Antifungal efficacy of Bacillus amyloliquefaciens ZK-9 against Fusarium graminearum and analysis of the potential mechanism of its lipopeptides.

Fusarium graminearum is a destructive fungal pathogen that seriously threatens wheat production and quality. In the management of fungal infections, biological control is an environmentally friendly and sustainable approach. Here, the antagonistic strain ZK-9 with a broad antifungal activity was identified as Bacillus amyloliquefaciens. ZK-9 could produce extracellular enzymes such as pectinase, protease, cellulase, and amylase, as well as plant growth-promoting substances including IAA and siderophore. Lipopeptides extracted from strain ZK-9 had the high inhibitory effects on the mycelia of F. graminearum with the minimum inhibitory concentration (MIC) of 0.8 mg/mL. Investigation on the action mechanism of lipopeptides showed they could change the morphology of mycelia, damage the cell membrane, lower the content of ergosterol and increase the relative conductivity of membrane, cause nucleic acid and proteins leaking out from the cells, and disrupt the cell membrane permeability. Furthermore, metabolomic analysis of F. graminearum revealed the significant differences in the expression of 100 metabolites between the lipopeptides treatment group and the control group, which were associated with various metabolic pathways, mainly including amino acid biosynthesis, pentose, glucuronate and glycerophospholipid metabolism. In addition, strain ZK-9 inhibited Fusarium crown rot (FCR) with a biocontrol efficacy of 82.14 % and increased the plant height and root length by 24.23 % and 93.25 %, respectively. Moreover, the field control efficacy of strain ZK-9 on Fusarium head blight (FHB) was 71.76 %, and the DON content in wheat grains was significantly reduced by 69.9 %. This study puts valuable insights into the antifungal mechanism of lipopeptides against F. graminearum, and provides a promising biocontrol agent for controlling F. graminearum.

Safety evaluation of an extension of use of the food enzyme glutaminase from the non-genetically modified Bacillus amyloliquefaciens strain AE-GT.

The food enzyme glutaminase (l-glutamine amidohydrolase; EC 3.5.1.2) is produced with the non-genetically modified Bacillus amyloliquefaciens strain AE-GT by Amano Enzyme Inc. A safety evaluation of this food enzyme was made previously, in which EFSA concluded that this food enzyme did not give rise to safety concerns when used in five food manufacturing processes. Subsequently, the applicant requested to extend its use to thirteen additional processes and to revise the use levels. In this assessment, EFSA updated the safety evaluation of this food enzyme when used in a total of eighteen food manufacturing processes. As the food enzyme-total organic solids (TOS) are removed from the final foods in two food manufacturing processes, the dietary exposure to the food enzyme-TOS was estimated only for the remaining sixteen processes. Dietary exposure was calculated to be up to 0.678 mg TOS/kg body weight per day in European populations. Based on the data provided for the previous evaluation and the revised dietary exposure in the present evaluation, the Panel concluded that this food enzyme does not give rise to safety concerns under the revised intended conditions of use.

Optimization, gene cloning, expression, and molecular docking insights for enhanced cellulase enzyme production by Bacillus amyloliquefaciens strain elh1.

