稻曲病菌厚垣孢子不同破壁方法的比较

为了探究稻曲病菌[Ustiloginoidea virens(Cooke)Takahashi]厚垣孢子的最佳破壁方法,研究采用4种破壁法对该病菌黄色和黑色厚垣孢子进行破壁,血球计数板计算破壁效果,并用考马斯亮蓝法测定不同破壁方法中厚垣孢子壁内可溶性蛋白含量。结果表明,在普通光学显微镜下观察,破壁后厚垣孢子多数为碎片,少数为孢壁内空圆球。4种破壁方法中液氮研磨-超声破碎法破壁效果最好,黄色和黑色厚垣孢子的破壁率均可达98%以上,用该法破壁测得的黄色和黑色厚垣孢子壁内可溶性蛋白质含量也最高。由此可见,液氮研磨-超声波破碎法是一种稻曲病菌厚垣孢子破壁的有效、简便、适宜在实验室应用的方法。     

孢子丝菌病快速诊断方法的探究

目的研究申克孢子丝菌DNA提取方法;探索申克孢子丝茵的种特异性引物,运用聚合酶链反应方法鉴定申克孢子丝菌;从而为临床孢子丝茵病的分子诊断奠定基础。方法用Viscozyme L酶替代液氮研磨破壁提取孢子丝菌的基因组DNA;根据申克孢子丝茵钙调蛋白基因序列设计一对寡核苷酸引物,分别对52株申克孢子丝菌及3种6株普通真茵的基因组DNA进行PCR扩增。结果用Viscozyme L酶替代液氮研磨破壁成功提取出孢子丝菌的基因组DNA。特异性引物对52株申克孢子丝菌可扩增出一条约430 bp的片段,而对念珠菌、曲霉、黑霉基因组DNA扩增结果均为阴性。结论用Viscozyme L酶替代液氮研磨破壁提取孢子丝菌基因组DNA与传统的CTAB法相比不仅操作更简便,而且避免了液氮研磨过程中难于避免的污染。运用我们所设计的特异性引物,结合PCR方法对申克孢子丝菌进行分子鉴定,结果显示不仅该引物对申克孢子丝菌特异、敏感而且该方法简便、快捷;可用于申克孢子丝菌病的临床诊断。     

一种快速提取丝状真菌染色体DNA的方法

介绍了一种适用于丝状真菌染色体DNA大片段的快速提取方法,该方法以(100mM Tris,100mM NaGl,50mM EDTA-Na2 2%SDS,pH值9.0)为提取液,经石英砂研磨破壁.应用该方法成功地提取了粗糙脉胞菌(Neurospora crassa)、米曲霉(Aspergillus oryzae)、产黄青霉(Penicillium chrysogenum)和头孢霉菌(Cep- halosporium sp.)等4种不同丝状真菌的染色体DNA大片段,且所提DNA片段均大于20kb,可直接用于限制性酶切、PCR等分子生物学研究.     

农杆菌介导的紫色红曲霉遗传转化体系的建立和优化

通过优化各种转化因素,建立了根癌农杆菌(Agrobacterium tumefaciens)介导红曲霉(Monascus)的高效转化体系:红曲霉在PDA培养基培养21 d后收集孢子,制备红曲霉孢子悬浮液,浓度为106个/mL,根癌农杆菌浓度为OD600值0.5,诱导剂AS浓度为100μmol/L,农杆菌与红曲霉在25℃共培养3 d。采用此转化体系构建了含有530多个转化子的红曲霉T-DNA插入突变体库。随机选取50株转化子菌株进行分子验证和稳定性检测,证明T-DNA成功插入红曲霉基因组DNA中,并能稳定遗传。最后,通过形态观察筛选出8株变异较大的菌株,为以后的红曲霉基因功能研究奠定了一定的基础。     

