枯草芽孢杆菌TF26抗菌蛋白抑菌活性的初步研究

目的:研究枯草芽孢杆菌TF26抗菌蛋白的抑菌活性和生物稳定性,为菌株及抗菌蛋白的应用提供理论依据.方法:采用硫酸铵盐析方法提取抗菌蛋白,采用菌丝生长速率法检测其对13种植物病原真菌的抑菌活性,采用抑菌圈方法对其生物稳定性进行分析.结果:抗菌蛋白粗提物能够抑制13种植物病原真菌的生长,平皿抑制率为74.3％ ～91.3％,对葵花菌核病菌、番茄和黄瓜枯萎病菌、黄瓜菌核病菌和立枯病菌、水稻恶苗病菌和大豆根腐病菌抑制作用较强.抗菌蛋白在100℃以下,pH＜ 10范围内抑菌活性稳定,对紫外线照射不敏感,室温(20℃)和4℃储存150d抑菌活性稳定.结论:抗菌蛋白具有较强的热、酸碱、紫外和储存稳定性以及广谱的抑菌活性.     

番茄灰霉病生防链霉菌筛选及鉴定

【背景】由灰葡萄孢侵染所致的番茄灰霉病是一类重要的真菌病害,生物防治具有环境友好、病原菌不易产生抗药性等特点,是果蔬灰霉病绿色防控的有效措施。【目的】筛选对番茄灰霉病具有防病作用且能促进番茄种子发芽的广谱拮抗性链霉菌,并明确该菌株种级分类地位。【方法】采用琼脂块法筛选拮抗番茄灰霉病菌的链霉菌菌株,采用对峙培养法和生长速率法检测菌株T22抑菌谱,通过产胞外酶活性检测、离体叶片防效和种子发芽试验明确该菌株的防病促生相关特性,根据形态学特征、生理生化特性和分子生物学方法对该菌株进行种类鉴定。【结果】从分离的56株放线菌中筛选到14株对番茄灰霉病菌具有拮抗效果的放线菌菌株,其中链霉菌T22对番茄灰霉病菌抑制作用最强,且具有较广抑菌谱,同时菌株T22具有产生纤维素酶和几丁质酶的能力。菌株T22无菌发酵滤液对番茄灰霉病菌、桃褐腐病菌、黄瓜枯萎病菌抑菌率分别为84.6%、81.5%和79.1%;其无菌发酵滤液原液对番茄灰霉病离体防效为55.1%;100倍稀释液处理番茄种子,胚轴、胚根和种子活力指数分别增加15.1%、29.7%和43.9%。根据形态学特征、生理生化特性和多基因聚类分析将链霉菌T22鉴定为白黑链霉菌(Streptomycesalboniger)。【结论】白黑链霉菌T22具有较强的抗真菌、产胞外酶、防病和促生活性,在番茄灰霉病生物防治中具有较好的开发应用潜力。     

猪场沼液对蔬菜病原菌的抑制作用

用不同贮存时间的猪场沼液对7种蔬菜病原菌的抑制作用进行了研究。结果表明,新鲜猪场沼液原液对番茄灰霉病菌、番茄早疫病菌、辣椒疫病菌、黄瓜炭疽病菌和茄子灰霉病菌有较强的抑制作用,沼液贮存对番茄灰霉病菌、番茄早疫病菌和茄子灰霉病菌的抑制效果没有显著影响,但对辣椒疫病菌和黄瓜炭疽病菌的抑制效果影响显著;新鲜猪场沼液滤液对番茄灰霉病菌和茄子灰霉病菌有较强的抑制作用,沼液滤液贮存对番茄灰霉病菌和茄子灰霉病菌的抑菌效果没有显著影响。     

大花金挖耳内生真菌H-18菌株的鉴定及其抑菌活性研究

对大花金挖耳内生真菌H-18菌株的菌丝提取物进行了抑菌活性研究并对菌株进行了鉴定.离体试验结果表明,H-18菌株菌丝体丙酮提取物在500mg/L浓度下对玉米大斑病菌、番茄灰霉病菌和辣椒疫霉病菌的菌丝生长抑制率均大于80%;组织法和盆栽试验结果表明,H-18菌株菌丝体丙酮提取物在2000mg/L浓度下对番茄灰霉病和辣椒疫霉病的保护效果和治疗效果均在50%以上.结合形态分类学和ITS-5.8S rDNA序列分析结果鉴定菌株H-18为同色镰孢菌(Fusarium concolor).     

