棣棠丛枝病相关植原体的分子鉴定

 植原体（Candidatus Phytoplasma）是一种没有细胞壁的原核微生物,主要由取食韧皮部的昆虫（叶蝉、飞虱等）传播, 也可由菟丝子寄生和嫁接等途径传播,常常引起植株黄化、丛枝、花器变态、萎缩等症状。迄今为止,世界上报道的植物植原体病害有1 000余种,仅我国就有100多种,造成巨大损失。     

Distinctions between phytoplasmas at the subgroup level detected by heteroduplex mobility assay

A total of 62 phytoplasma isolates were collected from North America, Europe and Asia and analysed by heteroduplex mobility assay (HMA) of the 16/23S spacer region amplified by the polymerase chain reaction. The results revealed wide genetic diversity among the phytoplasmas studied and a number of new phytoplasma strains were identified from known or new plant hosts in Alberta, Canada. Two distinctive subgroups were revealed by HMA in phytoplasmas associated with canola yellows, Chinese aster yellows, dandelion yellows and monarda yellows. In Alberta, two subgroups of the aster yellows group of phytoplasmas, I-A and I-B, were prevalent in naturally infected field crops and ornamentals in open gardens. The results indicated that HMA is a simple, but rapid and accurate, alternative method for the detection and estimation of genetic divergence of phytoplasmas when finer molecular characterization of phytoplasmas is required at the subgroup level.     

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.     

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.     

Detection of “Candidatus Phytoplasma asteris” associated with henon bamboo witches' broom in Korea

Witches' broom disease in bamboo (Phyllostachys nigra Munro var. henonis) was found in Yeoungyang, Korea. In transmission electron micrographs, phytoplasma-like bodies were detected in the phloem cells of diseased plants but not in those of healthy plants. The presence of phytoplasmas was confirmed by amplification of a 1.8-kb DNA fragment using a primer pair specific for the region containing a 16S rRNA gene and an intergenic spacer region between the 16S and 23S rRNA genes. Comparision of the 16S rRNA gene sequences showed that the causal phytoplasma belongs to “Candidatus Phytoplasma asteris,” and shared the highest degree of similarity with the sequence of the onion yellows (OY) isolate in Japan. This is the first phylogenetic identification of phytoplasma infection of bamboo in Korea.     

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.     

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。     

Molecular identification of a phytoplasma associated with Russian olive witches’ broom in Iran

Russian olive trees (Elaeagnus angustifolia) showing witches’ broom symptoms typical of phytoplasma infection were observed in the Urmia region of Iran. A phytoplasma named Russian olive witches’ broom phytoplasma (ROWBp-U) was detected from all symptomatic samples by amplification of the 16S rRNA gene and 16S/23S rDNA spacer region using the polymerase chain reaction (PCR) which gave a product of expected length. DNA from symptomless plants used as a negative control yielded no product. The sequence of the 16S rRNA gene and 16S/23S rDNA spacer region of ROWBp-U showed 99% similarity with the homologous genes of members of the aster yellows group. We also detected a phytoplasma in neighboring alfalfa plants (AlWBp-U) showing severe witches’ broom symptoms. An 1107 bp PCR product from the 16S rRNA gene showed 99% homology with the corresponding product in ROWBp-U, suggesting the presence of the same phytoplasma actively vectored in the area. Further observations showed that Russian olive trees with typical ROWB symptoms were present in an orchard near Tehran which is located over 530 km south-east of the original Urmia site. The corresponding sequence of this phytoplasma (ROWBp-T) showed 99% homology to that of the ROWBp-U. A sequence homology study based on the 16S rRNA gene and 16S/23S rDNA spacer region of ROWBp-U and other phytoplasmas showed that ROWBp-U is most closely related to the 16SrI group. To our knowledge, this is the first report of a phytoplasma infection in a member of the Elaeagnaceae.     

Electron microscopy and molecular identification of phytoplasmas associated with strawberry green petals in the Czech Republic

The presence of phytoplasma inFragaria ananassa x Duch cv Senga Sengana showing strawberry green petals symptoms was observed by electron microscopy of phloem tissue. No phytoplasmas were found in asymptomatic strawberry plants used as controls. Nucleic acids extracted from these plants were used in nested-PCR assays with primers amplifying 16S rRNA sequences specifie for phytoplasmas. Bands of 1.2 kb were obtained and the subsequent nested-PCR with specific primers and RFLP analyses allowed to classify the detected phytoplasmas in the aster yellows group (16SrI). They belonged to the subgroup I-C of which type strain is clover phyllody phytoplasma.     

