MYCOPLASMALIKE ORGANISMS FROM MILKWEED, GOLDENROD, AND SPIREA REPRESENT 2 NEW 16S RIBOSOMAL-RNA SUBGROUPS AND 3 NEW STRAIN SUBCLUSTERS RELATED TO PEACH X-DISEASE MLOS

Two strains of mycoplasmalike organisms (MLOs) from Asclepias syriaca (milkweed) and one strain each from Solidago rugosa (goldenrod) and Sp iraea tomentosa (spirea), transmitted to periwinkle (Catharanthus rose us), were characterized in terms of restriction fragment length polymo rphisms (RFLPs) in 16S rDNA and in random fragments of chromosomal DNA . A 1.2-kb fragment of MLO 16S rDNA was amplified by polymerase chain reaction (PCR) from each of these MLO strains and from four reference strains belonging to the 16S rRNA group III (peach X-disease and relat ed MLOs). RFLP analyses of the PCR products revealed that the milkweed MLOs were similar to clover yellow edge MLO (16S rRNA subgroup III-B) , whereas the goldenrod and spirea MLOs represented new subgroups (des ignated 16S III-D and III-E, respectively). Southern hybridizations of total DNA from infected plants, digested with EcoRI and HindIII, were performed with a probe cocktail of 21 cloned fragments of chromosomal DNA from two strains of X-disease MLOs. Numerous DNA fragments from e ach of the new MLO strains and reference strains in 16S rRNA group III hybridized with the probe cocktail, but little or no hybridization oc curred with DNA of nine other reference strains representing different 16S rRNA groups and strain clusters. Therefore, the new MLO strains w ere classified in the peach X-disease MLO strain cluster. Three new su bclusters were designated based on unique hybridization patterns in th e Southern analyses: subcluster TV for the two strains from milkweed ( MW1 and MW2), which had the same pattern; subcluster V for the goldenr od strain (GR1); and subcluster VI for the spirea strain (SP1).