Survival strategies of Bacillus spp. in saline soils: Key factors to promote plant growth and health.

Soil salinity is one of the most important abiotic factors that affects agricultural production worldwide. Because of saline stress, plants face physiological changes that have negative impacts on the various stages of their development, so the employment of plant growth-promoting bacteria (PGPB) is one effective means to reduce such toxic effects. Bacteria of the Bacillus genus are excellent PGPB and have been extensively studied, but what traits makes them so extraordinary to adapt and survive under harsh situations? In this work we review the Bacillus' innate abilities to survive in saline stressful soils, such as the production osmoprotectant compounds, antioxidant enzymes, exopolysaccharides, and the modification of their membrane lipids. Other survival abilities are also discussed, such as sporulation or a reduced growth state under the scope of a functional interaction in the rhizosphere. Thus, the most recent evidence shows that these saline adaptive activities are important in plant-associated bacteria to potentially protect, direct and indirect plant growth-stimulating activities. Additionally, recent advances on the mechanisms used by Bacillus spp. to improve the growth of plants under saline stress are addressed, including genomic and transcriptomic explorations. Finally, characterization and selection of Bacillus strains with efficient survival strategies are key factors in ameliorating saline problems in agricultural production.

Enhancement of sulfur metabolism and antioxidant machinery confers Bacillus sp. Jrh14-10-induced alkaline stress tolerance in plant.

Alkaline stress is a major environmental challenge that restricts plant growth and agricultural productivity worldwide. Plant growth-promoting rhizobacteria (PGPR) can be used to effectively enhance plant abiotic stress in an environment-friendly manner. However, PGPR that can enhance alkalinity tolerance are not well-studied and the mechanisms by which they exert beneficial effects remain elusive. In this study, we isolated Jrh14-10 from the rhizosphere soil of halophyte Halerpestes cymbalaria (Pursh) Green and found that it can produce indole-3-acetic acid (IAA) and siderophore. By 16S rRNA gene sequencing, it was classified as Bacillus licheniformis. Inoculation Arabidopsis seedlings with Jrh14-10 significantly increased the total fresh weight (by 148.1%), primary root elongation (by 1121.7%), and lateral root number (by 108.8%) under alkaline stress. RNA-Seq analysis showed that 3389 genes were up-regulated by inoculation under alkaline stress and they were associated with sulfur metabolism, photosynthetic system, and oxidative stress response. Significantly, the levels of Cys and GSH were increased by 144.3% and 48.7%, respectively, in the inoculation group compared to the control under alkaline stress. Furthermore, Jrh14-10 markedly enhanced the activities of antioxidant enzymes, resulting in lower levels of O2•-, H2O2, and MDA as well as higher levels of Fv/Fm in alkaline-treated seedlings. In summary, Jrh14-10 can improve alkaline stress resistance in seedlings which was accompanied by an increase in sulfur metabolism-mediated GSH synthesis and antioxidant enzyme activities. These results provide a mechanistic understanding of the interactions between a beneficial bacterial strain and plants under alkaline stress.

[Identification of Clinical Isolates of the Bacillus cereus Group and Their Characterization by Mass Spectrometry and Electron Microscopy].

Bacillus cereus is a spore-forming bacterium found in the environment mainly in soil. Bacillus spores are known to be extremely resistant not only to environmental factors, but also to various sanitation regimes. This leads to spore contamination of toxin-producing strains in hospital and food equipment and, therefore, poses a great threat to human health. Two clinical isolates identified as B. cereus and B. cytotoxicus were used in the present work. It was shown that their calcium ion content was significantly lower than that of the reference strains. According to electron microscopy, one of the SRCC 19/16 isolates has an enlarged exosporium, and the SRCC 1208 isolate has large electron-dense inclusions of an unclear nature during sporulation. We can assume that these contain a biologically active component with a cytotoxic effect and possibly play a role in pathogenesis. Comparative chemical, biochemical, physiological, and ultrastructural analysis of spores of clinical isolates and reference strains of B. cereus was performed. The results we obtained deepen our understanding of the properties of spores that contribute to the increased pathogenicity of B. cereus group species.

