基因标记枯草芽孢杆菌BS-68A在黄瓜上定殖

目的:野生型枯草芽孢杆菌Bacillus subtilisBS-68能有效地防治由Pythium spp.和Fusarium oxporum引起的黄瓜立枯病和枯萎病。为了探究该菌株的生防机制,利用该菌株的黄绿荧光蛋白基因和氯霉素抗性基因标记菌株BS-68A研究其在黄瓜植株各个部位的定殖能力、种群动态和在根围的分布。方法和结果:用基因标记菌株发酵液分别对黄瓜种子进行浸种和浇穴处理,播种后30d,该菌能在黄瓜根部和茎基部定殖,不能在茎部和叶部定殖。浸种处理,该菌在茎基部的种群数量为3.1×104cfu/株,大于根部的种群数量4.1×102cfu/株;浇穴处理,该菌在茎基部的种群数量8.0×103cfu/株,低于根部的种群数量2.5×104cfu/株。     

辣椒内生细菌BS-1和BS-2在植物体内的定殖及鉴定

以抗利福平为标记,用浸种、涂叶和灌根方法接种,测定菌株在植物体内的定殖。结果表明,来自辣椒体内的BS_2和BS_1菌株不仅可在辣椒体内定殖,也可在番茄、茄子、黄瓜、甜瓜、西瓜、丝瓜、小白菜等植物体内定殖,BS_2菌株还可在水稻、小麦及豇豆等植物体内定殖,BS_2菌株的内生定殖宿主范围比BS_1菌株的广;另外BS_2菌株可在辣椒和白菜体内较长期定殖。用常规方法、Biolog及16S rRNA序列比较,两菌株鉴定为枯草芽杆菌内生亚种（Bacillus subtilis subsp. endophyticus）。     

植物内生细菌的分离、分类、定殖与应用

植物内生细菌已成为国内外的研究热点,目前已从植物中分离得到许多不同种属的植物内生细菌,其中有的具有促生与防治病害等对宿主植物有利的作用,而一些则具有潜在的致病性。研究人员利用多种方法对植物内生细菌的内生定殖行为进行研究,发现了有意义的定殖规律,例如,某种植物内生细菌对其宿主的侵染能力明显强于另一种内生细菌。目前,尽管植物内生细菌还有不少问题有待解决,但它在农业上的巨大应用潜力业已彰显。     

蜡样芽孢杆菌B3-7在大田小麦根部的定殖动态及其对小麦纹枯病的防治效果

为了阐明蜡样芽孢杆菌B3-7在大田条件下的生态适应性以及对于小麦纹枯病的生防效果,通过利用绿色荧光蛋白编码基因gfp标记生防菌株B3-7,室内比较了GFP标记菌株和原始出发菌株在菌落形态、生长特性,生物薄膜产生以及在小麦根部定殖等方面的特性,结果发现GFP标记菌株和出发菌株在上述特性方面无明显差别。在此基础上,大田条件下测定了GFP标记菌株在小麦根部的定殖动态和对于小麦纹枯病的生防效果。结果发现,GFP标记菌株在小麦根部能够长期定殖,其存在量在小麦分蘖期最大,每克根重达到105CFU,拔节期后,该细菌数量一直维持在104CFU之上。同时发现,生防菌株能够有效降低小麦纹枯病的严重度和提高罹病小麦的产量。小麦分蘖期、孕穗期和灌浆期生防菌对于小麦纹枯病的防治效果分别达到60%、34%,34%,小麦成熟后产量提高13%—15%。结果表明,B3-7在大田条件下具有较好的生态适应性和防治小麦纹枯病的能力。     

绿色荧光蛋白基因标记野生型生防枯草芽孢杆菌的研究

根据绿色荧光蛋白基因和枯草芽孢杆菌木糖诱导型启动子PxylR 序列,分别设计两对特异引物primers PxyF/R和primers gfpF/R,扩增获得了完整的启动子PxylR和-gfp基因序列。进一步以上述产物混合物为模板,以primer PxyF/primer gfpR做引物进行重迭PCR,获得了PxylR-gfp重组翻译融合表达盒。经SphⅠ和KpnⅠ完全酶切后,将PxylR-gfp表达盒分别插入大肠杆菌_苏云金芽孢杆菌穿梭载体pHT315和大肠杆菌枯草芽孢杆菌穿梭载体pRP22。相应的重组表达质粒pGFP315和 pGFP22转化枯草芽孢杆菌感受态细胞。前者在标准菌株168中得到良好发光表型,后者则在标准菌株168和野生目标菌株B916中均得到良好的发光表型。室内平板抑菌实验结果显示B916生防效果与出发菌株没有明显差异,遗传稳定性研究表明连续稀释培养约175代后,工程菌株稳定性为94%,质粒丢失频率低于3.5×10^-4/代。     

