Degradation effects on dichlorvos by a biocontrol strain,Trichoderma atroviride T23

Excessive use of organophosphate pesticides(OP),such as dichlorvos,in farming system poses a threat to human health through potential contamination of environment.To date,biodegradation has been prospected most promising approach to eliminate environmental OP residues.Trichoderma species as a biological control microorganism is often exposed to the chemical pesticides applied in environments,so it is necessary to understand the mechanism of degradation of dichlorvos by Trichoderma.In this study,dichlorvos significantly inhibited the growth,sporulation and pigmentation of T.atroviride T23,and the dichlorvos degradation activity of T23 required the initial induction effect of dichlorvos and the culture conditions,including the nutrient and pH values of the medium.Various changed primary and secondary metabolites released from T23 in the presence of dichlorvos were speculated as the energy and antioxidants for the strain itself to tolerate dichlorvos stress.The results showed that T23 could produce a series of enzymes,especially the intracellular enzymes,to degrade dichlorvos.The activities of the intracellular enzyme generated by T23 were differentially changed along time course and especially relied on initial dichlorvos concentration,ammonium sulfate and phosphate added in the medium.In conclusion,some dichlorvos-induced chemical degradation related enzymes of T23 were proved to be involved in the degradation of dichlorvos.

Trichoderma viride菌生物量测定及其纤维素酶合成特征

利用HPLC法测定Trichodermaviride菌固态发酵曲中的麦角固醇含量。研究了麦角固醇与菌丝体间的关系。该菌固态曲中麦角固醇分离条件以 1∶2 5 (m/v)的丙酮抽提 1 5h为最佳。当固态发酵培养至 69h时 ,曲中的生物量达到最大值 ,为每克干曲中含有 0 5 75 g菌丝体。此时该菌所产生CMC酶和FP酶活力均达到最大值 ,呈现正相关性 ,说明这 2种酶的合成特征均为同步合成型 ,而C1 酶活力高峰滞后 ,出现在 72h。

Cloning of cDNA Fragment of Chitinase Gene from the Mycoparasite Trichoderma atroviride on Armandii Pine Blister Rust

[Objective] The aim of this study was to isolate chitinase gene from Trichoderma atroviride strain SS003. [Method] With the aeciospore wall of armandii pine blister rust as inducer, chitinase gene was induced to express in Trichoderma atroviride cells. The cDNA fragment of chitinase gene was cloned by RT-PCR approach. [Result] The activity of chitinase induced reached 40.17 μg/10 min; and the specific fragment amplified was 834 bp in length and proved to be the fragment of chitinase gene by sequencing and sequence analysis. [Conclusion] The result showed the feasibility of isolating the full length of chitinase gene and its transformation, and further producing chitinase.

绿色木霉(Trichoderma viride)_(867)产壳聚糖酶的发酵工艺条件的优化

系统研究了碳源、氮源、初始pH、培养温度、培养基装液量、接种量和培养时间等因素对绿色木霉867产壳聚糖酶的影响.结果表明,最佳碳、氮源分别为可溶性壳聚糖和蛋白胨,在初始pH5.0,培养温度28℃,培养基装量75 mL/250 mL,接种量6%和培养时间(180 r/min)40 h时最利于产酶.在此基础上通过均匀设计法优化了发酵培养基配方.优化后的培养基配方为:可溶性壳聚糖0.9%,氨基葡萄糖0.5%,蛋白胨0.9%,K2HPO40.016%,CaCl2.2H2O 0.055%.在该条件下,壳聚糖酶活为0.291 u/mL,比原基础培养条件下酶活提高29.9%.

生防木霉SS003菌株(Trichoderma atroviride)的固体发酵工艺研究

为了探索酷绿木霉SS003菌株固体发酵产孢子的较优工艺条件。分别以酷绿木霉SS003菌株为供试菌和以玉米秸秆、麦麸发酵基质,分别进行了固体发酵培养基配方和固体发酵条件的优化筛选,试验设计均采用单因索试验。试验最终检测方法采用血球计数板倍量稀释法镜检孢子量(108个/g)。分别获得了酷绿木霉SS003菌株产孢固体发酵的较优培养基配方和较优固体发酵条件,即80目的玉米秸秆与麦麸配比1∶3,接种1×106个/mL的SS003孢子液,保持含水量55%和充分通气,培养11 d后,酷绿木霉SS003菌株固体发酵产孢量达到最大(76.14×108个/g)。初步探索到绿木霉SS003菌株固体发酵产孢的较优工艺条件,为该菌的深入研发提供了有益的试验数据。

