In Vitro and In Vivo Anti-Clostridioides difficile Effect of a Probiotic Bacillus amyloliquefaciens Strain.

Clostridioides difficile infection (CDI) is a significant cause of hospital-acquired and antibiotic-mediated intestinal diseases and is a growing global public health concern. Overuse of antibiotics and their effect on normal intestinal flora has increased the incidence and severity of infections. Thus, the development of new, effective, and safe treatment options is a high priority. Here, we report a new probiotic strain, Bacillus amyloliquefaciens (BA PMC-80), and its in vitro/in vivo anti-C. difficile effect as a prospective novel candidate for replacing conventional antibiotics. BA PMC-80 showed a significant anti-C. difficile effect in coculture assay, and its cell-free supernatant (CFS) also exhibited a considerable anti-C. difficile effect with an 89.06 µg/ml 50% minimal inhibitory concentration (MIC) in broth microdilution assay. The CFS was stable and equally functional under different pHs, heat, and proteinase treatments. It also exhibited a high sensitivity against current antibiotics and no toxicity in subchronic toxicity testing in hamsters. Finally, BA PMC-80 showed a moderate effect in a hamster CDI model with reduced infection severity and delayed death. However, further studies are required to optimize the treatment condition of the hamster CDI model for better efficacy and identify the antimicrobial compound produced by BA PMC-80.

Genomic Analysis Reveals Potential Mechanisms Underlying Promotion of Tomato Plant Growth and Antagonism of Soilborne Pathogens by Bacillus amyloliquefaciens Ba13.

Bacillus amyloliquefaciens Ba13 is a plant beneficial bacterium isolated from loessial soil with notable biological activity. This study clarified potential mechanisms underlying the plant growth-promoting and antipathogenic effects of strain Ba13. A pot experiment was used to verify the plant growth-promoting effects of strain Ba13 on tomato, and the antipathogenic activity was tested in petri dishes. The underlying mechanisms were explored based on whole-genome sequencing of strain Ba13 and liquid chromatography-tandem mass spectrometry (LC-MS/MS) detection of plant hormones and biosynthetic intermediates. The results showed that exposure to strain Ba13 promoted tomato plant growth significantly. Compared with control treatment, bacterial treatment increased plant height and fresh weight by 10.98% and 20.15%, respectively, at 28 days after inoculation. Strain Ba13 exhibited antagonistic activity against all eight plant pathogens tested. The 3,861,210-bp genome of strain Ba13 was predicted to encode antibiotics (e.g., surfactin, bacillaene, bacillomycin D, bacilysin, and bacillibactin) and volatile gaseous compounds (e.g., 2,3-butanediol and acetoin). Genes were also predicted to encode extracellular phytase and ß-glucanase that are secreted through the secretory (Sec) system. Strain Ba13 could synthesize indole-3-acetic acid through the indole-3-pyruvic acid pathway. The results of this study indicate that B. amyloliquefaciens Ba13 has multiple effects on tomato plants and associated microorganisms, directly or indirectly promoting plant growth and controlling plant diseases. IMPORTANCE Microbial agents are considered the optimal alternative for chemical agents. Exploring the mechanisms underlying the beneficial effects of microbial agents is essential for rational applications in the field. In this study, we report a functional bacterial strain, Bacillus amyloliquefaciens Ba13, which exhibited plant growth-promoting and antipathogenic effects. The whole genome of strain Ba13 was sequenced, and functional genes of interest were predicted. Strain Ba13 could synthesize indole-3-acetic acid through the indole-3-pyruvic acid pathway.

Complete genome sequence of Bacillus velezensis NST6 and comparison with the species belonging to operational group B. amyloliquefaciens.

