一种优化的蜜环菌分离与纯化方法

从分离材料的采集、蜜环菌索的室内培养条件、培养基的改良和分离技术的优化4个方面对前人的蜜环菌分离方法进行了系统的完善和改进。结果表明：采用蜜环菌生长良好的木本植物组织为分离材料,在15℃保湿的条件下培养可以得到健壮的菌索;按玉米粉：黄豆粉：蔗糖：水=6：1：1：26的质量比例制作的培养基不仅能满足蜜环菌生长的营养需求,而且对于其他杂菌具有明显的阻滞作用,有助于菌株纯化;分离前对菌索表面进行适当干燥可显著提高分离的成功率。     

蜜环菌对镁的耐性和富集特性

研究了镁对蜜环菌生长的影响, 蜜环菌对于镁的耐性和富集规律, 以及高浓度镁胁迫下蜜环菌的抗氧化酶的变化情况。3?15 g/L的Mg浓度对于蜜环菌菌体的生长有促进作用。Mg浓度19 g/L以上时, 蜜环菌菌体的生长受到抑制。蜜环菌的子实体形成和子实体生物量在Mg的浓度为11 g/L以下时不受影响, 超过11 g/L则子实体不能萌发。皮壳状菌丝和菌索中Mg的含量随培养基中Mg浓度的增大而增大, 培养基Mg浓度达到16 g/L后, 菌丝、菌索中Mg的含量都不再上升。子实体对Mg的富集量比菌丝体小的多, 在培养基Mg浓度在9、10 g/L时, 子实体中Mg的含量与对照组有显著差异。随着培养基中Mg浓度的提高, 菌丝和菌索POD、CAT、SOD活性都有增加, 而且菌丝与菌索之间抗氧化酶活力的差异随着培养基中Mg浓度的提高而增大。     

蜜环菌菌索形成及分支过程中钙离子流的非损伤微测

为研究蜜环菌Armillaria mellea菌索形成及分支与钙离子的关系,以菌株Armillaria mellea 541为试验材料,采用离子非损伤微测技术,对在马铃薯琼脂糖培养基(PDA)、含0.2 g/L CaCl_2的PDA和NADPH氧化酶抑制剂200μmol/L氯化二亚苯基碘鎓(Diphenyleneiodonium chloride,DPI) PDA上培养的蜜环菌菌丝及菌索顶点、菌索分支点和成熟菌索与微环境间钙离子流进行测试。结果表明:菌株A. mellea 541菌丝和菌索的生长及菌索分支均与钙离子相关。添加0.02%CaCl_2或200μmol/L DPI后,菌丝及菌索生长速度均加快,菌丝及菌索顶端钙离子内流; 0.2 g/L CaCl_2能够促进菌株A. mellea 541菌索的形成和侧向分支,菌索数量和分支数量均显著增加,成熟菌索及菌索分支处均外排钙离子; 200μmol/L DPI则抑制菌索形成及侧向分支过程。推测钙离子与活性氧可能协同调节菌索分支。试验结果为深入揭示蜜环菌菌索形成及分支的机制提供了数据支持和研究方向。     

蜜环菌多糖的生物合成与发酵条件的相关性

培养基的碳、氮源,ＰＨ值直接影响蜜环菌多糖的生物合成,泥炭水解液的添加量占培养基的１０％～３９％（Ｖ／Ｖ）时,对蜜环菌多糖的生物合成有促进作用。液体振范培养时,蜜环菌生长形成球形菌丝体,菌丝生物量３．４５ｇ／Ｌ,菌丝多糖（精糖）得率为０．２８４％,而静置液体培养有利于菌索形成,菌素生物量１４．０５ｇ／Ｌ,菌索多糖（精糖）得率为０．５１５％,胞外多糖的得率分别为２９．４％和８．３％     

蜜环菌对锌的耐性和富集特性

研究了锌对蜜环菌Armillaria mellea生长的影响,蜜环菌对于锌的耐性和富集特性,以及锌胁迫下蜜环菌的抗氧化酶的变化情况。结果表明,锌浓度45mg/L下对于蜜环菌菌体的生长有显著促进作用（p＜0.05）,锌浓度90mg/L以上时,蜜环菌菌体的生长受到抑制（p＜0.05）。蜜环菌的子实体萌发和子实体生物量在锌的浓度为45mg/L以下时与对照组无显著性差异（p＞0.05）,锌的浓度超过90mg/L后子实体不能萌发。培养基锌含量在270mg/L以下时,皮壳状菌丝中锌的含量随培养基中锌浓度的增大而增大。培养基锌含量在135mg /L以下时,菌索中锌的含量随培养基中锌浓度的增大而增大。随着培养基中锌浓度的提高,菌丝和菌索POD、CAT、SOD活性都有增加,PPO的活性则是先升高后降低,而且菌丝与菌索之间抗氧化酶活力有显著差异（p＜0.05）。     

