strains are devastating plant pathogens with a global distribution, a wide host range, and genetic diversity, and they are now also referred to as species complex (RSSC). RSSC strains employ the quorum sensing (QS) system composed of the operon to regulate their virulence on plants. The RSSC strains previously examined produce either ()-methyl 3-hydroxymyristate (3-OH MAME) or ()-methyl 3-hydroxypalmitate (3-OH PAME) as their QS signals. Analogously, the phylogenetic analyses of the signal synthase PhcB and the signal receptor PhcS from 15 RSSC strains revealed that these proteins have two clades dependent on their QS signal types. However, the biochemical mechanism underlying this selectivity of QS signal production remains to be elucidated. We demonstrated that the PhcB methyltransferases synthesize QS signals from the cognate fatty acids ()-3-hydroxymyristic acid or ()-3-hydroxypalmitic acid. The RSSC strains used here produced both fatty acids, and thus the selectivity of QS signal production depends on the activity of PhcB enzymes. On the other hand, the enantioselective supply of the precursors functioned in the production of enantiopure QS signals. The opposite QS signals weakly induced the production of virulence factors in the RSSC strains. Furthermore, the complementation of gene encoding 3-OH PAME-type synthase to the -deletion mutant of 3-OH MAME-producing strain did not rescue its virulence on tomato plants. Taken together, we propose that the specific production of 3-OH MAME/3-OH PAME ensures the full virulence of RSSC strains.