BACKGROUND: In this study, we isolated a cellulase-producing bacterium, Bacillus amyloliquefaciens strain elh, from rice peel. We employed two optimization methods to enhance the yield of cellulase. Firstly, we utilized a one-variable-at-a-time (OVAT) approach to evaluate the impact of individual physical and chemical parameters. Subsequently, we employed response surface methodology (RSM) to investigate the interactions among these factors. We heterologously expressed the cellulase encoding gene using a cloning vectorin E. coli DH5α. Moreover, we conducted in silico molecular docking analysis to analyze the interaction between cellulase and carboxymethyl cellulose as a substrate. RESULTS: The bacterial isolate eh1 exhibited an initial cellulase activity of 0.141 ± 0.077 U/ml when cultured in a specific medium, namely Basic Liquid Media (BLM), with rice peel as a substrate. This strain was identified as Bacillus amyloliquefaciens strain elh1 through 16S rRNA sequencing, assigned the accession number OR920278 in GenBank. The optimal incubation time was found to be 72 h of fermentation. Urea was identified as the most suitable nitrogen source, and dextrose as the optimal sugar, resulting in a production increase to 5.04 ± 0.120 U/ml. The peak activity of cellulase reached 14.04 ± 0.42 U/ml utilizing statistical optimization using Response Surface Methodology (RSM). This process comprised an initial screening utilizing the Plackett-Burman design and further refinement employing the BOX -Behnken Design. The gene responsible for cellulase production, egl, was effectively cloned and expressed in E. coli DH5α. The transformed cells exhibited a cellulase activity of 22.3 ± 0.24 U/ml. The egl gene sequence was deposited in GenBank with the accession number PP194445. In silico molecular docking revealed that the two hydroxyl groups of carboxymethyl cellulose bind to the residues of Glu169 inside the binding pocket of the CMCase. This interaction forms two hydrogen bonds, with an affinity score of -5.71. CONCLUSIONS: Optimization of cultural conditions significantly enhances the yield of cellulase enzyme when compared to unoptimized culturing conditions. Additionally, heterologous expression of egl gene showed that the recombinant form of the cellulase is active and that a valid expression system can contribute to a better yield of the enzyme.

Enhancement of Green Production of Heme by Deleting Odor-Related Genes from Bacillus amyloliquefaciens Based on CRISPR/Cas9n.

Heme is a crucial component in endowing plant-based meat analogs with flavor and color. This study aimed to develop a green strategy for heme production by reducing fermentation off-odor and accelerating heme synthesis. First, an efficient CRISPR/Cas9n system was constructed in Bacillus amyloliquefaciens to construct the odor-reducing chassis cell HZC9nΔGPSU, and the odor substances including the branched-chain short fatty acids, putrescine, and ammonia were reduced by 62, 70, and 88%, respectively. Meanwhile, the hemA gene was confirmed to be the key gene for enhanced heme synthesis. Various hemA genes were compared to obtain the best gene dhemA, and the catalysis mechanism was explained by molecular docking simulation. After further expression of dhemA in HZC9nΔGPSU, the heme titer of HZC9nΔGPSU/pHY-dhemA reached 11.31 ± 0.51 mg/L, 1.70-fold higher than that of HZC9n/pHY-dhemA. The knockout of off-odor-related genes reduced the odor substances and enhanced the heme synthesis, which is promising for the green production of high-quality heme.

Safety evaluation of an extension of use of the food enzyme bacillolysin from the non-genetically modified Bacillus amyloliquefaciens strain AE-NP.

The food enzyme bacillolysin (EC 3.4.24.28) is produced with the non-genetically modified Bacillus amyloliquefaciens strain AE-NP by Amano Enzyme Inc. A safety evaluation of this food enzyme was made previously, in which EFSA concluded that this food enzyme did not give rise to safety concerns when used in thirteen food manufacturing processes. Subsequently, the applicant requested to extend its use to two additional processes. In this assessment, EFSA updated the safety evaluation of this food enzyme when used in a total of fifteen food manufacturing processes. As the food enzyme-total organic solids (TOS) are removed in two food manufacturing processes, the dietary exposure to the food enzyme-TOS was estimated only for the remaining thirteen processes. Dietary exposure was calculated to be up to 35.251 mg TOS/kg body weight per day in European populations. Based on the data provided for the previous evaluation and the revised dietary exposure in the present evaluation, the Panel concluded that this food enzyme does not give rise to safety concerns under the revised intended conditions of use.

Harnessing Bacillus amyloliquefaciens for Amazake Production: Comparison with Aspergillus oryzae Amazake for Metabolomic Characteristics, Microbial Diversity, and Sensory Profile.