Bacillus spp. play important roles in production of bioactive natural products with potential agricultural and medical applications. The three families of lipopeptides produced by Bacillus spp. have been most recognized for their antagonistic activity against other microbes, i.e. fengycin, iturin, and surfactin. A novel strain NST6 was isolated from soil and identified as B. velezensis based on phylogenomic analysis. Genome analysis revealed 21 putative biosynthetic gene clusters including the ones responsible for producing bacillomycin and surfactin. However, fengycin cluster was compromised with absence or partial disruption of three non-ribosomal peptide synthetases. Distribution of biosynthetic gene clusters showed that clusters for iturin families were well conserved in 327 genomes of the species belonging to the operational group B. amyloliquefaciens. However, clusters for fengycin and surfactin showed dynamic distribution at gene level. Comparative analysis of closely related species would provide new insights to the diversity in genetic elements for secondary metabolites.

A newly isolated Bacillus amyloliquefaciens SRB04 for the synthesis of selenium nanoparticles with potential antibacterial properties.

The aim of this study was to isolate and characterize marine bacterial strains capable of converting selenite to elemental selenium with the formation of Se nanoparticles (SeNPs). For the first time, a novel marine strain belonging to Bacillus amyloliquefaciens (GenBank accession no. MK392020) was isolated from the coast of the Caspian Sea and characterized based on its ability for transformation of selenite to SeNPs under aerobic conditions. The preliminary formation of SeNPs was confirmed via color changes and the products characterized by UV-Vis spectroscopy. The field-emission scanning electron microscopy (FESEM) together with energy-dispersive X-ray (EDX) analysis showed the presence of the spherical SeNPs on both the surface of the bacterial biomass and in the supernatant solution. Dynamic light scattering (DLS) analysis showed the SeNPs to have an average particle size (Z-average) around 45.4-68.3 nm. The X-ray diffraction (XRD) studies substantiated the amorphous nature of the biosynthesized SeNPs. Fourier-transform infrared spectroscopic (FTIR) studies of the SeNPs indicated typical proteinaceous and lipid-related bands as capping agents on the SeNPs. Different effective parameters corresponding the yield of SeNPs by B. amyloliquefaciens strain SRB04 were optimized under resting cell strategy. Results showed that the optimal process conditions for SeNP production were 2 mM of selenite oxyanion, 20 g/L of cell biomass, and 60 h reaction time. The synthesized SeNPs had a remarkable antibacterial activity on Staphylococcus aureus compared with chloramphenicol as a broad-spectrum antibiotic.

Characterization and application of a novel laccase derived from Bacillus amyloliquefaciens.

As the copper-containing enzymes, laccases demonstrate a promising potential in various environmental and industrial applications. In this study, a bacterial strain isolated from soil exhibited the laccase activity, which was subsequently characterized and named as Bacillus amyloliquefaciens TCCC 111018. The novel gene encoding CotA-laccase (lac) was amplified using the genome of B. amyloliquefaciens TCCC 111018 as the template and efficiently and actively expressed in Escherichia coli. The recombinant LAC (rLAC) exhibited its highest activity at 80 °C and pH 5.5 for 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) oxidization and 80 °C and pH 7.0 for 2,6-dimethoxyphenol (2,6-DMP) oxidization. rLAC was stable at up to 60 °C and within the pH ranging from 3.0 to 9.0 when using the substrate ABTS. Furthermore, rLAC demonstrated the relatively high tolerance to NaCl, SDS, and most metal ions. Moreover, rLAC was capable of decolorizing the structurally different azo, anthraquinone, and triphenylmethane with different mediator at 60 °C under pH 5.5, 7.0, and 9.0. Therefore, rLAC would be an ideal candidate for lots of biotechnological and industrial applications due to its stability in the extreme conditions, including but not limit to pH, high temperature, halides, heavy metals and detergents.

Differentiation between Bacillus amyloliquefaciens and Bacillus subtilis isolated from a South African sugarcane processing factory using ARDRA and rpoB gene sequencing.

A total of 104 exopolysaccharide (gum)-producing bacteria were isolated from the juice screen and juice tank in a sugarcane processing factory at times of low- and high dextran concentrations in the produced sugar. Dextran is an indicator of cane deterioration and sucrose loss after harvesting of the cane. The isolates were identified as Bacillus amyloliquefaciens (96 isolates) and Bacillus subtilis (eight isolates) based on restriction enzyme banding patterns of amplified 16S rRNA genes and rpoB gene sequence analysis. Exopolysaccharide production in sugarcane is normally associated with dextran produced by Leuconostoc mesenteroides. B. amyloliquefaciens, and to a lesser extent B. subtilis, could, however, also be responsible for exopolysaccharide (slime or gum) production in cane processing factories.

