Detection and molecular characterisation of an alfalfa phytoplasma in Argentina that represents a new subgroup in the 16S rDNA Ash Yellows group(‘<Emphasis Type="Italic">Candidatus</Emphasis> Phytoplasma fraxini’)

A phytoplasma infecting alfalfa crops was detected and characterised in Argentina, the Argentinean Alfalfa witches′-broom (ArAWB) phytoplasma. Typical witches′-broom symptoms were observed in diseased alfalfa plants from fields of the ‘Cuyo’ region in the Andean midwest. Pleomorphic bodies were observed by electron microscopy in sieve tubes of the diseased plants. The results obtained from the sequence homology, similarity coefficients derived from RFLP of the 16S rDNA and phylogenetic analysis led us to include this phytoplasma in the 16Sr VII (Ash Yellows) group. However, the ArAWB phytoplasma showed several differences when compared to other members of group 16Sr VII. The RFLP analysis of partial 16S rRNA gene of two ArAWB isolates, digested with 16 restriction enzymes, showed differences between the ArAWB and the reference strain (AshY1^T) in six enzyme patterns. Restriction patterns unique for the group and an exclusive HinfI restriction site were found in the ArAWB phytoplasma rDNA. Moreover, the similarity coefficients (0.92–0.86) were lower than those obtained among other group members. The significant differences detected suggested that this phytoplasma belonged to a subgroup different from those described so far. We propose therefore, that the ArAWB phytoplasma should be included in a new VII-C subgroup, closely related to the EriWB phytoplasma (VII-B) described in Brazil.     

‘Candidatus Phytoplasma aurantifolia’-related strain associated with tomato yellows disease in China

A new disease of tomato plants with typical phytoplasma disease symptoms such as stunting, yellows, auxiliary shoot proliferation and phyllody was observed in Yunnan Province, southwest China in 2011. By a nested-PCR, phytoplasma were detected using the phytoplasma universal primers specific for 16S rDNA. The results of the 16S rDNA sequencing, computer-simulated RFLP patterns and phylogenetic analysis indicated that the phytoplasma associated with the diseased tomato plants belongs to subgroup A of the peanut witches’-broom group. This is the first report of a 16SrII-A phytoplasma associated with a new tomato disease in China. This new disease was named tomato yellows.     

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.     

云南花生丛枝植原体16S rRNA和核糖体蛋白基因序列分析

 利用植原体16S rRNA基因及核糖体蛋白基因(ribosomal protein, rp)通用引物对发生在云南元谋的花生丛枝病病株DNA进行PCR扩增,并对扩增片段进行序列测定。扩增获得的云南元谋花生丛枝植原体(PnWB-YNym)16S rDNA、16S-23S rDNA和23S DNA片段总长1 806 bp,rp基因扩增片段长1 171 bp。云南株系与来源于台湾和海南的花生丛枝植原体均有较高同源性。比较16S rDNA片段,发现云南株系在5个位点上与来自台湾或海南的株系存在碱基差异,其中有1个位点的差异是云南元谋株系特异的;再分别比较核糖体蛋白rplV-rpsC 2个基因所编码的氨基酸序列,发现云南株系rpsC编码的第194位氨基酸与台湾和海南的株系存在差异。经16S rDNA片段系统进化及iPhyClassifier在线分析,表明PnWB-YNym在分类上属于16SrII-A亚组成员,与候选种‘Candidatus Phytoplasma australasiae’相关;基于rp基因构建的系统进化树表明,PnWB-YNym与16SrII-A亚组各成员聚为同一亚进化支(iii)。     

云南芒果变叶病植原体的分子鉴定

 植原体（phytoplasma）是一类没有细胞壁,不能离体培养的原核生物,对四环素敏感,主要存在于植物筛管细胞中。植原体主要通过叶蝉、飞虱等取食植物韧皮部的昆虫传播,也可通过菟丝子寄生和嫁接等方式传播。目前,全世界已发现1 000多种由植原体引起的植物病害,我国大陆已报道100余种与之相关的病害\[1\]。由植原体引起的病害症状主要表现为植株花器病态、小叶、丛枝、黄化等,从而导致植物产量和品质明显下降。     

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.     

Molecular characterization of the phytoplasmas associated with toon trees and periwinkle in India

In November 2008 in Himachal Pradesh and Chandigarh regions in India, toon trees and periwinkles were observed to have formed short internodes, small leaves and witches’-broom symptoms, typical of phytoplasma infection. The symptomatic toon and periwinkle samples were tested with universal PCR tests, and the 16S rRNA, rplB-rpsC, secA and secY genes were sequenced. The causal agents belonged to subgroup 16SrI-B of ‘Candidatus Phytoplasma asteris’, based on 16S rDNA, ribosomal protein gene, secA and secY phylogenetic analysis.     

