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Gaillardia aristata, Echinacea purpurea (Asteraceae) and Aquilegia coerulea (Ranunculaceae) are popular perennial garden plants. In 2017 and 2018, over 5% of G. aristata ‘Arizona Apricot’, E. purpurea ‘Cheyenne Spirit’ and A. coerulea ‘Biedermeier’ plants were affected by stunting, virescence and phyllody in a commercial nursery in Hungary. These symptoms were present in 182 out of 3500 Aquilegia, in 28 out of 520 Echinacea and 470 out of 10080 Gaillardia plantlets, with 5.2%, 5.38% and 4.66% infection rates, respectively. Leaf samples were collected from fifteen symptomatic and five symptomless plants of each species. Total DNA was extracted by CTAB method and nested PCR assays were performed using primer pair P1/P7 followed by R16F2n/R16R2 (Lee et al. 1998 and references therein). The 1.2 kb 16S rDNA fragment was amplified from each symptomatic plant separately. Restriction fragment length polymorphism analysis with AluI, HaeIII, HpaII, MseI and RsaI enzymes yielded restriction profiles identical to each other and to those of reference strain AY1 (GenBank accession number AF322644) belonging to the 16SrI-B subgroup (Lee et al. 1998). The P1/P7-amplified products obtained from G. aristata, E. purpurea and A. coerulea were cloned, sequenced and deposited in GenBank under accession numbers MK992773, MK992774 and MK992775, respectively. BLAST searches of GenBank yielded 99.8-100% sequence similarities to ‘Candidatus Phytoplasma asteris’ strain SAY (AF222063).
In subsequent experiments, the elongation factor Tu (tuf), ribosomal protein (rp) operon (rpl22 and rps3) and the molecular chaperonin groEL genes were examined according to Lee et al. (2004) and Mitrović et al. (2011). The PCR products obtained with primer pairs fTufl/rTufl, rpF1/rpR1 and AYgroelF/AYgroelR were cloned and sequenced (MN526022-MN526024). Identical sequences were obtained from the three plant species, sharing 100% identity with AY phytoplasma strain IRap (AJ271316), Onion proliferation phytoplasma strain OnP2 (GU228514) and AY phytoplasma strain AVUT (AB599686). The three phytoplasma isolates were classified into subgroups tufI-B, rpI-B and groELI-III. To our knowledge this is the first identification of ‘Ca. P. asteris’ in G. aristata, E. purpurea and A. coerulea in Hungary.
References
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Supplementary Fig. 1
Naturally infected Gaillardia aristata (a), Echinacea purpurea (b) and Aquilegia coerulea (c) showing symptoms of phytoplasma infection during field survey. (PNG 3718 kb)
Supplementary Fig. 2
RFLP analysis of 16S rDNA of AJ30 (MK992773), AJ33 (MK992774) and AJ6 (MK992775) phytoplasma isolates. R16F2n/R16R2 nested PCR products were digested with AluI, HaeIII, HpaII and RsaI enzymes and separated by electrophoresis in a 2% agarose gel stained with ethidium bromide. American aster yellows strain AAY (GenBank: X68373) was used as a reference, kindly provided by E. Seemüller. Thermo Scientific GeneRuler 50 bp DNA Ladder (SM0373) was used as a DNA size marker. Fragment sizes from top to bottom are 1000, 900, 800, 700, 600, 500, 400, 300, 250, 200, 150, 100 and 50 bp. (JPG 56 kb)
ESM 3
Phylogenetic trees constructed based on sequences of 16S rRNA (a), tuf (b), rp (c) and groEL (d) genes of ‘Candidatus Phytoplasma’ species using Acholeplasma laidlawii as outgroup. Strains isolated in this study are shown in bold. Multiple sequence alignments were made using ClustalX 2. Maximum likelihood analysis was conducted with the general time reversible nucleotide substitution model using the software package MEGA7. The percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. Scale bar indicates the number of nucleotide substitutions per site. (PDF 58 kb)
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Viczián, O., Fodor, J., Ágoston, J. et al. Molecular identification of phytoplasmas associated with three floricultural species in Hungary. J Plant Pathol 102, 1335–1336 (2020). https://doi.org/10.1007/s42161-020-00624-0
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DOI: https://doi.org/10.1007/s42161-020-00624-0