莜麦类甜蛋白Permatin抑制木霉菌活性的机制研究

类甜蛋白(thaumatin-like proteins,TLPs)是一个高度复杂的蛋白质家族,与植物、动物和真菌的宿主防御和发育过程有关。TLPs已在多种植物中被发现,具有广谱抗真菌活性。近年来,植物来源的抗真菌蛋白在植物抗病原菌中具有广泛的应用前景,然而关于莜麦来源的抗真菌蛋白研究比较少。本研究从莜麦中分离纯化得到莜麦类甜蛋白Permatin,通过带毒平板法测定了Permatin蛋白对木霉菌的半抑制浓度;然后利用Delta Vision显微镜观察了Permatin蛋白对木霉菌孢子萌发及菌丝生长的影响;进一步结合碘化丙啶(propidium Iodine,PI)染色研究Permatin蛋白对木霉菌菌丝及孢子细胞膜通透性的影响。结果显示,Permatin蛋白对木霉菌的半抑制浓度为0.73μmol/L,可以显著抑制木霉菌孢子的萌发及菌丝的生长,并且能够破坏真菌菌丝及孢子细胞膜的通透性。本研究为莜麦Permatin蛋白在农业领域的应用提供了理论基础。     

一株生防圆突起链霉菌的分类鉴定

菌株SCY311是从河南省凤凰山土壤样品中分离到的对多种植物病原真菌具有拮抗活性的一株放线菌。为了明确其分类地位, 在形态特征、培养特征、生理生化特征、细胞壁组分测定等传统分类学方法的基础上, 测定和分析了菌株的16S rRNA基因序列。结果表明, 菌株SCY311在高氏一号培养基上生长良好, 基内菌丝呈褐色; 气生菌丝灰色至鼠灰色, 不产生可溶性色素, 无吸水现象; 孢子链卷曲, 末端形成闭合或开放螺旋; 孢子椭圆或圆柱状, 表面形成结节状突起; 生理生化特征和在国际链霉菌计划(ISP)培养基上的培养特     

链霉菌30702的鉴定及其生防特性

对从海南粗榧根际分离的一株链霉菌菌株30702进行菌种鉴定和生物防治特性的研究。利用16S rRNA基因序列和多位点序列分析(MLSA),并结合形态和生理生化特征比较分析确定该菌株种水平的分类地位。该菌株鉴定为紫黑链霉菌进化分枝中的Streptomyces solisilvae;该菌株能够产生蛋白酶、纤维素酶、几丁质酶、β-葡聚糖酶、1-氨基环丙烷-1-羧酸(ACC)脱氨酶、铁载体,具有解磷活性和促进植物生长的特性,发酵代谢产物能够抑制山药炭疽菌孢子萌发和菌丝生长。菌株30702具有优良的生防特性,在植物病害的生物防治上具有潜在的开发利用价值。     

一株产蓝色素链霉菌18-A-5的鉴定与系统发育分析

从原始热带雨林土壤中,分离到一株产蓝色色素菌株18-A-5,对其进行了系统分类学研究。形态学特征观察表明,在高氏合成一号培养基上初产蓝绿色色素,日久为深蓝色,基内菌丝蓝色,气生菌丝灰白色,产灰色孢子,孢子丝直或柔曲,形成长孢子链,孢子圆柱形。其DNA的G＋C摩尔分数为62.4%,16S rDNA序列分析结果(GenBank登陆号为EU054353),18-A-5与生靛链霉菌Streptomyces indigoferus ATCC23924^T 、草绿色链霉菌Streptomyces herbaricolor ATCC23924^T具有极高的同源性,达100％,聚类分析表明,18-A-5与生靛链霉菌Streptomyces indigoferus、草绿色链霉菌Streptomyces herbaricolor两株菌聚类在一起,分支置信度为74%。结合生理生化特性、细胞壁化学组成分析、脂肪酸分析等将菌株18-A-5定名为草绿色链霉菌Streptomyces herbaricolor。并对该蓝绿色可溶性色素性质进行了耐酸碱性、热稳定性、抗菌谱等初步分析。     