小麦蓝矮植原体寄主范围的鉴定及RFLP分析

 小麦蓝矮是我国首次报道的小麦植原体病害。采用介体接种植物,症状观察和应用植原体16S rDNA基因通用引物对R16mF2/R16mR1进行PCR扩增,在接种小麦和传毒介体中均扩增出1.4kb的特异片段,鉴定出小麦蓝矮植原体新寄主7种。用巢式PCR方法对小麦蓝矮病田自然发病杂草进行分子检测,从表现症状的10种杂草中均扩增出1.2kb的特异片段。利用6种植原体特异性限制性内切酶对10种杂草的扩增片段进行RFLP(restriction fragment length polymor-phism)分析表明:扩增片段的RFLP图谱与目前已知的16Sr I组翠菊黄化植原体的RFLP图谱相近。鉴定出小麦蓝矮植原体田间自然新寄主10种。     

喜树丛枝植原体的分子鉴定及TaqMan探针实时荧光定量PCR检测方法的建立

 为确定在云南文山地区喜树上发生的疑似丛枝病的病原种类及快速检测喜树丛枝病,本研究利用植原体16S rDNA基因通用引物P1/P7和R16F2n/R16R2对感病喜树总DNA进行常规PCR和巢式PCR扩增、克隆和测序,通过系统进化分析,明确了喜树丛枝植原体属于16SrXXXII组。然后根据喜树丛枝病植原体16S rDNA基因保守区域设计并合成特异性引物和TaqMan探针,制备了喜树丛枝病植原体标准质粒,确定了最优引物浓度和最佳探针浓度,制作的标准曲线有极好的线性关系,决定系数(R²)达到0.999,建立的实时荧光定量PCR检测方法能够特异性地检测喜树丛枝植原体。本研究首次明确了喜树丛枝植原体的分类地位,优化和建立了喜树丛枝植原体TaqMan探针qPCR检测方法,为快速检测喜树丛枝病植原体提供参考。     

臭矢菜丛枝病植原体的分子鉴定研究

 本实验采用DAPI荧光显微镜、PCR、克隆和测序等技术,对海南臭矢菜丛枝病样进行了检测和鉴定。以染病臭矢菜总DNA为模板应用3对植原体特异性引物进行PCR扩增,获得PCR产物为16S rDNA(1 430 bp)、16S-23S rDNA(358bp)、rp DNA(1 294 bp)。应用DNA回收试剂盒获得了3个PCR扩增片断的纯化产物,并克隆到DH5α大肠杆菌中测序。应用DNAMAN和MEGA软件对获得的序列与NCBI数据库中植原体序列进行同源性分析和构建系统发育树。结果显示臭矢菜丛枝病植原体与花生丛枝病植原体序列同源性最高,16S rDNA的序列同源性为99.9%,16S-23S rDNA高达100%,rp为99.7%,因而将臭矢菜丛枝病植原体归为花生丛枝组(16SrⅡ),根据16S rDNA的RFLP分析,将其归为16SrⅡ-A亚组。     

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。     

Detection of ‘<Emphasis Type="Italic">Candidatus</Emphasis> Phytoplasma brasiliense’ in a new geographic region and existence of two genetically distinct populations’

‘Candidatus Phytoplasma brasiliense’, a phytoplasma taxon associated with hibiscus witches’ broom disease was first described in 2001 in Brazil. In September 2007, a peach tree (Prunus persica) displaying yellowing symptoms reminiscent of phytoplasma infection was sampled in Guba region of Azerbaijan. A phytoplasma was detected in the diseased peach tree by nested PCR amplification of its 16S rDNA with universal primers for phytoplasmas. Phylogenetical analyses of the amplified 16S rDNA showed that the phytoplasma infecting the peach tree corresponded to ‘Ca. P. brasiliense’, a species never reported in Euro-Mediterranean area. To set up a detection assay, cloning of a ‘Ca. P. brasiliense’ DNA fragment was undertaken by comparative RAPD. The amplified dnaK-dnaJ genetic locus was used to design a nested PCR assay able to amplify all ‘Ca. P. brasiliense’ isolates of the subgroup 16SrXV-A without amplifying the related members of the group 16SrII. This assay also allowed confirming the first detection of ‘Ca. P. brasiliense’ in diseased basil collected in south Lebanon.     

