Fungi are the most important phytopathogens that cause yield losses. The mycotoxins released by fungi cause spoilage of stored food consumed by humans and feed supplied to animals. Fungi-antagonistic microbes are gaining attention as potential biocontrol agents (BCAs). This study was designed to isolate bacterial isolates from different crops and evaluate their in vitro antifungal assay against three phytopathogens, plant growth promoting (PGP) characteristics, molecular identification, and in vivo efficiency against the most devastating phytopathogenic fungus Fusarium oxysporum Schltdl. In the in vitro experiment, the 3 isolates BA, GL-1, and 5a out of 360 isolates showed more than 60% inhibitory activity against the selected fungi in this study. On the basis of 16S rRNA sequencing and phylogenetic analysis, BA isolate was identified as Bacillus velezensis. All three isolates produced indole acetic acid (IAA), hydrogen cyanide (HCN), and cellulase enzymes, while the BA and GL-1 isolates also produced siderophores and the BA isolate also produced ammonia. BA was selected on basis of not only Biocontrol efficacy but also maximum PGPR activity compared to GL-1 and 5a. In vivo assay, the isolate BA showed a significant decrease in disease severity caused by Fusarium oxysporum by 64.97% after 100 days of inoculation on wheat (FD-08) seedlings in a greenhouse assay and enhanced the shoot root height, fresh and dry mass. The wide-ranging antagonistic action of Bacillus velezensis isolated from the phyllosphere of wheat crops showed promising fungicidal and plant growth-promoting capabilities, suggesting it can be used as a biofungicide.
Background
The ‘Microphenotron’ is an automated screening platform that uses 96-well microtiter plates to test the response of seedlings to natural products. This system allows monitoring the phenotypic effect of a large number of small molecules. Here, this model system was used to study the effect of phytohormones produced by plant growth-promoting rhizobacteria (PGPR) on the growth of wild-type and mutant lines of Arabidopsis thaliana.
Methods
In the present study, high-throughput screening based on ‘Microphenotron’ was used to screen PGPRs. Rhizobacteria were isolated from the rhizosphere of Acacia Arabica, which was growing in saline habitats. The phylogeny of these rhizobacteria was determined by 16S rRNA gene sequencing. Strains were screened for plant growth-promoting traits such as auxin production, 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, and phosphate solubilization. Ultra-Performance Liquid Chromatography (UPLC) was used to detect the presence of different indolic compounds. Finally, PGPR were evaluated to enhance the growth of A. thaliana in the ‘Microphenotron’ system and pot trials.
Results
Selected rhizobacteria strains showed positive results for multiple plant-growth promoting traits. For instance, strain (S-6) of Bacillus endophyticus exhibited the highest ACC-deaminase activity. UPLC analysis indicated the presence of different indolic compounds in bacterial extracts that included indole lactic acid (ILA), indole carboxylic acid (ICA), and indole-3-acetic acid (IAA). Two strains (S-7 and S-11) of Psychrobacter alimentarius produced the most IAA, ICA and ILA. A screening bioassay through 96-well microtiter plates with wild-type Col. N6000 showed an increase in root growth and proliferation. The highest twofold increase was recorded in root growth with B. thuringiensis S-26 and B. thuringiensis S-50. In pot trials, mutant lines of A. thaliana impaired for auxin signaling showed that B. endophyticus S-6, Psy. alimenterius S-11, Enterobacter asburiae S-24 and B. thuringiensis S-26 used auxin signaling for plant growth promotion. Similarly, for ethylene insensitive mutant lines (ein2.5 and etr1), Prolinoborus fasciculus S-3, B. endophyticus S-6, Psy. alimenterius S-7, E. asburiae S-24, and B. thuringiensis S-26 showed the involvement of ethylene signaling. However, the growth promotion pattern for most of the strains indicated the involvement of other mechanisms in enhancing plant growth. The result of Microphenotron assays generally agreed with pot trials with mutant and wild type A. thaliana varieties. Bacterial strains that induced the highest growth response by these cultivars in the ‘Microphenotron’ promoted plant growth in pot trials. This suggests that Microphenotron can accelerate the evaluation of PGPR for agricultural applications.
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