根虫瘟霉转寄主过程中毒力相关胞外蛋白酶系的诱导表达

根虫瘟霉Zoophthoraradicans是寄主范围较广的专性昆虫病原真菌。在该菌胞外蛋白酶系与毒力变化关系的研究中,不同寄主来源的根虫瘟霉菌株产胞外蛋白酶水平与其对小菜蛾(Plutellaxylostella)的毒力间无明显的相关性。但转寄主各代菌株随毒力逐步上升产胞外蛋白酶水平也有所增加,两者间有一定的相关性。经活性电泳检测显示,ARSEF1342原始菌株(R0)有分子量分别为148kD,153kD和162kD的三条蛋白酶条带,但转寄主传代菌株(R1~R4)的148kD蛋白酶条带突然消失,而153kD蛋白酶条带则随转寄主传代数增加逐步趋于明显,相关性分析发现153kD和148kD与毒力间具有较好的相关性。这表明根虫瘟霉菌株在转寄主过程中逐渐增加了对新寄主具有较高基质特异性的胞外蛋白酶的诱导表达,从而使转寄主菌株更适应对新寄主的入侵及致病。     

不同寄主来源的根虫瘟霉菌株对小菜蛾幼虫的毒力比较

在不同寄主来源的4株根虫瘟霉Zoophthora radicans对小菜蛾 Plutella xylostella 2龄幼虫的生物测定中,发现源于小菜蛾的菌株ARSEF1100毒力最强,在0.53～319.32/mm²的孢子剂量下,接种后第8 天累计死亡率为2.38%～97.44%,虫尸全部表现典型的虫瘟霉症状;源于叶蝉的ARSEF2699和F99101菌株的同日累计死亡率分别为2.38%～50.00% (剂量为1.56～314.84/mm²孢子)和2.38%～57.89% (剂量为1.84～484.08/mm²的孢子);而源于菜粉蝶的ARSEF1342菌株在3.54～633.0/mm²的孢子剂量下只引发6.52%～13.63%的累计死亡率,后3个菌株致死的小菜蛾幼虫仅部分表现典型症状。所获数据经时间剂量-死亡率模型模拟分析,剂量效应参数依次为ARSEF1100 (1.89) > F99101 (1.48) > ARSEF2699 (1.23) > ARSEF1342 (0.37),相互间差异均达极显著水平。接种后4～8 天内,ARSEF1100的LD₅₀值分别为231.68、113.08、71.41、40.87和35.30/mm²的孢子,其毒力远高于其余3个菌株;ARSEF2699的相应LD₅₀值为1344.43、922.39、555.58、410.06和397.07/mm²的孢子;F99101的LD₅₀值为666.86、451.64、413.82、350.65和332.57/mm²的孢子,而ARSEF1342的毒力太弱难以估计。这些结果表明,ARSEF1100菌株最有希望用于小菜蛾的微生物防治。     

稻纵卷叶螟根虫瘟霉的分离鉴定及其流行病研究

在广东省发现一种新的侵染稻纵卷叶螟幼虫的昆虫病原真菌,经鉴定该病原真菌为根虫瘟霉Zoophthora radicans,这是中国首次报道该菌侵染稻纵卷叶螟。在室内进行的温度和光周期对该菌生长的影响实验表明,该菌在15－25℃均可生长,最适生长温度为25℃;光周期对菌落生长影响不显著。根虫瘟霉主要侵染稻纵卷叶螟3－5龄幼虫,对3－5龄幼虫的侵染率分别为2.71%、24.32%和72.97%。在10－11月,根虫瘟霉可持续引发稻纵卷叶螟幼虫高强度流行病,侵染率高达95%。该菌以休眠孢子越冬,成为次年稻纵卷叶螟幼虫流行病的初侵染源。因此,根虫瘟霉是稻纵卷叶螟幼虫一种有效的自然控制因子。     

