Name: physical isolation of endospores from environmental samples by targeted lysis of vegetative cells
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作者承认瑞士国家科学基金会的批准号:31003A-一分之一十三万二千三百五十八，31003A_152972和No. 151948，和基金会皮埃尔·名士倒拉科学。

1.制备化学品和设备<ol><li>制成500毫升1％的六偏磷酸钠（SHMP）（磷酸钠3）N溶液和通过高压灭菌消毒。 </li><li>消毒硝化纤维素（NC）的过滤器（孔径0.22μm，直径47个毫米）通过高压灭菌在密闭的玻璃培养皿。 </li><li>通过高压在密闭玻璃培养皿消毒的NC滤波器（孔径0.22μm，直径25毫米）。 </li><li>称量并注意无菌50ml试管的空机重量（含上限）（每个样品一管）。 </li><li>制备的Tris-EDTA缓冲液（TE缓冲液）1×:使10毫摩尔Tris（ 三（羟甲基 ）氨基甲烷）和1mM EDTA缓冲溶液。调节pH至8，并通过高压灭菌消毒。 </li><li>通过在1ml的TE缓冲液中溶解0.02克溶菌酶制备溶菌酶溶液（20毫克/毫升）。理想的情况下，使新鲜每次。存放在4℃下不超过1周。 </li><li>通过将8克/升的氯化钠制备生理溶液（氯化钠）在蒸馏水。高压灭菌。 </li></ol>从沉积物2.分离生物质<ol><li>添加3克沉积物样品，以预先称重无菌使用乙醇火烧金属勺50ml试管。执行此在一个干净，UV消毒生物安全柜，以避免污染。 </li><li>加入15mL 1％的无菌（高压灭菌）SHMP溶液使用无菌刻度滴管样品。该SHMP溶液还可以进行筛选，以避免污染。 </li><li>均化沉渣和SHMP溶液与液体分散器/均化器（例如，超TURRAX均化）。 70％的乙醇 - 消毒或高压灭菌，使用前分散转子。运行同质化1分钟17,500转。让样品休息2分钟，在相同的转子速度重复均质化1分钟。 </li><li>让样品静置10分钟。在该步骤中，最重的粒子（矿物）会沉淀。将细胞和样品然而瓦特任何有机成分病留在溶液中。之后，转移上清液溶液（含有细胞生物量）到一个干净的50ml管中，同时小心不要扰乱沉积的颗粒。 </li><li>向沉淀添加粒料再次将15ml 1％的无菌（高压灭菌）SHMP使用无菌刻度滴管溶液。然后重复步骤2.3和2.4。这种重复可以确保电池和有机粒子的最大量，从沉淀物的矿物组分分离。此第二分离的上清液可以合并与来自第一分离上清液。 注:以下步骤都做对上清（含细胞生物量）。矿物组分（沉淀沉淀）可以被丢弃。 </li><li>离心样品以20×g离心1分钟。这一步增加了G力足以解决小矿粒，而生物细胞物质仍然在溶液中。离心后，转移上清液溶液（含有细胞的生物质）转化为干净的50ml管中。弃矿物颗粒。 </li><li>确定包含生物量通过称重样品溶液的最终体积。权重确定避免了必须将样品转移至量筒并降低污染的风险。 </li></ol> 3.生物质收集在滤膜<ol><li>制备过滤单元（47毫米直径的膜）和真空泵。或者通过高压灭菌或（如果派热克斯玻璃）用70％的乙醇喷洒和用本生灯燃烧它消毒过滤单元。让与协议，然后再继续它冷却下来。 </li><li>用乙醇 - 熄火灭菌镊子添加无菌NC膜的过滤单元。 </li><li>加入一半的上清液样品（来自步骤2.6）的到膜过滤单元和收集细胞上使用真空泵膜上。 </li><li>当液体已充分通过过滤器，停止真空泵，小心地用乙除去膜OL火焰消毒镊子。