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Comparison of different diagnostic methods for detection of Hop stunt viroid and Citrus exocortis viroid in citrus  [cached]
Cecilia Escobar Ponce de León,Ana I. Figueroa Castellanos,Julia Figueroa,Beatriz Stein
Revista Industrial y Agrícola de Tucumán , 2012,
Abstract: Viroids such as Hop stunt viroid (HSVd) and Citrus exocortis viroid (CEVd) are important pathogens in citrus plants. A comparative assay was performed to evaluate the viroid diagnostic techniques currently available at the Citrus Sanitation Center of Estación Experimental Agroindustrial Obispo Colombres. We compared the results obtained in the diagnosis of HSVd and CEVd in citrus using two molecular techniques: s-PAGE and RT-PCR and biological indexing. Biological indexing was performed in a greenhouse under controlled temperature, using Etrog citron Arizona 861-S1 grafted on rough lemon rootstock as the indicator plant. Tissue samples for s-PAGE were obtained from inoculated citrons used in the biological indexing tests, and DNA extraction was performed following published procedures. HSVd and CEVd specific primers were used for RT-PCR, and samples were taken directly from field plants and inoculated citrons. Two nucleic acid extraction methods were compared. A total of 12 viroid isolates collected from orange, citron, grapefruit, citrumelo and lime trees, mostly from North Western Argentina, were used in these assays. The three diagnostic methods compared gave similar results in 11 of 12 samples assayed. Several viroids were detected by s-PAGE and most of them were found as mixtures. HSVd and CEVd were identified by RT-PCR and no differences were found between the two extraction methods assayed. Sample R-407 was positive for exocortis with biological diagnosis and s-PAGE, but it was negative with RT-PCR. This was probably due to inefficient cDNA synthesis or to the presence of inhibitors that could interfere with the reaction. On the basis of the results obtained, it can be concluded that the three techniques assayed are complementary and useful tools for diagnosis and identification of HSVd and CEVd in citrus.
Diagnostics for Citrus exocortis and Hop stunt viroids associated with yellow corky vein disease in citrus  [cached]
Indian Phytopathology , 2012,
Abstract: The techniques of return polyacrylamide gel electrophoresis (R-PAGE), reverse transcriptase polymerase chain reaction (RT-PCR) and nucleic acid spot hybridization (NASH) were evaluated with special reference to yellow corky vein disease infecting Kagzi lime plants. The technology for quick and reliable detection of two different groups of viroids viz. Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd) infecting citrus has been standardized for the first time in the country. The tools developed will help to identify viroid-free rootstock for use in the budwod certification programme.
Cultivated Grapevines Represent a Symptomless Reservoir for the Transmission of Hop Stunt Viroid to Hop Crops: 15 Years of Evolutionary Analysis  [PDF]
Yoko Kawaguchi-Ito,Shi-Fang Li,Masaya Tagawa,Hiroyuki Araki,Masafumi Goshono,Shingen Yamamoto,Mayumi Tanaka,Masako Narita,Kazuaki Tanaka,Sheng-Xue Liu,Eishiro Shikata,Teruo Sano
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0008386
Abstract: Hop stunt was a mysterious disorder that first emerged in the 1940s in commercial hops in Japan. To investigate the origin of this disorder, we infected hops with natural Hop stunt viroid (HpSVd) isolates derived from four host species (hop, grapevine, plum and citrus), which except for hop represent possible sources of the ancestral viroid. These plants were maintained for 15 years, then analyzed the HpSVd variants present. Here we show that the variant originally found in cultivated grapevines gave rise to various combinations of mutations at positions 25, 26, 54, 193, and 281. However, upon prolonged infection, these variants underwent convergent evolution resulting in a limited number of adapted mutants. Some of them showed nucleotide sequences identical to those currently responsible for hop stunt epidemics in commercial hops in Japan, China, and the United States. Therefore, these results indicate that we have successfully reproduced the original process by which a natural HpSVd variant naturally introduced into cultivated hops was able to mutate into the HpSVd variants that are currently present in commercial hops. Furthermore, and importantly, we have identified cultivated grapevines as a symptomless reservoir in which HSVd can evolve and be transmitted to hop crops to cause epidemics.