灵芝孢子几种破壁方法比较分析

采用超临界破壁法、匀浆破壁法和超声破壁法对灵芝孢子进行了破壁试验研究,研究显示,采用超临界高压静止破壁法可显著提高灵芝孢子的破壁率。匀浆破壁法和超声破壁法虽灵芝孢子破碎率较低,显微镜观察大部分孢子外形完整,但细胞内容物已几乎消失,达到提取孢子内容物的目的。     

桑白盾蚧病原真菌Simplicillium lanosoniveum的致病性

从太原市白蜡树上采集的自然染病死亡的桑白盾蚧Pseudaulacaspis pentagona虫尸上分离获得一株病原真菌,菌株号为TYL001。经过形态观察和分子鉴定,该菌株为Simplicillium lanosoniveum。用该菌株5种浓度的孢子悬浮液(1.0×10~6孢子/m L、5.0×10~6孢子/m L、1.0×10~7孢子/m L、5.0×10~7孢子/m L、1.0×10~8孢子/m L)感染桑白盾蚧雌成虫,感染6d后,孢子悬浮液对桑白盾蚧的感染率分别为80.00%、83.33%、86.67%、90.00%、93.33%,说明该菌株对桑白盾蚧具有较强的感染力,且有浓度效应;感染症状出现的高峰期在72–120h之间,且孢子浓度越大,感染症状出现的越早;5种孢子浓度的感染中时IT50依次为106.95h、97.00h、91.46h、87.73h、79.38h,说明感染致病所用时间也具有浓度效应。本研究为生物防治桑白盾蚧提供了新菌种。     

定期转种法和低温冷冻法保藏致病真菌活性的能力比较

定期转种法和低温冷冻保存法是临床实验室最常用的两种真菌保存方法,为比较两种方法保藏致病真菌活性的能力,本研究使用两种保藏方法对实验室689株致病真菌保藏5年后进行检测。定期转种法是将菌落接种于马铃薯斜面培养基并将其储存在4℃冰箱,每6个月转种1次。低温冷冻法是挑取马铃薯斜面培养基上生长良好的菌落于无菌10%甘油中,放置在-80℃储存。保藏5年后,将两种方法保藏的菌株转种复苏,比较菌株的复活率。对于念珠菌属Candida、新生隐球菌Cryptococcus neoformans、毛癣菌属Trichophyton、曲霉属Aspergillus和孢子丝菌属Sporothirix真菌,两种方法的菌株复活率无统计学差异;对于小孢子菌属Microsporum真菌和马尔尼菲蓝状菌Talaromyces marneffei,使用低温冷冻法保藏的菌株复活率高于定期转种法保藏的菌株复活率;对于着色霉属Fonsecaea真菌,低温冷冻法保藏的菌株复活率低于定期转种法保藏的菌株复活率。因此,我们认为对于常见致病真菌的长期保藏,使用10%甘油作为保护剂的低温冷冻法优于定期转种法,但其不适用于着色霉属Fonsecaea真菌的长期保藏。     

1种小麦白粉病菌DNA基因组的微量简捷提取方法

为了寻找适合小麦白粉菌基因组DNA微量提取的方法,分别采用改进破壁法,液氮研磨法和溶菌酶消化法进行破壁,提取专性寄生菌小麦白粉菌DNA。结果表明,用改进的破壁方法,仅用3~10 mg的分生孢子粉所获得DNA的收率为（12.23±3.46）~（40.32±5.67）ng/mg,且OD260/OD280比值为1.71~1.92之间,说明该破壁方法获得的DNA收率大且纯度高。通过PCR反应获得了良好的效果。同时该方法也适用于小麦条锈菌和大麦白粉菌专性寄生菌DNA的提取。     

