Niğde İli Ceviz (Juglans spp.) Ağaçlarındaki Viral Etmenlerin Yüksek Kapasiteli Dizileme ile Araştırılması
Abstract
Niğde ilinde, Ekim 2023'te, virüs benzeri simptomlar gösteren ceviz ağaçları, viral enfeksiyonları araştırmak için yüksek kapasiteli dizileme kullanılarak analiz edilmiştir. Çift sarmallı RNA (dsRNA) ekstraksiyonu yapılmış, ardından dsRNA'lar cDNA'ya dönüştürülmüştür. Daha sonra metagenomik analizler için MID primerleri ile barkodlama kullanılarak PCR gerçekleştirilmiştir. B13, B22, B23 ve B21+B5 ceviz örneklerinin PCR ürünleri Illumina NovaSeq 6000 dizileme platformu kullanılarak dizilenmiştir. Dizileme verisi Geneious Prime ve CLC Workbench kullanılarak biyoinformatik analize tabi tutulmuştur. Bu karşılaştırmada, CLC Workbench'te daha fazla contig oluşturulmuş ve BLASTx analizi ile daha doğru sonuçlar elde edilmiştir. B21+B5 HTS verisinin referansa göre haritalama-analizlerine göre, 29.574 okuma cherry leaf roll virüsü RNA1 (CLRV) RefSeq NC_015414 ile ve 405 okuma CLRV RNA2 RefSeq NC_015415 ile eşleşmiştir. Ancak, B23 HTS verisindeki 4 okuma cucumber mosaic virus (CMV) RefSeq NC_001440 ile eşleşmiştir. Bu virüslerin varlığını doğrulamak amacıyla, cevizlerden ekstrakte edilen total RNA'yı test etmek için virüse özgü primerler kullanılmıştır. CLRV, B21 örneğinde tespit edilmiş ancak B5'te tespit edilmemiştir. CMV, B23 örneğinde tespit edilmemiştir. Yüksek kapasiteli dizileme kullanılarak viral ajanların tespitini arttırmak için nükleik asit ekstraksiyon yöntemlerinin iyileştirilmesi önerilmektedir.
References
- Baumgartnerova, H. (1996). First findings of plum pox virus in walnut trees (Juglans regia L.). Acta Virologica, 40(1), 59-60.
- Budak, Y. (2010). Ceviz Yetiştiriciliği, T.C. Samsun Valiliği, İl Tarım Müdürlüğü, Samsun.
- Caglayan, K., Gazel, M., Roumi, V., Kocabag, H. D., Tunç, B., Reynard, J. S., Ruiz-García, A. B., Olmos, A., & Candresse, T. (2020). Identification of pomegranate as a new host of Passiflora edulis symptomless virus (PeSV) and analysis of PeSV diversity. Agronomy, 10(11), 1821. https://doi.org/10.3390/agronomy10111821 .
- François, S., Filloux, D., Fernandez, E., Ogliastro, M., & Roumagnac, P. (2018). Viral metagenomics approaches for high-resolution screening of multiplexed arthropod and plant viral communities. Viral metagenomics: methods and protocols, 77-95. https://doi.org/10.1007/978-1-4939-7683-6_7 .
- Grieco, F., Alkowni, R., Saponari, M., Savino, V., & Martelli, G. P. (2000). Molecular detection of olive viruses. EPPO Bulletin, 30(3‐4), 469-473. https://doi.org/10.1111/j.1365-2338.2000.tb00931.x .
- Gürcan, K., Turan, S., Teber, S., Kılınçer, İ., Uz, İ., Tamisier, L., & Çağlayan, K. (2021). Molecular and biological characterization of a new mulberry idaeovirus. Virus Research, 298, 198411. https://doi.org/10.1016/ j.virusres.2021.198411 .
