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。     

Phylogenetic analysis of elongation factor Tu gene of phytoplasmas from Japan

The elongation factor Tu (tuf) gene from nine Japan phytoplasma isolates was amplified with the polymerase chain reaction, and the DNA sequences of the tuf gene were determined. The tuf gene from 14 phytoplasma isolates, including reference isolates and other bacteria, were phylogenetically analyzed. A nucleotide sequence of the tuf gene among seven aster yellows group (16Sr I-B and I-D) phytoplasmas had 97%–100% similarity, and the tuf gene of two phytoplasmas of the X-disease group (16Sr III-B) had 99% similarity. The tuf genes had lower homology than did the 16S rRNA gene in the phytoplasma groups. A phylogenetic tree of amino acid sequences of the tuf gene was nearly equal to that of the 16S rRNA gene but differed somewhat from the tree based on the 16S rRNA gene in that paulownia witches broom (PaW: 16Sr I-D) and American aster yellows (AAY: 16Sr I-B) were in a subclade.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB095495, AB095667, AB095668, AB095669, AB095670, AB095671, AB095672, AB095673 and AB095674     

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。     

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.     

Molecular and biologic characterization of a phytoplasma associated with <Emphasis Type="Italic">Brassica campestris</Emphasis> phyllody disease in Punjab province,Pakistan

A phytoplasma-associated disease was identified in Brassica campestris (sarson) plants during a survey conducted in Punjab province of Pakistan in 2014–2016. The symptomatic plants showed characteristic symptoms of phyllody and witches’ broom. Phytoplasma presence was detected by polymerase chain reaction on 16S ribosomal and tuf DNAs, followed by RFLP analysis and sequence comparison of the 16S rRNA and tuf genes. The phytoplasma detected was classified in a new ribosomal subgroup designed 16SrIX-H. The phytoplasma presence in phloem tissues of symptomatic sarson samples was also confirmed through light microscopy and transmission studies to healthy plants through dodder and the leafhopper Orosius albicinctus. This is the first report of identification of 16SrIX-H subgroup phytoplasma associated with sarson and its natural vector in Pakistan.     

First report of Almond witches’ broom phytoplasma1 (‘Candidatus Phytoplasma phoenicium’) causing a severe disease on nectarine and peach trees in Lebanon

Symptoms of shoot proliferation characteristic of phytoplasma diseases were observed on nectarine (Prunus persica var. nucipersica) and peach (P. persica) trees in the Sarada plain, south of Lebanon. The presence of phytoplasmas in the two orchards visited was confirmed by nested polymerase chain reaction using universal primers. The amplified DNA fragments were cloned and sequenced. Blast analysis of over 1000 nucleotides demonstrated the presence of ‘Candidatus Phytoplasma phoenicium’ which is considered to be the causal agent of Almond witches’ broom. This phytoplasma which belongs to the pigeon pea witches’ broom group (16SrIX) can be devastating since Almond witches’ broom has killed thousands of almond trees in Lebanon and Iran. Previous reports indicated that Almond witches’ broom may be transmitted by grafting to peach and nectarine under experimental conditions. This is the first report of a natural and epidemic spread of ‘Ca. Phytoplasma phoenicium’ in peach and nectarine. Farmers in the region were advised to eradicate the infected trees immediately. Further studies on the epidemiology of ‘Ca. Phytoplasma phoenicium’ and its vector(s) are recommended in order to develop successful eradication or disease management programmes.     

Molecular detection and identification of phytoplasma associated with pepper witches’ broom in China

Pepper witches’ broom (PWB) disease was observed in a field in Yangling, Shaanxi Province, China. The result of mechanical inoculation test for this disease was negative. Phytoplasma-like bodies were observed in ultrathin sections of petiole tissues of symptomatic samples. 16S rRNA gene and tuf gene of phytoplasma were amplified from the total DNA of symptomatic samples. Phylogeny analysis of the 16S rRNA gene and tuf gene suggested that the pepper witches’ broom associated phytoplasma belongs to the subgroup 16SrI-B, which was confirmed by the RFLP analysis of the 16S rRNA gene. The phytoplasma subgroup 16SrI-B was also detected in the vector Cicadella viridis trapped from the infected field. To our knowledge, this is the first report of 16SrI-B phytoplasma causing pepper witches’ broom in China.     