根虫瘟霉不同菌株对小菜蛾幼虫血淋巴酚氧化酶原的激活作用

在对小菜蛾Plutella xylostella幼虫血淋巴酚氧化酶原的存在部位及免疫激活作用特点研究的基础上,比较了根虫瘟霉Zoophthora radicans不同菌株对酚氧化酶原激活系统的免疫激化及防御作用的差异。研究发现, 酚氧化酶原主要位于小菜蛾幼虫血细胞膜及血细胞裂解液中,极少存在于血浆中。在免疫激活剂昆布多糖存在下,分别测得小菜蛾幼虫血细胞碎片、血细胞裂解液和血浆的酚氧化酶活性为26.80 U,16.68 U和2.53 U。酚氧化酶原显著地受血浆和昆布多糖同时存在的激活,但两者单独存在时对酚氧化酶原的激活作用较弱。根虫瘟霉菌丝裂解液对酚氧化酶原有不同程度的激活作用,其激活作用在有血浆存在时显著增强,其酚氧化酶活性可提高2.9～3.4倍。各菌株间对酚氧化酶原的激活作用则以ARSEF1342菌株最强,ARSEF2699和F99101菌株次之,ARSEF1100菌株最弱。被激活的酚氧化酶可粘附于根虫瘟霉菌丝上并能产生黑化反应,各菌株间酚氧化酶粘附于ARSEF1342菌株的能力最强,粘附于ARSEF2699和F99101菌株的次之,粘附于ARSEF1100菌株的最弱。但酚氧化酶粘附于昆布多糖的能力显著强于各虫霉菌株,表明各菌株在一定程度上能逃避寄主的免疫识别;各菌株激活酚氧化酶原及酚氧化酶粘附于菌株强弱,与对小菜蛾毒力呈负相关性,表明高毒力菌株具有易逃避寄主免疫识别的趋向。     

哈维氏弧菌胞外蛋白酶基因的克隆表达及免疫原性

根据实验室分离的大黄鱼弧菌病主要病原菌哈维氏弧菌(Vib rio harveyi)GYC1108-1株的胞外蛋白酶基因序列,设计胞外蛋白酶基因(ΔProA)特异性引物,扩增获得1552bp的ΔProA基因,克隆于pUC57-T载体;将ΔProA基因亚克隆到原核表达载体pET-28a进行融合表达,SDS-PAGE电泳检测发现,ΔProA融合蛋白经IPTG诱导后在大肠杆菌中以包涵体形式表达,分子量大小约55kD,诱导5h的表达蛋白产量约占细菌总蛋白的21%。Western blot分析,表达的55kD蛋白具有较高免疫原性。用纯化的ΔProA融合蛋白对大黄鱼进行免疫试验,结果免疫保护率达到75%。       

不同基质多代培养对蚧虫病原真菌蜡蚧霉致病力的影响

【目的】研究昆虫病原真菌蜡蚧霉Lecanicilliurn lecanii(Zimmermann.)菌株No.V3.4504在不同培养基上继代培养,对菌种的菌落生长特性、胞外酶活力和对蚧虫致病力的影响。【方法】试验菌种蜡蚧霉菌株No.V3.4504是从染病蚧虫上分离的。试验蚧虫是沙里院褐球蚧Rhodococcus sariuoni Borchsenius和日本龟蜡蚧Ceroplastes japonicus Green。采用7种培养基继代培养多代。观察菌落形态特征、测定生长速率、产孢量、胞外蛋白酶和几丁质酶活性及对蚧虫的致死率。【结果】在PDA培养基上,菌落生长速率最快,但产孢量最低,胞外蛋白酶和几丁质酶的活性均呈逐代下降趋势,对两种蚧虫致死率也最低;增加蛋白胨对改善菌种致病力没有明显效果;在增加蚧虫尸体的D、E、F培养基上,菌落生长速率虽然较慢,但产孢量上升为8.83×106-9.13×106孢子/cm2。蛋白酶和几丁质酶的活性平均达到2.16-2.13 U/g和1.01-1.03 U/g,对两种蚧虫的致死率分别在55%-58%和39%-42%;在活蚧虫上连续培养3代,蛋白酶和几丁质酶的活性最高,为3.08-2.92 U/g和1.45-1.42 U/g,是PDA培养基上的1.6倍。对两种蚧虫的致死率也最高,分别达到71.30%和58.89%。蛋白酶和几丁质酶的活性与蚧虫死亡率呈正直线相关关系。【结论】采用PDA培养基连续多代培养会引起菌株No.V3.4504明显退化;在培养基中加入蚧虫尸体,对于保持菌种活力有明显效果;在活蚧虫体上继代培养对复壮菌种,提高菌种毒力的效果最佳。     

