Field monitoring of plant-growth-promoting rhizobacteria by colony immunoblotting

Abstract

Inoculant plant-growth-promoting bacteria are emerging as an important component of sustainable agriculture. There is a need to develop inexpensive methods for enumerating these organisms after their application in the field, to better understand their survival and impacts on yields. Immunoblotting is one potential method to measure viable cells, but the high cost of the conventionally used nylon membranes makes this method prohibitive. In this study, less expensive alternative materials such as filter papers, glossy photo papers, and transparencies for the purpose of colony immunoblotting were evaluated and the best substance was chosen for further studies. Whatman filter paper No. 541 combined with a 0.01 mol.L⁻¹ H₂2SO₄ rinsing step gave similar results to nylon membranes but <20% of the overall cost of the original colony immunoblotting assay. The application of the modified immunoblot method was tested on nonsterile clay soil samples that were spiked with high numbers (>10⁷ CFU.g⁻¹) of the plant-growth-promoting bacteria 'Pseudomonas fluorescens', 'Azospirillum brasilense', or 'Rhizobium leguminosarum'. The modified protocol allowed the identification and recovery of over 50% of the inoculated cells of all three strains, amidst a background of the native soil microflora. Subsequently, the survival of 'P. fluorescens' was successfully monitored for several months after application to field-grown rice at Jerilderie, New South Wales, Australia, thus validating the procedure.