Spatial and temporal population dynamics of a phyllosphere colonizing Bacillus subtilis biological control agent of sugar beet cercospora leaf spot

This study examines the spatial and temporal variation of populations of the biological control agent (BCA) BacB, a Bacillus subtilis isolate, in the field and growth chamber in the presence of the fungus, Cercospora beticola, the causal agent of Cercospora leaf spot of sugarbeet. The use of the selective BCA support substrate β-glucan, applied at 0, 0.5, and 1.0% of the spray solution, did not influence differences in total population numbers (spores + vegetative cells) of a spontaneous rifampicin resistant isolate of BacB (Rif+) over a 14 day spray period. BacB Rif+, applied as a spore formulation, declined from 10,000 CFU/cm² on day 0.5–100 CFU/cm² on day 14 at the three levels of β-glucan tested. Distribution of BacB Rif+ populations was modeled on a leaf scale, with and without β-glucan. Higher populations of vegetative cells were more likely at 14 days with 1% β-glucan than with 0% β-glucan. BacB populations were more aggregated without β-glucan than with the nutrient substrate. There was no correlation between BacB density and Cercospora leaf spot disease severity, indicating that neither antibiosis nor parasitism is likely an important mechanism of disease control.     

Life cycle and spore resistance of spore-forming Bacillus atrophaeus

Bacillus endospores have a wide variety of important medical and industrial applications. This is an overview of the fundamental aspects of the life cycle, spore structure and factors that influence the spore resistance of spore-forming Bacillus. Bacillus atrophaeus was used as reference microorganism for this review because their spores are widely used to study spore resistance and morphology. Understanding the mechanisms involved in the cell cycle and spore survival is important for developing strategies for spore killing; producing highly resistant spores for biodefense, food and pharmaceutical applications; and developing new bioactive molecules and methods for spore surface display.     

Manganese Requirement for Sporulation and Other Secondary Biosynthetic Processes of Bacillus

Concentrations of manganese, considerably in excess of the quantity required for normal vegetative growth, are needed by Bacillus for (i) synthesis of such secondary metabolites and structures as antibiotics, D-glutamyl peptide, endospores, bacteriophage, and protective antigen; and (ii) longevity of vegetative cell cultures. No other biologically active element can substitute for manganese, and no secondary biosynthetic process of Bacillus has been found in which the requirement for manganese is absent. In the present study, manganese could induce sporulation of B. megaterium even when added to synthetic broth cultures as late as 100 hr after inoculation. When sub-bactericidal concentrations of various biologically active trace elements were supplied within 2 hr of manganese addition, suppression of spore formation occurred in cultures exposed to elements of group VI (chromium, molybdenum, tungsten, selenium, tellurium) and one of group VIII (nickel); of the six interfering elements, selenium and nickel were most potent.     

Evaluation of indigenous bacterial strains for biocontrol of the frogeye leaf spot of soya bean caused by Cercospora sojina

Aims: Assessment of biological control of Cercospora sojina, causal agent of frogeye leaf spot (FLS) of soya bean, using three indigenous bacterial strains, BNM297 (Pseudomonas fluorescens), BNM340 and BNM122 (Bacillus amyloliquefaciens). Methods and Results: From cultures of each bacterial strain, cell suspensions and cell‐free supernatants were obtained and assayed to determine their antifungal activity against C. sojina. Both mycelial growth and spore germination in vitro were more strongly inhibited by bacterial cell suspensions than by cell‐free supernatants. The Bacillus strains BNM122 and BNM340 inhibited the fungal growth to a similar degree (I～52–53%), while cells from P. fluorescens BNM297 caused a lesser reduction (I～32–34%) in the fungus colony diameter. The foliar application of the two Bacillus strains on soya bean seedlings, under greenhouse conditions, significantly reduced the disease severity with respect to control soya bean seedlings and those sprayed with BNM297. This last bacterial strain was not effective in controlling FLS in vivo. Conclusions: Our data demonstrate that the application of antagonistic bacteria may be a promising and environmentally friendly alternative to control the FLS of soya bean. Significance and Impact of the Study: To our knowledge, this is the first report of biological control of C. sojina by using native Bacillus strains.     

