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.     

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。     

Phylogenetic relationships among Flavescence dorée strains and related phytoplasmas determined by heteroduplex mobility assay and sequence of ribosomal and Nonribosomal DNA

Heteroduplex mobility assay (HMA) and DNA sequencing were performed on Flavescence dorée (FD) phytoplasma strains and related phytoplasmas belonging to the elm yellows group. Part of the ribosomal RNA gene operon and a nonribosomal DNA region were utilized for phylogenetic analyses. Two FD strains, FD92 and FD-D, detected in France and Italy, respectively, were identical in both DNA fragments, confirming previous results. Other FD strains were all very similar and most closely resembled ALY, an Italian alder phytoplasma. Phytoplasmas associated with German Palatinate grapevine yellows were shown to form a distinct subcluster, also different from the elm yellows phytoplasma subcluster. Strain disparities revealed by HMA and sequence data were mostly in agreement, highlighting the utility of HMA in differentiation and classification of phytoplasmas belonging to the same ribosomal RNA group.     

嵌套引物P1/P7-U3/U5检测葡萄黄化(stolbur)植原体时扩增出的额外片段分析

 当采用嵌套引物P1/P7(U5/P7) U3/U5的巢式PCR检测植原体时,伴随着正常的0.85kb片段还经常扩增出一条额外片段。与其它常见的非特异性片段不同,该额外片段十分稳定,与正常片段总是同步出现,二者的强度呈正比。经测定,额外片段的大小为0.36kb。迄今,这一现象仅在采用P1/P7(U5/P7)-U3/U5引物的巢式PCR中出现。在采用P1/P7、U5/P7或-U3/U5引物的常规PCR中从未出现过。另外,已知这一现象至少在葡萄黄化stolbur和榆黄化植原体的检测过程中出现。对该现象产生的原因进行了深入研究,方法是将额外片段从凝胶中分离出来,用不同的引物在不同的条件下进行重新扩增,同时结合已知的植原体16SrRNA基因序列进行综合分析判断。结果表明,该额外片段源于植原体16SrRNA基因上存在的一个与U5引物部分互补的位点。对额外片段的测序结果进一步证实了分析的正确性。据此,指出了该额外片段在植原体检测中的可能用途。     

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.     

Chromosome sizes of phytoplasmas composing major phylogenetic groups and subgroups

ABSTRACT Chromosome sizes of 71 phytoplasmas belonging to 12 major phylogenetic groups including several of the aster yellows subgroups were estimated from electrophoretic mobilities of full-length chromosomes in pulsed-field gels. Considerable variation in genome size, from 660 to 1,130 kilobases (kb), was observed among aster yellows phytoplasmas. Chromosome size heterogeneity was also observed in the stolbur phytoplasma group (range 860 to 1,350 kb); in this group, isolate STOLF contains the largest chromosome found in a phytoplasma to date. A wide range of chromosome sizes, from 670 to 1,075 kb, was also identified in the X-disease group. The other phytoplasmas examined, which included members of the apple proliferation, Italian alfalfa witches' broom, faba bean phyllody, pigeon pea witches' broom, sugarcane white leaf, Bermuda grass white leaf, ash yellows, clover proliferation, and elm yellows groups, all have chromosomes smaller than 1 megabase, and the size ranges within each of these groups is narrower than in the aster yellows, stolbur, and X-disease groups. The smallest chromosome, approximately 530 kb, was found in two Bermuda grass white leaf phytoplasma isolates. This not only is the smallest mollicute chromosome found to date, but also is the smallest chromosome known for any cell. More than one large DNA band was observed in several phytoplasma preparations. Possible explanations for the occurrence of more than one band may be infection of the host plant by different phytoplasmas, the presence of more than one chromosome in the same organism, or the presence of large extrachromosomal DNA elements.     

Detection of a Phytoplasma Associated with Grapevine Flavescence dorée in Clematis vitalba

About 40 different species of wild herbaceous and woody plants were collected in underbrush close to a vineyard where Flavescence dorée (FD) has been reported for several years. Polymerase chain reaction assays were carried out using DNA extracted from leaves of each species for the detection of the presence of phytoplasmas. Only samples of Clematis vitalba were found to be infected with phytoplasmas. Restriction fragment length polymorphism and sequencing data of the 16S ribosomal RNA gene and of a non-ribosomal DNA fragment FD9 revealed that the phytoplasma isolate was identical to that causing FD in the nearby vineyard. The isolate identified in the clematis is the same as the FD-C phytoplasma found in grapevine in north-east Italy.     

