泰安枣疯病植原体的分子鉴定

【目的】对3个枣疯病病原物泰安株系进行分子鉴定。【方法】采用植原体通用引物对R16F2n/R16R2,通过直接PCR技术,扩增枣疯病植原体16S rDNA基因,通过16S rDNA基因序列分析和在线模拟16S rDNA-RFLP分析,并将其16S rDNA基因序列提交到GenBank数据库。【结果】3个枣疯病病原物16S rDNA基因片段与16SrⅤ-B亚组中枣疯病植原体(AB052876和AF279272)、樱桃致死黄化植原体(AY197659)及杏卷叶植原体(FJ572660)的同源性高达99.5%99.7%,分别命名为枣疯病植原体泰安圆铃1号株系(Jujube witches’-broom phytoplasma strain Yuanling1,JWB-Yuanling1,TA)、枣疯病植原体泰安鲁北冬枣株系(Jujube witches’-broom phytoplasma strain Lubeidongzao,JWB-Lubeidongzao,TA)和枣疯病植原体泰安大白铃株系(Jujube witches’-broom phyto-plasma strain Dabailing,JWB-Dabailing,TA),基因登录号分别为:HM989946、HM989947和HM989948。【结论】3个枣疯病植原体泰安株系均归属于16SrⅤ-B亚组。     

枣疯病植原体tuf和rp基因的克隆与序列分析

【目的】枣疯病是一种重要的植原体病害,本研究旨在明确北京及河北地区枣疯病植原体的分类地位,为枣疯病在亚组水平上分类提供一定的参考依据。【方法】利用植原体通用引物fTufu/rTufu和rp(v)F1A/rp(v)R1A对北京和河北地区枣疯病植原体延伸因子tuf基因和核糖体蛋白基因(rp)进行PCR扩增并进行核苷酸序列测定及相似性分析。【结果】获得北京地区JWB-XFSZ株系、JWB-XFDO株系以及河北地区JWB-TXSZ株系的tuf基因片段均为824 bp;北京地区JWB-XFSZ株系的rp基因片段为1196 bp。经序列相似性比较表明:tuf基因与16SrV组的葡萄黄叶病(Flavescence dorée)相似性最高,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基因相似性较低,为57.29%;关于rp基因,北京地区枣疯病JWB-XFSZ株系与16SrV组的枣疯病泰山株系(JWB-Taishan)以及大麻丛枝病植原体(HFWB)相似性最高,均为99.83%,与16SrV组的成员相似性均在96%以上。【结论】北京与河北地区枣疯病植原体具有较高的相似性,而在tuf基因水平上,与陕西地区枣疯病植原体具有较大的差异;本研究中北京与河北两地区枣疯病植原体归属于16SrV组。     

杏褪绿卷叶病研究现状及展望

杏褪绿卷叶病(ACLR)是由ESFY植原体引发的一类昆虫媒传病害,在欧洲许多国家已将其列为检疫对象。近年来在我国新疆部分杏园首次发现类似欧洲ACLR的发病植株。简要介绍杏褪绿卷叶病的田间发病症状,疏理了国内外关于引起杏褪绿卷叶病的ESFY植原体的流行病学和病原检测以及防治方法的研究进展,特别对分子检测技术进行全面综述。另外,分析当前研究中尚存在的问题,并对今后的研究方向进行展望。     

新疆小叶白蜡丛枝病植原体的鉴定及16S rRNA基因序列分析

【目的】对新疆小叶白蜡丛枝病植原体进行检测,通过其16S rRNA基因分析确定其分类地位。【方法】利用苯胺蓝和4′,6-二脒基-2-苯基吲哚(DAPI)染色,在荧光显微镜下观察新疆小叶白蜡嫩茎横切片;采用植原体16S rRNA基因的通用引物对P1/P7和R16F2n/R16R2进行直接和巢式PCR扩增,对得到的16S rRNA基因的序列进行RFLP和构建系统进化树分析。【结果】表现丛枝病症状的新疆小叶白蜡中存在植原体,暂命名为Fraxinus sogdianaBunge witches’broom phytoplasma(Fraxinus sogdianaBunge WB);其16S rRNA基因的序列GenBank登录号为KF061042,RFLP图谱与16Sr V-B亚组的枣疯病植原体相同,系统进化地位与枣疯病菌株AB052876相同。【结论】新疆小叶白蜡丛枝病植原体为16Sr V-B亚组成员。     

