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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 (Tiszabog, Szombathely, and Kecskemet) 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
Seemuller 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 Seemuller 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.
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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 (Tiszabog, Szombathely, and Kecskemet) 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
Seemuller 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 Seemuller 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.
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