Development and validation of a multiplex RT-PCR method for the simultaneous detection of five grapevine viroids.

Grapevine yellow speckle viroid 1 (GYSVd-1), Grapevine yellow speckle viroid 2 (GYSVd-2), Australian grapevine viroid (AGVd), Hop stunt viroid (HSVd) and Citrus exocortis viroid (CEVd) are the five viroids known to infect naturally grapevines. We developed a multiplex RT-PCR (mRT-PCR) method for the simultaneous detection of these five viroids and the amplification of the cDNA fragment of a host-derived mRNA (actin mRNA) as an internal positive control. Specific primers for each targeted viroid were designed by taking into account the sequence variability within and between the viroid species and tested in silico. The method was validated by testing 57 grapevine samples from Iran and showed reliability and high sensitivity. The RT-PCR-negative samples were further assayed by Northern-blot hybridization. For this, a method was developed for the simultaneous detection of three different grapevine viroids on a single hybridization membrane. In this survey, HSVd, GYSVd-1, AGVd, and GYSVd-2 were detected in 100, 95, 93, and 65% of the samples tested, respectively, confirming the wide distribution of these viroids in Iran. CEVd was not detected in any of the samples collected. Based on these results, HSVd is proposed as a positive internal control for mRT-PCR in the areas where this viroid is widespread, so as to reduce the time and costs of DNase treatment, which is required when a host-derived internal control is used. The mRT-PCR method has the potential to be used routinely for large-scale surveys and certification programs.

Hajizadeh M ，Navarro B ，Bashir NS ，Torchetti EM ，Di Serio F ... -  《-》

Comprehensive diversity analysis of viroids infecting grapevine in China and Japan.

To date, several viroid species have been shown to infect grapevine, including Hop stunt viroid (HpSVd), Citrus exocortis viroid (CEVd), Australian grapevine viroid (AGVd), Grapevine yellow speckle viroid-1 (GYSVd-1), Grapevine yellow speckle viroid-2 (GYSVd-2) and a tentative new species, Grapevine yellow speckle viroid-3 (GYSVd-3). Here, we identified and analyzed the distribution, genetic diversity, and molecular properties of viroids infecting grapevine cultivated in China and Japan, including old grapevines. The analysis showed that all the five known viroids and a tentative species GYSVd-3 infecting grapevine exist in China, and three of them (HpSVd, GYSVd-1 and GYSVd-3) exist in Japan. The contrast in diversity of viroid species in old grapevines from China and Japan may reflect different history of viticulture between the two countries. In general, the species of viroids infecting grapevine in China, as well as those in Iran and Australia, were more diverse than in the other countries. The population structure of viroids infecting grapevine in China and Japan showed species-dependency; i.e., HpSVd shared similar population structures in both countries, but GYSVd-1, GYSVd-2, and AGVd showed regional disparity even within the same country, although the role of sequence diversity in the biology of viroids infecting grapevine, such as the pathogenicity and evolution, still needs further study.

Jiang D ，Sano T ，Tsuji M ，Araki H ，Sagawa K ，Purushothama CR ，Zhang Z ，Guo R ，Xie L ，Wu Z ，Wang H ，Li S ... -  《-》

Occurrence of grapevine yellow speckle viroid 2 and Australian grapevine viroid in Idaho grapevines.

Dahan J ，Orellana G ，Lee J ，Karasev AV ... -  《PLANT DISEASE》

Diagnostic real-time RT-PCR for the simultaneous detection of Citrus exocortis viroid and Hop stunt viroid.

Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd) are two important viroids known to infect several plant species worldwide. In this study, a real-time reverse transcription (RT) TaqMan polymerase chain reaction (PCR) assay was developed and optimized for the simultaneous detection of CEVd and HSVd. The assay's analytical and diagnostic sensitivity and specificity were evaluated using reference isolates. Two different RNA extraction methods and one rapid crude template preparation procedure were compared in terms of extraction purity and efficiency for PCR applications. Extraction method Q included a commercially available kit, whereas method C was a modified chloroform-phase extraction in house protocol. Procedure S involved blotting the sap extract on a positively charged nylon membrane and elution. The multiplex RT-TaqMan PCR assay successfully discriminated the two viroid species from all reference samples and its recorded diagnostic sensitivity (Dse) and specificity (Dsp) was 100%. On the contrary, in conventional RT-PCR tests, the overall Dse and Dsp were lower and estimated at 94 and 95% for CEVd, and 97 and 98% for HSVd, respectively. In a direct comparison, the developed assay presented 1000-fold more analytical sensitivity. Spectrophotometric results showed that RNA extraction methods Q and C, yielded the purest RNA, and gave the lowest mean Ct values. Alternative template preparation method S resulted in Ct values statistically similar to those obtained with methods Q to C when tested by RT-TaqMan PCR. The developed assay, using crude template preparation S, allows the simple, accurate and cost-effective testing of a large number of plant samples, and can be applied in surveys and certification schemes.

Papayiannis LC 《-》

Diversity of viroids infecting grapevines in the South African Vitis germplasm collection.

Seven viroid species and one putative viroid species have been reported to infect grapevine namely, hop stunt viroid (HSVd), grapevine yellow speckle viroid 1 (GYSVd-1), grapevine yellow speckle viroid 2 (GYSVd-2), Australian grapevine viroid (AGVd), Japanese grapevine viroid (JGVd), grapevine latent viroid (GLVd), and citrus exocortis viroid (CEVd), as well as a grapevine hammerhead viroid-like RNA (GHVd), so far. In this study, RNA sequence (RNA-Seq) data, from 229 Vitis accessions from the field-maintained vineyard of the South African Vitis germplasm collection, were analysed to determine the diversity of the viroids present. Five of the seven known grapevine-infecting viroids and one putative grapevine-infecting viroid species were very commonly found, with 214 of the 229 samples containing at least one viroid species. HSVd, GYSVd-1, GYSVd-2, AGVd, and JGVd, as well as GHVd, were identified in the RNA-Seq data of the samples and confirmed using RT-PCR and Sanger sequencing. The HSVd sequences indicated the presence of two variants, with one showing multiple nucleotide insertions. AGVd and GYSVd-2 did not display significant sequence diversity, confirming past international studies. GYSVd-1 occurs as four major variants worldwide and representatives of all four variants were identified in this vineyard. This is the first report on the diversity of viroids infecting grapevine in South Africa and the first report of JGVd outside of Japan and GHVd in South Africa. Further studies are needed to fully assess the population and to identify potentially new viroid species.

Morgan SW ，Read DA ，Burger JT ，Pietersen G ... -  《-》