灰色链霉菌YD3309菌丝体抗菌活性成分研究

采用生物活性跟踪法,从一株灰色链霉菌菌丝体中分离得到2个具有抑菌活性的化合物,通过核磁共振波谱和质谱等技术鉴定2个化合物的结构为新刺孢霉素A(1)和N-乙酰基色氨醇(2);化合物1首次从放线菌中分离得到。抑菌活性测定结果表明:化合物1和2对番茄灰霉病菌(Botrytis cinerea)、茄子黄萎病菌(Verticillium dahliae)和辣椒枯萎病菌(Fusarium oxysporum)等多种蔬菜病原真菌有抑制作用,其中化合物1对番茄灰霉病菌和番茄早疫病菌菌丝具有强烈的抑制作用,化合物2对茄子黄萎病菌的菌丝具有强烈的抑制作用;化合物1对番茄灰霉病菌和番茄早疫病菌菌丝生长的半抑菌浓度(IC_(50))分别为30.6和28.8 mg/L,化合物2对茄子黄萎病菌菌丝生长的半抑菌浓度(IC50)为34.3 mg/L。     

杀真菌素链霉菌AL-04的筛选与鉴定

对13株来源于青藏高原土壤的放线菌进行活化,再分别采用琼脂扩散法、生长速率法、孢子萌发抑制法和离体叶片法筛选出一株抗菌活性高且抗性稳定的菌株AL-04。抗菌活性结果表明,该菌株对4种常见的土传病害病原真菌:辣椒疫霉病菌(Phytophthora capsici)、黄瓜枯萎病菌(Fusarium oxysporum f.sp.cucumerinum)、西瓜枯萎病菌(Fusarium oxysporum f.sp.niveum)、番茄灰霉病菌(Botrytis cinerea)都有较强的抑制效果,抑菌率均在70.0%以上,其中对辣椒疫霉病菌(Phytophthora capsici)抑菌率高达93.0%。通过形态特征、生理生化特征分析和16S rDNA序列分析,将该菌株鉴定为杀真菌素链霉菌(Streptomyces fungicidicus)。     

枯草芽孢杆菌SN-02发酵液的抑菌谱及稳定性研究

目的:研究枯草芽孢杆菌SN-02发酵液的抑菌谱及稳定性。方法:以28种植物病原菌为供试菌,杯碟法测定SN-02菌发酵液的抑菌谱;以烟草靶斑病菌为指示菌,杯碟法测定发酵液的热稳定性、酸碱稳定性及传代稳定性。结果:SN-02菌发酵液对28种供试菌株的抑菌圈直径在20mm以上。将发酵液于120℃处理2.5h,-20℃处理25d抑菌活性没有明显变化;发酵液在pH 4~9时抑菌活性无明显变化,在pH 1~3和pH 10条件下抑菌活性明显下降;连续培养10代,发酵液抑菌活性没有下降。结论:SN-02菌发酵液抑菌谱较广,耐高温和低温,传代稳定性好,但在强酸和强碱条件下稳定性较差。     