柳树黄化病植原体的分子分类

[目的]柳树黄化病是一种重要的植原体病害,本研究旨在明确柳树黄化病植原体(Willow Yellow phytoplasma,WY)的分类地位,为进一步开展致病性和防治研究奠定基础.[方法]采用植原体特异引物通过PCR方法从患病植株DNA中扩增植原体16S rDNA基因和核糖体蛋白基因(ribosomal proteins gene,rp),对所得的序列进行分析,构建同源进化树,并用限制性片段长度多态性(RFTJP)对巢式PCR产物进行分析.[结果]首次从柳树黄化病植原体中分离出了16S rDNA基因和rp基因,大小分别为1246 bp和1212 bp.通过对植原体16S rDNA和rp基因的核苷酸同源性比较和RFLP分析,发现该分离物与16S rI组的核苷酸同源性均在99%以上,与16S rI-C亚组中的小麦蓝矮病植原体同源性高达99.8%(16Sr DNA)和99.6%(rp),且RFLP分析与16SrI-C亚组的植原体有相同的酶切条带.[结论]柳树黄化植原体应划分于16SrI-C亚组.     

坡柳(Dodonaea viscosa L.)丛枝植原体新病原的分子鉴定

从四川攀枝花芒果园中表现为丛枝、小叶和黄花等症状的坡柳植株发病样品中,利用植原体16S rDNA基因的通用引物R16m F2/R16m R1和R16F2n/R16R2,对发病植株总DNA进行巢式PCR检测,同时设计植原体抗原膜蛋白基因(AntMP)的保守引物AntMP-F/AntMP-R进行PCR验证。结果显示,坡柳样品巢式PCR的第一轮、第二轮DNA条带大小分别为1 400 bp和1 200 bp左右,经NCBI序列相似性比较均为植原体16S rDNA序列,Gen Bank登录号为KT957205和KT957206;PCR结果显示抗原膜蛋白基因大小约600 bp,与目标基因大小一致。将测得的16S rDNA基因序列与Gen Bank数据库中登录的16SrⅠ~ⅩⅤ组植原体16S rDNA序列进行同源性比对,构建系统进化树,结果显示四川坡柳丛枝植原体(DOVI-SC)属于16SrⅠ组(即翠菊黄花组),与已报道的5个16SrⅠ组(AY101386,AY566302,AY389822,L33760和KP662119)同属一个组。利用植原体亚组分类鉴定软件iPhy Classfier对获得的2条植原体16S rDNA序列进行虚拟RFLP分析,结果显示KT957205,KT957206与16SrⅠ-B亚组洋葱黄化植原体(NC-005303)相似度分为1.0、0.97,归属于16SrⅠ-B亚组。本研究对引起四川坡柳丛枝的植原体病原进行检测,为坡柳植原体病害的早期诊断、快速检测以及预防措施制定提供科学线索。     

仙人掌丛枝病植原体CWB1株系16SrRNA基因克隆及序列分析

对表现丛枝症状的仙人掌植株总DNA进行植原体 1 6SrRNA基因PCR扩增 ,得到一条约 1 5kb的特异片段 ,表明植株中有植原体存在 ,将此植原体株系命名为CWB1。把此特异片段与pGEM Teasy载体连接并转化到大肠杆菌JM1 0 9感受态细胞中 ,通过PCR鉴定、限制性内切酶 (EcoRI)酶切分析及核苷酸序列分析 ,均表明克隆成功。序列分析结果显示 ,此株系的 1 6SrRNA基因全长 1 489个碱基 ,与属于植原体 1 6SrⅡ C亚组的Fababeanphyllody植原体同源率最高 ,为 99 7%。通过 1 6SrRNA基因核酸序列同源性比较 ,认为该株系属于 1 6SrⅡ C亚组 ,基本确定了其分类地位。     

