Development and Validation of a Degenerate Primer and LNA Probe Set for Rapid, Reliable, and Sensitive Detection of grapevine rupestris stem pitting-associated virus (Foveavirus rupestris; GRSPaV) in Certification Programs

Year 2025, Volume: 35 Issue: 4, 742 - 757, 25.12.2025

Serkan Önder , Serpil Erilmez , İsmail Can Paylan , Metin Ceyhan

https://doi.org/10.29133/yyutbd.1655785

Abstract

Grapevine rupestris stem pitting-associated virus (GRSPaV) is a significant pathogen in certification programs, causing stem pitting in the trunks of grafted vines. Nevertheless, it remains latent in ungrafted varieties and rootstocks. Given its status as a certification agent, rapid, reliable, and sensitive detection of GRSPaV is of great importance. However, the identification of genetically distinct variants of GRSPaV has made reliable diagnosis using the current primer pair employed in certification analyses challenging, leading to false-negative results. In this study, degenerate primers and an LNA probe set were designed using 25 genomic sequences from five different GRSPaV strain groups to achieve rapid, reliable, and sensitive detection. Optimization studies were conducted using the Roche LightCycler® Nano Real-time PCR system. Real-time RT-PCR results carried out via 41 grapevine varieties and 81 rootstock samples demonstrated that the newly designed degenerate primer pair and LNA probe set yielded positive results in 37 out of 122 vine samples, whereas the existing primer pair detected only 5 positive samples. The findings indicate that the designed degenerate primer and LNA probe set can be successfully utilized for the rapid, reliable, and sensitive detection of GRSPaV in certification analyses. Given the high genetic variability of viruses like GRSPaV, robust detection requires continuously updated primers, rigorous strain monitoring, and skilled diagnosticians since a universally perfect, future-proof primer remains an unattainable ideal.

Keywords

GRSPaV , Certification analyses , Real-time PCR , Degenerate primer , LNA Probe

Ethical Statement

Ethical approval is not required for this study because a new detection method for diagnosis of GRSPaV was performed in this study.

Supporting Institution

The study was funded by Manisa Viticulture Research Institation that affiliated to Republic Of Türkiye Ministry Of Agriculture And Forestry General Directorate Of Agricultural Research And Policies.

Thanks

Thanks to Manisa Viticulture Research Institation that affiliated to Republic Of Türkiye Ministry Of Agriculture And Forestry General Directorate Of Agricultural Research And Policies for providing the plant material.