Viroid species associated with the bark-cracking phenotype of 'Tahiti' acid lime in the State of S?o Paulo, Brazil
Eiras, Marcelo;Silva, Simone R.;Stuchi, Eduardo S.;Flores, Ricardo;Daròs, José-Antonio;
Tropical Plant Pathology , 2010, DOI: 10.1590/S1982-56762010000500005
Abstract: viroids have been used as "graft transmissible dwarfing agents" (gtda) in several countries, mainly to reduce growth of citrus trees, thus increasing their density in orchards. in the state of s?o paulo, brazil, plants of the acid lime 'tahiti' are usually grafted with a complex of gtda, presumably viroids. the aim of the present work was the identification and molecular characterization of the viroids infecting trees of acid lime 'tahiti' displaying "quebra galho" (bark-cracking). viroids were identified and characterized by biological indexing in 'etrog' citron, northern-blot hybridization, rt-pcr, cloning and complete sequencing of the rna genomes. citrus exocortis viroid (cevd), hop stunt viroid (hsvd) and citrus dwarfing viroid (cdvd) were found in different combinations. although we have not been able to infer a direct relationship between the agronomical performance and symptom severity with the presence of a specific viroid or viroid combination, the differences in the severity of "quebra-galho" symptoms among different trees is probably associated with the presence (or absence) of cevd, with its interaction with other viroids perhaps determining the different phenotypes observed in the field.
Development of viroid free nucleus material for rootstock of citrus: an approach for management of yellow corky vein disease  [cached]
Indian Phytopathology , 2011,
Abstract: Yellow corky vein disease of citrus has been shown to be associated with two viroids,citrus exocortis viroid (CEVd) and hop stunt viroid (HSVd). The disease causes heavy yield reduction: Viroid-free nucleus material for rootstocks (rough lemon and trifoliate orange) have been developed after’ standardization of micropropagation protocol. Present study constitutes the first record of development of viroid-free rootstock plant for citrus through micropropagation. Growth pattern of two species are slightly different; initial shooting is higher in trifoliate orange, but callusing and rooting are much faster in rough lemon. Viroid free micropropagated plants were indexed by NASH test using radiolabelled cDNA probes to two groups of viroid viz. citrus exocortis viroid and hop stunt viroid.
Sequence analysis of three citrus viroids infecting a single Tunisian citrus tree (Citrus, reticulata, Clementine)
Elleuch, Amine;Khouaja, Fattouma Djilani;Hamdi, Imen;Bsais, Nabiha;Perreault, Jean-Pierre;Marrakchi, Mohamed;Fakhfakh, Hatem;
Genetics and Molecular Biology , 2006, DOI: 10.1590/S1415-47572006000400020
Abstract: we report the nucleotide sequences of three citrus viroids belonging to three different genera: citrus exocortis viroid (cevd), hop stunt viroid (hsvd) and citrus viroid-iii (cvd-iii) isolated from a single natural infected citrus reticulata var. clementine tree growing in a tree nursery in manouba (near tunis capital). we describe the sequence variability of these viroids from their natural host without using an alternative passage by an indicator host or an artificial inoculation. this work confirms that naturally occurring viroid infections contain a mixture of sequence variants. these are the first sequences of citrus viroids from africa.
A Current Overview of Two Viroids That Infect Chrysanthemums: Chrysanthemum stunt viroid and Chrysanthemum chlorotic mottle viroid  [PDF]
Won Kyong Cho,Yeonhwa Jo,Kyoung-Min Jo,Kook-Hyung Kim
Viruses , 2013, DOI: 10.3390/v5041099
Abstract: The chrysanthemum ( Dendranthema X grandiflorum) belongs to the family Asteraceae and it is one of the most popular flowers in the world. Viroids are the smallest known plant pathogens. They consist of a circular, single-stranded RNA, which does not encode a protein. Chrysanthemums are a common host for two different viroids, the Chrysanthemum stunt viroid (CSVd) and the Chrysanthemum chlorotic mottle viroid (CChMVd). These viroids are quite different from each other in structure and function. Here, we reviewed research associated with CSVd and CChMVd that covered disease symptoms, identification, host range, nucleotide sequences, phylogenetic relationships, structures, replication mechanisms, symptom determinants, detection methods, viroid elimination, and development of viroid resistant chrysanthemums, among other studies. We propose that the chrysanthemum and these two viroids represent convenient genetic resources for host–viroid interaction studies.
