A comparison of the fluorescent ELISA and antibiotic resistance identification techniques for use in ecological experiments with Rhizobium trifolii

Abstract

The fluorescent ELISA technique for the identification of bacteria was compared with antibiotic resistant mutants as marker systems for use with Rhizobium trifolii in root nodules and in soil. With an effective(CP3B) and an ineffective (R4) strain as a mixed 1:1 inoculum, there was a highly significant correlation (P < 0.001) between the two techniques when the plants were grown at pH 5.5 when the majority of nodules were inhabited by the effective strain. At pH 6.5, where the ineffective strain predominated in the nodules, there was no correlation. The reason was that 85% of R4 nodules had volumes less than 0.1 mm3 with bacterial numbers obviously below the necessary threshold for detection using the serological method. Both methods were efficient at enumerating rhizobia from soils although the recovery rate from a brown earth soil was significantly higher than from a peat soil. Fluorescent ELISA was able to detect rhizobia at 8.0 times 105 cells/ml soil suspension (1 g soil to 10 ml water) in the brown earth soil and at 2.0 times 105 cells/ml in the peat soil. The results are discussed in terms of the limitations of both techniques in ecological studies.

Stability of rifampicin resistance as a marker for root colonization studies of Pseudomonas putida in the field

“…During 41 months in the field, there was at least a 95% correlation between these two methods. Similar results were obtained by Renwick and Jones (1985), who reported a significant correlation between a fluorescent ELISA procedure and a combined streptomycin and spectinomycin resistance as methods to enumerate R. trifolii in root nodules and in soil.…”

Development of molecular methods for the detection of specific bacteria in the environment

“…Fluorochromes, such as fluorescein isothiocyanate, can be coupled to an antibody that binds directly with a target antigen on the cell or to a second anti body that recognizes an antibody produced against the micro-organism (for example, see Chantler & McIllmurray, 1988). Immunofluorescence detection has been used for various bacteria that are difficult to culture, for example methanogenic bacteria (Conway de Macario et al, 1982), methane oxidizers (Reed & Duggan, 1978), N ,-fixing organisms (Renwick & Gareth, 1985), nitrifiers in soil and marine habitats (Belser & Schmidt, 1978;Ward & Carlucci, 1985) and acidtolerant Thiobacillus ferrooxidans (Ape1 et ai., 1976). Successful application of fluorescent anti bodies can be affected by a range of factors, including specificity and cross-reactivity, autofluoresence (particularly when algae are the target micro-organisms), nonspecific staining, expression of the antigen-coding genes, stability of the antigen under environmental conditions, and the inability of the technique to distinguish viable and nonviable cells and be quantitative (Schmidt, 1974;Ford & Olsen, 1988).…”

The manipulation of white clover ‘host preference’ for strains of Rhizobium trifolii in an upland soil

“…This field experiment provided an excellent opportunity for further critical assessment of the fluorescent ELISA technique and also for the comparison of wild-type strains and their respective mutants in parallel studies. As shown by Renwick & Jones (1985), the main limiting factor of the fluorescent ELISA technique was its inability to identify the occupants of very small nodules (thus giving a 'blank' reaction). This is most important in experiments where the nodules are very small as with white clover.…”