Amazake is a traditional, sweet, non-alcoholic Japanese beverage typically produced through koji fermentation by the fungus Aspergillus oryzae. However, alternative microorganisms such as Bacillus amyloliquefaciens offer potential advantages and novel possibilities for producing similar fermented beverages. This study aimed to replicate the ancestral beverage of amazake by replacing A. oryzae (W-20) with B. amyloliquefaciens (NCIMB 12077) and comparing their fermentation processes and resulting products. Our results show that the production of amazake with B. amyloliquefaciens (ABA) is not only possible but also results in a beverage that is otherwise distinct from traditional amazake (AAO). Saccharification was achievable in ABA at higher temperatures than in AAO, albeit with lower reducing sugar and enzymatic activity values. Amino acids and organic acids were more abundant in AAO, with cysteine being uniquely present in AAO and shikimic acid only being present in ABA. The volatile aroma compound profiles differed between the two beverages, with AAO exhibiting a greater abundance of aldehydes, and ABA a greater abundance of ketones and alcohols. Interestingly, despite these compositional differences, the two beverages showed similar consumer panel acceptance rates. An analysis of their microbial communities revealed pronounced differences between the amazakes, as well as temporal changes in ABA but not in AAO. This study provides promising insights into harnessing the potential of B. amyloliquefaciens as the primary microorganism in the fermentation process of amazake-like beverages, marking an important advancement in the field of fermented low-alcohol beverage production, with possible applications in other fermented foods.

Biodegradation Characteristics and Mechanism of Aflatoxin B1 by Bacillus amyloliquefaciens from Enzymatic and Multiomics Perspectives.

Aflatoxin B1 (AFB1), a mycotoxin and natural carcinogen, commonly contaminates cereals and animal feeds, posing serious health risks to human and animal. In this study, Bacillus amyloliquefaciens ZG08 isolated from kimchi could effectively remove 80.93% of AFB1 within 72 h at 37 °C and pH 7.0. Metabolome and transcriptome analysis showed that metabolic processes including glycerophospholipid metabolism and amino acid metabolism were most affected in B. amyloliquefaciens ZG08 exposed to AFB1. The adaptation mechanism likely involved activation of the thioredoxin system to restore intracellular redox equilibrium. The key genes, tpx and gldA, overexpressed in Escherichia coli BL21, achieved degradation rates of 60.15% and 47.16% for 100 µg/kg AFB1 under optimal conditions of 37 °C and pH 8.0 and 45 °C and pH 7.0, respectively. The degradation products, identified as AFD1, were less cytotoxic than AFB1 in HepG2 cells. These findings suggest potential strategies for utilizing probiotics and engineered enzymes in AFB1 detoxification.

Spore germination and lactic acid combined treatment: A new processing strategy for the shelf-life extension of instant wet noodles.

Bacillus amyloliquefaciens (BAM) was identified as the predominant spoilage bacteria in instant wet noodles (IWNs). The utilization of industrial acid treatment as a long shelf-life strategy resulted in reduced consumer acceptance due to the acidic taste of the products. This study proposed a processing strategy that integrated spore germination (SG) and lactic acid (LA) treatment to effectively reduce the spore survival rate and extend the shelf life of IWNs. L-histidine, d-glucose, and sodium chloride were highly efficient and safe germinants for BAM spores. In IWNs, compound germinants (1.0 % L-histidine, 0.5 % d-glucose, and 1.0 % sodium chloride) boosted the SG rate by 3.61 times. With synergistic LA treatment, the spore lethality increased by 34.41 % -41.68 %. Under the SG and reduced acid-heat conditions of pH 2.30-2.50, the mortality of spores could reach 92.00 %-93.17 %, which was 14.11 %-15.28 % higher than the industrial acid-heat condition of pH 2.10. DPA, ATP, and membrane potential showed that germinants reduced the spore membrane permeability and promoted the occurrence of spore membrane damage under acid-heat conditions. Moreover, this strategy significantly extended the shelf-life of IWNs by 3.00-5.50 times and controlled the pH ≥ 5.50. Additionally, it improved color, texture, and overall sensory evaluation. Accordingly, this strategy solved the contradiction between the long shelf-life of IWNs and the unacceptable acidification in industrial production.

The Effects of Bacillus amyloliquefaciens SC06 on Behavior and Brain Function in Broilers Infected by Clostridium perfringens.