Potential of Bacillus amyloliquefaciens for biocontrol of bacterial canker of tomato incited by Clavibacter michiganensis ssp. michiganensis.

A total of 150 rhizobacteria and endorhizobacteria previously isolated from three different horticultural crops; strawberry, apple and apricot were screened for antagonistic activitiy against Clavibacter michiganensis ssp. michiganensis. Among them strain S1, exhibiting significantly higher antagonistic and plant growth promoting ability was characterized as Bacillus amyloliquefaciens based on morphological, biochemical and partial gene sequence analysis of 16S rRNA. B. amyloliquefaciens strain S1 showed maximum growth inhibition of C. michiganensis (12 mm). Moreover, B. amyloliquefaciens strain S1 exhibit significant phosphorus solubilization (94.16 %SEl) and indole acetic acid (27 µg ml-1) production under in vitro conditions. Antagonistic activity of Bacillus amyloliquefaciens strain S1 was compared with other four strains KU2S1, R2S(1), RG1(3) and AG1(7) against bacterial canker of tomato under net house conditions. Minimum bacterial canker disease incidence (30.0%) was recorded in B. amyloliquefaciens S1 followed by RG1(3) after 30 days of inoculation. The bio-control efficacy was higher in B. amyloliquefaciens S1 treated plants, followed by RG1(3).

Antifungal and plant growth promotion activity of volatile organic compounds produced by Bacillus amyloliquefaciens.

Fusarium wilt of watermelon, caused by F. oxysporum f.sp. niveum (FON), is a devastating disease that causes extensive losses throughout the world. Five bacterial strains (L3, h, ß, b, and L) isolated from the watermelon rhizosphere showed antagonistic activity against FON during in vitro tests. Strain L3 produced diffusible and volatile organic compounds (VOCs) which showed the strongest antifungal activity. Arabidopsis thaliana plantlets exposed to VOCs produced by strain L3 showed a 2.39-fold increase in biomass, 1.40-fold increase in primary root length, and 5.05-fold increase in number of lateral roots. Confocal laser scanning microscope showed that the GFP-labeled strain L3 could colonize along the elongation and differentiation zones of watermelon roots. In greenhouse pot experiments, the biocontrol efficiency of strain L3 against fusarium wilt of watermelon was up to 68.4% in comparison with the control treatment. In addition, inoculation of the strain L3 resulted in a 23.4% increase in plant fresh weight. Based on 16S rDNA sequence analysis, the strain L3 was identified as Bacillus amyloliquefaciens L3. Fourteen VOCs produced by strain L3 were identified through GC-MS analysis. Of nine VOCs tested, 2-nonanone and 2-heptanone were proved to have strong antifungal properties. Acetoin and 2,3-butanediol were found to promote plant growth. The results suggested B. amyloliquefaciens L3 was a potential biocontrol agent, and that VOCs produced by B. amyloliquefaciens L3 play important roles in the process of biocontrol and plant growth promotion.

Antimicrobial activity and spectroscopic characterization of surfactin class of lipopeptides from Bacillus amyloliquefaciens SR1.

A bacterial isolate screened from wet land soil sample, found to posses antimicrobial activity against an array of fungal plant pathogens viz., Rhizoctonia solani, Sclerotium rolfsii, Alternaria solani, Fusarium oxysporum under in vitro dual culture plate assay. Further the isolate was identified into Bacillus amyloliquefaciens based on 16S rRNA sequencing. The antimicrobial fraction from the extracellular supernatant of the isolate comprises chiefly of surfactin molecules and also iturin and fengycin group of compounds. The surfactins were partially purified by tangential flow ultra-filtration and quantified with liquid chromatography yielding 316.1 mg L-1. Further the surfactin molecules were characterized by HPLC separation, FT-IR, LC-MS spectroscopy and PCR amplification of antibiotic genes. The surfactin molecule with m/z 1022 performed for MS-MS fragmentation and produced two different patterns of ion dissociation.