- Güvenç, I., & Purlu, G. (2022). Projections of production and demand for walnut in the period 2020-2045 in Turkey. KSU Journal of Agrıculture and Nature 25(1), 57-65. https://doi.org/10.18016/ksutarimdoga.vi.848460
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- Ma, Y., Marais, A., Lefebvre, M., Theil, S., Svanella-Dumas, L., Faure, C., & Candresse, T. (2019). Phytovirome analysis of wild plant populations: comparison of double-stranded RNA and virion-associated nucleic acid metagenomic approaches. Journal of Virology, 94(1), 10-1128. https://doi.org/10.1128/jvi.01462-19 .
- MacKenzie, D. J., McLean, M. A., Mukerji, S., & Green, M. (1997). Improved RNA extraction from woody plants for the detection of viral pathogens by reverse transcription-polymerase chain reaction. Plant disease, 81(2), 222-226. https://doi.org/10.1094/PDIS.1997.81.2.222 .
- Marais, A., Faure, C., Bergey, B., & Candresse, T. (2018). Viral double-stranded RNAs (dsRNAs) from plants: alternative nucleic acid substrates for high-throughput sequencing. Viral metagenomics: methods and protocols, 45-53. https://doi.org/10.1007/978-1-4939-7683-6_4 .
- Maree, H. J., Fox, A., Al Rwahnih, M., Boonham, N., & Candresse, T. (2018). Application of HTS for routine plant virus diagnostics: state of the art and challenges. Frontiers in Plant Science, 9, 1082. https://doi.org/10.3389/fpls.2018.01082 .
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- Ozturk, M. O., Sipahioglu, H. M., Ocak, M., & Usta, M. (2008). Cherry leafroll virus in Juglans regia in the Lake Van Basin of Türkiye. Journal of Plant Pathology, 90(1), 75-79. https://www.jstor.org/stable/41998461 .
- Savino, V., Quacquarelli, A., Gallitelli, D., Piazzolla, P., & Marteli, G. P. (1976). Occurrence of Two Sap-Transmissible Viruses in Walnuts. In X International Symposium on Fruit Tree Virus Diseases 67 (pp. 327-327). https://doi.org/10.17660/ActaHortic.1976.67.44
- Sharma, P., Yadav, D., & Gaur, R. K. (Eds.). (2022). Bioinformatics in Agriculture: Next Generation Sequencing Era, Academic Press, 48-49.
- Sipahioglu, H. M., Tekin, Z., & Usta, M., (2011). Detection and partial characterization of two distinct walnut isolates of cherry leaf roll virus (CLRV). African Journal of Biotechnology , 10(14), 2579-2587.
- Sütyemez, M. Sağlıklı bir yasam tarzı için ceviz. KSÜ Doğa Bilimleri Dergisi, 11(1), 138-142.
- TUIK, (2023, April 3). Türkiye İstatistik Kurumu, https://www.tuik.gov.tr/
- Werner, R., Mühlbach, H. P., & Büttner, C. (1997). Detection of cherry leaf roll nepovirus (CLRV) in birch, beech and petunia by immuno‐capture RT‐PCR using a conserved primer pair. European Journal of Forest Pathology, 27(5), 309-318. https://doi.org/10.1111/j.1439-0329.1997.tb01085.x .
- Yegül, M., & Baloğlu, S. (2019). Ceviz ve Bademde Prune dwarf virus, Prunus necrotic ringspot virus ve Cherry leaf roll virus Yoğunluklarının Mevsimsel Değişimi. Çukurova Tarım ve Gıda Bilimleri Dergisi, 34(1), 37-50.
