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。     

来源于湖北省枣疯病植原体分离物16S rDNA和rp基因的序列分析

以田间采集的来源于我国湖北省枣树产业主产区随州市随县种植的表现为"枣疯病"症状的枣树分离株为试材,对其16S rDNA和核糖体蛋白(ribosomal protein,rp)基因采用Nested-PCR进行扩增以及序列分析。结果表明,湖北JWB-Hubei植原体分离物16S rDNA基因的核苷酸序列与我国山东、河南等地的分离株一致率均为99%以上,在进化树中位于同一亚组的不同进化分支;虚拟RFLP图谱分析表明,JWB-Hubei属于16SrV-B亚组一个成员,与其进化树分组结果一致。JWBHubei分离株rp基因的核苷酸序列也与我国山东、陕西等地区的分离株一致率均为99%以上,在进化树中聚为同一亚组,与报道的基于RFLP分类属于rpV-C亚组的中国枣疯病分离物(JWB)聚集于同一亚组不同分支。该研究结果明确了湖北省枣疯病植原体的分类地位以及与来源于我国不同地区枣疯病分离株之间的遗传进化关系,为进一步研究植原体的株系划分、基因遗传变异研究提供了理论基础。     

紫花苜蓿丛枝病植原体的分子检测及鉴定

 利用植原体16S rRNA基因通用引物对云南昆明发生的苜蓿丛枝病感病植株总DNA进行巢式PCR扩增,得到1.2kb的特异片段,从分子水平证实了苜蓿丛枝病的病原是植原体。从PCR产物的RFLP酶切图谱可看出,该植原体株系的酶切图谱与马里兰翠菊黄化植原体(AY1)相同。对扩增片段进行克隆及序列测定后,利用最小进化法做Bootstrap验证的系统进化树,表明苜蓿丛枝病植原体为Candidatus Phytoplasma asteris成员之一,与植原体16SrI-B亚组成员关系密切。     

卵叶山蚂蝗丛枝植原体的分子鉴定

2016年在生长于海南省澄迈县的卵叶山蚂蝗上发现了丛枝、小叶等疑似植原体感染症状。利用植原体16S rRNA基因的通用引物R16mF2/R16mR1,对卵叶山蚂蝗丛枝样品进行了PCR检测,并对检测到的植原体16S rRNA基因片段(1.4 kb)进行克隆测序、序列比对、虚拟限制性片段长度多态性分析和系统进化树构建分析。结果表明,卵叶山蚂蝗丛枝植原体(beggarweed witches'-broom phytoplasma,MK956144)为来檬植原体(Candidatus Phytoplasma aurantifolia,U15442)的相关株系,属于花生丛枝植原体组A亚组(16SrII-A),这是首次发现植原体感染卵叶山蚂蝗的报道。     

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。     

菊花绿变病植原体在中国的发生及分子鉴定

对内蒙古农业大学校园内表现花器绿变症状的菊花样品进行采集和DNA提取,应用植原体16S rRNA基因和rp基因的引物进行巢式PCR扩增,从感病样品中分别扩增得到了长度均约为1.2 kb的片段。序列一致性分析表明,菊花绿变植原体16S rRNA基因与翠菊黄化植原体匈牙利风信子株系(GenBank登录号MN080271)、印度玉米株系(KY565571)、印度繁缕株系(KC623537)和印度马铃薯株系(KC312703)的核酸一致性最高,为99.9%,rp基因序列与翠菊黄化植原体立陶宛洋葱株系(GU228514)的核酸一致性最高,为99.8%。基于16S rRNA基因和rp基因构建系统进化树时发现,菊花绿变植原体均与16SrI-B亚组成员聚为一起。16S rRNA基因相似性系数分析表明,菊花绿变植原体与洋葱黄化植原体(AP006628)的相似性系数最高为1.00,洋葱黄化植原体(AP006628)在分类上属于16SrI-B亚组。因此,我们可以确定该菊花绿变植原体属于16SrI-B亚组。这是我国首次报道菊花绿变病的发生。     

福建永安市辣椒丛枝病植原体的分子检测及鉴定

永安地区发病辣椒植株表现小叶、黄化、丛枝、簇芽等症状。利用植原体16SrDNA基因的通用引物R16mF2/R16mR2和R16F2n/R16R2,对发病辣椒植株总DNA进行巢式PCR检测,获得约1.2kb的特异性DNA片段。经测序并在GenBank数据库进行比对分析,共获得4条植原体特定的16SrDNA基因序列(CHY-C4-1、CHYY1-1、CHY-Y7-1、CHY-G1-1)。将测得的4条序列与已报道的植原体序列进行同源性比对,并构建系统进化树,结果显示获得的4条植原体序列均聚类到16SrI组,其中CHY-Y1-1、CHY-Y7-1、CHY-G1-1与16SrI-B亚组植原体聚类到同一支,而CHY-C4-1与已报道的16SrI组内的6个亚组均未聚类到一支,因此建议将CHY-C4-1命名为新的亚组。利用iPhyClassifier在线分析软件对获得的4条植原体序列进行虚拟RFLP分析,结果与进化树获得的结果一致。     

