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 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 et al. 1998 and references therein). Translocase protein (secY) 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 secY sequences available in Genbank. Elongation factor Tu (tuf) 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 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, 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 ‘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. (1997b) and Seemüller et al. (1998) in Germany, where phytoplasmas associated with a cyclamen disease were enclosed in the 16SrI-B subgroup. Other researches 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 diseases. The obtained secY and tuf 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 'Candidatus Phytoplasma asteris' in cyclamen in Hungary.
‘Candidatus Phytoplasma prunorum’ is causing ever increasing economic losses through the decline of apricot trees in European countries, e.g., Hungary. In this study, the pathogen was identified from plant tissues and insects by nested-PCR. The insect species were identified via morphology and molecular methods. The incidence of the pathogen was 29.6% in randomly selected apricot trees. Most of the infected trees with symptoms died within a year. These results show that phytoplasma is significantly present and causes damage in the investigated plantations. The only known insect vector of this phytoplasma is the plum psyllid, Cacopsylla pruni, which was regularly encountered in the sampled apricot orchards and in their surroundings. In a two-year study, several adults among the sampled specimens were observed to be infected by the pathogen. This observation further confirms the role of the plum psyllid in vectoring the phytoplasma. All the sampled plum psyllid adults belonged to the ‘B’ biotype. Besides C. pruni, Cacopsylla crataegi was abundant in the samples. Several adults of the latter species were also infected by the pathogen ‘Ca. Phytoplasma prunorum’. The rates of occurrence of this phytoplasma in male and female adults of the two psyllid species appeared to be similar. The examined C. crataegi individuals showed genetic differences from each other and from specimens included in a previous investigation.