Coupling of aromatic amines onto syringylglycerol β-guaiacylether using Bacillus SF spore laccase: A model for functionalization of lignin-based materials

The potential of Bacillus SF spore laccase for coupling aromatic amines to lignin model molecules as a way of creating a stable reactive surface was investigated. The Bacillus spore laccase was shown to be active within the neutral to alkaline conditions (pH 7–8.5) and was more resistant to common laccase inhibitors than fungal laccases. Using this enzyme, tyramine was successfully covalently coupled onto syringylglycerol β-guaiacylether via a 4-O-5 bond, leaving the –NH₂ group free for further attachment of functional molecules. This study demonstrates the potential of Bacillus SF spore laccase for application in lignocellulose surface functionalization and other coupling reactions which can be carried out at neutral to alkaline pH under extreme conditions which normally inhibit fungal laccases.     

The biocontrol agent Bacillus sp. CHEP5 primes the defense response against Cercospora sojina

Glycine max (soybean) production can be dramatically affected by frogeye leaf spot (FLS) caused by Cercospora sojina Hara. The inoculation of biocontrol agents may be an alternative strategy for C. sojina control. The native biocontrol bacterium Bacillus sp. CHEP5 reduced the severity of FLS in soybean by inducing systemic resistance. We suggest that the defense response was primed since the expression of the defense related gene GmAOS was enhanced in induced plants treated with both methyl jasmonate and C. sojina. Furthermore, as GmAOS is related to jasmonic acid biosynthesis, we assume that this phytohormone is involved in induced systemic resistance signaling defense pathway in soybean against C. sojina.     

Bacillus penetrans n. comb. Causing a virulent disease of plant-parasitic nematodes

The prokaryotic and bacterial nature of the incitant of a lethal disease of economically important plant-parasitic nematodes was confirmed by electron microscopy. The organism (formerly Duboscqia penetrans Thorne, Microsporea: Protozoa) was renamed Bacillus penetrans n. comb., Bacillaceae. B. penetrans produces an endospore typical of Bacillus spp., and its life cycle and spore morphology are similar to B. popilliae and B. lentimorbus, the “milky disease” organisms of insects. It differs from the latter pathogens in spore shape, in the dichotomously-branched colonies formed in the host pseudocoelom by nonseparating vegetative cells, and in that the spores are not known to be ingested by nematodes. Substantial reductions in Pratylenchus scribneri populations and in root-knot disease of tomato were obtained in glasshouse tests with the pathogen. Endospore production in vivo presents the first possibility of biological control of pest nematodes with inoculum of a highly specific virulent pathogen.     

Control of foliar diseases of mustard by Bacillus from reclaimed soil

Bacillus subtilis strain UK-9, an isolate from reclaimed soils, was studied for its biological control activity against Alternaria leaf spot disease of mustard. In dual culture, production of antifungal metabolites by the bacteria caused morphological alterations of vegetative cells and spores, disruption and lysis of their cell wall. The antagonist reduced spore germination on leaves and disease incidence of the pathogen in plant trial as well as it also demonstrated plant-growth-promoting ability.     

Optimizing Bacillus subtilis spore isolation and quantifying spore harvest purity

Investigating the biochemistry, resilience and environmental interactions of bacterial endospores often requires a pure endospore biomass free of vegetative cells. Numerous endospore isolation methods, however, neglect to quantify the purity of the final endospore biomass. To ensure low vegetative cell contamination we developed a quality control technique that enables rapid quantification of endospore harvest purity. This method quantifies spore purity using bright-field and fluorescence microscopy imaging in conjunction with automated cell counting software. We applied this method to Bacillus subtilis endospore harvests isolated using a two-phase separation method that utilizes mild chemicals. The average spore purity of twenty-two harvests was 88 ± 11% (error is 1σ) with a median value of 93%. A spearman coefficient of 0.97 correlating automated and manual bacterial counts confirms the accuracy of software generated data.     

