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.

Novel amylomacins from seaweed-associated Bacillus amyloliquefaciens as prospective antimicrobial leads attenuating resistant bacteria.

The rise in antibiotic-resistant bacterial strains prompting nosocomial infections drives the search for new bioactive substances of promising antibacterial properties. The surfaces of seaweeds are rich in heterotrophic bacteria with prospective antimicrobial substances. This study aimed to isolate antibacterial leads from a seaweed-associated bacterium. Heterotrophic Bacillus amyloliquefaciens MTCC 12716 associated with the seaweed Hypnea valentiae, was isolated and screened for antimicrobial properties against drug-resistant pathogens. The bacterial crude extract was purified and three novel amicoumacin-class of isocoumarin analogues, 11'-butyl acetate amicoumacin C (amylomacin A), 4'-hydroxy-11'-methoxyethyl carboxylate amicoumacin C (amylomacin B) and 11'-butyl amicoumacin C (amylomacin C) were isolated to homogeneity. The studied amylomacins possessed potential activities against Pseudomonas aeruginosa, vancomycin-resistant Enterococcus faecalis, Klebsiella pneumoniae, methicillin-resistant Staphylococcus aureus, and Shigella flexneri with a range of minimum inhibitory concentration values from 0.78 to 3.12 µg/mL, although standard antibiotics ampicillin and chloramphenicol were active at 6.25-25 µg/mL. Noticeably, the amylomacin compound encompassing 4'-hydroxy-11'-methoxyethyl carboxylate amicoumacin C functionality (amylomacin B), displayed considerably greater antagonistic activities against methicillin-resistant S. aureus, vancomycin-resistant E. faecalis, Vibrio parahaemolyticus, Escherichia coli, and K. pneumoniae (minimum inhibitory concentration 0.78 µg/mL) compared to the positive controls and other amylomacin analogues. Antimicrobial properties of the amylomacins, coupled with the presence of polyketide synthase-I/non-ribosomal peptide synthetase hybrid gene attributed the bacterium as a promising source of antimicrobial compounds with pharmaceutical and biotechnological applications.

Biochemical Characterization and Functional Analysis of Heat Stable High Potential Protease of Bacillus amyloliquefaciens Strain HM48 from Soils of Dachigam National Park in Kashmir Himalaya.

A novel temperature stable alkaline protease yielding bacteria was isolated from the soils of Dachigam National Park, which is known to be inhabited by a wide variety of endemic plant and animal species of Western Himalaya. This high-potential protease producing isolate was characterized and identified as Bacillus amyloliquefaciens strain HM48 by morphological, Gram's staining and biochemical techniques followed by molecular characterization using 16S rRNA approach. The extracellular protease of B. amyloliquefaciens HM48 was purified by precipitating with ammonium sulfate (80%), followed by dialysis and Gel filtration chromatography increasing its purity by 5.8-fold. The SDS-PAGE analysis of the purified enzyme confirmed a molecular weight of about ≈25 kDa. The enzyme displayed exceptional activity in a broad temperature range (10-90 °C) at pH 8.0, retaining its maximum at 70 °C, being the highest reported for this proteolytic Bacillus sp., with KM and Vmax of 11.71 mg/mL and 357.14 µmol/mL/min, respectively. The enzyme exhibited remarkable activity and stability against various metal ions, surfactants, oxidizing agent (H2O2), organic solvents and displayed outstanding compatibility with widely used detergents. This protease showed effective wash performance by exemplifying complete blood and egg-yolk stains removal at 70 °C and efficiently disintegrated chicken feathers making it of vital importance for laundry purpose and waste management. For functional analysis, protease gene amplification of strain HM48 yielded a nucleotide sequence of about 700 bp, which, when checked against the available sequences in NCBI, displayed similarity with subtilisin-like serine protease of B. amyloliquefaciens. The structure of this protease and its highest-priority substrate ß-casein was generated through protein modeling. These protein models were validated through futuristic algorithms following which protein-protein (protease from HM48 and ß-casein) docking was performed. The interaction profile of these proteins in the docked state with each other was also generated, shedding light on their finer details. Such attributes make this thermally stable protease novel and suitable for high-temperature industrial and environmental applications.

Dye-decolorization of a newly isolated strain Bacillus amyloliquefaciens W36.