Adaptive mechanisms of Bacillus to near space extreme environments.

Earth's near space is an extreme atmosphere environment with high levels of radiation, low atmospheric pressure and dramatic temperature fluctuations. The region is above the flight altitude of aircraft but below the orbit of satellites, which has special and Mars-like conditions for investigating the survival and evolution of life. Technical limitations including flight devices, payloads and technologies/methodologies hinder microbiological research in near space. In this study, we investigated microbial survival and adaptive strategies in near space using a scientific balloon fight mission and multi-omics analyses. Methods for sample preparation, storage, protector and vessel were optimized to prepare the exposed microbial samples. After 3 h 17 min of exposure at a float altitude of ~32 km, only Bacillus strains were alive with survival efficiencies of 0-10-6. Diverse mutants with significantly altered metabolites were generated, firstly proving that Earth's near space could be used as a new powerful microbial breeding platform. Multi-omics analyses of mutants revealed cascade changes at the genome, transcriptome and proteome levels. In response to environmental stresses, two mutants had similar proteome changes caused by different genomic mutations and mRNA expression levels. Metabolic network analysis combined with proteins' expression levels revealed that metabolic fluxes of EMP, PPP and purine synthesis-related pathways were significantly altered to increase/decrease inosine production. Further analysis showed that proteins related to translation, molecular chaperones, cell wall/membrane, sporulation, DNA replication/repair and anti-oxidation were significantly upregulated, enabling cells to efficiently repair DNA/protein damages and improve viability against environmental stress. Overall, these results revealed genetic and metabolic responses of Bacillus to the harsh conditions in near space, providing a research basis for bacterial adaptive mechanisms in extreme environments.

A toxin-mediated policing system in Bacillus optimizes division of labor via penalizing cheater-like nonproducers.

Division of labor, where subpopulations perform complementary tasks simultaneously within an assembly, characterizes major evolutionary transitions of cooperation in certain cases. Currently, the mechanism and significance of mediating the interaction between different cell types during the division of labor, remain largely unknown. Here, we investigated the molecular mechanism and ecological function of a policing system for optimizing the division of labor in Bacillus velezensis SQR9. During biofilm formation, cells differentiated into the extracellular matrix (ECM)-producers and cheater-like nonproducers. ECM-producers were also active in the biosynthesis of genomic island-governed toxic bacillunoic acids (BAs) and self-resistance; while the nonproducers were sensitive to this antibiotic and could be partially eliminated. Spo0A was identified to be the co-regulator for triggering both ECM production and BAs synthesis/immunity. Besides its well-known regulation of ECM secretion, Spo0A activates acetyl-CoA carboxylase to produce malonyl-CoA, which is essential for BAs biosynthesis, thereby stimulating BAs production and self-immunity. Finally, the policing system not only excluded ECM-nonproducing cheater-like individuals but also improved the production of other public goods such as protease and siderophore, consequently, enhancing the population stability and ecological fitness under stress conditions and in the rhizosphere. This study provides insights into our understanding of the maintenance and evolution of microbial cooperation.

Recent progress in the use of purple non-sulfur bacteria as probiotics in aquaculture.

The use of probiotics in aquaculture is widely recognized as an ecological and cost-effective approach to raising healthy, pathogen-tolerant aquatic animals, including fish and shrimp. In particular for shrimp, probiotics are viewed as a promising countermeasure to the recent severe damage to the shrimp industry by bacterial and viral pathogens. Purple non-sulfur bacteria (PNSB) are Gram-negative, non-pathogenic bacteria with wide application potential in agriculture, wastewater treatment, and bioenergy/biomaterials production. In aquaculture, lactic bacteria and Bacillus are the major probiotic bacteria used, but PNSB, like Rhodopseudomonas and Rhodobacter, are also used. In this review, we summarize the previous work on the use of PNSB in aquaculture, overview the previous studies on the stimulation of innate immunity of shrimp by various probiotic microorganisms, and also share our results in the probiotic performance of Rhodovulum sulfidophilum KKMI01, a marine PNSB, which showed a superior effect in promotion of growth and stimulation of immunity in shrimp at a quite low concentration of 1 × 103 cfu (colony forming unit)/ml in rearing water.

