Mirosława Cieślińska

Research Institute of Horticulture, Konstytucji 3 Maja 1/3, 96-100 Skierniewice, Poland

Aneliya Borisova

Institute of Agriculture – Kyustendil, 2500 Kyustendil, Bulgaria


During 2015, samples from 22 apple trees showing proliferation symptoms were collected in southwest Bulgaria and Central and South Poland and tested for phytoplasma presence. ‘Candidatus Phytoplasma mali’ was identified in 18 samples based on results of restriction fragment length polymorphism (RFLP) analysis of the 16S rRNA gene amplified in nested PCR using primer pair P1/P7 followed by R16F2n/R16R2 and F1/B6 primer pairs. The nitroreductase and rhodonase like genes and ribosomal protein genes rpl22 and rps3 were then analyzed using PCR-RFLP technique to study the genetic variability of the  phytoplasma strains. Two restriction profiles, P-I or P-II, were obtained for fragments of 16S rDNA plus 16S-23S spacer region digested with HpaII enzyme. Restriction fragment length polymorphism analysis of nitroreductase and rhodonase like genes using digestion with HincII endonuclease revealed that the all ‘Ca. P. mali’ strains belonged to subtype AP-15. Analysis of rpl22 and rps3 ribosomal protein genes digested with AluI enzyme resulted in classification of detected phytoplasma strains to rpX-A subgroup.


apple proliferation, PCR, RFLP, multilocus gene analyses

Bertaccini, A. (2014).

Bertaccini, A., Duduk, B., Paltrinieri, S., Contaldo, N. (2014). Phytoplasmas and phytoplasma diseases: a severe threat to agriculture. Am. J. Plant Sci., 5, 1763–1788. DOI: 10.4236/ajps.2014.512191

Borisova, A., Kamenova, I. (2016). Occurrence of phytoplasmas of the apple proliferation group in fruit trees in Kyustendil region of Bulgaria. Bulg. J. Agric. Sci., 22(3), 465–469.

Cainelli, C., Bisognin, C., Vindimian, M.E., Grando, M.S. (2004). Genetic variability of AP phytoplasma detected in apple growing area of Trentino (North Italy). Acta Hortic., 657, 425–430.

Casati, P., Quaglino, F., Tedeschi, R., Spiga, M.F., Alma, A., Spadone, P. (2010). Identification and molecular characterization of ‘Candidatus Phytoplasma mali’ isolates in North-western Italy. J. Phytopathol., 158, 81–87. DOI: 10.1111/j.1744-7348.2011.00461.x

Cieślińska, M., Hennig, E., Kruczyńska, D., Bertaccini, A. (2015). Genetic diversity of ‘Candidatus Phytoplasma mali’ strains in Poland. Phytopathol. Mediter., 54(3), 477–487. DOI: 10.14601/Phytopathol_Mediterr-15785

Cieślińska, M., Kruczyńska, D., Jaworska, K. (2012). Detection and characterization of phytoplasmas infecting apple trees in Poland and identification of their possible vectors. Petria, 22(3), 185.

Cieślińska, M., Kruczyńska, D. (2014). Detection and molecular characterization of phytoplasmas infecting apple trees in Poland. Hortic. Sci., 41(1), 27–33.

Danet, J., Bonet, P., Jarausch, W., Carraro, L., Skorić, D., Labonne, G., Foissac, X. (2007). Imp and secY, two new markers for MLST (multilocus sequence typing) in the 16SrX phytoplasma taxonomic group. Bull. Insectol., 60, 339–340.

Davis, R.E., Lee, I.M. (1993). Cluster-specific polymerase chain reaction amplification of 16S rDNA sequences for detection and identification of mycoplasma-like organisms. Phytopathology, 83, 1008–1011. DOI: 10.1094/Phyto-83-1008

Deng, S., Hiruki, C. (1991). Amplification of 16S rRNA genes from culturable and nonculturable mollicutes.

J. Microbiol. Methods, 14, 53–61. DOI: 10.1016/0167-7012(91)90007-D

Etropolska, A., Laginova, M. (2012). Monitoring of the distribution of fruit tree phytoplasmas in Bulgaria from 2007 until 2011. Petria, 22(3), 123–159.

Etropolska, A., Jarausch, W., Jarausch, B., Trenchev, G. (2015). Detection of European fruit tree phytoplasmas and their insect vectors in important fruit-growing regions in Bulgaria. Bulg. J. Agric. Sci., 21, 1248–1253.

Etropolska, A. (2012). COST Action FA0807 Integrated Management of Phytoplasma Epidemics in Different Crop Systems Short-term Scientific Mission (STSM) Report. Available:

Fránová, J., Koloniuk, I., Lenz, O., Sakalieva, D. 2018. Molecular diversity of ‘Candidatus Phytoplasma mali’ strains associated with apple proliferation disease in Bulgarian germplasm collection. Folia Microbiol.,

Fránová, J., Ludvíková, H., Paprštein, F., Bertaccini, A. (2013). Genetic diversity of Czech ‘Candidatus Phytoplasma mali’ strains based on multilocus gene analyses. Eur. J. Plant Pathol., 136, 675–688. DOI: 10.1007/s10658-013-0196-5

Gundersen, D.E., Lee, I.M. (1996). Ultrasensitive detection of phytoplasmas by nested-PCR assays using two universal primers. Phytopathol. Mediterr., 35, 144–151.

