Greenhouse and field trials of the bacterial antagonists in pelletformulations to suppress sheath blight of rice caused by Rhizoctoniasolani

Bacterial formulations, produced using both Bacillus megaterium andB. pumilus individually with pharmaceutical technology, were testedunder both greenhouse and field conditions. In the greenhouse testing,some bacterial formulations, for instance For 7 minus Lac and For 16 minusLac, performed as well as freshly prepared bacterial antagonists insuppress sheath blight disease. In the field testing, For 16 minus Lac wasnot effective in suppressing sheath blight development. Failure of the For16 minus Lac to suppress sheath blight disease in the field trial may be dueto the dilution and inactivation of antibiotics produced by B.megaterium in the aquatic environment in the rice field and climaticconditions during the formulation application.     

Molecular characterization of a lipase-producing <Emphasis Type="Italic">Bacillus pumilus</Emphasis> strain (NMSN-1d) utilizing colloidal water-dispersible polyurethane

Bacillus pumilus strain NMSN-1d isolated from polyurethane-contaminated water was found to grow in high salt concentration (NaCl 10%, w/v) and degrade Impranil-DLN, water-dispersible polyurethane. The genetic relatedness of the isolate has been established by standard molecular biological techniques and the enzyme(s) involved in polyurethane degradation were also studied. A total of nine bacterial strains were isolated from polyurethane-polluted sites and characterized by conventional, microbiological and biochemical methods. These isolates were subjected to 16S ribosomal RNA gene amplification by PCR using specific primers. The genetic relatedness of the isolates was also ascertained by ribotyping and BLAST analysis of the 16S ribosomal RNA gene sequences. The bacterial isolates were grown in yeast extract-salts minimal broth medium supplemented with water-dispersible polyurethane (Impranil DLN) as a sole source of carbon. The promising isolate utilizing polyurethane and producing lipase was identified as Bacillus pumilus NMSN-1d. The polyurethane degradation has been studied in polyurethane-Rhodamine-B and Luria-Bertani-polyurethane plate assays. The activity of hydrolytic enzymes such as lipase and esterase was confirmed on 2xYT-olive oil and tributyrin-Tween 20 plate assay. The newly isolated Bacillus pumilus appears promising in the management of polyurethane waste and in production of industrially important enzymes.     

Biocontrol of <Emphasis Type="Italic">Meloidogyne javanica</Emphasis> by <Emphasis Type="Italic">Rhizobium</Emphasis> and plant growth-promoting rhizobacteria on lentil

Biocontrol of the root-knot nematode Meloidogyne javanica was studied on lentil using plant growth-promoting rhizobacteria (PGPR) namely Pseudomonas putida, P. alcaligenes, Paenibacillus polymyxa and Bacillus pumilus and root nodule bacterium Rhizobium sp. Pseudomonas putida caused greater inhibitory effect on the hatching and penetration of M. javanica followed by P. alcaligenes, P. polymyxa and B. pumilus. Inoculation of any PGPR species alone or together with Rhizobium increased plant growth both in M. javanica-inoculated and -uninoculated plants. Inoculation of Rhizobum caused greater increase in plant growth than caused by any species of plant growth-promoting rhizobacteria in nematode-inoculated plants. Among PGPR, P. putida caused greater increase in plant growth and higher reduction in galling and nematode multiplication followed by P. alcaligenes, P. polymyxa and B. pumilus. Combined use of Rhizobium with any species of PGPR caused higher reduction in galling and nematode multiplication than their individual inoculation. Use of Rhizobium plus P. putida caused maximum reduction in galling and nematode multiplication followed by Rhizobium plus P. alcaligens. Pseudomonas putida caused greater root colonization and siderophore production followed by P. alcaligenes, P. polymyxa and B. pumilus. Analysis of the protein bands of these four species by SDS-PAGE revealed that P. putida had a different protein band profile compared to the protein profiles of P. alcaligenes, P. polymyxa and B. pumilus. However, the protein profiles of P. acaligenes, P. polymyxa and B. pumilus were similar.     