Crosstalk between in situ root exudates and rhizobacteria to promote rice growth by selenium nanomaterials.

Maximizing the potential of plant-microbe systems offers great opportunities to confront sustainability issues in agroecosystems. However, the dialog between root exudates and rhizobacteria remains largely unknown. As a novel nanofertilizer, nanomaterials (NMs) have significant potential to improve agricultural productivity due to their unique properties. Here, soil amendment with 0.1 mg·kg-1 selenium (Se) NMs (30-50 nm) significantly promoted rice seedling growth. Differences in root exudates and rhizobacteria were evident. At an earlier time point (3rd week), Se NMs increased the relative content of malic and citric acid by 15.4- and 8.1-fold, respectively. Meanwhile, the relative abundances of Streptomyces and Sphingomonas were increased by 164.6 % and 38.3 %, respectively. As the exposure time increased, succinic acid (40.5-fold) at the 4th week and salicylic acid (4.7-fold) and indole-3-acetic (7.0-fold) at the 5th week were enhanced, while Pseudomonas and Bacillus increased at the 4th (112.3 % and 50.2 %) and 5th weeks (190.8 % and 53.1 %), respectively. Further analysis indicated that (1) Se NMs directly enhanced the synthesis and secretion of malic and citric acids by upregulating their biosynthesis and transporter genes and then recruited Bacillus and Pseudomonas; (2) Se NMs upregulated the chemotaxis and flagellar genes of Sphingomonas for more interaction with rice plants, thereby promoting rice growth and stimulating root exudate secretion. This crosstalk of root exudates and rhizobacteria enhanced nutrient uptake, resulting in promoted rice growth. Our study offers insights into the crosstalk between root exudates and rhizobacteria by NMs and provides new insights into rhizosphere regulation in nano-enabled agriculture.

Identification of antarctic soil bacteria exhibiting antiproliferative activity against a colon cancer cell line / Identificación de bacterias de suelo antártico que presentan actividad antiproliferativa sobre una línea celular de cáncer de colon

SUMMARY: Cancer is the second leading cause of death in the world and colorectal cancer is the only cancer that has shown a sustained increase in mortality in the last decade. In the search for new chemotherapeutic agents against cancer, extremophilic microorganisms have shown to be a potential source to obtain molecules of natural origin and with selective cytotoxic action towards cancer cells. In this work we analyzed the ability of a collection of Antarctic soil bacteria, isolated on Collins Glacier from the rhizosphere of Deschampsia antarctica Desv plant, to secrete molecules capable of inhibiting cell proliferation of a colorectal cancer tumor line. Our results demonstrated that culture supernatants from the Antarctic bacteria K2I17 and MI12 decreased the viability of LoVo cells, a colorectal adenocarcinoma cell line. Phenotypic and genotypic characterization of the Antarctic bacteria showed that they were taxonomically related and nucleotide identity analysis based on the 16S rRNA gene sequence identified the bacterium K2I17 as a species belonging to the genus Bacillus.

El cáncer es la segunda causa de muerte en el mundo y el cáncer colorrectal es el único que presenta un aumento sostenido de la mortalidad en la última década. En la búsqueda de nuevos agentes quimioterapeúticos contra el cáncer, se ha propuesto a los microorganismos extremófilos como una fuente potencial para obtener moléculas de origen natural y con acción citotóxica selectiva hacia las células cancerígenas. En este trabajo analizamos la capacidad de una colección de bacterias de suelo antártico, aisladas en el glaciar Collins desde rizosfera de la planta de Deschampsia antarctica Desv, de secretar moléculas capaces de inhibir la proliferación celular de una línea tumoral de cáncer colorrectal. Nuestros resultados demostraron que los sobrenadantes de cultivo de las bacterias antárticas K2I17 y MI12 disminuyeron la viabilidad de la línea celular de adenocarcinoma colorrectal LoVo, en un ensayo de reducción metabólica de MTT. La caracterización fenotípica y genotípica de las bacterias antárticas, demostró que estaban relacionadas taxonómicamente y el análisis de la identidad nucleotídica en base a la secuencia del gen ARNr 16S identificó a la bacteria K2I17 como una especie perteneciente al género Bacillus.

