DETECTION OF RHIZOBIUM-MELILOTI CELLS IN-FIELD SOIL AND NODULES BY POLYMERASE CHAIN-REACTION

A genetically marked Rhizobium meliloti strain, R692, was prepared by insertion of a 1.7-kb DNA segment from Tn903 between the nifHDK and fi xABC genes in the nod megaplasmid. This DNA was used as a marker, dete ctable by polymerase chain reaction (PCR), for the specific identifica tion of bacteria in soil samples and alfalfa nodules. This detection t echnique was tested by applying different titres of the marked strain to field plots seeded with alfalfa. Samples of soil and nodules were a ssayed for the presence of the marker DNA fragment by PCR using primer s specific to the marker sequence. The experiments revealed that the b acteria could be detected directly in soil containing about 10(3)-10(4 ) bacteria/g, but greater sensitivity was prevented by potent PCR inhi bitors present in the samples. The titre of the bacteria in the soil d ecreased rapidly after inoculation, dropping about 10-fold per week. T ests of vertical location of the bacteria in soil cores showed that th e bacteria were initially dispersed to a depth of 18 cm, and subsequen tly retained viability in the top 2-8 cm. As few as 10 marked R. melil oti per gram of soil resulted in its establishment at detectable level s in nodules. Application of about 10(4)-10(5) bacteria/g soil was suf ficient to give the maximum number of nodules per plant and resulted i n 70-90% occupancy by the marked strain. Limited movement of the inocu lant was detected by analysis of nodules from plants adjacent to the s ites where the bacteria were applied, probably by movement in water. T he experiments demonstrated the advantages of PCR for the monitoring o f marked microorganisms in the environment.