Toxic Bacillus pumilus from indoor air, Recycled Paper Pulp,Norway Spruce, Food Poisoning Outbreaks and Clinical Samples

Forty-four B. pumilus isolates of food poisoning, clinical, environmental and industrial origins were investigated for toxin production using the boar spermatozoan motility assay, previously shown to be a sensitive method for detecting non-protein toxins from B. cereus and B. licheniformis. The three toxic isolates originated from live tree, indoor air and recycled paper pulp and were more toxic than the previously described food poisoning isolates of B. licheniformis, whereas the B. pumilus food poisoning and clinical isolates were lower in toxicity. The type strain also produced inhibitory substances. The toxic substances were insensitive to heat (100 °C, 20 min), to pH 2 or pH 10 and to digestion with pronase. The substances were readily soluble in methanol and chloroform, but less soluble in toluene. Exposure of boar spermatozoa to 1–10 μg ml^–1 (EC₅₀) of methanol soluble substance from the four strains disrupted the plasma membrane permeability barrier, induced abnormalities in the postacrosomal sheath, collapsed the mitochondrial and suppressed cytoplasmic NAD reduction. No change was observed in human peripheral blood lymphocytes exposed to concentrations of B. pumilus extract that affected spermatozoa. The toxin producing isolates were 99.4 to 99.6% similar in 16SrDNA (500 bp) to the type strain and could not be distinguished from the 41 non-toxic isolates by biochemical properties or whole cell fatty acid composition.     

Bacterial Antagonist as Seed Treatment to Control Leaf Blight Disease of Bambara Groundnut (Vigna subterranea)

Summay Soil samples were taken from 48 fields in the southern part of Thailand in which either bambara groundnut (Vigna subterranea) or groundnut (Arachis hypogeae) had been planted. Bacillus spp. were isolated using soil dilution plates and heat treatment to screen for endospore-producing bacteria. Among 342 Bacillus spp. isolates tested, 168 isolates were not antagonistic to Bradyrhizobium sp. strain NC-92 using dual culture technique. Further testing found 16 isolates of Bacillus spp. had the ability to inhibit mycelial growth of Rhizoctonia solani, a causal agent of leaf blight of bambara groundnut. Among these isolates, Bacillus spp. isolate TRV 9-5-2 had the greatest activity in anti-microbial tests against R. solani. This isolate was later identified as B. firmus. A powder formulation of B. firmus was developed by mixing bacterial endospores, talcum, sodium carboxymethylcellulose (SCMC) and polyvinylpyrolidone (PVP). The formulations contained bacterial levels ranging from 10⁸ to 10¹⁰ c.f.u./g and the viability of bacteria in all formulations remained high after 1 year storage at room temperature (26–32 °C). All formulations showed satisfactory effectiveness in vitro in suppressing mycelial growth of R. solani using dual culture technique. The application of formulations as seed treatment showed that these formulations did not cause abnormality of seedling shape and had no effect on the germination of bambara groundnut seeds.     

Keratinolytic activity of Bacillus megaterium F7-1, a feather-degrading mesophilic bacterium

The aim of this study was to investigate environmental conditions affecting chicken feather degradation and keratinolytic enzyme production by Bacillus megaterium F7-1, a feather-degrading mesophilic bacterium. B. megaterium F7-1 degraded whole chicken feather completely within 7 days. The bacterium grew with an optimum at pH 7.0–11.0 and 25–40 °C, where maximum keratinolytic activity was also observed. The production of keratinolytic enzyme by B. megaterium F7-1 was inducible with feather. Keratinolytic enzyme production by B. megaterium F7-1 at 0.6% (w/v) skim milk was 468 U/ml, which was about 9.4-fold higher than that without skim milk. The amount of keratinolytic enzyme production depended on feather concentrations. The degradation rate of autoclaved chicken feathers by cell-free culture supernatant was 26% after 24 h of incubation, but the degradation of untreated chicken feathers was unsuccessful. B. megaterium F7-1 effectively degraded feather meal, duck feather and human nail, whereas human hair and sheep wool showed relatively low degradation rates. B. megaterium F7-1 presented high keratinolytic activity and was very effective in feather degradation, providing potential use for biotechnological processes of keratin hydrolysis.     

