红火蚁病原真菌AUGM47早期发育超微结构研究

在前期筛选已获得对红火蚁Solenopsis invicta Buren高效致病真菌罗伯茨绿僵菌Metarhizium robertsii AUGM47的基础上,为进一步明确病原真菌对寄主昆虫的侵染机制。本试验在室内条件下,以红火蚁工蚁为侵染对象,利用荧光显微镜和透射电镜观察了罗伯茨绿僵菌AUGM47侵染单元分生孢子在体表附着萌发、穿透和体内增殖的早期发育过程。结果表明,菌株AUGM47分生孢子在红火蚁体表可萌发并形成附着胞侵入,接种后12 h观察到萌发,在36 h内普遍出现穿透结构穿透体壁。接种后48 h为菌体在血腔内的增殖阶段。菌丝体在穿透表皮和体腔内增殖过程中伴随着机械压力和酶的活动。接种后96 h,观察到自噬现象,菌体通过自噬降解并回收细胞器,为从体内穿出的晚期发育过程提供物质基础。本研究对罗伯茨绿僵菌AUGM47分生孢子在红火蚁体外至体内的发育进程研究证实了菌株的高致病性,为红火蚁生防真菌菌种改良和后续开发利用奠定理论基础。     

分离自赤豆的青霉新种

本文报道青霉属一个新种,分离自赤豆,特点是分生孢子无色,命名为无色青霉(Penicillium incoloratum sp.nov.).模式和来自模式的活培养物都保存在中国科学院微生物研究所。     

鱼类细胞自噬研究进展

自噬是广泛存在于真核生物中的生命现象,对于细胞的生长、分化、发育和维持细胞内环境稳态等方面具有重要意义。尽管对于作为低等脊椎动物的鱼类细胞自噬研究起步较晚,但近几年围绕其自噬的诱导、自噬相关基因表达及调控、鱼类病原诱导的自噬,特别是以斑马鱼为试验模型开展的自噬与发育调控关系等研究都取得了一些进展,就此进行综述。     

细胞衰老与细胞自噬的生物学关联及其意义

细胞衰老是指细胞生理功能的衰减,包括增殖能力下降、细胞周期停滞、对促凋亡应激不敏感、衰老相关基因和蛋白表达增加,伴有形态学衰老改变,渐趋死亡的现象,其至少可分为复制性衰老和应激诱导的衰老。细胞自噬属于细胞＂自食＂现象,是细胞依赖溶酶体的分解代谢过程,能降解受损蛋白、衰老或损伤的细胞器等细胞结构,可被多种应激所触发。细胞自噬的典型特征是形成自噬体并呈递给溶酶体,该过程在蛋白质和细胞器质量控制中起基础作用并维持了细胞能量的稳态。最新研究表明,自噬与细胞衰老密切相关,参与蛋白酶和自噬相关调节的BAG蛋白家族中BAG3/BAG1比值在复制性衰老时增高,且BAG3在细胞衰老时能介导自噬的激活。在Ras诱导的细胞衰老进程中亦可观察到较高的自噬活性。再者,自噬作为生物机体抗衰老的效应因子的遗传学证据已在低等真核生物中发现。还有研究证实,作为人类精液主要组分的亚精胺能够触发组蛋白H3脱乙酰基作用,此改变上调了自噬相关转录物的表达,继而引发自噬活性增强,从而延缓了多种细胞的衰老进程。另有研究显示,在P53/Arf的正常调节下,小鼠的衰老进程得以延缓,而Arf在细胞自噬过程的调节中亦是不可或缺的。总之,自噬活性的改变影响细胞衰老进程并可作为细胞衰老新的效应机理。     

一株绿僵菌的分离研究

利用马铃薯葡萄糖琼脂培养基,对一株绿僵菌进行了分离培养。对不同生长发育天数的菌落特征即颜色、大小、孢子堆的形成等特点进行了描述。在光学显微镜下观察了菌丝的形态,成熟孢子及分生孢子梗的形状、颜色及连接状况。利用扫描电镜观察了分生孢子和孢子梗的超微形态。经培养特征和形态学鉴定,确定该昆虫病原真菌是金龟子绿僵菌。     