摇床转速对淡紫拟青霉菌生长的影响

设置不同摇床转速来调节通气量,以淡紫拟青霉菌株NHPL03培养过程的OD值、pH值、菌丝重量、孢子量、毒力为指标,研究通气量对淡紫拟青霉菌NHPL03生长的影响。结果表明:淡紫拟青霉菌可在多种摇床转速(通气量)条件下生长。通气量不仅影响菌丝生长与孢子生成而且影响毒力产物分泌,菌丝生长与孢子生成的最佳摇床转速为100r/min,培养8d菌丝干重达到最高值1.10g/50mL,产孢量达28.5×106个/mL。从毒力强度看,低转速条件60r/min总体毒力水平最高,培养8d菌液对茎线虫校正致死率达到47.1%。     

一株链霉菌21-1的分离鉴定及其对禾谷镰刀菌的抑菌活性

【背景】由禾谷镰刀菌(Fusarium graminearum)引起的小麦赤霉病严重威胁我国的小麦生产。【目的】筛选对禾谷镰刀菌具有拮抗能力的链霉菌菌株,为生防菌剂开发提供理论基础。【方法】利用平板对峙法筛选对禾谷镰刀菌具有拮抗能力的链霉菌;通过形态特征、生理生化特征和16S rRNA基因序列分析对其进行鉴定;通过病原菌菌丝生长、孢子产生及萌发抑制试验分析其发酵液的抑菌活性;利用人工接种试验测定该菌株发酵液的防病效果。【结果】筛选到一株对禾谷镰刀菌具有较强拮抗活性的链霉菌21-1,抑菌率为59.5%。依据形态特征、生理生化特性和16S rRNA基因序列分析,将该菌株鉴定为黄三素链霉菌(Streptomycesflavotricini)。菌株21-1发酵液能够抑制禾谷镰刀菌的菌丝生长、孢子产生及萌发过程,而且可以降低禾谷镰刀菌菌丝中可溶性蛋白质的含量,并增加丙二醛的含量。菌株21-1可以产生蛋白酶及纤维素酶。菌株21-1菌液10倍稀释液对小麦赤霉病的防效最佳,为70.1%。此外,菌株21-1发酵液对其他8种植物病原菌均有较好的抑制作用。【结论】菌株21-1对禾谷镰刀菌有较好的抑菌活性,具...     

番茄灰霉菌拮抗放线菌LA-5的筛选及鉴定

利用稀释涂布法从番茄根际土壤中分离放线菌,并以番茄灰霉菌为靶标,利用对峙培养法和牛津杯法筛选拮抗放线菌,得到一株具有较强抑菌活性的放线菌LA-5.通过培养特征、生理生化特性及基于16S rDNA 序列系统进化分析,将菌株LA-5初步鉴定为链霉菌.复筛结果显示,LA-5发酵滤液对番茄灰霉菌孢子萌发及菌丝生长均有明显的抑制作用,其中100倍发酵滤液对孢子萌发抑制率和菌丝生长抑制率均在50%以上;受抑制菌落呈白色,气生菌丝萎缩稀疏,菌丝纤细、分支明显减少.离体防效试验显示,菌株LA-5发酵原液对番茄灰霉病防效可达83.4%.该菌株有望开发为防治番茄灰霉病的生防菌株.     

圈卷产色链霉菌分化及其特性的研究

链霉菌分化的分子生物学研究是一个饶有兴趣并富有挑战性的世界前沿研究课题.在原核生物的分化研究中,主要以枯草杆菌(Bacillus subtilis)作为模式系统,但枯草杆菌的生命周期尤其是分化过程远比链霉菌简单,不象链霉菌有基质菌丝、气生菌丝、菌丝螺旋和孢子分隔那样的发育分化过程[1].在真核生物中也有分化研究的报道,如构巢曲霉、酵母等.由于真核生物的基因结构比原核生物复杂得多,弄清分化中基因调控的关系就更加困难.因此,选用链霉菌作分化研究的材料有其独一无二的优越性.国际上有关链霉菌的分子生物学研究,近年来主要以链霉菌的抗生素生物合成基因和链霉菌的分化基因两个大的方面作为研究的热点和主攻方向,并展开了一些开拓性的研究.     

