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1.
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.  相似文献   

2.
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.  相似文献   

3.
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.  相似文献   

4.
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  相似文献   

5.
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.  相似文献   

6.
Polymerase chain reaction (PCR) assays were used to detect phytoplasmas in foliage samples from Chinaberry ( Melia azedarach ) trees displaying symptoms of yellowing, little leaf and dieback in Bolivia. A ribosomal coding nuclear DNA (rDNA) product (1·8 kb) was amplified from one or more samples from seven of 17 affected trees by PCR employing phytoplasma-universal rRNA primer pair P1/P7. When P1/P7 products were reamplified using nested rRNA primer pair R16F2n/R16R2, phytoplasmas were detected in at least one sample from 13 of 17 trees with symptoms. Restriction fragment length polymorphism (RFLP) analysis of P1/P7 products indicated that trees CbY1 and CbY17 harboured Mexican periwinkle virescence (16SrXIII)-group and X-disease (16SrIII)-group phytoplasmas, respectively. Identification of two different phytoplasma types was supported by reamplification of P1/P7 products by nested PCR employing X-disease-group-specific rRNA primer pair R16mF2/WXint or stolbur-group-related primer pair fSTOL/rSTOL. These assays selectively amplified rDNA products of 1656 and 579 bp from nine and five trees with symptoms, respectively, of which two trees were coinfected with both phytoplasma types. Phylogenetic analysis of 16S rDNA sequences revealed Chinaberry yellows phytoplasma strain CbY17 to be most similar to the chayote witches'-broom (ChWBIII-Ch10) agent, a previously classified 16SrIII-J subgroup phytoplasma. Strain CbY1 resembled the Mexican periwinkle virescence phytoplasma, a 16SrXIII-group member. The latter strain varied from all known phytoplasmas composing group 16SrXIII. On this basis, strain CbY1 was assigned to a new subgroup, 16SrXIII-C.  相似文献   

7.
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.  相似文献   

8.
The identity of the presumed phytoplasmal pathogen associated with cranberry false-blossom disease has never been fully clarified. In the present study a molecular-based procedure was employed to determine the identity of the phytoplasma. Tissues of cranberry plants exhibiting cranberry false-blossom symptoms were collected from multiple bogs on each of three randomly selected commercial cranberry farms in New Jersey. Leafhoppers, including the known vector Limotettix vaccinii (Van Duzee) (=Scleroracus vaccinii, Euscellis striatulus) and the sharp-nosed leafhopper Scaphytopius magdalensis (Provancher), a known vector of blueberry stunt disease, were collected from two different farms in New Jersey. Nested PCR assays and RFLP analysis of 16S rRNA gene sequences were employed for the detection and identification of the associated phytoplasmas. All of 20 cranberry plants sampled and five out of 14 batches of leafhoppers tested positive for phytoplasma. Virtual RFLP and sequence analyses revealed that all the associated phytoplasmas were members or variants of a new subgroup, 16SrIII-Y. Phylogenetic analysis of 16S rRNA sequences indicated that cranberry false-blossom phytoplasma strains represented a lineage distinct from other 16SrIII subgroups. This is the first report confirming that a new phytoplasma (designated as a new subgroup 16SrIII-Y) is associated with cranberry false-blossom disease and associated with both leafhopper species in New Jersey.  相似文献   