A novel phytoplasma associated with witches’ broom disease of Ligustrum ovalifolium in Turkey

California privet (Ligustrum ovalifolium Hassk.) plants exhibiting leaf yellowing, witches’ broom, dieback and decline symptoms were observed for two years (2010–2011) in three gardens at Adana region (Turkey). DNA isolated from symptomatic and healthy plants was used to amplify 16S rDNA fragments by direct and nested-PCR. Phytoplasmas were detected in 21 symptomatic plants, out of 30 samples collected, whilst no PCR amplifications were obtained from asymptomatic plants. BLAST analysis of the 16S rDNA showed that the phytoplasma found in L. ovalifolium from Turkey, denoted as Turkish Ligustrum witches’ broom phytoplasma (TuLiWB), most closely resembled members of group 16SrII (peanut witches’ broom group) and shared up to 92 % sequence identity. Based on in silico 16S rDNA RFLP analysis and automated calculation of the pattern similarity coefficient, TuLiWB showed molecular characteristics different from all previously described phytoplasma species to represent a new taxon. Similar indication also emerged from the phylogenetic tree which allocated it in a novel discrete subclade within the phytoplasma clade. This is the first report on the presence of a phytoplasma affecting L. ovalifolium and whether this novel phytoplasma is the same agent reported as a mycoplasma-like organism (MLO) and associated with witches’ broom disease of Ligustrum in Korea (1989) is yet to be determined.     

New natural host plants of ‘Candidatus Phytoplasma pini’ in Poland and the Czech Republic

The presence of phytoplasmas in seven coniferous plant species (Abies procera, Pinus banksiana, P. mugo, P. nigra, P. sylvestris, P. tabuliformis and Tsuga canadensis) was demonstrated using nested PCR with the primer pairs P1/P7 followed by R16F2n/R16R2. The phytoplasmas were detected in pine trees with witches’ broom symptoms growing in natural forest ecosystems and also in plants propagated from witches’ brooms. Identification of phytoplasmas was done using restriction fragment length polymorphism analysis (RFLP) of the 16S rDNA gene fragment with AluI, MseI and RsaI endonucleases. All samples showed RFLP patterns similar to the theoretical pattern of ‘Candidatus Phytoplasma pini’, based on the sequence of the reference isolate Pin127S. Nested PCR‐amplified products, obtained with primers R16F2n/R16R2, were sequenced. Comparison of the 16S rDNAs obtained revealed high (99·8–100%) nucleotide sequence identity between the phytoplasma isolates. The isolates were also closely related to four other phytoplasma isolates found in pine trees previously. Based on the results of RFLP and sequence analyses, the phytoplasma isolates tested were classified as members of the ‘Candidatus Phytoplasma pini’, group 16SrXXI.     

Molecular characterization,phylogenetic comparison and serological relationship of the Imp protein of several ‘Candidatus Phytoplasma aurantifolia’ strains

