Development of competitive-indirect ELISA for detection of nitrogen-fixing Klebsiella Spp. and Azospirillum spp.

Abstract

In order to detect, identify and quantitate nitrogen-fixing bacteria associated with rice root, antisera against Klebsiella strains R15, R17 and Azospirillum strain R25 were produced in rabbit yielding the agglutination titter of 1: 1,600, 1: 2,400 and 1: 3,200, respectively. These antisera were used successfully for the detection and identification of their corresponding antigens at the dilution of 1:100 by indirect fluorescent antibody technique (IND-FA). The environmental factors such as nutrients or high salinity could decrease the sensitivity of IND-FA. For the quantitation of associative diazotrophs, protocols of competitive-indirect-ELISA (COM-IND-ELISA) had been developed for each antiserum, providing the sensitivity of detecting homologous antigens at 1.95x106 cells.ml-1, with the accuracy shown by % recovery of 90-120. The precision of intra assay and inter assay were 3-10%CV and 3-15%CV, respectively. The specificity of both IND-FA and COM-IND-ELISA should be species specific because antisera against K. R15 and R17, which had been shown by chemotaxonomy to be very closely related strains, showed 96-98 % cross-reactivity with each other while showed 26-28% cross-reactivity with K. oxytoca NG13. In addition, antiserum of A. R25 showed 100% cross-reactivity only with A. lipòferum (FS and 34H), but not with other species of Azospirillum. Quantitation of R15, R17 and R25 inoculated in the rhizosphere of 8 rice cultivars by COM-IND-ELISA indicated that all 3 strains adhered to rice root within 1 h after inoculation and showed maximum colonization potential of 107-109 cells.g-1 root dry weight on day 12 after inoculation. The compatibility between rice and bacteria were also tested by measuring the net gain of colonization potential, N2-fixing potential, and plant vigor index comparing with uninoculated control plants. The plan t-bacterial interaction could be classified into 2 categories: 1) with net gain in colonization potential, N2-fixation and plant vigor index or root dry weight, the best pair is RD7-R15, and 2) with net gain in colonization potential and plant vigor index, the best pair is RD6-R25.