番茄灰霉病拮抗内生细菌的筛选、鉴定及其活性

对安徽省淮北市番茄植株根、茎、叶中内生细菌及其数量进行了调查和筛选,并测定了其抑菌活性。结果表明,番茄根、茎和叶中的内生细菌的数量分别为5.69×105、5.16×105和2.83×105CFU.g-1鲜重。根据分离部位和表型特征,从健康番茄植株体内分离到267株内生细菌,通过对峙实验,筛选到11株对番茄灰霉病菌有拮抗作用的菌株,占所分离内生细菌总数的4.12%。来自茎组织中的菌株XF136的抑菌效果最佳,抑菌带宽度达32.2mm。根据形态特征、生理生化特性、(G+C)mol%和16SrDNA序列分析,将菌株XF136鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。室内测定菌株XF136发酵滤液对灰霉病菌菌丝生长及分生孢子萌发的抑制作用,结果表明,菌株XF136发酵滤液可以抑制灰霉病菌菌丝生长和分生孢子萌发,且浓度越高,抑制能力越强;当发酵滤液浓度为20%时则完全抑制灰霉病菌菌丝生长和分生孢子萌发。盆栽防效试验结果表明,10%菌株XF136发酵滤液对番茄灰霉病防治效果与50%多菌灵600倍液相当,20%发酵滤液对番茄灰霉病的防治效果高于50%多菌灵600倍液。本研究表明,菌株XF136是防治番茄灰霉病潜在的优良生防菌株,具有良好的开发应用价值。     

番茄灰霉病拮抗内生放线菌的筛选、鉴定及其活性评价

对安徽省淮北市番茄植株根、茎、叶中内生放线菌进行了分离、筛选,并测定了其抑菌活性。结果表明:番茄根、茎和叶中的内生放线菌的数量分别为5.66×104、0.67×104和0.39×104CFU.g-1鲜重。根据分离部位和表型特征,从健康番茄植株体内分离到93株内生放线菌,通过对峙实验,筛选到7株对番茄灰霉病菌有拮抗作用的菌株,占所分离内生放线菌总数的7.5%。来自根组织中的菌株HNU-EA27的抑菌效果最佳,抑菌圈直径达28.3mm。根据形态特征、培养特征、生理生化特性、细胞壁组分和16SrDNA序列分析,将菌株HNU-EA27鉴定为毒三素链霉菌(Streptomyces toxytricini)。室内测定菌株HNU-EA27发酵滤液对灰霉病菌菌丝生长及分生孢子萌发的抑制作用,结果表明:菌株HNU-EA27发酵滤液可以抑制灰霉病菌菌丝生长和分生孢子萌发,且浓度越高,抑制能力越强;当发酵滤液浓度为30%时则完全抑制灰霉病菌菌丝生长和分生孢子萌发。盆栽防效试验结果表明:30%菌株HNU-EA27发酵滤液对番茄灰霉病的预防与治疗效果分别为80.6%和73.8%,均高于50%多菌灵可湿性粉剂600倍液。本研究表明,菌株HNU-EA27是防治番茄灰霉病潜在的优良生防菌株,具有良好的开发应用价值。     

桑树内生拮抗菌的分离鉴定及其对桑断枝烂叶病的生防初探

【目的】本研究从健康桑树茎中分离筛选对桑断枝烂叶病菌具显著拮抗作用的内生细菌,为该病生物防治奠定研究基础。【方法】采用组织培养法分离桑树内生菌,抑菌圈法和平板对峙法筛选抑菌活性稳定的内生拮抗菌;根据形态学、生理生化特征检测和基于16SrDNA、gyrA和gyrB基因的系统发育分析对拮抗菌进行菌种鉴定;利用抑菌圈法测定拮抗菌株活性发酵液热稳定性,菌丝生长速率法检测活性发酵液抑菌谱;并通过观察拮抗菌对桑断枝烂叶病菌BoeremiaexiguaGXH1菌株生长及菌丝形态的影响,扩增抑菌活性物质合成关键基因,以及采用酸沉淀法提取拮抗菌株脂肽类化合物并进行高效液相色谱串联质谱分析(LC-MS),初步探究可能的抑菌机制。【结果】从健康桑树茎中共分离获得17株桑树内生细菌,并从中筛选获得一株对桑断枝烂叶病菌B.exiguaGXH1有稳定拮抗作用的桑树内生细菌NPJ13菌株。该菌株形态学、生理生化特征与芽孢杆菌属一致,基于16SrDNA、gyrA和gyrB基因序列的系统发育分析结果显示该菌株与贝莱斯芽孢杆菌(Bacillusvelezensis)的亲缘关系最近,且处于系统发育树的最小分枝,故将NPJ13菌株鉴定为贝莱斯芽孢杆菌,命名为B. velezensis NPJ13。NPJ13菌株对灰霉病菌SWU5、核地杖菌SXSG-5、核盘菌PZ-2及烟草疫霉SWU20等12种病原真菌具有不同程度的拮抗作用,其活性发酵液具有较好的热稳定性。NPJ13菌株会导致桑断枝烂叶病菌GXH1菌丝发生扭曲、膨大、透明度增加、断裂等畸变现象;基因检测结果显示NPJ13菌株基因组中具有PKSI、NRPS、Sfp、ItuD、Srfc等5种抑菌活性物质合成关键基因,LC-MS检测结果表明菌株NPJ13脂肽类粗提物中含有表面活性素和伊枯草菌素。【结论】本研究分离筛选获得一株对桑断枝烂叶病菌具有显著拮抗作用的桑树内生细菌B. velezensis NPJ13菌株,为桑断枝烂叶病的生物防治提供了候选菌株。     