Transcriptome investigation on the multicellular behavior of Bacillus velezensis Bs916 anchoring surfactin.

AIMS: To provide valuable information for a comprehensive understanding of the multicellular behavior of Bacillus velezensis Bs916 regulated by surfactin and other natural signals by Transcriptome. METHODS AND RESULTS: Transcriptomics revealed a distinct effect on gene expression alterations caused by disruption of the surfactin gene cluster(Δsrf) and 100 µg/ml surfactin addition(Δsrf + SRF). A total of 1573 differential expression genes were identified among Bs916, Δsrf, and Δsrf + SRF and grouped into eight categories based on their expression profiles. RT-qPCR analysis of 30 candidate genes showed high consistency with those of transcriptome. Additionally, the expression of eight candidate genes regulated by surfactin in a dose-dependent manner was revealed by lacZ fusion. Based on the above evidence, we proposed that surfactin can act as an extracellular signal for monitoring biofilm formation in Bs916 by directly regulating the expression of AbrB, DegS-degU, and SinI-SinR, and indirectly regulating the phosphorylation of ComA and Spo0A. CONCLUSIONS: The biofilm of Δsrf was unable to restore significantly by surfactin addition, combined inclusion of surfactin (SRF), exopolysaccharide (EPS), and γ-poly-dl-glutamic acid (γ-PGA), results in significant restoration of Δsrf biofilm formation, thereby a preliminary model was presented about the molecular mechanism by which the signaling molecule surfactin regulates Bs916 multicellular behavior.

Isolation, identification, and evaluation of the biocontrol potential of a Bacillus velezensis strain against tobacco root rot caused by Fusarium oxysporum.

AIMS: Tobacco (Nicotiana tabacum) is an economically important crop. Root rot caused by Fusarium oxysporum has become a damaging disease in N. tabacum crops grown in Henan province of China. Therefore, the objectives of this study were to screen bacterial isolates against F. oxysporum from rhizosphere soils of tobacco growing areas and to evaluate their antifungal activities, biocontrol effects, and effects on plant growth. METHODS AND RESULTS: Nineteen strains with antifungal inhibition effects of >60% against F. oxysporum were obtained using the method of flat confrontation; the strain Ba-0321 was the strongest, with an antifungal effect of 75%. Moreover, this strain had broad spectrum antimicrobial activity to eight additional tobacco pathogens. The strain was identified as Bacillus velezensis by morphology and the 16S rDNA sequence. The B. velezensis strain Ba-0321 had strong UV resistance as well as tolerance to high temperatures and low nutrition. The bacteria inhibited spore germination and mycelial growth of F. oxysporum under in vitro co-culture conditions. In vivo assays demonstrated that the Ba-0321 strain significantly reduced the pathogenicity of F. oxysporum, resulting in a control effect on tobacco root rot of 81.00%. Simultaneously, the bacteria significantly promoted root development and the growth of tobacco plants. CONCLUSION: Our results confirmed that the B. velezensis strain Ba-0321 has a strong antifungal effect and stress resistance that enable it to be used as a biological control agent for tobacco root rot caused by F. oxysporum. SIGNIFICANCE AND IMPACT OF THE STUDY: Tobacco root rot caused by F. oxysporum has become a damaging disease in China. The B. velezensis strain Ba-0321 has promising application value for controlling tobacco root rot diseases, and it could provide a new biocontrol agent against root rot caused by F. oxysporum in other plant species.

Daqu microbiota adaptability to altered temperature determines the formation of characteristic compounds.

