海洋芽孢杆菌（Bacillus marinus）B-9987菌株抑制病原真菌机理的研究

摘要：【目的】：探讨海洋芽孢杆菌（Bacillus marinus）B-9987菌株的代谢产物BMME-1,对植物病原真菌茄链格孢菌的抑菌作用机理。【方法】分别使用分光光法、气相色谱-质谱GC-MS联用技术、红外光谱法等,检测了BMME-1处理病原真菌后,菌体渗透性、细胞壁及细胞膜成份的变化。【结果】BMME-1对茄链格孢菌的抑菌中浓度（MIC50）为6.2 mg/L,最小杀菌浓度（MFC）为50 mg/L,在MIC50浓度或高于此浓度处理靶标菌,将导致菌体蛋白质、核酸等大分子物质的外流;处理菌株葡聚糖结     

青蒿素衍生物抗菌机理研究

前期研究从臭蒿中提取分离出臭蒿抗菌流分Fr.5.2主要成分为青蒿素衍生物。为了研究青蒿素衍生物的抗菌机理,测试Fr.5.2和双氢青蒿素(Dihydroartemisinin,DHA)对烟草链格孢菌的抗菌活性、细胞膜完整度、胞内物质的泄露、菌体和孢子的影响。结果发现:Fr.5.2对烟草链格孢菌最小抑菌浓度(minimum inhibitory concentration,MIC)为1.25 mg/m L,最小杀菌浓度(minimum bactericidal concentration,MBC)为2.5 mg/m L,DHA对烟草链格孢菌的MIC为10 mg/m L,MBC为10 mg/m L。0.625 mg/m L Fr.5.2和2.5 mg/m L DHA作用于烟草链格孢菌,在0~3 h,细胞膜完整度不断下降,胞内核酸大分子不断流失;SEM观察发现菌丝体细胞膜细小、有空洞,孢子表面也有凹陷;Fr.5.2和DHA还可以抑制麦角甾醇的合成,与空白组相比,麦角甾醇的含量分别降低29.77%,28.99%,孢子平均密度提高,但是萌发率降低。这些实验结果表明,青蒿素衍生物能够抑制真菌麦角甾醇的合成,破坏细胞膜,阻碍真菌细胞的新陈代谢,破坏真菌孢子,能有效的抑制、杀死真菌。     

MIG1基因和葡萄糖对扣囊复膜孢酵母细胞形态变化的影响及机理探究

【目的】研究MIG1基因和葡萄糖对扣囊复膜孢酵母细胞形态变化的影响及其机理探究。【方法】扣囊复膜孢酵母在不同浓度葡萄糖的YPD培养基中培养,敲除MIG1基因菌株在常规YPD培养基中培养,研究细胞内葡聚糖酶和几丁质酶活性以及细胞壁β-葡聚糖和几丁质含量与细胞形态变化之间的关系。【结果】培养基中葡萄糖浓度越低,扣囊复膜孢酵母菌丝体越少,单细胞酵母越多,且葡聚糖酶和几丁质酶活性越高,β-葡聚糖和几丁质含量越低;葡萄糖浓度对敲除MIG1基因菌株没有显著影响,葡聚糖酶和几丁质酶活性始终保持在较高水平,β-葡聚糖和几丁质含量也较低,菌体多以单细胞酵母形式存在。【结论】MIG1基因和葡萄糖通过葡萄糖阻遏作用调节葡聚糖酶和几丁质酶活性,进而影响细胞壁的葡聚糖和几丁质含量,最终影响扣囊复膜孢酵母细胞的形态变化。     

