臭矢菜丛枝病植原体的分子鉴定研究

 本实验采用DAPI荧光显微镜、PCR、克隆和测序等技术,对海南臭矢菜丛枝病样进行了检测和鉴定。以染病臭矢菜总DNA为模板应用3对植原体特异性引物进行PCR扩增,获得PCR产物为16S rDNA(1 430 bp)、16S-23S rDNA(358bp)、rp DNA(1 294 bp)。应用DNA回收试剂盒获得了3个PCR扩增片断的纯化产物,并克隆到DH5α大肠杆菌中测序。应用DNAMAN和MEGA软件对获得的序列与NCBI数据库中植原体序列进行同源性分析和构建系统发育树。结果显示臭矢菜丛枝病植原体与花生丛枝病植原体序列同源性最高,16S rDNA的序列同源性为99.9%,16S-23S rDNA高达100%,rp为99.7%,因而将臭矢菜丛枝病植原体归为花生丛枝组(16SrⅡ),根据16S rDNA的RFLP分析,将其归为16SrⅡ-A亚组。     

金莲花绿变植原体的分子鉴定

本研究对河北省大面积发生的金莲花绿变病的病原进行检测和鉴定。以金莲花叶片的总DNA为模板,使用植原体16S rDNA和核糖体蛋白(ribosomal protein)基因rp的特异性引物进行PCR扩增,在感病金莲花样品中扩增到植原体的16S rDNA(1 432 bp)片段和rp基因(1 240 bp)片段。序列分析发现,获得的16S rDNA和rp基因片段与洋葱黄化植原体Onion yellows phytoplasma(GenBank登录号:AP006628)的相似度最高,分别为99.9%和99.3%,确定金莲花绿变病的病原为植原体,暂命名为金莲花绿变植原体Trollius chinensis virescence phytoplasma。对金莲花绿变植原体的16S rDNA进行虚拟RFLP分析,发现其酶切图谱与16SrⅠ-B亚组的洋葱黄化植原体的参照图谱完全一致,相似系数1.00。16S rDNA和rp基因的系统发育进化树显示,金莲花绿变植原体与16SrⅠ-B亚组的植原体聚为一支,属于植原体16S rⅠ-B亚组。     

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。     

介体条沙叶蝉传播小麦蓝矮病植原体特性研究

小麦蓝矮病植原体(wheat blue dwarf,WBD)属于翠菊黄化组三叶草绿变亚组植原体(16SrⅠ-C),由介体条沙叶蝉(Psammotettix striatus L.)专化性传播。通过电镜超微结构观察,在接种小麦、长春花和带毒条沙叶蝉体内有大量植原体,而在健康植物组织、无毒条沙叶蝉和带毒条沙叶蝉所产卵中未见植原体的存在。通过介体传毒试验和PCR检测发现,条沙叶蝉最适获毒期为7 d,植原体在虫体内的潜育期为1 5～1 7 d,接毒期为2～3 d。条沙叶蝉一旦获毒可终生持毒和传毒。不同虫态的条沙叶蝉带毒率没有明显的差异,但寄生植物的种类影响其带毒率。     

杏褪绿卷叶植原体新疆分离物16S rDNA基因克隆与序列分析

利用植原体16S rDNA基因通用引物对新疆轮台县疑似杏褪绿卷叶病植株总DNA进行巢氏PCR检测,扩增出大小约1.2 kb的特异性条带。对扩增产物克隆和测序,确定特异片段大小为1248 bp。序列同源性比较和系统进化分析表明,新疆杏褪绿卷叶植原体不同分离株16S rDNA基因序列同源性极高,达到99.8%~100%。与16SrⅤ组成员的同源性达到98.2%以上,其中与16SrⅤ-B亚组的枣疯病植原体山东宝山分离株,甜樱桃绿化植原体山东分离株同源性最高,达到99.4%~99.6%。进一步虚拟RFLP分析,结果表明该植原体属于榆树黄化组(16SrⅤ)的一个新的亚组,与其相似性最高的是16SrⅤ-B亚组,相似系数为0.94。本研究首次报道了新疆杏褪绿卷叶植原体16S rDNA的序列,确定了其分类地位,为杏褪绿卷叶病的早期诊断和检测提供了基础。     

