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First Report of ‘Candidatus Phytoplasma asteris’ Associated with Cyclamen Little Leaf in Hungary

    Affiliations
    Authors and Affiliations
    • Orsolya Viczián1
    • József Fodor1
    • János Ágoston2
    • Emese Mergenthaler1
    1. 1Plant Protection Institute, Centre for Agricultural Research, ELKH, 1022 Budapest, Hungary
    2. 2ELKH-SZE PhatoPlant-Lab, Széchenyi István University, 9200 Mosonmagyaróvár, Hungary

    Cyclamen (Cyclamen persicum) is a small perennial flowering plant with fragrant, showy flowers on long stems rising above the foliage. Between 2018 and 2022, about 6% of C. persicum plants belonging to diverse varieties showed stunting, leaf yellowing, virescence, and phyllody in commercial nurseries at three locations (Tiszabög, Szombathely, and Kecskemét) in Hungary. These symptoms are similar to those associated with the phytoplasma disease described in Italy as cyclamen little leaf (Bertaccini 1990) were observed in plants of six cyclamen cultivars: in 21 out of 352 plants of Super Serie Mini Winter Mix, 19 out of 286 plants of Super Serie Micro Mix, 12 out of 199 plants of Halios Mix, 3 out of 17 plants of Fantasia Purple, 1 out of 7 plants of Curly Early Mix Evolution, and 4 out of 66 plants of Halios Curly Rose plants. Total DNA was extracted from petioles collected when possible from 10 symptomatic and 5 symptomless plants from each cultivar by a CTAB method (Ahrens and Seemüller 1992) and used as templates for PCR. Phytoplasma 16S rDNA was amplified using the universal primers P1/P7 and R16F2n/R16R2 (Lee et al. 1998 and references therein). The translocase protein (secY) gene was amplified with the AYsecY_F-46 (5′-AAGCAGCCATTTTAGCAGTTG-3′) and AYsecY_R1450 (5′-AAGTAATCAGCTATCATTTGGTTAGT-3′) primer pair, which was designed on the basis of aster yellows (AY) phytoplasma secY sequences available in GenBank. The elongation factor Tu (tuf) was amplified with fTuf1/rTuf1 (Schneider and Gibb 1997) primer pairs. Thermocycler conditions consisted of 98°C for 2 min, 32 cycles at 98°C for 30 s, 60 or 55°C (in the case of tuf) for 30 s, and 72°C for 1 min, followed by a final extension of 72°C for 10 min with Phusion High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA). Amplicons of the expected sizes (P1/P7: 1.8 kb, R16F2n/R16R2: 1.1 kb, AYsecY_F-46/AYsecY_R1450: 1.5 kb, and fTuf1/rTuf1: 1.1 kb) were produced from all symptomatic plants but not from the asymptomatic ones. Amplified PCR products were gel purified and ligated into the pJET1.2/blunt cloning vector using a CloneJET PCR cloning kit (Thermo Fisher Scientific, Waltham, MA). The cloned PCR fragments (at least three from each PCR reaction) were sequenced from both directions by LGC Genomics (Berlin, Germany) using pJET1.2 forward and reverse primers, and the obtained sequences were deposited in GenBank. The 16S rRNA gene sequences (GenBank accession nos. ON594635 and ON594636) showed 100 and 99.95% identity, respectively, with the Onion yellows phytoplasma strain OY-M (GenBank AP006628) from the ‘Candidatus Phytoplasma asteris’ 16SrI-B subgroup. In iPhyClassifier analysis, the virtual RFLP pattern of 16S rDNA was identical (similarity coefficient = 1.00) to the reference pattern of 16Sr group I, subgroup B (GenBank AP006628). This is in agreement with the results of Schneider et al. (1997) and Seemüller et al. (1998) in Germany, where phytoplasmas associated with cyclamen disease were enclosed in the 16SrI-B subgroup. Other research studies in Italy (Alma et al. 2000) and Israel (Weintraub et al. 2007) revealed that phytoplasmas belonging to the 16SrI-C and 16SrXII-A groups have been associated with cyclamen disease. The obtained secY and tuf gene fragments (GenBank ON564432 and ON515746) shared 99.3 and 99.9% sequence identity, respectively, with the Onion yellows phytoplasma strain OY-M. To our knowledge, this is the first identification of ‘Ca. P. asteris’ in cyclamen in Hungary.

    The author(s) declare no conflict of interest.

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    Funding: This research was supported by the ELKH-SZE PhatoPlant-Lab (project no. 3200107).

    The author(s) declare no conflict of interest.