Temperature plays a critical role in the performance of microbial communities during traditional solid-state fermentation. However, it remains unknown how temperature shapes microbiota, metabolism, and their relationship in Daqu fermentation. Here, we investigated the response of Daqu microbiota and metabolites to temperature by actual Daqu fermentation and simulated fermentation. First, volatile organic compounds were similar in both fermentation systems. Seventy-nine shared volatile compounds accounted for 94.5 %-96.5 % in Daqu fermentation and 66 %-95.6 % in the end of simulated fermentation, indicating that the formation of compounds in Daqu fermentation could be repeated effectively by simulated fermentation. The simulated fermentation showed the temperature gradient of 17 °C-60 °C significantly affected the formation and accumulation of volatile compounds. Aldehydes, acids, and pyrazines positively correlated with temperature (p < 0.05). Eight compounds were identified as characteristic compounds in high temperature (50-60 °C), including tetramethylpyrazine, trimethylpyrazine, 2,3-dimethyl-5-ethylpyrazine, 3-hydroxy-2-butanone, 2,3-dimethylpyrazine, benzaldehyde, acetic acid, and isovaleric acid. Next, we explored the force of temperature on microbial assembly and microbial interaction in simulated fermentation. Temperature significantly affected the composition of bacterial community (ANOISM, R = 0.779, P = 0.001) and fungi community (ANOISM, R = 0.664, P = 0.001). At the genus level, Weissella, Lactobacillus, Pediococcus, Saccharomycopsis Saccharomyces and Monascus dominated in 17-40 °C while Bacillus, Kroppenstedtia, Oceanobacillus, Lentibacillus, Rasamsonia, Thermoascus, Candida and Aspergillus were predominant genera in 50-60 °C. The succession of Bacillales, Lactobacillales, Eurotiales and Saccharomycetales adapted to changes in temperature. High temperature promoted microbial network complexity and a significant variation in microbial interactions. Furthermore, Procrustes analysis revealed a significant correlation between microbial community and volatile compounds (M2 = 0.6035, P < 0.001). Bacillus, Lentibacillus, Kroppenstedtia, and Oceanobacillus were significant contributors correlated to characteristic compounds. This study revealed the temperature-driven Daqu microbiota functioned as a critical contributor to promoting flavor formation and provided the theoretical basis for regulating fermentation in spontaneous fermentation systems.

Dietary supplementation of two indigenous Bacillus spp on the intestinal morphology, intestinal immune barrier and intestinal microbial diversity of Rhynchocypris lagowskii.

This study evaluated the effects of dietary administration of two indigenous Bacillus (A: basal control diet; B: 0.15 g/kg of Bacillus subtilis; C: 0.1 g/kg of Bacillus subtilis and 0.05 g/kg of Bacillus licheniformis; D: 0.05 g/kg of Bacillus subtilis and 0.1 g/kg of Bacillus licheniformis; E: 0.15 g/kg of Bacillus licheniformis) on the digestive enzyme activities, intestinal morphology, intestinal immune and barrier-related genes relative expression levels, and intestinal flora of Rhynchocypris lagowskii. The results showed that the fold height, lamina propria width, and muscle layer thickness of midgut and hindgut in group C were significantly higher than that of group A (P < 0.05). The activities of protease, amylase, and lipase in group C were significantly higher than those of group A (P < 0.05). The relative expression levels of IL-1ß and IL-8 in the intestine of group C were significantly downregulated, and the relative expression levels of IL-10 and TGF-ß were significantly upregulated (P < 0.05). The relative expression levels of Claudin-2 in group A significantly increased and the relative expression levels of Claudin-4 in group A significantly reduced compared with other groups (P < 0.05). The relative expression levels of ZO-1 in groups C and D were significantly higher than those of other groups (P < 0.05). The Bacillus in the intestine of group C has the highest relative abundance among all groups. Overall, it can generally be concluded that dietary supplementation of indigenous Bacillus subtilis and Bacillus licheniformis (group C) can improve the intestinal morphology, digestion, and absorption enzyme activities, enhance intestinal mucosal immunity and barrier function, and maintain the intestinal microbial balance of R. lagowskii.

Maize growth response to different Bacillus strains isolated from a salt-marshland area under salinity stress.

