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area in the country. Grapevine yellows are a complex of diseases associated with the presence
of phytoplasmas. Phytoplasmas of at least five different groups can cause similar symptoms in
grapevines, and they can be distinguished only on a molecular basis (EPPO Bulletin 2016). One
of them, grapevine Flavescence doree phytoplasma (GFDP), which belongs to the 16SrV group,
is listed in Annex II, Part B, of the Commission Implementing Regulation (EU) 2019/2072 of 28
November 2019 as a Union quarantine pest known to occur in the Union territory. Official
surveys for GFDP in the Czech Republic have been carried out since 2007. In 2016, the first
occurrence of Scaphoideus titanus Ball, 1932, the main vector of GFDP, was reported in the
South Moravian region (EPPO Reporting Service 2016). This is a matter of concern because it
indicates that there is a risk of disease dissemination to other geographical locations. In
September 2021, a total of 250 samples of V. vinifera (preferentially focused on symptomatic
plants) and four samples of the wild plant host Clematis vitalba L. were collected from 50
vineyards in South Moravia. Total DNA was extracted using the High Pure PCR Template
Preparation Kit (Roche, Basel, Switzerland). For phytoplasma screening, a real-time PCR test
for generic detection of phytoplasmas was used (Christensen et al. 2004). Samples evaluated as
positive were further tested by PCR using phytoplasma universal P1 and P7 primers (Deng and
Hiruki 1991; Schneider et al. 1995), followed by nested PCR using the 16SrV group-specific
primers fB1 and rULWS1 (Smart et al. 1996). For identification of 16SrV phytoplasma, sequence
analysis of the secY-map genetic locus was performed. Two sets of primers were used:
FD9f5/MAPr1 primers for the first PCR and FD9f6/MAPr2 for the nested PCR (Arnaud et al.
2007) with PCRBIO TaqMix (PCR Biosystems, London, U.K.). The nested PCR products were
purified and sequenced (Eurofins Genomics, Ebersberg, Germany). The sequences were
compared with sequences from the GenBank database. Phytoplasma of the 16SrV group was
detected in three samples: V. vinifera cv. Gewurztraminer with symptoms of leaf reddening
with no rolling and no other typical symptoms, C. vitalba with leaf curling, and symptomless
C. vitalba. The obtained sequences of the secY-map locus of all three 16SrV-positive samples
were identical to the sequence of GFDP, isolate Vv-SI257 (accession no. FN811141), detected in
grapevine in Tuscany, Italy, which belongs to the 16SrV group. The sequence of the V.
vinifera cv. Gewurztraminer isolate was submitted to GenBank under accession number
OQ185203. This isolate belongs to the Map-FD3 cluster, and the genotype identified is M51,
which has already been found in C. vitalba and outbreaks of Flavescence doree in grapevines
in some other European countries (Malembic-Maher et al. 2020). Based on the abovementioned
results, this is the first report of GFDP in the Czech Republic.
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of phytoplasmas. Phytoplasmas of at least five different groups can cause similar symptoms in
grapevines, and they can be distinguished only on a molecular basis (EPPO Bulletin 2016). One
of them, grapevine Flavescence doree phytoplasma (GFDP), which belongs to the 16SrV group,
is listed in Annex II, Part B, of the Commission Implementing Regulation (EU) 2019/2072 of 28
November 2019 as a Union quarantine pest known to occur in the Union territory. Official
surveys for GFDP in the Czech Republic have been carried out since 2007. In 2016, the first
occurrence of Scaphoideus titanus Ball, 1932, the main vector of GFDP, was reported in the
South Moravian region (EPPO Reporting Service 2016). This is a matter of concern because it
indicates that there is a risk of disease dissemination to other geographical locations. In
September 2021, a total of 250 samples of V. vinifera (preferentially focused on symptomatic
plants) and four samples of the wild plant host Clematis vitalba L. were collected from 50
vineyards in South Moravia. Total DNA was extracted using the High Pure PCR Template
Preparation Kit (Roche, Basel, Switzerland). For phytoplasma screening, a real-time PCR test
for generic detection of phytoplasmas was used (Christensen et al. 2004). Samples evaluated as
positive were further tested by PCR using phytoplasma universal P1 and P7 primers (Deng and
Hiruki 1991; Schneider et al. 1995), followed by nested PCR using the 16SrV group-specific
primers fB1 and rULWS1 (Smart et al. 1996). For identification of 16SrV phytoplasma, sequence
analysis of the secY-map genetic locus was performed. Two sets of primers were used:
FD9f5/MAPr1 primers for the first PCR and FD9f6/MAPr2 for the nested PCR (Arnaud et al.
2007) with PCRBIO TaqMix (PCR Biosystems, London, U.K.). The nested PCR products were
purified and sequenced (Eurofins Genomics, Ebersberg, Germany). The sequences were
compared with sequences from the GenBank database. Phytoplasma of the 16SrV group was
detected in three samples: V. vinifera cv. Gewurztraminer with symptoms of leaf reddening
with no rolling and no other typical symptoms, C. vitalba with leaf curling, and symptomless
C. vitalba. The obtained sequences of the secY-map locus of all three 16SrV-positive samples
were identical to the sequence of GFDP, isolate Vv-SI257 (accession no. FN811141), detected in
grapevine in Tuscany, Italy, which belongs to the 16SrV group. The sequence of the V.
vinifera cv. Gewurztraminer isolate was submitted to GenBank under accession number
OQ185203. This isolate belongs to the Map-FD3 cluster, and the genotype identified is M51,
which has already been found in C. vitalba and outbreaks of Flavescence doree in grapevines
in some other European countries (Malembic-Maher et al. 2020). Based on the abovementioned
results, this is the first report of GFDP in the Czech Republic.
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