Properties of uracil transport by vegetative mycelium of Trichoderma viride

The transport of radioactively labelled uracil into submerged mycelium of T. viride was measured by means of a membrane filtration technique. It was found to be time-dependent (up to 90 min) and concentration-dependent (up to 8 mmol l-1). Its concentration dependence was biphasic and consisted from the saturatable part (at the uracil concentration below 0.2 mmol l-1) with KM = 0.08 +/- 0.02 mmol l-1 and Vmax = 1.74 +/- 0.3 nmol (mg dry wt.)-1 h-1, and from the region at higher uracil concentration which showed only a weak saturatability with the substrate. The transport measured in the saturatable part of the curve was also pH- and temperature-dependent. The optimal pH was between 5.4 and 6.4 and the optimal temperature was at 37 degrees C. The activation energy of 54 kJ mol-1 and the temperature quotient of Q10 = 2.1 could be calculated from the temperature dependence. The entry of uracil was in part inhibited by nucleobases and their analogues, nucleosides, nucleotides and amino acids. The inhibitors had similar inhibitory efficiency about 50% at 0.2 mmol l-1. 3,3',4',5-tetrachlorosalicylanilide (TCS), the uncoupling agent, significantly inhibited the uracil transport, but its inhibitory efficiency decreased upon increasing the uracil concentration. Ionophore antibiotics valinomycin and monensin also inhibited the uracil transport. Inhibitors of RNA-polymerase, rifamycin and rifampicin were without effect. The results suggest that at low uracil concentrations (below 0.2 mmol l-1), its transport is mediated by a carrier and is driven by the electrochemical potential of protons. At higher uracil concentrations, the transport may be driven by the concentration difference of uracil with the contribution of the protonmotive force. It is feasible that inhibitors of uracil transport tested exert their inhibition by the dissipation of the driving force rather than by the direct competition with the substrate-binding site.     

绿色木霉木聚糖酶的纯化和性质

绿色木霉木聚糖酶经分离纯化后,获得三个组分木聚糖酶,称为XⅠ,XⅡ和XⅢ,它们最反应温度分别为60℃、60℃、50℃,pH分别为5．5、5．0、0、4．5,pⅠ分别为XⅠ3．8,XⅡ3．4,XⅢ3．6。半失活温度分别为XⅠ37℃,XⅡ44℃,XⅢ40℃。     

绿色木霉代谢产物的植物毒性研究

通过人工固体培养和液体发酵研究发现绿色木霉的代谢产物对植物的幼苗生长有抑制作用,其代谢产物大量分泌到它所生长的环境中,在不同的营养基质中其代谢产物的抑制作用有差异．     

一个抗真菌蛋白在绿色木霉中的分泌表达

AFP(antifungalprotein)是在丝状真菌巨大曲霉 (AspergillusgiganteusMDH18894 )中分泌的一个抗真菌蛋白。其mRNA含长度为 4 30bp的开放阅读框 ,编码 94个氨基酸的AFP前体 ,而成熟的AFP为 5 1个氨基酸的多肽。根据推测 ,在巨大曲霉中 ,AFP前体可能经两步剪切去除前导序列 (4 3个氨基酸 ) ,并最终形成具有抗真菌活性的成熟AFP ,已有报道证实 ,在另一种丝状真菌绿色木霉 (Trichodermaviride)基因组中存在一个类似AFP基因但不表达的序列 ,该序列与没有内含子的AFPcDNA序列完全一样。为了解巨大曲霉AFP基因可否在绿色木霉中表达 ,将AFP基因开放阅读框插入真菌表达载体trpC基因的启动子和终止子之间 ,并成功的转化了绿色木霉。SDS PAGE和Western印迹分析表明 ,绿色木霉转化子分泌表达了具有抗真菌活性的成熟AFP。为研究在绿色木霉中分泌表达具有重要应用价值的异源真核蛋白质打下了基础。     

Biomass production from Trichoderma viride in nonconventional oat medium

Oatmeal, an alternative, renewable, and low‐cost substrate, was used for the production of Trichoderma viride spores by submerged fermentation. The nonconventional oat medium was only supplemented with potato peptone, which is a green source of nitrogen for the microorganism. Because particles are suspended in the nonconventional oat medium, the characterization was based on viscosity, average particle diameter, size distribution, and porosity of the particles. Because of the complexity of the fungal biomass extraction, the dry weight and protein content were used as methods for quantifying the growth of T. viride. The inversion between the proportion of mycelia and spores was captured in the microscopic image analysis during the fermentation process. After 60 h, spores began to appear, accounting for most of the form present at 120 h of fermentation. The decrease in pH and the increase in glucose concentration during fermentation indicate that glucan hydrolysis occurs and that glucose is released into the medium. The potential for industrial applications of submerged fermentation with oats for biomass production of T. viride is noted in the results. This simple and easily controllable process has several advantages, including the use of low‐cost substrates for the propagation of a microorganism that is widely used in scientific and commercial settings. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