The immunodominant membrane protein Imp of several phytoplasmas within the ‘Candidatus Phytoplasma aurantifolia’ (16Sr‐II) group was investigated. Eighteen isolates from Iran (11), East Asia (5), Africa (1) and Australia (1) clustered into three phylogenetic subgroups (A, B and C) based on the 16S rDNA and imp genes, regardless of geographic origin. The imp gene sequences were variable, with more non‐synonymous than synonymous mutations (68 vs 20, respectively), even though many of the non‐synonymous ones (75%) produced conservative amino acid replacements. Eight codon sites on the extracellular region of the protein were under positive selection, with most of them (75%) coding for non‐conservative amino acid substitutions. Full‐length (21 kDa) and truncated (16 kDa) Imp proteins of two economically important Iranian phytoplasmas [lime witches’ broom (LWB) and alfalfa witches’ broom (AlWB‐F)] were expressed as His‐tagged recombinant proteins in Escherichia coli. An antiserum raised against full‐length recombinant LWB Imp reacted in western blots with membrane proteins extracted from LWB‐infected periwinkle and lime, indicating that Imp (19 kDa) is expressed in infected plants and is a membrane‐associated protein. The same polyclonal antibody also detected native Imp in proteins from periwinkles infected by phytoplasmas closely related to LWB (subgroup C) only, confirming phylogenetic clustering based on 16S rDNA and imp genes. Imp proteins of LWB and AlWB‐F isolates were also recognized by an antiserum raised against an enriched preparation of AlWB‐F phytoplasma cells, demonstrating the antigenic properties of this protein.     

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.     

‘Candidatus Phytoplasma ziziphi’ associated with Sophora japonica witches’ broom disease in China

Chinese scholar tree (Sophora japonica) with witches’ broom symptoms was observed in Shandong Province in China. Phytoplasmas were detected in the diseased plants using 16S rDNA amplification with phytoplasma-specific universal primer pairs. On the basis of the results of 16S rDNA sequencing, virtual restriction fragment length polymorphism patterns and phylogenetic analyses, the phytoplasma found in S. japonica with witches’ broom symptoms was confirmed as a ‘Candidatus Phytoplasma ziziphi’-related strain belonging to the Elm yellows group 16SrV. This is the first report of ‘Ca. P. ziziphi’ infecting S. japonica plant with witches’ broom symptoms.     

Morphological, anatomical and molecular investigation into witches’ broom disease of mamejvo (Enicostemma axillare)

Mamejvo (Enicostemma axillare Raynal) is a perennial herb with diversified health benefits. It was found to be affected by a witches?? broom disease under the field conditions at Anand, Gujarat, India. Affected plants were 53.33% reduced in height due to shortened internodes, leading to typical witches?? broom symptoms. Flower size was reduced significantly in the affected plants compared with the healthy ones and their petal color gradually turned green (virescence). Affected plants produced 2.5 times more dry herbage than the normal plants but with less active ingredient (swertiamarin). Symptomatic plants died early while root suckers from such plants failed to establish after transplanting. DAPI stained transverse sections of affected shoot tips showed the presence of discrete greenish fluorescence in the phloem cells under UV light. Amplification of 1.2?kb phytoplasma specific rDNA fragment from diseased tissue confirmed the presence of the pathogen. Further characterization of the pathogen through virtual rDNA?CRFLP pattern and rDNA sequence based phylogeny suggested that the pathogen, Enicostemma witches?? broom phytoplasma, belonged to 16SrII?CC group of ??Ca Phytoplasma aurantifolia??.     

枣疯病植原体侵染对枣树叶绿素含量的影响

为明确枣疯病植原体侵染对枣树叶绿素含量的影响,本研究以田间‘赞皇大枣,健树和患枣疯病病树以及‘婆枣’枣疯病组培苗为材料,测定了健叶、病叶以及不同浓度生长调节剂处理后组培苗叶片的叶绿素含量.结果表明,在田间条件下枣疯病病树叶片的叶绿素a、b含量及叶绿素a/b值显著低于健康叶片;在培养基中添加IBA、6-BA可提高枣疯病组培苗叶片叶绿素a、b含量,明显改善患病组培苗的光合作用,但随着继代时间延长,这种影响逐渐消失.此结果表明枣疯病植原体、激素代谢及叶绿素合成之间存在密切联系.     

