Bacillus based biocontrol on Brassica

Many bacterial strains have been shown to mediate protection to biotic stress and promote growth of plants. Different bacteria can mediate protection in different ways e.g. by inhibition, competition or increasing plant resistance. Examples of bacteria that mediate protection to plants include different Pseudomonas, Serratia and Bacillus strains. Bacillus strains have one major advantage toward other biocontrol strains and that is the ability to form spores that are resilient against chemicals and mechanical damage. I have studied the effect of four closely related Bacillus strains on plants in two different projects, one concerned with oilseed rape (Brassica napus) and the other using Arabidopsis thaliana to allow mechanistic studies of the interaction. The bacterial strains are all classified as Bacillus amyloliquefaciens. These bacterial strains have been tested for phenological effects on plants and for plant protection towards pathogens like Alternaria brassicae, Botrytis cinerea, Leptosphaeria maculans, and Verticillium longisporum. Production of antifungal compounds by the strains and the effects on the different pathogens were investigated. Two potential candidates for biocontrol were identified. Both Bacillus strains were found to provide significant protection of oilseed rape against the four pathogens. The effects of Bacillus treatment on the B. napus transcriptome were studied using the cDNA-AFLP technique. Bacillus priming had strong systemic effects on leaf transcripts but small effects on roots. This far 65 differentially expressed plant genes have been identified due to Bacillus treatment, of which many seem related to metabolism. The effect of Bacillus seed treatment has also been studied on Arabidopsis. Significant protection was achieved also here using the same two strains toward Alternaria and Leptosphaeria as well as Pseudomonas syringae as pathogens. Arabidopsis signalling mutant studies showed that functional jasmonic acid (JA) and ethylene (Et) signalling as well as Npr1 were needed for Bacillus biocontrol. Expression levels of marker genes depending on these signalling pathways showed no increase upon Bacillus treatment, while an increase of the JA dependent marker occurred after Bacillus treated plants were infected by P. syringae. Altogether, Bacillus primed biocontrol seems to be based on induced systemic resistance (ISR)

Bacillus as Siderophore and Iron-bioremoval Bacteria

ome Bacillus strains can produce siderophore. Siderophore is a chelating agent for ferric iron as a response to low iron environment. Bacillus has ability as iron bioremoval. The aim of this research was to get siderophore Bacillus strain which could resist to iron and to know the ability of its bioremoval. This research used Bacillus isolated from Kalimas Surabaya ie: A6, DA11, and SS19. The strains were screened for siderophore bacteria in Fe-CAS agar medium. Ferric bioreduction was analysed on medium contained FeCl3.6H2O 50; 100; and 150 mg/L. Ferric bioremoval was measured by Atomic Absorption Spectroscopy method. Bacillus A6, DA11, and SS19 could produce siderophore and also stand to media containing 150 mg/L FeCl3.6H2O. Bacillus DA11 had the highest ability of ferric bioremoval, which was 26.841 mg/L from 33.365 mg/L concentration, with efficiency 80.5%

Developing Genetic Tools for Geobacillus

Bacillus and Geobacillus are the primary bacterium used in biotechnology industries due to their ability to excrete extracellular materials such as proteins, enzymes and other byproducts. Bacillus subtilis, has a well-characterized competence machinery, giving way to characterized genetic tools. However, a significant problem associated with working with Bacillus is the regulation of endospore formation. An alternative to Bacillus is Geobacillus, a thermophilic bacterium. Thermophiles offer significant advantages over other bacteria as host organisms in biofuel, bioremediation, and biocatalyst processes. However, the genetic tools and competency associated with Geobacillus is still unknown. Through, genetic engineering the ability of ComK to induce competency in Geobacillus is being studied. By inducing competency in this species biotechnology industries can be revolutionized

Evaluation of the Antimicrobial Activity of Endophytic Bacterial Populations From Chinese Traditional Medicinal Plant Licorice and Characterization of the Bioactive Secondary Metabolites Produced by Bacillus atrophaeus Against Verticillium dahliae