Poultry studies conducted on Clostridium perfringens (CP) mainly focus on the effects of intestinal health and productive performance. Notably, the probiotic Bacillus amyloliquefaciens SC06 (BaSC06) is known to play a role in preventing bacterial infection. However, whether CP could induce the changes in brain function and behaviors and whether BaSC06 could play roles in these parameters is yet to be reported. The aim of this study was to evaluate the effects of BaSC06 on stress-related behaviors and gene expression, as well as the brain morphology and mRNA sequence of the hypothalamus in broiler chickens. A total of 288 one-day-old chicks were randomly divided into four groups: (1) a control group with no treatment administered or infection; (2) birds treated with the BaSC06 group; (3) a CP group; and (4) a BaSC06 plus CP (Ba_CP) group. The results showed that stress and fear-related behaviors were significantly induced by a CP infection and decreased due to the treatment of BaSC06. CP infection caused pathological damage to the pia and cortex of the brain, while BaSC06 showed a protective effect. CP significantly inhibited hypothalamic GABA and promoted HTR1A gene expression, while BaSC06 promoted GABA and decreased HTR1A gene expression. The different genes were nearly found between the comparisons of control vs. Ba group and Ba vs. CP group, while there were a great number of different genes between the comparisons of control vs. Ba_CP as well as CP vs. Ba_CP. Several different gene expression pathways were found that were related to disease, energy metabolism, and nervous system development. Our results will help to promote poultry welfare and health, as well as provide insights into probiotics to replace antibiotics and reduce resistance in the chicken industry.

Biological Control of Root Rot of Strawberry by Bacillus amyloliquefaciens Strains CMS5 and CMR12.

Strawberry root rot caused by Fusarium solani is one of the main diseases of strawberries and significantly impacts the yield and quality of strawberry fruit. Biological control is becoming an alternative method for the control of plant diseases to replace or decrease the application of traditional chemical fungicides. To obtain antagonistic bacteria with a high biocontrol effect on strawberry root rot, over 72 rhizosphere bacteria were isolated from the strawberry rhizosphere soil and screened for their antifungal activity against F. solani by dual culture assay. Among them, strains CMS5 and CMR12 showed the strongest inhibitory activity against F. solani (inhibition rate 57.78% and 65.93%, respectively) and exhibited broad-spectrum antifungal activity. According to the phylogenetic tree based on 16S rDNA and gyrB genes, CMS5 and CMR12 were identified as Bacillus amyloliquefaciens. Lipopeptide genes involved in surfactin, iturin, and fengycin biosynthesis were detected in the DNA genomes of CMS5 and CMR12 by PCR amplification. The genes related to the three major lipopeptide metabolites existed in the DNA genome of strains CMS5 and CMR12, and the lipopeptides could inhibit the mycelial growth of F. solani and resulted in distorted hyphae. The inhibitory rates of lipopeptides of CMS5 and CMR12 on the spore germination of F. solani were 61.00% and 42.67%, respectively. The plant-growth-promoting (PGP) traits in vitro screening showed that CMS5 and CMR12 have the ability to fix nitrogen and secreted indoleacetic acid (IAA). In the potting test, the control efficiency of CMS5, CMR12 and CMS5+CMR12 against strawberry root rot were 65.3%, 67.94% and 88.00%, respectively. Furthermore, CMS5 and CMR12 enhanced the resistance of strawberry to F. solani by increasing the activities of defense enzymes MDA, CAT and SOD. Moreover, CMS5 and CMR12 significantly promoted the growth of strawberry seedlings such as root length, seedling length and seedling fresh weight. This study revealed that B. amyloliquefaciens CMS5 and CMR12 have high potential to be used as biocontrol agents to control strawberry root rot.

Effects of Dietary Bacillus amyloliquefaciens SCAU-070 (Based on a Woody Plant-Based Diet) on Antioxidation, Immune and Intestinal Microbiota of Tilapia (Oreochromis niloticus).