Experimental and molecular evidence of Reptalus panzeri as a natural vector of bois noir

Bois noir (BN) is an economically important grapevine yellows disease induced by the stolbur phytoplasma and principally vectored by the cixiid Hyalesthes obsoletus. This study addresses the involvement of other planthoppers and/or leafhoppers in BN epidemics in the South Banat district of northeastern Serbia, by performing transmission experiments and multilocus typing of stolbur phytoplasma isolates to determine the vector‐related characteristics of the disease. Transmission trials were conducted with adults of two cixiid congeners, Reptalus panzeri and R. quinquecostatus, which were found to harbour stolbur phytoplasma in the vineyards under study. A molecular characterization of stolbur phytoplasma isolates was performed by sequence analysis and/or RFLP typing of the two housekeeping genes tuf and secY and the two membrane proteins stamp and vmp1. Transmission trials with naturally infected R. panzeri adults from either the BN‐infected vineyards or maize redness (MR)‐affected maize fields revealed a high stolbur phytoplasma transmission efficiency to grapevines. In contrast, experiments conducted with stolbur‐positive R. quinquecostatus originating from BN‐infected vineyards, provided no evidence for a vector role of this species. Seven stolbur phytoplasma genotypes, all of which were tuf‐b types, were detected among the grapevine‐ and insect‐associated field samples according to the tuf/secY/vmp1/stamp typing. STOLg was the genotype most frequently found in naturally infected grapevine (42%), as well as R. panzeri originating from the vineyards (85%) and maize fields (98%). The same genotype was found in all experimental plants inoculated by R. panzeri, confirming its vectorship of the disease.     

泡桐丛枝植原体抗原膜蛋白抗血清的制备及应用

 根据报道的泡桐丛枝植原体(Paulownia witches’-broom phytoplasma,PaWB)抗原膜蛋白(antigenic membrane pro-tein,AMP)基因的核苷酸序列设计引物,提取发病泡桐总DNA,经PCR扩增并成功克隆泡桐丛枝植原体amp基因。序列分析表明,amp基因由696个核苷酸组成,编码231个氨基酸残基,与GenBank中登录的2个泡桐丛枝植原体的膜蛋白核苷酸序列同源性为100%。将amp基因91-604 nt部分序列(命名为ampd)克隆到原核表达载体pGEX-4T-3,诱导后,经SDS聚丙烯酰胺凝胶电泳分析,表明融合蛋白在大肠杆菌BL21(DE3)中得到了高效表达。以纯化的带GST标签的AMPD融合蛋白为抗原免疫德国大白兔,制备了PaWB-AMPD抗血清。利用该血清,通过Western印迹、点印迹、ELISA、间接免疫荧光和免疫捕获PCR试验均能在发病泡桐组织中特异检测到泡桐丛枝植原体。     

Ecology-based analysis of a recent association between Spartium junceum and 16SrV phytoplasma

An outbreak of Spartium witches’ broom (SpaWB) in Sicily prompted us to identify and characterize associated phytoplasmas. Over 80 samples of Spanish broom (Spartium junceum) and around 270 individuals of the potential vector Livilla spectabilis were collected and analysed. Single and mixed infections of 16SrV and ‘Candidatus Phytoplasma spartii’ were detected in Spanish broom samples and for the first time in L. spectabilis. The 16SrV isolates were further characterized by multilocus sequence typing (MLST) to determine their phylogenetic relationship with flavescence dorée phytoplasma (FDp) and to evaluate the risk of host-jumping to grapevine. Phylogenetic analysis of most of the analysed genes using the MLST approach grouped S. junceum 16SrV-C isolates with FDp isolates infecting grapevine and Scaphoideus titanus. Notably, phylogenetic analysis of the vmpA gene clustered the S. junceum isolates with FDp genotypes transmitted by S. titanus. This study extends the knowledge of SpaWB epidemiology, focusing on the possible risk of a 16SrV host jump from Spanish broom to grapevine. Spanish broom was identified as a reservoir and potential inoculum source of phytoplasmas that cause severe disease in cultivated crops. Furthermore, the L. spectabilis psyllid may be involved in the epidemiology of this 16SrV-C phytoplasma, although in the absence of in vivo transmission trials. The study further confirms the strong ability of phytoplasmas to adapt to new hosts and vectors, thus leading to potential phytosanitary emergencies.     