莲种子蛋白质提取方法比较与双向电泳分析

莲(Nelumbo nucifera Gaertn.)不仅是重要的水生蔬菜作物之一,而且是进行基础研究的好材料。本文采用4种蛋白质提取方法(新型TCA/丙酮法、传统TCA/丙酮法、改良的Tris-HCl法、Tris-饱和酚法)并结合双向电泳技术,对莲子蛋白质提取方法进行筛选与优化。双向电泳实验结果显示,所得蛋白质图谱与莲种子蛋白质组成分布特点一致。通过PDQuest软件分析表明,新型TCA/丙酮法适用于莲子叶和胚芽组织的双向电泳蛋白质提取,而传统TCA/丙酮法则适用于莲胚轴组织双向电泳的蛋白质提取。研究结果为进一步利用质谱进行莲子蛋白质组研究奠定了基础。     

食源米曲霉菌株的分离及其降解高效氯氰菊酯和3-苯氧基苯甲酸的特性研究

【目的】通过研究不同食源米曲霉菌株对高效氯氰菊酯(beta-cypermethrin,β-CP)及其必经代谢产物3-苯氧基苯甲酸(3-phenoxybenzoic acid,3-PBA)的降解特性,了解不同菌株的降解共性及差异性,为农副产品和发酵食品的农残减除提供理论基础和食品用安全微生物资源。【方法】以发酵食品为菌源,通过形态学鉴定、ITS测序和菌株产黄曲霉毒素B1 (AFB1)的测定筛选鉴定米曲霉菌株,采用高效液相色谱法(HPLC)、气相色谱-质谱法(GC-MS)、液相色谱-质谱法(LC-MS)对米曲霉模式菌株RIB40 (保藏编号:ATCC 42149)、米曲霉M4 (保藏编号:CGMCC 11645)和鉴定获得的米曲霉菌株的β-CP和3-PBA降解特性进行研究。【结果】鉴定获得15株不产AFB1的食源米曲霉,17株米曲霉在马铃薯液体培养基(PD)中振荡培养5d,对50mg/L的β-CP降解率为19.33%-50.29%不等,检测到降解产物3-PBA,对50 mg/L的3-PBA降解率为45.59%-99.67%不等;分别在添加50 mg/Lβ-CP和3-PBA的无机盐培养基(MM)中振荡培养5 d,米曲霉菌株均未生长,对β-CP和3-PBA无降解;在富集培养基(GM)中振荡培养2 d,对100 mg/L的3-PBA转化或降解率为69.28%-99.58%不等,检测到3-苯氧基苄醇(3-PBlc)和羟基-3-苯氧基苯甲酸(HO-3-PBA)。【结论】食源米曲霉具有共代谢降解β-CP和3-PBA的共性,3-PBA为β-CP降解中间产物,米曲霉对3-PBA普遍具有较高的降解率。在3-PBA降解初期,米曲霉可将其短暂还原生成毒性相对较低的3-PBlc,同时,3-PBA逐渐羟基化生成水溶性更强的HO-3-PBA参与下游降解。     

Relationship between agitation speed and the morphological characteristics of Verticillium lecanii CS-625 during spore production

Verticillium lecanii is recognized as an entomopathogenic fungus, and has high potential in the biological control of pests. In this study, it was investigated that the relationship between agitation speed in a 2.5 L stirred tank reactor (STR) at 25°C and initial pH 5.5, and the morphological characteristics of V. lecanii CS-625, such as hyphal length/width, spore length/width, and the number of tips during spore production. The agitation speed affected the hyphae patterns and the number of tips. The number of spores rapidly increased at 48 to 60 h of cultivation, and the highest spore productivity (2.5 × 10¹⁰ spore/L·h at 60 h) occurred with an agitation speed of 350 rpm and an aeration rate of 1.0 vvm. The number of tips increased in proportion to the increase in spore production during the same culture time. The highest number of tips (4.8 × 10⁸ tipJ.mL) was obtained at 72 h of cultivation. The shortest mean spore length (2.8 μm) was obtained at 60 h of cultivation. Therefore, it was determined that the increased number of tips and decreased mean spore length were closely related to the production of V. lecanii spores.     

