Genetic variation in Candidatus Phytoplasma australiense

This study examined whether genes that are less conserved than the 16S rRNA gene can distinguish Candidatus Phytoplasma australiense strains that are identical based on their 16S rRNA genes, with a view to providing insight into their origins and distribution, and any patterns of association with particular plant hosts. Sequence analysis of the tuf gene and rp operon showed that Ca . P. australiense strains could be differentiated into four subgroups, named 16SrXII-B ( tuf -Australia I; rp -A), 16SrXII-B ( tuf -New Zealand I; rp -B), 16SrXII-B ( tuf -New Zealand II) and 16SrXII-B ( rp -C). Strawberry lethal yellows 1, strawberry green petal, Australian grapevine yellows, pumpkin yellow leaf curl and cottonbush witches' broom phytoplasmas were designated members of the 16SrXII-B ( tuf -Australia I; rp -A) subgroup. The strawberry lethal yellows 2 and cottonbush reduced yellow leaves phytoplasmas were assigned to the 16SrXII ( tuf -New Zealand II; rp -B) subgroup. No relationship was observed between these phytoplasma subgroups and collection date, location or host plant. However, the study revealed evolutionary divergence in the 16SrXII group.     

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     

Phytoplasmas infecting sour cherry and lilac represent two distinct lineages having close evolutionary affinities with clover phyllody phytoplasma

Phytoplasmas infecting sour cherry and lilac in Lithuania were found to represent two lineages related to clover phyllody phytoplasma (CPh), a subgroup 16SrI-(R/S)C (formerly 16SrI-C) strain exhibiting rRNA interoperon sequence heterogeneity. 16S rDNAs amplified from the cherry bunchy leaf (ChBL) and lilac little leaf (LcLL) phytoplasmas were identical or nearly identical to those of operon rrnA and operon rrnB, respectively, of CPh. There was no evidence of 16S rRNA interoperon sequence heterogeneity in either LcLL or ChBL phytoplasma. Based on collective RFLP patterns of 16S rDNA, ChBL was classified in subgroup 16SrI-R, and LcLL was classified in new subgroup 16SrI-S. The ribosomal protein (rp) gene sequences from LcLL phytoplasma were identical to those of CPh, and strain LcLL was classified in rp subgroup rpI-C. By contrast, rp gene sequences from ChBL phytoplasma differed from those of subgroup rpI-C; based on RFLP patterns of rp gene sequences, ChBL was classified in new rp subgroup rpI-O. Single nucleotide polymorphisms (SNPs), designated here by a new SNP convention, marked members of rp subgroup rpI-C, and distinguished LcLL and CPh from ChBL and other non-rpI-C phytoplasmas in group 16SrI. The results raise questions concerning phytoplasma biodiversity assessment based on rRNA genes alone and encourage the supplemental use of a single copy gene in phytoplasma identification and classification, while drawing attention to a possible role of horizontal gene transfer in the evolutionary history of these lineages.     

西北旱区枣疯病植原体核糖体蛋白基因的生物信息学分析

枣疯病是枣树上的一种具有毁灭性的植原体病害,几乎分布于国内所有的枣树栽培区,造成了巨大的经济损失.对我国陕西、宁夏、甘肃3省枣疯病样品植原体核糖体蛋白基因进行克隆和测序,获得枣疯病植原体的核糖体基因片段为1 196bp,包含部分rps19,rpl22和rps3三个基因,其中rpl22和rps3大小分别354bp和753bp,分别编码118和251个氨基酸,且这两个基因为非重叠基因.序列同源性比较结果表明:我国陕西、宁夏、甘肃的枣疯病植原体的核糖体蛋白rp基因大小一致,归属于植原体16S rⅤ-B组;该植原体核糖体蛋白基因特性与樱桃致死黄化(CLY5)和桃树黄化印度分离株系(PY-In)植原体相似.首次报道了我国枣疯病核糖体蛋白基因rp基因的序列,把枣疯病植原体归到16S rⅤ-B组,为枣疯病植原体提供了新的分类依据.     

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.     

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。     

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.     

