出芽短梗霉细胞多形性及影响细胞分化因素探索

【背景】出芽短梗霉(Aureobasidium pullulans)是在生活史中有酵母状细胞生长阶段,并合成黑色素的一种黑酵母(Black yeast),具有典型的细胞多形性,可分化形成酵母状细胞(Yeast-like cell,YL)、膨大细胞(Swollen cell,SC)、厚垣孢子(Chlamydospore,CH)、菌丝(Hyphae,HY)、念珠状菌丝(Monilioid hyphae,MH)、有隔膜膨大细胞(Septate swollen cell,SSC)、分生组织状结构(Meristematic structure,MS),其中膨大细胞既可以作为生长的细胞类型,也可分化为其他的细胞类型。出芽短梗霉的形态分化是可调控的,调控因子有pH、温度、营养条件等。【目的】探究不同的氧气浓度、温度、盐浓度、营养水平对出芽短梗霉细胞形态的影响。【方法】利用显微镜、美兰染色等技术观察不同条件对出芽短梗霉细胞形态的影响。【结果】在完全无氧的试管底部菌体不能生长;在高层半固体表层(高氧气浓度),酵母状细胞(YL)在营养丰富的生长初期出芽繁殖,在养分匮乏的培养后期诱导酵母状细胞(YL)经过膨大细胞(SC)形成厚垣孢子(CH)并合成黑色素;在营养丰富的生长初期,半固体试管浅表层和中间层(微好氧)低浓度氧气诱导YL经过SC形成HY侵入性生长。养分差异对菌体细胞多形性分化影响显著,环境适宜养分丰富(Yeast extract peptone dextrose medium,YPD),以YL生长,不需要分化成HY;环境适宜养分不丰富(Potato dextrose agar,PDA),分化成SC或HY以适应或逃离环境;环境不适宜养分匮乏时(Malt extract agar,MEA),SC或HY分化成CH或MH进入休眠阶段。10%NaCl胁迫降低菌体生长速度,抑制色素合成、HY和MH的形成,并且细胞主要以YL生长繁殖。在相同质量浓度(10%)的KCl或Na2SO4渗透胁迫条件下,细胞多形性表型均为YL发达,HY及MH被抑制,说明高渗胁迫阻止了酵母状细胞向菌丝和厚垣孢子的分化。温度实验中,SC比YL耐高温,MS比SC耐高温。【结论】营养状态对出芽短梗霉细胞分化影响最大。     

出芽短梗霉膨大细胞分选及产糖分析

本研究运用Percoll密度梯度离心的方法对出芽短梗霉Aureobasidium pullulans的两种细胞形态进行分选,并对两种形态的细胞进行多糖产量的分析。通过对转速、分选时间、Percoll分离液浓度的优化,确定了两种细胞形态分选效果最佳的条件是Percoll分离液浓度为60%、转速为5 000r/min、离心时间为30min。经过光学显微镜和透射电子显微镜观察发现上层为酵母状细胞（YL）、下层为膨大细胞（SC）,并发现膨大细胞外有明显的薄膜包被,且产大量多糖。也为今后在相应状态下研究出芽短梗霉膨大细胞的其他代谢机理提供了可行的方法,满足后续研究的需要。     

出芽短梗霉的研究进展

出芽短梗霉是一类类酵母真菌,具有酵母样和真菌菌丝体两种形态,影响其形态的因素有碳源,氮源,离子种类及浓度和pH值等,出芽短梗霉的发酵产物多种多样,如多聚糖,酶,抗真菌素等,通过选育优良菌株可提高发酵产物的产量。     

出芽短梗霉在三种不同氮源的培养基中多糖积累以及形态变化

本研究旨在阐明出芽短梗霉在不同氮源培养基中形态和胞外多糖的积累及化学成分变化。采用摇瓶法培养出芽短梗霉。三种培养基的氮源分别为硝酸钠(培养基1,M1)、硫酸氨、酵母膏(培养基2,M2)和硫酸氨、蛋白胨和酵母膏(培养基3,M3)。M1培养基中,菌丝体和单细胞的生物量积累均比M2、M3低,但胞外多糖的产量则等于甚至略超过M2和M3。在指数生长的前期,白色菌丝体和酵母状细胞状态占优势。指数生长的后期,以厚垣孢子、肿大细胞和黑色菌丝体占优势。胞外多糖都能为茁霉多糖酶水解为麦芽糖和麦芽三糖,说明这些多糖的化学组成都具有(1→4,1→6)-α结构的茁霉多糖。但M1中产生的茁霉多糖结构单元为麦芽糖和麦芽三糖,且二者比例相当。M2中茁霉多糖的麦芽糖结构单元明显减少,而M3中144h后麦芽糖结构单元完全消失。这似乎表明氧化性的氮源和低溶解氧水平可能是造成茁霉多糖结构单元同时具有麦芽糖和麦芽三糖的原因。     