Identification and molecular characterization of a phytoplasma associated with sunflower in Argentina

Sunflower (Helianthus annuus L.) plants showing capitulum with virescence, phyllody and flower malformation, shortened internodes and abnormal branches were found in a field in Pedro Luro (Buenos Aires province, Argentina). Pleomorphic bodies resembling phytoplasmas were observed in sieve tube elements of symptomatic plants but not in healthy ones. DNA from all symptomatic sunflower plants analysed yielded, in direct PCR with phytoplasma universal primers P1/P7 and R16F2n/R2, fragments of expected size 1.8 kb and 1.2 kb, respectively. The phytoplasma associated with the disease, was named Sunflower Phyllody (SunPhy). Real and putative RFLP of the 16S rDNA showed the affiliation of SunPhy to 16SrIII (X-disease group), subgroup J. The 16S rDNA sequence from SunPhy showed the highest identity (99 %) with 16SrIII members and the phylogenetic tree confirmed a closer relationship to subgroup J of the 16SIII ribosomal group. This is the first report of a phytoplasma related to the 16SrIII group affecting sunflower.     

Detection and molecular characterization of a phytoplasma affecting Prunus persica L. in Jujuy, Argentina

Peach (Prunus persica L.) plants with symptoms of yellowing, reddening, curling and leaf necrosis, premature defoliation and internode shortening were observed in production fields in Jujuy province (Argentina). A phytoplasma was detected by PCR using the universal primer pairs P1/P7 and R16F2n/R16R2 in all the symptomatic samples analysed. The RFLP profile of PCR products, amplified with R16F2n/R16R2 primers, shows that this phytoplasma, named Argentinean Peach Yellows (ArPY), belongs to subgroup 16Sr III-B. The phylogenetic analysis of the 1244 bp 16S rDNA cloned sequence, grouped the ArPY phytoplasma into the X-disease group with a closer relationship with CFSD, PssWB and ChTDIII phytoplasmas. This is the first report of a phytoplasma infecting peach trees in Argentina.     

Molecular characterization of a phytoplasma causing Phyllody in Clover and other herbaceous hosts in Northern Italy

Red clover (Trifolium pratense) and Ladino clover (Trifolium repens) plants showing phytoplasma-associated symptoms (yellowing/reddening, virescence and phyllody) have been recovered in Friuli-Venezia Giulia, Italy. Using AluI RFLP analysis of PCR amplified 16S rDNA we showed that the disease can be caused independently by two phylogenetically distinct phytoplasmas. One of them showed the very typical 16S rDNA RFLP pattern of the agent of Clover Phyllody in Canada (CCPh). The 16S rDNA of the other phytoplasma (Italian Clover Phyllody phytoplasma, ICPhp) has been PCR amplified, cloned and sequenced. The sequence revealed high similarity (>98%) with phytoplasmas belonging to the X disease cluster, which includes organisms not reported to cause phyllody on their hosts. The analysis by AluI RFLP of the PCR amplified pathogen 16S rDNA from other herbaceous plants (Crepis biennis, Taraxacum officinale, Leucanthemum vulgare) collected nearby with phytoplasma-associated symptoms showed similar patterns. Southern blot hybridization of their EcoRI digested total DNA revealed identical RFLP patterns, suggesting that the causative agent may be the same organism.Abbreviations PCR Polymerase Chain Reaction - rDNA gene for the small subunit ribosomal RNA - RFLP Restriction Fragment Length Polymorphism     

Sensitive Detection of a Phytoplasma Associated with Little Leaf Symptoms in Withania somnifera

Withania somnifera is an important medicinal plant native to the Indian-sub continent. Owing to the presence of a number of precious alkaloids, flavonoids and withanolides, it is widely used in the Indian and African systems of medicines. It is severely affected by phytoplasma present in the sieve tubes of phloem. With a view to micropropagate phytoplasma-free W. somnifera plants, an efficient and effective nested PCR-based system was developed for detection of associated phytoplasmas. Universal primers, designed from the 16S rDNA sequences of phytoplasmas, were applied in direct/nested-PCR. Total DNA extracts from leaf tissues of 33 suspected symptomatic and 11 non-symptomatic plants were subjected to direct PCR. The direct PCR products were subsequently employed as templates in nested PCR. The nested PCR could reamplify direct PCR products yielding a DNA fragment of 1.4 kb. A phytoplasma was detected in all the diseased plants and not from the healthy looking plants. Further, it was sensitive enough to amplify phytoplasma DNA obtained from crude DNA diluted up to 2500 times from naturally infected plants and also from various stages of in vitro-propagated diseased plants. Identical restriction fragment polymorphism enzyme profiles were obtained following restriction enzyme digestion of nested PCR products, obtained from five different plants, by EcoRI, AluI and RsaI restriction endonucleases. The developed nested PCR based system should facilitate indexing of the phytoplasma in different stages of in vitro-generated plants and probably identification of, as yet unknown, hosts and vectors of phytoplasma associated with phytoplasma disease of W. somnifera.     