This study aimed to explore the effects of Bacillus amyloliquefaciens (BA) as one woody forage addition (as a probiotic, 1 × 107 CFU/g) on tilapia (Oreochromis niloticus). Woody forage is one kind of fishery feed that could significantly enhance the growth, feed utilization, and digestibility of tilapia. At first, tilapia was divided into eight groups and fed with control, control + BA, Moringa oleifera, M. oleifera + BA, Neolamarckia cadamba, N. cadamba + BA, Broussonetia papyrifera, and B. papyrifera + BA diets, respectively. After dieting for 8 weeks, the intestinal morphology of tilapia in the eight groups was observed, and the effects of the B. amyloliquefaciens addition and wordy forage on the intestine functions were analyzed by two-way ANOVA. As no significant negative effects were found on the woody forage on tilapia, the villus height, density and width, and epithelial goblet cells in the posterior intestines of tilapia with BA supplementation were greater than those in the groups without BA supplementation, suggesting B. amyloliquefaciens SCAU-070 could promote the growth and development of tilapia intestinal tracts. Furthermore, it was found that B. amyloliquefaciens SCAU-070 enhanced the antioxidation capacity of tilapia posterior intestine tissue by promoting the activity of superoxide dismutase and content of malondialdehyde. In addition, the result of high-throughput sequencing (16S rDNA) showed that the beneficial bacteria Cetobacterium and Romboutsia in the probiotic groups increased significantly, while the potential pathogenic bacteria Acinetobacter decreased significantly.

Whole-Genome Sequencing of Two Potentially Allelopathic Strains of Bacillus from the Roots of C. equisetifolia and Identification of Genes Related to Synthesis of Secondary Metabolites.

The coastal Casuarina equisetifolia is the most common tree species in Hainan's coastal protection forests. Sequencing the genomes of its allelopathic endophytes can allow the protective effects of these bacteria to be effectively implemented in protected forests. The goal of this study was to sequence the whole genomes of the endophytes Bacillus amyloliquefaciens and Bacillus aryabhattai isolated from C. equisetifolia root tissues. The results showed that the genome sizes of B. amyloliquefaciens and B. aryabhattai were 3.854 Mb and 5.508 Mb, respectively. The two strains shared 2514 common gene families while having 1055 and 2406 distinct gene families, respectively. The two strains had 283 and 298 allelochemical synthesis-associated genes, respectively, 255 of which were shared by both strains and 28 and 43 of which were unique to each strain, respectively. The genes were putatively involved in 11 functional pathways, including secondary metabolite biosynthesis, terpene carbon skeleton biosynthesis, biosynthesis of ubiquinone and other terpene quinones, tropane/piperidine and piperidine alkaloids biosynthesis, and phenylpropanoid biosynthesis. NQO1 and entC are known to be involved in the biosynthesis of ubiquinone and other terpenoid quinones, and rfbC/rmlC, rfbA/rmlA/rffH, and rfbB/rmlB/rffG are involved in the biosynthesis of polyketide glycan units. Among the B. aryabhattai-specific allelochemical synthesis-related genes, STE24 is involved in terpene carbon skeleton production, atzF and gdhA in arginine biosynthesis, and TYR in isoquinoline alkaloid biosynthesis. B. amyloliquefaciens and B. aryabhattai share the genes aspB, yhdR, trpA, trpB, and GGPS, which are known to be involved in the synthesis of carotenoids, indole, momilactones, and other allelochemicals. Additionally, these bacteria are involved in allelochemical synthesis via routes such as polyketide sugar unit biosynthesis and isoquinoline alkaloid biosynthesis. This study sheds light on the genetic basis of allelopathy in Bacillus strains associated with C. equisetifolia, highlighting the possible use of these bacteria in sustainable agricultural strategies for weed management and crop protection.

Bacillus amyloliquefaciens LM-1 Affects Multiple Cell Biological Processes in Magnaporthe oryzae to Suppress Rice Blast.

Magnaporthe oryzae, one of the most destructive rice pathogens, causes significant losses during the rice harvest every year. Bacillus amyloliquefaciens has been explored in many crops as a potential biocontrol agent. However, the mechanisms of B. amyloliquefaciens controled rice blast are not fully understood. Here, a biocontrol strain LM-1, isolated from a contaminated medium, was identified as B. amyloliquefaciens using morphological observation, physiological and biochemical tests, and 16S rDNA sequencing. LM-1 inhibited the growth and pathogenicity of M. oryzae and Bipolaris oryzae (Breda de Haan) Shoem. The mycelia of M. oryzae co-cultured with LM-1 were enlarged and broken by fluorescence microscopy using calcofluor white. LM-1 inhibited the mycelia of M. oryzae from producing conidia. Genes itu, srf, and fenB were detected in LM-1. Furthermore, the supernatant of LM-1 interfered with the appressorium formation of M. oryzae, blocked conidial cell death, and reduced autophagy degradation but did not affect the normal germination of rice seeds and seeding growth. Additionally, we observed hypersensitivity reactions, reactive oxygen species, and iron accumulation reduction in rice cells inoculated with supernatant. Our study reveals that LM-1 has a control effect on rice blast and affects cell wall integrity, sporulation, appressorium formation, cell death, and autophagy.

