First Report of Lychnis flos-cuculi as a New Host Plant for ‘Candidatus Phytoplasma asteris’ (Subgroup 16SrI-B)

Abstract

Lychnis flos-cuculi L. (Caryophyllaceae) is a widespread perennial herb occurring in wet grassland habitats throughout Europe (Galeuchet et al. 2005). A double-flowered cultivar, Petite Jenny, is now common as an ornamental in gardens throughout the world. In September 2018, approximately 46% of L. flos-cuculi ‘Petite Jenny’ exhibited symptoms of phytoplasma infection in a commercial nursery in central Hungary. The affected plants did not produce flowers, and their shoot tips and surrounding leaves showed symptoms of yellowing and a bushy appearance. Total DNA was extracted from stem phloem scrapings and whole leaves of five healthy and 20 diseased plants using a phytoplasma enrichment procedure (Ahrens and Seemuller 1992). The DNA was used as a template in direct PCR primed by the phytoplasma-universal primers P1/P7 followed by nested PCR primed by fU5/rU3 (Lorenz et al. 1995) with Phusion High-Fidelity DNA polymerase (NEB, Ipswich, MA). Reactions containing template DNA from the symptomatic plants yielded ribosomal RNA gene amplicons of the expected size (1.8 and 0.9 kb for P1/P7 and fU5/rU3, respectively) but not from asymptomatic plants. Products of P1/P7-primed PCR 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 were sequenced by LGC Genomics (Berlin, Germany) using pJET1.2 forward and reverse primers, and the obtained sequence was deposited in GenBank (MK992773). A BLASTn search revealed that the sequence obtained was 100% identical to 16S rDNA of aster yellows (AY) phytoplasma (AF222063) and shared 99.95% sequence similarity with the ‘Candidatus Phytoplasma asteris’ reference strain (AP006628). A phylogenetic analysis using MEGA7 (Tamura et al. 2013) confirmed that the phytoplasma clustered with 16SrI-B strains. In subsequent experiments, three representative samples were selected for PCR amplifications of the partial elongation factor Tu (tuf), ribosomal protein (rp) operon (rps19, rpl22, and rps3), and molecular chaperonin groEL genes with primer pairs fTufl/rTufl (Schneider et al. 1997), rpF1/rpR1 (Lim and Sears 1992), and AYgroelF/AYgroelR (Mitrovic et al. 2011), respectively. The generated amplicons were cloned and sequenced (GenBank accession nos. MN526022 to MN526024), and BLAST searches of the GenBank database showed 100% identity of the tuf sequence with AY phytoplasma strain IRap (AJ271316). The rp and groEL sequences were 100% identical to six and 11 GenBank entries, respectively (e.g., Onion proliferation phytoplasma strain OnP2 for rp [GU228514] and AY phytoplasma strain AVUT for groEL [AB599686]). Comparison with the reference strains (Lee et al. 2004; Mitrovic et al. 2011) allowed classification of the phytoplasma into AY subgroups tufI-B, rpI-B, and groELI-III. To our knowledge, this is the first report of an AY phytoplasma infecting L. flos-cuculi. Identification of subgroup 16SrI-B phytoplasma infection in L. flos-cuculi pointed to its broad host range, as has been reported earlier by several investigators (e.g., Marcone et al. 2016). Considering the serious symptoms and the high incidence of the disease in nursery-grown plant material, the infection may result in economic losses to the producers.