绿色木霉在啤酒糟基质上产纤维素酶条件的优化

对绿色木霉接种到啤酒糟固态发酵产纤维素酶的培养基和培养条件进行优化,考察发酵物料起始含水量、发酵时间、起始pH值等发酵条件,以及啤酒糟培养基中添加麸皮、氮源种类对产酶的影响。结果表明,以啤酒糟为发酵基质接种绿色木霉生产纤维素酶是可行的。经单因素和正交试验获得最适固态发酵的培养条件为:起始pH 5~6,培养温度28~30℃,发酵4 d;最佳发酵培养基组合为:麸皮比例30%,培养基起始含水量50%,(NH₄)₂SO₄添加量为2.0%~2.5%。     

pH控制下绿色木霉（Trichoderma viride）流加发酵生产纤维素酶

绿色木霉（Trichodermaviride）在pH控制发酵条件下,采用流加葡萄糖发酵策略,可显著提高综合滤纸酶活力（FPA）和内切酶（endo—β—1,4-glucanase,EG）、外切酶exo—β-1,4-glucanase,CBH）、纤维二糖酶（cellobiase,CB）酶活。在5L发酵罐中采用pH控制和流加葡萄糖工艺,可提高CB酶含量,改变酶组分之间的比例,使得FPA、EG、CB和CBH酶活分别达到50．0U／mL,210．0U／mL,4．0U／mL和2．5U／mL,比摇瓶发酵分别提高了6．7．4．2、19、2．5倍。     

绿色木霉菌合成金纳米颗粒的可能性及影响因素

【背景】金纳米颗粒(AuNPs)凭借其稳定性、抗氧化性能和生物相容性在许多领域有广泛应用。目前关于微生物合成金纳米颗粒的研究较少。【目的】对微生物合成金纳米颗粒的可能性以及影响因素进行探究,有利于揭示具体的合成机制,发现AuNPs的特性以及合成位置与菌丝和影响因素的关系。【方法】以绿色木霉菌(Trichoderma viride)菌株(GIM3.141)为菌种资源,通过目视检测法、紫外可见分光光度计、X射线衍射和透射电镜等手段分析合成AuNPs的特征。探讨细胞内生物合成金纳米颗粒(AuNPs)的可能性,研究生物量、初始金离子浓度、溶液pH等因素对细胞内合成AuNPs的影响。【结果】X射线衍射分析表明AuNPs以金纳米晶体形态存在。透射电镜分析表明AuNPs主要位于细胞壁膜间隙,一小部分附着在细胞壁上。紫外可见分光光度计分析表明,金纳米颗粒粒径随着生物量添加量和溶液pH的升高而变小,随着初始金离子浓度的升高而变大。【结论】非致病性真菌绿色木霉菌可以在细胞内合成AuNPs,其中包括伪球形、三角形、四边形和六边形等多种形状,粒径范围从几纳米到三百纳米,为大规模、低成本、无污染地生物合成纳米颗粒工艺提供了菌种资源。     

绿色木霉对小球藻细胞壁的酶解作用

【目的】探讨绿色木霉分泌液能否分解小球藻细胞壁。【方法】用海藻酸钠和氯化钙固定绿色木霉,游离绿色木霉和固定化绿色木霉分别培养一段时间,离心培养液,用分光光度计法检测上清液中纤维素酶活性。在上清液中加入浓缩的小球藻悬浮液,用显微镜计数细胞壁破碎的小球藻。【结果】绿色木霉能同时分泌内切葡聚糖酶、外切葡聚糖酶及β-1,4葡萄糖苷酶3种纤维素酶,其中外切葡聚糖酶活性最高。固定化绿色木霉反复使用5次后,分泌的纤维素酶活性能保持到初次的67.4%。市售纤维素酶、游离绿色木霉、固定化绿色木霉初次及第5次分解小球藻细胞壁的效率分别为47.3%、86.5%、81.5%、52.1%。【结论】市售纤维素酶、游离绿色木霉、固定化绿色木霉都能分解小球藻细胞壁,其中固定化绿色木霉因可重复使用,具有潜在的应用前景。     