Plant growth promotion abilities and formulation of <Emphasis Type="Italic">Bacillus megaterium</Emphasis> strain B 388 (MTCC6521) isolated from a temperate Himalayan location

Bacillus megaterium strain B388, isolated from rhizosphere soil of pine belonging to a temperate Himalayan location has been characterized. The plant growth promotion and biocontrol properties of the bacterium have been evaluated through petridish and broth based assays. The isolate solubilized tricalcium phosphate under in vitro conditions; maximum activity (166 μg/ml) was recorded at 28°C after 15 days of incubation. Production of indole acetic acid demonstrated in broth assays was another important plant growth promoting character. The bacterium produced diffusible and volatile compounds that inhibited the growth of two phytopathogens viz. Alternaria alternata and Fusarium oxysporum. The carrier based formulations of the bacterium resulted in increased plant growth in bioassays. The rhizosphere colonization and the viability of the cells entrapped in alginate beads were greater in comparison to coal or broth based formulations. The bacterium showed maximum similarity with Bacillus megaterium by 16S rRNA analysis.     

Water-soluble granules containing <Emphasis Type="Italic">Bacillus megaterium</Emphasis> for biological control of rice sheath blight: formulation,bacterial viability and efficacy testing

Endospores of B. megaterium were formulated in granule formulations with sodium alginate, lactose and polyvinylpyrrolidone (PVP K-30) by the wet granulation technique. The granule formulation exhibited good physical characteristics, such as high-water solubility and optimal viscosity, that would be suitable for spray application. The bacteria remained viable in the dry granule formulation at 10⁹ c.f.u./g after 24 months storage at room temperature. Under laboratory conditions, aqueous solutions of the formulation showed high activity against mycelial growth of R. solani (99.64 ± 0.14% mycelial inhibition). High viability of the bacterial antagonist on leaf sheath and leaf blade at day 7 after spraying with the formulation was observed (approximately 10⁶ c.f.u./g of plant). Application of an equivalent number of un-formulated endospores resulted in much loss of the bacterial endospores even 1 day after application. In a small pilot field study, an aqueous solution of the formulation (3%w/v) applied by spraying at days 1, 5 and 10 after pathogen inoculation of the rice plants was more effective in suppressing rice sheath blight disease than one application of a fungicide (Iprodione) at day 1. Additionally, rice plants sprayed with the aqueous solution of the granule formulation had higher panicle and whole kernel weights than those of fungicide-treated and control (untreated) plants.     

Quantification of indole-3-acetic acid from plant associated <Emphasis Type="Italic">Bacillus</Emphasis> spp. and their phytostimulatory effect on <Emphasis Type="Italic">Vigna radiata</Emphasis> (L.)

Sixteen Bacillus strains isolated from rhizosphere, histoplane and phyllosphere of different plant species were identified by 16S rDNA gene sequencing and evaluated for in vitro auxin production as well as growth stimulation of Vigna radiata (L.) Wilczek. Auxin production by Bacillus spp. in L-broth medium supplemented with 1,000 μg ml⁻¹ L-tryptophan ranges from 0.60 to 3.0 μg IAA ml⁻¹ as revealed by gas chromatography and mass spectrometric (GC–MS) analysis. Rhizospheric isolates exhibit relatively more IAA synthesis than histoplane and phyllosphere isolates. Plant microbe interaction experiments conducted under gnotobiotic conditions recorded 55.55, 46.46 and 46.20% increase in shoot length with Bacillus megaterium MiR-4, B. pumilus NpR-1 and B. subtilis TpP-1, respectively, over control. Bacillus inoculations also increased shoot fresh weight with B. megaterium MiR-4 (60.94%) and B. pumilus NpR-1 (37.76%). Highly significant positive correlation between auxin production analyzed by GC–MS and shoot length (r = 0.687**, P = 0.01) and shoot fresh weight (r = 0.703**, P = 0.01) was noted under gnotobiotic conditions. Similarly, significant correlation was also found between auxin production by Bacillus spp. (GC–MS analysis) and different growth parameters such as shoot length (r = 0.495*, P = 0.05), number of pods (r = 0.498*, P = 0.05) and grain weight (r = 0.537*, P = 0.05) at full maturity under natural wire house conditions. Results showed that auxin production potential of plant associated Bacillus spp. can be effectively exploited to enhance the growth and yield of V. radiata.     