萨克拉普奇尼亚菌串孢变种——北方根结线虫卵寄生真菌

分离自山东省牟平市塑料大棚种植的甜瓜根结线虫卵上的菌株CFCC84965,根据培养条件和形态特征鉴定为萨克拉普奇尼亚菌串孢变种Pochonia suchlasporia var.catenata,为中国新记录种。该菌生长较快,在CMA培养基培养15 d后菌落直径为27 ～43 mm,气生菌丝中等发达,白色至浅黄色,背面为黄色至淡褐色。分生孢子梗长,大多从菌丝上直立生长,多分支。分生孢子多串生,表面略粗糙。老熟菌丝上形成会聚的菌丝膨大细胞或形成少量的间生厚垣孢子。在琼脂培养基上可形成晶体。     

植物细胞自噬研究进展

细胞自噬是真核生物中进化保守的对细胞内物质进行周转的重要过程,该过程中一些损坏的蛋白或细胞器被双层膜结构的自噬小泡包裹后送入溶酶体(动物)或液泡(酵母和植物)中进行降解并得以循环利用。植物中通过序列比对鉴定了诸多自噬相关基因并分离到了部分细胞自噬功能缺陷的突变体,这些研究均推进了我们对植物细胞自噬机制和功能的了解。本文主要综述了植物细胞自噬分子机制和生理功能的研究进展。     

从白蚁中分离到具有纤维素酶活的贪噬菌

以黄胸散白蚁Reticulitermes flaviceps后肠为材料,分离培养具有降解纤维素能力的微生物,以进一步了解白蚁后肠微生物的种类。通过以羧甲基纤维素钠(CMC-Na)为唯一碳源的富集及选择培养基培养、筛选,获得一株具有纤维素酶活的菌株R3063。形态学鉴定、革兰氏染色观察及16S rDNA基因序列分析表明该菌株属于贪噬菌(Variovorax sp.)。目前尚未见贪噬菌具有纤维素酶活的报道。     

孤儿核受体Nur77对缺/复氧损伤中心肌细胞自噬的调节

目的:研究孤儿核受体Nur77对缺/复氧损伤中心肌细胞自噬的调节作用。方法:差速贴壁法分离乳鼠心肌细胞,经免疫荧光染色鉴定纯度。缺氧(1%O_2、5%CO_2和94%N_2)培养12 h后,常氧培养2 h构建心肌细胞缺/复氧损伤。实时定量PCR和western blot的方法检测Nur77的表达变化。通过siRNA转染抑制心肌细胞nur77表达,通过自噬标志蛋白表达改变作为细胞自噬水平的变化。结果:原代分离的心肌细胞纯度95%以上。缺氧12 h和缺/复氧(12 h/2 h)刺激后,心肌细胞中Nur77表达都明显升高(P0.01)。与缺氧组相比,缺/复氧组细胞质中的水平明显增加(P0.01),细胞核中Nur77水平无明显变化。抑制Nur77后,缺/复氧组自噬水平明显降低,缺氧组心肌细胞自噬水平无明显变化。结论:Nur77参与缺/复氧损伤中心肌细胞自噬水平的调节。     

一种优化小鼠成纤维细胞中自噬小体示踪的方法

目的为了提高对自噬小体示踪的精确度和清晰度,全面展示细胞自噬相关进程,优化基于绿色荧光蛋白-微管相关蛋白1轻链3β(GFP-LC3β)标记物的自噬小体示踪方法。方法组织块贴壁法原代培养小鼠耳成纤维细胞,脂质体转染过表达GFP-LC3β;GFP-LC3β充分表达后给予药物刺激以激活或阻断细胞自噬通路,然后进行免疫荧光染色。对染色操作进行优化:首先,延长多聚甲醛的固定细胞时间(从15min延长至45min);其次,提高Triton X-100处理时间,除第一次和最后一次使用PBS润洗,其余每一次使用的PBS都添加0.2%Triton X-100;最后,提升抗体稀释液中BSA和Triton X-100含量,BSA从3%提升至5%,TritonX-100的含量从0.1%提升至0.2%。结果拍照后统计自噬小体发现,新方法更加准确地标记GFP-LC3β指示物,自噬激活条件下示踪准确度显著提升(达4.2倍);新方法更加清晰地呈现自噬小体的形态和胞内分布,将自噬小体示踪的清晰度显著提升(达2.5倍);新方法还能使用不同荧光通道,同时标记其他自噬相关基因。结论本研究提供的一种优化小鼠成纤维细胞中自噬小体示踪的方法将为全面展示细胞自噬进程、深入了解细胞自噬相关机制提供有力的技术支持。     

