共查询到20条相似文献，搜索用时 31 毫秒
1.
2.
3.
The polyacrylamide gel electrophoresis (PAGE) test of Morris & Smith (1977) was evaluated for detection of potato spindle tuber viroid (PSTV) in breeding material. Number, density and mobility of nucleic acid bands in the electropherograms were influenced by genotype and growing temperature. So direct testing of genotypes was not reliable. After an intermediate viroid multiplication in tomato host plants at about 30oC and high irradiance, PSTV was reliably detectable with PAGE in inocula of potato samples of diverse origin. A 4-week incubation period proved to be suitable for inocula with low and high concentrations of a mild strain of PSTV (m-PSTV) as well as a severe strain of PSTV (s-PSTV). If incidence of PSTV is expected to be low, testing can be speeded up by bulking samples. With the combined tomato-intermediate/ PAGE assay, one m-PSTV or one s-PSTV infected leaf disk in 200 healthy ones was consistently detectable. Occasionally gels with a nucleic acid band of about the same relative mobility as the viroid band were found. Evidence that these bands were not caused by viroid is presented. A procedure to resolve such questionable test results is described. Infectivity of s-PSTV was higher than that of m-PSTV. Concentration of viroids in the inoculum influenced appearance of mild or severe symptoms and the rate of symptom production. 相似文献
4.
A rapid DNA extraction and loop‐mediated isothermal amplification (LAMP) procedure was developed and evaluated for the detection of two specific groups of phytoplasmas from infected plant material. Primers based upon the 16–23S intergenic spacer (IGS) region were evaluated in LAMP assays for amplification of group 16SrI (aster yellows group) and group 16SrXXII (Cape St Paul wilt group) phytoplasma strains. DNA could be extracted from leaf material (16SrI phytoplasmas) or coconut trunk borings (16SrXXII phytoplasmas) onto the membranes of lateral flow devices, and small sections of these membranes were then added directly into the LAMP reaction mixture and incubated for 45 min at 65°C. Positive reactions were detected through the hydroxyl napthol blue colorimetric assay within 1 h of the start of DNA extraction, and were confirmed by subsequent agarose gel electrophoresis of the LAMP products. The level of detection was comparable to that obtained by nested PCR using conventional 16S rDNA phytoplasma‐specific primers. Furthermore, the assays were specific for the phytoplasmas they were designed to detect – the 16SrI assay only detected 16SrI phytoplasmas and not those from any other phylogenetic groups, whilst the 16SrXXII assay only detected 16SrXXII phytoplasmas. The DNA extractions and LAMP assay are easy to perform, requiring minimal equipment, and may therefore form the basis of a rapid and reliable field‐detection system for phytoplasmas. 相似文献
5.
6.
Transmission of the coconut cadang-cadang viroid to six species of palm by inoculation with nucleic acid extracts 总被引：1，自引：0，他引：1
Seedlings of Areca catechu (betel nut palm), Corypha elata (buri palm), Adonidia merrillii (manila palm), Elaeis guineensis (oil palm), Chrysalidocarpus lutescens (palmera) and Oreodoxa regia (royal palm) were inoculated with nucleic acid extracts from coconut palms with cadang-cadang disease. Within 2 years of inoculation, analysis using a 32 P-labelled DNA probe complementary to the coconut cadang-cadang viroid (CCCV) showed that RNA sequences identical to CCCV were present in the inoculated seedlings. Electrophoresis in polyacrylamide gels showed that these palms also contained an RNA with mobility identical to CCCV. Four to five years after inoculation, the infected palms of four species were usually stunted compared with uninoculated palms, while betel nut and palmera were not stunted. Yellowing of leaflets was observed with defined spots or mottling of the older fronds in all except betel nut palms. All infected palms showed mild or severe yellow-leaf spotting. These results widen the known host range and. hence, the potential number of viroid reservoir species in the field. 相似文献
7.
