Confirmation of the Presence of Citrus Viroids in Citrus Orchards in Northwestern Argentina (original) (raw)
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Detection and Characterization of Citrus Viroids in Uruguay
International Organization of Citrus Virologists Conference Proceedings (1957-2010), 2000
Citrus trees in Uruguay are mainly grafted on trifoliate orange after the dissemination of citrus tristeza virus in the 1940s. Trifoliate orange is very susceptible to citrus exocortis viroid (CEVd), but the occurrence of typical rootstock symptoms are difficult to find in citrus orchards in Uruguay. Several samples from orange and grapefruit trees grafted on trifoliate orange, with dwarfing and rootstock bark scaling were chosen, as well as asymptomatic trees to determine the presence of citrus viroids. The detection of viroids was done by biological assays on Etrog citron Arizona 861, herbaceous hosts and sequential polyacryamide gel electrophoresis. Several cDNA probes labeled with 32 P or digoxigenin were tested and were hybridized with samples from different isolates collected from citrus orchards. The cDNA probe was produced from a severe Uruguayan CEVd isolate (CEVd-Uy1) which had been previously cloned and sequenced. The following citrus viroids were found: CEVd, CVd-Ia, CVd-Ib, CVd-IIa, CVd-IIIa and CVd-IIIb; no CVd-IV was found in the samples collected. Several viroid combinations were found in the same trees. Characterization of the citrus viroids is an important step for the control and prevention of these agents in Uruguayan citrus industry that is 90% grafted on a susceptible rootstock.
Phytopathology, 2002
Sequential polyacrylamide gel electrophoresis analyses showed many viroid-like RNAs in samples collected from citrus trees in Japan. Reverse transcription polymerase chain reaction and sequencing analyses of the amplified fragments verified that they were derived from variants of six citrus viroids, Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd) including CVd-I-LSS (a distinct variant of CBLVd), Hop stunt viroid, Citrus viroid III, Citrus viroid IV, and Citrus viroid OS. The samples induced symptoms with variable severity in Arizona 861-S1 ‘Etrog’ citrons (Citrus medica L.) likely due to the varying accumulation patterns produced by the different viroids. Some of the symptoms caused by the samples harboring the citrus viroids other than CEVd were as severe as those caused by CEVd. Some source citrus trees showing the severe bark scaling characteristic of exocortis disease in trifoliate orange (Poncirus trifoliata (L.) Raf.) rootstocks contained only citrus viroids o...
PCR Diagnosis of Citrus Viroids in Field Samples
Several procedures for rapid and sensitive detection of different citrus viroid-RNAs in field samples were evaluated. Bark tissue of different citrus species with single or mixed infections of CEVd, CVd-I1 and CVd-111 were collected a t different seasons. Nucleic acids were phenol extracted and purified by CF-11 cellulose chromatography or extracted with SDS-potas-sium acetate and analyzed by sequential polyacrylamide gel electrophoresis (sPAGE), reverse transcription (RT)-or multiplex reverse transcription (MRT)-polymerase chain reaction (PCR). Both PCR procedures were more sensitive than sPAGE, partially overcoming the difficulties derived from low viroid concentrations in winter and spring. However, in the presence of the three citrus viroid groups, these procedures didn't allow a full profile of viroid content. MRT-PCR, using two sets of primers, showed about the same sensitivity as RT-PCR, giving a simultaneous diagno-sis of two viroid groups in mixed infections but the amp...
Citrus Exocortis and Citrus Cachexia Viroids in Commercial Groves of Tahiti Limein México
International Organization of Citrus Virologists Conference Proceedings (1957-2010), 2000
A survey for citrus viroids was carried out on Tahiti lime in several citrus regions of México. Detection of citrus viroids was conducted by reverse transcription-polymerase chain reaction (RT-PCR) and sequential polyacrylamide gel electrophoresis (sPAGE). Both citrus exocortis (CEVd) and citrus cachexia (CCaVd) viroids were consistently detected in 33 out of 35 samples analyzed from Tamaulipas, Veracruz, Tabasco, and Yucatán citrus groves. There was no evidence of infection with other citrus viroids in the samples analyzed. Disease symptoms present in the groves included in the survey were severe bark cracking on main branches and trunk. These symptoms were frequently associated with differing degrees of tree deterioration and poor growth. In addition, infected trees which were grafted on Alemow rootstock had reduced growth in trunk diameter below the bud union along with brownish lesions on the wood. The consistent presence of both CEVd and CCaVd in Tahiti lime groves from diverse geographical origins in México indicates the continuing use of infected propagative budwood sources.