Detection of viroid infecting chrysanthemum in India  [cached]
Indian Phytopathology , 2012,
Abstract: In a survey of ornamental plants conducted in fields of IARI and some private nurseries in New Delhi, a large number of chrysanthemum plants were found showing symptoms of mild chlorosis on young leaves, stunting and delayed flowering. Electron microscopic' observations from infected leaves did not reveal the presence of virus particles. R-PAGE analysis of total nucleic acid extracts from symptomatic leaves from plants grown by cuttings, revealed the presence of RNA band while such a band was absent from healthy samples. The viroid nature of the RNA was confirmed by its similarity in electrophoretic mobility to potato spindle tuber viroid, resistance to high temperature and DNase but high sensitivity to RNase treatment. The nucleic acid extract from infected tomato cv. Rutgers, calendula and cineraria produced symptoms of leaf epinasty, veinal chlorosis and stunting in inoculated plants. Re-extraction of nucleic acid from inoculated
Viroides em citros
Eiras, Marcelo;Silva, Simone Rodrigues;Stuchi, Eduardo Sanches;Targon, Maria Luisa Penteado Natividade;Carvalho, Sérgio Alves;
Tropical Plant Pathology , 2009, DOI: 10.1590/S1982-56762009000500001
Abstract: viroids are the smallest known plant pathogens consisting of a non-encapsidated, circular, single-stranded rna that replicate autonomously in their host plants. viroids are classified into two families (pospiviroidae and avsunviroidae). all citrus viroids belong to the pospiviroidae family (species that present a central conserved region, replicate in the nucleus of infected cells and lack of ribozyme activity) with five citrus viroid species: citrus exocortis viroid, cevd (pospiviroid), hop stunt viroid, hsvd (hostuviroid), citrus bark cracking viroid, cbcvd (cocadviroid) and citrus bent leaf viroid, cblvd and citrus dwarfing viroid, cdvd (apscaviroid). in addition, citrus viroid original source (cvd-os) and, more recently, citrus viroid v (cvd-v) have been proposed as tentative species of the genus apscaviroid. citrus viroids are graft-transmitted and their dissemination occurs mainly by propagation of contaminated material. it is known that they have a broad host range, infecting species of citrus and plants of citrus-related genera. two important diseases in citrus are viroid-induced: (i) exocortis; (ii) and cachexia. symptoms vary from severe to asymptomatic, and their control is based on preventive measures as availability of viroid-free budwood as a source of propagation material follow by adequate indexing procedures. the purpose of this review is to present the reader the recent advances on citrus viroid research, mainly on taxonomy, geographical distribution, methods of detection, indexing and cleaning, epidemiology and control.
Identification and characterization of viroids in 'Navelina ISA 315' sweet orange
Marcelo Eiras,Simone R. Silva,Eduardo S. Stuchi,Sérgio A. Carvalho
Tropical Plant Pathology , 2013,
Abstract: To date, seven species of viroids have been described infecting Citrus. However, only Citrus exocortis viroid (CEVd), Hop stunt viroid (HSVd) and Citrus dwarfing viroid (CDVd) have been reported in this crop in Brazil. The aim of our work was to identify and characterize viroids infecting plants of 'Navelina ISA 315' sweet orange cultivar grafted on 'Rangpur' lime from field experiments located in Bebedouro, SP, that showed gumming in the bark and wood symptoms. Biological indexing was done on the indicator host 'Clemelin 1120' tangor plants that reacted with typical gumming symptoms. Viroid infection was assessed by graft-inoculation of buds from the field trees on 'Etrog' citron plants grafted on 'Rangpur' lime, followed by RNA extraction and sPAGE analysis. RNAs were subjected to RT-PCR with primers for citrus viroids. The full-length amplified products were sequenced and compared to those available in the GenBank. The trees were found to be infected with cachexia (Ca) variants of HSVd-Ca or HSVd-Ca plus CDVd. The results indicate that efforts have to be made to increase and stimulate the indexing programs, to maintain plants healthy and to develop sanitary programs focused on reducing the spread of viroids and other graft-transmissible agents.
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