三唑酮对玉米弯孢病菌超微结构和细胞化学的影响

三唑酮(triadimenfon)属于麦角甾醇类生物合成抑制剂(ergosterol biosynthesis inhibitors．EBI),具有较广的抗真菌谱,明确其对玉米弯孢菌发育的影响可为该杀菌剂的田间应用提供理论依据。利用电镜技术和细胞化学技术观察的结果表明,玉米率孢菌经三唑酮处理后,菌丝生长明显受到抑制,表现为菌落生长速度减慢、菌丝分枝增多,且不观则地肿大和缢缩,出现许多瘤状突起,处理菌丝明显畸形。透射电镜观察结果表明,三唑酮可引起菌丝细胞壁不规则增厚,特别是菌丝顶端细胞壁增厚尤为明显:菌丝细胞隔膜发育受阴而表现畸形;菌丝细胞外有大量电子染色深的外渗物质。细胞化学标记定位结果表明,真菌细胞壁主要成分β-1,3-葡聚糖和几丁质的含量在药剂处理后发生很大变化,其标记密度明显低于未处理的对照菌丝,表明病菌细胞壁的结构和功能受到明显的不利影响。论文对弯孢菌受三唑酮影响后胞壁成份变化与其它真菌不同的原因进行了讨论。     

AsSlt2基因对茄链格孢细胞壁完整性的调控

【背景】由茄链格孢(Alternaria solani)引起的马铃薯早疫病被普遍认为是马铃薯生产上的第二大叶部病害,在马铃薯各产区普遍发生,给马铃薯生产造成了巨大的经济损失。【目的】明确AsSlt2基因对茄链格孢细胞壁完整性的影响。【方法】在含有刚果红、细胞壁降解酶和十二烷基硫酸钠(sodiumdodecylsulfate,SDS)等细胞壁胁迫的培养基上观察ΔAsSlt2缺失突变株的生长情况,计算相对生长抑制率;通过实时荧光定量PCR (RT-qPCR)方法检测ΔAsSlt2菌株中细胞壁合成相关基因的表达情况;进一步检测ΔAsSlt2细胞壁中几丁质的含量及胞外酶活性。【结果】ΔAsSlt2缺失突变株对SDS、刚果红、细胞壁降解酶等细胞壁胁迫的敏感性增强,在加入细胞壁降解酶后突变株原生质体释放量显著增多;ΔAsSlt2对外源氧胁迫更敏感,突变株胞外过氧化物酶和漆酶活性均显著降低;进一步研究发现,ΔAsSlt2细胞壁中几丁质含量减少,几丁质合成相关基因与漆酶合成相关基因的表达量均明显降低。【结论】AsSlt2基因在茄链格孢细胞壁的完整性及抵御外界胁迫方面发挥重要作用。     

链霉菌S01菌株几丁质醇对植物病原真菌的拮抗作用

纯化后的链霉菌Ｓ０１菌株几丁质酶用环柱法在ＰＤＡ平板上对杨树腐烂病菌、葡萄孢菌（Ｂｏｔｒｙｔｉｓ ｓｐ．ＡＳ３．２６１６）、黄瓜黑腥病菌、辣椒疫病菌、棉花黄萎病菌、立枯丝核菌等植物病原真菌及产黄青霉、啤酒酵母的生长具有明显的抑制作用。在６株植物病原真菌的液体培养物中加入几丁质酶后,菌丝出现扭曲变形、细胞质聚集、外溢等异常现象。在高渗缓冲液中,经几丁质酶处理后,啤酒酵母、葡萄孢菌和产黄青霉的细胞壁受     

链格孢霉醇和链格孢霉醇单甲醚产生菌株的筛选

本文对分离自小麦、马铃薯、番茄和茄子上链格孢霉属(Alternaria)2个种(链格孢和茄链格孢)的96个菌株,用枯草杆菌生长抑制试验筛选链格孢霉醇(AOH)和链格孢霉醇单甲醚(AME)的产生菌株,有48株产生毒性作用(占所测菌株的50％)。18株产强、中毒性菌用高效液相色谱分析,有13株产AOH和AME(占所测菌株的72.2％)。链格孢的产毒素菌株率比茄链格孢低。但产毒素含量却是前者明显高于后者。其中产AOH和AME的最高含量,链格孢菌株XA-8分别为280和5140mg/kg,而茄链格孢菌株SA-10分别为95.9和94.3mg/kg。     