Screening of bacterial endophytes of larch against Neofusicoccum laricinum and validation of their safety.

In order to provide a highly feasible research pathway for the control of larch shoot blight, healthy larch branches and leaves were collected from 13 sampling sites in 8 provinces in China. The antagonistic endophytic bacteria obtained from the screening were used to carry out disease control experiments in potted seedlings. The safety evaluation test was conducted on the antagonistic bacteria. Subsequently, the strains with better preventive effect and high safety were identified by morphological and molecular methods. A total of 391 strains of endophytic bacteria were isolated from healthy larch branches and leaves. Seventy-eight strains of larch endophytic bacteria with antagonistic effect were obtained by primary sieving. Ten strains of endophytic bacteria with obvious antagonism were further obtained by secondary sieving, and all of them had an inhibition rate of more than 57%. Among them, strains YN 2, JL 6, NMG 23, and JL 54 showed the highest inhibition rate of 63.16%-65.08%, which was significantly different from the other treatments. The results of the pot test showed that 14 days after inoculation with the pathogen, strains YN 2 and JL 54 were more effective in the control of larch shoot blight, with the preventive effects reaching 57.7% and 50.0%, respectively. Strains JL 6 and JL 54 were biologically safe in the safety evaluation test. Therefore, strain JL 54 was selected for identification. It was identified as Bacillus amyloliquefaciens through morphological observation, 16S rDNA sequence, gyrB gene sequence and 16S rDNA-gyrB tandem feature sequence analysis. IMPORTANCE: Larch shoot blight is a widely distributed, damaging, and rapidly spreading fungal disease of forest trees that poses a serious threat to larch plantations. Endophytic bacteria have biological effects on host plants against pests and diseases, and they have a growth-promoting effect on plants. In this paper, we investigated for the first time the biocontrol effect of endophytic bacteria on larch shoot blight by screening endophytic bacteria with the function of antagonizing dieback fungi. Bacillus amyloliquefaciens JL 54 has a better prospect of biocontrol against larch shoot blight, which lays the foundation for the application of this bacterium in the future.

In vitro and genomic mining studies of anti-Clostridium perfringens Compounds Derived from Bacillus amyloliquefaciens.

Clostridium perfringens is an important opportunistic microorganism in commercial poultry production that is implicated in necrotic enteritis (NE) outbreaks. This disease poses a severe financial burden on the global poultry industry, causing estimated annual losses of $6 billion globally. The ban on in-feed antibiotic growth promoters has spurred investigations into approaches of alternatives to antibiotics, among which Bacillus probiotics have demonstrated varying degrees of effectiveness against NE. However, the precise mechanisms underlying Bacillus-mediated beneficial effects on host responses in NE remain to be further elucidated. In this manuscript, we conducted in vitro and genomic mining analysis to investigate anti-C. perfringens activity observed in the supernatants derived from 2 Bacillus amyloliquefaciens strains (FS1092 and BaD747). Both strains demonstrated potent anti-C. perfringens activities in in vitro studies. An analysis of genomes from 15 B. amyloliquefaciens, 11 B. velezensis, and 2 B. subtilis strains has revealed an intriguing clustering pattern among strains known to possess anti-C. perfringens activities. Furthermore, our investigation has identified 7 potential antimicrobial compounds, predicted as secondary metabolites through antiSMASH genomic mining within the published genomes of B. amyloliquefaciens species. Based on in vitro analysis, BaD747 may have the potential as a probiotic in the control of NE. These findings not only enhance our understanding of B. amyloliquefaciens's action against C. perfringens but also provide a scientific rationale for the development of novel antimicrobial therapeutic agents against NE.

