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1.
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   

2.
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   

3.
2022年首次在广州市发现园林植物雪花木小叶病病株, 采用分子生物学技术对其进行植原体的种类鉴定。以雪花木叶片总DNA为模板, 利用植原体16S rRNA通用引物P1/P7进行PCR扩增, 获得广东雪花木小叶病植原体(BLL-GD2022)16S rRNA基因片段(1 811 bp, GenBank登录号为OQ625536)。16S rRNA序列相似性显示, BLL-GD2022与16SrVI组植原体株系的相似性最高, 为97.05%~99.83%, 其中与隶属于16SrVI-D亚组的10个植原体株系相似性为99.21%~99.83%。系统进化分析显示, BLL-GD2022与16SrVI组各植原体株系聚类在一个大分支, 其中与16SrVI-D亚组成员聚类在一个小分支, 亲缘关系最近。基于16S rRNA序列的iPhyClassifier限制性内切酶虚拟RFLP分析表明, BLL-GD2022与16SrVI-D亚组的参考株系Brinjal little leaf phytoplasma (GenBank登录号为X83431)的酶切图谱一致, 相似系数为1.00。基于上述研究结果, 明确广州市雪花木小叶病植原体隶属16SrVI-D亚组成员。本研究首次在园林植物雪花木上检测到植原体, 通过16S rRNA序列分析明确为16SrVI-D亚组成员, 为开展16SrVI-D亚组植原体在蔬菜、花卉和园林植物的发生监测及病害防控提供科学依据。  相似文献   

4.
The 16S rRNA gene of plum leptonecrosis phytoplasma has been PCR-amplified, cloned and almost completely sequenced (1201 bp). The sequence analysis confirmed the close genetic relationship between plum leptonecrosis phytoplasma and the phytoplasmas associated with other stone-fruit diseases in Europe. By comparison with the 16S rDNA sequence of apple proliferation phytoplasma, two oligonucleotides were selected, differing by two nucleotides, which were specific for apple proliferation and plum leptonecrosis phytoplasmas, respectively. The oligonucleotides were labelled with digoxigenin and hybridized, in the presence of tetramethylammonium chloride, to 16S rDNA fragments amplified from apple and plum leaf samples. The results showed that, under the described hybridization conditions, the two phytoplasmas could clearly be distinguished. The advantage of the proposed technique over 16S rDNA restriction fragment length polymorphism is discussed.  相似文献   

5.
Davies 《Plant pathology》2000,49(1):86-88
Polymerase chain reaction using universal primers to sequences in the 16S rRNA gene, and group-specific primers to sequences in the 16S/23S spacer region, revealed two distinct phytoplasmas occurring in Rubus plants showing symptoms of rubus stunt. One phytoplasma appeared similar to phytoplasmas in the elm yellows group; the other appeared to fall into the X disease group. This finding was confirmed by RFLP analysis of PCR products. This is the first identification of phytoplasmas from either of these groups occurring in the UK, and the first report of a phytoplasma belonging to the X disease group in Rubus .  相似文献   

6.
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   

7.
Wang K  Hiruki C 《Phytopathology》2001,91(6):546-552
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.  相似文献   

8.
黄槐丛枝病植原体的检测及鉴定   总被引：1，自引：0，他引：1  
 应用植原体16S rRNA基因通用引物,对自然表现丛枝的黄槐植株进行巢式PCR检测,得到约1.2 kb的特异片段,证明此植株中存在植原体.将此特异片段与pGEM-T Easy载体连接并转化到大肠杆菌JM109感受态细胞中,通过PCR鉴定、序列测定及同源性比较分析,结果表明此植原体株系(STWB)16S rDNA片段G+C含量为45.8%,与榆树黄化植原体组(Elm yellows group,16SrV group)中的各株系最高同源率可达99.4%,而与其它组中的株系明显低于97.0%,故认为该植原体株系为榆树黄化植原体组中的成员之一.  相似文献   

9.

