白僵菌固态发酵试验研究*

以产孢量为指标,筛选出白僵菌固态发酵的培养基为:90%麦麸+5%玉米粉+5%黄豆粉,原孢粉的产孢量可达244.7亿/g。在25m³发酵罐内进行浅盘固态发酵,培养基的最适起始含水率为60%~65%;最佳通气条件为:前12h不通气,12~48h通气量为1:2,48~144h的通气量为1:1;而温度只需在12~48±8h内进行控制。常温干燥能最大限度地维持白僵菌的活孢率。     

分阶段调控关键因素提高白僵菌固态发酵产孢量

采用分阶段调控关键因素策略,对白僵菌固态发酵过程进行优化.优化后最佳发酵模式为:初始含水量为65％,发酵至48 h将含水量调为68％;发酵至20 h和40h分别适当翻料;0～48h(适应期)发酵温度为25℃,48 ～108 h(快速生长期)发酵温度为24℃,108 ～168 h(稳定期)发酵温度为28℃;同时,48 ～108 h按通气比1∶2通气,0～48 h和108 ～168 h不通气.采用上述关键因素调控策略后,白僵菌固态发酵产孢量达18亿孢子/g(干培养基),与优化前相比产孢量提高约360％,效果显著.     

球孢白僵菌固态罐发酵产孢培养的研究

建立了一套两升圆柱体玻璃发酵罐的固态通风培养装置及通风系统。以球孢白僵菌Bb371为生产菌,培养基220g(82％麸皮 4．5％黄豆粉 4．5％玉米粉 9％稻壳)作为生产原料,接种30ml,23℃培养温度下,分阶段调节通风量和空气湿度,培养5d,产孢量可达到116亿／g干培养基。     

球孢白僵菌Bb174固态发酵产几丁质酶产酶及酶学特征研究

对球孢白僵菌(Beauveria bassiana)Bb174产几丁质酶进行了固态发酵条件及酶学特征的研究.结果表明,以4:1麸皮:蚕蛹粉、蛋白胨1g·L^-1作为产酶最适培养基,在75g培养基中接种3ml液态种子,自然pH下28℃培养2d,酶活可达最高,为126U·g^-1(干培养基).粗酶液的最适反应温度为40℃,最适反应pH5.0,在30～70℃保温1h,得半失活温度48℃.在30～40℃、pH4～6范围内,酶的性质最稳定.根据Lineweaver-Burk作图法,得到该酶的动力学参数K_m为0.52mg·ml^-1,V_m为0.7△E₆₈₀·h^-1.     

固态发酵反应器

关于固态发酵反应器虽然已有多种形式,但真正用于饲料发酵的固态反应器由于工程放大等难题,目前国内外很少有定型产品,从文献报道固态发酵反应器有以下几种类型。 1 浅盘式发酵器此类反应器很早已应用,作为表面培养的抗生素生产工艺中曾广泛用于药厂。其又可分为带盖式、不带盖式及底部有强制通风和无强制通风等形式。虽然浅盘反应品操作简便,产率较高,产品均匀,但因占体积过大,耗费劳动力大,无法机械化操作等缺点,不适宜在工业生产中应用。     

球孢白僵菌Bb174固态发酵产几丁质酶产酶及酶学特征研究

对球孢白僵菌(Beauveria bassiana)Bb174产几丁质酶进行了固态发酵条件及酶学特征的研究．结果表明,以4：1麸皮：蚕蛹粉、蛋白胨1g·L^-1作为产酶最适培养基,在7．5g培养基中接种3ml液态种子,自然pH下28℃培养2d,酶活可达最高,为126U·g^-1(干培养基)．粗酶液的最适反应温度为40℃,最适反应pH5．0,在30-70℃保温1h,得半失活温度48℃．在30--40℃、pH4～6范围内,酶的性质最稳定．根据Lineweaver-Burk作图法,得到该酶的动力学参数Km为0．52mg·ml^-1,Vm为0．7△E680·h^-1．     