圈卷产色链霉菌分化相关基因——saw1的结构和功能

用双脱氧链终止法进行了分化基因——saw1的双链测序.结果表明在1500bp的DNA片段中有一个完整的开读框架(ORF),其编码区是在419bp至1252bp处.其产物与已知的天蓝色链霉菌whiG的氨基酸序列有89%的同源性.当把1500bp的saw1DNA片段插入到链霉菌表达质粒载体pIJ702后,构建的重组质粒转化天蓝色链霉菌孢子形成缺陷突变株C71,可使C71形成孢子和灰色色素.用基因破坏的策略进一步研究了该基因的生物学功能,结果表明saw1在圈卷产色链霉菌气生菌丝到孢子形成的发育转变中有重要作用,是分化中控制孢子发育起始的一个重要基因.     

一株拮抗链霉菌的鉴定及其多相分类特征

通过16S r RNA基因序列分析一株拮抗放线菌JXNU03的系统发育关系,通过生理生化试验分析其生理生化特征,利用菌丝生长率法和杯碟法测定其拮抗活性。结果发现,放线菌JXNU03在高氏一号培养基上培养时,基内菌丝发达,分支多且不断裂,气生菌丝发育良好,孢子丝直形或螺旋状,孢子表面带刺,细胞水解液中检测到葡萄糖,未检测出特征性糖,糖类型属于C型,细胞壁氨基酸中含有L,L-DAP,属于细胞壁Ⅰ型,16S r RNA基因序列与灭癌素链霉菌的同源性高达98%,其发酵液对革兰氏阳性细菌、革兰氏阴性细菌、酵母菌和霉菌等都具有较强的拮抗作用。因此放线菌JXNU03被鉴定为灭癌素链霉菌,记为灭癌素链霉菌JXNU03(Streptomyes gancidicus JXNU03),是一株对细菌、酵母菌和霉菌均有较强拮抗活性的链霉菌,具有潜在开发价值。     

拟核结合蛋白调控链霉菌形态分化和次级代谢的作用和机制

链霉菌具有独特而复杂的形态分化周期,涉及到染色体复制、浓缩和分离等多个步骤,并伴随着菌丝的分隔和片段化。拟核结合蛋白作为染色体高级结构的重要组成成分,在调控链霉菌的形态分化中发挥了重要作用,调控许多与DNA相关的过程,包括基因表达、DNA保护、重组/修复和拟核的形成与维持等。此外,拟核结合蛋白作为细菌重要的全局性调控因子,也广泛参与了链霉菌次级代谢的调控。本文总结了链霉菌拟核结合蛋白的结构和功能,特别是调控形态分化和次级代谢的最新研究成果。     

Litter N retention over winter for a low and a high phenolic species in the alpine tundra

Mycorrhizal fungus colonization of roots may modify plant metal acquisition and tolerance. In the present study, the contribution of the extraradical mycelium of an arbuscular mycorrhizal (AM) fungus, Glomus mosseae (BEG 107), to the uptake of metal cations (Cu, Zn, Cd and Ni) by cucumber (Cucumis sativus) plants was determined. The influence of the amount of P supplied to the hyphae on the acquisition and partitioning of metal cations in the mycorrhizal plants was also investigated. Pots with three compartments were used to separate root and root-free hyphal growing zones. The shoot concentration of Cd and Ni was decreased in mycorrhizal plants compared to non-mycorrhizal plants. In contrast, shoot Zn and Cu concentrations were increased in mycorrhizal plants. High P supply to hyphae resulted in decreased root Cu concentrations and shoot Cd and Ni concentrations in mycorrhizal plants. These results confirm that some elements required for plant growth (P, Zn, Cu) are taken up by mycorrhizal hyphae and are then transported to the plants. Conversely, Cd and Ni were transported in much smaller amounts by hyphae to the plant, so that arbuscular mycorrhizal fungus colonization could partly protect plants from toxic effects of these elements. Selective uptake and transport of plant essential elements over non-essential elements by AM hyphae, increased growth of mycorrhizal plants, and metal accumulation in the root may all contribute to the successful growth of mycorrhizal plants on metal-rich substrates. These effects are stimulated when hyphae can access sufficient P in soil.     

