Biological control of pre-harvest aflatoxin contamination in groundnut (Arachis hypogaea L.) with Bacillus subtilis G1

The antagonistic activity of Bacillus subtilis strain G1 was tested against various isolates of Aspergillus flavus in vitro. A talc-based powder formulation of B. subtilis strain G1 was prepared and evaluated to control A. flavus infection and aflatoxin B1 contamination in groundnut under greenhouse and field conditions. The results showed that B. subtilis strain G1 could inhibit the growth of all isolates of A. flavus tested in dual culture assay and the growth inhibition ranged from 93 to 100%. Results of greenhouse and field experiments indicated that B. subtilis strain G1 when applied to groundnut as seed treatment and soil application significantly suppressed A. flavus population in the soil, A. flavus infection and aflatoxin B1 content in kernels and increased the pod yield. These studies show that B. subtilis strain G1 has potential as a biocontrol agent for control of aflatoxin contamination in groundnut.     

Integrated control of Colletotrichum gloeosporioides on mango fruit in Taiwan by the combination of Bacillus subtilis and fruit bagging

Anthracnose disease caused by Colletotrichum gloeosporioides in Jingkwang mango grown in Taiwan was significantly reduced by the integration of fruit bagging with either B. subtilis strain LB5 or fungicides. The combined treatments were most effective in reducing early infection during the 2004 season, leading to 56.4 and 58.3% reduction, respectively, while in 2003 reduction accounted for 51 and 52.3%, respectively. Post-harvest application of B. subtilis strain LB5 cell suspensions on fruits already treated by bagging, bagging+LB5 and baggingfungicides in the field reduced anthracnose incidence significantly at all tested concentrations. These results indicate that biocontrol efficacy of B. subtilis LB5 may be due to the prevention of early fruit infection, thereby reducing significantly anthracnose incidence in ripening fruits to much lower levels than those obtained by using a conventional single post-harvest treatment.     

Biological control of anthracnose (Colletotrichum gloeosporioides) in yam by Streptomyces sp.MJM5763

Aim: To find a suitable biocontrol agent for yam anthracnose caused by Colletotrichum gloeosporioides. Methods and Results: An actinobacterial strain, MJM5763, showing strong antifungal activity, multiple biocontrol and plant growth‐promoting traits was isolated from a yam cultivation field in Yeoju, South Korea. Based on morphological and physiological characteristics and analysis of the 16S rDNA sequence, strain MJM5763 was identified as a novel strain of Streptomyces and was designated as Streptomyces sp. MJM5763. Treatment with MJM5763 and the crude culture filtrate extract (CCFE) was effective in suppressing anthracnose in detached yam leaves in vitro and reduced incidence and severity of anthracnose in yam plants under greenhouse conditions. The CCFE treatment was the most effective of all the treatments and reduced the anthracnose severity by 85–88% and the incidence by 79–81%, 90 days after inoculation with the pathogen. CCFE treatment was also effective under field conditions and showed a reduction of 86 and 75% of anthracnose severity and incidence, respectively. Conclusion: Streptomyces sp. strain MJM5763 was effective in biocontrolling anthracnose in yam caused by C. gloeosporioides. Significance and Impact of the Study: Streptomyces sp. MJM5763 is a potential alternative to chemical fungicides for reducing yield losses to anthracnose in yam.     

Emergence of resistance to glutaraldehyde in spores of Bacillus subtilis 168

Abstract The emergence of resistance to glutaraldehyde in spores of Bacillus subtilis 168 was examined. Resistance to an organic solvent (toluene), heat and lysozyme were included for comparison. A sequential development of resistance was observed, with toluene resistance occuring early on in sporulation (stages III and IV), thermal resistance at early stage V, lysozyme resistance at middle stage V and glutaraldehyde resistance arising late in stage V. Studies with sporulation mutants also indicate that glutaraldehyde resistance is acquired even later than lysozyme resistance and may therefore possibly be considered as a very late marker event for sporulation, characterizing late stages of B. subtilis 168 spore formation.     

