Characterization of X-Disease Phytoplasmas in Chokecherry from North Dakota by PCR-RFLP and Sequence Analysis of the rRNA Gene Region.

Abstract

Genetic variation of X-disease phytoplasma strains from chokecherry (ChX) in North Dakota and nearby sites, and their relatedness with three standard strains of the X-disease phytoplasma group, eastern X-disease (CX), western X-disease (WX), and goldenrod yellows (GR1) phyto-plasmas, were studied. Primer pairs were developed to amplify the 23S ribosomal RNA (rRNA) gene and the 16S/23S spacer region. The rRNA genes (16S rRNA, 23S rRNA, and two ribosomal protein [rp] genes) and the 16S/23S spacer region were amplified by polymerase chain reactions. The restriction fragment length polymorphism (RFLP) patterns of 16S rRNA, 23S rRNA, and rp genes, generated by digestion with four restriction enzymes (AluI, HpaII, MseI, and RsaI), showed no difference among 43 ChX phytoplasma isolates. Sequencing of the 441-bp 16S/23S spacer region revealed variation at four positions among 12 ChX phytoplasma strains. A tRNAIle and other conserved sequences were identified in the spacer region. Among X-disease subgroups, RFLP analysis indicated that ChX is similar to WX, closely related to CX, and easily distinguished from GR1. Sequencing indicated that ChX is closer to CX than to WX. Together, the analyses indicated that ChX phytoplasmas are genetically different from the standard strains of other X-disease phytoplasma subgroups.