Preliminary characterization of some Streptomyces species from four Tanzanian soils and their antimicrobial potential against selected plant and animal pathogenic bacteria

This study was undertaken to characterize Streptomyces strains occurring in some soils of Tanzania as well as to evaluate their potential to synthesize antimicrobial compounds. Six main classes of isolates were observed according to the colour of aerial mycelium. These were gray, cream, blue, pink, red, and white. The gray colour class dominated. About 65% of the isolates produced soluble pigments of various colours while about 33% of the isolates did not produce any soluble pigments. Brown coloured soluble pigments dominated. About 57% of the isolates had spiral spore chains. Some Streptomyces isolates displayed strong (> 30 mm inhibition zone), moderate (20–30 mm), or weak (< 20 mm) antibiosis against the plant/animal pathogenic bacteria tested. Other isolates did not show any antibiosis against any of the test pathogens. The plant pathogens CMM IPO 542 (Clavibacter michiganensis ssp. michiganensis) and Xanthomonas vascatoria were inhibited by most of the Streptomyces isolates. Xanthomonas oryzae pv. oryzae and X. campestris were inhibited by the least number of the Streptomyces isolates. Most of the animal pathogens tested seemed to show resistance to the antibiotics produced by some of the Streptomyces isolates which had shown high activity against the plant pathogens. This revised version was published online in July 2006 with corrections to the Cover Date.     

The Saccharomyces cerevisiae chitinase,encoded by the CTS1-2 gene,confers antifungal activity against Botrytis cinerea to Transgenic tobacco

The Saccharomyces cerevisiae chitinase, encoded by the CTS1-2 gene has recently been confirmed by in vitro tests to possess antifungal abilities. In this study, the CTS1-2 gene has been evaluated for its in planta antifungal activity by constitutive overexpression in tobacco plants to assess its potential to increase the plant's defence against fungal pathogens. Transgenic tobacco plants, generated by Agrobacterium-mediated transformation, showed stable integration and inheritance of the transgene. Northern blot analyses conducted on the transgenic tobacco plants confirmed transgene expression. Leaf extracts from the transgenic lines inhibited Botrytis cinerea spore germination and hyphal growth by up to 70% in a quantitative in vitro assay, leading to severe physical damage on the hyphae. Several of the F₁ progeny lines were challenged with the fungal pathogen, B. cinerea, in a detached leaf infection assay, showing a decrease in susceptibility ranging from 50 to 70%. The plant lines that showed increased disease tolerance were also shown to have higher chitinase activities.     

Peptidase activities during morphogenesis of Mucor racemosus

Multiple peptidase activities are expressed in the dimorphic fungus Mucor racemosus. Peptide hydrolysis was measured using an enzyme-coupled colorimetric assay. Aminopeptidase as well as carboxypeptidase activities increased during spore, swollen spore, and budding yeast-to-hyphae conversions, and activities achieved a maximum level prior to the period of rapid germ tube formation. These increases in peptidase activity were prevented by cycloheximide. Three distinct aminopeptidases (AP) and three distinct carboxypeptidases (CP) were partially purified by gel filtration column chromatography. AP1 (235 kDa), AP2 (112 kDa), and AP3 (70 kDa) were all expressed in spore, yeast, and hyphae. The activity levels of AP2 and AP3 decreased in hyphae entering stationary growth. CP1 (250 kDa) and CP3 (50 kDa) activities were expressed exclusively in hyphae, whereas CP2 (77 kDa) was expressed in spore, yeast, and hyphal forms. CP1 activity was most pronounced in hyphae entering stationary growth. We concluded that M. racemosus expresses a multiplicity of peptidases and that CP1 and CP3 are morphology-specific carboxypeptidases.     