A cell wall protein (YqgA) is genetically related to the cell wall-degrading dl-endopeptidases in Bacillus subtilis

The Gram-positive bacterium Bacillus subtilis has a thick cell wall. The cell wall contains various proteins, both for secretion and for peptidoglycan (PG) maintenance. Penicillin-binding proteins for PG synthesis, PG hydrolases (autolysins), and regulator proteins for the autolysins are the known components of the PG maintenance system. YqgA was identified as an abundant protein attached to the cell wall of B. subtilis through a proteomics analysis. The YqgA protein was localized at cell division sites during the transition period between the exponential and the stationary phases. YqgA localization was affected by mutations in the dl-endopeptidases (DLEPases), which are the autolysins involved in cell morphogenesis. Furthermore, yqgA mutations on a background of defective DLEPases led to delays in cell growth and cell morphological changes. These results demonstrate that yqgA is genetically related to the genes encoding DLEPases involved in cell morphogenesis.     

AbrB is a regulator of the sigma(W) regulon in Bacillus subtilis

The Bacillus subtilis global regulator AbrB was found to negatively control expression of sigW and genes of the sigma(W) regulon. AbrB bound to DNA regions in the autoregulatory sigW promoter and to some, but not all, of the other sigma(W)-dependent promoters in B. subtilis. Defects in antibiotic resistance properties caused by spo0A mutations are at least partially correlated with AbrB repression of the sigma(W) regulon.     

Biological control of postharvest diseases of apples,peaches and nectarines by Bacillus subtilis S16 isolated from halophytes rhizosphere

Penicillium expansum, Botrytis cinerea and Colletotrichum acutatum are the most common postharvest pathogens of apples, peaches and nectarines. In this study, 96 bacteria were isolated from halophytes rhizosphere and assayed for biocontrol activity under in vitro conditions. Among the 96 isolates tested, isolate S16 effectively inhibited the growth of P. expansum, B. cinerea and C. acutatum. The isolate S16 has reduced 78.33±1.53 to 82.98±2.13% of disease severity in apples, peaches and nectarines. Matrix-assisted laser desorption ionisation-time of flight mass spectrometry of the antifungal compounds revealed three lipopeptide complexes, namely surfactins, iturins and fengycins. Lipopeptides and hydrolytic enzymes produced by the isolate S16 play an important role in the antifungal activity. Polymerase chain reaction analysis using ituD, srfAD, fenD and fenE gene-specific primers showed that the isolate S16 carry sequences similar to ituD, srfAD, fenD and fenE genes. Based on the 16S rDNA sequencing, the effective isolate S16 was identified as Bacillus subtilis.     

Immunomodulatory effects of Bacillus subtilis (natto) B4 spores on murine macrophages

To investigate the immunomodulatory effects of Bacillus subtilis (B. subtilis) (natto) B4 spores on murine macrophage, RAW 264.7 cells were cultured alone or with B subtilis (natto) B4 spores at 37°C for 12 hrs, then both cells and culture supernatants were collected for analyses. Exposure of RAW 264.7 cells to B. subtilis (natto) B4 spores had no significant effects on macrophage viability and amounts of extracellular lactate dehydrogenase (LDH). However, it remarkably increased the activities of acid phosphatase (ACP), lactate dehydrogenase (LDH) and inducible nitric oxide synthase (iNOS) in cells and the amounts of nitric oxide (NO) and cytokines (tumor necrosis factor‐alpha, interferon‐gamma, interleukin [IL]‐1 beta, IL‐6, IL‐12, IL‐10 and macrophage inflammatory protein‐2) in culture supernatants. These results demonstrate that B. subtilis (natto) B4 spores are harmless to murine macrophages and can stimulate their activation through up‐regulation of ACP and LDH activities and enhance their immune function by increasing iNOS activity and stimulating NO and cytokine production. The above findings suggest that B. subtilis (natto) B4 spores have immunomodulatory effects on macrophages.     