喜树丛枝植原体的分子鉴定及TaqMan探针实时荧光定量PCR检测方法的建立

 为确定在云南文山地区喜树上发生的疑似丛枝病的病原种类及快速检测喜树丛枝病,本研究利用植原体16S rDNA基因通用引物P1/P7和R16F2n/R16R2对感病喜树总DNA进行常规PCR和巢式PCR扩增、克隆和测序,通过系统进化分析,明确了喜树丛枝植原体属于16SrXXXII组。然后根据喜树丛枝病植原体16S rDNA基因保守区域设计并合成特异性引物和TaqMan探针,制备了喜树丛枝病植原体标准质粒,确定了最优引物浓度和最佳探针浓度,制作的标准曲线有极好的线性关系,决定系数(R²)达到0.999,建立的实时荧光定量PCR检测方法能够特异性地检测喜树丛枝植原体。本研究首次明确了喜树丛枝植原体的分类地位,优化和建立了喜树丛枝植原体TaqMan探针qPCR检测方法,为快速检测喜树丛枝病植原体提供参考。     

3种检疫性葡萄黄化植原体的快速检测

 葡萄上的植原体病害由于引起叶片黄化而被称为葡萄黄化病。由于这一病害极为严重,葡萄黄化植原体被列为我国的植物检疫对象。其中,葡萄金黄化植原体(16SrV)、维吉尼亚葡萄黄化植原体(16Sr芋) 和澳大利亚葡萄黄化植原体 (16Sr狱) 是引起葡萄黄化病的主要3 个株系,它们导致的病害症状相似,难以区分。本文进行了3 个株系16S rRNA 基因 DNA 序列比对,而后根据同源性相对低的序列设计了43 条特异性引物、103 对引物对组合,对葡萄黄化植原体3 个株系各自的DNA 及混合DNA 进行PCR 扩增,从中筛选出来特异性较强的8 个引物对组合。这些引物对组合,能够同步、特异、快速地检测3 种葡萄黄化植原体。     

人工培养液在植原体昆虫介体筛选中的适用性

研究了一种人工培养液对各种常见的昆虫(主要是叶蝉)的亲和性和适用性.结果表明,该人工培养液适于本试验中大多数昆虫的人工饲养.用此方法,悬钩子广头叶蝉Macropsis.ftscula Zetterstedt和桤树广头叶蝉Oncopsis alniSchrank分别被再次确认为悬钩子矮化植原体和桤树黄化植原体的传播介体;田旋花麦蜡蝉Hyalesthes obsoletus Signoret再次被确认为葡萄黄化(stolbur)植原体的传播介体.此前,上述三种叶蝉已被传统的人工接种方法鉴定为相应植原体的传播介体.危害桤树的河谷树叶蝉Allygus modestus Scott尽管虫体DNA检测结果经常为阳性,但迄今其人工培养液的检测结果都是阴性,因此,我们认为河谷树叶蝉不是桤树黄化植原体的传播介体.Eppendorf管人工培养液饲养法不仅适用于潜在的植原体介体昆虫的筛选鉴定,而且可用于介体昆虫的生物防治研究.此外,本研究首次发现自然感染了葡萄上的一种被德国人称为"Vergi-lungskrankheit"植原体(AY组)的草地脊冠叶蝉Aprodes makarovi Zachvatkin.     

植原体16S rDNA RFLP指纹图谱分析软件研究

 植原体是一类寄生于植物韧皮部的原核生物。目前国际上主要根据植原体16S rDNA RFLP图谱的比对进行分类及鉴定。传统的PCR-RFLP试验步骤繁琐、重复性差,误差大,尤其在检测鉴定大量植原体病原时,耗时、耗力。通过对目前已公布的全部植原体16S rDNA序列进行比对整理,并生成16S rDNA RFLP电子指纹图谱库。在此基础上开发了指纹图谱分析软件DNA-FP Cluster1.0。该软件可基于序列或图谱形式进行比对,并自动输出比对结果。结果呈现形式为3种:即一种16S rDNA RFLP图谱与多种图谱间相似性分析;多种16S rDNA RFLP图谱间相似性分析;16S rDNA RFLP图谱间相似度树状图。该软件在不需要酶切试验操作的情况下实现了对植原体的快速分类与鉴定,并解决了因植原体材料不全而无法进行植原体酶切后比对的难题,为今后植原体候选种内的细化分类提供了方法与工具。     

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。     

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.     

黄槐丛枝病植原体的检测及鉴定

 应用植原体16S rRNA基因通用引物,对自然表现丛枝的黄槐植株进行巢式PCR检测,得到约1.2 kb的特异片段,证明此植株中存在植原体.将此特异片段与pGEM-T Easy载体连接并转化到大肠杆菌JM109感受态细胞中,通过PCR鉴定、序列测定及同源性比较分析,结果表明此植原体株系(STWB)16S rDNA片段G+C含量为45.8%,与榆树黄化植原体组(Elm yellows group,16SrV group)中的各株系最高同源率可达99.4%,而与其它组中的株系明显低于97.0%,故认为该植原体株系为榆树黄化植原体组中的成员之一.     

