原物为植原体,明确了其分类地位,为该病害流行学研究和防治提供了理论依据。     

小麦蓝矮病植原体16S rDNA基因片段的比较分析

 小麦蓝矮病是陕西乃至西北冬麦麦区的一个重要病害,由介体条沙叶蝉专化性传播。对小麦蓝矮病株叶片和带毒条沙叶蝉进行超薄切片及电镜观察,在叶片韧皮部和叶蝉后肠中均观察到大量典型植原体。利用植原体16S rDNA基因保守序列通用引物对Rm16F2/Rm16R1,应用PCR技术从小麦蓝矮病株叶片中扩增到1.4 kb的特异片段。通过对16S rDNA基因片段序列同源性比较,结果表明小麦蓝矮病病原与三叶草绿变、翠菊黄化、绣球花绿变、草莓矮化和番茄巨芽植原体亲缘关系较近,其同源率为99.2%~99.9%。据此可以判定小麦蓝矮病植原体是属于植原体16SrⅠ组,确定了其分类地位。     

New natural host plants of ‘Candidatus Phytoplasma pini’ in Poland and the Czech Republic

The presence of phytoplasmas in seven coniferous plant species (Abies procera, Pinus banksiana, P. mugo, P. nigra, P. sylvestris, P. tabuliformis and Tsuga canadensis) was demonstrated using nested PCR with the primer pairs P1/P7 followed by R16F2n/R16R2. The phytoplasmas were detected in pine trees with witches’ broom symptoms growing in natural forest ecosystems and also in plants propagated from witches’ brooms. Identification of phytoplasmas was done using restriction fragment length polymorphism analysis (RFLP) of the 16S rDNA gene fragment with AluI, MseI and RsaI endonucleases. All samples showed RFLP patterns similar to the theoretical pattern of ‘Candidatus Phytoplasma pini’, based on the sequence of the reference isolate Pin127S. Nested PCR‐amplified products, obtained with primers R16F2n/R16R2, were sequenced. Comparison of the 16S rDNAs obtained revealed high (99·8–100%) nucleotide sequence identity between the phytoplasma isolates. The isolates were also closely related to four other phytoplasma isolates found in pine trees previously. Based on the results of RFLP and sequence analyses, the phytoplasma isolates tested were classified as members of the ‘Candidatus Phytoplasma pini’, group 16SrXXI.     

Phytoplasmas associated with disease of coconut in Malaysia: phylogenetic groups and host plant species

Coconut palm ( Cocos nucifera ), oil palm ( Elaeis guineensis ), Bermudagrass ( Cynodon dactylon ) and Madagascar periwinkle ( Catharanthus roseus ) with symptoms indicative of phytoplasma disease were collected from different locations in Malaysia. PCR assays employing phytoplasma universal rRNA gene primers P1/P7 alone or P1/P7 followed by R16F2n/R16R2 detected phytoplasmas in eight out of 20 Malayan Red Dwarf (MRD), nine out of 12 Malayan Yellow Dwarf (MYD) and 12 out of 12 Malayan Tall (MT) coconut palms displaying coconut yellow decline symptoms. Positive detections were also obtained from six out of six oil palm seedlings showing symptoms of yellowing and necrosis, from 10 out of 10 Bermudagrass samples with white leaf symptoms, and from eight out of eight periwinkle plants showing phyllody, virescence, little leaf, proliferation and foliar yellowing. Phytoplasmas were not detected in any of the symptomless plants tested. Sequencing and phylogenetic analysis of PCR products determined that phytoplasmas infecting both MRD and MT coconuts and Bermudagrass in Serdang, Selangor State, were all members of the 16SrXIV ' Candidatus Phytoplasma cynodontis' group, whereas isolates in periwinkle in Serdang were all members of the 16SrI ' Ca. Phytoplasma asteris' group. However, the phytoplasmas detected in MYD coconuts and oil palms from Banting, Selangor State, and in periwinkle from Putrajaya were collectively very similar (99%), but shared <97·5% similarity with 16S rDNA sequences of all other known phytoplasmas, indicating that they represent a novel taxonomic group. Thus, at least two phylogenetically distinct phytoplasmas are associated with the coconut yellow decline syndrome in Malaysia, both of which were also detected in other plant species.     

Partial characterization of phytoplasmas associated with lettuce and wild lettuce phyllodies in Iran

Phytoplasmas associated with lettuce phyllody (LP) and wild lettuce phyllody (WLP) in southern Iran were partially characterized by molecular analyses and host-range studies. Agents of both diseases were transmitted by Neoaliturus fenestratus , a leafhopper colonizing lettuce and wild lettuce, to lettuce, wild lettuce, sowthistle and periwinkle, but not to safflower, sunflower, calendula and sesame. Both phytoplasmas induced bud proliferation, virescence, phyllody and witches' broom in infected plants. Total DNA extracted from infected lettuce and wild lettuce or from vector tissues was subjected to PCR using phytoplasma-specific primer pair P1/P7 or nested PCR using P1/P7 followed by R16F2n/R16R2. PCR product of nested PCR (1·2 kbp) was subjected to restriction fragment length polymorphism (RFLP). RFLP analysis of nested PCR product identified the LP, WLP and N. fenestratus -associated phytoplasmas as members of the pigeon pea witches' broom group, 16SrIX. Phylogenetic analysis of the 16S rRNA gene sequence also clustered LP and WLP phytoplasmas with other known members of the 16SrIX group. While no significant differences could be detected between LP and WLP phytoplasmas, both isolates differed from Lebanese wild lettuce phyllody in molecular properties.     

