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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.
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   

3.
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   

4.
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   

5.
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   

6.
甘蔗白叶病(sugarcane white leaf,SCWL)是由植原体引起的重要甘蔗病害[1],广泛分布在印度、泰国等许多国家[1,2].我国甘蔗产区的栽培品种也有SCWL的发生[3].甘蔗是无性繁殖作物,植原体可通过繁殖种苗进行传播,台湾斑纹叶蝉(Matsumuratetlix hiroglyhious)通过咬食感染甘蔗植株的韧皮部可引起该病害[4].  相似文献   

7.
Candidatus Phytoplasma prunorum was detected for the first time in almond (Prunus dulcis Mill.) cv. ‘Abiod’ in Tunisia. Infected trees showed emergence of new growth during dormancy and leafed out before flowers opened in addition to early defoliation in summer. Phytoplasma was detected by nested polymerase chain reaction (PCR) using universal phytoplasma primer pairs P1/P7 and F2n/R2. A band with expected size was observed in samples collected from five symptomatic, but not symptomless almond trees. PCR products (1.2 kbp) were used for restriction fragment length polymorphism (RFLP) analysis after digestion with endonucleases RsaI and SspI. RFLP patterns obtained were similar to those reported previously for the European stone fruit yellows (ESFY, 16SrX-B). Identification has been further confirmed by PCR using ESFY specific primer pairs (ECA1/ECA2). This is the first report of Ca. Phytoplasma prunorum infecting almonds in Tunisia.  相似文献   

8.
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   

9.
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.  相似文献   

10.
A conventional PCR and a SYBR Green real-time PCR assays for the detection and quantification of Phytophthora cryptogea, an economically important pathogen, have been developed and tested. A conventional primer set (Cryp1 and Cryp2) was designed from the Ypt1 gene of P. cryptogea. A 369 bp product was amplified on DNA from 17 isolates of P. cryptogea. No product was amplified on DNA from 34 other Phytophthora spp., water moulds, true fungi and bacteria. In addition, Cryp1/Cryp2 primers were successfully adapted to real-time PCR. The conventional PCR and real-time PCR assays were compared. The PCR was able to detect the pathogen on naturally infected gerbera plants and on symptomatic artificially infected plants collected 21 days after pathogen inoculation. The detection limit was 5 × 103 P. cryptogea zoospores and 16 fg of DNA. Real-time PCR showed a detection limit 100 times lower (50 zoospores, 160 ag of DNA) and the possibility of detecting the pathogen in symptomless artificially infected plants and in the re-circulating nutrient solution of closed soilless cultivation systems.  相似文献   

11.
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.  相似文献   

12.
In February 2007, sweet orange trees with characteristic symptoms of huanglongbing (HLB) were encountered in a region of S?o Paulo state (SPs) hitherto free of HLB. These trees tested negative for the three liberibacter species associated with HLB. A polymerase chain reaction (PCR) product from symptomatic fruit columella DNA amplifications with universal primers fD1/rP1 was cloned and sequenced. The corresponding agent was found to have highest 16S rDNA sequence identity (99%) with the pigeon pea witches'-broom phytoplasma of group 16Sr IX. Sequences of PCR products obtained with phytoplasma 16S rDNA primer pairs fU5/rU3, fU5/P7 confirm these results. With two primers D7f2/D7r2 designed based on the 16S rDNA sequence of the cloned DNA fragment, positive amplifications were obtained from more than one hundred samples including symptomatic fruits and blotchy mottle leaves. Samples positive for phytoplasmas were negative for liberibacters, except for four samples, which were positive for both the phytoplasma and 'Candidatus Liberibacter asiaticus'. The phytoplasma was detected by electron microscopy in the sieve tubes of midribs from symptomatic leaves. These results show that a phytoplasma of group IX is associated with citrus HLB symptoms in northern, central, and southern SPs. This phytoplasma has very probably been transmitted to citrus from an external source of inoculum, but the putative insect vector is not yet known.  相似文献   

