Novel primers and PCR protocols for the specific detection and quantification of Sphingobium suberifaciens in situ

The pathogen causing corky root on lettuce, Sphingobium suberifaciens, is recalcitrant to standard epidemiological methods. Primers were developed from 16S rDNA sequences to be useful for the specific detection and quantification of S.suberifaciens. Quantitative PCR (qPCR) protocols specifically amplified DNA from the type strain of S.suberifaciens (LMG 17323) and other members of this species but not from other members of the Sphingomonadaceae. The detection limit was as little as 100fg DNA (equivalent to 2×10²cells) in the qPCR. Detection was successful from soils inoculated with as little as 1×10³CFU/g soil. DNA isolated from naturally infested soils and diseased lettuce roots was amplified and sequenced fragments were identical or nearly identical to 16S rDNA sequences from S.suberifaciens. In growth chamber experiments, there was a positive correlation between disease severity and S.suberifaciens population levels in roots and soil, as detected by qPCR. Detection levels were below population levels of the pathogen necessary for disease development.