蜜环菌发酵液提取物对人参链格孢霉菌的抑制作用

为探究蜜环菌Armillaria mellea发酵液提取物的抗植物病原真菌作用,在预实验的基础上选取人参链格孢霉菌Alternaria panax作为供试靶标菌,确定蜜环菌发酵液乙酸乙酯提取物的抑菌效果及其作用机制。结果表明:蜜环菌发酵液乙酸乙酯提取物的MIC值为5 mg/mL,MFC值为20 mg/mL;人参链格孢霉菌孢子萌发抑制率与提取物浓度呈正相关,其孢子萌发EC50值为2.69 mg/mL;显微观察发现,蜜环菌乙酸乙酯提取物处理可明显破坏人参链格孢霉菌菌丝体微观结构。细胞膜通透性和相关生化指标研究结果表明:蜜环菌乙酸乙酯提取物处理后极大提升了人参链格孢霉菌细胞膜通透性,其孢外电导率、孢外核酸含量以及丙二醛(MDA)和过氧化氢(H2O2)含量显著提高,超氧化物歧化酶(SOD)与过氧化物酶(POD)活性在前期略升高后显著降低。据此推测蜜环菌发酵液提取物的抑菌机制为破坏链格孢霉菌菌丝细胞的膜系统,造成菌丝细胞成分的外流并导致胞内生化反应被破坏。     

地表球囊霉诱发番茄抗早疫病的机理

丛枝菌根可以改善植物营养状况,提高宿主植物的抗病性.本文研究了番茄幼苗预先接种丛枝菌根真菌(AMF)地表球囊霉后对番茄植株保护酶活性和防御反应基因表达,以及对番茄早疫病抗性的影响.结果表明:被AMF侵染的番茄植株在接种早疫病病原菌茄链格孢菌后,其叶片内的超氧化物歧化酶(SOD)和过氧化物酶(POD)活性迅速提高.其中SOD酶活性在接种后18h达到最高,比只接种地表球囊霉(G)、茄链格孢菌(A)以及未接种AMF和病原菌的对照(CK)分别高28.6％、79.2％和82.8％;POD酶活性在接种后65 h达到最高,分别比G、A处理和CK高762％、18.3％和1710％.经荧光定量PCR检测表明,AMF侵染后的番茄植株再接种病原菌,其叶片中PR1(病程相关蛋白基因)、PR2(β-1,3-葡聚糖酶基因)和PR3(几丁质酶基因)基因的最高转录水平达到CK的9.67、8.54和13.4倍.与CK相比,先接种地表球囊霉再接种茄链格孢菌的番茄植株(GA)的早疫病发病率和病情指数分别降低了36.3％和61.4％.预先接种AMF的番茄植株在遇到病原菌袭击时诱导的防御反应强而迅速,诱发(priming)可能是菌根真菌提高宿主植物抗病性的重要机制.     

环状芽孢杆菌几丁质酶基因序列分析、表达和生物活性测定

真菌病害一直是影响作物的主要病害之一 ,每年造成巨大经济损失。几丁质酶可水解许多病原真菌细胞壁所含有的主要成分—几丁质 ,是研究得最多的抗真菌蛋白质。许多几丁质酶基因已从微生物中克隆到 ,芽孢杆菌是一类重要的几丁质酶产生菌。环状芽孢杆菌可产生并分泌多种多糖降解酶类 ,包括几丁质酶、β 1 ,3 葡聚糖酶、β 1 ,6葡聚糖酶和半纤维素酶[1] 。Ｗａｔａｎａｂｅ克隆了环状芽孢杆菌ＷＬ 1 2菌株的几丁质酶基因ｃｈｉＡ和ｃｈｉＤ ,对该几丁质酶基因的结构和功能进行了深入研究[2～ 4 ] 。我国的陈三凤克隆了黄杆菌的几丁质酶基因 ,…     