Isolation of Endophytic Actinomycetes from Different Cultivars of Tomato and their Activities Against Ralstonia solanacearum in Vitro

The populations of endophytic actinomycetes from healthy and wilting tomato plants (tomato cultivars resistant and susceptible to Ralstonia solanacearum) grown in three different sites from Guangzhou, Guangdong Province, South China were investigated by cultivation methods. Most of the isolates belonged to streptomycetes. The Aureus group of Streptomyces was the most frequently isolated group. The population composition of Streptomyces varied according to tomato cultivars, physiological status and soil types. The proportions of antagonistic Streptomyces strains from healthy plants were higher than that from wilting plants (P < 0.05), although the difference among the proportions of antagonistic Streptomyces strains from different cultivars of healthy tomato was not significant, the similar result was found from wilting plants. No significant difference was found in the proportions of siderophere-producing Streptomyces strains from the same site (P > 0.05), but the difference was found from the different sampling sites (P < 0.05). The percentage of bacterial cell wall-degrading streptomycetes from wilting tomato was higher than that from healthy plants (P < 0.05). These results indicated that the cultivar of the host plant, physiological status and sampling sites would influence the proportion of endophytic streptomycetes with different physiological traits. Diversity of endophytic Streptomyces and their physiological diversity should be involved in developing potential biocontrol agents.     

The physiology of spore-negative and spore-positive nodules ofMyrica gale

The physiology of spore-negative and spore-positive root nodules was investigated inMyrica gale L. grown in water culture in a growth chamber. Spore(–) nodules were induced withFrankia cultures and spore(+) nodules with crushed nodules. Gas exchange was measured in a flow-through system.The time course of acetylene reduction following addition of acetylene was essentially the same in both spore(–) and spore(+) nodules with a stable maximum between 2 and 4 minutes followed by a steep decline to a minimum (37% of the maximum) between 9 and 30 minutes depending on the plant. The minimum was followed by a partial recovery. Nodule CO₂ evolution showed a similar pattern but the minimum rate (83% of the maximum) was not nearly as low.Plants nodulated with one spore(–) and one spore(+) strain were compared at 6, 8 and 10 weeks after inoculation. At 6 weeks the spore(–) plants had 52% greater specific nitrogenase activity and 46% more biomass than the spore(+) plants. At 8 and 10 weeks, however, the differences between plants with spore(–) and spore(+) nodules became smaller.Plants nodulated with 4 spore(–) and 5 spore(+) strains were compared at 8 weeks after inoculation. Collectively the spore(–) plants exhibited a 32% greater specific nitrogenase activity, a 15% lower energy cost of nitrogenase activity (CO₂/C₂H₄), and invested 31% less biomass in nodules than the spore(+) plants. The spore(–) plants also produced 16% more biomass indicating that spore(–) strains are generally more desirable than spore(+) strains. However, two spore(+) strains were as effective as the spore(–) strains.     