固态发酵

本世纪以来,由于人口不断增加,在世界范围内出现了粮食短缺,蛋白质极度不足的现象,这一矛盾在发展中国家变得愈来愈尖锐。本世纪初,科学家曾认为,大规模培养微生物作为人类食物或动物饲料中蛋白的一种直接来源,可根本解决这一矛盾。但由于技术及成本等等一系列问题,这一设想仅在欧洲少数国家中研究,并未真正形成世界性工业生产的规模。自60年代以来,由于发酵技术的迅速发展,如大规模的青霉素通气搅拌发酵普及以及由于谷氨酸、氨基酸发酵业形成,以代谢调控的发酵技术成熟,为大规模生产微生物蛋白奠定     

新型大规模纯种固态发酵研究进展

发酵工业是生物技术产业必要平台,但目前正面临节能减排的巨大压力.固态发酵技术天然具有节水、节能的优势,近年来固态发酵技术已逐渐成为研究的新热点.固态发酵总体发展趋势是菌株从最开始的自然混菌发酵和强化微生物混合发酵逐渐发展为纯种发酵和限定微生物混合发酵;发酵方式也从传统的静态培养发展到培养基的动态培养和气相动态培养;操作方式从批次发酵向连续发酵发展;在好氧发酵依然占主体的情况下,厌氧发酵也呈现了今后和好氧发酵并驾齐驱的潜力.而气相双动态好氧固态发酵技术与连续厌氧固态发酵技术平台的建立为现代固态发酵的广泛应用打下了很好的基础.     

球孢白僵菌Mars发酵牛奶产生紫红色物质的初步研究

球孢白僵菌Mars菌株发酵牛乳能使牛乳呈紫红色。但在查氏培养基、马铃薯葡萄糖培养基和牛乳固态培养基中没有紫红色物质产生。培养温度和pH对Mars菌株在牛乳培养基中产生的紫红色物质有明显的影响。28℃、pH在6．0～7．0的范围内Mars菌株能够产生大量的紫红色物质;对牛乳发酵液进行离心处理后,用不同萃取剂对沉淀进行萃取,最后用紫外分光光度计进行扫描,结果表明：沉淀萃取液在330nm左右有吸收峰且丙酮为最佳萃取刹;上清液在500nm左右有吸收峰。     

Extracellular production of L-glutaminase by alkalophilic Beauveria bassiana BTMF S10 isolated from marine sediment

Beauveria sp. BTMF S10 isolated from marine sediment produced extracellular L-glutaminase. Maximal L-glutaminase yield (46.9U/ml) was obtained in a medium supplemented with 1% (w/v) yeast extract and sorbitol, 9% (w/v) sodium chloride and 0.2% (w/v) methionine, initial pH 9.0 and at 27 ^°C after 108h. This enzyme was inducible and growth-associated.     

Transformation of cinoxacin by Beauveria bassiana

The ability of the fungus Beauveria bassiana ATCC 7159 to transform the antibacterial agent cinoxacin was investigated. Cultures in sucrose-peptone broth were dosed with cinoxacin, grown for 20 days, and then extracted with ethyl acetate. Two metabolites were detected and purified by high-performance liquid chromatography. The major metabolite was identified by mass and proton nuclear magnetic resonance spectra as 1-ethyl-1,4-dihydro-3-(hydroxymethyl)[1,3]dioxolo[4,5-g]cinnolin-4-one and the minor metabolite was identified as 1-ethyl-1,4-dihydro-6,7-dihydroxy-3-(hydroxymethyl)cinnolin-4-one. B. bassiana also reduced quinoline-3-carboxylic acid to 3-(hydroxymethyl)quinoline.     

球孢白僵菌对温室蚜虫的侵染生物学

研究了球孢白僵菌在不同浓度、施菌方式、温度和相对湿度下对温室蚜虫的侵染力以及接种后对蚜虫的侵染速率。结果表明,高(109个孢子.mL-1)、中(108个孢子.mL-1)、低(107个孢子.mL-1)3个浓度剂量对蚜虫都有较强的致病力,且浓度越大,蚜虫的死亡率越高,死亡时间越提前。用浸渍法和孢子浴法接种蚜虫,6 d的累积死亡率分别为100%和31.1%。在测试的3个温度(22、26、30℃)中,26℃时蚜虫侵染力最强,第3天出现死亡高峰,第5天时累积死亡率就达到100%,明显高于22、30℃的处理。相对湿度越大,球孢白僵菌的致病力越强,蚜虫死亡速度越快。在温湿度组合中相对湿度为95%时,温度对白僵菌的侵染力几乎无影响,但影响发病速度,相对湿度低于95%时,26℃的侵染力始终高于22和30℃时的侵染力。通过接种后不同时间段用0.2%百菌清处理蚜虫测定该菌株的侵染速率,结果表明接种后24 h是该球孢白僵菌有效侵染蚜虫的关键时期。     