桑树黄化型萎缩病植原体延伸因子基因的克隆及序列分析

利用已报道的引物对fTuf Ay/rTuf Ay,采用PCR技术对桑树黄化型萎缩病植原体延伸因子(EF-Tu)tuf基因片段进行了扩增、测序及序列分析。结果表明,从表现黄化型萎缩病症状的桑树样品中扩增得到预期大小的目的片段。经核苷酸序列测定,扩增得到的延伸因子基因片段为946 bp(GenBank登录号为:GQ268317)。对桑树黄化型萎缩病植原体及16Sr I组中的各亚组代表植原体的延伸因子tuf基因的相似性比较结果表明,桑树黄化型萎缩病植原体与16Sr I组中的MD、PRIVA亲缘关系最近,核苷酸的相似性为99.9%。该序列与已知的16Sr-I各亚组代表植原体构建的进化树表明,桑黄化型萎缩病植原体与tufI-B亚组聚类为一个亚组,归为翠菊黄化植原体组(Candidatus Phytoplasma asteris),即16Sr I组tufI-B亚组,该结果从亚组水平上进一步确定了桑树黄化型萎缩病植原体的分类地位。     

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)。     

山东省枣疯病植原体的鉴定及分子变异分析

 本研究对山东省11个地区的枣疯病样品进行了鉴定和分子变异分析。以样品总DNA为模板,经扩增和序列测定,分别得到16S rRNA (1 432 bp)、核糖体蛋白基因rp (1 196 bp)、转运蛋白基因secA (836 bp) 和secY (1 421 bp) 的序列,secA基因序列是首次从枣疯病植原体中扩增获得。对获得的序列与NCBI数据库中相关植原体序列进行聚类和核苷酸变异分析,结果显示山东省枣疯病植原体属于16SrⅤ-B、rpⅤ-C、secYⅤ-C亚组,相对于16S rRNA基因,rp,secA和secY变异更大,非同义突变更多,更利于对国内不同来源的枣疯病植原体的精细系统进化分析。     

三叶草绿变病植原体的分子鉴定

三叶草(Trifolium pratense Linn.)为车轴草属,蝶形花科多年生草本植物,原产亚洲南部和欧洲东南部,是一种世界性分布与栽培的优良牧草.因其花叶兼优、草姿美、绿期长而具有较高的观赏价值,近几年作为草坪用草被广泛种植.在自然条件下,三叶草很容易受到不同种植原体的侵染,国外已报道的侵染三叶草的植原体有:三叶草绿变植原体( Clover phyllody phytoplasma,CPh)和三叶草增殖植原体(Clover proliferation phytoplasma,CP)等,这些植原体分别属于16SrI组和16SrⅥ组[1,2].     

广东枣疯病植原体的鉴定

Several jujube plants with witches′ broom, little leaf, and big bud symptoms, which were likely infected by jujube witches′ broom (JWB) phytoplasma, were collected in Guangzhou, Guangdong Province. To identify the pathogen, PCR was performed using phytoplasma 16S rDNA universal primer pairs R16mF2/R1 and P1/P7 and SecA gene primer pair SecAfor1/rev3 with total DNA of the symptomatic plants as templates. Specific fragments, 1.4 kb, 1.8 kb, and 0.8 kb in length, were amplified from one of three symptomatic samples. Phylogenetic analysis based on 16S rDNA verified that the pathogen harming jujube plants in Guangzhou was jujube witches′ broom phytoplasma which belonged to 16SrV-B subgroup. Comparison results also showed that the 16S rDNA sequence of Guangzhou JWB phytoplasma shared the highest nucleotide identity (100%) with the reported jujube witches′ broom phytoplasma Japanese strain (AB442218) and JWB strain (AY197661) and shared the nucleotide identity ranging from 99.74% to 99.80% with the other JWB phytoplasma strains. In addition, phylogenetic analysis based on SecA also showed that Guangzhou jujube witches′ broom phytoplasma belonged to 16SrV-B subgroup and shared 99.28%-99.76% similarity with other phytoplasma strains. All these results suggested that jujube witches′ broom phytoplasma has infected jujube plants in Guangdong Province.     

Use of heteroduplex mobility assay for identification and differentiation of phytoplasmas in the aster yellows group and the clover proliferation group