Biological control of the soft rot bacterium Pectobacterium carotovorum by Bacillus amyloliquefaciens strain Ar10 producing glycolipid-like compounds.

Four hundred and fifty bacteria were evaluated for antagonistic activity against bacterial soft rot of potato caused by Pectobacterium carotovorum sp strain II16. A strain Ar10 exhibiting potent antagonist activity has been identified as Bacillus amyloliquefaciens on the basis of biochemical and molecular characterization. Cell free supernatant showed a broad spectrum of antibacterial activity against human and phytopathogenic bacteria in the range of 10-60 AU/mL. Incubation of P. carotovorum cells with increasing concentrations of the antibacterial compound showed a killing rate of 94.8 and 96% at MIC and 2xMIC respectively. In addition, the antibacterial agent did not exert haemolytic activity at the active concentration and has been preliminary characterized by TLC and GC-MS as a glycolipid compound. Treatment of potato tubers with strain Ar10 for 72 h significantly reduced the severity of disease symptoms (100 and 85.05% reduction of necrosis deep / area and weight loss respectively). The same levels in disease symptoms severity was also recorded following treatment of potato tubers with cell free supernatant for 1 h. Data suggest that protection against potato soft rot disease may be related to glycolipid production by strain Ar10. The present study affords new alternatives for anti-Pectobacterium carotovorum bioactive compounds against the soft rot disease of potato.

Antagonistic Activity against Dirty Panicle Rice Fungal Pathogens and Plant Growth-Promoting Activity of Bacillus amyloliquefaciens BAS23.

Bacterial strain BAS23 was isolated from rice field soil and identified as Bacillus amyloliquefaciens. Based on dual culture method results, the bacterium BAS23 exhibited potent in vitro inhibitory activity on mycelial growth against a broad range of dirty panicle fungal pathogens of rice (Curvularia lunata, Fusarium semitectum and Helminthosporium oryzae). Cell-free culture of BAS23 displayed a significant effect on germ tube elongation and mycelial growth. The highest dry weight reduction (%) values of C. lunata, H. oryzae and F. semitectum were 92.7%, 75.7% and 68.9%, respectively. Analysis of electrospray ionization-mass spectrometry (ESI-MS) and 1H nuclear magnetic resonance (NMR) spectroscopy revealed that the lipopeptides were iturin A with a C14 side chain (C14 iturinic acid) and with a C15 side chain (C15 iturinic acid), which were produced by BAS23 when it was cultured in nutrient broth (NB) for 72 h at 30°C.BAS23, the efficient antagonistic bacterium, also possessed in vitromultipletraits for plant growth promotionand improved rice seedling growth. The results indicated that B.amyloliquefaciens BAS23 represents a useful option either for biocontrol or as a plant growth-promoting agent.

Biological control of potato common scab by Bacillus amyloliquefaciens Ba01.

Potato common scab, which is caused by soil-borne Streptomyces species, is a severe plant disease that results in a significant reduction in the economic value of potatoes worldwide. Due to the lack of efficacious pesticides, crop rotations, and resistant potato cultivars against the disease, we investigated whether biological control can serve as an alternative approach. In this study, multiple Bacillus species were isolated from healthy potato tubers, and Bacillus amyloliquefaciens Ba01 was chosen for further analyses based on its potency against the potato common scab pathogen Streptomyces scabies. Ba01 inhibited the growth and sporulation of S. scabies and secreted secondary metabolites such as surfactin, iturin A, and fengycin with potential activity against S. scabies as determined by imaging mass spectrometry. In pot assays, the disease severity of potato common scab decreased from 55.6 ± 11.1% (inoculated with S. scabies only) to 4.2 ± 1.4% (inoculated with S. scabies and Ba01). In the field trial, the disease severity of potato common scab was reduced from 14.4 ± 2.9% (naturally occurring) to 5.6 ± 1.1% after Ba01 treatment, representing evidence that Bacillus species control potato common scab in nature.