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

绿色木霉(Trichoderma viride)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5 L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50.0 U/mL,210.0U/mL,4.0 U/mL和2.5 U/mL,比摇瓶发酵分别提高了6.7、4.2、19、2.5倍。

木霉属中国新记录种Trichoderma paratroviride

从山东保护地土壤中分离得到木霉菌Y15,结合ITS、tef1-α和形态学特征进行鉴定.结果发现Y15菌株为近深绿木霉（Trichoderma paratroviride）.该菌株在PDA上菌落稠密,老熟后毡状,灰绿色;分生孢子梗主轴长,着生短的二次分枝,再次分枝少,分枝成对或轮生.瓶梗通常2～4个轮状排列,烧瓶形或锥形,直或弯曲;分生孢子亚球形至卵圆形,绿色,光滑.tef1-α序列与近深绿木霉模式菌株S489和S385序列同源性高达99％,在系统进化树中与其亲缘关系也最近.该种在国内首次报道和描述,为木霉属中国新记录种.

绿色木霉(Trichoderma viride)基因组文库构建及其CBH I基因阳性克隆筛选

用绿色木霉核DNA部分酶解片段与噬菌体λEMBL3载体左右臂连接,并用高效价包装蛋白对重组分子进行体外包装,侵染宿主菌后,构建了含1.92×10_6个重组噬菌体的绿色木霉基因组文库。以李氏木霉纤维素酶CBH Ⅰ基因的5′、3′两个末端片段为探针,对所建立的基因组文库作轮回噬菌斑原位杂交,筛选到含绿色木霉CBH Ⅰ基因的阳性克隆7个,随机取其中三个克隆进一步用李氏木霉CBH Ⅰ、CBHⅡ基因的5′、3′四个末端片段探针作交叉斑点杂交,证明本实验确实克隆了很可能为全长的绿色木霉CBH Ⅰ基因,并提示CBH Ⅰ与CBHⅡ基因的末端序列之间不存在同源性。

Study on Fermentation Conditions of Trichoderma aureoviride sp.for the Production of Chitinase

[Objective] The paper was to study the best fermentation conditions of Trichoderma aureoviride sp. for the production of chitinase, thus provide new enzyme source for chitinsse industry. [Method] Using orthognnal experimental design, with the variation of sugar content after enzymatic hydrolysis measured by DNS method as the indicator, the fermentation conditions were optimized. [Result] Taking colloidal chitin as the carbon source and 2% peptone as the nitrogen source with the shaking speed of 170 r/rain, the optimum fermentation conditions of T. aureoviride for the production of chitinase were as follows ： initial pH value of medium, 5.0; inoculation amount, 8% ; bottle volume, 20 ml; 6 d cultivation at 28℃. [Conclusion] The optimum conditions for the production of chitinase were confirmed, which provided basis for the utilization of T. aureoviride.

Trichoderma viride Strains against Vegetable Grey Mold in Greenhouse

[Objective] The paper aimed to study the control effects of live spore preparations of Trichoderma viride strains against vegetable grey mold in greenhouse. [Method] Trichoderma viride strains NW-411 live spore preparations were prepared by solid-state fermentation,106-107 spore/g diluent was made to conduct field control experiment,traits change of cucumber and tomato plants inoculated grey mold were investigated,control effect was calculated. [Result] Cucumber and tomato plants without dilution treatment of T. viride spores could be infected with different changes in trait. T. viride spore preparations had a significant preventive effect on greenhouse cucumber and tomato gray mold,the optimal concentration of spores was in the range of 2.3×10^6-2.3×10^7 spore/g. The incidence of cucumber and tomato plants were reduced to 4.2% and 3.1%,the incidence rate decreased 29.8% and 39.1% compared with plants without treatment,biological control effect was over 87%,and the plant growth can be enhanced obviously. [Conclusion] Live spores preparation of T. viride not only had a significant effect on grey mold,but also significantly enhanced the plants growth in greenhouse,which is a safety and environmental protection biological agent,and worthy to be widely spread in large-scale green vegetable production.

