106(1)_str10

 

ISSN 1392-3196 / e-ISSN 2335-8947
Zemdirbyste-Agriculture, vol. 106, No. 1 (2019), p. 73–80
DOI  10.13080/z-a.2019.106.010

Multilocus sequence analysis of phytoplasmas detected in cherry trees in Poland

Mirosława CIEŚLIŃSKA, Terezia SMOLAREK

Abstract

During 2010–2013 and 2016, shoot samples were collected from symptomatic and asymptomatic 85 sour cherry and 80 sweet cherry trees grown in 16 orchards located in major cherry cultivation areas in Central and Western Poland. Some sour cherry trees showed shoot proliferation, whereas dieback symptoms and leaf roll and yellowing were observed on some sweet cherry trees. The universal phytoplasma‐specific primer pairs P1 and P7 derived from the ribosomal sequence within the 16S and 23S rRNA gene sequences and intergenic spacer region were used in direct polymerase chain reaction (PCR) followed by universal primers F1/B6 and primers R16(I)F1/R1 and R16(X)F1/R1 specific for 16SrI and 16SrX groups, respectively. Phytoplasmas were detected in two sweet cherry (‘Trzebnica’ and ‘Kordia II.14’) and one sour cherry (‘cherry XVI.12’) trees.

Restriction fragment length polymorphism (RFLP) analyses conducted after digestion of F1/B6 products (~1.65 kb) with HhaI, RsaI, SspI and MseI enzymes indicated that sweet cherry trees were infected with ‘Candidatus Phytoplasma prunorum’ (16SrX-B). The restriction profiles for sour ‘cherry XVI.12’ sample were indistinguishable from those of the reference strain AY-1 of ‘Candidatus Phytoplasma asteris’ (16SrI-B).

Multilocus sequence analysis of 16S DNA plus spacer region, secY and ribosomal protein (rp) operons confirmed the genetic diversity of phytoplasmas infecting cherry trees and showed the closest relationships ‘Trzebnica’ and ‘Kordia II.14’ isolates found in sweet cherry trees to the reference strains of ‘Ca. Ph. prunorum’ (16SrX-B). Depending on analysed sequenced region, the phytoplasma infecting sour ‘cherry XVI.12’ revealed close genetic relationship to phytoplasmas assigned to the different subgroups of aster yellows group (16SrI). SecY gene was the more informative marker for finer differentiation of the strains of phytoplasmas within 16SrI group and showed the highest genetic similarity of ‘cherry XVI.12’ isolate with ‘Ca. Ph. asteris’-related strains.

Key words: detection, identification, restriction analysis, phylogeny, sequencing.

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