出芽短梗霉F4的溶磷能力及机理

从安徽省铜陵市铜官山尾矿库木贼根际分离筛选出的出芽短梗霉F4,以磷酸钙、磷酸铝、磷酸铁和磷矿粉4种不同磷源进行液体培养,测定培养液的pH、水溶性磷、菌体磷及有机酸含量.结果表明： 菌株F4对不同磷源的溶磷能力为：磷酸铝>磷酸铁、磷酸钙>磷矿粉,溶磷量均高于200 mg·L^-1;培养液pH在48 h内迅速下降,以磷酸铝、磷酸铁为磷源的培养液pH下降幅度明显大于磷酸钙与磷矿粉.出芽短梗霉F4产生的有机酸主要为草酸、柠檬酸和酒石酸,其中,以草酸为主.菌株的溶磷能力与有机酸无显著相关性,而与pH呈显著相关.接种出芽短梗霉F4时加入葡萄糖,尾矿中速效磷含量显著增加,说明出芽短梗霉F4在尾矿生态修复中具有潜在的应用价值.
     

广西和海南的赛多孢属2新种

从广西和海南植物根系采集的土样经用头发富集后分离获得3株赛多孢菌株G79、EM65901和EM65901.2,采用系统发育和单倍型网络并结合形态特征对其分类地位进行确证,G79菌株提议为新种少孢赛多孢,其典型特征是:分生孢子顶生或侧生于菌丝上,产孢较少,孢子透明至半透明,倒卵形,5.4–10.8×3.2–5.4μm,无间生孢子和厚垣孢子;EM65901和EM65901.2两菌株为另一新种,定名为三亚赛多孢,其典型特征是:分生孢子顶生或侧生于菌丝或支撑细胞上,单生或1–4个着生于膨大或不膨大的支撑细胞上,透明至半透明,倒卵圆形,4–7.5×3–4μm,少数近球形,3–5μm,无间生孢子;厚垣孢子椭圆形,5–7.5×4–6.5μm,或近球形,5–7.5μm。     

曲霉制片法

曲霉为半知菌类,串珠霉目的一属。有少数具有性生殖阶段,归入子囊菌纲曲霉科。菌丝有隔而分枝。无性繁殖时,从营养菌丝的一个较为膨大和厚壁的细胞(即脚胞)上生出无隔和无分枝而向上生长的分生孢子梗,梗的顶端膨大呈球形或棒状,周围着生许多称为小梗的瓶状短枝,每个小梗上可能再生一、二个或二个     

粉红螺旋聚孢霉67‐1厚垣孢子生物学特性的研究

粉红螺旋聚孢霉(粉红粘帚霉)是多种植物病原真菌的重寄生菌,具有巨大的生防潜力。以实验室前期分离筛选的高效粉红螺旋聚孢霉67‐1菌株为材料,研究其厚垣孢子和分生孢子的生物学特性。结果表明,以厚垣孢子为接种体,其生长速度和菌丝鲜重与以分生孢子为接种体形成的菌落没有明显差异,但产孢水平提高6倍以上。粉红螺旋聚孢霉对高温和干燥的耐受程度较差,尤其分生孢子更为敏感。当加工温度范围在40–50℃时,厚垣孢子和分生孢子的活性存在显著差异(P<0.05)。菌株67‐1的厚垣孢子耐紫外线能力明显优于分生孢子。在无保护剂条件下,紫外灯照射60s,厚垣孢子活性保持在62.6%,是分生孢子活性1倍以上。土壤抑菌作用对真菌厚垣孢子的影响相对较小,24h孢子萌发率为47.2%,而分生孢子仅为4.6%。除了百菌清,菌株67‐1厚垣孢子对多种化学农药有较强的耐受力,在药剂田间使用浓度下,厚垣孢子萌发率均大于70%,其中对15%恶霉灵、1.8%阿维菌素和3%克百威的耐受力显著高于分生孢子(P<0.05),而对井冈霉素和两种杀虫剂没有显著提高。对菌株67‐1的寄生性测定结果表明,2种孢子对核盘菌菌核的寄生能力没有差异,菌核感染达到4级的占58.9%–60.0%。106/m L浓度厚垣孢子与分生孢子对黄瓜枯萎病的防效均达到65%以上,二者没有显著差异。     