Generation and characterization of monoclonal antibodies to Flavescence dorée phytoplasma: Serological relationships and differences in electroblot immunoassay profiles of Flavescence dorée and elm yellows phytoplasmas

Eleven stable hybridoma cell lines secreting monoclonal antibodies specific for FD-phytoplasma, the pathogenic agent of grapevine Flavescence dorée, were produced by fusing a non-secreting myeloma cell line with spleen cells from Balb/c mice immunized with Flavescence dorée phytoplasma purified by immunoaffinity. These monoclonal antibodies were characterized for their recognition of phytoplasma proteins by western blot. Six of eleven reacted specifically in ELISA and immunoblotting with Elm-yellows phytoplasma. These antibodies did not react either in ELISA or in western blot with preparations from periwinkles infected with phytoplasmas that cause GYU (Grapevine Yellows from Udine), AP (Apple Proliferation), EAY (European Aster Yellows) and StolC (Stolbur from France). Two of these hybridoma lines were used routinely for the immunodiagnosis of Flavescence dorée phytoplasma in diseased grapevines.Abbreviations ELISA Enzyme linked immunosorbent assay - FD Flavescence dorée - MLO Mycoplasmalike organism - EY Elm yellows     

Detection and Indentification of European Stone Fruit Yellows and Other Phytoplasmas in Wild Plants in the Surroundings of Apricot Chlorotic Leaf Roll-affected Orchards in Southern France

Between 1994 and 1998 a field study was conducted to identify plant hosts of the European stone fruit yellows (ESFY) phytoplasma in two apricot growing regions in southern and southwestern France where the incidence of apricot chlorotic leaf roll was high. A total of 431 samples from 51 different plant species were tested for the presence of phytoplasmas by PCR using universal and ESFY-specific primers. ESFY phytoplasma was detected in six different wild growing Prunus species exhibiting typical ESFY symptoms as well as in symptomless dog rose bushes (Rosa canina), ash trees (Fraxinus excelsior) and a declining hackberry (Celtis australis). The possible role of these plant species in the spread of ESFY phytoplasma is discussed. PCR-RFLP analysis of ribosomal DNA amplified with the universal primers was carried out to characterize the other phytoplasmas found. Thus, elm yellows phytoplasma, alder yellows phytoplasma and rubus stunt phytoplasma were detected in declining European field elm trees (Ulmus carpinifolia Gled), in declining European alder trees (Alnus glutinosa) and in proliferating Rubus spp. respectively. The presence of rubus stunt phytoplasma in great mallow (Malva sylvestris) and dog rose was demonstrated for the first time. Furthermore, the stolbur phytoplasma was detected in proliferating field bindweed (Convolvulus arvensis) and a previously undescribed phytoplasma type was detected in red dogwood (Cornus sanguinea). According to the 16S rDNA-RFLP pattern this new phytoplasma belongs to the stolbur phytoplasmas group.     

Occurrence, Distribution and Epidemiology of Grapevine Yellows in Spain

The main viticultural production areas in Spain were surveyed in 1994, 1995 and 1996 for the occurrence and incidence of Grapevine Yellows diseases associated to phytoplasmas. Samples from 300 plants showing symptoms of phytoplasma infection were collected from grapevine fields in the Spanish regions of Aragón, Catalonia and Navarra and analysed by PCR with specific primers for a non-ribosomal DNA of stolbur/Bois Noir (BN) and of Flavescence dorée (FD) phytoplasma. Nested PCR with universal primers P1/P7 and fU5/rU3 was also used. In the survey conducted in 1994 and 1995 only BN/stolbur phytoplasma was detected. The incidence of symptomatic plants was low in five plots of Catalonia from 3% to 18% in 1994 and 1995, respectively, and high in two plots of Navarra, from 60% to 80%. In the survey conducted in 1996 the incidence of symptomatic plants in Catalonia increased (6–80%) due to the presence of FD in five plots in the Northeastern Catalonia. An epidemiological study was carried out in two BN-affected plots of two regions from 1994 to 1997, with the evaluation of potential vectors and of host plants. The stolbur phytoplasma was found in individuals from different insect species belonging to the families Cicadellidae and Delphacidae. Some wild plants naturally infected with stolbur phytoplasma around the infected grapevines were: Convolvulus arvensis, Lavandula officinalis, Polygonum convolvulus, Solanum nigrum, and Thymus officinalis. The incidence of the disease in one BN-infected grapevine plot increased from 3.4% in 1994 to 18.40% in 1997.     

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.     