泡桐丛枝植原体tRNA异戊烯基焦磷酸转移酶基因克隆、原核表达及功能分析

【目的】为了鉴定植原体tRNA异戊烯基焦磷酸转移酶基因(tRNA-ipt)的表达及蛋白功能,探索植原体致病机理。【方法】对泡桐丛枝、桑萎缩、长春花绿变及苦楝丛枝植原体tRNA-ipt基因完整序列进行PCR扩增和生物信息学分析。对泡桐丛枝植原体tRNA-ipt基因进行原核表达并制备抗体。利用Western blot和FITC间接免疫荧光显微镜检测其在植原体中的表达。使用分光光度计分析该基因对大肠杆菌生长的影响,用ELISA测定转化菌株细胞分裂素含量。【结果】首次发现泡桐丛枝、桑萎缩、长春花绿变及苦楝丛枝植原体中完整tRNA-ipt基因,大小为876 bp,编码291个氨基酸,且N端均含有ATP/GTP结合位点保守序列(GPTASGKT)。4种植原体tRNA-IPT之间的氨基酸序列相似率为99.1%-99.5%,与同组植原体同源性在95.4%-99.3%,与其他组植原体同源性低于70%。SDS-PAGE结果显示tRNA-IPT蛋白在大肠杆菌中得到表达。首次获得泡桐丛枝植原体tRNA-IPT抗体并检测到该蛋白在泡桐发病组织中的特异表达。经过对转化菌株生长曲线及玉米素含量的测定,发现该基因能促进大肠杆菌后期生长和玉米素核苷的积累。【结论】4种植原体tRNA-ipt基因编码相同特性的功能蛋白,泡桐丛枝植原体tRNA-IPT蛋白能够在植原体中表达,根据该基因对异源菌株生长速率和激素合成的影响推断该蛋白可能参与植原体的细胞分裂素合成,在致病过程中起到重要作用。     

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

摘要：【目的】检测不同地区枣树品种上的枣疯植原体侵染及保守基因序列的变异。【方法】利用植原体16S rDNA的通用引物R16mF2／R16mR1、16S-23S间区序列(SR)的通用引物SR1/SR及secY基因引物FD9f/r,通过PCR检测采自国内7个地区14个枣树品种上的32个枣疯病和4个酸枣丛枝病样品。将PCR产物进行直接或克隆测序,结合已报导的测序数据,进行序列同源性和系统进化分析。【结果】所有枣疯病样品中均检测到植原体;皆属于榆树黄化16S rV-B亚组,与我国重阳木丛枝和樱桃致死黄化遗传关系     

茉莉C病毒侵染性克隆的构建及CP基序分析

【目的】构建茉莉C病毒(JaVC)福建分离物基因组全长cDNA侵染性克隆,克隆9省JaVC分离物的CP基因并比较分析基序差异,调查JaVC在我国茉莉产区的分布和传播情况。【方法】提取JaVC检测呈阳性的茉莉叶片总RNA,以反转录后的cDNA为模板扩增获得JaVC基因组全长序列并构建全长cDNA克隆pXT-JaVC-FJ;同时构建了外壳蛋白(coatprotein,CP)融合红色荧光蛋白mCherry的克隆(pXT-JaVC CP-mCherry)。利用农杆菌浸润法侵染本生烟,通过RT-PCR检测法和激光共聚焦扫描显微镜观察法验证JaVC侵染性。克隆其他8省JaVC分离物的3′末端包含CP的片段并测序分析CP基序差异。通过田间调查明确JaVC在茉莉上的发生情况和其传播介体。【结果】pXT-JaVC-FJ浸润本生烟可引起系统侵染,说明该克隆具有侵染活性。所有JaVC分离物的CP均编码296个氨基酸,JaVC中国台湾分离物的CP与各分离物核苷酸序列相似性为82.27%-91.36%,与广东分离物相似性最高;氨基酸序列相似性为92.23%-96.82%,与云南分离物相似性最高;各分离物CP的氨...     