蜜环菌菌索生物学特性的研究

为提高榛蘑人工栽培中出菇的稳定性,对蜜环菌菌索的生物学特性进行了研究,通过在不同生长时间、环境温度、培养基质的条件下对蜜环菌菌索进行培养。试验发现,菌索会由具有活性的黄色菌索逐渐角质化变成黑色丝状菌索,为保持蜜环菌菌索的活性状态,最佳培养条件:培养时间应控制在15²3 d,最佳培养温度为恒温25℃,最佳培养基配方为PDA+麦麸+锯末。     

猪苓、伴生菌及蜜环菌共培养的形态学研究

本文对猪苓、伴生菌及蜜环菌两两共培养及三者共培养进行了宏观形态观察及细胞学水平上的研究。结果表明,猪苓与伴生菌共培养时,在二者之间形成一致密拮抗线,猪苓菌落表面菌丝分化产生大量菌丝束;猪苓与蜜环菌共培养时,猪苓能阻止蜜环菌菌索对其自身的进一步侵袭,互作区中的双方菌丝及菌索均停止生长;蜜环菌与伴生菌共培养时,蜜环菌能穿透整个伴生菌菌落,在伴生菌菌落下方产生大量分枝;三者共培养后,猪苓对蜜环菌的防御能力有所下降,伴生菌对蜜环菌的耐受力有所提高,蜜环菌产生的新分枝均向伴生菌一侧生长,猪苓与伴生菌之间并不形成致密拮抗线,只可见双方菌丝的白色交融区。 猪苓与伴生菌均能在蜜环菌菌索皮层上形成侵入位点。     

蜜环菌(Armillaria mellea)内参基因的筛选

[背景]蜜环菌属(Armillaria)是一类营腐生或寄生生活的药食两用型真菌,研究其功能基因表达具有重要意义。[目的]筛选并获得蜜环菌(Armillaria mellea)最稳定的内参基因。[方法]以蜜环菌(A.mellea)541为研究对象,以马铃薯琼脂糖培养基培养的菌丝和菌索为对照组,以添加还原型烟酰胺腺嘌呤二核苷酸磷酸(nicotinamide adenine dinucleotide phosphate,NADPH)氧化酶抑制剂氯化二亚苯基碘鎓(diphenyleneiodonium chloride,DPI)培养的菌丝和菌索为抑制剂组,以添加木屑培养的菌丝和菌索为诱导剂组,利用RT-qPCR技术和BestKeeper程序系统评估候选内参基因ACT-1、α-TUB、β-TUB 1、γ-TUB、UBQ、EF-lγ、18S rRNA biogenesis protein基因(18S rRNA BP)和GAPDH的表达量稳定性。[结果]内参基因EF-1γ在对照组、DPI抑制剂组和木屑诱导剂组中的表达量稳定性最好。[结论]EF-1γ为蜜环菌(A.mellea)的最佳内参基因,为蜜环菌属真菌功能基因表达研究提供了参考。     

猪苓、伴生菌及蜜环菌共培养的形态学研究

本文对猪苓、伴生菌及蜜环菌两两共培养及三者共培养进行了宏观形态观察及细胞学水平上的研究。结果表明,猪苓与伴生菌共培养时,在二者之间形成一致密拮抗线,猪苓菌落表面菌丝分化产生大量菌丝束;猪苓与蜜环菌共培养时,猪苓能阻止蜜环菌菌索对其自身的进一步侵袭,互作区中的双方菌丝及菌索均停止生长;蜜环菌与伴生菌共培养时,蜜环菌能穿透整个伴生菌菌落,在伴生菌菌落下方产生大量分枝;三者共培养后,猪苓对蜜环菌的防御能力有所下降,伴生菌对蜜环菌的耐受力有所提高,蜜环菌产生的新分枝均向伴生菌一侧生长,猪苓与伴生菌之间并不形成致密拮抗线,只可见双方菌丝的白色交融区。猪苓与伴生菌均能在蜜环茵菌索皮层上形成侵入位点。     

蜜环菌分纯及深层液体培养条件初探

（Ａｒｍｉｌｌｅｒｉｅｌｌａ ｍｅｌｌｅａ）是一种有名的药用覃菌,对人类大有裨益。笔者对从云南采集的蜜环菌Ｓｈ－１４进行分离纯化,并对蜜环菌固体培养的营养、温度、水份等进行了研究。同时对蜜环菌深层液培养的一级种子及发酵培养基对蜜环菌菌丝体生长影响作了探讨。通过实验获得最佳一级种子培养基及发酵培养基。     

