No 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.

Abilities of rhizobacteria to promote mycorrhization of arbuscular mycorrhiza (AM) on maize roots have been documented. In this study, the rhizospheric bacterium, Brevibacillus sp. SUT47 was found to significantly promote spore number and root colonization of Acaulospora tuberculata. To understand how maize roots respond to SUT47, a comparative proteomics analysis was performed. We found that at 30 days after inoculation (dai), the proteins involved in plant defense mechanism and Reactive Oxygen Species (ROS)-scavenging enzymes were the main proteins altered in tested maize roots. Levels of salicylic acid, hydrogen peroxide, and the activity of superoxide dismutase were significantly decreased in AM+SUT47 roots at 7 dai, while the activities of peroxidase and ascorbate peroxidase increased especially in AM+SUT47 roots at 30 dai. Thus, this work showed the alteration of some plant defense-related compounds and antioxidative enzyme activities that are associated with an enhancing maize root colonization by AM when co-inoculated with SUT47.

The development of rhizobial inoculants with increased resistance to abiotic stress is critical to mitigating the challenges related to climate change. This study aims at developing a soybean stress-tolerant Bradyrhizobium inoculant to be used under the mixed stress conditions of acidity, high temperature, and drought. Six isolates of Bradyrhizobium with high symbiotic performance on soybean were tested to determine their growth or survival abilities under in vitro conditions. The representative stress-tolerant Bradyrhizobium isolates 184, 188, and 194 were selected to test their ability to promote soybean growth under stress conditions compared to the type strain Bradyrhizobium diazoefficiens USDA110. The plant experiment indicated that isolate 194 performed better in symbiosis with soybean than other Bradyrhizobium strains under stress conditions. Based on the stress tolerance index, soybeans inoculated with isolate 194 showed a high growth performance and significantly better nodulation competition ability than USDA110 under several stress conditions. Interestingly, supplementation of sucrose in the culture medium significantly enhances the survival of the isolate and leads to improved plant biomass under various stress conditions. Analysis of the intra-cellular sugars of isolate 194 supplemented with sucrose showed the accumulation of compatible solutes, such as trehalose and glycerol, that may act as osmoprotectants. This study indicates that inoculation of stress-tolerant Bradyrhizobium together with sucrose supplementation in a medium could enhance bacterial survival and symbiosis efficiency under stress conditions. Although it can be applied for inoculant production, this strategy requires validation of its performance in field conditions before adopting this technology.

The substrate-based production system has been widely applied for farm arbuscular mycorrhizal fungi (AMF) inoculum production. Plant growth promoting rhizobacterium (PGPR), Brevibacillus sp. SUT47 has been discovered to promote AMF spore production when co-inoculating on maize roots. However, the application of whole cell bacteria may cause the adverse outcome with contamination of bacterial cells in AMF inoculum. In order to avoid the bacterial cell contamination, we attempted to develop a new methodology using the bacterial secretion compounds as an optional technique to promote AMF spore production in maize roots. Secretion compounds of strain SUT47 were concentrated by freeze-drying and co-inoculated with the spores of Acaulospora tuberculata on maize seedling. The results showed that the bacterial secretion compounds promoted AMF spore production. The highest number of spores was produced when 360 mg of concentrated secretion compounds were applied. In contrast, the significant reduction of spore number was found when the secretion compounds at the concentration higher than 360 mg were applied. These results indicate the production of AMF spore was responded to concentration of bacterial secretion compounds which possibly contain an effective substance to promote AMF spore propagation.