放置膜进入无菌培养皿。 <ol><li>使用乙醇火烧消毒剪刀剪膜的一半。该膜的每一半添加到单独的2毫升管。膜的一个半部分将被用于DNA提取和整个细菌群落分析。滤波器的另一半将被保存在-80℃，并作为一个备份。 </li></ol></li><li>放置新的NC膜到过滤单元，并使用真空泵收集从样品体积（来自步骤2.6）的第二半的生物质。 </li><li>当液体已充分通过过滤器，停止真空泵，并仔细用乙醇 - 熄火灭菌镊子除去该膜。将整个膜到一个单独的2毫升管。该样品将被用于对生孢子从营养细胞分离的治疗。可将样品储存在-20℃直至使用。 </li></ol> 4.裂解营养细胞<ol><li>执行处理分离先前在NC膜（步骤3.6）收集的生物量营养细胞内生孢子。 <ol><li>如果膜被冻结，离开它在室温10分钟以解冻。然后放置在无菌培养皿的膜和使用乙醇火烧灭菌剪刀剪（约4倍）成更小的碎片。然后将所有的膜过滤片放入一个无菌的ml管。 </li></ol></li><li>加入900微升1×TE（的Tris-EDTA）缓冲液（见1.5），以含有样品膜管涡旋调匀。在此步骤中，所述生物质从膜进TE-缓冲液的溶液中除去。 </li><li>将管在培养箱在65℃下进行10分钟，80转。然后从孵化器中取出管，让它冷却15分钟。 </li><li>加入100微升新鲜制备的溶菌酶（见1.5），以达到2毫克/毫升的最终浓度。不要添加溶菌酶前将样品冷却到37℃，因为这可能DEG拉德的酶。 </li><li>孵育样品在37℃下60分钟，80转，对于溶菌酶的最佳条件以溶解营养细胞。 </li><li>裂解完成后，加入250微升3当量的氢氧化钠（氢氧化钠）和250微升6％十二烷基硫酸钠（SDS）溶液到样品中。通过添加此，样品体积达到1.5 ml和有0.5N的氢氧化钠的最终浓度和1％SDS的终浓度。 </li><li>孵育这种混合在室温进行60分钟和80转。加入碱和洗涤剂将有助于最终的细胞溶解。这些洗涤剂的浓度进行了优化，以不损害孢子，而裂解营养细胞。 </li><li>准备保存25mm直径的膜通过高压灭菌，或者（如果派热克斯玻璃）喷洒用70％乙醇和用本生灯燃烧它的无菌过滤单元。让它冷却下来。 </li><li>用乙醇 - 火焰灭菌镊子将过滤单元上的0.2微米的NC膜（25毫米直径）。&lt;/ LI&gt; <li>从步骤4.7添加示例到膜上，并使用真空泵过滤液体。当液体已通过，关闭真空泵。在该步骤中，裂解营养细胞材料被去除，因为它不保留在膜。只有内生孢子将留在该膜上。 </li><li>加入2毫升无菌生理溶液洗掉残留的清洁剂和用真空泵过滤液体。 </li><li>当液体完全过滤，关闭真空泵。离开过滤单元的膜。 </li></ol> 5. DNA酶处理注意:直接在过滤膜执行DNase处理。重要的是，过滤部不泄漏是重要的，所述真空泵是关闭的。 <ol><li>加入450微升无菌水，50微升DNA酶反应直接缓冲液（1x）和0.5微升DNA酶的酶附着在过滤器膜上，静置15分钟。如果可能的话，做到这一点消化，房间里稍微战争聚体比平均室温，由于酶的工作温度为25℃以上更好。 注意:保留本生灯一旁过滤单元也增加的温度和具有保持周围环境的无菌的附加好处，因此降低了样品的污染的风险。 </li><li>当DNA酶消化完成后，开启真空泵以从样品除去酶。 </li><li>通过添加和筛选1 ml的生理溶液洗掉残留的酶。 </li><li>如果液体已全面通过，关闭真空泵，并删除含有使用无菌镊子，并放入无菌培养皿内生孢子的过滤膜。 注意:分离内生孢子的样品现在准备用于下游分析。如果使用储存在-20℃下进行DNA提取或替代地，在4℃下，如果用于发芽和栽培。 </li></ol>

