First report of Flavescence Dorée-Related Phytoplasma in a Productive Vineyard in Germany

Flavescence dorée (FD) and Bois noir (BN) are the principal grapevine yellows in Europe caused by distinct phytoplasmas: BN by Candidatus Phytoplasma solani, FD by 16SrV-C and -D phytoplasmas (FDp) transmitted by the introduced Nearctic Deltocephalinae Scaphoideus titanus. FDp is listed as a quarantine pest in the European Union (Regulation (EU) 2019/2072). Black Alder (Alnus glutinosa) is a common asymptomatic host of 16SrV phytoplasmas in Europe and considered the original host of FDp (Malembic-Maher et al. 2020). Palatinate grapevine yellows (PGY) transmitted from alder to grapevine by the Macropsinae Oncopsis alni is not transmissible by S. titanus (Malembic-Maher et al. 2020). Germany is considered free from FD in grapevine and from its vector. A single case in a nursery in 2014 was eradicated (EPPO 2017), and FD was never before detected in a vineyard. Since S. titanus appeared in 2016 in the neighboring French Region of Alsace, monitoring of FD was carried out in Germany following a risk based strategy. It was focused on vineyard plots within a distance of 100 m from stands of alder. A geodata-based risk map (Jalke 2020) was used to localize those plots. All symptomatic vines sampled until September 2020 proved to be infected by BN or, occasionally, by PGY. Eight vines with typical symptoms were sampled in vineyards adjacent to alder stands in the winegrowing region of Rheinhessen in September 2020. Symptoms comprised leaf rolling and discoloration, incomplete lignification, and black pustules arranged in lines along the shoots. Diseased shoots were black and necrotic in December. Leaf midribs were sampled for total nucleic acids extraction. The phytoplasma 16S rRNA gene was amplified by generic primers R16F2/R2-mod followed by a nested PCR using 16Sr(V) group-specific primers R16(V)F1/R1, and primers R16(I)F1/R1 (Lee et al. 1995) to detect ‘Candidatus Phytoplasma solani’, associated with BN. While BN was detected in seven vines, one sample tested positive for 16SrV phytoplasma. This result was confirmed by triplex real-time Taq-Man assay based on rpl14 gene sequences (IPADLAB), by multiplex real-time PCR of map locus as well as by Loop-mediated isothermal amplification (LAMP) according to the EPPO diagnostic standard PM 7/079(2) (EPPO 2016). PCR-products of the map and the vmpA genes (Malembic-Maher et al. 2020) were sequenced and compared to reference sequences to distinguish between FD- and non-FD genotypes. The isolate from the diseased vine (GenBank MW 727272) exhibited 100% identity with map-M38 (GenBank LT221933), a genotype of the map-FD2 cluster. The same genotype was detected in A. glutinosa and Allygus spp. sampled at the infested site. A 234 bp sequence of the first repeat of the vmpA gene (GenBank MW727273) showed 100% identity with the homologous part of isolate FD-92 (GenBank LN680870) of the vmpA-II cluster. It can be concluded, that the symptomatic grapevine was infected by FD and not PGY This is the first report of FD in a productive vineyard in Germany. The infected vine of cv. Silvaner was 25 years old. While infected planting material is an unlikely source of the infection, a transmission of FDp from alder is highly probable. Finding a single FD-infection after several years of testing implies a low risk originating from the wild compartment, but the approach and possible establishment of S. titanus expected to be able to colonize the area (Jeger et al. 2016) justifies further monitoring activities. The infected vine was eradicated.

