Application of the enzyme-linked immunosorbent assay in the quality control of legume inoculants

Abstract

Samples of each batch of legume inoculant manufactured in South-Africa, are tested by the Plant Protection Research Institute for the number of viable Rhizobium and Bradyrhizobium cells per gram peat, strain identity and possible contamination before marketing. Possible replacement of plate counts and serological identification tests by a single indirect or DAS ELISA test, to assess the quality of legume inoculants, was investigated. Six of the nine strains of rhizobia investigated, are currently used for inoculant production. Somatic agglutination titres of most antisera, measured by tube agglutination not always reactivity in tests, lead ELISA were high. Use of these antisera did to effective ELISA systems, as low were obtained with some antisera with high somatic agglutination titres. Due to low reactivity of the four Rhizobium strains in ELISA, only Bradyrhizobium strains were used. Optimal concentrations of immunoreactants differed for each antigen-antibody combination tested in ELISA. Consequently, optimal ELISA concentrations had to be determined for each new batch of immunoglobulin, antigen and conjugate prepared. The plate count, as well as the indirect ELISA test, were conducted on inoculant suspensions containing each of two Bradyrhizobium strains. The indirect ELISA tests were negative, but centrifuged suspensions gave rise to weak, but inconsistant indirect ELISA reactions. Colour gradients were obtained in DAS ELISA with higher dilutions of inoculant suspension, containing each of three Bradyrhizobium strains. In the lowest dilutions tested, inhibition of the DAS ELISA reaction occurred. By allowing peat particles in the first dilution to settle for 3 h, the inhibition effect was markedly reduced. The lowest concentration of rhizobia significantly detected above the peat control differed for the three Bradyrhizobium strains and ranged from 6, 0 X 10 to 4,0 X 10 colony forming units per gram (cfu.g ) peat. Reproducible results were obtained with suspensions of each laboratory produced inoculant tested by the plate count technique and DAS ELISA. Statistical analysis indicated that the relationship between A 405 values and the number of (cfu.g) peat differed among packets tested.