Molasses supplementation promotes conidiation but suppresses aflatoxin production by small sclerotial Aspergillus flavus

AIMS: To find a supplemental ingredient that can be added to routinely used growth media to increase conidial production and decrease aflatoxin biosynthesis in small sclerotial (S strain) isolates of Aspergillus flavus. METHODS AND RESULTS: Molasses was added to three commonly used culture media: coconut agar (CAM), potato dextrose agar (PDA), and vegetable juice agar (V8) and production of conidia, sclerotia, and aflatoxins by A. flavus isolate CA43 was determined. The effect of nitrogen sources in molasses medium (MM) on production of conidia, sclerotia and aflatoxins was examined. Water activity and medium pH were also measured. Conidia harvested from agar plates were counted using a haemocytometer. Sclerotia were weighed after drying at 45 degrees C for 5 days. Aflatoxins B(1) and B(2) were quantified by high-performance liquid chromatography. Addition of molasses to the media did not change water activity or the pH significantly. Supplementing CAM and PDA with molasses increased conidial production and decreased aflatoxins. Two-fold increased yield of conidia was found on MM, which, like V8, did not support aflatoxin production. Adding ammonium to MM significantly increased the production of sclerotia and aflatoxins, but slightly decreased conidial production. Adding urea to MM significantly increased the production of conidia, sclerotia and aflatoxins. CONCLUSIONS: Molasses stimulated conidial production and inhibited aflatoxin production. Its effect on sclerotial production was medium-dependent. Water activity and medium pH were not related to changes in conidial, sclerotial or aflatoxin production. Medium containing molasses alone or molasses plus V8 juice were ideal for conidial production by S strain A. flavus. SIGNIFICANCE AND IMPACT OF THE STUDY: Insight into molecular events associated with the utilization of molasses may help to elucidate the mechanism(s) that decreases aflatoxin biosynthesis. Targeting genetic parameters in S strain A. flavus isolates may reduce aflatoxin contamination of crops by reducing the survival and toxigenicity of these strains.     

Relationship between number and type of adhesions of Penicillium oxalicum conidia to tomato roots and biological control of tomato wilt

We studied (a) the extent adhesion of Penicillium oxalicum conidia to tomato roots after application of P. oxalicum conidial formulations with or without stickers, (b) the relationship between the extent of conidial adhesion to roots and biocontrol of the conidial formulations against tomato wilt, and (c) colonisation of roots by P. oxalicum. Adhesion of P. oxalicum conidia to tomato roots occurred within the first minute of contact between the root and the conidial formulation and the bonding strength was sufficiently strong to prevent conidial removal from the roots. In addition, some formulations with stickers that increased conidial adhesion to roots improved the biocontrol of tomato wilt, when compared to that of formulations without stickers. A “dried conidia without stickers” with 0.025% Nu-Film 17 had no effect on the biocontrol of tomato wilt, despite good adherence of the conidia to the roots. The numbers of P. oxalicum conidia that adhered to the roots was constant for 60 days after application of a “dried conidia without stickers” conidial formulation. The significance of these results (speed of adhesion, number of adhered conidia, and variability of conidial external surface) are discussed in relation to the biocontrol success of tomato wilt using different types of conidial formulations with and without stickers.     

生防真菌分生孢子抗氧化能力与多糖含量的关系

作为重要的丝孢类昆虫病原真菌,球孢白僵菌和玫烟色棒束孢因其易于生产和环境友好等优点而在害虫生防防治中受到广泛青睐。为初步探求孢子耐氧化力及其与孢子多糖含量的关系,球孢白僵菌和玫烟色棒束孢11株菌经胁迫后的残存指数随氧化剂H2O2浓度增加而减小。所有菌株的残存指数均能良好地与Logistic方程拟合,并计算出各菌株在氧化胁迫条件下的半致死浓度。结果显示玫烟色棒束孢孢子的耐氧化力强于球孢白僵菌。两种真菌的分生孢子耐氧力与各自多糖含量呈现良好线性正相关。培养基碳源成分和浓度变化可影响球孢白僵菌孢子耐氧化力,但耐氧化力与多糖含量依旧呈现线性正相关。由此可见,生防真菌分生孢子的耐氧化力的确与多糖积累有关,并在一定程度上受培养条件的调节。研究结果有望为提高生防真菌孢子环境稳定性提供新的策略。     