References

• Anonymous, (2006). Seed Law (Law No. 5553 of October 31, 2006), Türkiye . https://www.wipo.int/wipolex/en/legislation/details/10909. Access date:08.02.2025
• Anonymous, (2010). Veterinary Services, Plant Health, Food, and Feed Law No. 5996. https://www.tarimorman.gov.tr/Belgeler/ENG/Legislation/law_veterinary_services.pdf. Access date:15.02.2025
• Basso, M. F., Fajardo, T. V. M., Eiras, M., Ayub, R. A., & Nickel, O. (2010). Molecular detection and identification of virus associated with symptomatic and symptomless grapevines. Ciência Rural, 40(11), 2249–2255. https://doi.org/10.1590/S0103-84782010001100001
• Çelen, H., İnce, E., & Özdemi̇r, M. (2020). Turkish Sapling Certifıcation System; Evaluations and Recommendations. Journal of Agriculture, 3(2), 10–22. https://doi.org/10.46876/ja.788397
• Çelik, H., Çelik, S., Kunter, B. M., Söylemezoğlu, G., Boz, Y., Özer, C., & Atak, A. (2005). Bağcılıkta gelişme ve üretim hedefleri. VI. Türkiye Ziraat Mühendisliği Teknik Kongresi, 3(7).
• Chen, R., Gao, X.-B., Yu, X.-L., Song, C.-X., & Qiu, Y. (2016). Novel multiplex PCR assay using locked nucleic acid (LNA)-based universal primers for the simultaneous detection of five swine viruses. Journal of Virological Methods, 228, 60–66. https://doi.org/10.1016/j.jviromet.2015.11.018
• Chousalkar, K. K., Cheetham, B. F., & Roberts, J. R. (2009). LNA probe-based real-time RT-PCR for the detection of infectious bronchitis virus from the oviduct of unvaccinated and vaccinated laying hens. Journal of Virological Methods, 155(1), 67–71. https://doi.org/10.1016/j.jviromet.2008.09.028
• Diaz-Lara, A., Golino, D., Preece, J. E., & Al Rwahnih, M. (2020). Development of RT-PCR degenerate primers to overcome the high genetic diversity of grapevine virus T. Journal of Virological Methods, 282, 113883. https://doi.org/10.1016/j.jviromet.2020.113883
• Glasa, M., Predajňa, L., Šoltys, K., Sihelská, N., Nagyová, A., Wetzel, T., & Sabanadzovic, S. (2017). Analysis of Grapevine rupestris stem pitting-associated virus in Slovakia reveals differences in intra-host population diversity and naturally occurring recombination events. The Plant Pathology Journal, 33(1), 34.
• Gökmen, D., Genç, Y., Atakurt, Y., Yağmurlu, B. (2004). Comparison of Two Correlated Proportions for Clustered Data. Journal of Statistical Research, 3(3), 21-29.
• Gustafson, K. S. (2008). Locked Nucleic Acids Can Enhance the Analytical Performance of Quantitative Methylation-Specific Polymerase Chain Reaction. The Journal of Molecular Diagnostics, 10(1), 33–42. https://doi.org/10.2353/jmoldx.2008.070076
• Ha, C., Coombs, S., Revill, P. A., Harding, R. M., Vu, M., & Dale, J. L. (2008). Design and application of two novel degenerate primer pairs for the detection and complete genomic characterization of potyviruses. Archives of Virology, 153(1), 25–36. https://doi.org/10.1007/s00705-007-1053-7
• Hasiów-Jaroszewska, B., Rymelska, N., & Borodynko, N. (2015). LNA probe-based assay for the detection of Tomato black ring virus isolates. Molecular and Cellular Probes, 29(1), 78–80. https://doi.org/10.1016/j.mcp.2014.12.002
• Jiménez-Montenegro, L., Mendizabal, J. A., Alfonso, L., Azparren, L., & Urrutia, O. (2022). Development of a duplex qPCR assay with locked nucleic acid probes for A, B and E kappa-casein variants detection. Scientific Reports, 12(1), 16387. https://doi.org/10.1038/s41598-022-20586-w
• Johnson, M. P., Haupt, L. M., & Griffiths, L. R. (2004). Locked nucleic acid (LNA) single nucleotide polymorphism (SNP) genotype analysis and validation using real‐time PCR. Nucleic Acids Research, 32(6), e55-e55.
• Kim, H. Y. (2017). Statistical notes for clinical researchers: Chi-squared test and Fisher's exact test. Restorative Dentistry & Endodontics, 42(2), 152.
• Kummert, J., Rufflard, G., & Marinho, V. L. (1996). Use of degenerate primers for RT-PCR detection of apple and pear tree viruses. In COST823. New technologies to improve phytodiagnosis. Workshop of the nucleic acid-based technology working group.
• Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, P. A., McWilliam, H., Valentin, F., Wallace, I. M., Wilm, A., Lopez, R., Thompson, J. D., Gibson, T. J., & Higgins, D. G. (2007). Clustal W and Clustal X version 2.0. Bioinformatics, 23(21), 2947–2948. https://doi.org/10.1093/bioinformatics/btm404
• Lim, S. J., Rosario, K., Kernbach, M. E., Gross, A. J., Furman, B. T., & Breitbart, M. (2023). Limited potexvirus diversity in eastern Gulf of Mexico seagrass meadows. Biorxiv, https://doi.org/10.1101/2023.12.11.571111
• Lima, Alkowni, R., Uyemoto, J. K., Golino, D., Osman, F., & Rowhani, A. (2006). Molecular analysis of a California strain of Rupestris stem pitting-associated virus isolated from declining Syrah grapevines. Archives of Virology, 151, 1889–1894.
• Lima, Alkowni, R., Uyemoto, J. K., & Rowhani, A. (2009). Genomic study and detection of a new variant of Grapevine rupestris stem pitting associated virus in declining California Pinot Noir grapevines. Journal of Plant Pathology, 155–162.
• Makhtar, S. T., Tan, S. W., Nasruddin, N. A., Abdul Aziz, N. A., Omar, A. R., & Mustaffa-Kamal, F. (2021). Development of TaqMan-based real-time RT-PCR assay based on N gene for the quantitative detection of feline morbillivirus. BMC Veterinary Research, 17, 1-11.