Investigation of Viral Agents in Walnut (Juglans spp.) Trees by High Throughput Sequencing from Niğde province, Türkiye
Abstract
In October 2023, virus-like symptomatic walnut trees in Niğde province were investigated using high-throughput sequencing (HTS) to investigate viral infections. Double-stranded RNA (dsRNA) extraction was conducted, followed by conversion of dsRNA into cDNA. PCR was then performed using barcoding with MID primers for metagenomic analyses. The PCR products from walnut samples B13, B22, B23, and B21 + B5 were sequenced using the Illumina NovaSeq 6000 sequencing platform. The sequencing data underwent bioinformatics analysis using Geneious Prime and CLC Genomic Workbench. In this comparison, more contigs were constructed in CLC Genomic Workbench, resulting in more precise outcomes with BLASTx analysis. According to the map, to reference analyses of B21+B5 HTS data, 29,574 reads matched with cherry leaf roll virus RNA1 (CLRV) RefSeq NC_015414, and 405 reads matched with CLRV RNA2 RefSeq NC_015415. However, 4 reads matched with cucumber mosaic virus (CMV) RefSeq NC_001440 from B23 HTS data. To confirm the presence of these viruses, virus-specific primers were used to test the total RNA extracted from walnuts. CLRV was detected in sample B21 but not in B5. CMV was not detected in sample B23. Improving nucleic acid extraction methods is recommended to enhance the detection of viral agents using high-throughput sequencing.
References
- Baumgartnerova, H. (1996). First findings of plum pox virus in walnut trees (Juglans regia L.). Acta Virologica, 40(1), 59-60.
- Budak, Y. (2010). Ceviz Yetiştiriciliği, T.C. Samsun Valiliği, İl Tarım Müdürlüğü, Samsun.
- Caglayan, K., Gazel, M., Roumi, V., Kocabag, H. D., Tunç, B., Reynard, J. S., Ruiz-García, A. B., Olmos, A., & Candresse, T. (2020). Identification of pomegranate as a new host of Passiflora edulis symptomless virus (PeSV) and analysis of PeSV diversity. Agronomy, 10(11), 1821. https://doi.org/10.3390/agronomy10111821 .
- François, S., Filloux, D., Fernandez, E., Ogliastro, M., & Roumagnac, P. (2018). Viral metagenomics approaches for high-resolution screening of multiplexed arthropod and plant viral communities. Viral metagenomics: methods and protocols, 77-95. https://doi.org/10.1007/978-1-4939-7683-6_7 .
- Grieco, F., Alkowni, R., Saponari, M., Savino, V., & Martelli, G. P. (2000). Molecular detection of olive viruses. EPPO Bulletin, 30(3‐4), 469-473. https://doi.org/10.1111/j.1365-2338.2000.tb00931.x .
- Gürcan, K., Turan, S., Teber, S., Kılınçer, İ., Uz, İ., Tamisier, L., & Çağlayan, K. (2021). Molecular and biological characterization of a new mulberry idaeovirus. Virus Research, 298, 198411. https://doi.org/10.1016/ j.virusres.2021.198411 .
- Güvenç, I., & Purlu, G. (2022). Projections of production and demand for walnut in the period 2020-2045 in Turkey. KSU Journal of Agrıculture and Nature 25(1), 57-65. https://doi.org/10.18016/ksutarimdoga.vi.848460
- Kreuze, J. F., Perez, A., Untiveros, M., Quispe, D., Fuentes, S., Barker, I., & Simon, R. (2009). Complete viral genome sequence and discovery of novel viruses by deep sequencing of small RNAs: a generic method for diagnosis, discovery and sequencing of viruses. Virology, 388(1), 1-7. https://doi.org/10.1016/j.virol.2009.03.024
- Ma, Y., Marais, A., Lefebvre, M., Theil, S., Svanella-Dumas, L., Faure, C., & Candresse, T. (2019). Phytovirome analysis of wild plant populations: comparison of double-stranded RNA and virion-associated nucleic acid metagenomic approaches. Journal of Virology, 94(1), 10-1128. https://doi.org/10.1128/jvi.01462-19 .
- MacKenzie, D. J., McLean, M. A., Mukerji, S., & Green, M. (1997). Improved RNA extraction from woody plants for the detection of viral pathogens by reverse transcription-polymerase chain reaction. Plant disease, 81(2), 222-226. https://doi.org/10.1094/PDIS.1997.81.2.222 .