Dye-decolorization is one of the most important steps in dye-polluted wastewater treatment. The dye-decolorization bacteria were isolated from active sludge collected from wastewater treating pond of a dyeing and printing plant using serial dilution method. Among the 44 bacteria isolates from the active sludge, the strain Bacillus amyloliquefaciens W36 was found to have strong ability in dye-decolorization. The effects of carbon source, nitrogen sources, C/N, metal ions, temperature, pH, and rotation speed for dye-decolorization were investigated. The optimum decolorization conditions were that the strain was grown in enriched mineral salt medium (EMSM) using maltose 1 g/L, (NH4)2SO4 1 g/L as carbon and nitrogen source respectively, supplemented with 100 mg/L different dyes (pH 6.0), at 30 °C, 200 rpm from 48 to 96 h. The bacteria could aerobically decolorize dyes, such as Coomassie brilliant blue (95.42%), Bromcresol purple (93.34%), Congo red (72.37%) and Sarranine (61.7%), within 96 h. The dyes decolorization products were analyzed by ultra-violet and visible (UV-vis) spectroscopy before and after decolorization, which indicated that the four dyes were significantly degraded by the strain. The results indicated that the bacteria Bacillus amyloliquefaciens W36 could be used in dye-polluted wastewater treatment.

Seaweed-associated heterotrophic bacteria: new paradigm of prospective anti-infective and anticancer agents.

Ever since the development of the first antibiotic compound with anticancer potential, researchers focused on isolation and characterization of prospective microbial natural products with potential anti-infective and anticancer activities. The present work describes the production of bioactive metabolites by heterotrophic bacteria associated with intertidal seaweeds with potential anti-infective and anticancer activities. The bacteria were isolated in a culture-dependent method and were identified as Shewanella algae MTCC 12715 (KX272635) and Bacillus amyloliquefaciens MTCC 12716 (KX272634) based on combined phenotypic and genotypic methods. Further, the bacteria were screened for their ability to inhibit drug-resistant infectious pathogens and prevent cell proliferation of human liver carcinoma (HepG2) and breast cancer (MCF7) cell lines, without affecting the normal cells. Significant anti-infective activity was observed with bacterial cells and their organic extracts against broad-spectrum multidrug-resistant pathogens, such as vancomycin-resistant Enterococcus faecalis, methicillin-resistant Staphylococcus aureus, Klebsiella pneumonia and Pseudomonas aeruginosa with minimum inhibitory concentration ≤ 3.0 µg mL-1 as compared to the antibiotic agents' chloramphenicol and ampicillin, which were active at ≥ 6.25 mg mL-1. The extracts also exhibited anticancer activity in a dose-responsive pattern against HepG2 (with IC50, half maximal inhibitory concentration ~ 78-83 µg mL-1) and MCF7 (IC50 ~ 45-48 µg mL-1) on tetrazolium bromide screening assay with lesser cytotoxic effects on normal fibroblast (L929) cell lines (IC50 > 100 µg mL-1). The results revealed that seaweed-associated heterotrophic bacteria could occupy a predominant role for a paradigm shift towards the development of prospective anti-infective and anticancer agents.

Bacillus amyloliquefaciens TL106 protects mice against enterohaemorrhagic Escherichia coli O157:H7-induced intestinal disease through improving immune response, intestinal barrier function and gut microbiota.

AIMS: This study evaluated the effects of Bacillus amyloliquefaciens TL106, isolated from Tibetan pigs' faeces, on the growth performance, immune response, intestinal barrier function, morphology of jejunum, caecum and colon, and gut microbiota in the mice with enterohaemorrhagic Escherichia coli (EHEC)-induced intestinal diseases. METHODS AND RESULTS: In all, 40 female C57BL/6J mice were randomly divided into four groups: mice fed a normal diet (Control), mice oral administration of TL106 daily (Ba), mice challenged with EHEC O157:H7 on day 15 (O157) and mice oral administration of TL106 daily and challenged with EHEC O157:H7 on day 15 (Ba+O157). The TL106 was administrated to mice for 14 days, and mice were infected with O157:H7 at day 15. We found that TL106 could prevent the weight loss caused by O157:H7 infection and alleviated the associated increase in pro-inflammatory factors (TNF-α, IL-1ß, IL-6 and IL-8) and decrease in anti-inflammatory factor (IL-10) in serum and intestinal tissues of mice caused by O157:H7 infection (P < 0·05). Additionally, TL106 could prevent disruption of gut morphology caused by O157:H7 infection, and alleviate the associated decrease in expression of tight junction proteins (ZO-1, occludin and claudin-1) in jejunum and colon (P < 0·05). In caecum and colon, the alpha diversity for bacterial community analysis of Chao and ACE index in Ba+O157 group were higher than O157 group. The TL106 stabilized gut microbiota disturbed by O157:H7, including increasing Lachnospiraceae, Prevotellaceae, Muribaculaceae and Akkermansiaceae, and reducing Lactobacillaceae. CONCLUSIONS: We indicated the B. amyloliquefaciens TL106 can effectively protect mice against EHEC O157:H7 infection by relieving inflammation, improving intestinal barrier function, mitigating permeability disruption and stabilizing the gut microbiota. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacillus amyloliquefaciens TL106 can prevent and treat intestinal disease induced by EHEC O157:H7 in mice, which may be a promising probiotic for disease prevention in animals.