9.
Twelve Argentinean 16SrIII (X-disease)-group phytoplasma strains were analyzed. Ten of them, detected in daisy (Bellis perennis), garlic (Allium sativum), ‘lagaña de perro’ (Caesalpinia gilliesii), periwinkle (Catharanthus roseus), ‘rama negra’ (Conyza bonariensis), ‘romerillo’ (Heterothalamus alienus), summer squash (Cucurbita maxima var. zapallito) and tomato (Solanum lycopersicum), are new phytoplasma strains while two strains, detected in garlic and China tree (Melia azedarach), have been previously described. The plants showed typical symptoms of phytoplasma diseases, such as leaf size reduction, proliferation, stunting and virescence. The identification and genetic diversity analysis of the phytoplasmas were performed based on 16S rDNA and ribosomal protein gene sequences. The classification into 16Sr groups and subgroups was established by actual and virtual RFLP analysis of the PCR products (R16F2/R16R2) compared with reference strains. According to the classification scheme, strains HetLL and ConWB-A and B represent two new subgroups 16SrIII-W and X, respectively. On the other hand, strains CatLL, TomLL and CaesLL are related to subgroup 16SrIII-B, and strains BellVir, TomRed, CucVir and GDIII-207 are related to subgroup 16SrIII-J. Ribosomal protein genes were amplified using primers rpF1/rpR1 and rpIIIF1/rpIIIR1. RFLP analysis performed with AluI, DraI and Tru1I (MseI isoschizomer) distinguished three new rp profiles within subgroup 16SrIII-B, one for subgroup 16SrIII-J, and one shared with strains of the new subgroups 16SrIII-W and X. The phylogenetic analysis based on 16S rDNA and ribosomal protein gene sequences confirmed the separation of HetLL and ConWB strains in two new subgroups and the close relatedness among subgroup J phytoplasmas, which have been detected only in South America.  相似文献   

10.
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.  相似文献   

11.
三叶草(Trifolium pratense Linn.)为车轴草属,蝶形花科多年生草本植物,原产亚洲南部和欧洲东南部,是一种世界性分布与栽培的优良牧草.因其花叶兼优、草姿美、绿期长而具有较高的观赏价值,近几年作为草坪用草被广泛种植.在自然条件下,三叶草很容易受到不同种植原体的侵染,国外已报道的侵染三叶草的植原体有:三叶草绿变植原体( Clover phyllody phytoplasma,CPh)和三叶草增殖植原体(Clover proliferation phytoplasma,CP)等,这些植原体分别属于16SrI组和16SrⅥ组[1,2].  相似文献   

12.
 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。  相似文献   

13.
Phytoplasma: ecology and genomic diversity   总被引：1，自引：0，他引：1  
ABSTRACT The recent development of molecular-based probes such as mono- and polyclonal antibodies, cloned phytoplasma DNA fragments, and phytoplasma-specific primers for polymerase chain reaction (PCR) has allowed for advances in detection and identification of uncultured phytoplasmas (formerly called mycoplasma-like organisms). Comprehensive phylogenetic studies based on analysis of 16S ribosomal RNA (rRNA) or both 16S rRNA and ribosomal protein gene operon sequences established the phylogenetic position of phytoplasmas as members of the class Mollicutes, and the revealed phylogenetic interrelationships among phytoplasmas formed a basis for their classification. Based on restriction fragment length polymorphism (RFLP) analysis of PCR-amplified 16S rRNA gene sequences, phytoplasmas are currently classified into 14 groups and 38 subgroups that are consistent with groups delineated based on phylogenetic analysis using parsimony of 16S rRNA gene sequences. In the past decades, numerous phyto-plasma strains associated with plants and insect vectors have been identified using molecular-based tools. Genomic diversity of phytoplasma groups appears to be correlated with their sharing common insect vectors, host plants, or both in nature. The level of exchange of genetic information among phytoplasma strains in a given group is determined by three-way, vector-phytoplasma-plant interactions. A putative mechanism for the creation of new ecological niches and the evolution of new ecospecies is proposed.  相似文献   