Mass production of spores of lactic acid‐producing Rhizopus oryzae NBRC 5384 on agar plate

Mass production of sporangiospores (spores) of Rhizopus oryzae NBRC 5384 (identical to NRRL 395 and ATCC 9363) on potato‐dextrose‐agar medium was studied aiming at starting its L (+)‐lactic acid fermentation directly from spore inoculation. Various parameters including harvest time, sowed spore density, size of agar plate, height of air space, and incubation mode of plate (agar‐on‐bottom or agar‐on‐top) were studied. Ordinarily used shallow Petri dishes were found out to be unsuitable for the full growth of R. oryzae sporangiophores. In a very wide range of the sowed spore density, the smaller it was, the greater the number of the harvested spores was. It was also interesting to find out that R. oryzae grown downward vertically with a deep air space in an agar‐on‐top mode gave larger amount of spores than in an agar‐on‐bottom mode at 30°C for 7‐day cultivation. Scale‐up of the agar plate culture from 26.4 to 292 cm² was studied, resulting in the proportional relationship between the number of the harvested spores/plate and the plate area in the deep Petri dishes. The number of plates of 50 cm in diameter needed for 100 m³ industrial submerged fermentation started directly from 2 × 10⁵ spores/mL inoculum size was estimated as about 6, from which it was inferred that such a fermentation would be feasible. Designing a 50 cm plate and a method of spreading and collecting the spores were suggested. Bioprocess technological significance of the “full‐scale industrial submerged fermentation started directly from spore inoculation omitting pre‐culture” has been discussed. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:876–881, 2013     

响应面法优化爪哇虫草菌的培养条件及其对斜纹夜蛾的毒力和保护酶活性影响

【目的】优化爪哇虫草菌Bd01的固态发酵培养条件,测定分生孢子对斜纹夜蛾3龄幼虫的毒力,研究被爪哇虫草菌侵染后寄主体内的保护酶活性变化。【方法】采用单因素试验确定爪哇虫草菌Bd01最佳的固态培养基及培养条件,利用Box-Behnken响应面法优化发酵参数,采用浸渍法测定分生孢子对斜纹夜蛾3龄幼虫的毒力,同时利用分光光度计法测定斜纹夜蛾3龄幼虫体内酶活性变化。【结果】以产孢量为指标,通过响应曲面法优化的爪哇虫草菌Bd01最佳产孢条件为：培养基营养成分含量为30.24g/L,pH值为7.55,光照时长为12.06h,在该条件下,培养基的产孢量为2.78×10⁸孢子/mL。浓度为1×10⁸孢子/mL的爪哇虫草菌孢子液对斜纹夜蛾3龄幼虫具有一定毒力,处理7 d时致死中浓度(LT₅₀)为3.11 d,致死中时(LC₅₀)为4.68×10⁵孢子/mL,校正死亡率为88.68%。处理后未死亡的斜纹夜蛾幼虫体内保护酶活性与对照组相比发生显著变化。【结论】优化后的培养基能够显著增加爪哇虫草菌的产...     

Analysis of broth-cultured Bacillus atrophaeus and Bacillus cereus spores

Aims: To compare physical properties of spores that were produced in broth sporulation media at greater than 10⁸ spores ml⁻¹. Methods and Results: Bacillus atrophaeus reproducibly sporulated in nutrient broth (NB) and sporulation salts. Microscopy measurements showed that the spores were 0·68 ± 0·11 μm wide and 1·21 ± 0·18 μm long. Coulter Multisizer (CM3) measurements revealed the spore volumes and volume-equivalent spherical diameters, which were 0·48 ± 0·38 μm³ and 0·97 ± 0·07 μm, respectively. Bacillus cereus reproducibly sporulated in NB, sporulation salts, 200 mmol l⁻¹ glutamate and antifoam. Spores were 0·95 ± 0·11 μm wide and 1·31 ± 0·17 μm long. Spore volumes were 0·78 ± 0·61 μm³ and volume-equivalent spherical diameters were 1·14 ± 0·11 μm. Bacillus atrophaeus spores were hydrophilic and B. cereus spores were hydrophobic. However, spore hydrophobicity was significantly altered after treatment with pH-adjusted bleach. Conclusions: The utility of a CM3 for both quantifying Bacillus spores and measuring spore sizes was demonstrated, although the volume between spore exosporium and spore coat was not measured. This study showed fundamental differences between spores from a Bacillus subtilis- and B. cereus-group species. Significance and Impact of the Study: This is useful for developing standard methods for broth spore production and physical characterization of both living and decontaminated spores.     