Endophytic bacteria associated with medicinal plants possess unique strategies that enhance growth and suvival of host plants, many of which are mediated by distinctive secondary metabolites. These bacteria and their secondary metabolites are important subjects for both basic and applied research aimed at sustainable agriculture. In the present study, 114 endophytic strains isolated from the wild ethnomedicinal plant Glycyrrhiza uralensis (licorice) were screened for their in vitro antimicrobial activities against common fungal pathogens of tomato (Fusarium oxysporum f. sp., Fulvia fulva, Alternaria solani), cotton (Fusarium oxysporum f. sp. Vesinfectum, Verticillium dahliae), pomegranite (Ceratocystis fimbriata), Cymbidinium (Colletotrichum gloeosporioides), and Tsao-ko (Pestalotiopsis microspora and Fusarium graminearum) and the common bacteria Staphylococcus aureus, Bacillus cereus, Salmonella enteritidis, and Escherichia coli. Several Bacillus strains, particularly Bacillus atrophaeus and Bacillus mojavensis, had a broad spectrum of antifungal and antibacterial activity. A total of 16 strains, selected based on broad antimicrobial activity, were shown to contain at least one putative secondary metabolite-encoding gene (i.e., polyketide synthase or non-ribosomal peptide synthetase) and/or one lytic enzyme (i.e., protease, cellulase, lipase, chitinase), which may be important mediators of antagonistic activity against pathogens. Five strains, representing Bacillus atrophaeus and Bacillus mojavensis, were selected for plant growth chamber experiments based on strong in vitro antifungal activities. All five strains significantly reduced disease severity in Arabidopsis thaliana plants challenged with V. dahlia infection. Gas-chromatography/mass-spectrometry analysis of cell-free extracts of Bacillus atrophaeus strain XEGI50 showed that at least 13 compounds were produced only during co-cultivation with V. dahlia, including putative compounds known to have antimicrobial activity, such as 1,2-benzenedicarboxylic acid, bis (2-methylpropyl) ester; 9,12-octadecadienoic acid (Z,Z)-, methyl ester; 9-octadecenoic acid, methyl ester, (E)-; and decanedioic acid, bis(2-ethylhexyl) ester. To our knowledge, this study is the first to report that bacteria isolated from G. uralensis have biocontrol abilities. Our findings provide new insights into the antimicrobial activities of natural endophytes, particularly B. atrophaeus, and suggest this species may a promising candidate as a biocontrol agent to confer resistance to Verticillium wilt disease and other phytopathogens in cotton and other crops

Ecology and thermal inactivation of microbes in and on interplanetary space vehicle components

The experiments conducted to determine the heat resistance of Bacillus megaterium ATCC 6458 at 90 and 100 C were completed. Estimates from replicate experiments at eight percent relative humidities (less than 0.001 to 100% RH) for each temperature were computed. A Bacillus cereus strain with high heat resistance was cultured and the resistance determined in phosphate buffer (D sub 121.1 = 2.16 min and z = 8.7 C). The profile of the dry heat resistance of B. megaterium is summarized and the most resistant condition to the three spores (Bacillus subtilis var. niger, ATCC 29669, and Bacillus stearothermophilus, strain 1518) is compared

Impact of bacteria and yeast with probiotic properties on performance, digestibility, health status and gut environment of growing pigs in Vietnam

This thesis aimed to evaluate the effects of six lactic acid bacteria (LAB) strains and Bacillus subtilis H4, Saccharomyces boulardii Sb, and a microbial enzyme mixture, supplemented to basal diets on the performance, diet digestibility, health status, and gut environment of growing pigs under Vietnamese conditions. The results showed that three different 3-strain-LAB complexes, comprising combinations of Enterococcus faecium 6H2, Lactobacillus acidophilus C3, Pediococcus pentosaceus D7, L. plantarum 1K8 and L. plantarum 3K2 improved performance, digestibility, health status and gut environment of the LAB-supplemented piglets in the first two weeks post-weaning (Per I), but not in the following 3 weeks (Per II). Adding an LAB strain (L. fermentum NC1) alone or combined with the Bacillus, or combined with a Bacillus and yeast complex, to a 3-strain-LAB complex showed improvements in the performance, diet digestibility, health status and gut environment of the probiotics-supplemented piglets in both Per I and II. The inclusion of Bacillus resulted in higher nutrient digestibility, and the addition of Saccharomyces showed improvements in the scouring scores of the piglets. Supplementation with either a microbial enzyme mixture alone or a combination of a 3-strain-LAB complex and yeast in a weaner diet improved the performance and diet digestibility in piglets in Per I. In Per II, no changes in performance or digestibility were found in enzymes-fed piglets, while improved performance and diet digestibility were obtained in piglets fed the LAB-yeast diet. There was lack of response of piglets to this enzyme mixture when these enzymes were supplemented to the diet that contained the complex of LAB and yeast in both Per I and II. Dietary supplementation with the combination of Bacillus, Saccharomyces and 4-strain-LAB complex had positive effects on performance and digestibility in grower pigs, but not in finisher pigs, while supplementation with the Bacillus alone or combined with Saccharomyces did not affect the performance and digestibility in grower and finisher pigs. The results of these studies suggest that combinations of suitable strains of Bacillus, Saccharomyces and LAB can be used as an alternative to antibiotic feed additives in pig production under the conditions of Vietnam