Formation and Germination of Septoria tritici Secondary Conidia as Affected by Environmental Factors

The effects of medium, isolate, temperature, light and pH on the formation and germination of Septoria tritici secondary conidia were tested. Of the six media tested, the malt–yeast extract agar was the best and generated 1.82 × 10⁹ conidia/plate. The ten isolates tested showed different ability of conidia production. Darkness significantly reduced conidial formation and enhanced the transition of intermediates. The conidial germination and germ tube growth was strongly inhibited at 30°C. The suitable pH for conidial budding in malt–yeast broth (MYB) was between 5 and 9. At pH 2, 10 and 11, almost no new conidia were formed. The number of conidia reached 1.27 × 10⁸ conidia/ml after 7 days in MYB, significantly more than that in potato dextrose broth, wheat leaf extract and H₂O.     

莲子草假隔链格孢SF 193菌株的复壮对其产孢量和致病性的影响

【背景】莲子草假隔链格孢是空心莲子草的重要生防菌,但长时间保存或继代培养会导致该菌株产孢量和致病性显著下降。【方法】通过对初始菌株回接、分离、纯化和鉴定相继获得复壮一代和二代菌株,分别在3、6、9d后对初始菌株、复壮一代和二代菌株进行产孢培养,比较其产孢量;同时比较分析了这3代菌株的液体发酵原液和1:10稀释液对空心莲子草的防除效果。【结果】获得了10株复壮一代和10株复壮二代菌株,20株菌对空心莲子草均有致病作用,且产生的分生孢子形态特征与初始菌株一致。与初始菌株相比,复壮菌株的产孢量显著提高且达到最大产孢量的时间显著短于初始菌株。其中,复壮二代菌株的孢子量是初始菌株的4.8倍,其次为复壮一代菌株,产孢量是初始菌株的4.1倍;复壮菌株达到最大产孢量的时间为3d,初始菌株为6d。复壮菌株一代和二代的原液对空心莲子草的致病性分别比初始菌株提高了4.65%和9.82%,1:10稀释液的致病性分别提高了25.79%和16.55%。【结论与意义】对长期保存或继代培养的空心莲子草生防菌复壮可显著提高其产孢量和致病性,对维持生防菌遗传性状的稳定性和提高对空心莲子草的防效具有重要意义。     

Ornamentation types of conidia and conidiogenous structures in fasciculate Penicillium species using scanning electron microscopy

KOZAKIEWICZ, Z., 1988. Ornamentation types of conidia and conidiogenous structures in fasciculate Penicillium species using scanning electron microscopy. As part of a multidisciplinary approach to the taxonomy of the fasciculate Penicillium species, the conidial ornamentation of 320 strains was examined in order to determine the types and range of ornamentation and assess the stability of this feature as a taxonomic character.
Five conidial ornamentation classes were identified: echinulate, tuberculate, reticulate, microtuberculate and microverrucate. The technique of cryoscanning was used for examining the penicillus, and assessing such characters as roughening, shape and orientation of the branches. The method provides a fast means of examining freshly frozen material in the absence of artefacts associated with air-drying, freezing and critical-point drying techniques.
Studies on large numbers of conidia from the same isolate and from isolates referred to the same species showed the scanning electron microscopy results to be reproducible and provide an objective and accurate method for comparing micromorphological features. The ornamentation types can be of particular value in species identification.     

GENETIC CONTROL OF CONIDIATION IN ASPERGILLUS RUGULOSUS

The potential for the production of abundant conidia was suggested when an essentially non-conidial isolate of Aspergillus rugulosus, obtained from dune sand, was grown at 25 C rather than at the usual 37 C. Ultraviolet-light-induced conidial strains were obtained from the original non-conidial isolate. Evidence is presented which suggests that the production of conidia is under the control of a single nuclear gene. The data, with a single exception, suggest that induced conidial mutations represent alterations at the same locus. Crosses with isolates from various localities indicate that the genes controlling conidiation in these strains may be linked to or independent of the genes studied in the original dune-sand isolate. No evidence has been obtained which would suggest involvement of a diffusible substance in the control of conidiation.     