Detection of ‘<Emphasis Type="Italic">Candidatus</Emphasis> Phytoplasma brasiliense’ in a new geographic region and existence of two genetically distinct populations’

‘Candidatus Phytoplasma brasiliense’, a phytoplasma taxon associated with hibiscus witches’ broom disease was first described in 2001 in Brazil. In September 2007, a peach tree (Prunus persica) displaying yellowing symptoms reminiscent of phytoplasma infection was sampled in Guba region of Azerbaijan. A phytoplasma was detected in the diseased peach tree by nested PCR amplification of its 16S rDNA with universal primers for phytoplasmas. Phylogenetical analyses of the amplified 16S rDNA showed that the phytoplasma infecting the peach tree corresponded to ‘Ca. P. brasiliense’, a species never reported in Euro-Mediterranean area. To set up a detection assay, cloning of a ‘Ca. P. brasiliense’ DNA fragment was undertaken by comparative RAPD. The amplified dnaK-dnaJ genetic locus was used to design a nested PCR assay able to amplify all ‘Ca. P. brasiliense’ isolates of the subgroup 16SrXV-A without amplifying the related members of the group 16SrII. This assay also allowed confirming the first detection of ‘Ca. P. brasiliense’ in diseased basil collected in south Lebanon.     

广东桉树黄化(丛枝)病植原体分子鉴定与检测

从表现黄化(丛枝)症状的桉树上采集病叶,抽提主脉总DNA,采用植原体通用引物与巢式引物进行PCR和巢式PCR扩增,对扩增产物进行克隆和序列测定,获得了植原体的近全长16S rRNA基因及部分16～23S rRNA基因间隔区序列.序列分析揭示,所获得的序列与已知植原体基因组相应区段的序列高度同源,与柳叶菜变叶植原体(epilobium phyllody)和白腊树丛枝植原体(ash witches'-broom)相应序列(GenBank登录号:AY101386和AY566302)同源率为99.9%,与白腊树黄化植原体(aster yellows BD2)相应序列和番茄巨芽植原体(tomato big bud)相应序列同源率分别为99.6%和99.3%.该序列构建的系统进化树表明,引起我国广州地区桉树黄化(丛枝)病的植原体属于16SrI组(即翠菊黄化组),将其暂命名为桉树黄化(丛枝)植原体广东株系(Eucalyp-tus yellowing and witches'-broom phytoplasma strain Guangdong,EYWB-Gd).建立了桉树植原体巢式PCR检测方法,对疑似病样及桉树组培苗进行了检测,多数疑似病样检测结果为阳性,供试的10株组培苗未发现阳性样品.     

New Group 16SrIII Phytoplasma Lineages in Lithuania Exhibit rRNA Interoperon Sequence Heterogeneity

Previously undescribed phytoplasmas were detected in diseased plants of dandelion (Taraxacum officinale) exhibiting virescence of flowers, thistle (Cirsium arvense) exhibiting symptoms of white leaf, and a Gaillardia sp. exhibiting symptoms of stunting and phyllody in Lithuania. On the basis of restriction fragment length polymorphism (RFLP) analysis of 16S rDNA amplified in PCR, the dandelion virescence (DanVir), cirsium whiteleaf (CirWL), and gaillardia phyllody (GaiPh) phytoplasmas were classified in phylogenetic group 16SrIII (X-disease phytoplasma group), new subgroups III-P and III-R and subgroup III-B, respectively. RFLP and nucleotide sequence analyses revealed 16S rRNA interoperon sequence heterogeneity in the two rRNA operons, rrnA and rrnB, of both DanVir and CirWL. Results from phylogenetic analysis based on nucleotide sequences of 16S rDNA were consistent with recognition of the two new subgroups as representatives of distinct new lineages within the group 16SrIII phytoplasma subclade. The branching order of rrnA and rrnB sequences in the phylogenetic tree supported this interpretation and indicated recent common ancestry of the two rRNA operons in each of the phytoplasmas exhibiting interoperon heterogeneity.     

Differential detection and genetic relatedness of phytoplasmas in papaya

The genetic relatedness of phytoplasmas associated with dieback (PDB), yellow crinkle (PYC) and mosaic (PM) diseases in papaya was studied by restriction fragment length polymorphism (RFLP) analysis of the 16S rRNA gene and 16S rRNA/23S rRNA spacer region (SR). RFLP and SR sequence comparisons indicated that PYC and PM phytoplasmas were identical and most closely related to members of the faba bean phyllody strain cluster. By comparison the PDB phytoplasma was most closely related to Phormium yellow leaf (PYL) phytoplasma from New Zealand and the Australian grapevine yellows (AGY) phytoplasma from Australia. These three phytoplasmas cluster with the stolbur and German grapevine yellows (VK) phytoplasmas within the aster yellows strain cluster. Primers based on the phytoplasma tuf gene, which amplify gene products from members of the AY strain cluster, also amplified a DNA product from the PDB phytoplasma but not from either the PYC or PM phytoplasmas. Primers deduced from the 16S rRNA/SR selectively amplified rDNA sequences from the PDB and AGY phytoplasmas but not from other members of the stolbur strain cluster. Similarly, primers designed from 16S rRNA/SR amplified rDNA from the PYC and PM phytoplasmas but not from the PDB phytoplasma. These primers may provide for more specific detection of these pathogens in epidemiological studies.     