Comparison of the ribosomal RNA operon from Texas Phoenix decline and lethal yellowing phytoplasmas

A comparison was made of the two palm yellows phytoplasmas affecting palms to determine if the entire ribosomal RNA operon portion of the phytoplasma genome, or portions thereof, could account for the observed palm host differences. Polymerase chain reaction (PCR) was used to amplify a 5.0?kb DNA fragment consisting of the entire ribosomal RNA operon from a subgroup 16SrIV-D phytoplasma that causes Texas Phoenix palm decline (TPD) in cabbage (Sabal palmetto) palm in west central Florida and from a subgroup 16SrIV-A phytoplasma that causes lethal yellowing (LY) in coconut (Cocos nucifera) palm in Jamaica. Before the PCR reaction, we sequenced by 454 sequencing a draft genome of the coconut LY phytoplasma, strain LYFL, that infects C. nucifera in Florida, and obtained from this draft sequence both copies of the entire ribosomal operon. Sequence analysis of the ribosomal RNA operons from both the LY and TPD phytoplasmas revealed the gene composition and orientation for the operons to be 5′16S rRNA-tRNA^Ile-23S rRNA-5S rRNA3′ and a tRNA^Val3′ downstream of the 5S rRNA gene. Based on molecular comparisons using the sequences of the ribosomal RNA operon, the TPD (16SrIV-D) strain was 98?% similar to the LY (16SrIV-A) strains.     

Electron Microscopy and Molecular Characterization of Phytoplasmas Associated with Strawflower Yellows in the Czech Republic

Strawflower (Helichrysum bracteatum) with symptoms resembling those associated to phytoplasma infection were observed in several areas in the Czech Republic during the period 1994–2001. Plants with leaf bronzing, reddening and necrosis, proliferation of secondary shoots, flower abnormalities and dwarfing died in advanced stages of the disease. The disease incidence ranged from 2% to 70% and caused significant loss to the flower and seed production. Transmission electron microscopy showed phytoplasmas in sieve cells of affected plants, but not in healthy ones. Association of phytoplasmas with the disease was confirmed by polymerase chain reaction using phytoplasma universal ribosomal primers R16F2n/R16R2. An amplification product of the expected size (1.2 kb) was observed in all samples of the symptomatic strawflowers. The restriction profiles obtained following separate digestion with three endonucleases (AluI, HhaI, MseI) showed that phytoplasmas infecting strawflowers from different localities in the Czech Republic were uniform and undistinguishable from aster yellows (subgroup 16SrI-B). Sequence analysis of 1771 bp of the ribosomal operon amplified with primers P1/U3, R16F2n/R2 and 16R758/P7 indicated that the closest related phytoplasmas were those associated with 'Rehmannia glutinosa var. purpurea', both originating from Bohemia. This is the first report on the occurrence of a phytoplasma-associated disease of strawflower in the Czech Republic.     

A Leafhopper (Hishimonus sellatus) Transmits Phylogenetically Distant Phytoplasmas: Rhus Yellows and Hovenia Witches' Broom Phytoplasma

Phytoplasmas causing a severe decline of three tree species, i.e., Rhus javanica, Hovenia tomentella and Zizyphus jujuba, in Japan were examined for their transmissibility by a leafhopper species Hishimonus sellatus, and for their phylogenetic relatedness. By H. sellatus, Rhus yellows (RhY) phytoplasma was transmissible to white clover and periwinkle seedlings, causing typical symptoms in these plants. Jujube witches' broom (JWB) phytoplasma was also transferred to the host plant, Z. jujuba, by the leafhopper. Because JWB phytoplasma was transmitted to Hovenia tomentella and caused the same symptoms as Hovenia witches' broom (HWB), JWB phytoplasma may be very closely related to HWB phytoplasma. RFLP analysis of the PCR products of 16S rDNA revealed that RhY phytoplasma belongs to the Aster yellows (AY) group, and JWB and HWB phytoplasmas belong to a different group (possibly Elm yellows group). Thus, we found that one species of leafhopper can carry phylogenetically distant phytoplasmas. Received 23 April 2001/ Accepted in revised form 29 October 2001     

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.     