Practical Field Detection of Citrus Viroids in Florida by RT-PCR
International Organization of Citrus Virologists Conference proceedings, 2002
A practical approach to the detection of the three common citrus viroids in Florida citrus from field-collected tissue by RT-PCR was developed and tested. Reverse transcriptions were done with total nucleic acid extracts prepared by a SDS-potassium acetate extraction of small amounts of tissue pulverized in Tris buffer. PCR amplifications were done using previously described primer pairs specific for Citrus exocortis viroid (CEVd), Citrus viroid II (CVd-II) (Hop stunt viroid) and Citrus viroid III (CVd-III), and the products were analyzed by agarose gel electrophoresis. Effects of cultivar, tissue location and sampling time were investigated. CEVd, CVd-II, and CVd-III were all consistently detected in tissue from experimentally infected orange, lemon, and lime cultivars, but detection from grapefruit and mandarins was less consistent, especially for CEVd. CEVd and CVd-II were not detected in Meiwa kumquat and detection of CVd-III was rare. Bark tissue from woody, budwood-sized twigs was the best tissue source, and samples collected in warm weather yielded better results than those collected in winter. Field surveys of several hundred trees in commercial groves and test plots with varying viroid profiles were conducted. The correlation between results from RT-PCR and biological indexing exceeded 90%. RT-PCR was especially effective for detection of CVd-II, the citrus viroid most difficult to detect biologically or by sequential PAGE. Several isolates that caused moderate symptoms in Etrog citron were not amplified by our CEVd, CVd-II and CVd-III primers. One was identified as Citrus viroid IV , and this is the first report of this viroid in Florida. The others were identified as sequence variants of CVd-III. The methods developed have been used successfully by personnel in three different laboratories.
Viroid Diseases of Citrus in India
International Organization of Citrus Virologists Conference Proceedings (1957-2010)
Exocortis disease of citrus in India has been known to occur for more than two decades but studies on its causal viroid have not been accomplished. The results of analysis of samples showing exocortis-like symptoms in R-PAGE revealed presence of two viroid species, one belonging to Indian Tomato bunchy top viroid (ITBVd) and the other to citrus B (CVd-IIa) group of viroids. This is for the first time in India that viroids have been found infecting citrus plantings.
A Viroid Different from Citrus Exocortis Viroid Found in Commercial Citrus in Sicily
International Organization of Citrus Virologists Conference Proceedings (1957-2010)
A low molecular weight RNA, named citrus "B" viroid (CBV), has been detected alone or associated with citrus exocortis viroid (CEV) in many commercial citrus species and varieties. The electrophoretic mobility of CBV on 5% polyacrylarnide gels under denaturing conditions is between those of CEV and the fast form of coconut cadang cadang viroid (CCCV) RNA1. CBV replicates in zucchini squash, a common host for CEV, but not in ten other herbaceous hosts of CEV. Citrus nucleic acid extracts containing CBV were reacted with full-length CEV-, potato spindle tuber viroid (PSTV)-cDNA probes and with CEV-, PSTV-, tomato apical stunt viroid (TASV)-and tomato planta macho viroid (TPMV)-RNA probes. CBV did not hybridize with any of the probes. Inoculations of partially purified CBV onto different citrus indicator plants gave variable epinasty on Etrog citron, but no symptoms in other indicators.
A Definition of Citrus Viroid Groups and Their Relationship to the Exocortis Disease
Journal of General Virology, 1988
Nucleic acid extracts from citrons (Citrus medica cv. Etrog) displaying mild and moderate symptoms associated with the exocortis disease were analysed by sequential and denaturing PAGE which revealed the presence of several viroids. A comparison was made of electrophoretic patterns displaying one or more distinct citrus viroids from field isolates of citrus with exocortis. Citrus viroids were characterized by the physical parameters of electrophoretic mobility, chromatography on CF-11 cellulose and hybridization to cDNA probes of the well characterized citrus viroids, citrus exocortis viroid, CV-Ib from the 'citron variable viroid' isolate, and citrus cachexia viroid. These characteristic properties combined with biological distinctions in the host range and symptom expression suggested a scheme for the organization of the citrus viroids into five major groups. The association of the symptoms induced by these citrus viroids in citron cv. Etrog, their organization into individual viroid groups and their presumed relationship to the exocortis disease of citrus are discussed.