Natural occurrence, identification and transmission of the phytoplasma associated with flax phyllody and stem fasciation in Pakistan

Flax plants (Linum usitatissimum) of the white (album) flower variety exhibiting typical phytoplasma-like symptoms were found for the first time in Pakistan during 2011. The symptoms included floral virescence, phyllody, little leaf, stunting and stem fasciation. Light microscopy of hand-cut stem sections treated with Dienes’ stain showed blue areas in the phloem region of symptomatic plants. To confirm phytoplasma infection, total DNA was extracted separately from five plants showing virescence/phyllody and from five others showing fasciation, and was amplified by nested PCR using universal 16S rDNA phytoplasma primers P1/P7 followed by R16F2n/R16R2. All samples from plants with virescence/phyllody and fasciation yielded a 1,250 bp PCR product, and identical RFLP profiles using the enzymes AluI and HpaII. Direct sequencing of the 16S rDNA of one representative PCR amplicon (GenBank Accession No. JX567504 for phyllody and Accession No. JX567505 for fasciation) showed highest sequence identity (99%) with 16SrII ‘Candidatus Phytoplasma aurantifolia’ phytoplasmas, and phylogenetic analysis placed the phytoplasma in subgroup 16SrII-D. Disease was successfully transmitted by grafting and by the leafhopper Orosius albicinctus. To our knowledge, flax is a new natural host for 16SrII-D phytoplasmas in Pakistan.     

Molecular characterization of a new isolate of phytoplasma associated with malformation and twisting of floral spikes of <Emphasis Type="Italic">Gladiolus</Emphasis> in India

In 2007–2009, severe virescence, malformation and twisting of flower spikes and yellowing of entire plants were observed in various Gladiolus cultivars growing in the gardens of the National Botanical Research Institute, Lucknow, India. The disease symptoms were very similar to symptoms in Gladiolus caused by the Aster yellows phytoplasma identified from Poland. Disease incidence was low (1.1–3.4%), but the severity of symptoms was high. A phytoplasma infection was detected in nine of 13 cultivars by PCR followed by nested PCR using universal phytoplasma primers P1/P6 or R16F2n/R16R2, respectively. An amplicon of ~1.2 kb obtained from the nested PCR was cloned and sequenced. Sequence analysis of the PCR amplicon revealed high (94–98%) identities and the closest phylogenic relationships with several isolates of Aster yellows phytoplasma of ‘Candidatus Phytoplasma asteris’ (16SrI group). Thus, the phytoplasma isolate of Gladiolus was identified as a new isolate of ‘Ca. P. asteris’ (16SrI group). In silico analysis of the phytoplasma isolate clearly indicated that the isolate was distinct from other Indian isolates of this phytoplasma.     

Molecular identification of a phytoplasma associated with Ajwain (<Emphasis Type="Italic">Trachyspermum ammi</Emphasis>) in India

A new disease of Trachyspermum ammi, commonly known as Ajwain in India, was observed in Lucknow, India. The symptoms included small chlorotic leaves, highly proliferating shoots, witches’ broom appearance, shortened internodes and an overall stunted growth. Poor flower heads and fruit setting caused considerable yield losses for farmers. In transmission electron microscopy, pleomorphic bodies were detected in the phloem cells of diseased plants but not in those of healthy plants. The disease etiology was investigated using direct and nested polymerase chain reaction with phytoplasma-specific primers, DNA sequencing, and phylogenetic analysis. Phylogenetic analysis indicated that this phytoplasma clustered in the 16SrVI group. A 1,249 bp sequence (FJ970035) of the 16S rRNA gene from the phytoplasma showed 99% homology with the 16S rRNA gene, (FJ427295) of Ca. Phytoplasma trifolii strain A6 belonging to the phytoplasma group VI (reported from Iran). This is the first report of phytoplasma infection affecting Ajwain (T. ammi).     

Detection and identification of the maize bushy stunt phytoplasma in corn plants in Brazil using PCR and RFLP

Plants of corn (Zea mays L.) exhibiting symptoms of stunting and leaf reddening were assayed for the presence of phytoplasma gene sequences through the use of phytoplasma rRNA and ribosomal protein gene and maize bushy stunt (MBS) phytoplasma-specific oligonucleotide primers in polymerase chain reactions (PCR). Polymorphisms in 16S rDNA amplified from diseased plants were those characteristic of phytoplasmas classified in the16S rRNA gene group 16SrI, subgroup IB, of which MBS phytoplasma is a member. Amplification of ribosomal protein (rp) gene sequences in PCR primed by phytoplasma-specific primers confirmed presence of a phytoplasma in the diseased plants. Restriction fragment length polymorphism (RFLP) patterns of the amplified phytoplasma rp gene sequences were similar or identical to those observed for a known strain of MBS phytoplasma. In separate PCR, an MBS-specific oligonucleotide pair primed amplification of a MBS-characteristic DNA from templates derived from the diseased corn. Our data provide the first firm evidence for the presence of maize bushy stunt phytoplasma in corn in Brazil.