枯草芽孢杆菌在抑制植物病原菌中的研究进展

枯草芽孢杆菌是芽孢杆菌中比较具应用潜力的菌种之一。近年来国内外对于芽孢杆菌各方面应用的研究日益增多,枯草芽孢杆菌作为一种生防细菌越来越引起人们的关注。主要综述了枯草芽孢杆菌在抑制植物病原菌生物防治领域的研究进展,阐述了枯草芽孢杆菌的控病作用机制,包括竞争作用、拮抗作用、溶茵作用、诱导植物产生抗性及促进植物生长5个方面。简要介绍了枯草芽孢杆菌及其制剂在国内外的应用情况及在植物病害防治应用中存在的问题、解决措施及发展前景。     

枯草芽孢杆菌ZH-8对黄瓜霜霉病防治效果的研究

枯草芽孢杆菌ZH-8分离于黄瓜根际,将该菌株发酵后,添加助剂制成液体菌剂。该液体菌剂在温室试验和田间试验中表现出较好的防治效果,对黄瓜霜霉病的防效分别达75．61％和69．12％。该菌剂可以有效抑制黄瓜霜霉病菌孢子囊的萌发,并且能够在黄瓜叶片上形成一层保护膜,阻止病原菌孢子的侵入。同时对其抗利福平标记菌株进行了在黄瓜叶片上定殖的研究,研究表明,该生防菌株在黄瓜叶片上具有较强的定殖能力,定殖时间达21d。     

类芽孢杆菌QHZ11-gfp在马铃薯植株上的定殖特征及促生效果

[背景] 生防菌在作物根系的有效定殖是其功能发挥的前提,而直观的跟踪技术和有效的定量方法是研究生防菌根系分布规律的重要工具。[目的] 研究马铃薯黑痣病病原菌立枯丝核菌（Rhizo ctonia solani） JT18的拮抗菌QHZ11在马铃薯植株上的定殖特征及对马铃薯的促生效果。[方法] 采用绿色荧光蛋白（Green Fluorescent Protein,GFP）对QHZ11进行标记,将标记菌株菌悬液、生物有机肥和无菌水分别接种至灭菌土壤,通过激光共聚焦显微技术和实时荧光定量PCR等方法观察和测定标记菌株在马铃薯植株不同部位的定殖特征、数量变化及对马铃薯的促生效果。[结果] pHAPII质粒成功导入QHZ11并可稳定遗传40代,记为QHZ11-gfp;菌株标记前后的菌落形态、生长曲线和对R.solani JT18的拮抗能力等基本一致。从第7天开始,相继在马铃薯芽上和根上发现了绿色荧光,说明QHZ11-gfp成功定殖到了马铃薯的芽、根等部位。QHZ11-gfp在根系和匍匐茎的定殖数量均呈现先升高至块茎形成期达到峰值后下降的趋势,并且在整个生育期根系的定殖数量始终大于匍匐茎。菌悬液和生物有机肥处理均显著促进了马铃薯根系的生长,并通过增加株高等农艺性状提高了块茎产量。其中,生物有机肥处理在各部位的荧光强度、定殖数量和对马铃薯的促生效果均显著优于菌悬液。[结论] QHZ11-gfp可在马铃薯植株上成功定殖并对马铃薯有良好的促生效果,将其制成生物有机肥促进了其定殖,使促生效果也更好。     