1株枯草芽胞杆菌体外拮抗6种肠道致病菌的研究

目的研究枯草杆菌BS-3株对大肠埃希菌等6种肠道致病菌的拮抗作用。方法通过在体外BS-3菌株分别与大肠埃希菌等6种致病菌混合培养后,观察不同时间内各菌的菌量变化。结果BS-3菌株与6种肠道致病菌混合培养24、48、72和96h,其菌量逐渐增加;6种致病菌的菌量随着培养时间延续逐渐减少,其中产毒性大肠埃希菌、致病性大肠埃希菌和宋内志贺菌与对照组比较差异更明显。结论BS3菌株在培养生长过程中,可抑制大肠埃希菌等6种肠道致病菌的生长。     

Isolation and characterization of antagonistic Bacillus subtilis strains from the avocado rhizoplane displaying biocontrol activity

AIM: This study was undertaken to isolate Bacillus subtilis strains with biological activity against soil-borne phytopathogenic fungi from the avocado rhizoplane. METHODS AND RESULTS: A collection of 905 bacterial isolates obtained from the rhizoplane of healthy avocado trees, contains 277 gram-positive isolates. From these gram-positive isolates, four strains, PCL1605, PCL1608, PCL1610 and PCL1612, identified as B. subtilis, were selected on the basis of their antifungal activity against diverse soil-borne phytopathogenic fungi. Analysis of the antifungal compounds involved in their antagonistic activity showed that these strains produced hydrolytic enzymes such as glucanases or proteases and the antibiotic lipopeptides surfactin, fengycin, and/or iturin A. In biocontrol trials using the pathosystems tomato/Fusarium oxysporum f.sp. radicis-lycopersici and avocado/Rosellinia necatrix, two B. subtilis strains, PCL1608 and PCL1612, both producing iturin A, exhibited the highest biocontrol and colonization capabilities. CONCLUSIONS: Diverse antagonistic B. subtilis strains isolated from healthy avocado rhizoplanes have shown promising biocontrol abilities, which are closely linked with the production of antifungal lipopeptides and good colonization aptitudes. SIGNIFICANCE AND IMPACT OF THE STUDY: This is one of the few reports dealing with isolation and characterization of B. subtilis strains with biocontrol activity against the common soil-borne phytopathogenic fungi F. oxysporum f.sp. radicis-lycopersici and R. necatrix.     