Identification of Armillaria field isolates using isozymes and mycelial growth characteristics

This research was conducted to develop procedures based on mycelial growth characteristics and patterns of esterase (EST) and polyphenol oxidase (PPO) production by diffuse mycelia for identification of Armillaria field isolates from Quercus-Carya-Pinus forests in the Ozark Mountains (central USA). The 285 isolates collected were first identified by standard diploid-haploid pairing tests as A. gallica, A. mellea, or A. tabescens. A strong PPO band was diagnostic for A. gallica. All A. mellea isolates tested and 91% of the A. tabescens isolates tested were distinguished based on production of EST bands in three standardized R f ranges. A procedure based on mycelial growth and morphology on tannic acid medium (TA) at 24 °C and on malt extract medium (ME) at 33 °C correctly identified 98% of A. gallica isolates and all A. mellea and A. tabescens isolates. On TA, A. gallica grew slowest. On ME, A. mellea grew slowest: mycelial morphology differed among species; A. gallica typically stained the agar and produced an appressed/submerged growth pattern with concentric bands of decreasing hyphal density, A. mellea typically did not stain the agar and produced round mycelia with smooth margins and abundant aerial hyphae, A. tabescens typically stained the agar and grew appressed/submerged with very irregular margins and patchy hyphal density. These are the first published systems evaluating the potential for identifying Armillaria field isolates based on their mycelial growth characteristics and EST and PPO complements. This revised version was published online in June 2006 with corrections to the Cover Date.     

超滤膜技术在多糖提取方面的应用

超滤膜技术广泛应用于各类多糖的分离、浓缩、纯化等研究中,包括中药药源多糖如灵芝多糖、大黄多糖、六味地黄汤多糖、黄芪多糖、紫芝多糖、人参多糖,海洋活性多糖如鲨鱼软骨粘多糖、褶纹冠蚌多糖、紫菜多糖、褐藻糖胶、卡拉胶,发酵多糖如蜜环菌菌索多糖、PS-9415发酵液多糖、冬虫夏草多糖,食用植物多糖如茶多糖、香菇多糖、金针菇多糖、芦荟多糖、枸杞多糖等.采用超滤膜技术处理多糖具有收率高、不易破坏多糖的生物活性、能耗低等特点,适于工业化生产.     

Cytological Studies on the Process of Armillaria Mellea Infection Through the Sclerotia of Grifola Umbellata

Adhering to the sclerotium of Grifola umbellata, Armillaria mellea could invade the sclerotium in a manner of rhizomorph without capsule, after which the sclerotium formed a deep coloured stereoscopic septate cavity outside of the rhizomorph. At the early stage of infection, segmentation was visualized either in the cortex or the apex of A. mellea rhizomorph to form a new rhizomorph which penetrated another parts of G. umbellata sclerotium. At the late stage of infection, the cortical hyphae of A. mellea rhizomorph produced a branch to invade the wall of the septate cavity of G. umbellata sclerotium and, in a manner of hyphae, it could further form new rhizomorph after its penetration through that wall. An alternate way of expanding A. mellea infection in sclerotium was to form a invading band which was composed of a few rolls of round ceils derived from cortical hyphae of A. mellea rhizomorph. The band could invade sclerotia to a farther distance and then could connect with each other.     

Efficient isolation method for high‐quality genomic DNA from cicada exuviae

In recent years, animal ethics issues have led researchers to explore nondestructive methods to access materials for genetic studies. Cicada exuviae are among those materials because they are cast skins that individuals left after molt and are easily collected. In this study, we aim to identify the most efficient extraction method to obtain high quantity and quality of DNA from cicada exuviae. We compared relative DNA yield and purity of six extraction protocols, including both manual protocols and available commercial kits, extracting from four different exoskeleton parts. Furthermore, amplification and sequencing of genomic DNA were evaluated in terms of availability of sequencing sequence at the expected genomic size. Both the choice of protocol and exuvia part significantly affected DNA yield and purity. Only samples that were extracted using the PowerSoil DNA Isolation kit generated gel bands of expected size as well as successful sequencing results. The failed attempts to extract DNA using other protocols could be partially explained by a low DNA yield from cicada exuviae and partly by contamination with humic acids that exist in the soil where cicada nymphs reside before emergence, as shown by spectroscopic measurements. Genomic DNA extracted from cicada exuviae could provide valuable information for species identification, allowing the investigation of genetic diversity across consecutive broods, or spatiotemporal variation among various populations. Consequently, we hope to provide a simple method to acquire pure genomic DNA applicable for multiple research purposes.     