Bacillus amyloliquefaciens ssp. plantarum F11 isolated from Algerian salty lake as a source of biosurfactants and bioactive lipopeptides.

In this study, we identified a new Bacillus strain isolated from an Algerian salty lake that produces metabolites that are active against Gram-positive and Gram-negative bacteria, as well as fungal pathogens. The draft genome sequence of the strain is presented herein. Genome sequence analysis identified the strain to be B. amyloliquefaciens subspecies plantarum F11, and showed that the strain carries the gene clusters for the production of a number of bioactive and surface-active compounds. These include the lipopeptides surfactin and fengycin, antibacterial polyketides macrolactin and bacillaene, and a putative novel lanthipeptide, among others. Through an activity-guided purification method using hydrophobic interaction chromatographic techniques, we confirmed the ability of the strain to produce fengycin lipopeptides. The identities of the isolated fengycin homologs were ascertained through tandem mass spectrometry.

Effect of the biocontrol bacterium Bacillus amyloliquefaciens on the rhizosphere in ginseng plantings.

Panax ginseng is an important medicinal herb due to its ability to strengthen the human immune system. However, due to the increasing needs of ginseng in medicine, the continuous cropping of ginseng has become more common and has resulted in increased problems with fungal decay. Thus, chemical fungicides are commonly used in ginseng plantings, which have caused fungicide residue problems. As an alternative control measure, biocontrol bacteria can be used to manage fungal pathogens. Additionally, these bacteria are environmentally friendly and can also improve stress tolerance in plants. In this study, an antifungal bacterial strain, TB6, that possesses ACC deaminase activity was isolated from the rhizosphere of ginseng plants. This strain was identified as Bacillus amyloliquefaciens. TB6 was applied to 2-year-old ginseng seedlings for a 2-year period, and its impact on the soil rhizosphere was evaluated. The results revealed that strain TB6 decreased fungal abundance and diversity; improved urease, catalase, and phosphatase activities; and decreased the cellulase activity of the rhizosphere soil. In addition, strain TB6 also promoted root growth and increased the fresh weight of ginseng roots, in addition to increasing polyphenol oxidase and catalase activities. These results may have practical implications for the use of biocontrol bacteria in ginseng plantings.

Sequence comparison of phoR, gyrB, groEL, and cheA genes as phylogenetic markers for distinguishing Bacillus amyloliquefaciens and B. subtilis and for identifying Bacillus strain B29.

Given the close genetic relationship between Bacillus amyloliquefaciens and B. subtilis, distinguishing the two solely based on their physiological and biochemical characteristics and 16S rRNA sequences is difficult. Molecular identification was used to discover suitable genes for distinguishing the two bacteria, and to identify the bio-controlling strain B29, due to molecular identification has been paid more and more attention. The similarity of four genes, cheA, gyrB, groEL and phoR, of the two species was compared by the software BLASTN and MAGA, and phylogenetic tree was constructed. The B29 strain was re-identified by using the screened genes. The similarities of the four genes, gyrB, groEL, cheA and phoR, of the two species were 93-95%, 82-84%, 76-78% and 76-77%, respectively. The homologies of the four genes of the strain B29 and the strains of B. amyloliquefaciens strains were more than 95%. We determined how well the phoR and cheA genes could be used to differentiate B. amyloliquefacien and B. subtilis. The previously isolated biological control strain B29, initially classified as B. subtilis, was re-classified as B. amyloliquefaciens. Our data indicate that other than the phoR gene, the cheA gene might be a useful phylogenetic marker for differentiating B. subtilis and B. amyloliquefaciens.

Antimicrobial peptide isolated from Bacillus amyloliquefaciens K14 revitalizes its use in combinatorial drug therapy.