Studies on Biological Control of Postharvest Rot in Yams (Dioscorea spp.) using Trichoderma viride

From in vitro and in vivo screening tests for antagonism by isolates of Trichoderma against postharvest pathogens of yams (Dioscorea spp.), an isolate of Trichoderma viride Pers. ex S.F. Gray was selected as the most promising candidate for the biocontrol of postharvest rot of yams. Inoculation of white yam (Dioscorea rotundata Poir.) with conidiaspores of T. viride and subsequent storage of the tubers under the ambient environment conditions of a traditional yam barn, resulted in a drastic reduction in the frequency of occurrence of the normal tuber surface mycoflora over a 4‐month (December‐April) storage period. Trichoderma viride on the other hand, maintained a high frequency of occurrence during the same period. Furthermore, whereas up to 52.0%) rot was found among groups of tubers that were artificially inoculated with the postharvest pathogens of yams, Aspergillus niger Van Tiegh., Botryodiplodia theobromae Pat. or Penicillium oxalicum Currie and Thom, and also the group that was not inoculated with any organism (control), among groups of tubers that were inoculated with T. viride the rot was either totally suppressed or only a low percentage of rot occurred. The significance of the findings is discussed in relation to yam storage especially by farmers with limited resources.     

利用木霉与根霉两步发酵秸秆制备L-乳酸研究

以秸秆为原料进行生物转化大量制备有机酸意义重大.在秸秆汽爆法预处理的基础上,以绿色木霉为菌种转化制备秸秆糖,对降解单糖接种米根霉进行二次发酵制备L-乳酸.试验结果表明,第一步绿色木霉固态培养制备纤维素酶时,控温30℃、通气0.12L/(L.min)、发酵40h后制备干曲,后按10g干曲/L汽爆液的配比进行55℃酶解36h,五、六碳糖累积浓度达到86g/L.第二步米根霉发酵时,控制温度32℃、通气0.4L/(L·min)、转速450r/min,发酵48h,最终产L-乳酸累积浓度为81.6g/L.秸秆制备L-乳酸的两步发酵法发酵工艺具有推广价值.     

Enhanced rhizosphere competence of Trichoderma viride grown in solid state fermentation on corn cob residue

Abstract

A process has been disclosed in the present investigation for the production of a formulation of mycopesticide with enhanced shelf life of Trichoderma viride. A comparative evaluation of talc-based and corn cob formulations in the form of seed coat and soil treatment brought to light that the soil treatment significantly enhanced the plant growth performance of pea, green gram and pigeon pea. Among the two formulations, the corn cob formulation proved to be better as the rhizosphere competence of corn cob formulation was superior to that of talc formulation. Our results suggest that corn cob residue is a better substrate owing to high cellulase and spore production. Solid-state fermentation in batch cultures. The biocontrol ability of both formulations was tested against Fusarium in Cajanus species and it was found that the disease incidence was reduced by 86% when the corn cob formulation was given as soil application.     

Alleviation of Fusarium oxysporum induced oxidative stress in wheat by Trichoderma viride

Trichoderma viride (Pers.) pre-inoculated wheat seedlings infected with Fusarium oxysporum Schlecht. (co-stressed) did not show wilting symptoms compared to Fusarium infected seedlings. Antagonistic activity of T. viride could be demonstrated against Fusarium infection by dual culture experiment. After seven days post infection, morphological and physiological parameters such as, root and shoot length, fresh and dry weight, relative water content, total soluble protein, total chlorophyll and carotenoid contents were observed to be increased in co-stressed compared to Fusarium infected seedlings. Accumulation of hydrogen peroxide was enhanced in Fusarium infected tissues compared to co-stressed. Trichoderma mediated activation of antioxidant enzymes such as, catalase, guaiacol peroxidase, ascorbate peroxidase, superoxide dismutase in co-stressed seedlings indicated their involvement in enhanced resistance against Fusarium infection, which is suggestive of playing crucial role in mitigating cellular toxicity developed due to excess H₂O₂. Thus, Trichoderma pre-inoculation protected wheat against Fusarium infection by stabilising oxidative stress.     

