Abstract
A simple and reliable method for the detection of specific nitrogen-fixing bacteria in both free-living and bacteroid forms is essential for the development and application of biofertilizer. Traditionally, a polyclonal antibody generated from an immunized rabbit was used for detection. However, the disadvantages of using a polyclonal antibody include limited supply and cross-reactivity to related bacterial strains. This is the first report on the application of phage display technology for the generation of a rabbit recombinant monoclonal antibody for specific detection and monitoring of nitrogen-fixing bacteria in both free-living form and in plant nodules. Bradyrhizobium sp. DOA9, a broad host range soil bacteria, originally isolated from the root nodules of Aeschynomene americana in Thailand was used as a model in this study. A recombinant single-chain fragment variable (scFv) antibody library was constructed from the spleen of a rabbit immunized with DOA9. After three rounds of biopanning, one specific phage-displayed scFv antibody, designated bDOA9rb8, was identified. Specific binding of this antibody was confirmed by phage enzyme-linked immunosorbent assay (phage ELISA). The phage antibody could bind specifically to DOA9 in both free-living cells (pure culture) and bacteroids inside plant nodules. In addition to phage ELISA, specific and robust immunofluorescence staining of both free-living and bacteroid forms could also be observed by confocal-immunofluorescence imaging, without cross-reactivity with other tested bradyrhizobial strains. Moreover, specific binding of free scFv to DOA9 was also demonstrated by ELISA. This recombinant antibody can also be used for the study of the molecular mechanism of plant–microbe interactions in the future.
Highlights
Bradyrhizobium is one of the soil bacteria that can fix nitrogen in symbiosis with specific legumes and convert nitrogen gas into ammonia to be used as fertilizer in plants
Bradyrhizobium strain DOA9 and other bradyrhizobia were isolated from A. americana in our laboratory [1], while the reference strains of B. diazoefficiens USDA110 and Bacillus subtilis 168 were obtained from the School of Biotechnology, SUT
In each round of affinity selection, 192 phage clones were randomly picked and amplified to confirm the binding to DOA9 by phage enzyme-linked immunosorbent assay (ELISA)
Summary
Bradyrhizobium is one of the soil bacteria that can fix nitrogen in symbiosis with specific legumes and convert nitrogen gas into ammonia to be used as fertilizer in plants. DOA9 was originally isolated from the root nodules of Aeschynomene americana in Thailand. This strain was classified as B. yuanmingense based on multilocus DNA sequence analysis of 16S rRNA and housekeeping genes (dnaK, recA, and glnB) [1]. DOA9 is a divergent nod-containing strain that can fix nitrogen under free-living conditions. This strain was able to colonize and efficiently nodulate the roots of most plants ranging from the Dalbergioid, Millettioid, and Robinioid tribes [2]. Due to its broad host range ability, DOA9 is an interesting strain to be further developed as a multi-purpose inoculant as well as to explore its mechanism of interaction with plants at the molecular level
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