中国一新记录属——埃里砖格孢

<正> 1.埃里砖格孢属 中国新记录属 Embellisia Simmons,Mycologia 63,1971 菌丝体典型的丛梗状,光滑或粗糙。菌丝体可产生厚垣孢子。分生孢子梗从菌丝或厚垣孢子上生出,分枝或少分枝,有隔膜,直立或在产孢处曲膝状弯曲。孔生孢子褐色,光滑或稍粗糙,单生,倒卵形,卵圆形、椭圆形或近圆柱形,直或弯曲,具横隔膜和斜隔膜,偶见纵隔膜。隔膜比孢壁加厚、     

农杆菌介导的出芽短梗霉遗传转化及高效筛选聚苹果酸高产菌株

建立根癌农杆菌介导的出芽短梗霉遗传转化方法及T-DNA突变库,高效筛选聚苹果酸高产菌株及功能基因。通过含潮霉素和草铵磷抗性基因的农杆菌转化出芽短梗霉,抗性压力筛选及PCR验证建立根癌农杆菌介导的出芽短梗霉遗传转化方法,结合发酵液p H与聚苹果酸含量响应变化,微孔板高效筛选高产聚苹果酸的T-DNA插入突变株,基因组步移确定T-DNA插入位点及功能基因。结果获得遗传稳定的抗性基因菌株,每107个细胞可获得80-120个转化子,出芽短梗霉H27号T-DNA突变株聚苹果酸摇瓶发酵产量提高24.5%,基因组步移证实糖酵解途径磷酸甘油酸变位酶基因被破坏。成功建立了根癌农杆菌介导的出芽短梗霉遗传转化方法和T-DNA插入突变库,结合高效筛选方法为聚苹果酸合成功能基因挖掘及高产机制解析奠定基础。     

Production of pigment-free pullulan by swollen cell in <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> NG which cell differentiation was affected by pH and nutrition

A black yeast strain “NG” was isolated from strawberry fruit and identified as Aureobasidium pullulans. Strain NG displayed yeast-like cell (YL), swollen cell (SC), septate swollen cell (SSC), meristematic structure (MS), and chlamydospore (CH) morphologies. pH was the key factor regulating cell morphogenesis of strain NG. Differentiation of YL controlled by extracellular pH had no relationship with nutrition level. YL was maintained at pH >6.0, but was transformed into SC at pH ∼4.5. SC, a stable cell type of A. pullulans, could bud, septate, or transform into MS or CH, in response to nutrition level and low pH. SC produced swollen cell blastospores (SCB) at pH 2.1 with abundant nutrition, and could transform into MS at lower pH (1.5). SC was induced to form CH by low level nutrition and pH <3, and this transition was suppressed by adjusting pH to ∼4.5. Crude polysaccharides without pigment (melanin) were produced by SC of strain NG. Pullulan content of the polysaccharides was very high (98.37%). Fourier-transform infrared spectroscopy confirmed that chemical structures of the polysaccharides and standard pullulan were identical. Swollen cells produced 2.08 mg/ml non-pigmented polysaccharides at 96 h in YPD medium. Controlling pH of fermentation is an effective and convenient method to harvest SC for melanin-free pullulan production.     

寄主抗药性对菜蛾绒茧蜂生物学特性的影响

以小菜蛾Plutella xylostella敏感品系SP作寄主饲育的菜蛾绒茧蜂Cotesia plutellae SC品系分别寄生于小菜蛾的SP品系(SC-SP组合)或抗性RP品系(SC-RP组合),还以小菜蛾RP品系作寄主饲育的菜蛾绒茧蜂RC品系分别寄生于小菜蛾的SP品系(RC-SP组合)或RP品系(RC-RP组合),均在幼虫中期施用氰戊菊酯,考察了药剂对该蜂生物学特性的影响。结果发现：在不施用杀虫剂时,SC-RP组合中蜂的结茧率为45.8%,显著低于其它组合, 所结茧长0.76 mm,育出雌蜂前翅和后足胫节长分别为3.28 mm和2.33 mm,也分别小于其它各组合的结果,表明寄主抗药性对该蜂有不利影响;施用杀虫剂后,RC-RP、SC-RP组合中,蜂的结茧率分别为95.5%和37.8%,显著高于SC-SP和RC-SP组合中蜂的结茧率（22.5%,25.8%）,表明寄主抗性能保护其体内的幼蜂少受杀虫剂的影响;RC-SP组合在受到和未受到杀虫剂作用时茧的羽化率分别为95.2%和93.6%,无显著差异,卵+幼虫及雌蛹的发育历期在处理和对照间也无显著差异,表明用抗性寄主饲育的菜蛾绒茧蜂在寄生敏感寄主时仍表现一定的耐药性,有利于该蜂抗药性的发展,即寄主 寄生蜂之间在抗药性方面存在协同进化。     

