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First Report of Forsythia suspensa, Spiraea vanhouttei, and Viburnum lantana as New Natural Plant Hosts of ‘Candidatus Phytoplasma mali’, the Causal Agent of Apple Proliferation Disease, in Lithuania

    Authors and Affiliations
    • A. Abraitienë , Institute of Biotechnology, Vilnius University, Lithuania
    • A. Bevilacqua
    • A. Scarafoni , Department of Food and Environmental Sciences, University of Milan, Italy
    • F. Quaglino , Department of Agricultural and Environmental Sciences, University of Milan, Italy.

      Candidatus Phytoplasma mali’, associated with apple proliferation (AP) disease, is a quarantine pathogen on the list A2 of EPPO since 1975. ‘Ca. P. mali’ is transmitted to apple by oligo/polyphagous insect vectors that could acquire it also from other plant hosts (e.g., hawthorn) (Tedeschi and Alma 2007); thus, the knowledge of natural plant hosts of ‘Ca. P. mali’ is crucial for planning AP containment strategies. In this study, during a survey conducted in 2017 in the district of Kaunas (Lithuania), phytoplasma-like symptoms were observed in one plant of the species Forsythia suspensa (leaf reddening), Viburnum lantana (witches’ broom), and Spiraea vanhouttei (decline) grown in a private garden. DNA of leaf samples collected from each plant was extracted using the DNeasy Plant Mini Kit (Qiagen, Germany) and used as a template in polymerase chain reactions (PCRs). Nested PCRs, carried out using the primer pairs P1/P7 and R16F2n/R16R2 (F2n/R2) (Gundersen and Lee 1996), allowed the amplification of phytoplasmal 16S rRNA gene fragments from the analyzed samples. No amplification was obtained in negative control (PCR mixture devoid of DNA). The F2n/R2 amplicons were sequenced (Institute of Biotechnology, Vilnius University), assembled with the software BioEdit, and compared with the NCBI database. BlastN analyses revealed that phytoplasma strains, identified in F. suspensa (accession no. MG739445), V. lantana (MG739446), and S. vanhouttei (MG739447), showed the best identity versus the reference strains of the species ‘Ca. P. mali’ (AJ542541) (99.9 to 100%), ‘Ca. P. pyri’ (AJ542543), and ‘Ca. P. prunorum’ (AJ542544) (98.7 to 99.2%). Owing to the high (>97.5%) 16S rDNA sequence similarity with reference strains of three species previously described, further analyses were performed for obtaining an accurate taxonomic classification (IRPCM 2004). Restriction fragment length polymorphism analysis, carried out on F2n/R2 amplicons with the enzymes RsaI and SspI (Lee et al. 1998), revealed that phytoplasma strains infecting F. suspensa, V. lantana, and S. vanhouttei in Lithuania showed the same profiles, indistinguishable from those of ‘Ca. P. mali’ (subgroup 16SrX-A) and distinct from those of ‘Ca. P. prunorum’ (subgroup 16SrX-B) and ‘Ca. P. pyri’ (subgroup 16SrX-C). Moreover, 16S rDNA nucleotide sequences of such phytoplasma strains carried the ‘Ca. P. mali’ unique signature sequence (5′-AATACTCGAAACCAGTA-3′) (Seemüller and Schneider 2004) and single nucleotide polymorphisms distinguishing ‘Ca. P. mali’ from ‘Ca. P. pyri’ and ‘Ca. P. prunorum’: nucleotide positions 20(A), 119(C), 215(A), 375(T), and 432(G) from the annealing site of the primer F2n. Minimum evolution phylogenetic analysis, performed using the software MEGA7, showed that phytoplasma strains, here identified in Lithuania, clustered together with ‘Ca. P. mali’ in a subclade clearly distinct from other phytoplasmas of the 16SrX taxonomic group. All this evidence was consistent with the attribution of phytoplasma strains identified in F. suspensa, V. lantana, and S. vanhouttei in Lithuania to ‘Ca. P. mali’ (16SrX-A). To the best of our knowledge, such plant species were never reported before as ‘Ca. P. mali’ hosts; thus, further studies are needed to determine their possible role in the epidemiology of AP.