Molecular Identification of a Phytoplasma Associated with Witches'-Broom Disease of Black Raspberry in Oregon and Its Classification in Group 16SrIII, New Subgroup Q.

Abstract

Plants of Rubus occidentalis (black raspberry) 'Munger' exhibiting symptoms of black raspberry witches'-broom (BRWB) disease were observed in commercial fields in Oregon (1). Symptoms were often severe, leading to death of infected plants, and a phytoplasma (mycoplasmalike bodies) was observed in ultrathin sections of diseased plants (1). In the current work, the association of phytoplasma with BRWB was assessed using the polymerase chain reaction (PCR) for specific amplification of phytoplasmal rDNA. DNA template for use in the PCR was extracted from plants as described elsewhere (2). Phytoplasmal 16S rDNA was amplified from diseased black raspberry plants in PCR primed by primer pair P1/P7 and reamplified in nested PCR primed by primer pair R16F2n/R2 (F2n/R2) by a method described previously (2). These results indicated the presence of a phytoplasma, designated BRWB phytoplasma, in the diseased plants. Identification of BRWB phytoplasma was accomplished by restriction fragment length polymorphism (RFLP) analysis of DNA amplified in PCR primed by F2n/R2. Phytoplasma classification was done according to the system of Lee et al. (3). On the basis of collective RFLP patterns of the amplified 16S rDNA, the BRWB phytoplasma was classified as a member of group 16SrIII (group III, X-disease phytoplasma group). The HhaI RFLP pattern of BRWB 16S rDNA differed from that of its close relative, clover yellow edge (CYE) phytoplasma. The RsaI RFLP pattern of BRWB rDNA differed from that of rDNA from all phytoplasmas previously described in group III. Based on these results, BRWB phytoplasma was classified in a new subgroup, designated subgroup Q (III-Q) within group III. The 1.8 kbp DNA product of PCR primed by primer pair P1/P7 was cloned and its nucleotide sequence determined. The sequence was deposited in GenBank under Accession no. AF302841. Results from putative restriction site analysis of the cloned and sequenced rDNA were in excellent agreement with the results from enzymatic RFLP analysis of uncloned rDNA amplified from BRWB diseased black raspberry. Sequence similarity between the 1.8 kbp rDNA of BRWB phytoplasma and that of CYE phytoplasma was 99.4%. The nucleotide sequence data support the conclusion that the BRWB phytoplasma is related to, but is distinct from, other strains that are classified in group III. These findings contribute knowledge about the diversity of phytoplasmas affiliated with group III and provide information to aid the diagnosis of BRWB disease.