Assessment of potential antagonists for anthracnose (Colletotrichum gloeosporioides) disease of mango (Mangifera indica L.) in North Western Ethiopia (Pawe)

Mango (Mangifera indica L.) is considered as one of the most popular fruits among millions of people in the tropical area and increasingly in the developed countries. Anthracnose, caused by the fungus Colletotrichum gloeosporioides, is the most important pre- and post-harvest disease of mango. The objective of this research was to evaluate the prevalence of different promising antagonistic Trichoderma and Bacillus spp. on phyloplane of mango in Ethiopia and to evaluate their antagonistic potential against the pathogen. A total of 19 mango fields were surveyed and anthracnose affected all fields. Culture studies on potato dextrose agar for evaluation of antibiosis activity of Trichoderma spp. and Bacillus spp. revealed that they have inhibitory and lytic effect on C. gloeosporioides, which is an indication of their potential biocontrol agent for management of mango anthracnose as an alternative to chemical control. Significant differences (p?<?0.05) were observed among Bacillus isolates in causing lysis of pathogen mycelium, when inoculated on actively growing colony of C. gloeosporioides. Maximum reduction in growth rate of pathogen was observed with Bacillus spp. (B50), which restricted the growth to 2.7?mm compared to 8.3?mm in the control with 67.5% efficacies. There were similar effects (p?<?0.05) among Trichoderma spp. in formation of inhibition zones and lysis by varying degrees up to 59.7% efficacies in reducing linear growth of the pathogen in dual culture.     

Bacillus subtilis AB1 controls Aeromonas infection in rainbow trout (Oncorhynchus mykiss, Walbaum)

AIM: To develop a probiotic with effectiveness against Aeromonas sp., which was pathogenic to rainbow trout. METHODS AND RESULTS: When Bacillus subtilis AB1, which was obtained from fish intestine, was administered for 14 days to rainbow trout in feed at a concentration of 10(7) cells per gram either as viable, formalized or sonicated cells or as cell-free supernatant, the fish survived challenge with the pathogen. AB1 stimulated immune parameters, specifically stimulating respiratory burst, serum and gut lysozyme, peroxidase, phagocytic killing, total and alpha1-antiprotease and lymphocyte populations. CONCLUSIONS: Bacillus subtilis AB1 was effective as a probiotic at controlling infections by a fish-pathogenic Aeromonas sp. in rainbow trout. SIGNIFICANCE AND IMPACT OF THE STUDY: Disease control in fish is possible by means of the oral application of live and inactivated cells and their subcellular components with the mode of action reflecting stimulation of the innate immune response.     

Expression of chloramphenicol acetyltransferase in Bacillus subtilis under the control of a phytoplasma promoter

A cloned putative promoter region upstream of the 16S rRNA gene of the western X-disease phytoplasma was inserted behind the promoterless chloramphenicol acetyltransferase gene of plasmid pPL603. The DNA construct was used to transform Bacillus subtilis cells. The transformants were assayed for chloramphenicol acetyltransferase activity, showing that the phytoplasma promoter is efficiently expressed in a B. subtilis background.     

A Mechanistic Study of Au(III) Removal from Solution by Bacillus subtilis

Bacteria–Au interactions control the fate of Au in a variety of geologic systems. Although previous studies have determined that non-metabolizing Bacillus subtilis cells can remove Au(III) from solution via cell surface adsorption reactions, and that upon removal Au(III) is rapidly reduced to Au(I) and remains bound to the cell surface, the mechanism of Au(III) removal by B. subtilis is poorly understood. This study provides further constraints on the mechanisms responsible for Au(III) removal by B. subtilis by conducting batch Au(III) removal experiments as a function of pH and Au loading (Au:biomass ratio) using biomass with and without two different types of treatment: (1) a treatment to remove extracellular polymeric substances (EPS) from the biomass, and (2) a treatment to irreversibly block surface sulfhydryl sites from Au binding. The experimental results suggest that Au(III) removal can be attributed primarily to Au complexation with bacterial sulfhydryl sites, but that Au–amino binding is also important under some conditions. Our experiments also suggest that Au–sulfhydryl binding occurs predominantly on EPS molecules produced by B. subtilis, and that Au–amino binding is also important and is located within the bacterial cell envelope. These findings are the first to constrain the location of sulfhydryl-binding sites for B. subtilis biomass, and they are the first to demonstrate the important role played by bacterial EPS in the process of Au adsorption and reduction by bacteria.     