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First Report of an Elm Yellows Subgroup 16SrV-C Phytoplasma Infecting Grapevine in Serbia

Plant Disease ◽

10.1094/pdis.2003.87.5.599c ◽

2003 ◽

Vol 87 (5) ◽

pp. 599-599 ◽

Cited By ~ 7

Author(s):

B. Duduk ◽

M. Ivanovic ◽

N. Dukic ◽

S. Botti ◽

A. Bertaccini

Keyword(s):

Leaf Roll ◽

Restriction Enzymes ◽

Stem Bark ◽

Rrna Gene ◽

Universal Primer ◽

First Report ◽

Flavescence Dorée ◽

Scaphoideus Titanus ◽

Two Samples ◽

Elm Yellows

During a 2002 survey in Serbia, samples of grapevine (Vitis vinifera) were collected from plants showing typical phytoplasma-like symptoms: leaf roll, leaf redness, vein chlorosis and necrosis, and absence of lignification. The material was collected from one viticultural region (Zupa Aleksandrovac), where the disease was recorded in 2000 and showed an increasing percentage of symptomatic plants every year. Total nucleic acid was extracted separately from leaf midveins and stem bark collected from 10 symptomatic and 2 asymptomatic plants. Phytoplasma infection was detected using polymerase chain reaction (PCR) assays with universal primer pair P1/P7 for the amplification of phytoplasma 16S rRNA gene, and primer pair FD9f2/FD9r followed by FD9f3/FD9r2 in nested PCR for specific amplification of the FD9 nonribosomal DNA fragment of the EY-group (1). Phytoplasmas were detected in 9 of 10 midvein extracts from symptomatic grapevines (three of cv. Plovdina, two of cv. Smederevka, and four of cv. Gamé). Also, 6 of 10 bark preparations representing stem collections from the same plants were positive (two samples of cv. Plovdina, both samples of cv. Smederevka, and two samples of cv. Gamé). Both collections of midveins and bark tissues from asymptomatic plants were negative. Fragments amplified with universal P1/P7 primers (16S-23S rDNA) were analyzed by restriction fragment length polymorphism with TruI and TaqI restriction enzymes. The phytoplasmas produced identical restriction profiles to those of 16SrV Elm Yellows group and 16SrV-C Flavescence doreé subgroup (2). To our knowledge, this is the first report of phytoplasma infecting grapevines in Serbia, and the first survey in progress to verify the presence of Scaphoideus titanus to determine if this grapevine yellows could be defined as Flavescence dorée. References: (1) E. Angelini et al. Vitis 40:79, 2001. (2) M. Martini et al. Mol. Cell. Probes 16:197, 2002.

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First Report of Alder Yellows Phytoplasma Associated with Common Alder (Alnus glutinosa) in the Republic of Macedonia

Plant Disease ◽

10.1094/pdis-03-14-0315-pdn ◽

2014 ◽

Vol 98 (9) ◽

pp. 1268-1268 ◽

Cited By ~ 5

Author(s):

B. Atanasova ◽

D. Spasov ◽

M. Jakovljević ◽

J. Jović ◽

O. Krstić ◽

...

Keyword(s):