土壤中可编码乌头酸异构酶的芽胞杆菌菌株筛选及鉴定

【目的】以芽胞杆菌(Bacillus)为筛选对象,分离土壤中可编码乌头酸异构酶(aconitate isomerase,AI)的革兰氏阳性(Gram positive,G+)菌株,以丰富对AI分布的科学认识,为其生物学功能研究奠定理论与材料基础。【方法】采用土样高温预处理法、含反式乌头酸(trans-aconitic acid,TAA)唯一碳源的ACO固体平板培养法,结合16S rDNA基因序列同源性分析,筛选能够编码AI的芽胞杆菌目的菌株。【结果】共分离得到22株能够利用TAA碳源的细菌菌株,成功鉴定了其中的16株,分别为巨大芽胞杆菌(Bacillus megaterium) 2株,阿氏芽胞杆菌(Bacillus aryabhattai) 7株,短小芽胞杆菌(Bacillus pumilus) 1株,未鉴定到种的芽胞杆菌(Bacillus sp.) 6株;且它们所含AI编码基因与已知AI基因在序列上存在差异。【结论】首次证明可编码AI的芽胞杆菌细菌种类具有多样性,暗示G+细菌广泛编码AI的可能性,更新了AI几乎只在G–细菌中分布的观点,为后续深入挖掘AI基因及其生物学功能研究提供更多可用微生物资源。     

Conjugal transfer of enterococcal transposons in Bacillus megaterium

The conjugative enterococcal transposons Tn916 and Tn919 were introduced into Bacillus megaterium by a filtermating technique. The transfer frequencies obtained ranged from 1.3×10^-6 to 6.6×10^-7. The transposons integrated stably into the B. megaterium chromosome. Tn916 could generate auxotrophs and was transferred from B. megaterium Tn916 transconjugants to other species.     

Typification of seabeach amaranth, Amaranthus pumilus (Amaranthaceae)

Amaranthus pumilus is known from coastal Massachusetts to South Carolina and from ballast in Philadelphia, Pennsylvania. It is currently listed by the United States Fish and Wildlife Service as a threatened species.Amaranthus pumilus was originally described by Rafinesque in 1808 from southern New Jersey, but he did not cite specimens. Here, the nameA. pumilus is lectotypified using the only known original element, a Rafinesque specimen at the herbarium of The Academy of Natural Sciences, Philadelphia (PH).     

Speciation of <Emphasis Type="Italic">Bacillus</Emphasis> spp. in honey produced in Northern Ireland by employment of 16S rDNA PCR and automated DNA sequencing techniques

Phenotypic speciation of foodborne Bacillus spp. remains problematic in terms of obtaining a reliable identification. In this study, we wished to identify several bacterial isolates from honey produced in Northern Ireland, and which belonged to the genus Bacillus, through employment of a molecular identification scheme based on PCR amplification of universal regions of the 16S rRNA operon in combination with direct automated sequencing of the resulting amplicons. Seven samples of honey and related materials (propolis) were examined microbiologically and were demonstrated to have total viable counts (TVC) ranging from <100 to 1700 colony-forming units/g. No yeasts or filamentous fungi were isolated from the honey materials. Several bacterial isolates were identified using this method, yielding two different genera (Paenibacillus and Bacillus), as well as four Bacillus species, namely Bacillus pumilus, B. licheniformis, B. subtilis and B. fusiformis, with B. pumilus the most frequently identified species present. When the use of molecular identification methods is justified, employment of partial 16S rDNA PCR and sequencing provides a valuable and reliable method of identification of Bacillus spp. from foodstuffs and negates associated problems of conventional laboratory and phenotypic identification.     