Optimization of spore laccase production by Bacillus amyloliquefaciens isolated from wastewater and its potential in green biodecolorization of synthetic textile dyes.

The spore laccase enzyme production by B. amyloliquefaciens was optimized. It was characterized and tested for its textile dye decolorization potential. LB medium was found to be the most promising growth medium with addition of glucose (1-2%), yeast extract (0.1%), FeCl3 (0.01 mM) and MnCl2 (0.001 mM). The optimum spore laccase production was at pH 8, 30 °C, 1:5 medium to air ratio, 2% inoculum size and 7 days incubation. The characterization study of the enzyme showed the maximum activity at 60 °C and pH 6-7.5. It was induced by Ca+2, Mg+2, Fe+3, Zn+2, Cu+2 and Na+ at 1 mM concentration. Also, it was stable in the presence of methanol, ethanol, acetone and chloroform. In addition, it enhanced about 34% by 5 mM H2O2 and it was nearly stable at 10-20 mM H2O2. Furthermore, mediators such as ABTS, syrengaldazine and 2, 6 dimethyl phenol enhanced the spore laccase activity. The spore laccase enzyme efficiently decolorized direct red 81 and acid black 24 after 24 h. Phytotoxicity of the direct red 81 solution after decolorization by tested spore laccase was lower than that of the untreated dye solution. Finally, this study added a promising spore laccase candidate for ecofriendly and cost-effective dye wastewater bio-decolorization.

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.

Potential role of compost mixed biochar with rhizobacteria in mitigating lead toxicity in spinach.

Consumption of heavy metals, especially lead (Pb) contaminated food is a serious threat to human health. Higher Pb uptake by the plant affects the quality, growth and yield of crops. However, inoculation of plant growth-promoting rhizobacteria (PGPR) along with a mixture of organic amendments and biochar could be an effective way to overcome the problem of Pb toxicity. That's why current pot experiment was conducted to investigate the effect of compost mixed biochar (CB) and ACC deaminase producing PGPR on growth and yield of spinach plants under artificially induced Pb toxicity. Six different treatments i.e., control, Alcaligenes faecalis (PGPR1), Bacillus amyloliquefaciens (PGPR2), compost + biochar (CB), PGPR1 + CB and PGPR2 + CB were applied under 250 mg Pb kg-1 soil. Results showed that inoculation of PGPRs (Alcaligenes faecalis and Bacillus amyloliquefaciens) alone and along with CB significantly enhanced root fresh (47%) and dry weight (31%), potassium concentration (11%) in the spinach plant. Whereas, CB + Bacillus amyloliquefaciens significantly decreased (43%) the concentration of Pb in the spinach root over control. In conclusion, CB + Bacillus amyloliquefaciens has the potential to mitigate the Pb induced toxicity in the spinach. The obtained result can be further used in the planning and execution of rhizobacteria and compost mixed biochar-based soil amendment.

A chemical screening method for menaquinone-producing strains based on HPLC-UV technology.

Despite menaquinones (MKs)-4 and - 7 being known to have extensive biological activities and applications, less attention has been paid to the other MKs. Thus, to obtain a range of MKs to further explore their pharmacological activities, structure-activity relationships, and applications, a chemical screening method for MK-producing strains was established based on high-performance liquid chromatography-ultraviolet (HPLC-UV) technology. Considering that Bacillus strains have proven to be an important MK-producing bioresource, twenty-nine putative Bacillus isolates previously sought from a fermented soybean sample were used for the validation of the chemical screening method, which ultimately led to the discovery of sixteen MK-producing strains. Among them, Bacillus subtilis DC-1 presented excellent ability to produce MKs. Another, a purchased strain of B. amyloliquefaciens was discovered to be an MK-producing strain. These results indicated this screening method was simple and highly efficient for the discovery of MK-producing strains, especially those producing a range of MK structures.