Characterisation of a 16SrII phytoplasma strain associated with bushy stunt of hawkweed oxtongue (Picris hieracioides) in south-eastern Serbia and the role of the leafhopper Neoaliturus fenestratus (Deltocephalinae) as a natural vector

A 2-year study of host association, molecular characterisation and vector transmission of a phytoplasma related to the 16SrII group in a vineyard of south-eastern Serbia was conducted. Grapevine, eight common weeds and 31 Auchenorrhyncha species were collected and analysed for phytoplasma presence. PCR-RFLP analyses of the 16S rRNA gene identified the presence of a new strain of phytoplasma related to the 16SrII group in P. hieracioides with symptoms of stunting or bushy stunting. Grapevine samples, all without symptoms, were negative for phytoplasma presence. Plants of Erigeron annuus, Cynodon dactylon, Daucus carota and P. hieracioides, either exhibiting symptoms of yellowing or without symptoms, were positive for the presence of stolbur phytoplasma. Among the tested cicada species, seven were infected with phytoplasmas from the aster yellows group, two with stolbur phytoplasma, two with 16SrII phytoplasma, and one with the 16SrV-C phytoplasma subgroup. The phytoplasma strain of the 16SrII group was recorded in approximately 50?% of the collected leafhopper species Neoaliturus fenestratus and in a few specimens of the planthopper Dictyophara europaea. The vector status of N. fenestratus was tested using the second generation of the planthopper in two separate transmission trials with P. hieracioides and periwinkle seedlings. In both tests, the leafhopper successfully transmitted 16SrII phytoplasma to exposed plants, proving its role as a natural vector of this phytoplasma in Europe. A finer molecular characterisation and phylogenetic relatedness of the 16SrII phytoplasma strain by sequence analyses of the 16S rRNA and ribosomal protein genes rpl22-rps3 indicated that it was most closely related to the 16SrII-E subgroup.     

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.     

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。     

Molecular characterization and phylogeny of phytoplasma associated with bunchy top disease in its new host Okra (<Emphasis Type="Italic">Abelmoschus esculentus</Emphasis>) in India reveal a novel lineage within the 16SrI group

Okra plants with bunchy top disease were found to be prevalent during the period of August–October 2009 in New Delhi, India. The common symptoms observed were shortening of internodes, aggregation of leaves at the apical region, reduced leaf lamina, stem reddening, fruit bending, phyllody and stunting of plants. The disease incidence ranged from 2–60% accompanied by significant reductions in production of both flowers and seeds. Nested polymerase chain reaction targeting phytoplasma specific 16S rDNA and rp genes revealed all symptomatic plants to be positive for phytoplasma. Homology searches depicted its closest identity to phytoplasmas of 16SrI ‘Candidatus Phytoplasma asteris’, like the Sugarcane yellows and Periwinkle phyllody phytoplasmas. Profiles for 16S rDNA obtained with 10 restriction endonucleases, differed in TaqI sites for two phytoplasma isolates (BHND5 & 10) from the standard pattern of 16SrI-B subgroup, the latter was seen in the case of isolate BHND1. Restriction fragment analysis of rp genes with AluI, Tsp509I matched with patterns of the rpI-B phytoplasmas. Phylogenetic reconstruction of rp genes revealed okra bunchy top phytoplasma (BHND1) as a divergent isolate, the subsequent sequence analysis of which showed the presence of a novel BslI site. These significant differences suggest that multiple phytoplasma strains are affecting okra, one of which is a diverging lineage within the 16SrI-B group while others represent a new 16SrI subgroup not reported so far. Additionally, this is the first report of a phytoplasma associated disease in okra plants worldwide.     

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.     

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.     

First report of a 16SrIII-B subgroup phytoplasma associated with leaf reddening, virescence and phyllody of purple coneflower

Purple coneflower plants showing leaf reddening and flower abnormalities were observed in South Bohemia (Czech Republic). Transmission electron microscopy observations showed phytoplasmas in sieve cells of symptomatic plants but not in healthy ones. Polymerase chain reactions with universal and group specific phytoplasma primers followed by restriction fragment length polymorphism analyses of 16S rDNA allowed us to classify the detected phytoplasmas into the X-disease group, ribosomal subgroup 16SrIII-B. Sequence analyses of the 16S-23S ribosomal operon (1684 bp), ribosomal protein L15, and protein translocase genes (1566 bp) confirmed the closest relationship with phytoplasmas belonging to the 16SrIII ribosomal group, specifically the 16SrIII-B subgroup. The current study reports purple coneflower as a new host for the X-disease phytoplasma group in the Czech Republic and worldwide.