目前, 我国梅树上的病毒种类及发生情况仍不完全清楚。本研究从北京、武汉、南京和无锡的梅园中采集了64份疑似感染病毒的叶片样品, 通过RT-PCR和斑点杂交, 对7种病毒和2种类病毒进行了检测。共检测到6种病毒和1种类病毒。其中, 李属坏死环斑病毒(prunus necrotic ringspot virus, PNRSV)和桃潜隐花叶类病毒(peach latent mosaic viroid, PLMVd)为我国梅树上的首次检出。PNRSV、亚洲李属病毒2号(Asian prunus virus 2, APV2)、桃叶痘伴随病毒(peach leaf pitting-associated virus, PLPaV)的检出率高于30%。综合考虑病毒的分布及检出率, PLPaV、APV2、PNRSV和李树皮坏死茎痘伴随病毒(plum bark necrosis stem pitting-associated virus, PBNSPaV)是武汉、南京和无锡梅树上的主要病毒。此外, 通过克隆和测序, 获得了PLMVd和梅树病毒A(mume virus A, MuVA)的基因组, PLPaV的RNA1组分和PNRSV外壳蛋白(CP)基因序列。序列比较分析显示, 我国PLMVd梅分离物和PNRSV梅分离物与我国桃分离物亲缘关系最近, 表明PLMVd和PNRSV可能在梅和桃树间交互侵染;我国MuVA梅分离物序列与日本梅分离物序列的相似性高达98.56%;PLPaV梅分离物与我国桃分离物之间序列变异较大。上述结果不仅进一步明确了我国梅树上的病毒及类病毒种类和分布情况, 而且有助于深入了解它们的流行与传播。 相似文献
8.
A viroid was detected in Chrysanthemum plants showing symptotns of stunting in a commercial field in Brazil. Analysis by return polyacrylamide gel electrophoresis (R-PAGE) of the nucleic acid preparations of leaves and flowers revealed the presence of a nucleic acid of low molecular weight with mobility within the range of viroids. The viroid-like band was completely eliminated by ribonuclease treatment or alkaline hydrolysis. The Chrysanthemum viroid was readily transmissible to Chrysathemum , tomnato and Gynura , which suggests that it may be an isolate of chrysanthemum stunt viroid. 相似文献
9.
10.
The fast growth of the human population forces us to produce more food, but higher crop production also leads to the fast spread of diseases. Plant pathology deploys a wide range of methods that do not provide an adequate solution to all disease losses. In the case of viroids, therapeutic means of control are not available; therefore control strategies are more focused on the development of reliable detection methods to quickly exclude the infected plant material. Although viroids are the smallest and simplest plant pathogens, their identification and detection is not straightforward. Each viroid–host combination is specific, and for reliable identification, all steps from sampling to final detection must be performed accurately. In this review, several methods for viroid detection in various host plants are discussed, including their advantages and disadvantages. Even though relatively new molecular methods enable fast and sensitive detection of viroids, a combination of different methods gives the most reliable identification. Techniques based on nucleic acids may be the future for viroid detection but they still cannot replace biological indexing, which is usually essential in epidemiological and aetiological studies. 相似文献
11.
DNA amplification by polymerase chain reaction (PCR) was used specifically to detect the mycoplasma-like organism (MLO) associated with lethal yellowing disease of palms in Florida. For PCR, a pair of oligonucleotide primers was synthesized according to partial sequences of a cloned 1·3 kbp fragment of lethal yellowing MLO-specific genomic DNA isolated from a diseased windmill palm ( Trachycarpus fortunei ). A DNA product of about 1 kbp was specifically amplified by PCR in reaction mixtures containing template DNA derived from either heart, inflorescence or leaf tissues of lethal yellowing-affected palms. PCR performed for 35 cycles with as little as 5 pg of DNA template, in some instances, was sufficient consistently to amplify the same lethal yellowing MLO DNA product from hearts of 11 species comprising 30 symptomatic palms. Similar reliable and reproducible detection of the lethal yellowing MLO in palm inflorescence spikelets was also achieved after 35 cycles of PCR. When template DNA for PCR was derived from tissues of the the most immature emerging leaf, a 40-cycle reaction was sufficient for consistent foliar detection of the pathogen in all coconut palms including palms with earliest visible symptoms of disease. 相似文献
12.