水稻内生枯草芽孢杆菌G87抗菌蛋白的分离纯化及理化特性

【目的】为得到枯草芽孢杆菌(Bacillus subtilis)G87的抗菌蛋白,明确其蛋白理化特性。【方法】采用硫酸铵沉淀和柱层析法进行分离纯化。【结果】获得单一抗菌活性蛋白(峰6-2-1),此抗菌蛋白分子量为50.8 kDa,等电点为5.90。经初步分析,抗菌蛋白不含脂,而含有少量(0.62%)糖;其蛋白部分具有脯氨酸或羟脯氨酸,但不含芳香族氨基酸。抗菌蛋白在高温(≥60℃)和较碱(pH8)环境下活性明显下降,但较抗紫外线、氯仿和胰蛋白酶、蛋白酶K、胃蛋白酶。【结论】枯草芽孢杆菌G87的抗菌蛋白为不含芳烃的糖蛋白,对高温和碱性条件敏感,而对蛋白酶类和紫外线等不敏感。     

微流控芯片监测绿色荧光蛋白在枯草芽孢杆菌中的表达

目前主要使用激光共聚焦扫描显微镜观察绿色荧光蛋白的表达,但需要昂贵的仪器并耗费大量时间。本研究开发了一种新型激光诱导的微流芯片检测系统来监测绿色荧光蛋白在枯草芽孢杆菌中的表达。该系统主要由激光装置、光路系统、微流控芯片、光电倍增管和计算机处理系统等5部分组成。对该系统的测试结果显示,随着诱导强度的增强监测信号峰也随之增强,并且与激光共聚焦显微镜观察的结果一致。利用该芯片系统能够快速准确地筛选和鉴定用绿色荧光蛋白作为标记的细胞克隆,可以替代PCR鉴定方法。但该系统仅仅能够监测表达强度,不能够满足蛋白定位等高水平研究,因此,该系统适合应用于环境的微生物监测、药物筛选和其他无需观察蛋白定位等研究。     

Surfactin and fengycin contribute to the protection of a Bacillus subtilis strain against grape downy mildew by both direct effect and defence stimulation

Bacillus subtilis GLB191 (hereafter GLB191) is an efficient biological control agent against the biotrophic oomycete Plasmopara viticola, the causal agent of grapevine downy mildew. In this study, we show that GLB191 supernatant is also highly active against downy mildew and that the activity results from both direct effect against the pathogen and stimulation of the plant defences (induction of defence gene expression and callose production). High-performance thin-layer chromatography analysis revealed the presence of the cyclic lipopeptides fengycin and surfactin in the supernatant. Mutants affected in the production of fengycin and/or surfactin were thus obtained and allowed us to show that both surfactin and fengycin contribute to the double activity of GLB191 supernatant against downy mildew. Altogether, this study suggests that GLB191 supernatant could be used as a new biocontrol product against grapevine downy mildew.     

Rapid production of thermostable cellulase-free xylanase by a strain of Bacillus subtilis and its properties

A Bacillus subtilis strain isolated from a hot-spring was shown to produce xylanolytic enzymes. Their associative/synergistic effect was studied using a culture medium with oat spelts xylan as xylanase inducer. Optimal xylanase production of about 12 U ml⁻¹ was achieved at pH 6.0 and 50°C, within 18 h fermentation. At 50°C, xylanase productivity obtained after 11 h in shake-flasks, 96,000 U l⁻¹ h⁻¹, and in reactor, 104,000 U l⁻¹ h⁻¹ was similar. Increasing temperature to 55°C a higher productivity was obtained in the batch reactor 45,000 U l⁻¹ h⁻¹, compared to shake-flask fermentations, 12,000 U l⁻¹ h⁻¹. Optimal xylanolytic activity was reached at 60°C on phosphate buffer, at pH 6.0. The xylanase is thermostable, presenting full stability at 60°C during 3 h. Further increase in the temperature caused a correspondent decrease in the residual activity. At 90°C, 20% relative activity remains after 14 min. Under optimised fermentation conditions, no cellulolytic activity was detected on the extract. Protein disulphide reducing agents, such as DTT, enhanced xylanolytic activity about 2.5-fold. When is used xylan as substrate, xylanase production decreased as function of time in contrast, with trehalose as carbon source, xylanase production in maintained constant for at least 80 h fermentation.     