Fusarium andDidymella-neglected spores in the air

Summary This paper reports about the occurence ofFusarium- andDidymella spores in the air of Essen/FRG. During the spore season 1990, the spore concentration was measured on several days with a volumetric pollen trap by hourly analysis. The calculated amount of spores per hour is compared to the data of a pluviometer and the values of the relative humidity during the same period.The occurence of both spore types in the air and high relative humidity (>80%) are correlated in a highly significant way (P<0.001). The dispersion of spores starts when rain begins or directly after the precipitation.Didymella reaches higher concentrations thanFusarium in the air (Maximum values:Didymella 30000 spores/m³,Fusarium only 800 spores/m³). During the emission of the spores the temperature varied between 10°C und 20°C degrees. Didymella andFusarium must be an important allergenic source in the outdoor area, because of their allergen-loaded biological aerosols. The question of providing well defined extracts ofDidymella exitialis is given to the pharmaceutical industry.     

Possible involvement of Ca<Superscript>2+</Superscript>-dependent protein kinases in spore germination of the fern<Emphasis Type="Italic">Osmunda Japonica</Emphasis>

Fern gametophyte is a good model system to investigate signal transduction in plant cells. In this work, we examined whether CDPKs are involved in the mechanisms of spore germination of the fernOsmunda japonica. A protein extract from the spores included four CDPK isoforms with relative molecular weights of 56, 53, 49, and 47 kDa, as detected by immunoblot analysis, and they showed CDPK-like activities, as detected by in-gel protein-kinase assay. It was also found that the inhibitors effective on CDPKs, such as a general protein kinase inhibitor, K252a, and a calmodulin antagonist, W-7, largely suppressed the spore germination, and that many proteins of the spores were phosphorylated in vivo in a calcium dependent manner in the period when the spores require external Ca²⁺ for the germination. Furthermore, we showed that Sr²⁺ and Mn²⁺, which could substitute for Ca²⁺ in the spore germination, were also able to activate theOsmunda CDPKs. From these results, we concluded that CDPKs would participate in the spore germination ofO. japonica.     

Effects of the sporulation conditions on the lovastatin production by Aspergillus terreus

The production of biomass and lovastatin by spore-initiated submerged fermentations of Aspergillus terreus ATCC 20542 was shown to depend on the age of the spores used for inoculation. Cultures started from older spores produced significantly higher titers of lovastatin. For example, the lovastatin titer increased by 52% when the spore age at inoculation rose from 9 to 16 days. The lovastatin titer for a spore age of 16 days was 186.5±20.1 mg L⁻¹. The time to sporulation on surface cultures was sensitive to the light exposure history of the fungus and the spore inoculation concentration levels. A light exposure level of 140 μE m⁻² s⁻¹ and a spore concentration of 1,320 spore cm⁻² produced the greatest extent of sporulation within about 50 h of inoculation. Sporulation was slowed in the dark and with diluted inoculants. A rigorous analysis of the data of statistically designed experiments showed the above observations to be highly reproducible.     