链霉菌IMS002的分类鉴定及其抗真菌活性物质解析

【目的】对一株分离自植物根际土壤的具有抗真菌活性的链霉菌IMS002进行菌株分类鉴定,通过活性追踪分离纯化并鉴定有机相中的活性物质。【方法】通过16S rDNA和5个不同基因(atpD,gyrB,recA,rpoB,trpB)串联聚类分析以及生理生化实验分析,对链霉菌IMS002进行菌株分类鉴定,用扫描电子显微镜观察该株链霉菌的菌丝及孢子形态,以尖孢镰刀菌(Fusarium oxysporum)为指示菌进行生物活性追踪,通过硅胶柱层析、凝胶柱层析及高压液相色谱(HPLC)对活性物质进行分离和纯化,使用液质联用高分辨质谱仪、500 MHz核磁共振波谱仪以及圆二色光谱仪确定该物质的化学结构。【结果】IMS002经初步鉴定与产二素链霉菌(Streptomycesambofaciens)具有较近的亲缘关系,其发酵液对尖孢镰刀菌具有良好的抑菌效果,经分离和纯化以及现代波谱技术分析,确定有机相中的抑菌活性组分为Borrelidin。【结论】链霉菌IMS002能够产生化合物Borrelidin,该化合物对尖孢镰刀菌具有抑制活性。     

Metabolic engineering for the production of prenylated polyphenols in transgenic legume plants using bacterial and plant prenyltransferases

Prenylated polyphenols are secondary metabolites beneficial for human health because of their various biological activities. Metabolic engineering was performed using Streptomyces and Sophora flavescens prenyltransferase genes to produce prenylated polyphenols in transgenic legume plants. Three Streptomyces genes, NphB, SCO7190, and NovQ, whose gene products have broad substrate specificity, were overexpressed in a model legume, Lotus japonicus, in the cytosol, plastids or mitochondria with modification to induce the protein localization. Two plant genes, N8DT and G6DT, from Sophora flavescens whose gene products show narrow substrate specificity were also overexpressed in Lotus japonicus. Prenylated polyphenols were undetectable in these plants; however, supplementation of a flavonoid substrate resulted in the production of prenylated polyphenols such as 7-O-geranylgenistein, 6-dimethylallylnaringenin, 6-dimethylallylgenistein, 8-dimethylallynaringenin, and 6-dimethylallylgenistein in transgenic plants. Although transformants with the native NovQ did not produce prenylated polyphenols, modification of its codon usage led to the production of 6-dimethylallylnaringenin and 6-dimethylallylgenistein in transformants following naringenin supplementation. Prenylated polyphenols were not produced in mitochondrial-targeted transformants even under substrate feeding. SCO7190 was also expressed in soybean, and dimethylallylapigenin and dimethylallyldaidzein were produced by supplementing naringenin. This study demonstrated the potential for the production of novel prenylated polyphenols in transgenic plants. In particular, the enzymatic properties of prenyltransferases seemed to be altered in transgenic plants in a host species-dependent manner.     

Evaluation of Streptomyces spp. for biocontrol of gummy stem blight (Didymella bryoniae) and growth promotion of Cucumis melo L.

Gummy stem blight of Cucumis melo L. (melon) caused by Didymella bryoniae is a serious disease in the major production area of northwest China. Two Streptomyces isolates (Streptomyces pactum A12 and S. globisporus subsp. globisporus C28) previously isolated from the Qinghai-Tibet Plateau were investigated regarding their biocontrol of gummy stem blight and growth promotion of melon under controlled conditions. Streptomyces A12 and C28 indicated obvious antagonistic activity against D. bryoniae in vitro. Both A12 and C28 significantly decreased disease severity and AUDPC (area under the disease progress curve) of melon gummy stem blight in vivo (P < 0.05). Ten-fold dilution of C28 culture filtrate was more effective in controlling the disease compared with other treatments, the disease reduction effects were 41.0–64.2%. The mean fresh weights were increased by 40.4% for plants, 44.2% for roots, and 40.3% for aerial parts, when A12 was applied in both nursery soil and transplanted soil. Streptomyces C28 also increased the mean fresh weights of melon plants by 18.4–49.0% compared with the control in pot trial. Streptomyces A12 and C28 showed substantial colonization abilities in the rhizosphere and on the rhizoplane of melon plants. Results demonstrated that Streptomyces A12 and C28 were of positive effect on the biocontrol of gummy stem blight and growth promotion of Cucumis melo L.