Detection and Identification of an Elm Yellows Group Phytoplasma Associated with Camellia in China

In 2012, yellowing of camellias was observed in Tai'an in Shandong province, China. Transmission electron microscopy (TEM) revealed phytoplasma in the phloem sieve tube elements of symptomatic plants. A specific fragment of phytoplasma 16S rRNA gene was amplified by polymerase chain reaction (PCR) using the universal phytoplasma primers P1/P7 followed by R16F2n/R16R2. Sequence and restriction fragment length polymorphism (RFLP) analyses allowed us to classify the detected phytoplasma into the elm yellows (EY) group (16SrV), subgroup 16SrV‐B. Sequence analyses of the ribosomal protein (rp) gene confirmed a close relationship with phytoplasmas belonging to the rpV‐C subgroup. Thus, the phytoplasma associated with yellows disease in camellia, designated as ‘CY’, is a member of the 16SrV‐B subgroup. This is the first report of phytoplasma associated with camellia.     

Occurrence of Phytoplasmas Related to Stolbur and to ‘Candidatus Phytoplasma japonicum’ in Woody Host Plants in China

During a survey in a limited area of the Shanxi province in China, phytoplasma symptoms were observed on woody plants such as Chinese scholar tree, apple, grapevine and apricot. The polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analyses on the phytoplasma 16S ribosomal gene confirmed that symptomatic samples from all these species were infected by phytoplasmas. The molecular characterization of the pathogen, performed also with sequencing of polymerase chain reaction amplified 16S rDNA, showed that the phytoplasmas detected in all plant species tested are closely related with stolbur, but two samples from a Chinese scholar tree were infected with phytoplasmas related to ‘Candidatus Phytoplasma japonicum’. The presence of RFLP polymorphism was found in the 16S rDNA amplicons with three of the six enzymes employed in the majority of phytoplasma strains studied.     

Sequence heterogeneity in the two 16S rRNA genes of Phormium yellow leaf phytoplasma.

Phormium yellow leaf (PYL) phytoplasma causes a lethal disease of the monocotyledon, New Zealand flax (Phormium tenax). The 16S rRNA genes of PYL phytoplasma were amplified from infected flax by PCR and cloned, and the nucleotide sequences were determined. DNA sequencing and Southern hybridization analysis of genomic DNA indicated the presence of two copies of the 16S rRNA gene. The two 16S rRNA genes exhibited sequence heterogeneity in 4 nucleotide positions and could be distinguished by the restriction enzymes BpmI and BsrI. This is the first record in which sequence heterogeneity in the 16S rRNA genes of a phytoplasma has been determined by sequence analysis. A phylogenetic tree based on 16S rRNA gene sequences showed that PYL phytoplasma is most closely related to the stolbur and German grapevine yellows phytoplasmas, which form the stolbur subgroup of the aster yellows group. This phylogenetic position of PYL phytoplasma was supported by 16S/23S spacer region sequence data.     

Characterization of a Phytoplasma Associated with Pear Decline in Iran

Symptoms of pear decline (PD) were observed in several pear growing regions of Iran. Pear trees with typical symptoms of PD from Estahban (Fars Province) were examined for phytoplasma infection using polymerase chain reaction (PCR) assay. Graft inoculation of healthy pear trees with scions from diseased trees resulted in production of PD symptoms and transmission of phytoplasma as verified by PCR. Target DNA was amplified from symptomatic pear trees with fO1/rO1, an apple proliferation (AP) group-specific primer pair. Physical and putative restriction fragment length polymorphism (RFLP) analyses of fO1/rO1 primed PCR products showed profiles corresponding to AP group, 16SrX-C subgroup ( Candidatus Phytoplasma pyri). Percent similarity values and phylogenetic analysis of fO1/rO1 primed sequences confirmed that, as a member of AP subclade, Estahban PD phytoplasma has a closer relationship to PD and peach yellow leaf roll phytoplasmas than to AP ( Ca . Phytoplasma mali) and European stone fruit yellows ( Ca . Phytoplasma prunorum) phytoplasmas. This is the first report of PD phytoplasma in the eastern Mediterranean.     