Modelling Beauveria bassiana horizontal transmission

1 We modelled horizontal transmission of Beauveria bassiana in Colorado potato beetle (CPB) between larval cadavers and soil inhabiting prepupae. 2 The rate of disease transmission, based on the probability of a prepupa contacting sporulating cadavers on the soil surface, is a non‐linear function of cadaver density and also dependent upon temperature. 3 The spatial pattern of cadavers is needed to estimate prepupal contacts with cadavers. Observational field studies determined Johnson distributions to model the spatial pattern of cadavers and prepupae in the field. The model also implicitly incorporates within‐field larval spatial pattern into estimates of field‐level horizontal transmission. 4 Potential for horizontal transmission is higher in simulations using weather data from the warmer year of 1995, than in simulations of the cooler growing season of 1993. 5 Simulations of CPB populations under northern Maine climatic conditions recorded in 1993 and 1995 suggest that horizontal transmission can range from 3 to 24% depending upon the timing of primary infection of larvae in the field. 6 Two simulated sequential applications of B. bassiana targeted at peak first instars resulted in maximum horizontal infection in both years. 7 Sensitivity analysis suggests that horizontal transmission is most sensitive to changes in the proportion of cadavers that sporulate and least sensitive to changes in the time between larval death and the onset of cadaver sporulation. 8 Field validation of the model indicates good prediction of one measure of horizontal infection, the proportion of prepupae which eventually sporulated after being released in controlled field experiments.     

Review on safety of the entomopathogenic fungi Beauveria bassiana and Beauveria brongniartii

The commercial use of entomopathogenic fungi and their products as mycoinsecticides necessitates their registration. Worldwide, several registration guidelines are available, however, most of them focus on similar or even the same safety issues. With respect to the two entomopathogenic fungi, Beauveria bassiana (Bals.-Criv.) Vuill. and Beauveria brongniartii (Sacc.) Petch, many commercial products have been developed, and numerous papers on different biological, environmental, toxicological and other safety aspects have been published during the past 30-40 years. The aim of the present review is to summarise these data. The following safety issues are presented: (1) identity of Beauveria spp.; (2) biological properties of Beauveria spp. (history, natural occurrence and geographical distribution, host range, mode of action, production of metabolites/toxins, effect of environmental factors); (3) analytical methods to determine and quantify residues; (4) fate and behaviour in the environment (mobility and persistence in air, water and soil); (5) effects on non-target organisms (non-target microorganisms, plants, soil organisms, aquatic organisms, predators, parasitoids, honey bees, earth worms and nontarget arthropods); (6) effects on vertebrates (fish, amphibia, reptiles and birds); and (7) effects on mammals and human health. Based on the present knowledge it is concluded that both Beauveria species are considered to be safe.     

防治西花蓟马的病原真菌-白僵菌微菌核的液体培养

微菌核是许多植物病原真菌产生的越冬结构,目前还没有关于白僵菌微菌核的报道。用含不同氮源的介质培养出了2株白僵菌菌株,RSB和SZ21的微菌核。在液体培养条件下,2株菌株不仅能产生典型的芽孢和菌丝,还能产生微菌核。在以玉米粉(RSB和SZ21菌株分别为6.5×102和6.3×102mL-1)为氮源的介质中产生的微菌核浓度高于以大豆粉(RSB和SZ21菌株分别为2.5×102和2.2×102mL-1)为氮源的介质。菌株SZ21产生的芽孢浓度(在以大豆粉和玉米粉为氮源的介质中,分别为17.9×105和7.4×105L-1)高于RSB菌株的芽孢浓度(在以大豆粉和玉米粉为氮源的介质中,分别为14.8×105和6.2×105L-1)。含有硅藻土的微菌核制剂能在真空干燥(<5%湿度)后存活下来,并且对于活力没有显著影响。干燥后的微菌核颗粒在水洋菜培养基上复水和培养后,能够萌发菌丝和产生高浓度的分生孢子。将微菌核颗粒预混到土壤中能引起西花蓟马Frankliniella occidentalis(Pergande)土栖阶段的高死亡率。这是首次报道白僵菌的微菌核的培养,并为用虫生真菌防治土栖昆虫提供了一个新的方法。     