大菜粉蝶根虫瘟霉菌株转染小菜蛾后侵染力变化及其与寄主血淋巴酚氧化酶活性的关系

源于大菜粉蝶Pieris brassicae的根虫瘟霉Zoophthora radicans菌株R₀通过反复转染小菜蛾Plutella xylostella而分别获得转染菌株R₁、R₃和R₅。用这些菌株对小菜蛾2龄幼虫进行生物测定,发现菌株对寄主的侵染力有随转染次数增加而增强的趋势。接种后第1～6 天,R₀的LC₂₀（孢子数/mm²）分别为14.7、14.5、9.0、7.1、6.0和5.5;R₁的LC₂₀分别为9.6、5.0、4.2、3.6、3.1和3.0;R₃的LC₂₀分别为4.6、2.9、2.8、2.5、2.4和2.2; R₅的LC₂₀分别为5.2、3.7、3.2、2.8、2.6和2.6,接种后同一天菌株 R₃的LC₂₀值最小即侵染力最强。各菌株感染小菜蛾幼虫后可显著激活寄主血淋巴中的酚氧化酶活性,但R₁、R₃和R₅对酚氧化酶的激活程度显著低于原始菌株R₀。各菌株对小菜蛾的侵染力强弱指标log₁₀ (LC₂₀)与其侵染后寄主血淋巴酚氧化酶活性呈明显正相关（0.852<0.95）,表明R0在对新寄主转染过程中逐渐获得了逃避或克服新寄主免疫防御的能力,从而增强对新寄主的侵染力。     

小菜蛾幼虫血淋巴中β-1,3-葡聚糖结合蛋白的分离及其对根虫瘟霉侵染的免疫活性

用亲和沉淀法从小菜蛾(Plutella xylostella)幼虫血淋巴中分离获得2种β-1,3-葡聚糖结合蛋白,分子质量分别为75.9 ku和83.2 ku,主要存在于幼虫血浆中,但血细胞中未检出.研究表明,β-1,3-葡聚糖结合蛋白能特异性地识别β-1,3-葡聚糖,并显著激活幼虫血淋巴中的酚氧化酶原（ProPO）,与昆布多糖共存时所激活的酚氧化酶（PO）活性显著高于两者单独存在时的PO活性.与4株根虫瘟霉(Zoophthora radicans)菌丝裂解液共存时,β-1,3-葡聚糖结合蛋白能激活幼虫血淋巴中的ProPO,使PO活力显著高于该菌原生质体裂解液所激活的PO活性.显然,β-1,3-葡聚糖结合蛋白只有特异性地识别根虫瘟霉细胞壁中的β-1,3-葡聚糖后才能激活幼虫血淋巴中的ProPO,表明虫霉原生质体可逃避寄主免疫反应.此外,β-1,3-葡聚糖结合蛋白对不同菌株所激活的PO活性存在差异,各菌株所激活的PO活性由高到低依次为：ARSEF1342＞ARSEF2699＞F99101＞ARSEF1100,这与各菌株对小菜蛾的毒力强弱相一致,即菌株逃避寄主免疫识别的能力与其毒力相关.     

出芽短梗霉Aureobasidium sp. SRF的菌株特性分析及胞外多糖结构鉴定

菌株SRF是1株从意大利树莓(Rubus corchorifolius)果实表面分离、可产胞外多糖的新菌株。在鉴定其分类归属的基础上,对其产生的胞外多糖进行了结构分析和发酵条件优化,为寻找微生物多糖提供新的菌株,为开发利用资源微生物提供借鉴。通过形态学和ITS序列对比分析进行菌株鉴定;通过薄层层析和红外光谱分析,确定胞外多糖结构;通过单因素检测试验,确定影响产糖量的主要因素;响应面Plackett-Burman和Box-Behnken设计筛选发酵产胞外多糖的最优条件。结果表明,出发菌株SRF隶属于出芽短梗霉属,命名为Aureobasidium sp. SRF;SRF所产胞外多糖为普鲁兰多糖;单因素检测表明,对多糖产量影响最大的因素为碳源浓度、氮源浓度、无机离子浓度,其次是碳源、氮源、无机离子、pH值;根据响应面结果确定最优发酵条件为麦芽糖8%(质量分数)、酵母提取物3%(质量分数)、钙离子0.3 g/L、pH 6,产糖量达5.93 g/L。SRF是1株来源于树莓浆果表面,可产胞外普鲁兰多糖的出芽短梗霉新菌株,是1株产微生物多糖的候选菌株。     