Jarausch, W., Saillard, C., Dosba, F., Bove, J.-M. (1994). 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. Appl. Environ. Microbiol., 60(8), 2916–2923.

Jarausch, W., Saillard, C., Helliot, B., Garnier, M., Dosba, F. (2000). Genetic variability of apple proliferation phytoplasmas as determined by PCR-RFLP and sequencing of a non-ribosomal fragment. Mol. Cell. Probe., 14, 17–24. DOI:10.1006/mcpr.1999.0279

Kamińska, M., Zawadzka, B. (1970). Badania nad proliferacją (miotlastością) jabłoni w Polsce. I. Objawy chorobowe, porażone odmiany i występowanie. Acta Agrobot., 23, 329–340.

Lee, I.M., Zhao, Y., Bottner, K.D. (2006). SecY gene sequence analysis for finer differentiation of diverse strains in the aster yellows phytoplasma group. Mol. Cell. Probe., 20, 87–91. DOI: 10.1016/j.mcp.2005.10.001

Makarova, O., Contaldo, N., Paltrinieri, S., Kawube, G., Bertaccini, A., Nicolaisen, M. (2012). DNA barcoding for identification of ‘Candidatus Phytoplasmas’ using a fragment of the elongation factor Tu gene. PLoS One, 7(12):e52092. DOI: 10.1371/journal.pone.0052092

Martini, M., Lee, I.M., Bottner, K.D., Zhao, Y., Botti, S., Bertaccini, A., Harrison, N.A, Carraro, L., Marcone, C., han, A.J, Osler, R. (2007). Ribosomal protein gene-based phylogeny for finer differentiation and classification of phytoplasmas. Int. J. Syst. Evol. Microbiol., 57(9), 2037–2051. DOI:10.1099/ijs.0.65013-0

Martini, M., Ermacora, P., Falginella, L., Loi, N., Carraro, L. (2008). Molecular differentiation of ‘Candidatus Phytoplasma mali’ and its spreading in Friuli Venezia Giulia Region (north-east Italy). Acta Hort,. 781, 395–402.

Milusheva, S., Terziev, I. (2013). Preliminary results from investigation on apple proliferation. Acta Hort., 981(2), 513–515.

Olivier, T., Fauche, F., Demonty, E. (2014). Distribution of ‘Candidatus Phytoplasma mali’ in infected apple trees in Belgium. Commun. Agric. Appl. Biol. Sci.,79(3),463–467.

Padovan, A.C., Gibb, K.S., Bertaccini, A., Vibio, M., Bonfiglioli, R.E., Magarey, P.A., Sears, B.B. (1995). Molecular detection of the Australian grapevine yellows phytoplasma and comparison with a grapevine yellows phytoplasma from Emilia-Romagna in Italy. Aust. J. Grape Wine Res., 1, 25–31. DOI: 10.1111/j.1755-0238.1995.tb00074.x

Paltrinieri, S., Duduk, B., Dal Molin, F., Mori, N., Comerlati, G., Bertaccini A. (2010). Molecular characterization of ‘Candidatus Phytoplasma mali’ strains in outbreaks of apple proliferation in north eastern Italy, Hungary, and Serbia. Julius-Kühn-Archiv, 427, 178–182.

Schneider, B., Seemüller, E. (2009). Strain differentiation of ‘Candidatus Phytoplasma mali’ by SSCP and sequence analyses of the hflB gene. J. Plant Pathol., 91, 103–112. DOI: 10.4454/jpp.v91i1.630

Schneider, B., Seemüller, E., Smart, C.D., Kirkpatrick, B.C. (1995). Phylogenetic classification of plant pathogenic mycoplasma-like organisms or phytoplasmas. In: Molecular Diagnostic Procedures in Mycoplasmology, Razin, R., Tully, J.G. (eds). Academic Press, San Diego, 369–380. DOI: 10.1016/B978-012583805-4/50040-6

Tedeschi, R., Bosco, D., Alma A. (2002). Population dynamics of Cacopsylla melanoneura (Homoptera: Psyllidae) a vector of apple proliferation phytoplasma in Northwestern Italy. J. Econ. Entomol., 95, 544–551. DOI: 10.1603/0022-0493-95.3.544

Trifonov, D. (1965). Apple proliferation witches’broom. Spread and damage. Hortic. Viticult. Sci., 2(4), 437–444 [in Bulgarian].



Mirosława Cieślińska 
Research Institute of Horticulture, Konstytucji 3 Maja 1/3, 96-100 Skierniewice, Poland
Aneliya Borisova 
Institute of Agriculture – Kyustendil, 2500 Kyustendil, Bulgaria



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