Molecular identification and monitoring of major apple tree viruses in Ukraine

Kateryna Hrynchuk, Ihor Antipov
Abstract

Viral diseases of apple trees cause significant economic losses in horticulture in Ukraine, which requires an updated research into their prevalence. The purpose of the study was to assess the prevalence and regional distribution of the main apple tree viruses in industrial plantings of Ukraine using molecular diagnostic methods. The study was conducted by reverse transcription and polymerase chain reaction on 350 samples of apple leaves and bark taken from industrial plantings in five regions of Ukraine. The specificity of detection of three target viruses was confirmed by sequencing the obtained amplicons. The study found that the overall infection rate of apple plantations was 70.6%, which indicates an epiphytic situation. The most prevalent was Apple stem pitting virus (46.0%), whereas Apple chlorotic leaf spot virus and Apple mosaic virus were detected in 36.0% and 13.7% of samples, respectively. Regional analysis revealed the maximum prevalence of viruses in the Zakarpattia (86.0%) and Kyiv (76.3%) regions, while the minimum indicators were recorded in the Kharkiv region (56.8%). A high level of mixed infections was found (25.4%), with a predominance of the combination of Apple stem pitting virus and Apple chlorotic leaf spot virus (18.3%). Also significant is the high percentage of asymptomatic infections (31.2%), which complicates phytosanitary control. The conclusions confirmed that the results obtained justify the need for urgent implementation of a system for molecular monitoring and certification of planting material. The results of the study can be used to develop a national programme for improving apple plantations in Ukraine

Keywords

polymerase chain reaction; apple stem pitting virus; apple chlorotic leaf spot virus; apple mosaic virus

