Comparative analysis of techniques for detection of grapevine and citrus viroids in Tunisia (original) (raw)
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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...
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.
Agriculture and Biology Journal of North America, 2010
Two extensive surveys and laboratory work were conducted to determine the occurrence of citrus exocortis viroid (CEVd) and hop stunt viroid (HSVd), the causal agent of cachexia (CVd-IIb) disease in the main citrus growing areas in three states in the northern part of Sudan, viz, Northern, River Nile and Khartoum. For CEVd, all, but one of the examined citrus trees failed to show typical exocortis symptoms. The only symptomatic orange tree encountered in the survey displayed the characteristic CEVd symptoms including tree stunting and bark cracking or bark splitting of the rootstock. While for the cachexia disease, most of the commercial citrus trees were found to be symptomless carriers except mandarin. Symptomatic mandarin trees which were grafted on sour orange rootstock displayed heavy gum impregnation (bark gumming), wood staining and deep bark cracking, in addition to severe stem pitting, bark pegging, twig dieback and yellowing of leaves. Pitting and gum development were usually encountered near the budunion but could spread to other sensitive portions of the infected tree. However, using viroid specific primers and reverse transcription polymerase chain reaction (RT-PCR) approach, both viroids were shown to exist in total RNA preparations from symptomatic and asymptomatic citrus species. Bands of 370 bp and 300 bp corresponding to the full length genomes of CEVd and CVd-IIb, respectively, were detected. The test was positive in 54 for CEVd and 20 for CVd-IIb out of 200 random samples tested from orange, grapefruit and mandarin. The results indicated that a considerably high percentage of citrus species in the northern part of Sudan, particularly in Khartoum State, were symptomless carriers of both CEVd and CVd-IIb. Being rapid, sensitive and in most cases reliable, the molecular approach could be considered indispensable for viroid detection and in testing programs to produce certified viroid-free planting materials.
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.
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.
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.
Advanced Research in Life Sciences
Citrus exocortis is a grafting disease caused by Citrus Exocortis Viroid (CEVd). The knowledge of the viroid’s incidence and distribution are necessary to further apply control measures. The objective of this work was to apply the real time PCR assay for the detection of CEVd in samples collected from symptomatic CEVd-infected plants in Mitidja (North Algeria). The assay showed an excellent diagnostic specificity where 38 out of 50 samples showed a positive reaction for CEVd, which revealed the presence of CEVd in citrus orchards with a prevalence of 76%. Consequently, this work offers a quick alternative to conventional methods for the early diagnosis and the prevalence assessment of CEVd.
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.
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.