16S rDNA-derived oligonucleotide probes for the differential diagnosis of plum leptonecrosis and apple proliferation phytoplasmas

The 16S rRNA gene of plum leptonecrosis phytoplasma has been PCR-amplified, cloned and almost completely sequenced (1201 bp). The sequence analysis confirmed the close genetic relationship between plum leptonecrosis phytoplasma and the phytoplasmas associated with other stone-fruit diseases in Europe. By comparison with the 16S rDNA sequence of apple proliferation phytoplasma, two oligonucleotides were selected, differing by two nucleotides, which were specific for apple proliferation and plum leptonecrosis phytoplasmas, respectively. The oligonucleotides were labelled with digoxigenin and hybridized, in the presence of tetramethylammonium chloride, to 16S rDNA fragments amplified from apple and plum leaf samples. The results showed that, under the described hybridization conditions, the two phytoplasmas could clearly be distinguished. The advantage of the proposed technique over 16S rDNA restriction fragment length polymorphism is discussed.     

Detection and identification of the maize bushy stunt phytoplasma in corn plants in Brazil using PCR and RFLP

Plants of corn (Zea mays L.) exhibiting symptoms of stunting and leaf reddening were assayed for the presence of phytoplasma gene sequences through the use of phytoplasma rRNA and ribosomal protein gene and maize bushy stunt (MBS) phytoplasma-specific oligonucleotide primers in polymerase chain reactions (PCR). Polymorphisms in 16S rDNA amplified from diseased plants were those characteristic of phytoplasmas classified in the16S rRNA gene group 16SrI, subgroup IB, of which MBS phytoplasma is a member. Amplification of ribosomal protein (rp) gene sequences in PCR primed by phytoplasma-specific primers confirmed presence of a phytoplasma in the diseased plants. Restriction fragment length polymorphism (RFLP) patterns of the amplified phytoplasma rp gene sequences were similar or identical to those observed for a known strain of MBS phytoplasma. In separate PCR, an MBS-specific oligonucleotide pair primed amplification of a MBS-characteristic DNA from templates derived from the diseased corn. Our data provide the first firm evidence for the presence of maize bushy stunt phytoplasma in corn in Brazil.     

Molecular identification of a new phytoplasma associated with alfalfa witches'-broom in oman

ABSTRACT Alfalfa (Medicago sativa) plants showing witches'-broom symptoms typical of phytoplasmas were observed from Al-Batinah, Al-Sharqiya, Al-Bureimi, and interior regions of the Sultanate of Oman. Phytoplasmas were detected from all symptomatic samples by the specific amplification of their 16S-23S rRNA gene. Polymerase chain reaction (PCR), utilizing phytoplasma-specific universal primer pairs, consistently amplified a product of expected lengths when DNA extract from symptomatic samples was used as template. Asymptomatic plant samples and the negative control yielded no amplification. Restriction fragment length polymorphism profiles of PCR-amplified 16S-23S rDNA of alfalfa using the P1/P7 primer pair identified phytoplasmas belonging to peanut witches'-broom group (16SrII or faba bean phyllody). Restriction enzyme profiles showed that the phytoplasmas detected in all 300 samples belonged to the same ribosomal group. Extensive comparative analyses on P1/P7 amplimers of 20 phytoplasmas with Tru9I, Tsp509I, HpaII, TaqI, and RsaI clearly indicated that this phytoplasma is different from all the other phytoplasmas employed belonging to subgroup 16SrII, except tomato big bud phytoplasma from Australia, and could be therefore classified in subgroup 16SrII-D. The alfalfa witches'-broom (AlfWB) phytoplasma P1/P7 PCR product was sequenced directly after cloning and yielded a 1,690-bp product. The homology search showed 99% similarity (1,667 of 1,690 base identity) with papaya yellow crinkle (PapayaYC) phytoplasma from New Zealand. A phylogenetic tree based on 16S plus spacer regions sequences of 35 phytoplasmas, mainly from the Southern Hemisphere, showed that AlfWB is a new phytoplasma species, with closest relationships to PapayaYC phytoplasmas from New Zealand and Chinese pigeon pea witches'-broom phytoplasmas from Taiwan but distinguishable from them considering the different associated plant hosts and the extreme geographical isolation.