- Marais, A., Faure, C., Bergey, B., & Candresse, T. (2018). Viral double-stranded RNAs (dsRNAs) from plants: alternative nucleic acid substrates for high-throughput sequencing. Viral metagenomics: methods and protocols, 45-53. https://doi.org/10.1007/978-1-4939-7683-6_4 .
- Maree, H. J., Fox, A., Al Rwahnih, M., Boonham, N., & Candresse, T. (2018). Application of HTS for routine plant virus diagnostics: state of the art and challenges. Frontiers in Plant Science, 9, 1082. https://doi.org/10.3389/fpls.2018.01082 .
- Moubset, O., François, S., Maclot, F., Palanga, E., Julian, C., Claude, L., Fernandez, E., Rott, P., Daugrois, J.-H., Antoine-Lorquin, A., Bernardo, P., Blouin, A. G, Temple, C., Kraberger, S., Fontenele, R, S., Harkins, G. V., Ma, Y., Marais, A., Candresse, A., Chéhida, S. B., Lefeuvre, P., Lett, J.-M., Varsani, A., Massart, S., Ogliastro, M., Martin, D. P., Filloux, D. & Roumagnac, P. (2022). Virion-associated nucleic acid-based metagenomics: a decade of advances in molecular characterization of plant viruses. Phytopathology, 112(11), 2253-2272. https://doi.org/10.1094/PHYTO-03-22-0096-RVW .
- Ozturk, M. O., Sipahioglu, H. M., Ocak, M., & Usta, M. (2008). Cherry leafroll virus in Juglans regia in the Lake Van Basin of Türkiye. Journal of Plant Pathology, 90(1), 75-79. https://www.jstor.org/stable/41998461 .
- Savino, V., Quacquarelli, A., Gallitelli, D., Piazzolla, P., & Marteli, G. P. (1976). Occurrence of Two Sap-Transmissible Viruses in Walnuts. In X International Symposium on Fruit Tree Virus Diseases 67 (pp. 327-327). https://doi.org/10.17660/ActaHortic.1976.67.44
- Sharma, P., Yadav, D., & Gaur, R. K. (Eds.). (2022). Bioinformatics in Agriculture: Next Generation Sequencing Era, Academic Press, 48-49.
- Sipahioglu, H. M., Tekin, Z., & Usta, M., (2011). Detection and partial characterization of two distinct walnut isolates of cherry leaf roll virus (CLRV). African Journal of Biotechnology , 10(14), 2579-2587.
- Sütyemez, M. Sağlıklı bir yasam tarzı için ceviz. KSÜ Doğa Bilimleri Dergisi, 11(1), 138-142.
- TUIK, (2023, April 3). Türkiye İstatistik Kurumu, https://www.tuik.gov.tr/
- Werner, R., Mühlbach, H. P., & Büttner, C. (1997). Detection of cherry leaf roll nepovirus (CLRV) in birch, beech and petunia by immuno‐capture RT‐PCR using a conserved primer pair. European Journal of Forest Pathology, 27(5), 309-318. https://doi.org/10.1111/j.1439-0329.1997.tb01085.x .
- Yegül, M., & Baloğlu, S. (2019). Ceviz ve Bademde Prune dwarf virus, Prunus necrotic ringspot virus ve Cherry leaf roll virus Yoğunluklarının Mevsimsel Değişimi. Çukurova Tarım ve Gıda Bilimleri Dergisi, 34(1), 37-50.
Details
| Primary Language | English |
|---|---|
| Subjects | Phytopathology |
| Journal Section | RESEARCH ARTICLE |
| Authors | |
| Early Pub Date | May 1, 2025 |
| Publication Date | |
| Submission Date | January 13, 2025 |
| Acceptance Date | February 26, 2025 |
| Published in Issue | Year 2025Volume: 28 Issue: 3 |
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