Genetic Locus ◽

Ribosomal Protein Gene ◽

Alnus Glutinosa ◽

Sensu Stricto ◽

Universal Primers ◽

Rrna Gene ◽

Significant Similarity ◽

First Report ◽

Flavescence Dorée ◽

Scaphoideus Titanus

Alder yellows phytoplasma (AldYp) is classified as a member of the 16SrV-group of phytoplasmas and is closely related to Flavescence dorée (FD), a quarantined pathogen of economic importance affecting vineyards across Europe. AldYp is associated with common (Alnus glutinosa) and grey alder (A. incana), and has been reported in France, Italy, Germany, Austria, Switzerland, the Baltic region, Serbia, and Montenegro (1,2,4). For Macedonian vineyards, so far, neither infection of grapevine with 16SrV-group of phytoplasmas nor the presence of the main FD phytoplasma vector, Scaphoideus titanus, has been recorded. However, the presence of FD-related phytoplasma was detected in wild Clematis vitalba. In September and October 2013, leaves with petioles from A. glutinosa exhibiting leaf discoloration and yellowing were collected from two sites (41°23′43″ N, 22°54′ E and 41°23′ N, 22°53′ E) in southeast Macedonia near the village of Smolare (Strumica district). Eight samples were collected from each site. Leaves of six asymptomatic alder seedlings collected from the same sites served as a control. Nucleic acids were extracted from fresh leaf midribs and petioles using a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Initial phytoplasma identification was carried out by nested PCR assay of the 16S rRNA gene, using universal primers P1/P7 and R16F2n/R16R2 followed by RFLP with MseI endonuclease (Fermentas, Vilnius, Lithuania), as previously reported (4). Characterization of detected phytoplasmas was performed by amplifying two genetic loci specific for the members of the 16SrV group phytoplasmas; the ribosomal protein gene operon (rp) using primers rp(V)F1/rpR1 and rp(V)F1A/rp(V)R1A (3), and the non-ribosomal metionine aminopeptidase (map) gene using primer set FD9f5/MAPr1 and FD9f6/MAPr2 (1). The PCR amplicons were sequenced and deposited in NCBI GenBank database under the accession numbers KJ605448 to 52 (map) and KJ605453 to 57 (rp). The obtained sequences were compared with reference sequences of the 16SrV-group phytoplasmas (1,3) using the neighbor-joining method in MEGA5 (5). The presence of phytoplasma was detected in 14 of 16 symptomatic alder samples, while all control plants tested negative. The MseI restriction profiles were identical among all 14 samples and with the reference strains of the 16SrV group phytoplasmas (EY1 - 16SrV-A, FD-C - 16SrV-C, and FD-D - 16SrV-D). The rp-based phylogeny enabled identification of four diverse phytoplasma strains among the AldYp strains from Macedonia. Three strains clustered within the rpV-E subgroup while one belonged to rpV-L subgroup. Phylogenetic analysis of the more variable genetic locus, map, showed the presence of five diverse phytoplasma strains. Four strains belonged to the map-FD2 (FD-D, FD92) cluster, while one grouped within the map-FD1 (FD70) cluster. To our knowledge, this is the first report of 16SrV phytoplasma group occurrence on alder in Macedonia. The significant similarity between AldYp strains and FD sensu stricto indicate the risk of pathogen exchange between the wild ecosystem and the grapevine (1). Alder trees naturally infected with the FDp-related strains could therefore represent a serious risk for FD outbreak in Macedonian vineyards if local S. titanus populations developed. References: (1) G. Arnaud et al. Appl. Environ. Microbiol. 73:4001, 2007. (2) T. Cvrković et al. Plant Pathol. 57:773, 2008. (3) M. Martini et al. Int. J. Syst. Evol. Microbiol. 57:2037, 2007. (4) S. Radonjić et al. Plant Dis. 97:686, 2013. (5) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011.

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First Report of Flavescence Dorée-Related Phytoplasma Affecting Grapevines in Croatia

Plant Disease ◽

10.1094/pdis-09-10-0664 ◽

2011 ◽

Vol 95 (3) ◽

pp. 353-353 ◽

Cited By ~ 7

Author(s):

M. Šeruga Musić ◽

D. Škorić ◽

I. Haluška ◽

I. Križanac ◽

J. Plavec ◽

...

Keyword(s):