Comparison of volatile sulphur compound production by cheese-ripening yeasts from methionine and methionine–cysteine mixtures

Production of volatile sulphur compounds (VSC) was assessed in culture media supplemented with l-methionine or l-methionine/l-cysteine mixtures, using five cheese-ripening yeasts: Debaryomyces hansenii DH47(8), Kluyveromyces lactis KL640, Geotrichum candidum GC77, Yarrowia lipolytica YL200 and Saccharomyces cerevisiae SC45(3). All five yeasts produced VSC with l-methionine or l-methionine/l-cysteine, but different VSC profiles were found. GC77 and YL200 produced dimethyldisulphide and trace levels of dimethyltrisulphide while DH47(8), KL640 and SC45(3) produced mainly methionol and low levels of methional. S-methylthioacetate was produced by all the yeasts but at different concentrations. DH47(8), KL640 and SC45(3) also produced other minor VSC including 3-methylthiopropyl acetate, ethyl-3-methylthiopropanoate, a thiophenone, and an oxathiane. However, VSC production diminished in a strain-dependent behaviour when l-cysteine was supplemented, even at a low concentration (0.2 g l⁻¹). This effect was due mainly to a significant decrease in l-methionine consumption in all the yeasts except YL200. Hydrogen sulphide produced by l-cysteine catabolism did not seem to contribute to VSC generation at the acid pH of yeast cultures. The significance of such results in the cheese-ripening context is discussed.     

Industrial Application of Artificially Induced Diploid Strains of Torulaspora delbrueckii

Diploid strains of Torulaspora delbrueckii were tested for industrial application. Because the cell volume of the diploid strain was three times as large as that of the parental haploid strain, collection and subsequent dehydration to make compressed yeast cakes were greatly improved with the diploid YL3. The time required for dehydration of the diploid strain was shortened to 1/2.5 that of the parent strain under conventional conditions. Moreover, for the diploid cells frequent filter changes for dehydration were not required, which was the case with parental cells. Fermentation activity and tolerance to freeze-thawing in dough were succesfully inherited by the diploid strains. The diploid YL3 showed nearly the same activity as the diploid F31 in bread making. However, the endurance period of yeast cakes when stored at 30°C without softening to lead to liquefaction was much longer in YL3 (199 h) than in F31 (132 h). This superiority was ascribed to the fact that YL3 was induced through direct diploidization and had no genetic defect on chromosomes because the wild-type strain was employed as the parent, whereas F31 was obtained through protoplast fusion from two auxotrophic mutants and carried at least two mutagenized genes that were masked by heterolallelism.     

我国蜜环菌分类单元间菌索的相互作用

蜜环菌菌索是天麻生长的主要营养来源,而蜜环菌生物种菌索之间可能存在种间相互作用,因此天麻栽培时蜜环菌种的混用可能对天麻的产量产生影响。为揭示我国蜜环菌分类单元间菌索的相互作用,以我国8个蜜环菌分类单元为研究对象,通过研究其共同培养时整体及单侧菌索的生长速率和单位长度内生长尖端个数来研究其菌索间的作用特性。结果表明：两者间相互拮抗的有CBS D-CBS F;仅有一个蜜环菌菌索未受到影响或受到协同作用的有：CBS A-CBS H中的CBS A、CBS F-CBS J中的CBS J、CBS A-CBS F中的CBS A、CBS A-CBS N中的CBS A、CBS F-CBS M中的CBS M、CBS J-CBS M中的CBS M、CBS B-CBS J中的CBS B;组合中两种蜜环菌菌索靠近侧协同生长的有：CBS A-CBS M;组合中对两者靠近的区域具有优势的有：CBS A-CBS B中的CBS B、CBS A-CBS J中的CBS J、CBS B-CBS F中的CBS F、CBS D-CBS H中的CBS H、CBS D-CBS N中的CBS N和CBS F-CBS M中的CBS F。本研究的开展为我国蜜环菌的鉴定、天麻栽培用蜜环菌种的选用提供理论指导。     

Oxidation of amines by yeasts grown on 1-aminoalkanes or putrescine as the sole source of carbon,nitrogen and energy