Chitinolytic and antifungal activity of a <Emphasis Type="Italic">Bacillus pumilus</Emphasis> chitinase expressed in <Emphasis Type="Italic">Arabidopsis</Emphasis>

The Bacillus pumilus SG2 chitinase gene (ChiS) and its truncated form lacking chitin binding (ChBD) and fibronectin type III (FnIII) domains were transformed to Arabidopsis plants and the expression, functionality and antifungal activity of the recombinant proteins were investigated. Results showed that while the two enzyme forms showed almost equal hydrolytic activity toward colloidal chitin, they exhibited a significant difference in antifungal activity. Recombinant ChiS in plant protein extracts displayed a high inhibitory effect on spore germination and radial growth of hyphae in Alternaria brassicicola, Fusarium graminearum and Botrytis cinerea, while the activity of the truncated enzyme was strongly abolished. These findings demonstrate that ChBD and FnIII domains are not necessary for hydrolysis of colloidal chitin but play an important role in hydrolysis of chitin–glucan complex of fungal cell walls. Twenty microgram aliquots of protein extracts from ChiS transgenic lines displayed strong antifungal activity causing up to 80% decrease in fungal spore germination. This is the first report of a Bacillus pumilus chitinase expressed in plant system.     

环境条件对芽孢杆菌B-9987菌株防御酶诱导效应的影响

研究室内外条件下,海洋芽孢杆菌不同处理物对番茄防御酶系的诱导作用。分别使用不同浓度芽孢杆菌B-9987发酵液和无细胞滤液喷施番茄植株,于室内外两种环境条件下养殖,测定了处理11d内番茄5种防御酶的变化趋势。两种处理诱导抗性结果显示,在两种环境条件下处理植株苯丙氨酸解氨酶（PAL）、多酚氧化酶（ppo）、过氧化物酶（POD）、过氧化氢酶（CAT）较空白对照植株酶活性均有不同程度增加。除超氧化物歧化酶（SOD）外,室内环境较室外更利于B-9987菌株处理液对番茄防御酶系的诱导作用发挥;相同环境条件下,发酵液的处理诱导活性较强。B-9987菌株对番茄防御酶系有较好的诱导作用,但诱导作用的发挥受环境条件影响明显。     

Natamycin blocks fungal growth by binding specifically to ergosterol without permeabilizing the membrane

Natamycin is a polyene antibiotic that is commonly used as an antifungal agent because of its broad spectrum of activity and the lack of development of resistance. Other polyene antibiotics, like nystatin and filipin are known to interact with sterols, with some specificity for ergosterol thereby causing leakage of essential components and cell death. The mode of action of natamycin is unknown and is investigated in this study using different in vitro and in vivo approaches. Isothermal titration calorimetry and direct binding studies revealed that natamycin binds specifically to ergosterol present in model membranes. Yeast sterol biosynthetic mutants revealed the importance of the double bonds in the B-ring of ergosterol for the natamycin-ergosterol interaction and the consecutive block of fungal growth. Surprisingly, in strong contrast to nystatin and filipin, natamycin did not change the permeability of the yeast plasma membrane under conditions that growth was blocked. Also, in ergosterol containing model membranes, natamycin did not cause a change in bilayer permeability. This demonstrates that natamycin acts via a novel mode of action and blocks fungal growth by binding specifically to ergosterol.     

