No 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