Optimizing Bacillus subtilis spore isolation and quantifying spore harvest purity

Investigating the biochemistry, resilience and environmental interactions of bacterial endospores often requires a pure endospore biomass free of vegetative cells. Numerous endospore isolation methods, however, neglect to quantify the purity of the final endospore biomass. To ensure low vegetative cell contamination we developed a quality control technique that enables rapid quantification of endospore harvest purity. This method quantifies spore purity using bright-field and fluorescence microscopy imaging in conjunction with automated cell counting software. We applied this method to Bacillus subtilis endospore harvests isolated using a two-phase separation method that utilizes mild chemicals. The average spore purity of twenty-two harvests was 88 ± 11% (error is 1σ) with a median value of 93%. A spearman coefficient of 0.97 correlating automated and manual bacterial counts confirms the accuracy of software generated data.     

Peptide Ligands That Bind Selectively to Spores of Bacillus subtilis and Closely Related Species

As part of an effort to develop detectors for selected species of bacterial spores, we screened phage display peptide libraries for 7- and 12-mer peptides that bind tightly to spores of Bacillus subtilis. All of the peptides isolated contained the sequence Asn-His-Phe-Leu at the amino terminus and exhibited clear preferences for other amino acids, especially Pro, at positions 5 to 7. We demonstrated that the sequence Asn-His-Phe-Leu-Pro (but not Asn-His-Phe-Leu) was sufficient for tight spore binding. We observed equal 7-mer peptide binding to spores of B. subtilis and its most closely related species, Bacillus amyloliquefaciens, and slightly weaker binding to spores of the closely related species Bacillus globigii. These three species comprise one branch on the Bacillus phylogenetic tree. We did not detect peptide binding to spores of several Bacillus species located on adjacent and nearby branches of the phylogenetic tree nor to vegetative cells of B. subtilis. The sequence Asn-His-Phe-Leu-Pro was used to identify B. subtilis proteins that may employ this peptide for docking to the outer surface of the forespore during spore coat assembly and/or maturation. One such protein, SpsC, appears to be involved in the synthesis of polysaccharide on the spore coat. SpsC contains the Asn-His-Phe-Leu-Pro sequence at positions 6 to 10, and the first five residues of SpsC apparently must be removed to allow spore binding. Finally, we discuss the use of peptide ligands for bacterial detection and the use of short peptide sequences for targeting proteins during spore formation.     

Endospores of Sporosarcina halophila: characteristics and ultrastructure

Sporosarcina halophila forms endospores. Electron micrographs revealed ultrastructural similarity to spores of S. ureae. Spore germination indicated by loss of refractility, darkening, swelling and formation of new vegetative cells was followed by phase contrast light microscopy. To induce spore germination, the endospores needed to be heat avtivated. After activation, they were inoculated into nutrient broth medium supplemented with sea-water. Double concentrated sea-water was found to be optimal for germination. Similar to other bacterial endospores, the spores were found to be resistant to heat and ethanol. An ultraviolet absorbing substance was isolated from suspensions of free spores; it was identified to be pyridine-2,6-dicarboxylic acid (DPA) usually present in bacterial spores. DPA was detected in amounts ranging from 5–7% of the spore dry weight; it was not detected in extracts of vegetative cells.Abbreviation DPA 2,6-pyridine-dicarboxylic acid     

GidA Expression in Salmonella is Modulated Under Certain Environmental Conditions

Bacillus subtilis endospores have applications in different fields including their use as probiotics and antigen delivery vectors. Such specialized applications frequently require highly purified spore preparations. Nonetheless, quantitative data regarding both yields and purity of B. subtilis endospores after application of different growth conditions and purification methods are scarce or poorly reported. In the present study, we conducted several quantitative and qualitative analyses of growth conditions and purification procedures aiming generation of purified B. subtilis spores. Based on two growth media and different incubations conditions, sporulation frequencies up to 74.2 % and spore concentrations up to 7 × 10⁹ spores/ml were achieved. Application of a simplified spore isolation method, in which samples were incubated with lysozyme and a detergent, resulted in preparations with highly purified spores at the highest yields. The present study represents, therefore, an important contribution for those working with B. subtilis endospores for different biotechnological purposes.     