14.
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.  相似文献   

15.
Foliar and root symptoms are described for Australian lucerne yellows (ALuY), a disease common in Australian lucerne seed crops. A phytoplasma was detected in plants exhibiting symptoms, but not in symptomless lucerne plants. Oligonucleotide primers specific to the phytoplasma 16S-23S rRNA intergenic spacer region (SR) were used in polymerase chain reaction (PCR) assays on DNA extracted from lucerne plants with and without symptoms. Identical restriction fragment length polymorphism (RFLP) enzyme profiles were obtained for PCR products amplified from 10 yellows-affected lucerne samples. RFLP profiles obtained for four restriction enzymes were different from those of the tomato big bud (TBB) phytoplasma. ALuY phytoplasma PCR products were sequenced to determine phylogeny and were found to fall within the faba bean phyllody phytoplasma group, or phytoplasma group 16srII. Transmission electron microscopy revealed phytoplasmas in the phloem of yellows-affected plant samples, but not in symptomless plant samples. Fungal, bacterial and viral agents in the aetiology of Australian lucerne yellows were ruled out.  相似文献   

16.
Phytoplasmas associated with lettuce phyllody (LP) and wild lettuce phyllody (WLP) in southern Iran were partially characterized by molecular analyses and host-range studies. Agents of both diseases were transmitted by Neoaliturus fenestratus , a leafhopper colonizing lettuce and wild lettuce, to lettuce, wild lettuce, sowthistle and periwinkle, but not to safflower, sunflower, calendula and sesame. Both phytoplasmas induced bud proliferation, virescence, phyllody and witches' broom in infected plants. Total DNA extracted from infected lettuce and wild lettuce or from vector tissues was subjected to PCR using phytoplasma-specific primer pair P1/P7 or nested PCR using P1/P7 followed by R16F2n/R16R2. PCR product of nested PCR (1·2 kbp) was subjected to restriction fragment length polymorphism (RFLP). RFLP analysis of nested PCR product identified the LP, WLP and N. fenestratus -associated phytoplasmas as members of the pigeon pea witches' broom group, 16SrIX. Phylogenetic analysis of the 16S rRNA gene sequence also clustered LP and WLP phytoplasmas with other known members of the 16SrIX group. While no significant differences could be detected between LP and WLP phytoplasmas, both isolates differed from Lebanese wild lettuce phyllody in molecular properties.  相似文献   

17.
Coconut palm ( Cocos nucifera ), oil palm ( Elaeis guineensis ), Bermudagrass ( Cynodon dactylon ) and Madagascar periwinkle ( Catharanthus roseus ) with symptoms indicative of phytoplasma disease were collected from different locations in Malaysia. PCR assays employing phytoplasma universal rRNA gene primers P1/P7 alone or P1/P7 followed by R16F2n/R16R2 detected phytoplasmas in eight out of 20 Malayan Red Dwarf (MRD), nine out of 12 Malayan Yellow Dwarf (MYD) and 12 out of 12 Malayan Tall (MT) coconut palms displaying coconut yellow decline symptoms. Positive detections were also obtained from six out of six oil palm seedlings showing symptoms of yellowing and necrosis, from 10 out of 10 Bermudagrass samples with white leaf symptoms, and from eight out of eight periwinkle plants showing phyllody, virescence, little leaf, proliferation and foliar yellowing. Phytoplasmas were not detected in any of the symptomless plants tested. Sequencing and phylogenetic analysis of PCR products determined that phytoplasmas infecting both MRD and MT coconuts and Bermudagrass in Serdang, Selangor State, were all members of the 16SrXIV ' Candidatus Phytoplasma cynodontis' group, whereas isolates in periwinkle in Serdang were all members of the 16SrI ' Ca. Phytoplasma asteris' group. However, the phytoplasmas detected in MYD coconuts and oil palms from Banting, Selangor State, and in periwinkle from Putrajaya were collectively very similar (99%), but shared <97·5% similarity with 16S rDNA sequences of all other known phytoplasmas, indicating that they represent a novel taxonomic group. Thus, at least two phylogenetically distinct phytoplasmas are associated with the coconut yellow decline syndrome in Malaysia, both of which were also detected in other plant species.  相似文献   

18.
臭矢菜丛枝病植原体的分子鉴定研究   总被引：1，自引：0，他引：1  
 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。  相似文献   

19.
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.  相似文献   

20.

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.  相似文献   

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