A real-time PCR assay for detection and absolute quantitation of Citrus exocortis viroid in two sensitive and tolerant rootstocks (original) (raw)
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Virology, 2011
Citrus exocortis viroid (CEVd) exists as populations of heterogeneous variants in infected hosts. In vivo generated CEVd progeny variants (CEVd-PVs) populations from citrus protoplasts, seedlings and mature plants, following inoculation with transcripts of a single CEVd cDNA-clone (wild-type, WT), were studied. The CEVd-PVs population in protoplasts was heterogeneous and became progressively more homogeneous in seedlings and mature plants. The infectivity and pathogenicity of selected CEVd-PVs was evaluated in citrus and herbaceous experimental hosts. The CEVd-PVs U30C, G128A and U182C were not infectious; G50A and 108U+ were infectious but reverted back to WT and 62A+, U129A and U278A were infectious, genetically stable and more severe than WT. The 62A+ and U278A and U129A accumulated at higher levels than WT in protoplasts and seedlings respectively. The effect of specific mutations on the predicted secondary structure of the CEVd-PVs' RNA coupled with the infectivity and replication studies suggested complex structure-tofunction relationships for CEVd.
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.
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...
Diagnosis of Citrus Exocortis and Hop Stunt-Homologous Citrus Viroids by Oligonucleotide Probes
International Organization of Citrus Virologists Conference Proceedings (1957-2010)
Three CEVd and two HSVdoligonucleotides have been evaluated asprobes to diagnose citrus viroids in electroblot hybridization analyses in comparison with full-length CEVd and HSVd cDNA probes. CEVd-3 probe was the only oligo probe specifically reacting with different CEVd isolates in citrus or tomato and with CSVd (chrysanthemum stunt viroid) in tomato, but not with healthy controls. HSVd-5 oligo recognized only CVd-I1 infected samples. HSVd-3 oligo probe recognized CVd-IIa RNA, but not CVd-IIb, and allowed to discriminate CVd-IIa.
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.
The Citrus Exocortis Disease: A Complex of Viroid-RNAs
International Organization of Citrus Virologists Conference Proceedings (1957-2010), 1988
Citrons inoculated with different field sources, displayed a variety of symptoms ranging from very mild leaf bending and necrosis to the severe reaction normally associated with exocortis disease. Nucleic acid preparations from shoot samples were analyzed by sequential polyacrylamide gel electrophoresis. All source from both California and Spain contained one to four viroids with distinct physical and biological properties. The size range was estimated from 371 nucleotides for the citrus exocortis viroid (CEV) to 275 for the smallest viroid. The recovery of single viroids suggested a relationship between the distinct viroids and the symptom reaction expressed in citron.
Construction and application of infectious citrus viroids for biological indexing
Journal of Citrus Pathology, 2016
Viroid species identified in citrus induce a range of symptoms in this host as well as in non-citrus hosts. Currently, 7 citrus viroid species are recognized including Citrus bent leaf viroid (CBLVd), Hop stunt viroid (HSVd), Citrus dwarfing viroid (CDVd), Citrus bark cracking viroid (CBCVd), Citrus viroid V (CVd-V), Citrus viroid VI (CVd-VI) and Citrus exocortis viroid (CEVd). Cachexia-inducing variants of HSVd and CEVd, which causes exocortis, are considered severe pathogens of citrus, whereas other viroid species induce less severe symptoms such as stunting, either singly or in combination. Some viroid species, such as CDVd, have previously been used to deliberately induce stunting for high density planting of citrus, but studies on the effects of commercial orchards are limited. Research on the effect of viroid species requires the isolation of single viroid species. Biological isolation of single species from naturally infected citrus is challenging since viroids seldom occur as single infections and are often found in combination with various other pathogens. The production of single viroid species clones for in vitro transcription enables the generation of a single viroid species inoculum for research applications and circumvents the need for maintenance of sources in plants. Complete genomes of 7 viroid strains including CBLVd, 2 HSVd variants (CVd-IIa and CVd-IIb), CDVd, CBCVd, CVd-V and CEVd were cloned with a leading T7 promoter sequence to facilitate in vitro transcription. Circularized RNA transcripts were successfully used to transfect 'Etrog' citron (Citrus medica) by slashinoculation that developed typical citrus viroid symptoms.