Co-operational PCR coupled with dot blot hybridization for detection and 16SrX grouping of phytoplasmas

A protocol based on Co-operational PCR has been successfully applied to the detection of phytoplasmas. A triprimer reaction coupled with hybridization using general and specific probes permitted detection of ' Candidatus Phytoplasma mali', ' Ca . Phytoplasma prunorum' and ' Ca . Phytoplasma pyri', and their identification as members of 16S ribosomal quarantine group X. The sensitivity of this method was at least one hundred times greater than conventional PCR and similar to that achieved by nested PCR and real-time PCR. The method was validated by testing field samples collected from Malus , Prunus and Pyrus spp. and Olea europaea and compared with seven phytoplasmas maintained in Catharanthus roseus .     

Detection and molecular characterization of a phytoplasma affecting Prunus persica L. in Jujuy, Argentina

Peach (Prunus persica L.) plants with symptoms of yellowing, reddening, curling and leaf necrosis, premature defoliation and internode shortening were observed in production fields in Jujuy province (Argentina). A phytoplasma was detected by PCR using the universal primer pairs P1/P7 and R16F2n/R16R2 in all the symptomatic samples analysed. The RFLP profile of PCR products, amplified with R16F2n/R16R2 primers, shows that this phytoplasma, named Argentinean Peach Yellows (ArPY), belongs to subgroup 16Sr III-B. The phylogenetic analysis of the 1244 bp 16S rDNA cloned sequence, grouped the ArPY phytoplasma into the X-disease group with a closer relationship with CFSD, PssWB and ChTDIII phytoplasmas. This is the first report of a phytoplasma infecting peach trees in Argentina.     

Natural occurrence, identification and transmission of the phytoplasma associated with flax phyllody and stem fasciation in Pakistan

Flax plants (Linum usitatissimum) of the white (album) flower variety exhibiting typical phytoplasma-like symptoms were found for the first time in Pakistan during 2011. The symptoms included floral virescence, phyllody, little leaf, stunting and stem fasciation. Light microscopy of hand-cut stem sections treated with Dienes’ stain showed blue areas in the phloem region of symptomatic plants. To confirm phytoplasma infection, total DNA was extracted separately from five plants showing virescence/phyllody and from five others showing fasciation, and was amplified by nested PCR using universal 16S rDNA phytoplasma primers P1/P7 followed by R16F2n/R16R2. All samples from plants with virescence/phyllody and fasciation yielded a 1,250 bp PCR product, and identical RFLP profiles using the enzymes AluI and HpaII. Direct sequencing of the 16S rDNA of one representative PCR amplicon (GenBank Accession No. JX567504 for phyllody and Accession No. JX567505 for fasciation) showed highest sequence identity (99%) with 16SrII ‘Candidatus Phytoplasma aurantifolia’ phytoplasmas, and phylogenetic analysis placed the phytoplasma in subgroup 16SrII-D. Disease was successfully transmitted by grafting and by the leafhopper Orosius albicinctus. To our knowledge, flax is a new natural host for 16SrII-D phytoplasmas in Pakistan.     

Association of 'Candidatus Phytoplasma australiense' with green petal and lethal yellows diseases in strawberry

The identity of phytoplasmas detected in strawberry plants with green petal (SGP) and lethal yellows (SLY) diseases was determined by RFLP analysis of the 16S rRNA gene and adjacent spacer region (SR). RFLP and sequence comparisons indicated that the phytoplasmas associated with SGP and SLY were indistinguishable and were most closely related to ' Candidatus Phytoplasma australiense', the phytoplasma associated with Australian grapevine yellows, papaya dieback and Phormium yellow leaf diseases. This taxon lies within the aster yellows strain cluster. Primers based on the phytoplasma tuf gene, which amplify only members of the AY strain cluster, amplified a DNA product from the SGP and SLY phytoplasmas. Primers deduced from the 16S rRNA/SR of P. australiense that amplify only members of this taxon amplified rDNA sequences from the SGP and SLY phytoplasmas. Primers that selectively amplify members of the faba bean phyllody (FBP) phytoplasma group, the most commonly occurring phytoplasma group in Australia, did not amplify rDNA from the SGP and SLY phytoplasmas.     

小麦蓝矮病植原体转运蛋白secY基因片段序列分析

 Wheat blue dwarf(WBD) is a disease caused by phytoplasma and only reported from China. A fragment about 1.3 kb in protein translocation gene, secY was amplified by PCR from the total DNA of di-seased wheat sample with primer pair secYF/secYR, which was designed based on secY gene sequence of known 16SrI group members. Nucleotide acid sequence analysis of amplified fragment indicated that the length was 1 240 bp. A phylogenetic tree based on secY gene sequences was constructed and showed that wheat blue dwarf phytoplasma was clustered into the Candidatus Phytoplasma asteris, subgroup 16SrI-C. Wheat blue dwarf phytoplasma showed high homology with clover phyllody phytoplasma strains based on sequence comparison and phylogenetic analysis.     

First report of ‘Candidatus Phytoplasma asteris’ infecting hydrangea showing phyllody in Japan

Phytoplasma-induced floral malformations such as virescence, phyllody, and proliferation were observed on hydrangeas in Gunma Prefecture, Japan. Phylogenetic analyses based on 16S rRNA, secY, groEL, and amp gene sequences indicated that the affected hydrangea plants were associated with phytoplasmas belonging to ‘Candidatus Phytoplasma asteris’, but not to ‘Ca. P. japonicum’, which occurs in hydrangeas showing phyllody in Japan. This is the first molecular evidence of an association of ‘Ca. P. asteris’ with hydrangea plants in Japan.