ABSTRACT This paper describes the identification and differentiation of phytoplasmas by a highly sensitive diagnostic technique, DNA heteroduplex mobility assay (HMA). Closely related phytoplasma isolates of clover proliferation (CP), potato witches'-broom (PWB), and alfalfa witches'-broom (AWB) were collected from the field from 1990 to 1999. The entire 16S rRNA gene and 16/23S spacer region were amplified by polymerase chain reaction (PCR) from the field samples and standard CP, PWB, and AWB phytoplasmas and were subjected to restriction fragment length polymorphism (RFLP) analysis and HMA. Two subgroups (I and II) of phytoplasmas in the CP group were identified by HMA but not by RFLP analysis. The results were confirmed by 16/23S spacer region sequence data analysis. After HMA analyses of the PCR-amplified 16/23S spacer region, 14 phytoplasma isolates from field samples were classified into two aster yellows subgroups: subgroup I, phytoplasma isolates from China aster (Callistephus chinensis) yellows, French marigold (Tagetes patula) yellows, cosmos (Cosmos bipinnatus cv. Dazzler) yellows, clarkia (Clarkia unguiculata) yellows, California poppy (Eschscholzia californica cv. Tai Silk) yellows, monarda (Monarda fistulosa) yellows, and strawflower (Helichrysum bracteatum) yellows; and subgroup II, phytoplasma isolates from zinnia (Zinnia elegans cv. Dahlia Flower) yellows, Queen-Annes-Lace (Daucus carota) yellows, scabiosa (Scabiosa atropurpurea cv. Giant Imperial) yellows, Swan River daisy (Brachycombe multifida cv. Misty Pink) yellows, pot marigold (Calendula officinalis) yellows, purple coneflower (Echinacea purpurea) yellows, and feverfew (Chrysanthemum parthenium) yellows. The results indicate that HMA is a simple, rapid, highly sensitive and accurate method not only for identifying and classifying phytoplasmas but also for studying the molecular epidemiology of 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.     

Characterization of phytoplasmas detected in Chinaberry trees with symptoms of leaf yellowing and decline in Bolivia

Polymerase chain reaction (PCR) assays were used to detect phytoplasmas in foliage samples from Chinaberry ( Melia azedarach ) trees displaying symptoms of yellowing, little leaf and dieback in Bolivia. A ribosomal coding nuclear DNA (rDNA) product (1·8 kb) was amplified from one or more samples from seven of 17 affected trees by PCR employing phytoplasma-universal rRNA primer pair P1/P7. When P1/P7 products were reamplified using nested rRNA primer pair R16F2n/R16R2, phytoplasmas were detected in at least one sample from 13 of 17 trees with symptoms. Restriction fragment length polymorphism (RFLP) analysis of P1/P7 products indicated that trees CbY1 and CbY17 harboured Mexican periwinkle virescence (16SrXIII)-group and X-disease (16SrIII)-group phytoplasmas, respectively. Identification of two different phytoplasma types was supported by reamplification of P1/P7 products by nested PCR employing X-disease-group-specific rRNA primer pair R16mF2/WXint or stolbur-group-related primer pair fSTOL/rSTOL. These assays selectively amplified rDNA products of 1656 and 579 bp from nine and five trees with symptoms, respectively, of which two trees were coinfected with both phytoplasma types. Phylogenetic analysis of 16S rDNA sequences revealed Chinaberry yellows phytoplasma strain CbY17 to be most similar to the chayote witches'-broom (ChWBIII-Ch10) agent, a previously classified 16SrIII-J subgroup phytoplasma. Strain CbY1 resembled the Mexican periwinkle virescence phytoplasma, a 16SrXIII-group member. The latter strain varied from all known phytoplasmas composing group 16SrXIII. On this basis, strain CbY1 was assigned to a new subgroup, 16SrXIII-C.     

Identification of a phytoplasma causing yellows of Monarda

Monarda yellows occurring in southern Alberta was found to be associated with a phytoplasma. Using two pairs of universal primers, 16S ribosomal DNA fragments (about 1.5 and 1.2 kb) were amplified separately by polymerase chain reaction (PCR) from DNA samples that had been extracted from infected monarda. No such DNA bands were observed using DNA samples from uninfected monarda. The DNA fragment (1.2 kb) amplified by nested-PCR was analysed and compared with western aster yellows (AY27, Canada), eastern aster yellows (EAY, USA), French hydrangea aster yellows (AYHF), Belgium hydrangea aster yellows (AYHB), clover proliferation (CP, Canada) and potato witches'-broom (PWB, Canada) by means of restriction fragment length polymorphism (RFLP) using endonucleases Alu I, Mse I, Hpa II, Sau 3AI, Kpn I and Rsa I. The results showed that monarda yellows phytoplasma belongs to the aster yellows subclade and is different from CP and PWB. This is the first report of aster yellows phytoplasma infecting monarda.     

海南长春花黄化病植原体的16S rDNA序列分析研究

 Periwinkle(Catharanthus roseus) yellows is a common disease in Hainan. Periwinkle's leaf tissue with symptoms was assayed for phytoplasma infection by using PCR assay employing phytoplasma universal 16S rRNA gene primers (Rl6mF2/Rl6mR1). A PCR product (about 1.4 kb) was amplified from periwinkle showed yellows. Nucleotide sequencing and phylogenetic tree analysis showed that the amplified 16S rDNA contained 1 432 nucleotides, the most homology was 98.1% with the members of elm yellows group (16S r Ⅴ) and clustered in the same clade, while it was under 96.1% with other phytoplasma groups. Our results suggested that the phytoplasma sample belonged to 16S rⅤgroup and was tentatively named as Hainan periwinkle yellows phytoplasma (PY-Hn). This is the first report of existence of 16S r Ⅴ group phytoplasma in naturally infected periwinkle.