离子注入选育高效植物病原真菌拮抗菌JA

Bacillus subtitles JA能够对多种植物病原菌如小麦赤霉菌、西瓜枯萎病菌、黄瓜黑星、油菜菌核、小麦白粉等有良好的抑制效果,本文利用离子注入诱变筛选高效植物病原真菌拮抗菌并且对JA菌株的发酵特性作了进一步的研究,获得了两株高效拮抗菌。其发酵水平的抑菌圈直径从16.62mm提高到21.23mm,经传代实验表明,其遗传性较为稳定。     

枯草芽孢杆菌B-332产抗稻瘟病菌的活性物质分析

枯草芽孢杆菌B-332能够产生具有稻瘟病菌拮抗活性的物质。通过等电点法提取该抗菌液粗提物,并利用高效液相色谱-质联用仪对粗提物进行分离纯化,共得到5个组分,它们的质荷比(m/z)分别为1 045.0、1 057.8、1 072.3、1 017.5和1 031.1,是Bacillomycin D(C14-C17)的同系物(质荷比(m/z)为1 017.5的除外),它们结构间相差1个或几个-CH2-基团;各组分均对稻瘟病菌有抑制作用,其中质荷比(m/z)为1 045.0、1 057.8、1 072.3组分的抑菌效果最强。致畸作用试验表明,这3种组分的致畸作用均为使稻瘟病菌的附着孢膨大破裂,与B-332菌株的致畸作用相同,证实了这3种组分是B-332菌株产生具有抑制稻瘟病菌作用的主要活性成分。     

Diarrhoeal enterotoxin production by strains of Bacillus thuringiensis isolated from commercial Bacillus thuringiensis-based insecticides

Abstract Strains of Bacillus cereus and B. thuringiensis were tested by the Tecra VIA kit for the ability to produce a diarrhoeal enterotoxin. The strains of B. thuringiensis were isolated from commercial B. thuringiensis -based insecticides (Bactimos^TM, DiPel^TM, Florbac^TM FC, Foray^TM 48B, Novodor^TM FC, Turex^TM, VecTobac^TM, XenTari^TM). The production of diarrhoeal enterotoxin varied by a factor of more than 100 among the different strains tested. B. cereus (F4433/73) produced the highest amount of enterotoxin and the B. thuringiensis strain isolated from DiPel^TM the lowest. The products were tested for their content of diarrhoeal enterotoxin and all products, except MVP^TM which does not contain viable B. thuringiensis spores, contained diarrhoeal enterotoxins. The results indicates an potential risk for gastroenteritis outbreak caused by B. thuringiensis .     

Achieving improved control of foliar diseases of sesame (Sesamum indicum L.) intercropped with maize (Zea mays L.) through foliar spray of plant extracts

Field trials were conducted to evaluate the effect of foliar spray of aqueous extracts of Tithonia diversifolia or Ocimum gratissimum on Cercospora leaf spot (CLS) and Alternaria leaf blight (ALB) diseases of sesame intercropped with maize. Spraying regime was at 2 weeks interval from 3 weeks after planting (WAP) until 12 WAP. Extracts of T. diversifolia or O. gratissimum reduced the incidence and severity of both diseases. CLS incidence and severity as well as defoliation was significantly (p < 0.05) reduced below what obtained in the unsprayed intercrop. ALB lesion size was significantly (p < 0.05) reduced by T. diversifolia extract at 8.0% (w/v) from 154.7 mm² (sole crop) or 13.4 mm² (unsprayed intercrop) to 4.9 mm² (sprayed intercrop). T. diversifolia extract at 8.0% (w/v) enhanced higher values of grain yield/plant and incidence of normal seeds, and lower incidence of fungal infection of seeds than in the unsprayed intercrop.     

Identity of hemolysins produced by Bacillus thuringiensis and Bacillus cereus

A hemolysin (Bt-hemolysin) produced by Bacillus thuringiensis var. kurstaki HD-1 producing crystalline toxin(s) was purified by successive treatments of ammonium sulfate (45-65%) and column chromatography using DEAE-cellulose, Sephadex G-75 and KB-002 (a hydroxyapatite column for fast protein liquid chromatography). A hemolysin (Bc-hemolysin) produced by B. cereus HG-6A was also purified by the same procedure. The purified Bt-hemolysin and Bc-hemolysin, both of which are thiol-activated hemolysins, were biologically, physicochemically and immunologically identical. These findings provide further evidence of the similarity of B. thuringiensis, which is being used as a biological insecticide, to B. cereus, a toxigenic organism of food poisoning.     