Characterization and synthetic biology of lipase from Bacillus amyloliquefaciens strain.

Lipases with high tolerance to temperature play a significant role in industry from food manufacturing to waste management systems. Thus, there is a need to investigate these enzymes from different geographical areas to look out for a more thermo-stable one. Characterization of lipases through experimental approaches is time consuming process and sometimes the results are ambiguous due to errors. However, integration of computational technologies is quite useful for prediction of optimized conditions. Such technologies can be applied as synthetic biology, which has many major applications in engineered biological approaches for accurate prediction of effects of different physical and chemical parameters on the system. In this study, cloning and expression of a lipase gene from Bacillus amyloliquefaciens, isolated from a novel geographical region of Pakistan, in Escherichia coli DH5α cells followed by sequencing was carried out. To isolate thermostable lipase producing strains, all the samples were kept at 50 °C. Genomic DNA was isolated and signal peptide (1-32 residues) sequence was chopped (ΔSPLipase). The ΔSPLipase was amplified and expressed in Linearized p15TV-L vector. The purified lipase appeared as single band of approximately 26 kDa. Suitable conditions of factors required for maximum lipase activity such as temperature, pH, substrate, organic solvent, detergents and metal ions were predicted through synthetic biology approach and further confirmed in wet lab. The predicted suitable factors for enzyme were almost similar to those determined experimentally. The optimum enzyme activity was recorded at pH 8 and 50 °C temperature. Interestingly, the activity of enzyme was found on a number of solvents, metal ions, detergents, and surfactants. The predicted optimum values and their experimental confirmations highlights the importance of integrated synthetic biology approaches in wet lab experiments. The characterized lipase of B. amyloliquefaciens at molecular level from Pakistani strains displayed good activity on a range of factors that implies this strain to be used for application in industrial level production.

Plant growth promoting and antifungal activity in endophytic Bacillus strains from pearl millet (Pennisetum glaucum).

Bacterial endophytes are well known inhabitants of living plant system and perform important assignments in maintaining plant growth and health. Currently, limited reports are available on the endophytes of pearl millet (Pennisetum glaucum) reflecting antagonistic and plant growth promoting (PGP) attributes. Therefore, the major objectives of current investigation were to identify antagonistic strains of endophytic Bacillus from pearl millet and further illustrate their PGP capabilities. In this study, 19 endophytic Bacillus strains (EPP5, EPP21, EPP30, EPP32, EPP35, EPP42, EPP49, EPP55, EPP62, EPP65, EPP70, EPP71, EPP74, EPP78, EPP83, EPP86, EPP93, EPP100, and EPP102) displaying antagonistic activity towards Rhizoctonia solani (RS), Sclerotium rolfsii (SR), and Fusarium solani (FS) were isolated from different sections (root, leaf, stem, and root) of pearl millet. Phenotypic (shape, colony, gram staining reaction, endospore formation, and motility) and biochemical features (catalase, oxidase, citrate, gelatinase, urease, Voges Proskauer's, methyl red, indole, and nitrate reduction), along with the similarly comparison of 16S rRNA gene sequence with type strains identified eight antagonistic endophyhtes as B. amyloliquefaciens (EPP35, EPP 42, EPP62, and EPP 102), Bacillus subtilis subsp. subtilis (EPP65), and Bacillus cereus (EPP5, EPP71, and EPP74). The production of indole acetic acid and siderophores was varied among the isolated endophytes. Besides displaying enzymatic activities, these isolates varied in solubilizing capabilities of phosphate, potassium, and zinc. The presence of three antimicrobial peptide genes (ituD, bmyC, and srfA) also confirmed their antifungal nature. Further, single treatment of three promising strains (EPP5, EPP62, and EPP65) offered protection ranging from 35.68 to 45.74% under greenhouse conditions. However, microbial consortium (EPP5+ EPP62 + EPP65) provided the highest protection (71.96%) against root rot and wilt infection with significant increase in plant biomass. Overall, the current study indicated that pearl millet plant harbors various species of endophytic Bacillus that possess excellent biocontrol and growth promotion activities.

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.