Identification and molecular characterization of a novel Bacillus strain capable of degrading Tween-80

A Tween-80-degrading novel marine Bacillus strain, N10, has recently been isolated in Alexandria University, Egypt. The taxonomic position of this endospore forming bacterium was investigated on the basis of fatty acid analysis and 16S rRNA gene sequencing. Comparative computer database analyses revealed that the bacterium is a Bacillus subtilis strain. The gene encoding the small acid-soluble protein gamma-type (SASP-B), sspE, was successfully utilized in this study as a tool for discrimination between the two B. subtilis subspecies W23 and 168. Based on the alignment of 16S rRNA sequences and analysis of SASP-B relatedness, it has been demonstrated that the novel marine B. subtilis strain N10 is more closely related to the B. subtilis reference strain W23 than to 168. The strain, N10, has been deposited in the Bacillus Genetic Stock Center (BGSC) and assigned the accession number 3A17.     

枯草芽孢杆菌活菌体外拮抗6种肠道致病菌的研究

对枯草芽孢杆菌BS 3株在体外对 6种常见肠道致病菌 (肠产毒性大肠杆菌、肠侵袭性大肠杆菌、肠致病性大肠杆菌、鼠伤寒沙门菌、福氏志贺菌和宋内志贺菌 )的拮抗作用进行了研究。结果表明 ,枯草芽孢杆菌BS 3株对宋内志贺菌、肠致病性大肠杆菌及肠产毒性大肠杆菌拮抗作用较为明显。     

1株生防细菌的筛选、鉴定及其抑菌特性的初步研究

从鲜牛奶中筛选1株对番茄灰霉病菌具有拮抗作用的菌株A9,经16S rDNA序列分析,鉴定为枯草芽胞杆菌(Bacillus subtilis)。抑菌特性研究表明,该菌株代谢产物会造成番茄灰霉病菌菌丝畸形,同时抑制其孢子生长,使孢子细胞壁破裂;代谢粗提物经过DEAE-52离子交换层析及Sephadex-G50凝胶柱层析后,电泳检测到具有抑菌活性且分子量约为17ku的单一条带,经验证该抑菌物具有蛋白酶活性。     

枯草芽孢杆菌对杨树水泡溃疡病菌菌丝生长的抑制作用

测定了枯草芽孢杆菌BS-2菌株对杨树水泡溃疡病菌菌丝生长的影响,发现BS-2菌株能够抑制病菌菌丝的生长,并导致菌丝形态畸变,表现为菌丝伸长生长受抑制、扭曲、原生质浓缩、局部膨大成球形或椭圆形以及菌丝断裂和菌丝细胞变成空泡等。BS-2发酵液对病菌菌丝生长也有明显的抑制作用,培养时间越长,其培养液的抑菌作用越强;在不同培养基中获得的发酵液对病菌的抑制效果不同。     

Biocontrol of Bacillus subtilis against infection of Arabidopsis roots by Pseudomonas syringae is facilitated by biofilm formation and surfactin production

Relatively little is known about the exact mechanisms used by Bacillus subtilis in its behavior as a biocontrol agent on plants. Here, we report the development of a sensitive plant infection model demonstrating that the bacterial pathogen Pseudomonas syringae pv tomato DC3000 is capable of infecting Arabidopsis roots both in vitro and in soil. Using this infection model, we demonstrated the biocontrol ability of a wild-type B. subtilis strain 6051 against P. syringae. Arabidopsis root surfaces treated with B. subtilis were analyzed with confocal scanning laser microscopy to reveal a three-dimensional B. subtilis biofilm. It is known that formation of biofilms by B. subtilis is a complex process that includes secretion of surfactin, a lipopeptide antimicrobial agent. To determine the role of surfactin in biocontrol by B. subtilis, we tested a mutant strain, M1, with a deletion in a surfactin synthase gene and, thus, deficient in surfactin production. B. subtilis M1 was ineffective as a biocontrol agent against P. syringae infectivity in Arabidopsis and also failed to form robust biofilms on either roots or inert surfaces. The antibacterial activity of surfactin against P. syringae was determined in both broth and agar cultures and also by live-dead staining methods. Although the minimum inhibitory concentrations determined were relatively high (25 microg mL(-1)), the levels of the lipopeptide in roots colonized by B. subtilis are likely to be sufficient to kill P. syringae. Our results collectively indicate that upon root colonization, B. subtilis 6051 forms a stable, extensive biofilm and secretes surfactin, which act together to protect plants against attack by pathogenic bacteria.     