Immunohistochemical and immunocytochemical detection of SchS34 antigen in <Emphasis Type="Italic">Stachybotrys chartarum</Emphasis> spores and spore impacted mouse lungs

The purpose of this study was to evaluate the distribution of a 34 kD antigen isolated from S. chartarum sensu lato in spores and in the mouse lung 48 h after intra-tracheal instillation of spores by immuno-histochemistry. This antigen was localized in spore walls, primarily in the outer and inner wall layers and on the external wall surfaces with modest labelling observed in cytoplasm. Immuno-histochemistry revealed that in spore impacted mouse lung, antigen was again observed in spore walls, along the outside surface of the outer wall and in the intercellular space surrounding spores. In lung granulomas the labelled antigen formed a diffusate, some 2–3× the size of the long axis of spores, with highest concentrations nearest to spores. Collectively, these observations indicated that this protein not only displayed a high degree of specificity with respect to its location in spores and wall fragments, but also that it slowly diffuses into surrounding lungs.     

Ultrastructural changes in the spore and mycelia of Ascosphaera apis after treatment with benomyl (Benlate 50 W)

In Ascosphaera apis, after 8 days growth in darkness at 28° C, numerous sporocysts were observed, within which mature spores were seen aggregated into a spore ball. The mature spore of A. apis had a thick spore wall with an electron-opaque outer layer, a spore membrane with many depressions, and sporoplasm containing numerous ribosomes and mitochondria. In the cytoplasm of the mycelium, mitochondria with well-defined cristae and numerous ribosomes were observed. At a concentration of 1 g/ml of culture medium, benomyl appeared to inhibit colony growth of A. apis, but some sporocysts containing deformed spores were found. Deformed spores possessed a thick spore wall with a grainy matrix, and depressions were no longer detected in the spore membrane. Ribosomes were lacking in the sporoplasm and mitochondria appeared degenerate. The mycelium from the treated culture contained mitochondria with an electron-lucid matrix and no well defined cristae, while ribosomes were completely depleted. The significance of these observations in relation to the use of benomyl to control chalkbrood disease in the honey bee is discussed.     

The impact of nutrition on spore yields for various fungal entomopathogens in liquid culture

Spore yields were measured for various fungal entomopathogens grown in six nutritionally different liquid media with low and high carbon concentrations (8 and 36 g l^–1, respectively) at carbon-to-nitrogen (C:N) ratios of 10:1, 30:1 and 50:1. Six fungi were tested: two Beauveria bassiana strains, three Paecilomyces fumosoroseus strains and one Metarhizium anisopliae strain. Spore yields were examined after 2, 4 or 7 days growth. In general, highest spore yields were obtained in media containing 36 g/l and a C:N ratio of 10:1. After 4 days growth, highest spore yields were measured in the three Paecilomyces isolates (6.9–9.7 × 10⁸ spores ml^–1). Spore production by the B. bassiana isolates was variable with one isolate producing high spore yields (12.2 × 10⁸ spores ml^–1) after 7 days growth. The M. anisopliae isolate produced low spore concentrations under all conditions tested. Using a commercial production protocol, a comparison of spore yields for the coffee berry borer P. fumosoroseus and a commercial B. bassiana isolate showed that highest spore concentrations (7.2 × 10⁸ spores ml^–1) were obtained with the P. fumosoroseus isolate 2-days post-inoculation. The ability of the P. fumosoroseus strain isolated from the coffee berry borer to rapidly produce high concentrations of spores prompted further testing to determine the desiccation tolerance of these spores. Desiccation studies showed that ca. 80% of the liquid culture produced P. fumosoroseus spores survived the air-drying process. The virulence of freshly produced, air-dried and freeze-dried coffee berry borer P. fumosoroseus blastospores preparations were tested against silverleaf whiteflies (Bemisia argentifolii). While all preparations infected and killed B. argentifolii, fresh and air-dried preparations were significantly more effective. These results suggest that screening potential fungal biopesticides for amenability to liquid culture spore production can aid in the identification of commercially viable isolates. In this study, P. fumosoroseus was shown to possess the production and stabilization attributes required for commercial development.