日本根霉IFO5318胞外β—葡萄糖苷酶的纯化及部分特性

采用硫酸铵沉淀及柱层析等步骤纯化了日本根霉IFO5318的β—葡萄糖苷酶,回收率为22％。该酶分子量约为4.0×10~5,由四个相同大小的亚基组成;最适反应温度55℃,最适反应pH5.5;对热较敏感,但能在较大的pH范围内保持稳定。用对硝基苯基—β-D-吡喃葡糖苷为底物,测得的K_m和V_(max)值分别为0.825mg·ml~(-1)和135.4μmol·min~(-1)·mg~(-1)。该酶对纤维二糖的水解能力最强,SDS、Fe~(3 )、Hg~2 )等对酶活力有抑制作用。     

Extracellular protease activity of different Pseudomonas strains: dependence of proteolytic activity on culture conditions

AIMS: This study investigated the effect of growth conditions on proteolytic activity of a Pseudomonas strain, named Pseudomonas sp. LBSA1, isolated from bulk raw milk. It was compared with three Pseudomonas chlororaphis and one Pseudomonas fluorescens strain from culture collections. METHODS AND RESULTS: Bacteriae were grown in a minimal salt medium. For all the strains, addition of 1% (v/v) skim milk to the growth medium was sufficient to induce protease production in 48-h culture. Addition of 1 mmol l(-1) calcium chloride permitted the detection of proteolytic activity of four strains in 48-h cultures but not for Pseudomonas sp. LBSA1. The five strains presented two patterns of proteolytic activity when grown in the minimal salt medium supplemented with 2% (v/v) skim milk at various temperatures for 48 h. Two electrophoretic protease patterns were also obtained from the zymogram of extracellular medium for the five strains. CONCLUSIONS: The growth conditions permitting protease production are variable and do not depend on the genus of the producing strain. SIGNIFICANCE AND IMPACT OF THE STUDY: For the first time a study on proteolytic activity of P. chlororaphis strains is reported. Among the tested criteria, zymograms of extracellular medium were the only ones that permitted distinguishing the P. chlororaphis strains from the P. fluorescens strain.     

Extracellular proteases produced by the wood-degrading fungus Phanerochaete chrysosporium under ligninolytic and non-ligninolytic conditions

When subjected to nitrogen limitation, the wood-degrading fungus Phanerochaete chrysosporium produces two groups of secondary metabolic, extracellular isoenzymes that depolymerize lignin in wood: lignin peroxidases and manganese peroxidases. We have shown earlier the turnover in activity of the lignin peroxidases to be due in part to extracellular proteolytic activity. This paper reports the electrophoretic characterization of two sets of acidic extracellular proteases produced by submerged cultures of P. chrysosporium. The protease activity seen on day 2 of incubation, during primary growth when nitrogen levels are not known to be limiting, consisted of at least six proteolytic bands ranging in size from 82 to 22 kDa. The activity of this primary protease was strongly reduced in the presence of SDS. Following the day 2, when nitrogen levels are known to become limiting and cultures become ligninolytic, the main protease activity (secondary protease) consisted of a major proteolytic band of 76 kDa and a minor band of 25 kDa. The major and minor secondary protease activities were inhibited by phenylmethylsulfonyl fluoride and pepstatin A, respectively. When cultures were grown in the presence of excess nitrogen (non-ligninolytic condition), the primary protease remained the principal protease throughout the culture period. These results identify and characterize a specific proteolytic activity associated with conditions that promote lignin degradation.     

Use of a Novel Sporulation Monitor to Quantify the EVects of Formulation and Storage on Conidiation by Dried Mycelia of the Entomopathogenic Fungus Zoophthora radicans

A novel piece of equipment, the sporulation monitor, is described for the comparison of conidia production from mycelia receiving diVerent formulation and storage treatments. This equipment was used to compare the viability of Zoophthora radicans mycelial samples treated either with 10% maltose solution or with distilled water before drying and storage for 0-12 weeks at 4oC. Freshly dried maltose-treated mycelial mat samples produced significantly more conidia for a significantly longer time than distilled water-treated mat samples of the same age. Very few conidia were produced from mats in either treatment after storage for 4 weeks or longer. There were great diVerences in conidia production from mycelial mat samples produced in diVerent fermenter runs. These results are discussed in relation to the potential for the use of dried mycelia in biological control programmes.     