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内生孢子外部攻击性理化因素 （如温度或清洁剂）的电阻来设计一种方法，从营养细胞环境样品中分离的细菌芽孢。这是第一个全面的方法来隔离环境样品的内生孢子在非破坏性的方式。以前的方法来定量，检测或分析样品中内生孢子是根据特定的代理内生孢子诸如吡啶二羧酸或特定的标记基因的测定。与此相反，与协议这里介绍，比生孢子其他样品组分被去除，所以内生孢子最终仍作为...

这项工作的目的是提供一种协议，用于细菌内生孢子的从营养细菌细胞环境样品中分离。细菌内生孢子的形成是一个生存策略，通常由饥饿触发，在多个属于脊索动物门厚壁菌门1细菌群体的发现。形成内生孢子的细菌被很好的研究，主要是因为一些菌株是病原体，因此医学重要（例如， 炭疽芽孢杆菌或艰难梭菌 ）。形成内生孢子菌的环境菌株已分离出几乎所有的环境（土壤，水，沉淀物，空气，冰，人体肠道，内脏动物，和更多）1-3。因此，厚壁菌在培养物保藏4第二个最丰富的门。 由于其耐寒外皮层和保护核心蛋白，内生孢子能够生存极端的环境条件范围FROM干燥高辐射，极端温度和有害化学物质5。这一显着性使它成为一个挑战提取芽孢6-8 DNA。这可能解释了为什么他们被忽视了环境测序研究9,10。其他方法，如环境样品中内生孢子的荧光抗体11靶向，吡啶二羧酸的定量（DPA）中土壤12和沉积物13，流式细胞仪14或巴氏灭菌和随后的培养15,16已被用于在检索或量化生孢子环境样品。在最近几年，优化的DNA提取方法以及具体的分子的引物靶向生孢子特异性基因序列已经被开发10,17-20。这有助于揭示这组细菌21之间更多的生物多样性，并且也导致了内生孢子的检测应用在工业和医药中，例如在奶粉19。 这里介绍的协议是基于到有害的物理化学条件（例如热和洗涤剂）相对于营养细胞细菌内生孢子的电阻的差别。以破坏营养细胞的样品中，我们连续施加热，溶菌酶和低浓度的洗涤剂。这些治疗的时间和强度都得到了优化，以便不破坏孢子，但以裂解所有营养细胞。一些细胞在一个环境室池比其它更耐磨，所以为了提高破坏所有营养细胞的概率，我们应用了三种不同的治疗方法。这种方法的优点和新颖性在于仍保持原样，在处理后的内生孢子可用于进一步下游分析。这些包括DNA提取，定量PCR（qPCR的）和扩增子或宏基因组测序（专门针对组内生孢子的，从而减少ðiversity，同时增加覆盖）。的内生孢子也可以用于下行种植或定量由荧光显微镜，流式细胞仪，或检测DPA的。这种方法的一个重要特征是，通过未处理样品与处理过的样品进行比较，可以推断出内生孢子的数量和多样性环境样品中除了对应于营养细胞的组件。

<table><tbody><tr><td>Whatman nitrocellulose membrane filters, 0.2 &amp;#956;m pore size, 47 mm diameter</td><td>Sigma-Aldrich</td><td>WHA7182004 Aldrich&amp;nbsp;</td><td></td></tr><tr><td>Tris(hydroxymethyl)aminomethane</td><td>Sigma-Aldrich</td><td>252859 Sigma-Aldrich</td><td>CAS 77-86-1</td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E9884 Sigma-Aldrich</td><td>CAS&amp;nbsp; 60-00-4&amp;nbsp;</td></tr><tr><td>Lysozyme from chicken egg white</td><td>Sigma-Aldrich</td><td>62971 Fluka</td><td>powder, CAS&amp;nbsp; 12650-88-3&amp;nbsp;</td></tr><tr><td>Ultra-Turrax homogenizer T18 basic</td><td>IKA</td><td>3720000</td><td></td></tr><tr><td>Glass filter holders for 47&amp;nbsp;mm membranes, Pyrex glass</td><td>EMD Millipore</td><td>XX10 047 00</td><td></td></tr><tr><td>Chemical duty vacuum pump</td><td>Millipore</td><td>WP6122050</td><td>220 V/50 Hz</td></tr><tr><td>Manifold sampling filtration for 25&amp;nbsp;mm membranes</td><td>Millipore</td><td>1225 Sampling Manifold</td><td>polypropylene</td></tr><tr><td>Dnase</td><td>New England Biolabs</td><td>M0303S</td><td>Rnase free</td></tr><tr><td>NaOH</td><td>Sigma-Aldrich</td><td>S5881 Sigma-Aldrich</td><td>CAS&amp;nbsp; 1310-73-2&amp;nbsp;</td></tr><tr><td>Sodium dodecyl sulfphate (SDS)</td><td>Sigma-Aldrich</td><td>L3771 Sigma&amp;nbsp;</td><td></td></tr><tr><td>Whatman nitrocellulose membrane filters, 0.2 &amp;#956;m pore size, 25 mm diameter</td><td>Sigma-Aldrich</td><td>WHA7182002 Aldrich</td><td></td></tr></tbody></table>

physical isolation of endospores from environmental samples by targeted lysis of vegetative cells