The present study was performed to evaluate the antibacterial activities of an antimicrobial peptide (CSpK14) and the synergies thereof with ß-lactams against vancomycin-resistant Staphylococcus aureus (VRSA) and Enterococci (VRE). Our strain was isolated from fermented food (kimchi), which is 99.79 % homologous with Bacillus amyloliquefaciens subsp. plantarum FZB42(T). CSpK14 was purified to homogeneity by diammonium sulfate precipitation, concentration, dialysis, and followed by two-stage chromatographic separation, i.e., Sepharose Cl-6B and Sephadex G-25 chromatography, and had a molar mass of ~4.6 kDa via Tricine SDS-PAGE and in situ examination. It was stable at pH 6.0-11.5 and temperature up to 80 °C. In addition, it was also stable with various metal ions, solvents, and proteases. The N-terminal amino acid sequence was H-Y-D-P-G-D-D-S-G-N-T-G and did not show any significant homology with reported peptides. However, it shows some degrees of identity with alpha-2-macroglobulin and ligand-gated channel protein from different microorganisms. CSpK14 significantly reduced the minimum inhibitory concentrations (MICs) of ß-lactams and had no effect on non-ß-lactams against VRSA and VRE. MICs of CSpK14/oxacillin and CSpK14/ampicillin were reduced by 8- to 64-fold and 2- to 16-fold, respectively. The time killing assay between CSpK14/oxacillin (2.29-2.37 Δlog10CFU/mL at 24 h) and CSpK14/ampicillin (2.30-2.38 Δlog10CFU/mL at 24 h) being >2-fold and fractional inhibitory concentration index ˂0.5 revealed synergy. Furthermore, the biofilms formed by VRSA and VRE were reduced completely. CSpK14 was simple to purify, had low molecular mass, was stable over a wide pH range or tested chemicals, had broad inhibitory spectrum, and possessed potent synergistic antimicrobial-antibiofilm properties. CSpK14 synergistically enhanced the efficacy of ß-lactams and is therefore suitable for combination therapy.

Bacillus amyloliquefaciens SB14 from rhizosphere alleviates Rhizoctonia damping-off disease on sugar beet.

The use of biocontrol strains recently has become a popular alternative to conventional chemical treatments. A set of bacteria isolated from sugar beet rhizosphere and from roots and shoots of apple and walnut were evaluated for their potential to control sugar beet seedling damping-off caused by R. solani AG-4 and AG2-2.The results of in vitro assays concluded that three isolates, SB6, SB14, SB15, obtained from rhizosphere of sugar beet and five isolates, AP2, AP4, AP6, AP7, AP8, obtained from shoots and roots of apple were the most effective antagonists that inhibited the mycelial growth of both R. solani isolates. Combination of several biochemical tests and partial sequencing of 16S rRNA and gyrBgenes revealed that eight efficient bacterial isolates could be assigned to the genus Bacillus and all could tolerate high temperatures and salt concentrations in their vegetative growth. The potential biocontrol activity of the eight bacterial antagonists were tested in greenhouse condition. The results indicated that four strains,B. amyloliquefaciens SB14, B. pumilus SB6,B. siamensis AP2 and B. siamensisAP8 exerted a significant influence on controlling of seedling damping-off and performed significantly better than others.However, the treatment of the seeds with bacteria was most effective when the isolate SB14 was used, which significantly controlled damping-off disease by 58% caused by R. solani AG-4 and by 52.5% caused by R. solani AG-2-2. This indicates that the use of beneficial bacterial native to the host plant may increase the success rate in screening biocontrols, because these microbes are likely to be better adapted to their host and its associated environmental conditions than are strains isolated from other plant species grown in different environmental conditions. We can infer from the results reported here that sugar beet plantsmay recruitbeneficial microbes to the rhizosphere to help them solve context-specific challenges.

Antifungal Activity of Isolated Bacillus amyloliquefaciens SYBC H47 for the Biocontrol of Peach Gummosis.