Maize (Zea mays) growth performance has been hindered due to the high soil salinity. Salinity is one of the most severe abiotic stresses that has led to growth imbalance and profitability of harvests in arid and semi-arid regions. Plants have taken advantage of salt-tolerant bacteria as plant growth-promoters to enhance growth and reduce the adverse effects of salinity through the regulation of some biochemical, physiological, and molecular features. Preferences for non-chemical, eco-friendly, and economical approaches have caused the inquiry of the Bacillus genus as a joint group of plant growth-promoting rhizobacteria known to alleviate salt-stress impacts. In the present study, halotolerant Bacillus strains were isolated from salt-marshland soil and characterized for their physiological, molecular, and biochemical properties. Twenty-four bacterial isolates collected from high saline fields of salt marshland were analyzed by MALDI-TOF MS proteome analysis, which confirmed the taxonomic affiliation with Bacillus cereus, Bacillus subtilis, Bacillus atrophaeus, and Bacillus thorngiensis. Applying the isolates on maize plants as bio-inoculant bacteria obviously increased the growth parameters (P < 0.01). Pot experiments showed that isolates 74 and 90 were the most prominent strains to minimize the harmful effects of salinity. Its effects are heightening the potassium/sodium ratio and K-Na selectivity in shoots and roots measured by flame atomic absorption photometry (AAS). Accordingly, Bacillus cereus isolate 74 showed a maximum increase in dry weights of the shoot (133.89%), root (237.08%), length of the shoot (125%), and root (119.44%) compared to the control condition. Our findings suggest that bacteria isolated from marshland may be an economical and simple means to increase plant growth and resistance to high salinity soil conditions.

Maize Root Exudates Recruit Bacillus amyloliquefaciens OR2-30 to Inhibit Fusarium graminearum Infection.

Bacillus spp. can exert plant growth-promoting effects and biocontrol effects after effective colonization, and bacterial chemotaxis toward plant root exudates is the initial step to colonize. Under biotic stress, plants are able to alter their root exudates to attract or avoid different types of microbes. Hence, Bacillus chemotaxis toward root exudates after pathogen infection is crucial for exerting their beneficial effects. In this study, the Bacillus amyloliquefaciens OR2-30 strain, which exhibited greater chemotaxis ability toward maize root exudates after Fusarium graminearum infection, was screened from 156 rhizosphere microorganisms. The infected maize root exudates were further confirmed to improve the swarming and biofilm formation ability of the OR2-30 strain. Chemotaxis, swarming, and biofilm formation ability were able to influence bacterial colonization. Indeed, the the OR2-30 strain displayed more effective colonization ability in the maize rhizosphere after F. graminearum inoculation. Moreover, lipopeptides produced by OR2-30 were identified as iturins and responsible for suppressing F. graminearum growth. Further study showed that lipopeptides suppressed the growth of F. graminearum by inhibiting conidia formation and germination, inducing reactive oxygen species production and causing cell death in mycelium. Eventually, the OR2-30 strain increased maize resistance against F. graminearum. These results suggested that maize root exudates could recruit B. amyloliquefacines OR2-30 after F. graminearum infection, and that OR2-30 then suppresses the F. graminearum by producing lipopeptides, such as iturins, to protect maize.

Bacillus altitudinis HNH7 and Bacillus velezensis HNH9 promote plant growth through upregulation of growth-promoting genes in upland cotton.

AIMS: The potential of endophytic Bacillus strains to improve plant growth and yield was evaluated. METHODS AND RESULTS: Endophytic Bacillus altitudinis HNH7 and Bacillus velezensis HNH9 were evaluated for their growth-promoting traits. In an in vitro plate assay, HNH7 and HNH9 exhibited proteolytic, amylolytic, lipolytic and cellulolytic activity. HNH7 and HNH9 were able to solubilize iron by producing siderophores but were unable to solubilize insoluble phosphate. PCR confirmed the presence of four growth-promoting genes viz. pvd, budA, asbA and satA in the genome of HNH7, while HNH9 also possessed the same genes except for budA. In a greenhouse experiment, HNH7 and HNH9 promoted the growth of upland cotton plants by upregulating the expression of growth-linked genes, EXP6, ARF1, ARF18, IAA9, CKX6 and GID1b. However, the expression of genes involved in ethylene biosynthesis, that is ERF and ERF17 was downregulated after treating the plants with HNH7 and HNH9 compared to the control. Furthermore, cotton plants treated with HNH7 and HNH9 exhibited a significantly higher rate of photosynthesis and stomatal conductance. CONCLUSION: HNH7 and HNH9 showed a promising potential to promote the growth of cotton plants. SIGNIFICANCE AND IMPACT OF STUDY: Research on plant growth-promoting Bacillus strains can lead to the formation of biofertilizers.