Antifungal quinazolinones from marine-derived Bacillus cereus and their preparation

Two new quinazolinones alkaloids, R(+)-2-(heptan-3-yl)quinazolin-4(3H)-one (1) and (2R,3′R)+(2S,3′R)-2-(heptan-3-yl)-2,3-dihydroquinazolin-4(1H)-one (2) (a pair of epimers), as well as seven known analogues, 2-methylquinazolin-4(3H)-one (3), 2-benzylquinazolin-4(3H)-one (4), cyclo-(Pro-Ile), cyclo-(Pro-Leu), cyclo-(Pro-Val), cyclo-(Pro-Phe), and cyclo-(Tyr-Pro) were isolated from the n-butyl alcohol extract of the marine-derived bacterium Bacillus cereus 041381. The new compounds were identified by spectroscopic analysis and chemical synthesis. Four optical isomers 5−8 were also synthesized. Compounds 1−8 all showed moderate antifungal activity against Candida albicans with MIC values of 1.3−15.6 μM. Compound 5 exhibits the most powerful antifungal activity, which may reveal that S-configuration and 2,3-double bond were necessary for antifungal activity, and the racemization at C-2 and C-3′ reduced the antifungal activity.     

Production of an antifungal protein for control of Colletotrichum lagenarium by Bacillus amyloliquefaciens MET0908

A plant pathogenic fungus, Colletotrichum lagenarium, causing watermelon anthracnose, was isolated from naturally infected leaves, stems, and fruits of watermelon. A bacterial strain, MET0908, showing a potent antifungal activity against C. lagenarium, was isolated from soil. An antifungal protein was purified by 30% ammonium sulfate saturation and concentrated using Centricon 10, DEAE-Sepharose(TM) Fast Flow column and Sephacryl S-100 gel filtration chromatography. The molecular weight of the purified protein was estimated as 40 kDa by SDS-PAGE. The purified protein was stable at 80 degrees C for 20 min and exhibited a broad spectrum of antifungal activity against various plant pathogenic fungi. Confocal microscopy image analysis and scanning electron microscopy showed that the protein acted on the cell wall of C. lagenarium. The purified antifungal protein exhibited beta-1,3-glucanase activity. The N-terminal amino acid sequence of the purified protein was determined as Ser-Lys-Ile-x-Ile-Asn-Ile-Asn-Ile-x-Gln-Ala-Pro-Ala-Pro-x-Ala. A search of the sequence with NCBI BLAST showed no significant homology with any known proteins, suggesting that the purified protein may be novel.     

Cloning in Bacillus subtilis of temperature-dependent promoter fragments from Bacillus stearothermophilus and Bacillus subtilis

Abstract Using promoter-probe plasmids, more than 200 promoter-containing fragments from Bacillus stearothermophilus and Bacillus subtilis were cloned in B. subtilis . Among these, 15 promoter fragments were highly temperature-dependent in activity compared to the promoter sequence (TTGAAA for the −35 region, TATAAT for the −10 region) of the amylase gene, amyT , from B. stearothermophilus . Some fragments exhibited higher promoter activities at elevated temperature (48°C), others showed higher activities at lower temperature (30°C). Active promoter fragments at higher and lower temperatures were obtained mainly from the thermophile ( B. stearothermophilus ) and the mesophile ( B. subtilis ), respectively. A promoter fragment active at high temperature was sequenced, and the feature of the putative promoter region was discussed.     

Transformation of Bacillus subtilis by DNA bound on clay in non-sterile soil

Abstract Chromosomal DNA from Bacillus subtilis and different forms of plasmid pHV14 (covalently closed circular (CCC), linear monomer (M), and linear multimer (LM)) were adsorbed and bound on the clay mineral montmorillonite. After extensive washing of the clay-DNA complexes with DNA buffer (pH 7.5), approx. 25% of the chromosomal DNA, and approx. 30, 90, and 5%, respectively, of the CCC, M and LM form remained bound. Chromosomal and plasmid DNA bound on clay were capable of transforming competent cells, with different specific activities. The clay-DNA complexes persisted in non-sterile soil and retained transforming ability up to 15 days after their addition to the soil. DNA bound on montmortillonite was protected from the activity of Eco RI, supporting the evidence that DNA adsorbed on soil components was resistant to degradation by nucleases.     