The maximum growth rate of Trichosporon cutaneum CBS 8111 in chemostat cultures was 0.185 h^-1 on ethylamine and 0.21 h^-1 on butylamine, that of Candida famata CBS 8109 was 0.32 h^-1 on putrescine.The amine oxidation pattern of the ascomycetous strains studied, viz. Candida famata CBS 8109, Stephanoascus ciferrii CBS 4856 and Trichosporon adeninovorans CBS 8244 was independent of the amine that had been used as the growth substrate. It resembled that of benzylamine/putrescine oxidase found in other ascomycetous yeasts. However, differences in pH optimum and substrate specificity were observed between the amine-oxidizing systems of these three species.The amine oxidation pattern of cell-free extracts of Trichosporon cutaneum CBS 8111 varied with the amine that was used as growth substrate. The enzyme system produced by Cryptococcus laurentii CBS 7140 failed to oxidize isobutylamine and benzylamine, and showed a high pH optimum.The synthesis of amine oxidase in the four yeast strains studied was not repressed by ammonium chloride and was weakly repressed by glucose but was strongly repressed if both compounds were present in the growth medium.     

The properties of citrate transport catalyzed by CitP of Lactococcus lactis ssp. lactis biovar diacetylactis

Abstract The SC3 hydrophobin gene of Schizophyllum commune was disrupted by homologous integration of an SC3 genomic fragment interrupted by a phleomycin resistance cassette. The phenotype of the mutant was particularly clear in sealed plates in which formation of aerial hyphae was blocked. In non-sealed plates aerial hyphae did form but these were hydrophilic and not hydrophobic as in wild-type strains. Complementation with a genomic SC3 clone restored formation of hydrophobic aerial hyphae in sealed plates. In a dikaryon homozygous for the SC3 mutation normal sporulating fruiting bodies were produced but aerial hyphae were hydrophilic.     

A phosphatidylinositol/phosphatidylcholine transfer protein is required for differentiation of the dimorphic yeast Yarrowia lipolytica from the yeast to the mycelial form

The SEC14SC gene encodes the phosphatidylinositol/phosphatidylcholine transfer protein (PI/PC-TP) of Saccharomyces cerevisiae. The SEC14SC gene product (SEC14pSC) is associated with the Golgi complex as a peripheral membrane protein and plays an essential role in stimulating Golgi secretory function. We report the characterization of SEC14YL, the structural gene for the PI/PC-TP of the dimorphic yeast Yarrowia lipolytica. SEC14YL encodes a primary translation product (SEC14YL) that is predicted to be a 497-residue polypeptide of which the amino- terminal 300 residues are highly homologous to the entire SEC14pSC, and the carboxyl-terminal 197 residues define a dispensible domain that is not homologous to any known protein. In a manner analogous to the case for SEC14pSC, SEC14pYL localizes to punctate cytoplasmic structures in Y. lipolytica that likely represent Golgi bodies. However, SEC14pYL is neither required for the viability of Y. lipolytica nor is it required for secretory pathway function in this organism. This nonessentiality of SEC14pYL for growth and secretion is probably not the consequence of a second PI/PC-TP activity in Y. lipolytica as cell-free lysates prepared from delta sec14YL strains are devoid of measurable PI/PC-TP activity in vitro. Phenotypic analyses demonstrate that SEC14pYL dysfunction results in the inability of Y. lipolytica to undergo the characteristic dimorphic transition from the yeast to the mycelial form that typifies this species. Rather, delta sec14YL mutants form aberrant pseudomycelial structures as cells enter stationary growth phase. The collective data indicate a role for SEC14pYL in promoting the differentiation of Y. lipolytica cells from yeast to mycelia, and demonstrate that PI/PC-TP function is utilized in diverse ways by different organisms.     

Capture of mites and rotifers by four strains of Dactylella gephyropaga known as a nematophagous hyphomycete

Four strains of Dactylella gephyropaga obtained from Japan and Indonesia captured mites, as well as nematodes, by means of the adhesive hyphal network composed of columnar processes and rectangular meshes of hyphae, in which each of the meshes was made by additional growth of apex of a columnar process toward that of neighboring process and anastomosis with each other. This is the first report showing that a fungus captured and consumed mites. When immersed under water, the four strains captured rotifers also with the columnar processes by adhesion. The CBS178.37 used for comparison was not the strain of D. gephyropaga, and its adhesive network was produced only by repeating development and anastomosis of curved hyphae that captured neither mites and rotifers but only nematodes.