黄绿木霉菌(Trichoderma aureoviride)产纤维素酶研究

通过分离获得一株纤维素分解菌——黄绿木霉(Trichoderma aueroviride),在对其发酵条件的研究中发现:以稻草粉与麸皮为发酵固体培养基的条件下,最适稻草与麸皮比例为3:2或2.5:2.5;最适氮源为氯化铵;最适产酶温度为27～30℃,产酶高峰为发酵4d,通气条件变化,产酶能力变化不明显。液体培养中该菌有较高产量的β-葡萄糖苷酶形成,产酶活性高于绿色木霉As3.3711菌株。

纤维素酶高产菌株Trichoderma atroviride HP35-3原生质体制备及转化

旨在优化深绿木霉(Trichoderma atroviride)菌株HP35-3原生质体制备和转化条件,便于对该菌株进行遗传操作以提高其纤维素酶产量。分别对制备深绿木霉原生质体的菌龄、酶解时间、酶组分及比例和转化条件进行优化。结果显示,利用3mg/m L蜗牛酶、3 mg/m L溶菌酶和3 mg/m L裂解酶酶组分酶解菌龄10 h的菌丝2 h,获得的原生质浓度达到3.5×107个/m L以上,原生质体再生率为61%。利用原生质体进行PEG介导转化,当原生质体浓度为1×108个/m L、外源DNA为5μg时,转化率达到35个转化子/μg DNA。建立的高效原生质体制备及转化体系可用于深绿木霉的遗传转化及菌株改造。

Utilization of winery wastes for Trichoderma viride biocontrol agent production by solid state fermentation

Biocontrol agents are safe and environmental friendly alternatives for pesticides in agriculture application. Trichoderma viride WEBL0703 performed a high level of antagonistic activity toward a broad spectrum of phytopathogens and was determined as a biocontrol agent, which was produced by solid state fermentation using grape marc and wine lees. The maximum yield of T. viride conidia was up to 6.65 × 10^9 CFU/g initial dry substrate （IDS） after 10 d fermentation. As important enzymes for protecting plants from disease, chitinase, β-glucanase, and pectinase yields were 47.8 U/g IDS, 8.32 U/g IDS and 9.83 U/g IDS, respectively. These results show that it is feasible to convert winery wastes to a value-added and environmental friendly biocontrol agent.

Purification and characterization of the kinetic parameters of cellulase produced from wheat straw by Trichoderma viride under SSF and its detergent compatibility

This paper reports the purification and characterization of kinetic parameters of cellulase produced from Trichoderma viride under still culture solid state fermentation technique using cheap and an easily available agricultural waste material, wheat straw as growth supported substrate. Trichoderma viride was cultured in fermentation medium of wheat straw under some previously optimized growth conditions and maximum activity of 398±2.43U/mL obtained after stipulated fermentation time period. Cellulase was purified 2.33 fold with specific activity of 105U/mg in comparison to crude enzyme extract using ammonium sulfate precipitation, dialysis and Sephadex-G-100 column chromatography. The enzyme was shown to have a relative low molecular weight of 58kDa by sodium dodecyl sulphate poly-acrylamide gel electrophoresis. The purified enzyme displayed 6.5 and 55oC as an optimum pH and temperature respectively. Using carboxymethyl cellulose as substrate, the enzyme showed maximum activity (Vmax) of 148U/mL with its corresponding KM value of 68μM. Among activators/inhibitors SDS, EDTA, and Hg2+ showed inhibitory effect on purified cellulase whereas, the enzyme activated by Co2+ and Mn2+ at a concentration of 1mM. The purified cellulase was compatible with four local detergent brands with up to 20 days of shelf life at room temperature suggesting its potential as a detergent additive for improved washing therefore, it is concluded that it may be potentially useful for industrial purposes especially for detergent and laundry industry.

Insertion Mutagenesis of Trichoderma atroviride by Restriction Enzyme-mediated DNA integration

Restriction enzyme-mediated integration(REMI) of DNA has been recently received attention as a new technique for the generation of mutants by transformation in fungi. Trichoderma atroviride strain T23 was transformed with linearized plasmid pV2, conferring resistance to hygromycin B, in the presence of restriction enzyme used to linearize the plasmid. A total of 172 regeneration transformants were detected by successive inoculation for seven times subcultivation on fresh PDA plate containing hygromycin B. The plasmid was integrated stably into the chromosome DNA, which was confirmed by PCR and southern analysis. The difference between 172 transformants and the parent strain was confirmed in colonial color, sporulation and growth rate. The results showed that the significant difference appeared in above mentioned characters between transformants and parent strain is sporulation capability. Transformants TC6, TD5, TE7, TF1 and TK1 produced higher amounts of conidia than the parent strain T23. In addition, transformants TK1and TC6 showed stronger inhibition to the growth rate of the cucumber wilt pathogen (Fusarium oxyporum) in vitro.

