Study of primers for the diagnosis of phytoplasmas from the Apple proliferation group

One of the most dangerous pests for plants are phy­toplasmas. Phytopathogens from the Apple prolifera­tion group (16SrX) cause phytoplasmoses in pome and stone fruit crops, which lead to huge losses of fruit and berry products around the world. The 16SrX group in­cludes, in particular, such uncultivated bacteria as: Candidatus Phytoplasma mali, causing apple proliferation; Candidatus Phytoplasma pyri, resulting in pear decline; Candidatus Phytoplasma prunorum, leading to European stone fruit yellows. The first two species are included in the Common List of Quarantine Pests of the Eurasian Economic Union (EAEU). Although they be­long to the same group, these phytoplasmas have dif­ferent natural hosts, insect vectors, and a different set of characteristic symptoms in infected plants. The arti­cle presents data on the study of the analytical charac­teristics (specificity and sensitivity) of specific primers for the detection of phytopathogenic microorganisms from the Apple proliferation group by real-time poly­merase chain reaction (PCR) and for subsequent spe­cies identification of the causative agent of apple pro­liferation Candidatus Phytoplasma mali using specific primers classical PCR method. During the experiments, high-quality specific amplification products were ob­tained. It has been established that using the studied pair of primers for classical PCR, it is possible to diag­nose and identify the causative agent of apple proliferation in infected plant material. In this work, the analy­sis of the studied specific primers was carried out using the NCBI Primer-BLAST online service. For research, we used a collection of phytoplasma DNA from various groups: Apple proliferation, Stolbur, Foxtail palm yel­low decline, Peanut witches’ broom and Elm yellows.

1. Bashkirova I., Matyashova G., Gins M. Detection and identification of pathogens of phytoplasmoses of the Apple proliferation group on fruit crops [Vyyavleni­ye i identifikatsiya vozbuditeley fitoplazmozov gruppy Apple proliferation na plodovykh kulturakh]. Russian agricultural science, 2018a; 3: 10–14 (in Russian).

2. Bashkirova I., Matyashova G., Zavriev S., Ryazantsev D., Shneyder Yu. Approval of the test systems for the detection of the apple and pear phytoplasma. Plant protection and quarantine, 2018b; 7: 40–41.

3. Bashmakova E. Detection of single nucleo­tide polymorphisms based on derivatives of the Ca2+-regulated photoprotein obelin [Vyyavleniye odnonuk­leotidnykh polimorfizmov na osnove proizvodnykh Ca2+-reguliruyemogo fotoproteina obelina]: extended abstract of Cand. Biol. Sci. Dissertation: 03.01.06. Kras­noyarsk, 2017, 22 p. (in Russian).

4. Girsova N., Kastalyova T., Mozhaeva K. Method for determining phytoplasmas using molecular diag­nostic methods: PCR and RFLP [Metodika opredeleniya fitoplazm s ispol'zovaniyem molekulyarnykh metodov diagnostiki: PCR i RFLP]. M.: Rosselkhozakademiya, 2013, 23 p. (in Russian).

5. Elshin N., Petrov A. Possibilities of qPCR con­trol of mycoplasma contamination of cell cultures [Izucheniye vozmozhnosti ispolzovaniya metoda qPCR dlya kontrolya ot·sut·stviya mikoplazmennoy kontaminatsii v kletochnykh kulturakh]. – BIOpreparations. Prevention, Diagnosis, Treatment, 2017; 17 (3): 173–179 (in Russian).

6. Karimova E., Prikhodko Yu., Shneyder Yu. Phytoplasma – pathogenic agents of stone fruit crops diseases [Fitoplazmy – vozbuditeli bolezney kostochkovykh plodovykh kultur]. Plant Protection and Quarantine, 2019; 5: 35–39 (in Russian).

7. Matyashova G., Morozova O. Guidelines for the detection and identification of the causative agent of pear depletion Candidatus Phytoplasma pyri (Pear de­cline) [Metodicheskiye rekomendatsii po vyyavleniyu i identifikatsii vozbuditelya istoshcheniya grushi Can­didatus Phytoplasma pyri (Pear decline)]. M.: VNIIKR, 2016, 39 p. (in Russian).

8. ISPM 27. International standards for phytosanitary measures. Diagnostic protocols for regulated pests. DP 12: Phytoplasmas. 2018, 18 p.

9. Prikhodko Yu., Matyashova G. Guidelines for the detection and identification of the causative agent of apple proliferation Candidatus Phytoplasma mali (Apple proliferation) [Metodicheskiye rekomendatsii po vyyavleniyu i identifikatsii vozbuditelya proliferatsii yabloni Candidatus Phytoplasma mali (Apple proliferation)]. M.: VNIIKR, 2015, 80 p.

10. Sviridova L., Vankova A. Mycoplasmas are plant pathogens [Mikoplazmy – patogeny rasteniy]. Niva Povolzhya, 2012; 4 (25): 26–32 (in Russian).

11. Shneyder Yu., Prikhodko Yu., Shneyder E., Kuleshova Yu. Assessment of pest risk analysis as­sociated with import, export and movement of seed­lings, rootstocks and cuttings of stone fruit crops [Otsenka fitosanitarnykh riskov vrednykh organizmov, svyazannykh s importirovaniyem, eksportirovaniyem i peremeshcheniyem sazhentsev, podvoyev i cheren­kov kostochkovykh plodovykh kultur] (ed. by T.V. Arte­myeva). M.: VNIIKR, 2017, 503 p.