Immigration of Bacillus thuringiensis to bean leaves from soil inoculum or distal plant parts

AIMS: We addressed the process of immigration of Bacillus thuringiensis from soil to leaves and its capacity to grow on bean diffusate medium (BDM), a medium designed to simulate the nutrient composition of the phylloplane. METHODS AND RESULTS: Two different B. thuringiensis strains were inoculated into soils, onto seeds or onto lower leaves of bean plants to determine if they were able to disperse to upper leaves under controlled conditions. While B. thuringiensis isolates were commonly recovered from leaves exposed to such inocula, populations were very low (<10 CFU cm(-2) of leaf). In addition, the number of cells of B. thuringiensis recovered decreased with increasing distance from the soil or from the inoculated leaves. Moreover, B. thuringiensis colonies did not grow well on BDM. CONCLUSIONS: This indicates that B. thuringiensis disperses poorly from the soil or the seed to the leaves or between leaves of the same plant under controlled conditions. Bacillus thuringiensis apparently has greater nutrient requirements than other bacterial species that are prominent inhabitants of the phylloplane. SIGNIFICANCE AND IMPACT OF THE STUDY: Finding the mechanisms that favour bacteria colonization on leaves will in turn help to improve the efficacy of biocontrol agents against the target pests.     

利用ERIC-PCR对苏云金芽胞杆菌与蜡状芽胞杆菌进行鉴定的研究

为了探索ERIC-PCR技术在苏云金芽胞杆菌和蜡状芽胞杆菌的鉴定及分型中的应用价值,本研究采用PCR方法初步检测苏云金芽胞杆菌杀虫晶体蛋白基因的组成,并对苏云金芽胞杆菌和蜡状芽胞杆菌的总DNA进行ERIC-PCR扩增,分析ERIC-PCR指纹图谱的特点并采用NTSYS2.10软件对其进行聚类。结果显示,各菌株的ERIC指纹图谱表现出不同程度的多态性,但图谱与菌株所含cry基因的类型存在一定的相关性。聚类分析结果显示,含有相同或相近cry基因类型的Bt菌株在进化树上趋向聚为一类,而不含cry基因的蜡状芽胞杆菌趋向于与不含cry基因的Bt菌株聚为一类或单独聚类。若在多种模式菌株的参考下,该方法可用于苏云金芽胞杆菌的初步鉴定和分型。     

巨大芽孢杆菌青霉素G酰化酶基因在枯草杆菌中的高表达

用ＰＣＲ方法从巨大芽孢杆菌的基因组ＤＮＡ中扩增到青霉素Ｇ酰化酶基因,并装载到枯草杆菌质粒ｐＰＺＷ１０３中,将其转化到枯草杆菌ＤＢ１０４中进行了分泌表达,重组菌株产酶无需苯乙酸诱导。在３７℃培养２４ｈ,菌液酶活力可达６ｕ／ｍｌ。１０天的连续传代实验表明重组菌株的稳定性很高。     

Differential identification of Bacillus anthracis from environmental Bacillus species using microarray analysis

AIMS: To determine whether microarray analysis could be employed for the differential identification of a range of environmental Bacillus sp. from four strains of Bacillus anthracis. METHODS AND RESULTS: Oligonucleotide probes were designed that were specific to virulence factor genes of B. anthracis (pag, lef and cap), the variable number tandem repeat region of the B. anthracis vrrA gene and to the 16S-23S rRNA intergenic transcribed spacer region (ITS) and pleiotropic regulator (plcR) regions of the Bacillus cereus subgroup species. Generic probes were also designed to hybridize with conserved regions of the 16S rRNA genes of Bacillus (as a positive control), Neisseria sp., Pseudomonas sp., Streptococcus sp., Mycobacterium sp. and to all members of the Enterobacteriaceae to allow simultaneous detection of these bacteria. Identification of B. anthracis was found to rely entirely on hybridization of DNA specific to regions of the pag, lef and cap genes. Cross-reaction was observed between B. anthracis and other Bacillus species with all the other Bacillus probes tested. Results obtained using microarray hybridizations were confirmed using conventional microbiological techniques and found to have very high comparability. CONCLUSIONS: Microarray-based assays are an effective method for the identification of B. anthracis from mixed-culture environmental samples without problems of false-positivity that have been observed with conventional PCR assays. SIGNIFICANCE AND IMPACT OF THE STUDY: Identification of environmental Bacillus sp. by conventional PCR is prone to potential for reporting false-positives. This study provides a method for the exclusion of such isolates.     