Real-time PCR detection systems for Flavescence dorée and Bois noir phytoplasmas in grapevine: comparison with conventional PCR detection and application in diagnostics

A new real-time PCR detection system was developed for grapevine yellows (GY) using TaqMan minor groove binder probes and including two amplicons for group-specific detection of Flavescence dorée (FD) and Bois noir (BN) phytoplasmas, plus a universal phytoplasma amplicon. FD and BN amplicons were designed to amplify species-specific genomic DNA fragments and the universal amplicon to amplify the 16S ribosomal DNA region. Efficiency of PCR amplification, limit of detection, range of linearity and dynamic range were assessed for all three amplicons. The specificity of detection systems was tested on several other isolates of phytoplasmas and bacteria and on healthy field grapevine and insect samples. No cross-reactivity with other phytoplasma strains, plant or insect DNA was detected. The assay was compared with conventional PCR on more than 150 field grapevine, insect and field bindweed samples. Real-time PCR showed higher sensitivity as phytoplasmas were detected in several PCR-negative and in all PCR-positive samples. A data-mining analysis of results from both detection approaches also favoured real-time PCR over conventional PCR diagnostics. The developed procedure for detection of phytoplasmas in grapevine also included amplification of plant DNA co-extracted with phytoplasmic DNA, providing additional quality control for the DNA extraction and PCR amplification for each sample. The newly developed assay is a reliable, specific and sensitive method easily applicable to high-throughput diagnosis of GY.     

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.     

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。     

Detection and identification of phytoplasmas in yellows-diseased weeds in Italy

Yellows-diseased plants of Crepis setosa (hawksbeard), Knautia arvensis (field scabious), Convolvulus arvensis (field bindweed), Picris echioides (bristly oxtongue), Echium vulgare (blueweed) and Calendula officinalis (pot marigold) collected in central and southern Italy were examined for phytoplasma infection by means of polymerase chain reaction (PCR) technology using universal phytoplasma primers directed to ribosomal sequences. The detected phytoplasmas were characterized and differentiated using restriction fragment length polymorphism analysis of PCR-amplified DNA. The phytoplasma detected in diseased pot marigold plants was identified as a member of the aster yellows group and proved indistinguishable from a strain of the American aster yellows phytoplasma. The phytoplasma identified in diseased field bindweed plants is a putative new type of the stolbur group that differed from the typical stolbur phytoplasma. Phytoplasmas detected in diseased hawksbeard, blueweed and field scabious plants are all putative new members of the sugarcane white leaf group while the phytoplasma detected in diseased bristly oxtongue plants represents a new member of the faba bean phyllody group. For hawksbeard and field scabious this is the first report on the occurrence of phytoplasma diseases, whereas phytoplasmas infecting bristly oxtongue and blueweed have never been characterized before.     

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.     

Comparison of phytoplasmas infecting winter oilseed rape in the Czech Republic with Italian Brassica phytoplasmas and their relationship to the aster yellows group

Winter oilseed rape grown in several areas in South Bohemia showed symptoms of stunting, leaf reddening and extensive malformation of floral parts. Phytoplasmas were consistently observed by using electron microscopy only in phloem tissue of symptomatic plants. DNA isolated from infected and healthy control plants was used in PCR experiments. Primer pairs R16F2/R2, P1/P7 and rpF2/R2, amplifying, respectively, 16S rDNA, 16S rDNA plus spacer region and the beginning of the 23S and ribosomal protein gene L22 specific for phytoplasmas, were used. According to RFLP and sequence analyses of PCR products, the phytoplasma from rape was classified in the aster yellows phytoplasma group, subgroup 16SrI-B. The PCR products from the Czech phytoplasma-infected rape also had RFLP profiles identical to those of phytoplasma strains from Italian Brassica . This first molecular characterization of phytoplasmas infecting rape compared with strains from Brassica does not, however, clearly indicate differences among isolates of the same 16SrI-B subgroup. Further studies on other chromosomal DNA portions could help the research on host specificity or on geographical distribution of these phytoplasmas.     

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。     

Characterization and classification of phytoplasmas from wild and cultivated plants by RFLP and sequence analysis of ribosomal DNA

Restriction fragment length polymorphism and sequence analysis of PCR-amplified ribosomal DNA were used to identify and classify phytoplasmas associated with diseases of various wild and cultivated plants. The diseases examined were either not known before or the presumable causal agents were not yet identified and characterized or were only known from other geographic areas. New diseases examined were those causing virescence and phyllody of Bunias orientalis and Cardaria draba. Both were associated with strains of the aster yellows phytoplasma. The same type of aster yellows phytoplasma was also found to be associated with yellows and phyllody diseases of Portulaca oleracea, Stellaria media, Daucus carota ssp. sativus, and Cyclamen persicum. In German and French DNA samples from diseased Trifolium repens, the clover phyllody phytoplasma was identified, which could clearly be distinguished from other phytoplasmas of the aster yellows group. Strains of the stolbur phytoplasma were detected in big bud-affected tomatoes and almost exclusively in Convolvulus arvensis. In Cirsium arvense and Picris echioides two distinct phytoplasmas were identified which showed relationship to the sugarcane white leaf phytoplasma group but may represent a new group or subgroup. In Conyza (syn.: Erigeron) canadensis a phytoplasma of the X-disease group was detected. A strain from Gossypium hirsutum showed the same restriction profiles as the faba bean phyllody phytoplasma.     

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)。