Grapevine yellows in Northern Italy: molecular identification of Flavescence dorée phytoplasma strains and of Bois Noir phytoplasmas

AIMS: Verify the presence and the molecular identity of phytoplasmas in Northern and Central Italy vineyards where yellows diseases are widespread. METHODS AND RESULTS: Phytoplasma presence and identity were determined by PCR/RFLP analyses on 16S ribosomal gene testing 1424 symptomatic samples. The 65% of samples resulted phytoplasma infected; in particular 256 samples were found positive to phytoplasmas belonging to group 16SrV (mainly Flavescence dorée associated), and the remaining 37% was infected by phytoplasmas belonging to ribosomal subgroup 16SrXII-A (Stolbur or Bois Noir associated). 16SrV ribosomal group representative strains were further typed for variability in SecY and rpS3 genes. The results showed the presence of phytoplasmas belonging to 16SrV-C, 16SrV-D and to a lesser extent, 16SrV-A subgroup. CONCLUSIONS: Possible relationships between genetic polymorphisms of phytoplasma strains belonging to subgroup 16SrV-C and their geographic distribution and/or epidemic situations were detected. SIGNIFICANCE AND IMPACT OF THE STUDY: Bois Noir and Flavescence dorée phytoplasmas are present in significant percentages in the areas under investigation. Molecular tools allowed to identify phytoplasma-infected plants and the genes employed as polymorphism markers resulted useful in distinguishing and monitoring the spreading of the diseases associated with diverse phytoplasmas belonging to 16SrV subgroup in vineyards.     

Artemisia vulgaris,a new host of 16SrV-C phytoplasma related strains infecting black alder in Poland

Yellowing of leaf tissue and strongly deformed shoots were observed in common mugwort (Artemisia vulgaris L.) growing in a nature reserve in Southern Poland. Similar foliage chlorosis together with abnormal shoot proliferation was noticed on alder tree (Alnus glutinosa Gaertn.) growing next to the common mugwort. DNA specific fragments coding 16S rRNA and ribosomal proteins (rp) were amplified from mugwort and alder samples using direct and nested PCR (Polymerase Chain Reaction) assays. Phylogenetic relationships inferred from 16S and rps3 genes indicated that strains infecting mugwort and alder were most closely related to phytoplasmas of subgroups 16SrV-C and 16SrV-D. Based on the restriction fragment length polymorphism (RFLP) analysis of the 16S rDNA, the investigated phytoplasma strains were classified to subgroup 16SrV-C. Two sequence variants of the rps3 gene which differed by a single nucleotide were detected in all analysed samples by pairwise analysis of the aligned reads. Taking into account that this single-nucleotide polymorphism (SNP) occurs among 16SrV-C and 16SrV-D related phytoplasmas and that the phytoplasmas have a single copy of rp operon, we concluded that each plant species was infected by two distinct, closely related phytoplasma strains. To the best of our knowledge, this is the first report of group 16SrV-C related phytoplasmas infecting common mugwort worldwide, adding a new host species that is possibly linked to the spread of the alder pathogen in Eastern Europe. Although alder yellows phytoplasma has been frequently found in Europe, this is the first detection of phytoplasmas associated with alder in Poland.     

The phytoplasma associated with purple woodnettle witches'‐broom disease in Taiwan represents a new subgroup of the aster yellows phytoplasma group

In October 2013, a new disease affecting purple woodnettle, Oreocnide pedunculata, plants was found in Miaoli County, Taiwan. Diseased plants exhibited leaf yellowing and witches'‐broom symptoms. Molecular diagnostic tools and electron microscopic cell observation were used to investigate the possible cause of the disease with a specific focus on phytoplasmas. The result of polymerase chain reaction with universal primer pairs indicated that phytoplasmas were strongly associated with the symptomatic purple woodnettles. The virtual restriction fragment length polymorphism (RFLP) patterns and phylogenetic analysis based on 16S rDNA and ribosomal protein, rplV‐rpsC region revealed that purple woodnettle witches'‐broom phytoplasma (PWWB) belongs to a new subgroup of 16SrI and rpI group and was designated as 16SrI‐AH and rpI‐Q, respectively, herein. RFLP analysis based on tuf gene region revealed that the PWWB belongs to tufI‐B, but phylogenetic analysis suggested that PWWB should be delineated to a new subgroup under the tufI group. Taken together, our analyses based on 16S rRNA and rplV‐rpsC region gave a finer differentiation while classifying the subgroup of aster yellows group phytoplasmas. To our knowledge, this is the first report of a Candidatus Phytoplasma asteris‐related strain in 16SrI‐AH, rpI‐Q and tufI‐B subgroup affecting purple woodnettle, and of an official documentation of purple woodnettle as being a new host of phytoplasmas.     