Air-sampled Filter Analysis for Endotoxins and DNA Content

Outdoor aerosol research commonly uses particulate matter sampled on filters. This procedure enables various characterizations of the collected particles to be performed in parallel. The purpose of the method presented here is to obtain a highly accurate and reliable analysis of the endotoxin and DNA content of bio-aerosols extracted from filters. The extraction of high molecular weight organic molecules, such as lipopolysaccharides, from sampled filters involves shaking the sample in a pyrogen-free water-based medium. The subsequent analysis is based on an enzymatic reaction that can be detected using a turbidimetric measurement. As a result of the high organic content on the sampled filters, the extraction of DNA from the samples is performed using a commercial DNA extraction kit that was originally designed for soils and modified to improve the DNA yield. The detection and quantification of specific microbial species using quantitative polymerase chain reaction (q-PCR) analysis are described and compared with other available methods.     

Characterization of Armillaria isolates from tea (Camellia sinensis) in Kenya

Armillaria is a primary root rot pathogen of tea (Camellia sinensis) in Kenya. The main species presently described in this country are A. mellea and A. heimii. A survey covering fourteen districts of Kenya was carried out and forty-seven isolates of Armillaria collected. Cultural morphology, rhizomorph characteristics, somatic incompatibility and features of basidiomata were used to characterize the isolates, together with molecular analysis based on randomly amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR), restriction fragment length polymorphism (RFLP) of the internal transcribed spacer (ITS) and the intergenic spacer (IGS) regions and sequence of the IGS region. It can be concluded that two Armillaria species were present and they were different from A. mellea. The first group was morphologically similar to A. heimii but this was contradicted by the molecular data, suggesting that A. heimii could be a complex of several species. The second group was different from the first and morphological and molecular data strongly suggest that it could be a new Armillaria species.     

蜜环菌菌种分离新法——天麻组织分离法

报道了一蜜环菌菌种分离方法——天麻组织分离法,并对此法与常用分离法——菌索分离法进行比较试验。结果发现,天麻组织分离法分离成功率高（78％）,远高于常用的菌索分离法（16％）,且前者操作简便、难度低,所得菌种生活活力、生长形态均优于后法。     

猪苓与蜜环菌的关系

蜜环菌Armilla riella mcllea(Vahl：Ff．)Kgtst·侵入猪苓Gri[ola umbe』zd‘4(PetsFr．)PilOt)菌核,激活了猪苓菌抵御异体侵染免疫反应的本能,猪苓菌丝细胞木质化,形成与菌核表皮结构相似的隔离腔,将蜜环菌素和部分猪苓菌丝包围。在隔离腔中蜜环菌消化分隔在腔中的猪苓菌丝,另外猪苓菌丝也可侵入或附着在蜜环菌索及侵染带细胞间隙吸收其代谢产物,猪苓菌核即可萌发出新苓正常生长。当隔离腔中的猪苓菌丝被消化后,蛮环菌生活力也减弱,解体后被猪苓菌吸收利用,隔离腔变成空腔。从广义角度看,仍可把蜜环菌与猪苓菌寄生与反寄生的营养关系概括为共生关系。     

Nutrient Source of Sclerotia of Grifola umbellata and Its Relationship to Armillaria mellea

Ways nutrient-uptake of sclerotia of Grifola umbellata and the relationship between G. umbellata and Armillaria mellea were studied. At the primary stage of sclerotia of G. umbellate infected by A. mellea, the hyphae of G. umbellata could obtain nutrients by invading the one- to three-layer-cells of A. mellea cortex which existed in the sclerotia of G. umbellata, ar late stage of A. mellea infection, the nutrient source of sclerotia of G. umbellata mainly depended on its hyphae, adhering on the intercellular space of A. mellea, to suck the metabolic products of A. mellea. After being nourished the hyphae of sclerotia of G. umbellata outside of the rhizomorph of A. mellea began to reproduce, as their nuclear divisions were well observed. The results suggested that the mutual assimilation between G. umbellata and A. meIlea could be defined as a form of special Symbiotic relation.     

微波法快速提取放线菌基因组DNA

原有的放线菌基因组DNA提取方法费时、费力、费用高,且对极端环境放线菌成功率较低。利用微波热振惊提取固体培养基表面放线菌菌落基因组DNA,具有快速、简便、费用低廉等优点。所得的基因组DNA可作为PCR反应的模板进行16S rRNA基因有效扩增。这为大量放线菌菌株的快速鉴别和系统分类创造了条件。