Endospore formation is a survival strategy found among some bacteria from the phylum Firmicutes. During endospore formation, these bacteria enter a morpho-physiological resting state that enhances survival under adverse environmental conditions. Even though endospore-forming Firmicutes are one of the most frequently enriched and isolated bacterial groups in culturing studies, they are often absent from diversity studies based on molecular methods. The resistance of the spore core is considered one of the factors limiting the recovery of DNA from endospores. We developed a method that takes advantage of the higher resistance of endospores to separate them from other cells in a complex microbial community using physical, enzymatic and chemical lysis methods. The endospore-only preparation thus obtained can be used for re-culturing or to perform downstream analysis such as tailored DNA extraction optimized for endospores and subsequent DNA sequencing. This method, applied to sediment samples, has allowed the enrichment of endospores and after sequencing, has revealed a large diversity of endospore-formers in freshwater lake sediments. We expect that the application of this method to other samples will yield a similar outcome.

这里介绍的结果前面已经10,21公布。请参阅那些条数据的环境解释和讨论。 整体过程总结在图1中，对应于三个主要步骤:首先，从沉淀物或任何其他环境矩阵生物质的分离;第二，营养细胞的破坏;和第三，分离的内生孢子的下游分析。下游分析可以由，例如，DNA提取和扩增子测序以确定多样性。的DNA提取需要优化以保证内生孢子的裂解。在沉积物样品，我?...

Watch this Scientific Journal Video about Physical Isolation of Endospores from Environmental Samples by Targeted Lysis of Vegetative Cells at JoVE.com

Physical Isolation of Endospores from Environmental Samples by Targeted Lysis of Vegetative Cells

A method to single out bacterial endospores from complex microbial communities was developed to perform tailored culture or molecular studies of this group of bacteria.

从环境样品中的内生孢子通过无性细胞定向裂解物理隔离

Endospore formation is a survival strategy found among some bacteria from the phylum Firmicutes. During endospore formation, these bacteria enter a ...

physical-isolation-endospores-from-environmental-samples-targeted

Research

JoVE Journal

Environment

14.1K 次观看.  Macquarie University. A method to single out bacterial endospores from complex microbial communities was developed to perform tailored culture or molecular studies of this group of bacteria.

视频: Physical Isolation of Endospores from Environmental Samples by Targeted Lysis of Vegetative Cells

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a Gram-positive, anaerobic, sporogenic bacterium that is primarily responsible for antibiotic associated diarrhea (AAD) and is a significant nosocomial pathogen. C. difficile is notoriously difficult to isolate and cultivate and is extremely sensitive to even low levels of oxygen in the environment. Here, methods for isolating C. difficile from fecal samples and subsequently culturing C. difficile for preparation of glycerol stocks for long-term storage are presented. Techniques for preparing and enumerating spore stocks in the laboratory for a variety of downstream applications including microscopy and animal studies are also described. These techniques necessitate an anaerobic chamber, which maintains a consistent anaerobic environment to ensure proper conditions for optimal C. difficile growth. We provide protocols for transferring materials in and out of the chamber without causing significant oxygen contamination along with suggestions for regular maintenance required to sustain the appropriate anaerobic environment for efficient and consistent C. difficile cultivation.

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a pathogenic bacterium that is a strict anaerobe and causes antibiotic associated diarrhea (AAD). Here, methods for isolating, culturing and maintaining C. difficile vegetative cells and spores are described. These techniques necessitate an anaerobic chamber, which requires regular maintenance to ensure proper conditions for optimal C. difficile cultivation.

培养和维护<em&gt;难辨梭状芽孢杆菌</em&gt;在厌氧环境

Clostridium  difficile is a Gram-positive, anaerobic, sporogenic bacterium  that is primarily responsible for antibiotic associated diarrhea (AAD) and is ...