BbRho5对球孢白僵菌生长速率的作用研究

为阐明BbRho5对球孢白僵菌生防潜能的作用,构建了Bbrho5单基因敲除菌株ΔBbrho5,以野生型菌株WT作为对照,在不同培养基上测定菌落生长速率,并测定了菌株对多菌灵胁迫耐受性及对大蜡螟幼虫体壁侵染能力。进一步获取和分析了ΔBbrho5和WT细胞内基因转录组数据。结果表明,BbRho5蛋白功能缺陷显著抑制球孢白僵菌菌丝生长速率,同时微弱影响其多菌灵胁迫抗逆性及生防能力。相较于WT,ΔBbrho5中具有770个差异表达基因(DEGs),其中上调基因395个,下调基因375个。GO分析显示,ΔBbrho5 VS WT中DEGs主要富集于氧化还原酶活力(oxidoreductase activity)和单加氧酶活力(monooxygenase activity)功能。KEGG通路富集结果显示,DEGs主要富集于氮代谢及多种氨基酸代谢通路。在氮代谢通路中富集到7个功能基因,其中有5个上调,2个下调,说明敲除菌株可能采用增强氮源利用及谷氨酸合成以应对Bbrho5缺陷引起的生长迟缓。以上研究结果揭示了球孢白僵菌中小GTP酶BbRho5对球孢白僵菌生长速率具有重要影响,且氮代谢和氨基酸代谢可能为其重要的响应代谢通路。     

蜡蚧轮枝菌和球孢白僵菌的共发酵

【目的】球孢白僵菌(Beauveria bassiana)和蜡蚧轮枝菌(Verticillium lecanii)是国内外目前研究应用最广泛的杀虫生防真菌,欲扩大其防治范围、增强防治效果、降低生防成本。【方法】采用共发酵技术,通过组合菌株产孢能力和杀虫毒力比较试验,确定蜡蚧轮枝菌和球孢白僵菌共发酵的可行性。【结果】蜡蚧轮枝菌L-31和球孢白僵菌Q-55共发酵的最佳配比为1:1时,按10%总量接种于发酵培养液中(培养液按酵母膏5.0 g/L、葡萄糖20.0 g/L、麦芽糖提取物5.0 g/L、KH2PO43.0 g/L、黄小米200.0 g/L,pH 6.5配制),23.0°C±0.1°C恒温静置发酵12 d,共发酵液的含孢量可达1×109CFU/mL以上,杀虫毒力比较强,其对温室白粉虱和菜青虫可同时显效,处理9 d后的致死中浓度LC50分别为2.09×104±0.12 CFU/mL和3.17×105±0.11 CFU/mL,发酵液浓度为1×108CFU/mL时的致死中时间LT50分别为2.11±0.14 d和4.27±0.43 d,温室小区试验校正防效在80%以上,与其各单一菌株发酵液的防效之间存在显著性差异。【结论】通过两株生防真菌的共发酵研究,为杀虫真菌的扩谱增效以及植物害虫的有效防治提供科学依据和有效途径。     

Cloning of a cuticle-degrading protease from the entomopathogenic fungus, Beauveria bassiana

Abstract A Beauveria bassiana extracellular subtilisin-like serine endoprotease is a potential virulence factor by virtue of its activity against insect cuticles. A cDNA clone of the protease was isolated from mycelia of B. bassiana grown on cuticle/chitin cultures. The amino acid sequence of this gene was compared to that of Metarhizium anisopliae Pr1, the only pathogenicity determinant so far described from an entomopathogenic fungus, and proteinase K, isolated from Tritirachium album , a saprophytic fungus. The cDNA sequence revealed that B. bassiana Prl is synthesized as a large precursor ( M _r 37 460) containing a signal peptide, a propeptide and the mature protein predicted to have an M _r of 26 832.