First report of ' Phytoplasma asteris' associated with cyclamen little leaf in Hungary.

Cyclamen () is a small perennial flowering plant with fragrant, showy flowers on long stems rising above the foliage. Between 2018 and 2022, about 6% of 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 known 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 universal primers P1/P7 and R16F2n/R16R2 (Lee . 1998 and references therein). Translocase protein () gene was amplified with 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 sequences available in Genbank. Elongation factor Tu () was amplified with fTuf1/rTuf1 (Schneider et al. 1997a) primer pairs. Thermocycler conditions consisted of 98°C for 2 min, 32 cycles at 98°C for 30 s, 60°C or 55°C (in case of ) 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, USA). Amplicons of the expected sizes (P1/P7: 1.8 kb, R16F2n/R16R2: 1.1 kb, AYsecY_F-46/AYsecY_R1450: 1.5 kb, 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 sequence was deposited in GenBank. The 16S rRNA gene sequences (GenBank Accession Nos. ON594635 and ON594636) showed 100% and 99.95% identity, respectively, with Onion yellows phytoplasma strain OY-M (GenBank AP006628) from the ' Phytoplasma asteris' 16SrI-B subgroup. . In PhyClassifier 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 . (1997b) and Seemüller . (1998) in Germany, where phytoplasmas associated with a cyclamen disease were enclosed in the 16SrI-B subgroup. Other researches in Italy (Alma ., 2000) and Israel (Weintraub ., 2007) revealed that phytoplasmas belonging to the 16SrI-C and 16SrXII-A groups have been associated with cyclamen diseases. The obtained and gene fragments (GenBank ON564432 and ON515746) shared 99.3% and 99.9% sequence identity, respectively, with Onion yellows phytoplasma strain OY-M. To our knowledge this is the first identification of ' Phytoplasma asteris' in cyclamen in Hungary.