13.
14.
Changes in carbohydrate metabolism in coconut palms infected with the lethal yellowing phytoplasma 总被引：1，自引：0，他引：1
ABSTRACT Lethal yellowing (LY), a disease caused by a phytoplasma, is the most devastating disease affecting coconut (Cocos nucifera) in Mexico. Thousands of coconut palm trees have died on the Yucatan peninsula while plantations in Central America and on the Pacific coast of Mexico are severely threatened. Polymerase chain reaction assays enable identification of incubating palm trees (stage 0+, phytoplasma detected but palm asymptomatic). With the development of LY, palm trees exhibit various visual symptoms such as premature nut fall (stage 1), inflorescence necrosis (stages 2 to 3), leaf chlorosis and senescence (stages 4 to 6), and finally palm death. However, physiological changes occur in the leaves and roots prior to onset of visual symptoms. Stomatal conductance, photosynthesis, and root respiration decreased in stages 0+ to 6. The number of active photosystem II (PSII) reaction centers decreased during stage 2, but maximum quantum use efficiency of PSII remained similar until stage 3 before declining. Sugar and starch concentrations in intermediate leaves (leaf 14) and upper leaves (leaf 4) increased from stage 0- (healthy) to stages 2 to 4, while root carbohydrate concentrations decreased rapidly from stage 0- to stage 0+ (incubating phytoplasma). Although photosynthetic rates and root carbohydrate concentrations decreased, leaf carbohydrate concentrations increased, suggesting inhibition of sugar transport in the phloem leading to stress in sink tissues and development of visual symptoms of LY. 相似文献
15.
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
16.
Frank Van Der Wilk Marja Korsman Frans Zoon 《European journal of plant pathology / European Foundation for Plant Pathology》1994,100(2):109-122
An assay, based on amplification of cDNA synthesized from genomic viral RNA, has been developed to detect tobacco rattle virus in infected plant material and viruliferous nematodes. A range of different TRV strains could be detected using the procedure developed. The presence of one to three viruliferous nematodes in a nematode suspension was sufficient for the detection of TRV. The minimum amount of purified virus detectable in the assay was 15 fg, indicating an increased sensitivity of the PCR-based assay as compared to serological detection methods, like ELISA. A dot-blot hybridization procedure was developed for the detection of the PCR products, making agarose gel electrophoresis dispensable. 相似文献
17.
Xuefeng Wang Changyong Zhou Kezhi Tang Yan Zhou Zhongan Li 《European journal of plant pathology / European Foundation for Plant Pathology》2009,124(1):175-180
Citrus plants are natural hosts of five viroid species and large numbers of sequence variants. In this paper a simple and
sensitive one step multiplex RT-PCR protocol with an internal control was utilised to simultaneously detect and differentiate
five citrus viroids: Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd), Hop stunt viroid (HSVd), Citrus viroid-III (CVd-III) and Citrus viroid-IV (CVd-IV). In addition, a micro and rapid total nucleic acid extraction method was developed and the protocol applied to evaluate
the occurrence and distribution of citrus viroids in China. 相似文献
18.
T. Kapari-Isaia A. Kyriakou L. Papayiannis D. Tsaltas S. Gregoriou I. Psaltis 《Plant pathology》2008,57(2):348-353
A new laboratory technique combining shoot-tip grafting in vitro and biological indexing on indicator plants was explored for the detection of citrus exocortis and related viroids. Τhree in vitro laboratory methods were used and compared with the classical biological method. With the classical in vivo method, diagnosis is based on the expression of symptoms on indicators 11–14 weeks after inoculation. In contrast, with the first in vitro method, microindexing in vitro of citron seedlings by graft inoculation, diagnosis was possible 12 days after inoculation; with the second method, microindexing in vitro of citron cuttings by graft inoculation, 20 days after inoculation; and with the third method, microindexing in vitro of citron cuttings by injection inoculation, 40 days after inoculation. Inoculated Etrog citron plantlets grown in vitro and tested by RT-PCR showed the same viroid content as the source plants. Of the three in vitro viroid indexing methods, microindexing on cuttings by grafting was easier and more reliable than microindexing either on seedlings or on cuttings by injection. 相似文献
19.