科研

免疫与感染

Isolation, Propagation, and Identification of Bacterial Species with Hydrocarbon Metabolizing Properties from Aquatic Habitats

Hydrocarbon pollutants are recalcitrant to degradation and their accumulation in the environment is toxic to all life forms. Bacteria encode numerous catalytic enzymes and are naturally capable of metabolizing hydrocarbons. Scientists harness biodiversity in aquatic ecosystems to isolate bacteria with biodegradation and bioremediation potential. Such isolates from the environment provide a rich set of metabolic pathways and enzymes, which can be further utilized to scale up the degradation process at an industrial scale. In this article, we outline the general process of isolation, propagation, and identification of bacterial species from aquatic habitats and screen their ability to utilize hydrocarbons as the sole carbon source in vitro using simple techniques. The present protocol describes the isolation of various bacterial species and their subsequent identification using the 16S rRNA analysis. The protocol also presents steps for characterizing the hydrocarbon degrading potential of bacterial isolates. This protocol will be useful for researchers trying to isolate bacterial species from environmental habitats for their biotechnological applications.

We present the process of isolating, propagating, and characterizing hydrocarbon-degrading bacteria from aquatic habitats. The protocol outlines bacterial isolation, identification by the 16S rRNA method, and testing of their hydrocarbon-degrading potential. This article would help researchers in characterizing microbial biodiversity in environmental samples, and specifically screen for microbes with bioremediation potential.

从水生栖息地分离、繁殖和鉴定具有碳氢化合物代谢特性的细菌种类

Hydrocarbon pollutants are recalcitrant to degradation and their accumulation in the environment is toxic to all life forms. Bacteria encode numerous ...

环境科学

Detecting Cortex Fragments During Bacterial Spore Germination

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming bacterium, Bacillus subtilis. Germination is triggered by small molecule germinants and occurs without the need for macromolecular synthesis. Though differences exist between the mechanisms of spore germination in species of Bacillus and Clostridium, a common requirement is the hydrolysis of the peptidoglycan-like cortex which allows the spore core to swell and rehydrate. After rehydration, metabolism can begin and this, eventually, leads to outgrowth of a vegetative cell. The detection of hydrolyzed cortex fragments during spore germination can be difficult and the modifications to the previously described assays can be confusing or difficult to reproduce. Thus, based on our recent report using this assay, we detail a step-by-step protocol for the colorimetric detection of cortex fragments during bacterial spore germination.

Herein, we describe a colorimetric assay to detect the presence of reducing sugars during bacterial spore germination.

检测皮质片段在细菌孢子萌发

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming ...

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence microscopy. Super-resolution three-dimensional Structured Illumination Microscopy (3D-SIM) is a promising tool to overcome this hurdle and reveal the molecular details of the process of germination of Bacillus subtilis (B. subtilis) spores. Here, we describe the use of a modified SIMcheck (ImageJ)-assistant 3D imaging process and fluorescent reporter proteins for SIM microscopy of B. subtilis spores&#8217; germinosomes, cluster(s) of germination proteins. We also present a (standard)3D-SIM imaging procedure for FM4-64 staining of B. subtilis spore membranes. By using these procedures, we obtained unsurpassed resolution for germinosome localization and show that &#62;80% of B. subtilis KGB80 dormant spores obtained after sporulation on defined minimal MOPS medium have one or two GerD-GFP and GerKB-mCherry foci. Bright foci were also observed in FM4-64 stained spores&#8217; 3D-SIM images suggesting that inner membrane lipid domains of different fluidity likely exist. Further studies that use double labeling procedures with membrane dyes and germinosome reporter proteins to assess co-localization and thus get an optimal overview of the organization of Bacillus germination proteins in the inner spore membrane are possible.

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

Germinant receptor proteins cluster in &#8216;germinosomes&#8217; in the inner membrane of Bacillus subtilis spores. We describe a protocol using super resolution microscopy and fluorescent reporter proteins to visualize germinosomes. The protocol also identifies spore inner membrane domains that are preferentially stained with the membrane dye FM4-64.

枯草芽孢杆菌中细菌体和内膜的可视化

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence ...

生物工程

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

用活细胞显微镜研究低压等离子体灭菌对枯草芽孢杆菌孢子存活的不利影响

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...