Suggested citation
Hrynchuk, K., & Antipov, I. (2025). Molecular identification and monitoring of major apple tree viruses in Ukraine. Biological Systems: Theory and Innovation, 16(4), 57-70. https://doi.org/10.31548/biologiya/4.2025.57
References
  1. Barros, H.L., Moresco, G., & Stefani, V. (2018). A simple protocol for the isolation, quantification and quality assessment of DNA and RNA. Revista Virtual de Quimica, 10(5), 1119-1126. doi: 10.21577/1984-6835.20180079.
  2. Bogner, P.N., & Killeen, A.A. (2006). Extraction of nucleic acids. In W.B. Coleman & G.J. Tsongalis (Eds.), Molecular diagnostics: For the clinical laboratorian (pp. 25-30). Totowa: Humana Press. doi: 10.1385/1-59259-928-1:025.
  3. Canales, C., Morán, F., Olmos, A., & Ruiz-García, A.B. (2021). First detection and molecular characterisation of Apple stem grooving virusApple chlorotic leaf spot virus, and Apple hammerhead viroid in loquat in Spain. Plants, 10(11), article number 2293. doi: 10.3390/plants10112293.
  4. Convention on Biological Diversity. (2025). Retrieved from https://www.cbd.int/convention/.
  5. Crossley, B.M., Bai, J., Glaser, A., Maes, R., Porter, E., Killian, M.L., Clement, T., & Toohey-Kurth, K. (2020). Guidelines for Sanger sequencing and molecular assay monitoring. Journal of Veterinary Diagnostic Investigation, 32(6), 767-775. doi: 10.1177/1040638720905833.
  6. Deben, C., Zwaenepoel, K., Boeckx, C., Wouters, A., Pauwels, P., Peeters, M., Lardon, F., Baay, M., & Deschoolmeester, V. (2013). Expression analysis on archival material revisited: Isolation and quantification of RNA extracted from FFPE samples. Diagnostic Molecular Pathology, 22(1), 59-64. doi: 10.1097/PDM.0b013e318269de3b.
  7. EFSA Panel on Plant Health (PLH), et al. (2021). Commodity risk assessment of Malus domestica plants from Ukraine. EFSA Journal, 19(11), article number e06909. doi: 10.2903/j.efsa.2021.6909.
  8. Franco Ortega, S., Prencipe, S., Gullino, M.L., & Spadaro, D. (2020). New molecular tool for a quick and easy detection of apple scab in the field. Agronomy, 10(4), article number 581. doi: 10.3390/agronomy10040581.
  9. Gritsenko, D.A., Aubakirova, K.P., Voitsekhovskiy, I., Soldatova, I., & Galiakparov, N.N. (2020). Simultaneous detection of five apple viruses by RT-PCR. International Journal of Biology & Chemistry, 13(1). doi: 10.26577/ijbch.2020.v13.i1.13.
  10. Hao, L., Xie, J., Chen, S., Wang, S., Gong, Z., Ling, K.-S., Guo, L., Fan, Z., & Zhou, T. (2016). A multiple RT-PCR assay for simultaneous detection and differentiation of latent viruses and apscarviroids in apple trees. Journal of Virological Methods, 234, 16-21. doi: 10.1016/j.jviromet.2016.04.003.
  11. Heo, S., & Chung, Y.S. (2020). Development of real-time quantitative PCR assay based on SYBR Green I and TaqMan probe for detection of apple viruses. The Korean Journal of Crop Science, 65(4), 496-507. doi: 10.7740/kjcs.2020.65.4.496.
  12. Ji, Z., Zhao, X., Duan, H., Hu, T., Wang, S., Wang, Y., & Cao, K. (2013). Multiplex RT-PCR detection and distribution of four apple viruses in China. Acta Virologica, 57(4), 435-441. doi: 10.4149/av_2013_04_435.
  13. Jiao, J., et al. (2020). Field detection of multiple RNA viruses/viroids in apple using a CRISPR/Cas12a‐based visual assay. Plant Biotechnology Journal, 19(2), 394-405. doi: 10.1111/pbi.13474.
  14. Kairova, G., Daulet, N., Solomadin, M., Sandybayev, N., Orkara, S., Beloussov, V., Kerimbek, N., Gritsenko, D., & Sapakhova, Z. (2023). Identification of apple varieties resistant to fire blight (Erwinia amylovora) using molecular markers. Horticulturae, 9(9), article number 1000. doi: 10.3390/horticulturae9091000.
  15. Kanapiya, A., Amanbayeva, U., Tulegenova, Z., Abash, A., Zhangazin, S., Dyussembayev, K., & Mukiyanova, G. (2024). Recent advances and challenges in plant viral diagnostics. Frontiers in Plant Science, 15, article number 1451790. doi: 10.3389/fpls.2024.1451790.
  16. Karanfil, A. (2021). Prevalence and molecular characterization of Turkish isolates of the rose viruses. Crop Protection, 143, article number 105565. doi: 10.1016/j.cropro.2021.105565.
  17. Kerimbek, N., Kapytina, A.I., & Taskuzhina, A.K. (2024). Assessment of occurrence and diversity of apple viruses in South Kazakhstan. Science and Education, 1(2(75)), 79-85. doi: 10.52578/2305-9397-2024-2-2-79-85.
  18. Kim, N.-Y., Lee, H.J., Kim, H.S., Lee, S.H., Moon, J.S., & Jeong, R.D. (2021). Identification of plant viruses infecting pear using RNA sequencing. The Plant Pathology Journal, 37(3), 258-267. doi: 10.5423/PPJ.OA.01.2021.0009.
  19. Kwon, Y.H., Lee, G.R., Eom, H., Kim, H.K., Yoo, S.E., & Park, S. (2024). Efficacy of antiviral agents and thermotherapy in eliminating viruses from in vitro plantlets derived from apical meristems of fire blight resistant apple rootstocks G11 and G30. Korean Journal of Plant Resources, 37(6), 579-588. doi: 10.7732/kjpr.2024.37.6.579.