Spatial distribution and antifungal interactions of a Bacillus biological control agent on wheat surfaces

The proximity of a biological control agent and its associated anti-microbial metabolites to pathogens on plant surfaces can determine the outcome of disease control. In this study we investigated whether deficiencies in inoculum deposition and localization could explain the inability of the biological control agent Bacillus amyloliquefaciens strain TrigoCor to consistently control Fusarium head blight in the field, despite producing effective and consistent disease control in greenhouse settings. Using epifluorescent stereomicroscopy and confocal laser scanning microscopy, we visualized the coverage of wheat spike surfaces by Bacillus post-application in greenhouse and field environments, and determined that there are large unprotected areas on wheat spikes sprayed with commercial-scale field equipment, as compared to typical greenhouse applications. Additionally, we found that in conditions of low relative humidity, antifungal compounds produced by Bacillus were not able to diffuse across wheat surfaces in biologically relevant amounts, further suggesting that the inadequate coverage of wheat surfaces by Bacillus could be directly limiting disease control. Bacillus cells were easily rinsed off wheat surfaces within 8 h of application, indicating that rainfastness might be an additional limitation of biological control in field settings. Finally, we observed the inhibition of Fusarium graminearum spore germination by TrigoCor inoculum on wheat surfaces, confirming this as a mode of action for TrigoCor biocontrol. Future optimization efforts for biological control agents applied to above-ground plant parts should focus on enhancing the rainfastness, quantity, and spatial coverage of the inoculum on plant surfaces.     

Efficacy of Bacillus subtilis,Moringa oleifera seeds extract and potassium bicarbonate on Cercospora leaf spot on sugar beet

Cercospora leaf spot caused by Cercospora beticola are among the most dangerous plant diseases on sugar beet plants. It causes heavy economic losses, whether on the yield of roots, the percentage of sugar in them, or the quality of sugar produced. In addition to the economic cost caused by chemical control, these chemical pesticides cause an imbalance in the ecosystem and harm the health of humans and animals. In an attempt to search for a safer method than pesticides and environmentally friendly, an evaluation of using biocontrol agents, Bacillus subtilis as cell suspension (10⁸ cell/ml), was conducted in this study. Seeds extract of Moringa oleifera with two concentrations (25 and 50 g/L) and potassium bicarbonate at (5 and10 g/L (compared to fungicide Montoro 30% EC (Propiconazole 15% + Difenoconazole 15%). The evaluation results for twenty-five sugar beet varieties showed a significant discrepancy between these varieties in the extent of their susceptibility to infection with the disease under investigation. In-Vitro, B. subtilis induced an antagonist to C. beticola, and both M. oleifera seeds extract and potassium bicarbonate significantly reduced the linear growth of this pathogen. Under field conditions, the treatments used have given positive results in controlling Cercospora leaf spots. They significantly decreased the severity of disease and prevented C. beticola from creating conidiophores and conidiospores, along with examining their cell walls with the formation of plasmolysis of the fungus cells and reducing both the number and diameter of the spots on the surface leaves; this was demonstrated using a scanning electron microscope (SEM). It is worth noting that the best results obtained were most often when treated with M. oleifera seeds extract, followed by potassium bicarbonate, then cell suspension of B. subtilis. In addition, the percentage of the content of beet roots from total soluble solids and sucrose has improved significantly due to spraying sugar beet plants with the substances mentioned earlier. These treatments also contributed to a significant improvement in the enzymes polyphenol oxidase, peroxidase, and phenylalanine ammonia-lyase.     

Detection technologies for Bacillus anthracis: Prospects and challenges

Bacillus anthracis is a Gram-positive, spore-forming bacterium representing the etiological agent of acute infectious disease anthrax, a lethal but rare disease of animals and humans in nature. With recent use of anthrax as a bioweapon, a number of techniques have been recently developed and evaluated to facilitate its rapid detection of B. anthracis in the environment as well as in point-of-care settings for humans suspected of exposure to the pathogen. Complex laboratory methods for B. anthracis identification are required since B. anthracis has similarities with other Bacillus species and its existence in both spore and vegetative forms. This review discusses current challenges and various improvements associated with anthrax agent detection.     