应用PCR方法快速检测黄瓜细菌性角斑病菌 总被引：1，自引：0，他引：1
黄瓜细菌性角斑病是黄瓜上的一种重要细菌病害,其病原为丁香假单胞菌黄瓜致病变种(Pseudomonas syringae pv.lachrymans),目前未见到该病害特异性PCR检测方法的报道。通过分析丁香假单胞菌(P.syringae)不同致病变种glyceraldehyde-3-phosphate dehydrogenase 1(gap1)基因序列设计得到一对Psl特异性PCR引物。利用该引物对丁香假单胞菌不同致病变种、假单胞菌属其他种及其他属的共46株菌株进行了PCR扩增,结果表明,所有不同来源的12株黄瓜细菌性角斑病菌均得到179bp的目标片段,而所有其他参试菌株均无扩增条带,PCR检测的灵敏度为7.5×103cfu/mL。利用该方法可从接种后发病的黄瓜叶片总DNA中检测到特异条带,而健康叶片无条带。该引物的PCR检测方法可直接用于植株总DNA的检测,无需进行病原菌的分离培养,快速简便,适用于进出境检验检疫及种苗健康检测等。 相似文献
20.
Vernière C Perrier X Dubois C Dubois A Botella L Chabrier C Bové JM Vila ND 《Phytopathology》2006,96(4):356-368
ABSTRACT Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd), a noncachexia variant of Hop stunt viroid (HSVd), Citrus viroid III (CVd-III), and Citrus viroid IV (CVd-IV) were co-inoculated as two-, three-, four-, and five-viroid mixtures to Clementine trees grafted on trifoliate orange to evaluate their effect on symptom expression, tree growth, and fruit yield. Most trees infected with CEVd-containing viroid mixtures developed exocortis scaling symptoms, as did CEVd alone, whereas most trees infected with HSVd- or CVd-IV-containing mixtures developed bark-cracking symptoms. Trees infected with mixtures containing both CEVd and CVd-IV revealed the existence of antagonism between these two viroids in terms of the expected bark-scaling and cracking symptoms. Synergistic interactions also were identified in trees infected with certain viroid combinations that, in spite of lacking CEVd, expressed exocortis-like scaling symptoms. Viroid interactions also affected the expected response of trees in terms of vegetative growth and fruit yield. Trees infected with viroid combinations containing CEVd or CVd-III were smaller and produced less fruit than trees infected with mixtures not containing these viroids. Viroid interactions on scion circumference and cumulative fruit yield, in terms of additivity of their effects, were statistically confirmed using a factorial analysis of variance model with two mean estimation approaches. In single-viroid infections, CEVd, CVd-III, and, to a lesser extent, CBLVd consistently and significantly reduced tree size and fruit yield. Conversely, HSVd and CVd-IV slightly increased fruit yield and reduced scion circumference. Rare and not consistent significant interactions were detected with the five-, four-, and three-viroid combinations. Antagonistic interactions between CEVd and CVd-III or CBLVd and CVd-III were revealed over the years with consistent significance. The antagonistic interaction between CEVd and CVd-IV was highly significant over the years when additional viroids were present; however, this antagonism appeared much later in the case of an exclusive interaction. HSVd and CVd-IV showed a consistent and significant synergistic interaction on yield only when both viroids were exclusively present. These results demonstrate antagonistic or synergistic relationships between citrus viroids depending on the viroid mixtures present in the host. 相似文献