  20. Lee, H.J., & Jeong, R.D. (2022). A reliable reverse transcription loop-mediated isothermal amplification assay for detecting Apple stem grooving virus in pearResearch in Plant Disease (식물병연구), 28(2), 92-97.
  21. Ma, L., et al. (2021). Incidence of major pome fruit tree viruses and viroids in commercial pear orchards in China and in Pyrus betulifolia seedlings. Plant Pathology, 70(6), 1467-1475. doi: 10.1111/ppa.13375.
  22. Mandal, A., Mukherjee, A., & Bandyopadhyay, R. (2024). Plant virus disease management strategies: Conventional versus modern techniques. In Detection and management of new and emerging mystery plant virus sources (pp. 207-238). New York: Apple Academic Press.
  23. Mishchenko, L.T., Dunich, A.A., Mishchenko, I.A., Dashchenko, A.V., Kozub, N.O., Kyslykh, T.M., & Molodchenkova, O.O. (2022). Wheat dwarf virus in Ukraine: Occurrence, molecular characterization and impact on the yield. Journal of Plant Diseases and Protection, 129(1), 107-116. doi: 10.1007/s41348-021-00552-w.
  24. Mohammadi, N., Guo, Y., Wang, K., & Granato, D. (2023). Macroporous resin purification of phenolics from Irish apple pomace: Chemical characterization, and cellular antioxidant and anti-inflammatory activities. Food Chemistry, 437, article number 137815. doi: 10.1016/j.foodchem.2023.137815.
  25. Nabi, S.U., Baranwal, V.K., Yadav, M.K., & Rao, G.P. (2020). Association of Apple necrotic mosaic virus (ApNMV) with mosaic disease in commercially grown cultivars of apple (Malus domestica Borkh) in India. 3 Biotech, 10, article number 122. doi: 10.1007/s13205-020-2117-6.
  26. Noorani, M.S., Baig, M.S., Khan, J.A., & Pravej, A. (2023). Whole genome characterization and diagnostics of prunus necrotic ringspot virus (PNRSV) infecting apricot in India. Scientific Reports, 13, article number 4393. doi: 10.1038/s41598-023-31172-z.
  27. Pavliuk, L., Udovychenko, K., Riaba, I., & Bublyk, M. (2021). Detection of sour and sweet cherry viruses in Ukraine. Agronomy Research, 19(1), 199-209. doi: 10.15159/ar.20.238.
  28. Shi, W., Yao, R., Sunwu, R., Huang, K., Liu, Z., Li, X., Yi, Y., & Wang, J. (2020). Incidence and molecular identification of Apple necrotic mosaic virus (ApNMV) in Southwest China. Plants, 9(4), article number 415. doi: 10.3390/plants9040415.
  29. Srivastava, S., Upadhyay, D.J., & Srivastava, A. (2020). Next-generation molecular diagnostics development by CRISPR/Cas tool: Rapid detection and surveillance of viral disease outbreaks. Frontiers in Molecular Biosciences, 7, article number 582499. doi: 10.3389/fmolb.2020.582499.
  30. Tsvigun, V.O., Levishko, A.S., Gumeniuk, I.I., & Mazur, S.O. (2024). Monitoring of viral infections in vegetable crops in agroecosystems of Ukraine. Microbiological Journal, 86(5), 87-101. doi: 10.15407/microbiolj86.05.087.
  31. Wang, Y.M., Ostendorf, B., Gautam, D., Habili, N., & Pagay, V. (2022). Plant viral disease detection: From molecular diagnosis to optical sensing technology – a multidisciplinary review. Remote Sensing, 14(7), article number 1542. doi: 10.3390/rs14071542.
  32.  Wright, A.A., Cross, A.R., & Harper, S.J. (2020). A bushel of viruses: Identification of seventeen novel putative viruses by RNA-seq in six apple trees. PLoS One, 15(1), article number e0227669. doi: 10.1371/journal.pone.0227669.
  33. Xiao, H., Hao, W., Storoschuk, G., MacDonald, J.L., & Sanfaçon, H. (2022). Characterizing the virome of apple orchards affected by rapid decline in the Okanagan and Similkameen valleys of British Columbia (Canada). Pathogens, 11(11), article number 1231. doi: 10.3390/pathogens11111231.
  34. Xing, F., Gao, D., Liu, H., Wang, H., Habili, N., & Li, S. (2020). Molecular characterization and pathogenicity analysis of prunus necrotic ringspot virus isolates from China rose (Rosa chinensis Jacq.). Archives of Virology, 165, 2479-2486. doi: 10.1007/s00705-020-04739-8.
  35. Yin, L., Man, S., Ye, S., Liu, G., & Ma, L. (2021). CRISPR-Cas based virus detection: Recent advances and perspectives. Biosensors and Bioelectronics, 193, article number 113541. doi: 10.1016/j.bios.2021.113541.
  36. Zikeli, K., Berwarth, C., Born, U., Leible, T., Jelkmann, W., & Hagemann, M.H. (2025). Prevalence, genetic diversity, and molecular detection of the apple hammerhead viroid in Germany. Frontiers in Microbiology, 16, article number 1592572. doi: 10.3389/fmicb.2025.1592572
  37. Zuļģe, N., Gospodaryk, A., & Moročko-Bičevska, I. (2023). Genetic diversity and phylogenetic relationships of Apple chlorotic leaf spot virus isolates from Malus, Pyrus and Prunus hosts in Latvia. Plant Pathology, 72(5), 900-911. doi: 10.1111/ppa.13712.