木霉几丁质酶基因克隆及植物转化载体构建

利用PCR技术从绿色木霉LTR-2(Trichoderma virede)基因组DNA中扩增到一段序列,测序结果表明,该编码基因片段大小为 1 508 bp,其中包括一个 1 459 bp的开放阅读框,起始密码子位于 45 bp,终止密码子位于 1 501 bp,共编码氨基酸 424 个.在Genbank中进行序列比对,发现该序列同已发表的Trichoderma viride 42 ku几丁质酶氨基酸序列具有99%的同源性.将该片段同pCAMBIA1300中的35S启动子和35S-polyA终止子连接后,插入载体pCAMBIA1302多克隆位点中,构建成植物转化载体,最后将构建好的载体pCHI1302-42通过转化导入根癌农杆菌LBA4404中,为进一步构建转基因植物奠定了基础.     

Biocontrol of yellow disease of Brassica campestris caused by Fusarium oxysporum with Trichoderma viride under field conditions

Trichoderma viride was selected from three species of Trichoderma and was applied to the soil for biocontrol of yellow disease of Brassica campestris caused by Fusarium oxysporum. The population density of F. oxysporum averaged 10³ to 10⁴ cfu/g of soil in eight greenhouses. Moreover, the results of application of T. viride showed that the yellow disease of the first, second, and third crops in greenhouse A was 33%, 48%, and 35% in control plot, while it was 1.4%, 11.5%, and 3.0% in T. viride plots, respectively. Also in greenhouse B, the yellow disease of the first crop was inhibited by T. viride compared with control. However, the suppressive effect of T. viride declined on the second crop. Therefore, the third crop was not cultured in greenhouse B. The population density of T. viride in greenhouse A, in which yellow disease was successfully controlled, was higher than that in greenhouse B.     

Addition of aluminum oxide microparticles to Trichoderma viride My preculture enhances cellulase production and influences fungal morphology

Morphological engineering techniques have recently become popular, since they are used to increase the production of a variety of metabolites and enzymes when fungi are grown in submerged cultures. This study aimed to facilitate cellulase production by adding aluminum oxide to Trichoderma viride My precultures.

The results showed that the highest cellulase activity was achieved when aluminum oxide at 10 g/L was used, and the activities of cellulase for filter paper and endoglucanase activity assays increased from 519.11 to 607.35 U/mL by 17.1%, and from 810.08 U/mL to 917.59 U/mL by 13.3%, compared with the control, respectively. Addition of aluminum oxide decreased the size of T. viride My pellets and increased the final pH. The changes in pellet diameter after the addition of different concentrations of aluminum oxide were fitted using a modified exponential decay model, which could precisely predict the pellet size by controlling aluminum oxide concentration.

The optimum concentration of microparticles, and therefore pellet size, could significantly improve cellulase production, which is an encouraging step towards commercial cellulase production.

     

两种不同来源的绿色木霉降解木质纤维素研究

利用纤维素酶高产菌绿色木霉Trichoderma viride降解木质纤维素是实现废料资源化的重要手段。本研究选取来自不同生境的两株T. viride,分别以玉米秸秆和甘草药渣为基质,测定两者滤纸纤维素酶（filter paper cellulase,FPase）活性和还原糖产量。从时间、温度、水分、pH 4个方面比较两株T. viride的环境适应性和不同基质的差异性。结果表明,以玉米秸秆为基质,T. viride XJ最适初始料液比为1:4-1:5.5,T. viride AG最适初始料液比为1:5-1:5.5。初始料液比1:5.5时,T. viride AG产FPase活性显著高于T. viride XJ。两株T. viride最适发酵温度均为28℃,各温度处理下不同菌株间无显著差异。两株T. viride均表现为还原糖消耗。以甘草药渣为基质,T. viride XJ最适初始料液比为1:2-1:2.5,T. viride AG最适初始料液比为1:3-1:3.5。料液比高于1:3,T. viride AG产FPase活性显著高于T. viride XJ。T. viride AG最适发酵温度为28℃,T. viride XJ最适发酵温度为23-28℃。温度低于28℃,T. viride XJ产FPase活性显著高于T. viride AG。两株T. viride均表现为还原糖积累。两株T. viride最适初始pH均为6-7,最适发酵时间均为3d。最优发酵条件下FPase活性：T. viride AG>T. viride XJ。对T. viride产FPase诱导能力：甘草药渣>玉米秸秆。变差分解表明两株T. viride产FPase活性差异主要源于菌株对生境的生态适应。比较分析菌种来源、基质类型、环境条件对T. viride发酵效果的影响,将有助于该菌大规模应用性研究。