13.
Chickpea (Cicer arietinum L.) plants showing typical symptoms of infection by a phytoplasma that causes phyllody disease have been commonly observed in recent years in parts of south India. The symptoms included pale green leaves, bushy appearance due to excessive stunting of shoots, reduced internodal length and excessive axillary proliferation. The causal agent of the phyllody disease was identified based on symptoms, amplification of 16S rDNA of the phytoplasma by polymerase chain reaction (PCR) from infected samples, as well as by sequencing and phylogenetic analysis. First round PCR and nested-PCR protocols were standardized for improved efficiency and reliability of the diagnostic protocols. Using the primers P1/P7 and R16F2n/R16R2, 1,800?bp and 1,200?bp size products were amplified in first round PCR and nested-PCR protocols, respectively. The PCR product was cloned and sequenced and compared with the reference phytoplasma sequences from the database (NCBI). The Indian chickpea phyllody phytoplasma 16S rDNA sequences shared the highest nucleotide identity (>98%) with the 16S rII group phytoplasma candidates, also infecting chickpea from Australia and Pakistan. This is the first report of a phytoplasma of the 16SrII-group infecting chickpea from India. The genetic similarities and the potential threat of this new disease to chickpea cultivation in India are discussed.  相似文献   

14.
The phytoplasmas of groups 16SrI (‘Candidatus Phytoplasma asteris’) and 16SrVII (‘Ca. Phytoplasma fraxini’) have been associated with phytoplasma diseases in several urban tree species in Bogotá, Colombia and surrounding areas. The insect vectors responsible for this phytoplasma transmission are unknown. The objectives of this study were to test for the presence of phytoplasmas in leafhopper species (Cicadellidae) collected in areas with diseased trees and to determine the phytoplasma transmission ability of two of these species. Leafhoppers of nine species were collected at two sampling sites and tested by nested or double nested PCR using primers for the 16S rRNA gene. The amplicons were subjected to RFLP and/or sequencing analysis. Phytoplasmas of group 16SrI were detected in morphospecies MF05 (Haldorus sp.), group 16SrVII in MF07 (Xestocephalus desertorum), MF08 (Empoasca sp.) and MF09 (Typhlocybinae), and both groups 16SrI and 16SrVII in MF01 (Empoasca sp.), MF02 (Typhlocybinae), MF03 (Scaphytopius sp.), MF04 (Amplicephalus funzaensis) and MF06 (Exitianus atratus). Transmission tests to uninfected bean plants (Phaseolus vulgaris) were performed using field collected A. funzaensis and E. atratus individuals in separate assays. After 5 weeks, the test plants exposed to individuals of both species of leafhoppers showed symptoms, suggesting phytoplasma infection. Phytoplasma groups 16SrI and 16SrVII were detected in the two groups of exposed plants, indicating that A. funzaensis and E. atratus were able to transmit both groups of phytoplasmas. This is the first report of insect vectors for phytoplasmas of group 16SrVII in the world and of 16SrI in South America.  相似文献   

15.
This study investigated the potential of seed transmission of Cape St. Paul wilt disease (CSPWD) in coconuts. PCR amplification was used to assess the distribution of phytoplasmas in parts of West African Tall (WAT) palms infected with CSPWD. Employing phytoplasma universal primer pair P1/P7 in standard PCR, or followed with a nested PCR using CSPWD–specific primer pair G813f/AwkaSR, phytoplasma infection was detected in the trunks, peduncles, spikelets, male and female flowers of four infected WAT coconut palms. Through nested PCR, phytoplasma was also detected in four of 19 embryo DNA samples extracted individually from fruits harvested from three of the four infected palms and was confirmed as CSPWD by cloning and sequencing. Subsequently, CSPWD phytoplasma was again detected in five of 33 embryos from nine infected palms, and in one of eight fruits from two symptomless palms. Fruits from infected palms recorded higher percentage germinations in two field nurseries (average of 71·0%) compared to fruits from healthy palms (average of 57·6%), and matured fruits that had dropped from infected palms showed the same levels of germination as those harvested directly from the palms. This indicates that infected fruits retain the ability to germinate whether harvested or dropped. No phytoplasmas were detected in any of the resulting seedlings and plantlets obtained through embryo in-vitro culture. Therefore, although phytoplasma DNA can be detected in embryos, there is as yet no evidence that the pathogen is seed transmitted through to the seedling to cause disease in progeny palms.  相似文献   