Biocontrol of Phyllosticta citricarpa by Bacillus spp.: biological and chemical aspects of the microbial interaction.

Citrus fruits are the most produced fruits in the world, but they are threatened by several pathogens, including the fungus Phyllosticta citricarpa, the causal agent of citrus black spot (CBS). The fungus affects most citrus species and the infection results in economic losses in citrus-producing areas. This disease causes the aesthetic depreciation of fresh fruit, impairing its commercialization. As an alternative to the use of synthetic fungicides to control the pathogen, the biological control, using bacteria of the genus Bacillus, is highlighted. Such microorganisms enable biocontrol by the production of volatile organic compounds (VOC) or non-volatile. Therefore, this work aimed to evaluate the production of VOC by isolates of Bacillus spp. grown in different culture media; to evaluate the effects of these compounds on the evolution of CBS lesions in orange fruits; to study the effects of VOC on resistance induction in orange fruits; to evaluate the effects of VOC on P. citricarpa morphology in CBS lesions, and to identify the produced VOC. Tryptone soya agar (TSA) and tryptone soya broth (TSB) media used to culture the bacterium resulted in up to 73% pathogen inhibition by VOC. Volatile compounds from Bacillus spp. ACB-65 and Bacillus spp. ACB-73 when cultured in TSB culture medium provided 86% inhibition of freckles that evolved to hard spots. The volatile fractions produced by the bacteria were identified as alcohols, ketones, amines, ethers, aldehydes and carboxylic acids that can serve as arsenal against the phytopathogen. The present work demonstrated the potential of VOC produced by Bacillus spp. in the control of P. citricarpa.

Physiological, proteomic, and metabolomic analysis provide insights into Bacillus sp.-mediated salt tolerance in wheat.

KEY MESSAGE: Herein, the inoculation with strain wp-6 promoted the growth of wheat seedlings by improving the energy production and conversion of wheat seedlings and alleviating salt stress. Soil salinization decreases crop productivity due to high toxicity of sodium ions to plants. Plant growth-promoting rhizobacteria (PGPR) have been demonstrated to alleviate salinity stress. However, the mechanism of PGPR in improving plant salt tolerance remains unclear. In this study, physiological analysis, proteomics, and metabolomics were applied to investigate the changes in wheat seedlings under salt stress (150 mM NaCl), both with and without plant root inoculation with wp-6 (Bacillus sp.). Under salt stress, root inoculation with strain wp-6 increased plant biomass (57%) and root length (25%). The Na+ content was reduced, while the K+ content and K+/Na+ ratio were increased. The content of malondialdehyde was decreased by 31.94% after inoculation of wp-6 under salt stress, while the content of proline, soluble sugar, and soluble protein were increased by 7.48%, 12.34%, and 4.12%, respectively. The peroxidase, catalase, and superoxide dismutase activities were increased after inoculation of wp-6 under salt stress. Galactose metabolism, phenylalanine metabolism, caffeine metabolism, ubiquinone and other terpenoid-quinone biosynthesis, and glutathione metabolism might play an important role in promoting the growth of salt-stressed wheat seedlings after the inoculation with wp-6. Interaction analysis of differentially expressed proteins and metabolites found that energy production and transformation-related proteins and six metabolites (D-arginine, palmitoleic acid, chlorophyllide b, rutin, pheophorbide a, and vanillylamine) were mainly involved in the growth of wheat seedlings after the inoculation with wp-6 under salt stress. Furthermore, correlation analysis found that inoculation with wp-6 promotes the growth of salt-stressed wheat seedlings mainly through regulating amino acid metabolism and porphyrin and chlorophyll metabolism. This study provides an eco-friendly method to increase agricultural productivity and paves a way to sustainable agriculture.