Nested Pcr ◽

Reference Strain ◽

Simultaneous Detection ◽

Pinot Noir ◽

Vitis Vinifera L ◽

Rrna Gene ◽

Pcr Assay ◽

Direct Pcr ◽

Flavescence Dorée ◽

Scaphoideus Titanus

Flavescence dorée (FD) and Bois noir (BN) phytoplasmas are principal grapevine yellows (GY) agents in the wider Euro-Mediterranean Region. While BN phytoplasma belongs to the ribosomal subgroup 16SrXII-A, the FD agents belong either to the ribosomal subgroups 16SrV-C or -D. During the official GY survey in 2009, 40 symptomatic grapevines (Vitis vinifera L.) were sampled throughout grapevine-growing regions in Croatia. Typical GY symptoms of leaf yellowing or reddening were evident on white and red varieties, respectively. Leaf rolling as well as irregular lignification of the shoots and withering of clusters were also observed. Phloem tissue from cuttings and leaf veins from mature vines were sampled for total DNA extraction and amplification of phytoplasma 16S rRNA gene by using generic primers P1/P7 in a direct PCR assay followed by a nested PCR using primer pair R16F2n/R2 (2). Phytoplasma ribosomal group affiliation was determined by restriction fragment length polymorphism (RFLP) analysis of the nested PCR products with enzyme Tru1I (Fermentas, Vilnius, Lithuania). These initial findings were validated and augmented by a triplex real-time PCR assay targeting the nonribosomal map gene. This assay enables simultaneous detection of BN and FD (16SrV-C and -D) phytoplasmas in grapevine (3). Assay results revealed the majority of GY positive vines (19 of 40) contained BN phytoplasma which is widespread. For the first time in Croatia, two red variety samples, Pinot Noir and Plemenka Crvena, from the vicinity of Ozalj (Vivodina) and Zagreb (Brezje), respectively, were found to harbor FD-related phytoplasmas. Fragments amplified by P1/P7 primers from latter samples were cloned and sequenced. Sequence analyses using online interactive tool iPhyClassifier (4) revealed that the phytoplasma under study from Pinot Noir sample (GenBank Accession No. HQ712064) is a member of 16SrV-C subgroup and shares 99.87% similarity with 16S rDNA sequence of the reference strain (GenBank Accession No. AF176319). The sequence from the Plemenka Crvena sample (GenBank Accession No. HQ712065) shares 99.54% similarity with the reference strain and has the most similar virtual RFLP pattern to the one of the 16SrV-C subgroup (GenBank Accession No. AY197642). These findings are currently limited to vineyards in northwestern Croatia. Even so, the presence of FD principal cicadellid vector Scaphoideus titanus in the country and the occurrence and distribution of FD in neighboring countries (1,2) are factors indicating that the spread of FD in Croatia is highly probable. References: (1) L. Filippin et al. Plant Pathol. 58:826, 2009. (2) S. Kuzmanović et al. Vitis 47:105, 2008. (3) C. Pelletier et al. Vitis 48:87, 2009. (4) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

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Identification and Epidemic Distribution of Two Flavescence Dorée—Related Phytoplasmas in Veneto (Italy)

Plant Disease ◽

10.1094/pdis.1999.83.10.925 ◽

1999 ◽

Vol 83 (10) ◽

pp. 925-930 ◽

Cited By ~ 56

Author(s):

Marta Martini ◽

Ermanno Murari ◽

Nicola Mori ◽

Assunta Bertaccini

Keyword(s):

16S Rdna ◽

Fragment Length Polymorphism ◽

The European Union ◽

Direct Pcr ◽

Nested Polymerase Chain Reaction ◽

Flavescence Dorée ◽

Scaphoideus Titanus ◽

Polymerase Chain ◽

Elm Yellows ◽

Infected Grapevine

Grapevine yellows associated with phytoplasmas of the elm yellows group (16SrV), better known as flavescence dorée (FD), is a serious quarantine problem in some important grapevine growing regions in the European Union. A survey was carried out in 1997 to 1998 in Veneto region (Italy) where a serious outbreak of FD was in progress. Phytoplasma identification by nested polymerase chain reaction (PCR)/restriction fragment length polymorphism (RFLP) analyses on 275 grapevine samples and on batches of Scaphoideus titanus was carried out. RFLP analyses of the 16S rDNA/spacer region with TaqI detected the presence of two distinct elm yellows phytoplasma subgroups designated 16SrV-C and 16SrV-D in 77 FD-infected grapevine samples. Only phytoplasmas of the 16SrV-D subgroup were detected in S. titanus. In 1997, the two phytoplasma subgroups appeared to be located in two diverse geographic areas; but in 1998, the 16SrV-D type also was detected in the provinces where in 1997 only 16SrV-C type was identified. The sequencing of a 400-bp fragment at the 3′ end of 16S rDNA plus spacer region allowed a specific primer construction that was successfully employed for detection of both FD types in grapevine by direct PCR.