16.
A transposon‐like element, A3aPro, with multiple copies in the Phytophthora sojae genome, was identified as a suitable detection target for this devastating soyabean root rot pathogen. The PCR primers TrapF1/TrapR1 were designed based on unique sequences derived from the transposon‐like sequence. A 267‐bp DNA fragment was amplified using this primer pair, the specificity of which was evaluated against 118 isolates of P. sojae, 72 isolates of 25 other Phytophthora spp., isolates of Pythium spp. and isolates of true fungi. In tests with P. sojae genomic DNA, detection sensitivities of 10 pg and 10 fg DNA were achieved in standard PCR (TrapF1/TrapR1) and nested PCR (TrapF1/TrapR1 and TrapF2/TrapR2), respectively. Meanwhile, PCR with TrapF1/TrapR1 primers detected the pathogen at the level of a single oospore, and even one zoospore. These primers also proved to be efficient in detecting pathogens from diseased soyabean tissues, residues and soils. In addition, real‐time quantitative PCR (qPCR) assays coupled with the TrapF1/TrapR1 primers were developed to detect and quantify the pathogen. The results demonstrated that the TrapF1/TrapR1 and TrapF2/TrapR2 primer‐based PCR assay provides a rapid and sensitive tool for the detection of P. sojae in plants and in production fields.  相似文献   

17.
 当采用嵌套引物P1/P7(U5/P7) U3/U5的巢式PCR检测植原体时,伴随着正常的0.85kb片段还经常扩增出一条额外片段。与其它常见的非特异性片段不同,该额外片段十分稳定,与正常片段总是同步出现,二者的强度呈正比。经测定,额外片段的大小为0.36kb。迄今,这一现象仅在采用P1/P7(U5/P7)-U3/U5引物的巢式PCR中出现。在采用P1/P7、U5/P7或-U3/U5引物的常规PCR中从未出现过。另外,已知这一现象至少在葡萄黄化stolbur和榆黄化植原体的检测过程中出现。对该现象产生的原因进行了深入研究,方法是将额外片段从凝胶中分离出来,用不同的引物在不同的条件下进行重新扩增,同时结合已知的植原体16SrRNA基因序列进行综合分析判断。结果表明,该额外片段源于植原体16SrRNA基因上存在的一个与U5引物部分互补的位点。对额外片段的测序结果进一步证实了分析的正确性。据此,指出了该额外片段在植原体检测中的可能用途。  相似文献   

18.
Pear decline (PD) is an important phytoplasmal disease that occurs mainly in Europe and North America. In 1994, pear trees exhibiting symptoms typical of PD disease were observed in orchards of central Taiwan. The sequence of 16S rDNA and 16S–23S rDNA intergenic spacer region (ISR) of the causative agent of pear decline in Taiwan (PDTW) were amplified with polymerase chain reaction (PCR) using a DNA template prepared from the diseased leaves. Sequence analysis of 16S rDNA revealed that the PDTW agent was closely related to the phytoplasmas of the apple proliferation group that cause diseases in stone fruits, pear and apple. Consistent with the result of 16S rDNA sequence analysis, sequence analysis of the 16S–23S rDNA ISR and putative restriction site analyses of 16S rDNA and 16S–23S rDNA ISR sequences provided further support for the view that the PDTW phytoplasma causing pear decline in Taiwan may represent a new subgroup of the apple proliferation group. According to the rDNA sequence of PDTW phytoplasma, two specific PCR primer pairs, APf2/L1n and fPD1/rPDS1, were designed in this study for the detection of the etiological agent in pear trees and insect vectors. Based on the sequence analyses of the PCR-amplified fragments, two species of pear psyllas, Cacopsylla qianli and Cacopsylla chinensis, were found to carry PDTW phytoplasma.  相似文献   

19.
‘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.  相似文献   

20.
Fusarium oxysporum f. sp. cubense is the causal agent of Panama disease of banana. A rapid and reliable diagnosis is the foundation of integrated disease management practices in commodity crops. For this diagnostic purpose, we have developed a reliable molecular method to detect Foc race 4 isolates in Taiwan. By PCR amplification, the primer set Foc-1/Foc-2 derived from the sequence of a random primer OP-A02 amplified fragment produced a 242 bp size DNA fragment which was specific to Foc race 4. With the optimized PCR parameters, the molecular method was sensitive and could detect small quantities of Foc DNA as low as 10 pg in 50 to 2,000 ng host genomic DNA with high efficiency. We also demonstrated that by using our PCR assay with Foc-1/Foc-2 primer set, Foc race 4 could be easily distinguished from other Foc races 1 and 2, and separated other formae speciales of F. oxysporum. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.  相似文献   

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