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1.
Mesta (Hibiscus sabdariffa) is an important bast fiber crop. In August 2011, there was an outbreak of a phytoplasma-like disease on H. sabdariffa in different villages of the northern coastal mesta-growing region of Andhra Pradesh, India, covering mainly two districts – Srikakulam and Vijayanagaram. The infected plants showed characteristic symptoms such as phyllody and reddening of leaves. PCR with P1/P7 universal primer pair of 16 S rDNA yielded amplicons of 1850 bp from all symptomatic mesta leaf samples similar to samples of brinjal little leaf (phytoplasma positive reference control). However, asymptomatic samples were not amplified. Multiplex nested-PCR showed simultaneous amplification of DNA fragments with phytoplasma specific primers, viz., P1/P7 universal primer pair of 16 S rDNA, nested primer pair R16F2n/R2, uvrB and DegV gene-specific uvrB-degVF/R primer generating amplicons of 1850 bp, 1200 bp and 1023bp, respectively. However, SecY-map gene specific primer SecY-mapF/R was not amplified. The 1023 bp nucleotide sequence of uvrB and DegV gene of the phytoplasma was deposited in the GenBank (NCBI) with the accession no. JX975061. NCBI BLASTn analysis of the 1023 bp products showed that the phytoplasma strain belonged to elm yellows group (16SrV-D). This is the first report that Hibiscus sabdariffa is infected by a phytoplasma and we named it mesta phyllody disease (MPD).  相似文献   

2.
 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   

3.
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.  相似文献   

4.
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   

5.
小麦蓝矮病植原体16S rDNA序列分析研究   总被引：5，自引：3，他引：2  
 小麦蓝矮病是我国西北地区冬小麦上一种重要病害。本研究利用植原体16S rDNA通用引物对小麦蓝矮病患病植株全DNA进行nest-PCR扩增,获得1.2 kb的特异片段,并对扩增产物进行核苷酸序列测定,从分子水平证明了小麦蓝矮病的病原是植原体。利用最大简约法构建了16S rDNA系统演化树,系统演化关系分析表明:小麦蓝矮病植原体应该归属于翠菊植原体(Candidatus Phytoplasma asteris);小麦蓝矮病植原体与三叶草变叶病植原体(CPh)关系密切,被聚类为同一亚组(16Sr I-C),但是它们在寄主范围和传播介体等生物学性状方面差异很大。  相似文献   

6.
安徽桑黄花型萎缩病植原体16S rDNA序列分析及分子检测   总被引：1，自引：0，他引：1  
 Mulberry yellow dwarf(MYD)disease is an quarantine disease and the causal agent is a phytoplasma.Two pairs of published universal primer, P1/P7 and Rm16F2/Rm16R1, based on the 16S-23S rDNA sequence of phytoplasma and total DNA extracted from infected mulberry tissues were employed for PCR and nested-PCR detection.The results revealed that a phytoplasma-specific 1 830 bp fragment with a G+C content of 46.01% was sequenced(GenBank accession No.GQ249410).The sequence shared 99.7% and 99.8% identity with aster yellows, the representatiive phytoplasma in 16SrI group, and mulberry dwarf phytoplasma classified into subgroup B in 16SrI group and named as the MYD phytoplasma strain Anhui(MYD-Anh).A phylogenetic tree based on 16S rDNA sequences was constructed and showed that MYD-Anh was clustered into 16SrI group.Identity of 16S rDNA sequence between MYD-Anh and mulberry yellow dwarf phytoplasma strain Zhenjiang(MD-zj) was nearly 100%, and they might belong to the same strain.Nested-PCR was used to detect the pathogenic phytoplasma from the differential tissues of mulberry infected with MYD-Anh.The results showed that a phytoplasma-specific 1.4 kb fragment was amplified with total DNA extracted from bark and vein.Nested-PCR was more sensitive than PCR for detecting MYD phytoplasma.  相似文献   

7.
Berberis thunbergii atropurpurea, also known as Red barberry, is a small ornamental shrub in the family Berberidaceae. In recent years, a phyllody symptom has been observed frequently, spreading in the shrubs in northwestern China. A phytoplasma 16S rDNA specific fragment was amplified by PCR from Berberis plants with the phyllody symptoms. DNA sequencing and restriction fragment length polymorphism analysis revealed the phytoplasma belongs to 16SrV-B subgroup. This is the first report that Berbegis thunbergii atropurpurea is a host for 16SrV-B phytoplasma. The disease was named Berberis phyllody.  相似文献   