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Acquisition capability of the grapevine Flavescence dorée by the leafhopper vector Scaphoideus titanus Ball correlates with phytoplasma titre in the source plant

Journal of Pest Science ◽

10.1007/s10340-014-0593-3 ◽

2014 ◽

Vol 87 (4) ◽

pp. 671-679 ◽

Cited By ~ 31

Author(s):

Luciana Galetto ◽

Dimitrios Miliordos ◽

Chiara Roggia ◽

Mahnaz Rashidi ◽

Dario Sacco ◽

...

Keyword(s):

Leafhopper Vector ◽

Flavescence Dorée ◽

Scaphoideus Titanus ◽

Source Plant

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Models Applied to Grapevine Pests: A Review

Insects ◽

10.3390/insects12020169 ◽

2021 ◽

Vol 12 (2) ◽

pp. 169

Author(s):

Federico Lessio ◽

Alberto Alma

Keyword(s):

Lobesia Botrana ◽

Grape Berry ◽

Homalodisca Vitripennis ◽

Flavescence Dorée ◽

Or Groups ◽

Scaphoideus Titanus ◽

Different Types ◽

Computer Based ◽

Biological Aspects ◽

Definition Of

This paper reviews the existing predictive models concerning insects and mites harmful to grapevine. A brief conceptual description is given on the definition of a model and about different types of models: deterministic vs. stochastics, continuous vs. discrete, analytical vs. computer-based, and descriptive vs. data-driven. The main biological aspects of grapevine pests covered by different types of models are phenology, population growth and dynamics, species distribution, and invasion risk. A particular emphasis is put on forecasting epidemics of plant disease agents transmitted by insects with sucking-piercing mouthparts. The most investigated species or groups are the glassy-winged sharpshooter Homalodisca vitripennis (Germar) and other vectors of Xylella fastidiosa subsp. fastidiosa, a bacterium agent of Pierce’s disease; the European grape berry moth, Lobesia botrana (Denis and Schiffermuller); and the leafhopper Scaphoideus titanus Ball, the main vector of phytoplasmas agents of Flavescence dorée. Finally, the present and future of decision-support systems (DSS) in viticulture is discussed.

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Real-Time PCR Assay for Detection and Enumeration of Dekkera bruxellensis in Wine

Applied and Environmental Microbiology ◽

10.1128/aem.69.12.7430-7434.2003 ◽

2003 ◽

Vol 69 (12) ◽

pp. 7430-7434 ◽

Cited By ~ 77

Author(s):

Trevor G. Phister ◽

David A. Mills

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Pcr Primers ◽

Rrna Gene ◽

Dekkera Bruxellensis ◽

Pcr Assay ◽

Specific Pcr ◽

Spoilage Yeast ◽

Pcr Method ◽

26S Rrna

ABSTRACT Traditional methods to detect the spoilage yeast Dekkera bruxellensis from wine involve lengthy enrichments. To overcome this difficulty, we developed a quantitative real-time PCR method to directly detect and enumerate D. bruxellensis in wine. Specific PCR primers to D. bruxellensis were designed to the 26S rRNA gene, and nontarget yeast and bacteria common to the winery environment were not amplified. The assay was linear over a range of cell concentrations (6 log units) and could detect as little as 1 cell per ml in wine. The addition of large amounts of nontarget yeasts did not impact the efficiency of the assay. This method will be helpful to identify possible routes of D. bruxellensis infection in winery environments. Moreover, the time involved in performing the assay (3 h) should enable winemakers to more quickly make wine processing decisions in order to reduce the threat of spoilage by D. bruxellensis.

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