A Bacillus subtilis sensor kinase involved in triggering biofilm formation on the roots of tomato plants

The soil bacterium Bacillus subtilis is widely used in agriculture as a biocontrol agent able to protect plants from a variety of pathogens. Protection is thought to involve the formation of bacterial communities - biofilms - on the roots of the plants. Here we used confocal microscopy to visualize biofilms on the surface of the roots of tomato seedlings and demonstrated that biofilm formation requires genes governing the production of the extracellular matrix that holds cells together. We further show that biofilm formation was dependent on the sensor histidine kinase KinD and in particular on an extracellular CACHE domain implicated in small molecule sensing. Finally, we report that exudates of tomato roots strongly stimulated biofilm formation ex planta and that an abundant small molecule in the exudates, (L) -malic acid, was able to stimulate biofilm formation at high concentrations in a manner that depended on the KinD CACHE domain. We propose that small signalling molecules released by the roots of tomato plants are directly or indirectly recognized by KinD, triggering biofilm formation.     

解淀粉芽孢杆菌生防菌BS-3全基因组测序及生物信息分析

【背景】解淀粉芽孢杆菌BS-3是从健康橡胶树树根中分离获得的一株对真菌具有较强抗菌活性的内生细菌,有作为生物农药的潜力。【目的】解析菌株BS-3的基因组序列信息,以深入研究该菌株防病促生机制及挖掘次级代谢产物基因资源。【方法】采用第二代BGISEQ与第三代Pac Bio平台相结合的测序技术,对生防菌BS-3进行全基因组测序,并对测序数据进行基因组组装、基因预测与功能注释、共线性分析及次级代谢产物合成基因簇预测等。【结果】BS-3全基因组大小为3 870 130 bp,平均GC含量为46.88%,共编码4 161个基因;含有92个t RNA基因、28个r RNA、10个sRNA;含有122个串联重复序列、98个小卫星DNA、2个微卫星。在COG、GO、KEGG、NR和Swiss-Prot数据库分别注释到基因2 875、2 620、1 885、4 040和3 328个。同时,预测到BS-3中有10个次级代谢产物合成基因簇,编码表面活性素、丰原素、多烯类、儿茶酚型嗜铁素等抑菌物质。基因组测序数据提交至NCBI获得GenBank登录号为CP060384。【结论】为基因组层面上解析菌株BS-3具有良好防病效果的内在原因提供基础数据,为深入了解解淀粉芽孢杆菌次级代谢合成途径提供参考信息,对菌株BS-3后续相关研究具有重要意义。     

Characterization of Mycolytic Enzymes of Bacillus Strains and Their Bio-Protection Role Against Rhizoctonia solani in Tomato

Four antagonists bacteria namely, Bacillus megaterium MB3, B. subtilis MB14, B. subtilis MB99 and B. amyloliquefaciens MB101 were able to produce chitinase, β-1,3-glucanase and protease in different range with the presence of Rhizoctonia solani cell wall as a carbon source. Amplification of chitinase (chiA) gene of 270 bp and β-1, 3-glucanase gene of 415 bp was given supportive evidence at molecular level of antibiosis. After in vitro screening, all antagonists were tested against R. solani under greenhouse conditions. Root treatment of Bacillus strains showed superior defense during pathogen suppression in terms of chitinase, glucanase, peroxidase, poly phenol oxidase, phenylalanine ammonia-lyase activity and total phenolic content in leaves of tomato. All these enzymes accumulated high in tomato leaves as compared to roots. Pathogenesis-related proteins and defense-related enzymes accumulation was directly correlated with plant protection and greenhouse results indicated that B. amyloliquefaciens MB101- and B. subtilis MB14-treated plants offered 69.76 and 61.51 % disease reductions, respectively, over the infected control. These results established that these organisms have the potential to act as biocontrol agents. This study could be highlighted a mutual importance of liquid formulation of antagonistic Bacillus spp. against root associated sclerotia former pathogen R. solani.