Bio-Priming with Bacillus Isolates Suppresses Seed Infection and Improves the Germination of Garden Peas in the Presence of Fusarium Strains.

Seed infection caused by Fusarium spp. is one of the major threats to the seed quality and yield of agricultural crops, including garden peas. The use of Bacillus spp. with multiple antagonistic and plant growth-promoting (PGP) abilities represents a potential disease control strategy. This study was performed to evaluate the biocontrol potential of new Bacillus spp. rhizosphere isolates against two Fusarium strains affecting garden peas. Six Bacillus isolates identified by 16S rDNA sequencing as B. velezensis (B42), B. subtilis (B43), B. mojavensis (B44, B46), B. amyloliquefaciens (B50), and B. halotolerans (B66) showed the highest in vitro inhibition of F. proliferatum PS1 and F. equiseti PS18 growth (over 40%). The selected Bacillus isolates possessed biosynthetic genes for endoglucanase (B42, B43, B50), surfactin (B43, B44, B46), fengycin (B44, B46), bacillomycin D (B42, B50), and iturin (B42), and were able to produce indole-3-acetic acid (IAA), siderophores, and cellulase. Two isolates, B. subtilis B43 and B. amyloliquefaciens B50, had the highest effect on final germination, shoot length, root length, shoot dry weight, root dry weight, and seedling vigor index of garden peas as compared to the control. Their individual or combined application reduced seed infection and increased seed germination in the presence of F. proliferatum PS1 and F. equiseti PS18, both after seed inoculation and seed bio-priming. The most promising results were obtained in the cases of the bacterial consortium, seed bio-priming, and the more pathogenic strain PS18. The novel Bacillus isolates may be potential biocontrol agents intended for the management of Fusarium seed-borne diseases.

Safety evaluation of a food enzyme containing bacillolysin and subtilisin activities from the non-genetically modified Bacillus amyloliquefaciens strain AR-383.

The food enzyme with two declared activities, bacillolysin (EC 3.4.24.28) and subtilisin (EC 3.4.21.62), is produced with the non-genetically modified Bacillus amyloliquefaciens strain AR-383 by AB Enzymes GmbH. The food enzyme is intended to be used in nine food manufacturing processes. Since residual amounts of total organic solids (TOS) are removed in the production of distilled alcohol, dietary exposure was calculated only for the remaining eight food manufacturing processes. Exposure was estimated to be up to 1.958 mg TOS/kg body weight per day in European populations. As the production strain qualifies for the qualified presumption of safety approach to safety assessment and no issues of concern arising from the production process of the food enzyme were identified, the Panel considered that no toxicological studies other than the assessment of allergenicity were necessary. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made, and 30 matches were found, including one food allergen (melon). The Panel considered that, under the intended conditions of use, the risk of allergic reactions by dietary exposure to this food enzyme cannot be excluded, but for individuals sensitised to melon, this would not exceed the risk of consuming melon. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Screening and identification of an aflatoxin B1-degrading strain from the Qinghai-Tibet Plateau and biodegradation products analysis.

This research aimed to address the issue of aflatoxin B1 (AFB1) contamination, which posed severe health and economic consequences. This study involved exploring unique species resources in the Qinghai-Tibet Plateau, screening strains capable of degrading AFB1. UPLC-Q-Orbitrap HRMS and NMR were employed to examine the degradation process and identify the structure of the degradation products. Results showed that Bacillus amyloliquefaciens YUAD7, isolated from yak dung in the Qinghai-Tibet Plateau, removed 91.7% of AFB1 from TSB-AFB1 medium with an AFB1 concentration of 10 µg/mL (72 h, 37°C, pH 6.8) and over 85% of AFB1 from real food samples at 10 µg/g (72 h, 37°C), exhibiting strong AFB1 degradation activity. Bacillus amyloliquefaciens YUAD7's extracellular secretions played a major role in AFB1 degradation mediated and could still degrade AFB1 by 43.16% after boiling for 20 min. Moreover, B. amyloliquefaciens YUAD7 demonstrated the capability to decompose AFB1 through processes such as hydrogenation, enzyme modification, and the elimination of the -CO group, resulting in the formation of smaller non-toxic molecules. Identified products include C12H14O4, C5H12N2O2, C10H14O2, C4H12N2O, with a structure consisting of dimethoxyphenyl and enoic acid, dimethyl-amino and ethyl carbamate, polyunsaturated fatty acid, and aminomethyl. The results indicated that B. amyloliquefaciens YUAD7 could be a potentially valuable strain for industrial-scale biodegradation of AFB1 and providing technical support and new perspectives for research on biodegradation products.