Distinct immunomodulatory properties of extracellular vesicles released by different strains of Acanthamoeba

Free living amoeba of the genus Acanthamoeba are opportunist protozoan involved in corneal, systemic, and encephalic infections in humans. Most of the mechanisms underlying intraspecies variations and pathogenicity are still unknown. Recently, the release of extracellular vesicles (EVs) by Acanthamoeba was reported. However, comparative characterization of EVs from distinct strains is not available. The aim of this study was to evaluate EVs produced by Acanthamoeba from different genotypes, comparing their proteases profile and immunomodulatory properties. EVs from four environmental or clinical strains (genotypes T1, T2, T4, and T11) were obtained by ultracentrifugation, quantitated by nanoparticle tracking analysis and analyzed by scanning and transmission electron microscopy. Proteases profile was determined by zymography and functional properties of EVs (measure of nitrite and cytokine production) were determined after peritoneal macrophage stimulation. Despite their genotype, all strains released EVs and no differences in size and/or concentration were detected. EVs exhibited a predominant activity of serine proteases (pH 7.4 and 3.5), with higher intensity in T4 and T1 strains. EVs from the environmental, nonpathogenic T11 strain exhibited a more proinflammatory profile, inducing higher levels of Nitrite, tumor necrosis factor alpha and interleukin-6 via TLR4/TLR2 than those strains with pathogenic traits (T4, T1, and T2). Preincubation with EVs treated with protease inhibitors or heating drastically decreased nitrite concentration production in macrophages. Those data suggest that immunomodulatory effects of EVs may reflect their pathogenic potential depending on the Acanthamoeba strains and are dependent on protease integrity.     

Enhancement and regulation of extracellular protein production by Bacillus brevis 47 through manipulation of cell culture conditions

Bacillus brevis 47 was cultivated in 2 liter fermentors in semidefined media containing polypeptone with or without glucose or fructose. Neither sugar was essential for growth or extracellular (S-layer) protein production, and 2.5 to 3.0 g/L protein was accumulated in the medium. When present, glucose was used very slowly, however, fructose was used much more quickly. Dramatic changes in metabolic indicators (dissolved oxygen and pH) were seen when fructose became depleted, and protease was produced, decreasing the amount of protein ultimatelv accumulated in the medium. Using the change in dissolved oxygen as a marker for the time of addition, polypeptone, fructose, or both were used to stimulate protein production. With the addition of polypeptone, on stimulation was achieved, but protease production was suppressed. Addition of fructose did result in a small stimulation of protein production (to 5 g/L) if added once. Further additions resulted in more growth, but no increase in protein production. Various combinations of polypeptone and fructose were also used, with the most effective combination (fructose added early, fructose and polypeptone added later) resulting in an accumulation of 15 g/L protein in the medium. This is comparable to that seen when B. brevis 47 is grown in a complex glucose medium and stimulated with polypeptone addition at 21 hours. These results are discussed with respect to the structure and function of S-layer proteins, as well as the use of this organism for the production of heterologous proteins.     

Stomatal and cuticular traits on carnation tissue culture under different ventilation conditions

Establishment of microplants is related to the moisture vapourtransmission of the culture vessel lid. In this respect, stomatal andcuticular physiology were characterized in detached leaves from Dianthuscaryophyllus grown in the glasshouse or in vitro at different rates ofventilation. In vitro plants grown in non-ventilated culture vessels hadless waxes and therefore higher RWL compared to in vitro plants grown at Vr0.86 changes.h^–1. The improvement of stomatal function inleaves obtained in ventilated vessels can be due to a performance of ionicrelations between guard and subsidiary cells, mainly by an increasingK⁺ concentration in the guard cells as ventilation decreases.Moreover, data showthat there is an increase in free ABA in the leavesfromventilated culture vessels to compensate for the conjugated ABA lostduring desiccation. If the proliferation stage proceeds in ventilatedculture vessels, the physiological characteristics of the plants producedare better than those obtained in non-ventilated culture vessels, confirmedby higher survival after soil transplantion.     

不同增菌和培养条件下沙门氏菌检出效果的比较

通过在鸡精样本中添加低含量的鼠伤寒沙门氏菌和干扰菌(弗氏柠檬酸杆菌和奇异变形杆菌),参考国标和美国药典中沙门氏菌的定性方法,研究比较了3种选择性增菌液、4个培养时间段以及4种选择性平板对于沙门氏菌的不同检出效果。结果表明,在RV肉汤及四硫磺酸钠煌绿增菌液(TTB)中培养18 h～20 h,使用沙门显色培养基及亚硫酸铋琼脂(BS平板)进行选择性培养沙门氏菌的检出效果最好。