Enhancement of surfactin production in iron-enriched media by bacillus subtilis ATCC 21332

Production of a lipopeptide surfactant, surfactin, by Bacillus subtilis ATCC 21332 was highly enhanced when iron concentration in the medium was raised from 4 μ to the few m level. This iron enrichment of the medium also resulted in increased biomass concentration. With 1.7 m iron sulfate added to the media of different initial iron concentrations or at different stages of growth, surfactin concentration can be raised to levels of several hundred mg l⁻¹ in general. Acidification of the broth was usually observed following the iron addition, and surfactin in the broth disappeared rapidly due to precipitate as soon as the pH level dropped below 5.0; however, if this acidification phenomenon was delayed, avoided (for reasons still unknown), or reversed (by alkaline addition), surfactin production was highly enhanced to as high as 3,500 mg l⁻¹ which was almost ten times over the previously reported level for this strain and higher than most reported values for genetically improved strains.     

内生细菌在荔枝体内的定殖及其防病保鲜功能

用含有GFP基因标记的内生细菌菌株BS-2-gfp和TB2-gfp,采用喷雾接种的方法,研究其在荔枝叶片、花、幼果及成熟果实果皮内的定殖动态及其与防病保鲜之间的关系.结果表明:内生细菌BS-2-gfp和TB2-gfp能在荔枝叶片、花、幼果及采后果皮上定殖,能在各组织内繁殖并可在花和幼果间传导.内生细菌在荔枝叶片上的定殖因季节和荔枝生长期的不同而异,与秋季相比,春季定殖期限长,定殖量大.内生细菌在荔枝不同部位的定殖时间和定殖量也不同,在叶片上接种37d后还可分离回收到接种的2种目标细菌,在花上接种10d后就回收不到BS-2-gfp,而成熟荔枝果皮上2种菌的定殖量最大.防病试验表明,当荔枝霜疫病的病情指数急剧上升时,TB2-gfp在荔枝果皮的定殖量达到最大,为1.90×106CFU.g-1FM;保鲜试验发现,TB2-gfp菌株的保鲜效果优于BS-2-gfp,菌株在荔枝果皮内的定殖量也高于菌株BS-2-gfp,表明供试内生细菌在荔枝果皮的定殖量与其防病及保鲜效果呈正相关.     

Biological control against bacterial wilt and colonization of mulberry by an endophytic Bacillus subtilis strain

Forty-five bacterial isolates were collected from surface-sterilized leaves of mulberry ( Morus alba L.). By screening their antagonistic activities against Ralstonia solanacearum in vitro , four isolates showed a remarkable inhibitory effect. The evaluation of the antagonistic strains against bacterial wilt of mulberry indicated that the strain Lu144 effectively reduced disease incidence. In the greenhouse, Lu144 displayed effective biological control against bacterial wilt of mulberry when it was applied to sterile or nonsterile soil before the infection by the pathogen. Based on bacteriological properties and 16S rRNA gene sequencing, Lu144 was identified as a strain of Bacillus subtilis . The endophytic population and infection process of Lu144 in mulberry seedlings was explored following recovery of the green fluorescent protein (GFP)-labeled Lu144 and examination of the labeled strain by confocal laser scanning microscopy. Interestingly, the infection of GFP-labeled Lu144 cells into the mulberry seedlings occurred through the cracks formed at the lateral root junctions and the zone of differentiation and elongation, and the cells were able to develop and transfer in mulberry and mainly in the intercellular spaces of different tissues. The population of the GFP-labeled Lu144 inoculant was larger and more stable in leaves than that in roots and stems.     

枯草杆菌ylmK基因的荧光标记及Ylmk蛋白的亚细胞定位

目的：对枯草杆菌ylmK基因进行3’端荧光标记以便对其产物YlmK蛋白在菌体中的位置进行初步观察。方法：以BS168菌株基因组DNA为模板,PCR扩增ylmK基因的3’端序列,并将其克隆到载体pSGll64中,形成ylmK’-gfpmutl融合,构建重组载体pNC-424;将pNG424转化枯草杆菌168菌株,单交换形成完整的功能性3’端gfpmutl标记的ylmK基因,菌落PCR对阳性转化子BS362进行鉴定,用表面荧光显微镜技术对BS362进行观察。结果：荧光检测结果表明GFP标记的YlmK分布于菌体的外周,在位置上靠近细胞膜并与之平行排列。结论：YlmK是一种膜蛋白。