Detection and Characterization of Phytoplasmas Infecting Ornamental and Weed Plants in Iran

During field surveys in 2004, ornamental and weed plants showing symptoms resembling those caused by phytoplasmas were observed in Mahallat (central Iran). These plants were examined for phytoplasma infections by polymerase chain reaction (PCR) assays using universal phytoplasma primers directed to ribosomal DNA (rDNA). All affected plants gave positive results. The detected phytoplasmas were characterized and differentiated through restriction fragment length polymorphism (RFLP) and sequence analysis of PCR‐amplified rDNA. The phytoplasmas detected in diseased Asclepias curassavica and Celosia argentea were identified as members of clover proliferation phytoplasma group (16SrVI group) whereas those from the remaining plants examined proved to be members of aster yellow phytoplasma group (16SrI group) (‘Candidatus Phytoplasma asteris’). In particular, following digestion with AluI, HaeIII and HhaI endonucleases, the phytoplasma detected in Limonium sinuatum showed restriction profiles identical to subgroup 16SrI‐C; phytoplasmas from Gomphocarpus physocarpus, Tanatacetum partenium, Lactuca serriola, Tagetes patula and Coreopsis lanceolata had the same restriction profiles as subgroup 16SrI‐B whereas Catharanthus roseus‐ and Rudbeckia hirta‐infecting phytoplasmas showed restriction patterns of subgroup 16SrI‐A. This is the first report on the occurrence of phytoplasma diseases of ornamental plants in Iran.     

Identification of Phytoplasmas Associated with Grapevine Yellows in Serbia

The molecular identification and characterization of phytoplasmas from infected grapevines in four locations in Serbia are reported. Phytoplasmas were detected and identified by restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction (PCR) amplified 16S rDNA. Grapevine yellows were associated with three molecularly distinguishable phytoplasmas: Flavescence dorée phytoplasmas (elm yellows group: 16SrV‐C subgroup) were present only in the Župa Aleksandrovac region; Bois noir phytoplasmas (stolbur group: 16SrXII‐A subgroup) were detected in the other surveyed regions; a mixed infection of European stone fruit yellows (apple proliferation group: 16SrX‐B subgroup) and Bois noir phytoplasmas was identified in one sample. A finer molecular characterization by RFLP analysis of rpS3 and SecY genes of Flavescence dorée phytoplasmas from Župa Aleksandrovac confirmed that the Serbian genotype is indistinguishable from a strain from the Veneto region, Italy. Characterization of the tuf gene of Bois noir phytoplasmas showed lack of amplification of samples from Erdevik. HpaII profiles of tuf gene PCR products of samples from Pali and Radmilovac were identical, and were indistinguishable from one of the two profiles produced by samples from Italian grapevines used as reference strains.     

Detection and Characterization of Phytoplasma and Sugarcane Yellow Leaf Virus Associated with Leaf Yellowing of Sugarcane

Leaves from sugarcane were collected from Egyptian plantation fields and tested for phytoplasma (Sugarcane yellows phytoplasma, SCYP) and Sugarcane yellow leaf virus (SCYLV) using nested PCR (with different primers) and RT‐PCR, respectively. These results showed significant differences in the amplification of the PCR assays. The primer MLO‐X/MLO‐Y, which amplified the 16S‐23S rDNA spacer region, was the most precise to detect the phytoplasma in sugarcane plants. Sequencing and restriction fragment length polymorphism analysis revealed that all tested phytoplasmas belonged to the 16SrI (aster yellows phytoplasma) group, with the exception of cultivar G84‐47 belonged to the 16SrXI (Rice yellow dwarf phytoplasma) group. Three Egyptian sugarcane cultivars were phytoplasma free. Phylogenetic analyses of 34 screened accessions of 16S ribosomal DNA gene sequences of Candidatus phytoplasma including the ones collected from Egypt used in this study and those extracted from GenBank showed that they split into two distinct clusters. The phylogenetic analyses indicated that these phytoplasmas are closely related and share a common ancestor. All tested Egyptian sugarcane plants were infected by SCYLV with the exception of cultivar Phil‐8013 which was virus free.