Influence of Culture Conditions on Growth and Survival of Conidia of Trichoderma spp. Coated on Seeds

Five Trichoderma strains were grown on rice, on vermiculite plus potato-dextrose broth (PDB), on potato-dextrose agar (PDA) or in liquid cultures supplemented with glycerol, KCl or polyethylene glycol (PEG) at -1 MPa or - 2 MPa. Conidia were coated on seeds through a methyl cellulose coating or through an industrial film-coating process. The conidial yield decreased with glycerol, KCl or PEG compared with PDB alone. The percentage viability was from 23 to 44% after methyl cellulose coating, regardless of the culture conditions for conidial production. In general, the industrial coating resulted in lower numbers of living conidia. The viability during storage was enhanced when vermiculite, rice or PDA were used as substrates for fungal growth. Nevertheless, temperature of storage was found to be more critical to spore survival than the substrate used for spore production; conidial viability on seeds did not exceed 4 months at 15 C. Solid and liquid cultures produced conidia able to control R. solani and P. ultimum when applied to seeds through industrial film coating. The level of disease suppression varied with the number of viable conidia/seed and with the culture medium used for conidial production. The three main conditions for further industrial application-high yields, longevity and biocontrol effectiveness-might be optimized by selecting the appropriate medium (liquid or solid), water potential and solutes used.     

Effect of nutrition on growth and virulence of the entomopathogenic fungus Beauveria bassiana

Three isolates of the entomopathogen Beauveria bassiana along with one strain of Metarhizium anisopliae were cultured on seven media with different carbon/nitrogen (C/N) ratios. The effect of nutrition on virulence of the isolates was evaluated via measurement of colony growth, spore yield, germination speed, conidial C/N ratio and Pr1 (a serine protease) activity. 'Osmotic stress' medium produced the lowest colony growth with low numbers of conidia in all isolates. However, these conidia showed a high germination rate and virulence. However, conidial Pr1 activity was low in some isolates. In most but not in all cases conidia from 1% yeast extract, 2% peptone and low (10 : 1) C/N medium had higher Pr1 activity compared with conidia from other media. However, in some instances we could not conclude that there was a relationship among germination rate, conidial Pr1 activity and virulence. C/N ratio of conidia was statistically different among various media and fungal isolates. Conidia with lower C/N ratio generally produced lower LT(50) (lowest median lethal time) values (more virulent). Insect-passaged conidia from different media had lower C/N ratio compared with similar conidia from artificial cultures. Therefore, they should be more virulent than in vitro produced conidia. As germination rate, conidial Pr1 activity and C/N ratio are independent of host, it seems that host-related determinants such as insect cuticle and physiology and environmental conditions may influence host susceptibility and therefore fungal isolate virulence towards host insects.     

Effects of culture media on hydrophobicity and thermotolerance of Bb and Ma conidia, with description of a novel surfactant based hydrophobicity assay

Hypocrealean entomopathogenic fungal conidia are made up of multi-aged groups given their chronological conidiogenesis. Most thermotolerance assays have been conducted using mixed-age conidia. The present work exploited a polysiloxane polyether copolymer (siloxane) (Silwet L-77®) mediated conidial collection method, validated by a hydrophobicity assay. This was done to divide mixed-age conidia into two groups based on hydrophobicity and test their thermotolerance, relying on the relationship of conidial age with hydrophobicity. Beauveria bassiana GHA and ERL1170 and Metarhizium anisopliae ERL1171 and ERL1540 conidia, produced on millet agar, whey permeate agar, and ¼SDAY were subjected to hydrophobicity assays that included data on yield of conidia/unit of surface area. Conidia were also collected using 0.01% siloxane, and those remaining with 0.08% siloxane. Hydrophobicity was correlated with percent conidia collected in the two siloxane solutions and yield, suggesting a relationship between percent conidia collected and conidial age (maturation). The conidial suspensions were exposed to 45 °C for 45 min, and conidial germination was examined. Overall, conidia which were collected in 0.08% siloxane had lower germination after heat exposure than those collected in the 0.01% solution. Conidia of both fungi produced by incubation on millet or whey permeate for 14 d were more hydrophobic and exhibited greater thermotolerance than those produced on ¼SDAY. These results suggest that conidia can be divided into two groups with different thermotolerance by using a siloxane-mediated conidial collection method based on hydrophobicity. This depends on the types of substrates used that could influence conidial maturation.