Isolation of the Bacillus subtilis antimicrobial peptide subtilosin from the dairy product-derived Bacillus amyloliquefaciens

Aims: To purify and characterize an antimicrobial protein (bacteriocin) isolated from the dairy product‐derived Bacillus amyloliquefaciens. Methods and Results: An unknown bacterial species cultured from the Yogu Farm? probiotic dairy beverage was identified through 16S ribosomal RNA analysis as B. amyloliquefaciens, a phylogenetically close relative of Bacillus subtilis. The cell‐free supernatant (CFS) of overnight cultures was active against Listeria monocytogenes and also against clinical isolates of Gardnerella vaginalis and Streptococcus agalactiae. At the same time, several isolates of vaginal probiotic Lactobacilli were resistant to the CFS. The nature of the compound causing inhibitory activity was confirmed as proteinaceous by enzymatic digestion. The protein was isolated using ammonium sulfate precipitation, and further purified via column chromatography. PCR analysis was conducted to determine relatedness to other bacteriocins produced by Bacillus spp. Conclusion: The antimicrobial protein isolated from B. amyloliquefaciens was shown to be subtilosin, a bacteriocin previously reported as produced only by B. subtilis. Significance and Impact of the Study: This is the first report of intra‐species horizontal gene transfer for subtilosin and the first fully characterized bacteriocin isolated from B. amyloliquefaciens. Finally, this is the first report on subtilosin’s activity against bacterial vaginosis‐associated pathogens.     

Plasmid transfer in bacilli by a self-transmissible plasmid p19 from a Bacillus subtilis soil strain

The cryptic 95-kb plasmid p19 of the Bacillus subtilis 19 soil strain promotes the transfer of a small kanamycin resistance plasmid pUB110. To facilitate direct selection for p19 transfer, a plasmid derivative carrying the chloramphenicol resistance gene was constructed. The frequency of transfer of the large plasmid between cells of B. subtilis 19 approached 100% but was more than two orders of magnitude lower when the strain B. subtilis 168 was a recipient. However, when the restriction-deficient strain B. subtilis 168 was a recipient, the transfer efficiency was almost completely recovered. The effectiveness of pUB110 mobilization was virtually not altered in all these cases. pC194 was not mobilized by p19. The kinetics of p19 conjugative transfer is also presented.     

中国新分离碱性耐热芽孢杆菌的木聚糖酶产酶特征

目的：检测由中国新分离碱性耐热芽孢杆菌[1]产生的粗木聚糖酶的酶活。方法：通过DNS法测定粗木聚糖酶的酶活。结果：实验表明,以橡树木聚糖为底物培养的新分离菌株在30-50℃处理2h酶活不丧失。其中,XJU-1菌株在60、70和80℃时粗酶酶活分别丧失是最初酶活的1.54%、19.09%和72.59%;而XJU-80的粗酶酶活分别是3.59%、26.43%和72.59%。两个菌株产生的粗木聚糖酶的最适pH是7.5-8.0。将该粗酶在pH 7.0-9.0（50℃）处理24h后,酶活几乎均降低最初酶活的18%。结论：由XJU-1和XJU-80产生的木聚糖酶是生化领域有用的嗜碱耐热酶。     

Isolation, purification and properties of new restriction endonucleases from Bacillus badius and Bacillus lentus

We tentatively named two enzymes as BbaI and BleI, which were isolated and purified from Gram-positive mesophilic bacteria Bacillus badius 1458 and Bacillus lentus 1689 respectively, by ammonium sulphate precipitation, phosphocellulose and heparin-sepharose column chromatography. SDS-PAGE protein profiles for BbaI and BleI showed denatured molecular weights of 52 and 48 kDa, respectively. BbaI hydrolyzed pUC18 DNA into 1900 and 700 bp, pBR322 DNA into two fragments of 2800 and 1500 bp and Φ×174 DNA into 3800 and 1600 bp. BleI hydrolyzed pUC18 DNA into 1800 and 800 bp, pBR322 DNA into two fragments of 2700 and 1600 bp and Φ×174 DNA into 3700 and 1700 bp. The effects of temperature, ionic strength, pH and Mg²⁺ ion concentrations were studied to demonstrate some biochemical properties of BbaI and BleI. Maximum activities of these enzymes were observed at 37 °C (pH 8.0) with 100 mM NaCl and 10 mM Mg²⁺ concentrations.