8.
从表现黄化(丛枝)症状的桉树上采集病叶,抽提主脉总DNA,采用植原体通用引物与巢式引物进行PCR和巢式PCR扩增,对扩增产物进行克隆和序列测定,获得了植原体的近全长16S rRNA基因及部分16～23S rRNA基因间隔区序列.序列分析揭示,所获得的序列与已知植原体基因组相应区段的序列高度同源,与柳叶菜变叶植原体(epilobium phyllody)和白腊树丛枝植原体(ash witches'-broom)相应序列(GenBank登录号:AY101386和AY566302)同源率为99.9%,与白腊树黄化植原体(aster yellows BD2)相应序列和番茄巨芽植原体(tomato big bud)相应序列同源率分别为99.6%和99.3%.该序列构建的系统进化树表明,引起我国广州地区桉树黄化(丛枝)病的植原体属于16SrI组(即翠菊黄化组),将其暂命名为桉树黄化(丛枝)植原体广东株系(Eucalyp-tus yellowing and witches'-broom phytoplasma strain Guangdong,EYWB-Gd).建立了桉树植原体巢式PCR检测方法,对疑似病样及桉树组培苗进行了检测,多数疑似病样检测结果为阳性,供试的10株组培苗未发现阳性样品.  相似文献   

9.
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   

10.
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.  相似文献   

11.
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   

12.
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   

13.
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   

14.
小麦蓝矮病植原体16S rDNA基因片段的比较分析   总被引：21，自引：3，他引：18  
 小麦蓝矮病是陕西乃至西北冬麦麦区的一个重要病害,由介体条沙叶蝉专化性传播。对小麦蓝矮病株叶片和带毒条沙叶蝉进行超薄切片及电镜观察,在叶片韧皮部和叶蝉后肠中均观察到大量典型植原体。利用植原体16S rDNA基因保守序列通用引物对Rm16F2/Rm16R1,应用PCR技术从小麦蓝矮病株叶片中扩增到1.4 kb的特异片段。通过对16S rDNA基因片段序列同源性比较,结果表明小麦蓝矮病病原与三叶草绿变、翠菊黄化、绣球花绿变、草莓矮化和番茄巨芽植原体亲缘关系较近,其同源率为99.2%~99.9%。据此可以判定小麦蓝矮病植原体是属于植原体16SrⅠ组,确定了其分类地位。  相似文献   

15.
 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。  相似文献   

16.
A disease on parthenium weed (Parthenium hysterophorus L.) was observed in June 2008 in Danzhou of Hainan Province. Infected weeds showed phytoplasma-like associated symptoms such as severe stunting, excessive proliferation of shoots, inflorescence-clustering, green petal, small leaves and witches’-broom. The original cause of phytoplasma was further confirmed by polymerase chain reaction (PCR). PCR products of 1.8 kb were obtained using the universal primers pair (P1/P7) designed to amplify the entire 16S rDNA and the 16/23S intergenic spacer region in a direct PCR assay. The primers pair R16F2n/R2 was used to amplify a PCR product of 1.2 kb. Restriction fragment length polymorphism (RFLP) was used to analyze the partial 16S rDNA sequences (1.2 kb) of all phytoplasma DNA digested with five endonucleases (Kpn I, Hpa II, Taq I, Rsa I, EcoR I). The RFLP patterns of the strain were found to be identical with that of the reference peanut witches’-broom phytoplasma. Based on the RFLP data, it is suggested that the phytoplasma strain belongs to subgroup 16SrII-A. This is the first demonstration of a 16SrII-A group phytoplasma associated with parthenium weed.  相似文献   

17.
Sugarcane is an important cash crop in Vietnam and has been widely promoted at national and provincial level. In 2006, a new disease was discovered in sugarcane in the Nghean Tate&Lyle Sugar Mill in Nghean Province in north-central Vietnam. The key symptoms were the formation of green grassy shoots around the base of mature stools. We applied nested polymerase chain reaction (PCR) using P1/P7 and R16F2n/R16R2 for detection and characterization of phytoplasma from the symptomatic tissues. PCR products of the expected size (approx. 1200?bp) were obtained from the 16S rDNA of the phytoplasma. The restriction fragment length polymorphism profiles indicated that all samples were infected by the same phytoplasma. Phylogenetic analysis confirmed that the SCGS phytoplasma from Vietnam belong to the 16SrXI group, formerly Rice Yellow Dwarf group.  相似文献   

18.
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   

19.
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   

20.
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.  相似文献   

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