Identification and molecular characterization of 16SrV group phytoplasmas with flowering cherry in China

Abstract

Flowering cherry (Prunus serrulata) trees showing symptoms of excessive axillary growth, stunting, short internodes and decline were observed in Yantai City, Shandong province, China. The samples with the flowering cherry decline were designated as FcD-YT phytoplasma. Sequencing results, restriction fragment length polymorphism (RFLP) and phylogenetic analysis of sequences from the 1248-bp R16F2n ⁄ R16R2 products and partial ribosomal protein operon genes revealed the FcD-YT phytoplasma as a member of subgroup 16SrV-B, rpV-C. Two RFLP patterns were observed in recombinant colonies from the cloned 16S rDNA gene with endonucleases HpaII and MseI. To our knowledge, this is the first report of 16SrV group phytoplasma declining flowering cherry trees in China.The expected PCR product fragment sizes of 1.8 kb using the universal P1/P7 primers, and 1.25 kb using the nested PCR primers R16F2n/R2, were obtained from two symptomatic flowering cherry trees, but not from the asymptomatic sample ( Fig S1). Representative AluI, RsaI, Sau3AI, HinfI, EcoRI, HpaII, MseI and BfaI RFLP patterns of the 16S rDNA amplified from the two symptomatic plants and the reference JWB-Hubei phytoplasma are illustrated (Fig.S2). The collective RFLP patterns for the positive samples were identical to each other and to that of the JWB-Hubei strain (GenBank Accession No. AF305240), a member of the 16SrV-B subgroup. RFLP profiles from 16S rDNA PCR products of 10 clones randomly selected were compared with those obtained from direct 16S rDNA PCR products digested with RsaI, TaqI, SspI, HpaI, HinfI, HhaI, HaeIII, BfaI, AluI and BstUI. The resulting profiles were identical to those of the 16SrV-B subgroup strains described by Lee et al. (2004). Two variants (I and II) of the collective RFLP patterns from the cloned 16S rRNA gene sequences were observed with MseI and HpaII digestions (Fig. S3). One RFLP variant (I) showed five bands with HpaII and seven bands with MseI in one of 10 clones. The other RFLP variant (II) showed four bands with HpaII and six bands with MseI in 9 of 10 clones, matching the RFLP pattern of the uncloned 16Sr DNA PCR products of the FcD-YT phytoplasma (Figs S2 and FcD-YT phytoplasma-Flower cherry#1(China) Apricot leaf roll phytoplasma YS (FJ572660 China) 16SrV-B FcD-YT phytoplasma-Flower cherry#4(China) Cherry lethal yellows CLY-5 (AY197659 China) 16SrV-B JWB (AY197661 China) 16SrV-B Ca Phytoplasma ulmi EY1 T (AY197655 USA) 16SrV-A

List of non‐EU phytoplasmas of Cydonia Mill., Fragaria L., Malus Mill., Prunus L., Pyrus L., Ribes L., Rubus L. and Vitis L.

“…reported to be present in the EU -Species description: (Davis et al, 2016); Fragaria: (Jomantiene et al, 1998;Fernandez et al, 2015) Species description: (Verdin et al, 2003;EPPO, 2017); Vitis: (Ghayeb Zamharir et al, 2017); Prunus in Italy: (Nigro et al, 2020) 6 Candidatus Phytoplasma pruni Species description and Fragaria: (Hiruki and Wang, 2004); Prunus: (Zirak et al, 2010b); Czech Republik: (Pribylova et al, 2009); Austria: (Borroto Fernandez et al, 2007) 9 Candidatus Phytoplasma ziziphi Species description: (Jung et al, 2003); Malus: (Li et al, 2014); Prunus: (Zhu et al, 1998;Wang et al, 2014Wang et al, , 2018; Prunus in Italy: (Paltrinieri et al, 2006); Olea europea in Italy: (Pasquini et al, 2000) 10 Species description: (Montano et al, 2001); Prunus: (Balakishiyeva et al, 2011) 12 Species description: (Davis et al, 2013); Fragaria: (Jomantiene et al, 2002); in clover and Lithuania (Staniulis et al, 2000) 13 Schneider, 2004;and Morton et al, 2003, it is listed here as Ca. P. pyrirelated strain Species description: (Davis et al, 2013); Pyrus in Croatia: (Jezic et al, 2016); Pyrus in Italy: (Lee et al, 1995); Rubus in UK: (Davies, 2000); Rubus in Poland: (Cieslinska, 2011); Vitis in Croatia: (Jezic et al, 2013); Prunus in Lithuania: (Valiunas et a...…”

“…P. solani’ and ‘ Ca . P. asteris’ and the other one has no further characterization beside PCR and RFLP (restriction fragment length polymorphism) analysesPaltrinieri et al., ( Prunus in Italy: only a Congress abstracts, no further details); Pasquini et al., ( Olea europea in Italy: phytoplasma detected based only on PCR and RFLP analyses of ribosomal operon)Species description: (Jung et al., ); Malus : (Li et al., ); Prunus : (Zhu et al., ; Wang et al., , ); Prunus in Italy: (Paltrinieri et al., ); Olea europea in Italy: (Pasquini et al., )10 Unclassified Buckland valley grapevine yellows phytoplasma–XXIII–––––––YesNot reported to be present in the EU–Species description: (Constable et al., )…”

Elm yellows: A phytoplasma disease of concern in forest and landscape ecosystems

“…P. ulmi" is a member of the EY phytoplasma group or 16SrV group, subgroup 16SrV-A (Lee et al, 2004). Other members of this group are phytoplasmas causing mainly diseases of woody plants such as flavescence dorée of grapevine, alder yellows, Palatinate grapevine yellows, spartium witches' broom, rubus stunt, eucalyptus little leaf, cherry lethal yellows, flowering cherry decline, peach yellows in India, jujube witches' broom, Japanese raisin witches' broom and sophora japonica witches' broom ( Figure 1) (Arnaud et al, 2007;Gao et al, 2011;Jung et al, 2003;Kamala-Kannan et al, 2011;Lee et al, 2004;Malembic-Maher et al, 2011;Marcone et al, , 1997Ren et al, 2014;Wang et al, 2014).…”