Expression of phytoplasma‐induced witches’ broom disease symptoms in acid lime (Citrus aurantifolia) trees is affected by climatic conditions

Witches’ broom disease (WBD), caused by ‘Candidatus Phytoplasma aurantifolia’, is a serious disease of acid lime (Citrus aurantifolia) in Oman and the UAE. However, little is known about the distribution of phytoplasma and the expression of WBD symptoms in different geographical locations. A survey was carried out in 18 districts in Oman and the UAE covering 143 orchards and 5823 acid lime trees. ‘Candidatus Phytoplasma aurantifolia’ was detected in acid lime in all the 18 surveyed districts. However, the development of typical symptoms of WBD was only observed in 12 districts. Districts in which the phytoplasma was present but symptoms were not expressed were located either in desert areas or in areas characterized by semitropical conditions. Phylogenetic analysis of 16 phytoplasma isolates from trees developing WBD symptoms and six phytoplasma isolates from trees with no WBD symptoms showed that all isolates share an identical 16S rRNA sequence, belonging to subgroup II‐B. Quantitative PCR analysis showed that the concentration of phytoplasma is significantly higher (8800–801 000 copies) in leaves developing WBD symptoms compared to 2–268 copies in symptomless leaves from the same trees and 8–874 copies in acid lime trees from areas where disease symptoms were not expressed. The lack of expression of WBD symptoms under certain environmental conditions may suggest that symptom development and phytoplasma are affected by certain unfavourable environmental conditions. These findings could provide a basis for managing WBD through encouraging lime cultivation under climatic conditions less conducive to WBD symptom expression.     

竹丛枝植原体16SrDNA片段克隆与序列分析

利用植原体16SrRNA基因序列设计合成的引物,对表现丛枝的竹子植株总DNA进行直接PCR及巢式PCR扩增,得到长1.2kb的目的片段。将此片段与pGEMTEasy载体连接并转化到大肠杆菌DH5α感受态细胞中。通过酶切、PCR鉴定,对筛选得到的重组阳性克隆进行核酸序列测定及同源性比较分析,结果表明其与植原体16SrⅠ组中的西方翠菊黄化植原体(SAY)同源率为99%。依据16SrDNA序列建立了竹子丛枝病植原体株系的系统进化树。对云南竹子丛枝病植原体株系分类鉴定与已报道的结果相似。     

Identification of wheat blue dwarf phytoplasma effectors targeting plant proliferation and defence responses

The identification of effectors from pathogenic microbes is one of the most important subjects for elucidating infection mechanisms. Wheat blue dwarf (WBD) phytoplasma causes dwarfism, witches' broom, and yellow leaf tips in wheat plants, resulting in severe yield loss in northwestern China. In this study, 37 candidate effector proteins were transiently expressed in Nicotiana benthamiana. Plants expressing the SAP11‐like protein SWP1 exhibited typical witches' broom. Interestingly, another protein, SWP11, induced both cell death and defence responses, including H₂O₂ accumulation and callose deposition. Analysis by qRT‐PCR was used to show that a marker gene of the hypersensitive response, HIN1, and three pathogenesis‐related genes, PR1, PR2 and PR3, were significantly up‐regulated in leaves of N. benthamiana expressing SWP11. In addition, SWP12 and SWP21 (TENGU‐like) were shown to suppress SWP11‐, BAX‐, and/or INF1‐induced cell death. These results indicated that SWP21 has a distinct role in virulence compared with TENGU and that WBD phytoplasma possesses effectors that target plant proliferation and defence responses. The ability of these effectors to trigger or suppress plant immunity provides new insights into the phytoplasma–plant interaction.