12. Bashkirova I., Bondarenko G., Kornev K. Study of methods for detecting quarantine phyto­plasma’s from the apple proliferation group on the territory of Russia. Phytopathogenic Mollicutes, 2019; 9 (1): 211–212. URL: https://doi.org/10.5958/2249-4677.2019.00106.3.

13. Duduk B. Molecular characterization of phy­toplasmas detected in agronomically relevant crops in Serbia. 2009, 127 p.

14. IRPCM Phytoplasma/Spiroplasma Working Team – Phytoplasma Taxonomy Group. Correspondence G. Firrao. ‘Candidatus Phytoplasma’, a taxon for the wall-less, non-helical prokaryotes that colonize plant phlo­em and insects. International Journal of Systematic and Evolutionary Microbiology, 2004; No. 54: 1243–1255. URL: https://doi.org/10.1099/ijs.0.02854-0.

15. Jarausch W., Saillard C., Dosba F., Bové J.M. Differentiation of mycoplasmalike organisms (MLOs) in European fruit trees by PCR using specific primers derived from the sequence of a chromosomal fragment of the apple proliferation MLO. Applied and Environmen­tal Microbiology, 1994; 60 (8): 2916–2923. URL: https://doi.org/10.1128/aem.60.8.2916-2923.1994.

16. Jomantiene R., Davis R.E., Valiunas D., Almi­naite A. New group 16SrIII phytoplasma lineages in Lithuania exhibit interoperon sequence heterogeneity. European Journal of Plant Pathology, 2002; 108 (6): 507–517. URL: https://doi.org/10.1023/A:1019982418063.

17. Lee I.-M., Davis R.E., Gundersen-Rindal D.E. Phytoplasma, phytopathogenic mollicutes. Annual Re­view of Microbiology, 2000; 54 (1): 221–255. URL: https://doi.org/10.1146/annurev.micro.54.1.221.

18. Mehle N., Nikolić P., Gruden K., Ravnikar M., Dermastia M. Real-time PCR assays for specific detec­tion of three phytoplasmas from apple proliferation group. Phytoplasma: Methods and Protocols, Methods in molecular biology, 2013; Vol. 938: 269–281. URL: https://doi.org/10.1007/978-1-62703-089-2_23.

19. Mehle N., Ravnikar M., Seljak G., Knapic V., Dermastia M. The most widespread phytoplasmas, vec­tors and measures for disease control in Slovenia. Phy­topathogenic Mollicutes, 2011; 1 (2): 65–76. URL: https://doi.org/10.5958/j.2249-4669.1.2.012.

20. Nikolić P., Mehle N., Gruden K., Ravnikar R., Dermastia M. A panel of real-time PCR assays for spe­cific detection of three phytoplasmas from the apple proliferation group. Molecular and Cellular Probes, 2010; 24 (5): 303–309. URL: https://doi.org/10.1016/j.mcp.2010.06.005.

21. Picard C., Afonso T., Benko-Beloglavec A., Karadjova O., Matthews-Berry S., Paunovic S.A., Pi­etsch M., Reed P., van der Gaag D.J., Ward M. Recom­mended regulated non-quarantine pests (RNQPs), as­sociated thresholds and risk management measures in the European and Mediterranean region. Bulletin OEPP/EPPO Bulletin, 2018; 48 (3): 552–568. URL: https://doi.org/10.1111/epp.12500.

22. PM 7/62 (3). ‘Candidatus Phytoplasma mali’, ‘Ca. P. pyri’ and ‘Ca. P. prunorum’. Bulletin OEPP/EPPO Bulletin, 2020; 50 (1): 69–85.

23. Seemüller E., Schneider B. ‘Candidatus Phy­toplasma mali’, ‘Candidatus Phytoplasma pyri’ and ‘Candidatus Phytoplasma prunorum’, the causal agents of apple proliferation, pear decline and European stone fruit yellows, respectively. International Journal of Systematic and Evolutionary Microbiology, 2004; Vol. 54: 1217–1226. URL: https://doi.org/10.1099/ijs.0.02823-0.

24. Wei W., Lee I.-M., Davis R.E., Suo X., Zhao Y. Automated RFLP pattern comparison and similari­ty coefficient calculation for rapid delineation of new and distinct phytoplasma 16Sr subgroup lineages. In­ternational Journal of Systematic and Evolutionary Micro­biology, 2008; Vol. 58: 2368–2377. URL: https://doi.org/10.1099/ijs.0.65868-0.

25. Weintraub P.G., Beanland L. Insect vectors of phytoplasmas. Annual Review of Entomology, 2006; Vol. 51: 91–111. URL: https://doi.org/10.1146/annurev.ento.51.110104.151039.

26. Woese C. Interpreting the universal phyloge­netic tree. Proceedings of the National Academy of Sciences of the USA, 2000; Vol. 97: 8392–8396. URL: https://doi.org/10.1073/pnas.97.15.8392.

27. EPPO Global Database. URL: https://gd.eppo.int (last accessed: 19.05.2022).

28. The National Center for Biotechnology In­formation. URL: https://www.ncbi.nlm.nih.gov/tools/primer-blast/ (last accessed: 21.04.2022).