作用于细胞壁的抗真菌药物研究进展

与人类细胞相比,细胞壁为真菌的特有结构,因此作用于细胞壁的抗真菌药物相较于其他类型抗真菌药物而言具有高效、低毒的特点,是迄今为止安全性最高的一类抗真菌药物。本文对作用于细胞壁的抗真菌药物进行综述,根据作用机制及靶点的不同分别介绍葡聚糖合成酶抑制剂、几丁质合成酶抑制剂及糖基磷脂酰肌(glycosylphosphatidylinositol,GPI)锚定蛋白抑制剂,对其进行总结和归纳,为相关药物的研发及将来的临床应用前景提供参考。     

Phenotype analysis of Saccharomyces cerevisiae mutants with deletions in Pir cell wall glycoproteins

Proteins with internal repeats (Pir) belong to a minor group of covalently linked yeast cell wall proteins. They are not essential for viability but important for cell wall strength, reduced permeability against plant antifungal enzymes and maintenance of osmotic stability. Here we show the importance of Pir proteins of Saccharomyces cerevisiae for growth at low pH and in presence of various inhibitors. Cell wall analysis of Deltapir1,2,3,4 deletion strain revealed slightly increased chitin content and changes in relative proportion of alkali-soluble and insoluble glucan and chitin fractions. Activation of the cell wall integrity pathway was indicated by increased levels of double phosphorylated Mpk1p/Slt2p in the pir deletants.     

The fungal cell wall and the mechanism of action of anidulafungin

The fungal cell wall is a structure with a high plasticity that protects the cell from different types of environmental stresses including changes in osmotic pressure. In addition to that, the cell wall allows the fungal cell to interact with its environment, since some of its proteins are adhesins and receptors. Some of its components are highly immunogenic. The structure of the fungal cell wall is unique to the fungi, and it is composed of glucan, chitin and glycoproteins. Since humans lack the components present in the cell walls of fungi, this structure is an excellent target for the development of antifungal drugs. Anidulafungin, like the rest of echinocandins acts on beta-1,3-D-glucan synthase inhibiting the formation of beta-1,3-D-glucan and causing, depending on the type of fungus, a fungicidal or either a fungistatic effect.     

Multiple effects of a novel compound from Burkholderia cepacia against Candida albicans

A novel compound (named CF66I) produced by Burkholeria cepacia CF-66 strain was investigated for its antifungal activity against Candida albicans. This compound exhibited excellent antifungal activity in a dose- and time-dependent manner. Uptake analysis revealed that the compound preferentially acted against the fungal cell wall, and was also able to enter the cells. Transmission electron microscopy indicated that this compound caused loosening of the cell wall and a significant increase in the cell wall thickness was noted; however, no alterations were observed in the contents of the cell wall components. CF66I probably affected the normal assembly and integration of fungal cell wall components by interrupting the weak interactions between them, such as hydrogen and hydrophobic bonds. Propidium iodide (PI) staining indicated that on exposure to CF66I C. albicans cells became permeable to PI. Marked alterations in lipid and sterol contents were observed, and the major changes were a depletion of total lipids and ergosterol, concomitant with an increase in lanosterol content. These observations suggested that the novel compound CF66I may have considerable potential for development of a new class of antifungal agents.     

Evidence from mycelial studies for differences in the sterol biosynthetic pathway of Rhizoctonia solani and Phytophthora cinnamomi.

Phytophthora cinnamomi, a member of the Pythiacease, does not synthesize sterols. Small amounts of squalene, but no squalene epoxide or sterol, were isolated from the dried mycelium of this fungus after growth in sterol-free medium. The dried mycelium of Rhizoctonia solani, a sterol-synthesizing fungus grown under the same conditions, contained small amounts of squalene and squalene epoxide and large amounts of ergosterol. When the two organisms were grown in the presence of [14C]acetate, only labelled geraniol, farnesol and squalene were recovered from the P. cinnamomi mycelium, whereas labelled geraniol, farnesol, squalene, squalene epoxide and ergosterol were recovered from the R. solani mycelium. Similar results were obtained when the organisms were incubated in the presence of [2(-14)C]mevalonate; in this case, labelled lanosterol was also detected in the R. solani mycelium. Both organisms, when incubated in the presence of unlabelled squalene, squalene epoxide or lanosterol, incorporated these compounds into their mycelia; however, only the R. solani mycelium was able to convert these substrates into products further along the sterol pathway. It appears that squalene is the terminal compound in the sterol biosynthetic pathway of P. cinnamomi.