Diagnosis of Citrus Exocortis and Hop Stunt-Homologous Citrus Viroids by Oligonucleotide Probes (original) (raw)
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International Organization of Citrus Virologists Conference Proceedings (1957-2010), 1991
Nucleic acids extracted from viroid-infected commercial citrus trees were analyzed by bidirectional electrophoresis in 7% polyacrylamide gels with the second-direction run performed under denaturing conditions either at homogeneous pH (8.3) or at discontinuous pH (8.3-6.5). Electrotransfer of gel samples to nylon membranes, followed by molecular hybridization tests with citrus exocortis viroid (CEVd)-or hop stunt viroid (HSVd)-specific nucleic acid probes revealed that, of all the viroids detected, only CEVd reacted with the CEVd-specific probes, whereas two viroids reacted with the HSVd-specific probes. Identity of these viroids is discussed in comparison with results of other investigators.
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...
Crop Protection, 2019
Citrus exocortis viroid (CEVd) is the causal agent of exocortis disease, the economically important viroid disease of citrus. In this research, CEVd copy number was quantified in sour orange (Citrus aurantium L.) and citrange (Poncirus trifoliata L. Raf. × Citrus sinensis L.) seedlings by absolute real-time PCR. A full-length in vitro transcript of a single-sequence CEVd-S1 variant was synthesized and mechanically inoculated into sour orange and Troyer citrange seedlings as tolerant and sensitive hosts, respectively. CEVd copy number was subsequently monitored at different times after inoculation in the green bark of infected seedlings for up to 16 weeks post inoculation (wpi). CEVd was detected at 3 wpi and totally increased up to 16 wpi. The copy number of CEVd per milligram of bark tissue in both host species was in the range of 10 3 to 10 4 at the third and sixth weeks post inoculation, without any significant differences. Although the viroid titer was remarkably higher in citrange (sensitive host) than in sour orange (tolerant host) at 10-16 wpi, we demonstrated that CEVd titer differed in citrus species depending on the time of sampling and host plant species.
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.
Real time RT-PCR assay for quantitative detection of Citrus viroid III in plant tissues
Plant Pathology, 2009
A rapid and sensitive real time reverse transcription-PCR (RT-PCR) assay based on SYBR Green I chemistry was developed for the quantitative detection of Citrus viroid III (CVd-III) in citrus samples. CVd-III titre was determined at different times in green bark of sour orange, Troyer citrange, trifoliate orange and alemow seedlings inoculated with a CVd-IIIb source. Ten weeks after inoculation the viroid was detected in the four species, without substantial differences in viroid titre among them. Nine weeks later an overall increase of viroid titre was observed. The copy number of CVd-III in sour orange and Troyer citrange was monitored up to 52 weeks after inoculation and a further increase of viroid titre was observed at 35 weeks. For validation purposes, field samples were tested from 58 citrus trees with mixed infections of CVd-III, Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd), as well as from healthy controls. Based on the sensitivity (100%), specificity (96·7%), accuracy (99·2%) and repeatability (Cohen's kappa index 0·98) of the assay, it is suggested that its employment in breeding programmes would be helpful in the evaluation of host resistance and viroid accumulation in plants.
Nucleic Acids Research, 1987
The primary structure of a grapevine viroid (GVs) isolated in Spain was determined. The sequence consisted of 369 nucleotide residues forming a circular molecule. GVs presented extensive homology with viroids of the potato spindle tuber viroid (PSTV) group, that was specially high in the case of citrus exocortis viroid (CEV) both with variants found in isolates inducing severe (92* with CEV-A) and mild (89* with CEV-DE26) symptoms on tomato. The secondary structure proposed for GVs showed that the changes in the sequence in relation to CEV-A generated modifications of the secondary structure particularly Important in the left terminal (Tl), variable (V) and pathogenesis (P) viroid domains that have been postulated. Nevertheless it was noted 1n GVs a central core in the P domain that is conserved in the class A sequence variants characteristic of severe isolates, but not 1n the class B ones found in mild isolates of CEV. These observations indicate that GVs should be considered as a severe isolate of CEV from grapevine (CEV-g), a suggestion that correlates with the biological properties of CEV-g both in tomato and in Gynura aurantiaca. The presence of this central core in the P domain seems to characterize alI the variants of CEV Inducing severe symptoms in tomato.
The Use of RT-PCR in the Florida Citrus Viroid Indexing Program
International Organization of Citrus Virologists Conference proceedings, 2002
Reverse transcription polymerase chain reaction (RT-PCR) has been incorporated as an adjunct to Florida's biological indexing program for viroids, especially to test for Citrus viroid II (CVd-II) and all viroids where rapid results are needed. The sensitivity of RT-PCR for testing of composite samples was evaluated using 5, 10, 25, 50, 100 and 200 tree composites. Single positive trees were detected consistently in the 5-and 10-tree composite samples; these 5 and 10 tree composites are routinely used to test for CVd-II using Etrog citron indicators. When a composite sample is found to be positive, the individual trees comprising the composite are retested. Viroid infected source trees, identified by biological indexing, were tested by RT-PCR for CVd-II, Citrus viroid III (CVd-III) and Citrus exocortis viroid (CEVd) using tissue collected from both the citron indicators and the corresponding field trees to evaluate the validity of RT-PCR testing. Reliable detection of CVd-II, III and CEVd from field samples by RT-PCR is important for shortening testing time and reducing costs. RT-PCR also allows rapid diagnosis of field samples and testing of trees near a citrus canker quarantine zone without exposing our biological test facilities to potential sources of citrus canker inoculum. Agarose gel electrophoresis was adequate for visualization of CVd-II RT-PCR products, but the sensitivity of CEVd and CVd-III detection has been increased by visualization of RT-PCR products by using either polyacrylamide gels with GelStar stain or Metaphor agarose gels. Improved primers have increased our confidence in CEVd detection by RT-PCR. Screening PCR products from 32 Florida viroid isolates causing a mild citron reaction with oligonucleotide probes specific for CVd-IIIa or CVd-IIIb revealed that CVd-IIIa and IIIb occur both separately and as mixed infections.
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...
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.