Use of Mycoplasmalike Organism (MLO) Group-Specific Oligonucleotide Primers for Nested-PCR Assays to Detect Mixed-MLO Infections in a Single Host Plant

Oligonucleotide primer pairs R16(I)F1/R1, R16(III)F2/R1, and R16(V)F1/R1 for polymerase chain reactions (PCRs) were designed on the basis of mycoplasmalike organism (MLO) 16S rRNA sequences. The primer pair R16(I)F1/R1 specifically initiated amplification of 16S rDNA sequences among MLO strains in the MLO 16S rRNA group I, which includes aster yellows MLO and related strains; R16(III)F2/R1 specifically initiated amplification in the MLO 16S rRNA group III, which includes peach X-disease MLO and related strains; and R16(V)F1/R1 specifically initiated amplification in the MLO 16S rRNA group V, which includes elm yellows MLO and related strains. None of the primer pairs initiated amplification of 16S rDNA sequences from MLO strains in other groups or from other prokaryotes, including animal Mollicutes and plant pathogenic bacteria. An MLO group-specific primer pair allows sensitive detection and simultaneous classification of specific MLO strains from plant and insect sources. Nested-PCR assays using the universal primer pair R16F2/R2 and a group-specific primer pair further increased sensitivity in MLO detection. These specially designed assay procedures allowed for the first time detection of a secondary, cryptic MLO(s) associated with a single host plant.