Characterization of Bacillus amyloliquefaciens BA-4 and its biocontrol potential against Fusarium-related apple replant disease.

Apple replant disease (ARD), caused by Fusarium pathogens, is a formidable threat to the renewal of apple varieties in China, necessitating the development of effective and sustainable control strategies. In this study, the bacterial strain BA-4 was isolated from the rhizosphere soil of healthy apple trees in a replanted orchard, demonstrating a broad-spectrum antifungal activity against five crucial apple fungal pathogens. Based on its morphology, physiological and biochemical traits, utilization of carbon sources, and Gram stain, strain BA-4 was tentatively identified as Bacillus amyloliquefaciens. Phylogenetic analysis using 16S rDNA and gyrB genes conclusively identified BA-4 as B. amyloliquefaciens. In-depth investigations into B. amyloliquefaciens BA-4 revealed that the strain possesses the capacity to could secrete cell wall degrading enzymes (protease and cellulase), produce molecules analogous to indole-3-acetic acid (IAA) and siderophores, and solubilize phosphorus and potassium. The diverse attributes observed in B. amyloliquefaciens BA-4 underscore its potential as a versatile microorganism with multifaceted benefits for both plant well-being and soil fertility. The extracellular metabolites produced by BA-4 displayed a robust inhibitory effect on Fusarium hyphal growth and spore germination, inducing irregular swelling, atrophy, and abnormal branching of fungal hyphae. In greenhouse experiments, BA-4 markedly reduced the disease index of Fusarium-related ARD, exhibiting protective and therapeutic efficiencies exceeding 80% and 50%, respectively. Moreover, BA-4 demonstrated plant-promoting abilities on both bean and Malus robusta Rehd. (MR) seedlings, leading to increased plant height and primary root length. Field experiments further validated the biocontrol effectiveness of BA-4, demonstrating its ability to mitigate ARD symptoms in MR seedlings with a notable 33.34% reduction in mortality rate and improved biomass. Additionally, BA-4 demonstrates robust and stable colonization capabilities in apple rhizosphere soil, particularly within the 10-20 cm soil layer, which indicates that it has long-term effectiveness potential in field conditions. Overall, B. amyloliquefaciens BA-4 emerges as a promising biocontrol agent with broad-spectrum antagonistic capabilities, positive effects on plant growth, and strong colonization abilities for the sustainable management of ARD in apple cultivation.

Bacillus amyloliquefaciens attenuates the intestinal permeability, oxidative stress and endoplasmic reticulum stress: transcriptome and microbiome analyses in weaned piglets.

Endoplasmic reticulum (ER) stress is related to oxidative stress (OS) and leads to intestinal injury. Bacillus amyloliquefaciens SC06 (SC06) can regulate OS, but its roles in intestinal ER stress remains unclear. Using a 2 × 2 factorial design, 32 weaned piglets were treated by two SC06 levels (0 or 1 × 108 CFU/g), either with or without diquat (DQ) injection. We found that SC06 increased growth performance, decreased ileal permeability, OS and ER stress in DQ-treated piglets. Transcriptome showed that differentially expressed genes (DEGs) induced by DQ were enriched in NF-κB signaling pathway. DEGs between DQ- and SC06 + DQ-treated piglets were enriched in glutathione metabolism pathway. Ileal microbiome revealed that the SC06 + DQ treatment decreased Clostridium and increased Actinobacillus. Correlations were found between microbiota and ER stress genes. In conclusion, dietary SC06 supplementation increased the performance, decreased the permeability, OS and ER stress in weaned piglets by regulating ileal genes and microbiota.