Abstract
A multiplex RT-PCR reaction was developed and validated for the simultaneous detection of two viruses and two viroids: grapevine fleck virus, grapevine Pinot gris virus, grapevine yellow speckle viroid 1, and hop stunt viroid. The multiplex reaction specificity was verified on a set of 210 grapevines from the Czech Republic. The results showed the occurrence of hop stunt viroid in 94.3% of the examined plants, while incidence of the other pathogens was lower: grapevine Pinot gris virus (64.8%), grapevine yellow speckle viroid 1 (52.4%), and grapevine fleck virus (15.2%).
This is a preview of subscription content, log in via an institution to check access.
References
Aloisio M, Morelli M, Elicio V, Saldarelli P, Ura B, Bortot B, Severini GM, Minafra A (2018) Detection of four regulated grapevine viruses in a qualitative, single tube real-time PCR with melting curve analysis. J Virol Methods 257:42–47
Bruisson S, Lebel S, Walter B, Prevotat L, Seddas S, Schellenbaum P (2017) Comparative detection of a large population of grapevine viruses by TaqMan® RT-qPCR and ELISA. J Virol Methods 240:73–77
Eichmeier A, Komínková M, Komínek P, Baránek M (2016a) Comprehensive virus detection using next generation sequencing in grapevine vascular tissues of plants obtained from the wine regions of Bohemia and Moravia (Czech Republic). PLoS One 11(12):e0167966
Eichmeier A, Peňázová E, Pavelková R, Mynarzová Z, Saldarelli P (2016b) Detection of grapevine Pinot gris virus in certified grapevine stocks in Moravia, Czech Republic. J Plant Pathol 98:155–157
Gambino G (2015) Multiplex RT-PCR method for the simultaneous detection of nine grapevine viruses. Methods Mol Biol 1236:39–47
Giampetruzzi A, Roumi V, Roberto R, Malossini U, Yoshikawa N, La Notte P, Terlizzi F, Credi R, Saldarelli P (2012) A new grapevine virus discovered by deep sequencing of virus-and viroid-derived small RNAs in cv Pinot gris. Virus Res 163:262–268
Glasa M, Predajňa L, Komínek P, Nagyová A, Candresse T, Olmos A (2014) Molecular characterization of divergent Grapevine Pinot gris virus isolates and their detection in Slovak and Czech grapevines. Arch Virol 159:2103–2107
Hajizadeh M, Navarro B, Sokhandan Bashir N, Torchetti EM, Di Serio F (2012) Development and validation of a multiplex RT-PCR method for the simultaneous detection of five grapevine viroids. J Virol Methods 179:62–69
Komínek P (2008) Distribution of grapevine viruses in vineyards of the Czech Republic. J Plant Pathol 90:357–358
Komínek P, Komínková M, Jandová B (2016) Effect of repeated Ribavirin treatment on grapevine viruses. Acta Virol 60(4):400–403
López-Fabuel I, Wetzel T, Bertolini E, Bassler A, Vidal E, Torres LB, Yuste A, Olmos A (2013) Real-time multiplex RT-PCR for the simultaneous detection of the five main grapevine viruses. J Virol Methods 188:21–24
Matoušek J, Orctová L, Patzak J, Svoboda P, Ludvíková I (2003) Molecular sampling of hop stunt viroid (HSVd) from grapevines in hop production areas in the Czech Republic and hop protection. Plant Soil Environ 49:168–175
Nassuth A, Pollari E, Helmeczy K, Stewart S, Kofalvi SA (2000) Improved RNA extraction and one-tube RT-PCR assay for simultaneous detection of control plant RNA plus several viruses in plant extracts. J Virol Methods 90:37–49
Osman F, Rowhani A (2008) Real-time RT-PCR (TaqMan®) assays for the detection of viruses associated with Rugose wood complex of grapevine. J Virol Methods 154:69–75
Osman F, Hodzic E, Omanska-Klusek A, Olinek T, Rowhani A (2013) Development and validation of a multiplex quantitative PCR assay for the rapid detection of Grapevine virus A, B and D. J Virol Methods 194:138–145
Sahana AB, Adkar-Purushothama CR, Chennappa G, Zhang ZX, Sreenivasa MY, Sano T (2013) First report of Grapevine yellow speckle viroid-1 and Hop stunt viroid infecting grapevines (Vitis vinifera) in India. Plant Dis 97:1517
Saldarelli P, Giampetruzzi A, Morelli M, Malossini U, Pirolo C, Bianchedi P, Gualandri V (2015) Genetic variability of Grapevine Pinot gris virus and its association with grapevine leaf mottling and deformation. Phytopathology 105:555–563
Szychowski JA, Credi R, Reanwarakorn K, Semancik JS (2007) Population diversity in Grapevine yellow speckle viroid-1 and the relationship to disease expression. Virology 248:432–444
Ward LI, Burnip GM, Liefting LW, Harper SJ, Clover GRG (2011) First report of Grapevine yellow speckle viroid 1 and Hop stunt viroid in grapevine (Vitis vinifera) in New Zealand. Plant Dis 95:617
Acknowledgements
The authors wish to thank Peter Lemkin for improvements in the English.
Funding
This work was supported by institutional support MZE-RO0418 from the Ministry of Agriculture of the Czech Republic.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
The authors declare they carefully followed the COPE guidelines.
The authors declare that they have no conflicts of interest.
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
ESM 1
(XLSX 24 kb)
Rights and permissions
About this article
Cite this article
Komínková, M., Komínek, P. Development and validation of RT-PCR multiplex detection of grapevine viruses and viroids in the Czech Republic. J Plant Pathol 102, 511–515 (2020). https://doi.org/10.1007/s42161-019-00452-x
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s42161-019-00452-x