Aphicidal activity of Bacillus amyloliquefaciens strains in the peach-potato aphid (Myzus persicae).

Myzus persicae Sulzer (Hemiptera: Aphididae), is a generalist cosmopolitan insect that infests more than 400 plant species of 40 different families and is one of the major pests infesting potato crops. It causes direct damage and also spread plant viruses. The intensive use of synthetic insecticide to control aphids has led to resistant populations. Therefore, there is a need to develop biopesticides for effective control that minimizes environmental hazards. The bacteria Bacillus amyloliquefaciens is recognized as a producer of a variety of bioactive compounds. The aim here was to evaluate the aphicidal effect of B. amyloliquefaciens strains, CBMDDrag3, PGPBacCA2, and CBMDLO3, and their metabolites on the mortality and fecundity of M. persicae. Cells suspensions, heat-killed cell suspensions, cell-free supernatants, or isolated lipopeptide fractions from B. amyloliquefaciens strains were offered to aphids through artificial diets. The isolated lipopeptide fractions composed mainly of kurstakins, surfactins, iturins, and fengycins, when were administrated through diets, had no aphicidal effect against M. persicae. However, aphids fed on diets with whole cell suspensions and its cell-free supernatant of all three bacteria strains resulted in 100% mortality of adult aphids and nymphs. Specially, B. amyloliquefaciens CBMDLO3, has an effective aphicidal effect on M. persicae, used both bacterial cells and their metabolites. Moreover, heat-killed cells of B. amyloliquefaciens CBMDLO3 also had aphicidal action, although the aphid mortality was lower than on diet with living bacteria. Therefore, these results propose that B. amyloliquefaciens, could function as a novel eco-friendly biopesticide for the control of M. persicae.

Di-n-butyl phthalate degrading endophytic bacterium Bacillus amyloliquefaciens subsp. strain JR20 isolated from garlic chive and its colonization in a leafy vegetable.

Di-n-butyl phthalate (DBP) is one of the primary PAEs (phthalate acid esters) pollutants. DBP can be absorbed by plants and threaten human health via the food chain. Some DBP-degrading bacteria have been successfully isolated from the environment (water, soil, etc.). However, only a few DBP-degrading plant endophytes have been isolated. In this study, an endophytic bacterium, Bacillus amyloliquefaciens subsp. strain JR20, which was found capable of degrading DBP, was isolated from garlic chive. We found that strain JR20 metabolized 89.74% of DBP at a 5 mg/L concentration within 4 d in liquid mineral salts medium (MSM). The optimized conditions for maximum removal of DBP were as follows: DBP concentration, 5 mg/L; pH, 7-8; temperature, 30-40 °C. The colonization of strain JR20 significantly improved the degradation rate of DBP in the roots, stems and leaves of leafy vegetables.

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.

Screening, plant growth promotion and root colonization pattern of two rhizobacteria (Pseudomonas fluorescens Ps006 and Bacillus amyloliquefaciens Bs006) on banana cv. Williams (Musa acuminata Colla).

Banana is the second largest export crop in Colombia. To meet the demand of international markets, high amounts of chemical fertilizers are required, which represent high costs and can be hazardous to the environment. Plant growth promoting rhizobacteria (PGPR) can, at least partially, replace chemical fertilizers. In this paper, we evaluated the effect of nine PGPR of the genera Bacillus and Pseudomonas on banana growth. Banana seedlings were produced through tissue culture and acclimatized in the greenhouse core. Plants were inoculated with the rhizobacteria and growth parameters (plant height, leaf number, leaf area, pseudostem thickness, root and shoot fresh weight, root and shoot dry weight) were assessed after 55 days. The two best performing PGPR, Bs006 and Ps006 previously identified as Bacillus amyloliquefaciens and Pseudomonas fluorescens, respectively, promoted banana growth similarly or even slightly superior to 100% chemical fertilization, and were selected for further characterization of root colonization by both eletron microscopy and confocal microscopy of fluorescence in situ hybridization (FISH)-stained root tissues. Both P. fluorescens Ps006 and B. amyloquifaciens Bs006 showed ability to colonize banana roots, but Bs006 appeared faster than Ps006 in the colonization dynamics. This work demonstrated that inoculation of rhizobacteria Bacillus amyloliquefaciens Bs006 and Pseudomonas fluorescens Ps006 could partially replace the chemical fertilization of tissue cultured banana plants, and therefore could be used for the formulation of a new biofertilizer.

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.