PCR as an ecological tool to determine the establishment and persistence of Rhizobium strains introduced into the field as seed inoculant Diane

Abstract

Appraisal of the establishment and persistence of inoculant strains, and the diversity of the rhizobial populations in 2 field experiments, required the precise characterisation of isolates from individual nodules. The polymerase chain reaction (PCR) method of generating randomly amplified polymorphic DNA (RAPD) from simple bacterial cell lysates was applied to nodule isolates obtained from field plots of clover (Trifolium spp.) and medic (Medicago spp.) plants inoculated with Rhizobium leguminosarum bv. Trifolium and Rhizobium meliloti. Comparison of the PCR method with gel immune diffusion serology was applied to selected nodule isolates obtained from the clover trial. Agreement between the methods was high (97·6%). Further characterisation of nodule isolates from the 2 field trials proceeded using PCR amplification profiles only, which allowed a large number of isolates to be quickly and precisely identified. Mean recovery of inoculant strains in the first season of the clover trial was 76·5% across 10 hosts, and 45·3% in the second season sampling. Recovery of inoculant strains in the medic trial was assessed only during the second season of growth, which recorded a mean recovery of 53% across 8 hosts. In addition to providing a rapid and reliable means of identifying inoculant strains of R. leguminosarum bv. Trifolium and R. meliloti directly in association with a range of different host plants, the PCR approach also allowed inoculant strain types to be readily identified when recovered as isolates from plots in which they had not been introduced. Analysis of the non-inoculant strains by PCR also indicated that the naturalised populations of rhizobia at both sites were highly diverse.