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   

10.
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   

11.
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.  相似文献   

12.
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.  相似文献   

13.
In the United States, yellow starthistle (Centaurea solstitialis) is an annual invasive weed with Mediterranean origins. Malformed plants displaying witches' broom, fasciations, abortion of buds and flower virescence symptoms were observed in central Italy. Attempts to transmit the causal agent from the natural yellow starthistle host to periwinkle by grafting, resulted in typical symptoms of a phytoplasma, i.e. yellowing and shortening of internodes. The detection of phytoplasmas was obtained from both symptomatic yellow starthistle and periwinkle by the specific amplification of their 16S-23S rRNA genes. PCR amplification of extracted DNA from symptomatic plant samples gave a product of expected size. Asymptomatic plants did not give positive results. An amplicon obtained by direct PCR with universal primers P1/P7 was cloned and sequenced. The homology search using CLUSTALW program showed more than 99% similarity with Illinois elm yellows (ILEY) phytoplasma from Illinois (United States) and 97% with Brinjal little leaf (BLL) phytoplasma from India. Digestion of the nested-PCR products with restriction enzymes led to restriction fragment length polymorphism patterns referable to those described for phytoplasmas belonging to the clover proliferation (16S-VI) group. Since this is a previously undescribed disease, the name Centaurea solstitialis virescence has been tentatively assigned to it. This is a new phytoplasma with closest relationships to ILEY and BLL, but distinguishable from them on the basis of 16S rDNA homology, the different associated plant hosts and their geographical origin.  相似文献   

14.
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.  相似文献   

15.
竹丛枝植原体16SrDNA片段克隆与序列分析   总被引：7，自引：0，他引：7       下载免费PDF全文
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。  相似文献   

16.
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   

17.
本研究对河北省大面积发生的金莲花绿变病的病原进行检测和鉴定。以金莲花叶片的总DNA为模板,使用植原体16S rDNA和核糖体蛋白(ribosomal protein)基因rp的特异性引物进行PCR扩增,在感病金莲花样品中扩增到植原体的16S rDNA(1 432 bp)片段和rp基因(1 240 bp)片段。序列分析发现,获得的16S rDNA和rp基因片段与洋葱黄化植原体Onion yellows phytoplasma(GenBank登录号:AP006628)的相似度最高,分别为99.9%和99.3%,确定金莲花绿变病的病原为植原体,暂命名为金莲花绿变植原体Trollius chinensis virescence phytoplasma。对金莲花绿变植原体的16S rDNA进行虚拟RFLP分析,发现其酶切图谱与16SrⅠ-B亚组的洋葱黄化植原体的参照图谱完全一致,相似系数1.00。16S rDNA和rp基因的系统发育进化树显示,金莲花绿变植原体与16SrⅠ-B亚组的植原体聚为一支,属于植原体16S rⅠ-B亚组。  相似文献   

18.
DNA primers, based on the ribosomal sequences of lethal yellowing-type disease (LYD) phytoplasmas, were used to analyse genetic variation within the lethal yellowing-type diseases of coconut in East Africa. Samples were collected from palms in Kenya, Mozambique and high, medium and low disease incidence areas of Tanzania. The mollicute-specific primer pair P1 and P6 amplified a 1.5 kbp product from all diseased palms and no product from symptomless palms, indicating that phytoplasmas were associated with all of these diseases. However, the Rohde forward and Rohde reverse primers (a second rRNA primer pair designed to detect East African LYD-associated phytoplasmas) only amplified products from Tanzanian and Kenyan diseased palms and not from those of Mozambique. Conversely, primers Ghana 813 and AK-SR, designed for specific detection of coconut-associated phytoplasmas in West Africa, amplified products only from the Mozambique palms, indicating that the phytoplasma associated with LYD in Mozambique is more closely related to those from West Africa. This was supported by restriction enzyme digestion of PCR products. DNA sequencing of PCR products from phytoplasmas within Tanzania revealed no detectable differences in the rDNA sequences of isolates from high, medium and low incidence areas.  相似文献   

19.
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   

20.
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.  相似文献   

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