The gummosis disease is caused by Botryosphaeria dothidea (Moug. ex. Fr) Ces. et de Not., and it is one of the most important diseases of stone fruits worldwide. The use of biocontrol as an alternative approach to synthetic chemical fungicides has aroused general concern about how to control plant diseases that are caused by phytopathogens. The aim of this study is to isolate Bacillus strains from raw honeys with the capacity to inhibit B. dothidea and to explore the mechanisms by which they could be used in the biocontrol of peach gummosis. Bacillus amyloliquefaciens SYBC H47 was isolated and identified on the basis of its physiological and biochemical characteristics and its 16S rRNA and gyrB gene sequences. The cell suspension and the cell-free supernatant of its culture showed significant antifungal activity against Aspergillus niger, Mucor racemosus, Fusarium oxysporum, Penicillium citrinum, and Candida albicans by agar-diffusion assays. The primary antifungal substances were bacillomycin L, fengycin, and surfactin, which were analyzed by HPLC LC/ESI-MS/MS. Bacillomycin L showed the best inhibitory effect against conidial germination of B. dothidea, followed by fengycin and surfactin. Surfactin had limited effects on mycelial growth, contrary to those of bacillomycin L and fengycin. However, a mixture of the three lipopeptides had a synergistic effect that disrupted the structure of the conidia and mycelia. In order to reduce the production cost, the use of waste frying peanut oil and soy oil as the sole carbon source increased the lipopeptide yield levels by approximately 17% (2.42 g/L) and 110% (4.35 g/L), respectively. In a field trial, the decreases in the infected gummosis rate (IGR) and the disease severity index (DSI) through cell suspension treatments were 20% and 57.5% (in 2014), respectively, and 40% and 57.5% (in 2015), respectively, in comparison with the control. In conclusion, B. amyloliquefaciens SYBC H47 could inhibit the germination of conidia and the growth of mycelia from B. dothidea; therefore, this strain behaves as a potential biocontrol agent against the gummosis disease.

Gene expression regulation in the plant growth promoting Bacillus atrophaeus UCMB-5137 stimulated by maize root exudates.

Despite successful use of Plant Growth Promoting Rhizobacteria (PGPR) in agriculture, little is known about specific mechanisms of gene regulation facilitating the effective communication between bacteria and plants during plant colonization. Active PGPR strain Bacillus atrophaeus UCMB-5137 was studied in this research. RNA sequencing profiles were generated in experiments where root exudate stimulations were used to mimic interactions between bacteria and plants. It was found that the gene regulation in B. atrophaeus UCMB-5137 in response to the root exudate stimuli differed from the reported gene regulation at similar conditions in B. amyloliquefaciens FZB42, which was considered as a paradigm PGPR. This difference was explained by hypersensitivity of UCMB-5137 to the root exudate stimuli impelling it to a sessile root colonization behavior through the CcpA-CodY-AbrB regulation. It was found that the transcriptional factor DegU also could play an important role in gene regulations during plant colonization. A significant stress caused by the root exudates on in vitro cultivated B. atrophaeus UCMB-5137 was noticed and discussed. Multiple cases of conflicted gene regulations showed scantiness of our knowledge on the regulatory network in Bacillus. Some of these conflicted regulations could be explained by interference of non-coding RNA (ncRNA). Search through differential expressed intergenic regions revealed 49 putative loci of ncRNA regulated by the root exudate stimuli. Possible target mRNA were predicted and a general regulatory network of B. atrophaeus UCMB-5137 genome was designed.

Formation of Guaiacol by Spoilage Bacteria from Vanillic Acid, a Product of Rice Koji Cultivation, in Japanese Sake Brewing.

The formation of guaiacol, a potent phenolic off-odor compound in the Japanese sake brewing process, was investigated. Eight rice koji samples were analyzed, and one contained guaiacol and 4-vinylguaiacol (4-VG) at extraordinarily high levels: 374 and 2433 µg/kg dry mass koji, respectively. All samples contained ferulic and vanillic acids at concentrations of mg/kg dry mass koji. Guaiacol forming microorganisms were isolated from four rice koji samples. They were identified as Bacillus subtilis, B. amyloliquefaciens/subtilis, and Staphylococcus gallinarum using 16S rRNA gene sequence. These spoilage bacteria convert vanillic acid to guaiacol and ferulic acid to 4-VG. However, they convert very little ferulic acid or 4-VG to guaiacol. Nine strains of koji fungi tested produced vanillic acid at the mg/kg dry mass koji level after cultivation. These results indicated that spoilage bacteria form guaiacol from vanillic acid, which is a product of koji cultivation in the sake brewing process.