Construction of biological control strain of Trichoderma viride and study of their ability to induce plant disease resistance

Plant diseases heavily affct plant growth and crop yield even in modern agriculture. Control its difficult because pathogens mutate frequently, and this leads in frequent breaking of disease resistance in commercial cultivars. The excessive application of chemical pesticides is not only producing pesticide-resistant pathogens, but it is harming the environment threatening the health of human beings. Therefore, the use of biological control agents (BCA) may provide an environmental friendly alternative to chemicals for plant disease control. Hypersensitive response (HR) and systemic acquired resistance (SAR) are the typical expressions of plant defense reactions. Once SAR is established,, the plants exhibits a broad-spectrum of disease resistance against pathogen attack. Researchers have identified elicitor proteins, such as elicitins and harpins, which activate plant defense reactions. It would be useful to explore the possibility of using biological control agents to induce a status of SAR in crop plants. Trichoderma viride is an ubiquitous soil saprophyte and a biological control agent acting by competition for nutrients, antibiosis, and mycoparasitism. If T. viride could be used as a producer and carrier of an elicitor protein, it may be used as a novel BCA specifically active on some plants. To test this possibility, we used cryptogein, a proteinaceous elicitor secreted by Phytophthora cryptogea, to bio-engineering T. viride . The plasmid containing the Crypt gene or its mutated form, was introduced into T. viride genome by using the restriction enzyme mediated integration (REMI) method. The transformed T. viride was able to produce the Crypt protein and to improve disease resistance when the mutants were applied on tobacco plants. In summary our study included: 1. Construction of pCSNTCC and pCSNTCCm plasmids: Crypt gene was mutated by changing the K at position 13 of Crypt into a V (the mutated form was named CryK13V) as described elsewhere. In order allow secretion of the transgenic protein in T. viride cells, a signal sequence of a chitinase gene from Trichoderma (ThChi) was fused to the 5’ end of Crypt and CryK13V. The chimeric genes were placed under the control of trpC promoter in the vector pCSN43. A hygromycin resistant gene was introduced into the vectors, thus obtaining the plasmids pCSNTCC (for Crypt gene) and pCSNTCCm (CrypK13V) . 2. Establishment of a T. viride transformation system:The optimum conditions for T. viride protoplasts isolation and regeneration from were determined. For protoplast isolation, 24 hours-old hyphae of T. viride were digested with 4 mg/mL of Glucanex in phosphate buffer (pH 6.98) for 4 hours at 30 ℃, with a protoplast yield of 4.7×107 colony forming unit/mL. The maximum regeneration rate (14.5%) was obtained in the CM medium containing 0.3 mol/L KCl and 0.3 mol/L inositol. Plasmids pCSNTCC and pCSNTCCm were transformed into the protoplasts of T. viride by a Xho I restriction enzyme-mediated integration, with an efficiency of 1-2 transformants per microgram of DNA. Thirty transformants were obtained, TV-1 to TV-20 for Crypt gene and TV-21 to TV-30 for CrypK13V gene. The presence of the hygromycin resistance gene in the transformants was determined by polymerase chain reactions. The elicitor protein was detected in the culture media by western blot analysis but not inside the cells. The result indicated that the exogenous gene was expressed in T. viride , but the transgenic protein was entirely secreted into the culture media. 3. Expression of Crypt gene in T. viride enhanced plant disease resistance:Tobacco plants (4-6 week-old) were treated with spores of the transgenic or the wild-type T. viride applied to the soil. After ten days the plants or detached leaves were inoculated with Phytophthora parasitica var nicotianae, Alternaria alternata, Pseudomonas syringae pv. tabaci (Pst), or Tobacco mosaic virus (TMV). The lesions caused by TMV were suppressed by the treatment with the transgenic T. viride as compared with the wild-type

Enhancing rice resistance to fungal pathogens by transformation with cell wall degrading enzyme genes from Trichoderma atroviride