Role of spore coat proteins in the resistance of Bacillus subtilis spores to Caenorhabditis elegans predation

Bacterial spores are resistant to a wide range of chemical and physical insults that are normally lethal for the vegetative form of the bacterium. While the integrity of the protein coat of the spore is crucial for spore survival in vitro, far less is known about how the coat provides protection in vivo against predation by ecologically relevant hosts. In particular, assays had characterized the in vitro resistance of spores to peptidoglycan-hydrolyzing enzymes like lysozyme that are also important effectors of innate immunity in a wide variety of hosts. Here, we use the bacteriovorous nematode Caenorhabditis elegans, a likely predator of Bacillus spores in the wild, to characterize the role of the spore coat in an ecologically relevant spore-host interaction. We found that ingested wild-type Bacillus subtilis spores were resistant to worm digestion, whereas vegetative forms of the bacterium were efficiently digested by the nematode. Using B. subtilis strains carrying mutations in spore coat genes, we observed a correlation between the degree of alteration of the spore coat assembly and the susceptibility to the worm degradation. Surprisingly, we found that the spores that were resistant to lysozyme in vitro can be sensitive to C. elegans digestion depending on the extent of the spore coat structure modifications.     

Predicting favorable conditions for early leaf spot of peanut using output from the Weather Research and Forecasting (WRF) model

Early leaf spot of peanut (Arachis hypogaea L.), a disease caused by Cercospora arachidicola S. Hori, is responsible for an annual crop loss of several million dollars in the southeastern United States alone. The development of early leaf spot on peanut and subsequent spread of the spores of C. arachidicola relies on favorable weather conditions. Accurate spatio-temporal weather information is crucial for monitoring the progression of favorable conditions and determining the potential threat of the disease. Therefore, the development of a prediction model for mitigating the risk of early leaf spot in peanut production is important. The specific objective of this study was to demonstrate the application of the high-resolution Weather Research and Forecasting (WRF) model for management of early leaf spot in peanut. We coupled high-resolution weather output of the WRF, i.e. relative humidity and temperature, with the Oklahoma peanut leaf spot advisory model in predicting favorable conditions for early leaf spot infection over Georgia in 2007. Results showed a more favorable infection condition in the southeastern coastline of Georgia where the infection threshold were met sooner compared to the southwestern and central part of Georgia where the disease risk was lower. A newly introduced infection threat index indicates that the leaf spot threat threshold was met sooner at Alma, GA, compared to Tifton and Cordele, GA. The short-term prediction of weather parameters and their use in the management of peanut diseases is a viable and promising technique, which could help growers make accurate management decisions, and lower disease impact through optimum timing of fungicide applications.     

Inactivation of Bacillus Endospores in Envelopes by Electron Beam Irradiation

The anthrax incidents in the United States in the fall of 2001 led to the use of electron beam (EB) processing to sanitize the mail for the U.S. Postal Service. This method of sanitization has prompted the need to further investigate the effect of EB irradiation on the destruction of Bacillus endospores. In this study, endospores of an anthrax surrogate, B. atrophaeus, were destroyed to demonstrate the efficacy of EB treatment of such biohazard spores. EB exposures were performed to determine (i) the inactivation of varying B. atrophaeus spore concentrations, (ii) a D₁₀ value (dose required to reduce a population by 1 log₁₀) for the B. atrophaeus spores, (iii) the effects of spore survival at the bottom of a standardized paper envelope stack, and (iv) the maximum temperature received by spores. A maximum temperature of 49.2°C was reached at a lethal dose of ~40 kGy, which is a significantly lower temperature than that needed to kill spores by thermal effects alone. AD₁₀ value of 1.53 kGy was determined for the species. A surface EB dose between 25 and 32 kGy produced the appropriate killing dose of EB between 11 and 16 kGy required to inactivate 8 log₁₀ spores, when spore samples were placed at the bottom of a 5.5-cm stack of envelopes.