• Martelli, G. P. (1993). Graft-transmissible diseases of grapevines: Handbook for detection and diagnosis. Food and Agriculture Organization of The United Nations. 263 pp. https://books.google.com/books?hl=tr&lr=&id=G1IVY1kbUiwC&oi=fnd&pg=PR2&dq=Grafttransmissible+diseases+of+grapevines,+handbook+for+detection+and+diagnosis.+&ots=ExaP6Nizvz&sig=ygKSIHSoOFKGqBMOO17_OuB5hTg. Access date:10.01.2025
• Martelli, G. P. (2017). An Overview on Grapevine Viruses, Viroids, and the Diseases They Cause. In B. Meng, G. P. Martelli, D. A. Golino, & M. Fuchs (Eds.), Grapevine Viruses: Molecular Biology, Diagnostics and Management. Springer International Publishing, 31-46. https://doi.org/10.1007/978-3-319-57706-7_2
• Matus, J. T., Vega, A., Loyola, R., Serrano, C., Cabrera, S., & Arce-Johnson, P. (2008). Phytoplasma and virus detection in commercial plantings of Vitis vinifera cv. Merlot exhibiting premature berry dehydration. Electronic Journal of Biotechnology, 11(5), 7-8.
• Meena, R. P., Prabha, K., & Baranwal, V. K. (2020). Development of RT-PCR degenerate primers for the detection of two mandariviruses infecting citrus cultivars in India. Journal of Virological Methods, 275, 113753. https://doi.org/10.1016/j.jviromet.2019.113753
• Meng, B. and Rowhani, A. (2017). Grapevine rupestris stem pitting-associated virus. In: Meng, B., Martelli, G.P., Golino, D.A. and Fuchs, M. (Eds.) Grapevine Viruses: Molecular Biology, Diagnostics and Management. Springer International Publishing, 257–287.
• Meng, B., Credi, R., Petrovic, N., Tomazic, I., & Gonsalves, D. (2003). Antiserum to Recombinant Virus Coat Protein Detects Rupestris stem pitting associated virus in Grapevines. Plant Disease, 87(5), 515–522. https://doi.org/10.1094/PDIS.2003.87.5.515
• Meng, B., & Gonsalves, D. (2007). Grapevine rupestris stem pitting-associated virus: Plant Viruses.
• Meng, B., Johnson, R., Peressini, S., Forsline, P. L., & Gonsalves, D. (1999). Rupestris Stem Pitting Associated Virus-1 is Consistently Detected in Grapevines that are Infected with Rupestris Stem Pitting. European Journal of Plant Pathology, 105, 191-199.
• Meng, B., & Li, C. (2010). The capsid protein of Grapevine rupestris stem pitting-associated virus contains a typical nuclear localization signal and targets to the nucleus. Virus Research, 153(2), 212–217. https://doi.org/10.1016/j.virusres.2010.08.003
• Meng, B., Pang, S. Z., Forsline, P. L., McFerson, J. R., & Gonsalves, D. (1998). Nucleotide sequence and genome structure of grapevine rupestris stem pitting associated virus-1 reveal similarities to apple stem pitting virus. Journal of General Virology, 79(8), 2059–2069. https://doi.org/10.1099/0022-1317-79-8-2059
• Meng, B., Rebelo, A. R., & Fisher, H. (2006). Genetic diversity analyses of grapevine Rupestris stem pitting-associated virus reveal distinct population structures in scion versus rootstock varieties. Journal of General Virology, 87(6), 1725-1733.
• Minafra, A., Casati, P., Elicio, V., Rowhani, A., Saldarelli, P., Savino, V., & Martelli, G. P. (2000). Serological detection of Grapevine rupestris stem pitting-associated virus (GRSPaV) by a polyclonal antiserum to recombinant virus coat protein. VITIS-Journal of Grapevine Research, 39(3), 115.
• Nakaune, R, K. Inoue, H. Nasu, K. Kakogawa, H. Nitta, M. Nakano. 2006. Etiology of rugosewood disease in Japanese grapevines. In Extended Abstracts of the 15th Meeting of the ICVG, Stellenbosch, South Africa. Apr., 3(7), 237–238.
• Orfanidou, C. G., Moraki, K., Panailidou, P., Lotos, L., Katsiani, A., Avgelis, A., ... & Maliogka, V. I. (2021). Prevalence and genetic diversity of viruses associated with rugose wood complex in Greek vineyards. Plant Disease, 105(11), 3677-3685.
• Rojas, M. R. (1993). Use of Degenerate Primers in the Polymerase Chain Reaction to Detect Whitefly-Transmitted Geminiviruses. Plant Disease, 77(4), 340. https://doi.org/10.1094/PD-77-0340
• Rowhani, A., Zhang, Y. P., Chin, H., Minafra, A., Golino, D. A., & Uyemoto, J. K. (2000). Grapevine rupestris stem pitting-associated virus: Population diversity, titer in the host and possible transmission vector. Extended Abstracts of the 13th Meeting of ICVG, Adelaide, 12-17 March, 37. https://cir.nii.ac.jp/crid/1574231874738864512
• Sanyürek, N. K., Çakır, A., & Söylemezoğlu, G. (2021). Optimization of Meristem Culture to Obtain Virus-Free Clonal Basic Material of Grape Cultivars. Yuzuncu Yıl University Journal of Agricultural Sciences, 31(3), 617-628.
• Terlizzi, F., Li, C., Ratti, C., Qiu, W., Credi, R., & Meng, B. (2011). Detection of multiple sequence variants of Grapevine rupestris stem pitting-associated virus using primers targeting the polymerase domain and partial genome sequencing of a novel variant. Annals of Applied Biology, 159(3), 478–490. https://doi.org/10.1111/j.1744-7348.2011.00512.x
• Uysal, H., & Karabat, S. (2017). Forecasting and evaluation for raisin export in turkey. In BIO Web of Conferences 9, 03002. EDP Sciences.
• Wang, Q., Wang, X., Zhang, J., & Song, G. (2012). LNA real-time PCR probe quantification of hepatitis B virus DNA. Experimental and Therapeutic Medicine, 3(3), 503–508. https://doi.org/10.3892/etm.2011.442
• Zhang, Y.-P., Uyemoto, J. K., Golino, D. A., & Rowhani, A. (1998). Nucleotide Sequence and RT-PCR Detection of a Virus Associated with Grapevine Rupestris Stem-Pitting Disease. Phytopathology, 88(11), 1231–1237. https://doi.org/10.1094/PHYTO.1998.88.11.1231