Characterization of the 5' subtilisin (aprE) regulatory region from Bacillus subtilis

The poly(ADP-ribose) polymerase-like thermozyme purified from Sulfolobus solfataricus was characterised with respect to some physico-chemical properties. The archaeal protein exhibited a scarce electrophoretic mobility at both pH 2.9 and pH 7.5. Determination of the isoelectric point (pI=7.0-7.2) allowed us to understand the reason for the limited migration at pH 7.5, while amino acid composition analysis showed a moderate content of basic residues, which reduced mobility at pH 2.9. With respect to the charge, the archaeal enzyme behaved differently from the eukaryotic thermolabile poly(ADP-ribose) polymerase, described as a basic protein (pI=9.5). Well known inhibitors of the mesophilic polymerase like Zn(2+), nicotinamide and 3-aminobenzamide exerted a smaller effect on the enzyme from S. solfataricus, reducing the activity by at most 50%. Mg(2+) was a positive effector, although in a dose-dependent manner. It influenced the fluorescence spectrum of the archaeal protein, whereas NaCl had no effect.     

Purification, characterization and functional analysis of an endo-arabinanase (AbnA) from Bacillus subtilis

Bacillus subtilis synthesizes at least one arabinanase encoded by the abnA gene that is able to degrade the polysaccharide arabinan. Here, we report the expression in Escherichia coli of the full-length abnA coding region with a His6-tag fused to the C-terminus. The recombinant protein was secreted to the periplasmic space and correctly processed by the E. coli signal peptidase. The substrate specificity of purified AbnA, the physico-chemical properties and kinetic parameters were determined. Functional analysis studies revealed Glu 215 as a key residue for AbnA hydrolytic activity and indicated that in addition to AbnA B. subtilis secretes other enzyme(s) able to degrade linear 1,5-alpha-l-arabinan.     

The effects of growth conditions on cell wall composition and cell morphology in a temperature-sensitive tag-B mutant of Bacillus subtilis

The relationship between wall anionic polymer synthesis and cell morphology has been studied in Bacillus subtilis 168 and its temperature-sensitive tagB mutant strain BR19-200B. The amount and type of anionic polymer synthesized varied under different growth conditions, as did the morphology of the bacteria. Anionic polymer synthesis was affected by the phosphate supply. It was also found that teichuronic acid synthesis was temperature-sensitive in wild-type bacteria. Teichuronic acid synthesis was affected by the tagB lesion, previously thought to affect only teichoic acid synthesis. A relationship was observed between synthesis of the alternative polymers, such that suppression of teichuronic acid synthesis is accompanied by an increase in the synthesis of teichoic acid. Variation in anionic polymer content was accompanied by variation in cell shape. Differences in shape were related to differences in total anionic polymer rather than to differences in individual polymer type.     

Analysis of non-polar deletion mutations in the genes of the spo0K (opp) operon of Bacillus subtilis

The spo0K (opp) operon of Bacillus subtilis encodes an oligopeptide permease that is required for uptake of oligopeptides, development of genetic competence, and initiation of sporulation. We made in-frame, non-polar deletion mutations in each of the first four genes of the five-gene spo0K operon and tested effects on oligopeptide transport, sporulation, and expression of competence genes. spo0KA, B, C, and D were required for sporulation, competence development, and oligopeptide transport. Disruption of spo0KE caused a less severe phenotype than did disruption of any of the other genes of the operon.     

Efficient production of menaquinone (vitamin K2) by a menadione-resistant mutant of Bacillus subtilis

Efficient production of menaquinone (MK) by Bacillus subtilis was achieved. An edible strain of B. subtilis, isolated from the traditional Japanese food natto, was mutated to improve MK productivity. A menadione-resistant mutant producing 30% more MK than its parent strain was obtained. Soybean extract and glycerol were the best nitrogen and carbon sources, respectively, among the sources tested. Addition of yeast extract also increased MK productivity. The maximum concentration of MK reached about 35.0 mg/l after 4 days of culture in a jar fermenter. The pH of the medium decreased to 5.5 after the start of cultivation, then spontaneously increased to 7.7–8.0. This pH change might be important in the production of MK because only small amounts of MK were obtained when pH was controlled at 5.7, 6.0, 7.0, 7.5 or 8.0. Journal of Industrial Microbiology & Biotechnology (2001) 26, 115–120. Received 24 April 2000/ Accepted in revised form 14 August 2000