生物学

Investigating Teliospore Germination Using Microrespiration Analysis and Microdissection

Smut fungi are the etiological agents of several devastating agricultural diseases. They are characterized by the production of teliospores, which are thick-walled dispersal agents. Teliospores can remain dormant for decades. The dormancy is characterized by low metabolic rates, paused macromolecular biosynthesis and greatly reduced levels of respiration. Upon receiving required environmental signals, teliospores germinate to produce haploid cells, which can initiate new rounds of infection. Teliospore germination is characterized by the resumption of macromolecular biosynthesis, increased respiration and dramatic morphological changes. In order to precisely measure changes in cellular respiration during the early stages of germination, we have developed a simple protocol employing a Clark-type respirometer. The later stages of germination are distinguished by specific morphological changes, but germination is asynchronous. We developed a microdissection technique that enables us to collect teliospores at distinct germination stages.

Smut fungi cause many devastating agricultural diseases. They are dispersed as dormant teliospores that germinate in response to environmental cues. We outline two methods to investigate molecular changes during germination: measuring respiration increase to detect metabolic activation and assessing changing molecular events by isolating teliospores at distinct morphological stages.

利用 Microrespiration 分析和显微解剖研究 Teliospore 萌发

Smut fungi are the etiological agents of several devastating agricultural diseases. They are characterized by the production of teliospores, which are ...

Isolation and Selection of Entomopathogenic Fungi from Soil Samples and Evaluation of Fungal Virulence against Insect Pests

Entomopathogenic fungi (EPF) are one of the microbial control agents for integrated pest management. To control local or invasive pests, it is important to isolate and select indigenous EPF. Therefore, the soil bait method combined with the insect bait (mealworm, Tenebrio molitor) system was used in this study with some modifications. The isolated EPF were then subjected to the virulence test against the agricultural pest Spodoptera litura. Furthermore, the potential EPF strains were subjected to morphological and molecular identifications. In addition, the conidia production and thermotolerance assay were performed for the promising EPF strains and compared; these data were further substituted into the formula of effective conidia number (ECN) for laboratory ranking. The soil bait-mealworm system and the ECN formula can be improved by replacing insect species and integrating more stress factors for the evaluation of commercialization and field application. This protocol provides a quick and efficient approach for EPF selection and will improve the research on biological control agents.

Here we present a protocol based on the&#160;mealworm (Tenebrio molitor)-bait system that was used for isolating and selecting entomopathogenic fungi (EPF) from soil samples. An effective conidia number (ECN) formula is used to select high stress tolerant EPF based on physiological characteristics for pest microbial control in the field.

土壤样本中昆虫病原真菌的分离与选择及真菌对害虫的毒力评价

Entomopathogenic fungi (EPF) are one of the microbial control agents for integrated pest management. To control local or invasive pests, it is important ...

Comparison of Methods for Isolating Entomopathogenic Fungi from Soil Samples

The goal of the present study is to compare the effectiveness of using insect baits versus artificial selective medium for isolating entomopathogenic fungi (EPF) from soil samples. The soil is a rich habitat for microorganisms, including EPF particularly belonging to the genera Metarhizium and Beauveria, which can regulate arthropod pests. Biological products based on fungi are available in the market mainly for agricultural arthropod pest control. Nevertheless, despite the high endemic biodiversity, only a few strains are used in commercial bioproducts worldwide. In the present study, 524 soil samples were cultured on potato dextrose agar enriched with yeast extract supplemented with chloramphenicol, thiabendazole, and cycloheximide (CTC medium). The growth of fungal colonies was observed for 3 weeks. All Metarhizium and Beauveria EPF were morphologically identified at the genus level. Additionally, some isolates were molecularly identified at the species level. Twenty-four out of these 524 soil samples were also surveyed for EPF occurrence using the insect bait method (Galleria mellonella and Tenebrio molitor). A total of 51 EPF strains were isolated (41 Metarhizium spp. and 10 Beauveria spp.) from the 524 soil samples. All fungal strains were isolated either from croplands or grasslands. Of the 24 samples selected for comparison, 91.7% were positive for EPF using Galleria bait, 62.5% using Tenebrio bait, and 41.7% using CTC. Our results suggested that using insect baits to isolate the EPF from the soil is more efficient than using the CTC medium. The comparison of isolation methods in addition to the identification and conservation of EPF has a positive impact on the knowledge about biodiversity. The improvement of EPF collection supports scientific development and technological innovation.

Entomopathogenic fungal colonies are isolated from tropical soil samples using Tenebrio bait, Galleria bait, as well as selective artificial medium, i.e., potato dextrose agar enriched with yeast extract supplemented with chloramphenicol, thiabendazole, and cycloheximide (CTC medium).