Three genes encoding for fungal cell wall degrading enzymes (CWDEs), ech42, nag70 and gluc78 from the biocontrol fungus Trichoderma atroviride were inserted into the binary vector pCAMBIA1305.2 singly and in all possible combinations and transformed to rice plants. More than 1800 independently regenerated plantlets in seven different populations (for each of the three genes and each of the four gene combinations) were obtained. The ech42 gene encoding for an endochitinase increased resistance to sheath blight caused by Rhizoctonia solani, while the exochitinase-encoding gene, nag70, had lesser effect. The expression level of endochitinase but exochitinase was correlated with disease resistance. Nevertheless, exochitinase enhanced the effect of endochitinase on disease resistance when the two genes co-expressed in transgenics. Resistance to Magnaporthe grisea was found in all kinds of regenerated plants including that with single gluc78. A few lines expressing either ech42 or nag70 gene were immune to the disease. Transgenic plants are being tested to further evaluate disease resistance at field level. This is the first report of multiple of expression of genes encoding CWDEs from Trichoderma atroviride that result in resistance to blast and sheath blight in rice.

Using of green fluorescent reporter gene (GFP) to monitor the fate of Fusarium moniliforme mycoparasitized by Trichoderma viride

Fusarium moniliforme Sheld.is a rice pathogenic fungus and causes the disease called Bakanae,which has increasingly damaged rice production in the recent years. Trichoderma spp. has been one of the most widely used biological control agent of plant disease. By geneticaly labelling F. moniliforme with the GFP reporter gene, we have studied the antagonistic action of Trichoderma viride against this pathogenic fungus. The binary GFP reporter vector pCHF3-35S∷GFP was constructed, which carries the gfp gene driven by the CaMv35S promoter. The vector was transformed into F. moniliforme via Agrobacterium.The mycoparasitism of T.viride against F.moniliforme was tested by dual culture and examined with fluorescence microscope. The result of the dual culture showed that the T.viride maintained a strong competitive ability against F. moniliforme , by growing on the top of the pathogen colony. Fluorescence microscope observation indicated that attacked hyphae of F. moniliform were distorted, swollen or broken. This indicate an enzymatic by T.viride to degrade the host cell walls and used the cell contents as a source of nutrients (Fig 1) .

Characterization of purified <i>β</i>-glucosidase produced from <i>Trichoderma viride</i>through bio-processing of orange peel waste

In the present study, solid state fermentation was carried out using orange peel waste to produce β-glucosidase from Trichoderma viride. A locally isolated fungal strain T. viride was cultured in the solid state medium of orange peel (50% w/w moisture) under optimized fermentation conditions and maximum activity of 515 ± 12.4 U/mL was recorded after 4th day of incubation at pH 5.5 and 30℃. Indigenously produced β-glucosidase was subjected to the ammonium sulfate precipitation and Sephadex-G-100 gel filtration chromatography. In comparison to the crude extract β-glucosidase was 5.1-fold purified with specific activity of 758 U/mg. The enzyme was shown to have a relative molecular weight of 62 kDa as evidenced by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The purified β-glucosidase displayed 6 and 60℃ as an optimum pH and temperature respectively.

金属离子对Trichoderma longibrachiatum UN32石斛碱型生物碱产量的影响

石斛碱是传统植物金钗石斛(Dendrobium nobile)的特征药用活性成分,结构上属于石斛型生物碱(dendrobine-type total alkaloids,DTTAs)。石斛碱的积累受周期和环境的影响,仅从植物中获取难以满足需求。该研究的对象Trichoderma longibrachiatum UN32是实验室前期通过复合诱变获得的产DTTAs的正突变株。为提高UN32的生物碱产量,使产物表达水平趋于稳定,该研究从UN32发酵培养基中无机盐的添加量出发,利用响应面法确定金属离子的最优配比。根据Plackett-Burman法设计筛选出对响应结果影响程度最显著的3个因素分别为Zn^(2+)、Cu^(2+)、Fe^(2+),其最佳添加时间和添加浓度分别是Cu^(2+)0.5 mmol/L(第6天)、Fe^(2+)0.4 mmol/L(第6天)和Zn^(2+)0.7 mmol/L(第3天)。响应面统计的结果显示,当模型响应值生物碱量达最大值时,Cu^(2+)、Zn^(2+)和Fe^(2+)的浓度分别为0.54、0.69、0.39 mmol/L,此时生物碱产量达到(317.36±6.48)μg,比初始培养基提高了72.68%。综上,金属离子及其不同组合对菌株UN32生物碱的积累具有促进作用,该方法具有较好的开发价值和应用前景。