Evidence of an association between poly(3-hydroxybutyrate) accumulation and phosphotransbutyrylase expression in<Emphasis Type="Italic"> Bacillus megaterium</Emphasis>

Molecular analysis of a genomic region of Bacillus megaterium, a polyhydroxybutyrate (PHB)-producing microorganism, revealed the presence of a gene coding for the enzyme phosphotransbutyrylase (Ptb). Enzyme activity was measured throughout the different growth phases of B. megaterium and was found to correlate with PHB accumulation during the late-exponential growth phase. Ptb expression was repressed by glucose and activated by the branched amino acids isoleucine and valine. Overexpression of Act_Bm, a ⁵⁴ regulator from B. megaterium whose gene is located upstream from ptb, caused an increase in Ptb activity and PHB accumulation in B. megaterium.     

Evaluation of microbial strains for linoleic acid hydroxylation and reclassification of strain ALA2

In previous studies, a new microbial strain ALA2 was isolated which produced many new products from linoleic acid [Gardner H.W., Hou C.T., Weisleder D. and Brown W. 2000. Lipids 35: 1055–1060; Hou C.T. 1998. 12,13,17-Trihydroxy-9(Z)-Octodecenoic acid and derivatives and microbial isolate for production of the acid. US Patent No. 5, 852, 196]. Strain ALA2 was preliminary identified as Clavibacter sp. based on its physiological and fatty acid profiles. To determine if strain ALA2 is the optimal strain for industrial applications, other related strains were screened for their abilities to convert linoleic acids. Two strains from Clavibacter and 20 type strains from the phylogenetically related genus Microbacterium were studied. Surprisingly, all of these strains tested showed very little or no activity in converting linoleic acid. On reexamination of the identification of strain ALA2, the sequence of the 16S ribosomal RNA gene of ALA2 was found to be 99% identical to that of Bacillus megaterium and the strain was also found to have 76.3% DNA homology to the B. megaterium type strain. Therefore, strain ALA2 is now reclassified as B. megaterium. Screening of 56 strains of B megaterium strains showed that many of them were able to produce reasonable amounts of hydroxyl fatty acids from linoleic acid, although strain ALA2 possessed the greatest activity.     

Effect of Carbon Source on the Antimicrobial Activity of the Air Flora

Summary In an attempt to screen for air flora producing new potent antimicrobial substances, Bacillus megaterium NB-3, Bacillus cereus NB-4, Bacillus cereus NB-5, Bacillus subtilis NB-6 and Bacillus circulans NB-7, were isolated and were found to be antagonistic to bacteria and/or fungi. Production of antimicrobial substances by the bacterial strains was greatly influenced by variation of carbon sources. Glycerol strongly enhanced the antimicrobial activity of strains NB-3 and NB-6, whereas glucose increased the antimicrobial activity of strains NB-4 and NB-5. The maximum antibiotic yield of NB-7 was achieved with fructose as a carbon source. Starch (Bacillus megaterium NB-3), maltose (Bacillus cereus NB-5), glycerol (Bacillus circulans NB-7), arabinose, ribose (Bacillus cereus NB-4) and arabinose, fructose, glucose, ribose and sucrose (Bacillus subtilis NB-6) repressed the production of antimicrobial substances by the respective bacterial strains.     