Bacillus toyonensis SAU-19 Ameliorates Hepatic Insulin Resistance in High-Fat Diet/Streptozocin-Induced Diabetic Mice.

Insulin resistance (IR) is a hallmark of type 2 diabetes mellitus (T2DM). This study was performed to investigate the antidiabetic effect of Bacillus toyonensis SAU-19 and its possible mechanisms of action in mice with type 2 diabetes mellitus (T2DM). Thirty SPFKM mice were randomly assigned to three groups: control, diabetic model, and diabetes + Bacillus toyonensis SAU-19 group. After 35 days, blood was collected for biochemical analysis and liver tissue samples for histopathological analysis using H&E staining, qPCR, and ELISA. The results showed that the administration of B. toyonensis SAU-19 significantly improved the blood glucose, hepatic insulin resistance, and morphological changes of the liver characterized by significant improvement of dyslipidemia, glycogen synthesis, and antioxidant status (p < 0.05), indicating the strains' ameliorating effects on hepatic insulin resistance in T2DM. In conclusion, the probiotic strain (B. toyonensis SAU-19) inhibits T2DM by reducing insulin resistance, improving antioxidant status, and downregulating genes related to glucose synthesis; hence, it may be used in treating diabetes and other metabolic disorders. This study provides the basis for further studies into the molecular mechanisms of B. toyonensis SAU-19 in treating T2DM.

Bacillus tequilensis strain 'UPMRB9' improves biochemical attributes and nutrient accumulation in different rice varieties under salinity stress.

Soil salinity exert negative impacts on agricultural production and regarded as a crucial issue in global wetland rice production (Oryza sativa L.). Indigenous salt-tolerant plant growth-promoting rhizobacteria (Bacillus sp.) could be used for improving rice productivity under salinity stress. This study screened potential salt-tolerant plant growth-promoting rhizobacteria (PGPR) collected from coastal salt-affected rice cultivation areas under laboratory and glasshouse conditions. Furthermore, the impacts of these PGPRs were tested on biochemical attributes and nutrient contents in various rice varieties under salt stress. The two most promising PGPR strains, i.e., 'UPMRB9' (Bacillus tequilensis 10b) and 'UPMRE6' (Bacillus aryabhattai B8W22) were selected for glasshouse trial. Results indicated that 'UPMRB9' improved osmoprotectant properties, i.e., proline and total soluble sugar (TSS), antioxidant enzymes like superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT). Moreover, 'UPMRB9' inoculated rice plants accumulated higher amount of nitrogen and calcium in tissues. Therefore, the indigenous salt-tolerant PGPR strain 'UPMRB9' could be used as a potential bio-augmentor for improving biochemical attributes and nutrient uptake in rice plants under salinity stress. This study could serve as a preliminary basis for future large-scale trials under glasshouse and field conditions.

Distribution, Genetic Diversity and Biocontrol of Aflatoxigenic Aspergillus flavus in Serbian Maize Fields.

Maize is one of the leading export products in the Republic of Serbia. As a country where economic development depends on agriculture, maize production plays a critical role as a crop of strategic importance. Potential aflatoxin contamination of maize poses a risk to food and feed safety and tremendous economic losses. No aflatoxin contamination of maize samples harvested in 2019 and 2020 in different localities in the Republic of Serbia was detected by the Enzyme-Linked Immunosorbent Assay (ELISA) test and High-Performance Liquid Chromatography (HPLC) method. On the other hand, the Cluster Amplification Patterns (CAP) analyses of the isolated Aspergillus flavus strains from 2019 maize samples confirmed the presence of key biosynthesis genes responsible for aflatoxin production. Artificial inoculation and subsequent HPLC analysis of the inoculated maize samples confirmed the high capacity of the A. flavus strains for aflatoxin production, pointing to a high risk of contamination under favorable conditions. Prevention of aflatoxin contamination is primarily based on A. flavus control, where biocontrol agents play a significant role as sustainable disease management tools. In this study, antagonistic activity screening of the novel strains belonging to the Bacillus genus indicated superior suppression of A. flavus strains by two Bacillus strains isolated from the rhizosphere of Phaseolus vulgaris.