Genetic and serological analyses of elongation factor EF‐Tu of paulownia witches’‐broom phytoplasma (16SrI‐D)

This study determined the tuf gene sequence of the phytoplasma specific to paulownia witches’‐broom from Nanyang, China (hereby designated PaWB‐Ny). The PaWB‐Ny tuf gene was 1185 nucleotides in length and confirmed that the phytoplasma belongs to subgroup 16SrI‐D of aster yellows. Three characteristic GTP‐binding protein motifs were identified based on the peptide deduced from the tuf gene sequence. Results suggested that the elongation factor EF‐Tu was localized in the cytoplasm and lacked hydrophobic transmembrane domains. Antibodies against PaWB‐Ny EF‐Tu were prepared by rabbit immunization with glutathione‐S‐transferase (GST)‐tagged EF‐Tu fusion protein expressed in Escherichia coli. EF‐Tu exhibited a molecular weight of ～43 kDa and was detected in PaWB‐infected paulownia plants by western blot analysis. Indirect enzyme‐linked immunosorbent assays (ELISA) and dot blotting analyses were performed with freezing and thawing treatments during antigen preparation. Dilution of extracts to an appropriate scale significantly reduced non‐specific reactions. The resultant PaWB EF‐Tu antibody reacted with antigens from plants infected with periwinkle virescence and chinaberry tree witches’‐broom phytoplasmas, but not those infected with jujube witches’‐broom or bishopwood witches’‐broom phytoplasma. The EF‐Tu was characteristically localized within the phytoplasmal cytoplasm of infected plant phloem tissues.     

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。     

我国几种植物植原体的快速分子鉴别与鉴定的研究

 选用桑萎缩病(Mulberry dwarf,MD)、枣疯病(Jujube witches'-broom,JWB)、酸枣丛枝病(Wild jujube witches'-broom,WJWB)、泡桐丛枝病(Paulownia witches'-broom,PaWB)和苦楝丛枝病(Chinaberry tree witches'-broom,CWB)5种不同植物植原体和来源于3个不同地区PaWB和JWB材料进行16S rDNA和23S rDNA PCR扩增、异源双链迁移率分析(HMA)、PCR产物的RFLP分析和16S rDNA的克隆和测序等比较研究,建立了一种快速确定未知植原体种类和分类地位的分子鉴别与鉴定优化程序;并可对田间采集的各种植物植原体样品进行快速鉴定和鉴别。16S rDNA PCR产物HMA分析结果显示,JWB与CWB、MD和PaWB皆可形成明显的异源杂交双链;而CWB、MD和PaWB植原体之间未能形成异源双链。JWB和PaWB不同地区样品之间、JWB和WJWB之间也未发现异源杂交双链的形成。而23S rDNA PCR产物HMA分析则可以将MD与PaWB区分开。进一步对未知分类地位的CWB序列测定及与其它植原体16S rDNA的RFLP和同源性比较结果显示,CWB与PaWB同源性为99.5%,其中与MD的同源性高达99.7%,因而应将CWB归为翠菊黄花组16Sr I-B,16Sr I-B (rp-B)。     

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.     

An Antibody Against the SecA Membrane Protein of One Phytoplasma Reacts with Those of Phylogenetically Different Phytoplasmas

ABSTRACT Antisera raised against phloem-limited phytoplasmas generally react only with the phytoplasma strain used to produce the antigen. There is a need for an antiserum that reacts with a variety of phytoplasmas. Here, we show that an antiserum raised against the SecA membrane protein of onion yellows phytoplasma, which belongs to the aster yellows 16S-group, detected eight phytoplasma strains from four distinct 16S-groups (aster yellows, western X, rice yellow dwarf, and elm yellows). In immunoblots, approximately 96-kDa SecA protein was detected in plants infected with each of the eight phytoplasmas. Immunohistochemical staining of thin sections prepared from infected plants was localized in phloem tissues. This antiserum should be useful in the detection and histopathological analysis of a wide range of phytoplasmas.     

Reclassification of aster yellows group phytoplasmas in Korea

Aster yellows group phytoplasmas were reclassified by analysis of the 16S rRNA gene sequence, their phylogeny and the presence of interoperon heterogeneity. Nine phytoplasmas were classified into subgroups 16SrI-B and 16SrI-D using the 16S rRNA gene sequence. Then, based on the presence of interoperon heterogeneity, subgroup 16SrI-B phytoplasmas were differentiated into three subunits as 16SrI-B(a): mulberry dwarf, sumac witches’ broom and porcelain vine witches’ broom; 16SrI-B(b): angustata ash witches’ broom and Japanese spurge yellows; and 16SrI-B(c): onion yellow dwarf, water dropwort witches’ broom and hare’s ear yellow dwarf phytoplasma.     

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.     

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。     

Molecular characterization of diverse phytoplasmas of subgroups 16SrI-A, 16SrI-B, 16SrI-L, and 16SrI-M infecting ornamental plants in Lithuania

Trade in ornamental plant species comprises a significant segment in the economies of countries in Europe, North America and Asia. Since the quality of ornamental plants is adversely affected by diseases attributed to phytoplasmas, we surveyed plant collections in botanical gardens and floriculture farms in Lithuania for phytoplasmal diseases. Seventeen ornamental species belonging to nine plant families exhibited disease symptoms including general yellowing and stunting, proliferation of shoots, phyllody, virescence and reduced size of flowers, and reddening of leaves. Analysis of the phytoplasmal 16S rRNA gene sequences amplified by PCR revealed that the plants were infected by phytoplasmas belonging to four distinct subgroups (16SrI-A, 16SrI-B, 16SrI-L, and 16SrI-M) of group 16SrI (aster yellows phytoplasma group) and indicated the presence of sequence-heterogeneous 16S rRNA genes in newly recognized strains belonging to subgroups 16Sr-L and 16SrI-M. Infections by these diverse phytoplasmas in a wide array of plant species and families suggests that unidentified, polyphagous insect vectors may actively transmit phytoplasmas threatening the Baltic region's ornamental plant industry.     

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。