从土壤样品中分离昆虫致病真菌的方法比较

The goal of the present study is to compare the effectiveness of using insect baits versus artificial selective medium for isolating entomopathogenic ...

Isolation of Culturable Yeasts and Molds from Soils to Investigate Fungal Population Structure

Soil is host to an incredible amount of microbial life, with each gram containing up to billions of bacterial, archaeal, and fungal cells. Multicellular fungi such as molds and unicellular fungi, broadly defined as yeasts, fulfill essential roles in soil ecosystems as decomposers of organic material and as food sources for other soil dwellers. Fungal species diversity in soil is dependent on a multitude of climatic factors such as rainfall and temperature, as well as soil properties including organic matter, pH, and moisture. Lack of adequate environmental sampling, especially in regions of Asia, Africa, South America, and Central America, hinders the characterization of soil fungal communities and the discovery of novel species. 
We characterized soil fungal communities in nine countries across six continents using ~4,000 soil samples and a protocol developed in the laboratory for the isolation of yeasts and molds. This protocol begins with separate selective enrichment for yeasts and the medically relevant mold Aspergillus fumigatus, in liquid media while inhibiting bacterial growth. Resulting colonies are then transferred to solid media and further processed to obtain pure cultures, followed by downstream genetic characterization. Yeast species identity is established via sequencing of their internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene cluster, while global population structure of A. fumigatus is explored via microsatellite marker analysis. 
The protocol was successfully applied to isolate and characterize soil yeast and A. fumigatus populations in Cameroon, Canada, China, Costa Rica, Iceland, Peru, New Zealand, and Saudi Arabia. These findings revealed much-needed insights on global patterns in soil yeast diversity, as well as global population structure and antifungal resistance profiles of A. fumigatus. This paper presents the method of isolating both yeasts and A. fumigatus from international soil samples.

This protocol is an effective, speedy method of culturing yeasts and the mold Aspergillus fumigatus from large sets of soil samples in as little as 7 days. The methods can be easily modified to accommodate a range of incubation media and temperatures as needed for experiments.

从土壤中分离可培养酵母和霉菌以研究真菌种群结构

Soil is host to an incredible amount of microbial life, with each gram containing up to billions of bacterial, archaeal, and fungal cells. Multicellular ...

Isolation, Characterization, and Total DNA Extraction to Identify Endophytic Fungi in Mycoheterotrophic Plants

Mycoheterotrophic plants present one of the most extreme forms of mycorrhizal dependency, having totally lost their autotrophic capacity. As essential as any other vital resource, the fungi with which these plants intimately associate are essential for them. Hence, some of the most relevant techniques in studying mycoheterotrophic species are the ones that enable the investigation of associated fungi, especially those inhabiting roots and subterranean organs. In this context, techniques for identifying culture-dependent and culture-independent endophytic fungi are commonly applied. Isolating fungal endophytes provides a means for morphologically identifying them, analyzing their diversity, and maintaining inocula for applications in the symbiotic germination of orchid seeds. However, it is known that there is a large variety of non-culturable fungi inhabiting plant tissues. Thus, culture-independent molecular identification techniques offer a broader cover of species diversity and abundance. This article aims to provide the methodological support necessary for starting two investigation procedures: a culture-dependent and an independent one. Regarding the culture-dependent protocol, the processes of collecting and maintaining plant samples from collection sites to laboratory facilities are detailed, along with isolating filamentous fungi from subterranean and aerial organs of mycoheterotrophic plants, keeping a collection of isolates, morphologically characterizing hyphae by slide culture methodology, and molecular identification of fungi by total DNA extraction. Encompassing culture-independent methodologies, the detailed procedures include collecting plant samples for metagenomic analyses and total DNA extraction from achlorophyllous plant organs using a commercial kit. Finally, continuity protocols (e.g., polymerase chain reaction [PCR], sequencing) are also suggested for analyses, and techniques are presented here.

The current article aims to provide detailed and adequate protocols for the isolation of plant-associated endophytic fungi, long-term preservation of isolates, morphological characterization, and total DNA extraction for subsequent molecular identification and metagenomic analyses.

分离、表征和总 DNA 提取以鉴定真菌异养植物中的内生真菌

Mycoheterotrophic plants present one of the most extreme forms of mycorrhizal dependency, having totally lost their autotrophic capacity. As essential as ...