Molecular Identification of a New Phytoplasma Strain Associated with the First Observation of Jujube Witches'-Broom Disease in Northeastern China.

Jujube (Zizyphus jujuba Mill.) is a deciduous tree that is native to northern Africa and Syria. Because of its tolerance to a broad range of climatic conditions, jujube has attained a wide natural distribution from southeastern Europe to eastern Asia (3). Jujube has a long history of cultivation, especially in Asia, for its valuable medicinal properties, strong wood, and nutritious fruits. Jujube trees are susceptible to phytoplasma infections and develop jujube witches'-broom (JWB) disease. To date, JWB diseases have been reported in Korea, Japan, and central China (1,4). In this communication, we describe a new phytoplasma strain associated with the first observation of JWB disease in northeastern China. In the summer of 2006, six jujube trees exhibiting pronounced witches'-broom symptoms were observed in suburban Dalian, Liaoning Province. The trees developed dense clusters of highly proliferating branches with shortened internodes. Leaves on the affected branches were chlorotic and significantly reduced in size. A DNA segment characteristic of phytoplasma rRNA partial operons was amplified from DNA samples extracted from leaves of all diseased trees in polymerase chain reactions (PCR) using phytoplasma-universal primer pair P1/P7 (2). No PCR product was obtained from DNA samples extracted from two symptomless jujube trees in the same region. The PCR-amplified DNA segment, spanning a near full-length 16S rRNA gene, a 16S-23S rRNA intergenic spacer, a tRNA-Ile gene, and a partial 23S rRNA gene was cloned and sequenced to achieve 4× coverage per base position in sequencing both strands (GenBank Accession No. EF661852). Results from analysis of the sequence data indicated that the six jujube trees were infected by a phytoplasma of elm yellows group (16SrV), to which other reported JWB phytoplasma strains belong. However, the JWB phytoplasma strain identified in the current study, hereby designated as JWB-DL, displayed sequence variations within the partial rRNA operon compared with those of other JWB strains (GenBank Accession Nos. AY072722, AF305240, and AY197661), indicating that JWB-DL is a distinct strain. To further characterize the JWB-DL phytoplasma, a genomic segment covering full-length ribosomal protein genes rplV and rpsC was PCR-amplified using primer pair rp(V)F2A/rpR1 (2), cloned, and sequenced (GenBank Accession No. EF661581). The nucleotide sequence of the JWB-DL phytoplasma rplV-rpsC locus is identical to that of hemp fiber witches'-broom phytoplasma (GenBank Accession No. EF029093) rather than to those of JWB phytoplasma strains described previously. To our knowledge, this is the first report of a JWB disease in northeastern China, and JWB-DL represents a new, distinct 'Candidatus Phytoplasma ziziphi'-related strain. References: (1) H.-Y. Jung et al. Int. J. Syst. Evol. Microbiol. 53:1037, 2003. (2) I.-M. Lee et al. Int. J. Syst. Evol. Microbiol. 54:337, 2004. (3) W. H. Outlaw et al. Econ. Bot. 56:198, 2002. (4) J. B. Tian et al. Hortic. Sci 35:1274, 2000.