The effects of traits of Bacillus megaterium onseed and root colonization and their correlation withthe suppression of Rhizoctonia root rot of soybean

Bacillus megaterium strainB153-2-2 is a potential bacterial biocontrol agentagainst Rhizoctonia solani isolate 2B12(ISG-2B). To study the role of antagonism (Ant),chemotaxis (Che), motility (Mot), and sporulation(Spo) of the biocontrol agent during seed and rootcolonization and the correlation between rootcolonization and the suppression of soybean (Glycine max) root rot caused by R. solani,strain B153-2-2(Che⁺Mot⁺Ant⁺⁺Spo⁺⁺) and the sevenderived mutants with altered antagonism, chemotaxis,motility, and/or sporulation were used. The bacterialcells were introduced into soil separately either asa soybean seed coating or soil application. Two soilmixtures defined as coarse and fine soil were used. The bacterial cell chemotactic response to soybeanroot and seed exudates and antagonism to R.solani were significantly (p = 0.05) correlatedwith root and seed colonization in some but not alltreatments. The sporulation-defective mutants had lowcell populations immediately after application and,therefore, reduced root colonization. The differencesin root colonization diminished among the mutants andstrain B153-2-2 when R. solani was present inthe soil or, as seedlings grew older. Soybean seedlingroots grown in coarse soil had significantly greatercolonization by B153-2-2 or its mutants and a lowerdisease index than that in fine soil. There was asignificant positive correlation (r ² = 0.78)between root colonization by strain B153-2-2 or itsmutants and suppression of Rhizoctonia root rot.     

Biochemical characterisation of grain mould resistant and susceptible genotypes and PGPR-induced resistance in the host to Curvularia lunata and Fusarium proliferatum

Resistance to biotic stresses in plants is either due to the presence of preformed biochemical compounds or induced in response to external stimulus. In this study, 13 grain mould resistant and seven susceptible lines of sorghum were analysed for biochemical defence mechanism. The levels of total phenols and phenylalanine ammonia lyase were almost same in the resistant and susceptible genotypes. However, two additional isoforms of peroxidase were found in the three of the 13 resistant genotypes. The isoform peroxidase corresponding to the R _f value of 0.25 was found in the resistant genotypes IS 13969, ICSB 377 and IS 8219-1, and two genotypes IS 13969 and ICSB 377 had an additional isoform corresponding to the R _f value of 0.32. The results indicated the genotype specific association of peroxidases with grain mould resistance in sorghum. Nine bacterial strains (Bacillus pumilus SB 21, Bacillus megaterium HiB 9, Bacillus subtilis BCB 19, Pseudomonas plecoglossicida SRI 156, Brevibacterium antiquum SRI 158, B. pumilus INR 7, P. fluorescens UOM SAR 80, P. fluorescens UOM SAR 14, B. pumilus SE 34) were tested to induce systemic resistance in sorghum cultivars 296B and Bulk Y against the highly pathogenic grain mould pathogens Curvularia lunata and Fusarium proliferatum, respectively. The bacterial isolates were effective in inducing resistance in sorghum. Among the strains tested, SRI 158 was found highly effective in reducing grain mould severity in both the genotypes.     

Sequential anaerobic–aerobic degradation of munitions waste

A sequential anaerobic–aerobic biodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) was studied. The results demonstrated that: (i) a complete degradation of RDX was achieved within 20 days using a consortium of bacteria from a wastewater activated sludge, (ii) RDX degradation did not occur under aerobic conditions alone, (iii) RDX-degrading bacterial strain that was isolated from the activated sludge completely degraded RDX within 2 days, and (iv) RDX- induced protein expressions were observed in the RDX-degrading bacterial strain. Based on fatty acid composition and a confirmation with a 16S rRNA analysis, the RDX-degrading bacterial strain was identified as a Bacillus pumilus—GC subgroup B.     

短小芽胞杆菌的益生作用及其生物降解功能

短小芽胞杆菌(Bacillus pumilus)是一种重要的微生物资源,能够分泌活性较强的代谢产物,在农业、工业、医药等领域具有良好的应用前景.就目前短小芽胞杆菌对动植物的抗菌、抗病毒、改善农作物品质等益生作用及其生物降解功能进行综述,总结并分析降解过程中可能存在的问题,为进一步研究短小芽胞杆菌的生物学功能及其在多个领...