Glutamate biosynthesis in Bacillus azotofixans. 15N NMR and enzymatic studies

Pathways of ammonia assimilation into glutamic acid in Bacillus azotofixans, a recently characterized nitrogen-fixing species of Bacillus, were investigated through observation by NMR spectroscopy of in vivo incorporation of 15N into glutamine and glutamic acid in the absence and presence of inhibitors of ammonia-assimilating enzymes, in combination with measurements of the specific activities of glutamate dehydrogenase, glutamine synthetase, glutamate synthase, and alanine dehydrogenase. In ammonia-grown cells, both the glutamine synthetase/glutamate synthase and the glutamate dehydrogenase pathways contribute to the assimilation of ammonia into glutamic acid. In nitrate-grown and nitrogen-fixing cells, the glutamine synthetase/glutamate synthase pathway was found to be predominant. NADPH-dependent glutamate dehydrogenase activity was detectable at low levels only in ammonia-grown and glutamate-grown cells. Thus, B. azotofixans differs from Bacillus polymyxa and Bacillus macerans, but resembles other N2-fixing prokaryotes studied previously, as to the pathway of ammonia assimilation during ammonia limitation. Implications of the results for an emerging pattern of ammonia assimilation by alternative pathways among nitrogen-fixing prokaryotes are discussed, as well as the utility of 15N NMR for measuring in vivo glutamate synthase activity in the cell

Understanding the Evolutionary Relationships and Major Traits of \u3cem\u3eBacillus\u3c/em\u3e through Comparative Genomics

Background: The presence of Bacillus in very diverse environments reflects the versatile metabolic capabilities of a widely distributed genus. Traditional phylogenetic analysis based on limited gene sampling is not adequate for resolving the genus evolutionary relationships. By distinguishing between core and pan-genome, we determined the evolutionary and functional relationships of known Bacillus. Results: Our analysis is based upon twenty complete and draft Bacillus genomes, including a newly sequenced Bacillus isolate from an aquatic environment that we report for the first time here. Using a core genome, we were able to determine the phylogeny of known Bacilli, including aquatic strains whose position in the phylogenetic tree could not be unambiguously determined in the past. Using the pan-genome from the sequenced Bacillus, we identified functional differences, such as carbohydrate utilization and genes involved in signal transduction, which distinguished the taxonomic groups. We also assessed the genetic architecture of the defining traits of Bacillus, such as sporulation and competence, and showed that less than one third of the B. subtilis genes are conserved across other Bacilli. Most variation was shown to occur in genes that are needed to respond to environmental cues, suggesting that Bacilli have genetically specialized to allow for the occupation of diverse habitats and niches. Conclusions: The aquatic Bacilli are defined here for the first time as a group through the phylogenetic analysis of 814 genes that comprise the core genome. Our data distinguished between genomic components, especially core vs. pan-genome to provide insight into phylogeny and function that would otherwise be difficult to achieve. A phylogeny may mask the diversity of functions, which we tried to uncover in our approach. The diversity of sporulation and competence genes across the Bacilli was unexpected based on previous studies of the B. subtilis model alone. The challenge of uncovering the novelties and variations among genes of the non-subtilis groups still remains. This task will be best accomplished by directing efforts toward understanding phylogenetic groups with similar ecological niches