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First Report of ‘Candidatus Phytoplasma rubi’ Associated with Rubus Stunt Disease of Raspberry and Blackberry in the Czech Republic

    Affiliations
    Authors and Affiliations
    • Jana Fránová1
    • Jaroslava Přibylová1
    • Rostislav Zemek2
    • Jiunn Luh Tan2 3
    • Zhibo Hamborg4
    • Dag-Ragnar Blystad4
    • Ondřej Lenz1
    • Igor Koloniuk1
    1. 1Biology Centre CAS, Institute of Plant Molecular Biology, 370 05 České Budějovice, Czech Republic
    2. 2Biology Centre CAS, Institute of Entomology, 370 05 České Budějovice, Czech Republic
    3. 3Faculty of Science, University of South Bohemia, 370 05 České Budějovice, Czech Republic
    4. 4Division of Biotechnology and Plant Health, Norwegian Institute of Bioeconomy Research, Ås, Norway

    Raspberries (Rubus idaeus L.), occurring in the temperate zone of the Northern Hemisphere, and blackberries (R. fruticosus L.), cultivated and growing all over the world, are plant species of the family Rosaceae. These species are susceptible to phytoplasma infections, which cause Rubus stunt disease. It spreads uncontrollably by vegetative propagation of plants (Linck and Reineke 2019a) and by phloem-sucking insect vectors, especially Macropsis fuscula (Hemiptera; Cicadellidae) (de Fluiter and van der Meer 1953; Linck and Reineke 2019b). During a survey in a commercial field in June 2021, over 200 raspberry bushes cultivar Enrosadira exhibiting typical symptoms of Rubus stunt were observed in Central Bohemia. Symptoms included dieback, leaf yellowing or reddening, stunted growth, severe phyllody, and fruit malformations. Most diseased plants were growing in the edge rows of the field (about 80%). No symptomatic plants were observed in the middle of the field. Similar symptoms were observed in private gardens in South Bohemia on raspberry cultivar Rutrago and blackberry (unknown cultivar) in June 2018 and August 2022, respectively. DNA was extracted using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) from flower stems and parts affected by phyllody of seven symptomatic plants as well as flower stems, leaf midribs, and petioles of five asymptomatic field plants. The DNA extracts were analyzed by a nested polymerase chain reaction assay using the universal phytoplasma P1A/P7A primers followed by R16F2m/R1m and the group-specific R16(V)F1/R1 primers (Bertaccini et al. 2019). All samples from the symptomatic plants yielded an amplicon of the expected size, whereas no product was amplified in the asymptomatic plants. The P1A/P7A amplicons from three selected plants (two raspberries and one blackberry, each from a different location) were cloned and bidirectionally Sanger sequenced (GenBank accession nos. OQ520100 to OQ520102). The sequences spanned nearly the full length of the 16S rRNA gene, the 16S-23S rRNA intergenic spacer, the tRNA-Ile gene, and a partial 23S rRNA gene. A BLASTn search revealed the highest sequence identity (99.8 to 99.9%; 100% query coverage) to the ‘Candidatus Phytoplasma rubi’ strain RS (GenBank accession no. CP114006). To further characterize the ‘Ca. P. rubi’ strains, all three samples were subjected to multigene sequence analysis. Sequences from a major portion of the tuf, rplV-rpsC, rpsH-rplR, uvrB-degV, and rplO-SecY-map genes (accession nos. OQ506112 to OQ506126) were obtained as described previously (Fránová et al. 2016). Comparison with GenBank sequences confirmed their highest identity (99.6 to 100%; 100% query coverage) with the ‘Ca. P. rubi’ RS strain, regardless of their geographic location and host (raspberry or blackberry). Recently, Bertaccini et al. (2022) suggested the 98.65% ‘Ca. Phytoplasma’ strain identity threshold within 16Sr RNA sequences. In this survey, all three strains sequenced shared ≥99.73% sequence identity to the analyzed 16S rRNA gene sequences and high identity in the other genes with the reference ‘Ca. P. rubi’ RS strain. To our knowledge, this is the first report of Rubus stunt disease in the Czech Republic as well as the first molecular identification and characterization of ‘Ca. P. rubi’ from raspberry and blackberry in our country. As Rubus stunt disease is of great economic importance (Linck and Reineke 2019a), the pathogen detection and prompt removal of the diseased shrubs are essential to mitigating the spread and impact of the disease.

    The author(s) declare no conflict of interest.

    References:

    Funding: This work was supported by the project NOBERRYVIRUSCZ funded by grants from Iceland, Liechtenstein, and Norway through the EEA Grants and the Technology Agency of the Czech Republic (TO01000295) and by institutional support from the Czech Academy of Sciences (RVO:60077344).

    The author(s) declare no conflict of interest.