Effect of oxygen on the germination and culturability of Bacillus atrophaeus spores

The effect of oxygen on the germination and culturability of aerobic Bacillus atrophaeus spores was investigated in this study. Under oxic or anoxic conditions, various nutritional and non-nutritional germinants were utilized to induce germination. Tb3+-dipicolinic acid fluorescence assay and phase-contrast microscopy were used to track the germination process. The final germination level, germination half time, and germination speed were used to define germination kinetics. Colony-forming unit enumeration was used to assess the culturability of germinated spores germinated with or without oxygen. The results show that in the absence of oxygen, the final germination level was unaffected, germination half time decreased by up to 35.0%, germination speed increased by up to 27.4%, and culturability decreased by up to 95.1%. It is suggested that oxygen affects some germinant receptor-dependent germination pathways, implying that biomolecules engaged in these pathways may be oxygen-sensitive. Furthermore, spores that have completed the germination process in either anoxic or oxic conditions may have different culturability. This research contributed to a better understanding of the fundamental mechanism of germination.

Determination of the Antagonistic Effects of Some Rhizospheric Bacteria against Macrophomina phaseolina under In Vitro Conditions

Macrophomina phaseolina (Tassi) Goid. is a fungal pathogen causes charcoal rot disease (Sin: Rhizoctonia bataticola) and is responsible for significant yield losses in many plants. In our study, we aimed to evaluate the antagonistic ability of 39 different bacteria, isolated from the fields of sugar beet in 2019, against the pathogen Macrophomina phaseolina isolated from sugar beet, beans and chickpeas. Approximately 31% of the bacteria showed antibiosis effect against the pathogen. It was determined that the effectiveness level of Lelliottia amnigena, Bacillus atrophaeus, B.pumilus and B. cereus (7 isolates) was moderate to high against Macrophomina phaseolina. Bacillus atrophaeus (PTo15-1a) showed the highest efficacy of 80%, 72.94% and 82.35% against Macrophomina phaseolina of chickpea, bean and sugar beet respectively. Lelliottia amnigena (Pto 14-1b) was moderately effective (57.78%) against the chickpea isolate of the pathogen. It was observed that of the seven Bacillus cereus isolates used in the experiment, three isolates (Pto14-1a, Pto12-1b, Pto17-1b) were highly effective against the chickpea pathogen, two (Pto12-1b, Pto14-2b) against bean pathogen, and one (Pto15-1b) against sugar beet isolate. Results have shown varied level of antagonism by different test bacterial against different Macrophomina phaseolina isolates, while the highest level of antibiosis shown by Bacillus atrophaeus against all pathogenic isolates indicated that it can be a potential future bioagent in managing the disease.

Novel Genetic Dysregulations and Oxidative Damage in Fusarium graminearum Induced by Plant Defense Eliciting Psychrophilic Bacillus atrophaeus TS1

This study elaborates inter-kingdom signaling mechanisms, presenting a sustainable and eco-friendly approach to combat biotic as well as abiotic stress in wheat. Fusarium graminearum is a devastating pathogen causing head and seedling blight in wheat, leading to huge yield and economic losses. Psychrophilic Bacillus atrophaeus strain TS1 was found as a potential biocontrol agent for suppression of F. graminearum under low temperature by carrying out extensive biochemical and molecular studies in comparison with a temperate biocontrol model strain Bacillus amyloliquefaciens FZB42 at 15 and 25 °C. TS1 was able to produce hydrolytic extracellular enzymes as well as antimicrobial lipopeptides, i.e., surfactin, bacillomycin, and fengycin, efficiently at low temperatures. The Bacillus strain-induced oxidative cellular damage, ultrastructural deformities, and novel genetic dysregulations in the fungal pathogen as the bacterial treatment at low temperature were able to downregulate the expression of newly predicted novel fungal genes potentially belonging to necrosis inducing protein families (fgHCE and fgNPP1). The wheat pot experiments conducted at 15 and 25 °C revealed the potential of TS1 to elicit sudden induction of plant defense, namely, H2O2 and callose enhanced activity of plant defense-related enzymes and induced over-expression of defense-related genes which accumulatively lead to the suppression of F. graminearum and decreased diseased leaf area.

Efficient Preparation of Chitooligosaccharide With a Potential Chitosanase Csn-SH and Its Application for Fungi Disease Protection

Chitosanase plays a vital role in bioactive chitooligosaccharide preparation. Here, we characterized and prepared a potential GH46 family chitosanase from Bacillus atrophaeus BSS. The purified recombinant enzyme Csn-SH showed a molecular weight of 27.0 kDa. Csn-SH displayed maximal activity toward chitosan at pH 5.0 and 45°C. Thin-layer chromatography and electrospray ionization–mass spectrometry indicated that Csn-SH mainly hydrolyzed chitosan into (GlcN)2, (GlcN)3, and (GlcN)4 with an endo-type cleavage pattern. Molecular docking analysis demonstrated that Csn-SH cleaved the glycoside bonds between subsites −2 and + 1 of (GlcN)6. Importantly, the chitosan hydrolysis rate of Csn-SH reached 80.57% within 40 min, which could reduce time and water consumption. The hydrolysates prepared with Csn-SH exhibited a good antifungal activity against Magnaporthe oryzae and Colletotrichum higginsianum. The above results suggested that Csn-SH could be used to produce active chitooligosaccharides efficiently that are biocontrol agents applicable for safe and sustainable agricultural production.

Isolation and identification of the bacterium producing antitumor and antimicrobial compounds derived from Iranian swamp frog (Rana ridibunda) skin

Background and Objectives: Cancer incidence and recurrence, antibiotic resistance, and overuse of antibiotics have become a global concern. The purpose of this study was to identify and isolate bacteria from the skin of the Rana ridibunda, Iranian swamp frog, which has produced antimicrobial compounds, and investigate its cytotoxic activity on the breast (MCF7) and glioblastoma (U87) cancer cell line. Materials and Methods: An antibiotic-producing bacterium was isolated from the frog skin. The bacterium was identified based on 16S rDNA sequencing and biochemical and morphological characteristics. Antimicrobial activity of the culture supernatant was examined by disc diffusion and MIC methods. Cytoplasmic and cell wall extracts of bacteria were prepared by sonication. SDS-PAGE was then used to examine protein contents of them. The cancer cell lines were treated with cytoplasmic and cell wall extracts at different concentrations. The effects of cytotoxicity were assessed by MTT assay at 24 and 48 h intervals. Finally, the results were analyzed by SPSS. Results: The isolated bacterium was identified as a new strain of Bacillus atrophaeus. MIC and disc diffusion methods showed that the Bacillus atrophaeus antimicrobial activity was broad spectrum. MTT assay showed IC50 values 30 μg/ml and 20 μg/ml for U87 and MCF7 cells after 24-48 h exposure, respectively. Conclusion: The cytoplasmic extracts of Bacillus atrophaeus has anticancer potential and can be used as an alternative or complementary candidate in the treatment of cancer. Further in vivo and in vitro mechanistic studies are suggested to confirm the biological activities.