Secretome analysis of thermophilic mould Myceliophthora thermophila cultivated on rice straw and hydrolysis of lignocellulosic biomass for bioethanol production

Abstract

Myceliophthora thermophila encodes for large number of carbohydrate-active enzymes (CAZymes) involved in lignocellulosic biomass degradation. The mould was grown on rice straw in solid state fermentation at pH 5.0 and 45?°C that produced high levels of cellulolytic and xylanolytic enzymes i.e. 2218.12, 515.23, 478.23, 13.34?U/g DMR for xylanase, CMCase, FPase and β-glucosidase, respectively. The secretome analysis of M. thermophila BJAMDU5 by mass spectroscopy, described 124 different proteins with majority of CAZymes consisting of glycosyl hydrolases (GH), lytic polysaccharide mono-oxygenases (LPMO), carbohydrate esterases (CE) and polysaccharide lyases (PL). Furthermore, the enzyme cocktail of the mould was evaluated for hydrolysis of steam treated rice straw that produced 184.59?mg/g substrate reducing sugars after 24?h, which was used for production of bioethanol by using fast fermenting yeast Saccharomyces cerevisiae resulting in high production of bioethanol.     

Encapsulation of Trichoderma harzianum conidia as a method of conidia preservation at room temperature and propagation in submerged culture

The objective of the present work was to develop a method for the preservation of T. harzianum conidia at room temperature and the immobilised conidia propagation in submerged culture. This was accomplished by immobilising the strain in sodium alginate capsules (white capsules) and subsequently propagating them in a column bubble reactor (green capsules). Three capsule diameters were tested (micro, medium and large capsules), which were produced by emulsion internal gelation and dripping methods. Tested variables were the immobilised conidia propagation in submerged culture for free conidia production, the immobilised conidia viability throughout the time (two years), the resistance of the encapsulated conidia to the UV irradiation of short and long wavelength, and the antagonistic effect of the encapsulated T. harzianum against four phytopathogenic fungi. It was found that the medium capsules (1.5?±?0.3?mm) favoured the massive production of released conidia in submerged culture and that the higher the density of conidia per capsule, the greater the protection against the ultraviolet irradiation. Regarding the conidia preservation in calcium alginate, a viability loss of around 30% was observed two years after storage at environmental temperature in both white and green capsules; along the two years that the viability of conidia was analyzed, the purity of the formulation was corroborated. The results presented here show the efficacy of the green and white capsules for T. harzianum preservation at room temperature for a long period of time.     

哈茨木霉几丁质酶cDNA基因的克隆及在酿酒酵母中的表达

为研究哈茨木霉 (Trichodermaharzianum)的生物防治机制并获得与生物防治相关基因 ,通过构建哈茨木霉菌丝生长期的cDNA文库及对部分表达序列标签序列的测定与生物信息学分析 ,成功获得了哈茨木霉几丁质酶v(ChiV)基因的全长cDNA序列。该基因的编码框长度为 1194bp ,编码 397个氨基酸 ,理论分子量为 4 4kD。将该基因构建到酿酒酵母诱导型表达载体pYES2上 ,转化到酿酒酵母H15 8菌株中 ,通过Northern杂交检验后 ,确定该基因在酿酒酵母转录水平上表达。在 β_半乳糖诱导下 ,转化子在培养 6 0h时产生的酶活活性最高 ,几丁质酶V最适活性温度为 37℃ ,在pH 6和pH 8时活性较高。     

Isolation and selection of cellulolytic fungi from palm oil mill effluent

Cellulolytic fungi, 34 strains, were isolated from samples taken from palm oil mill residues and effluent, and high cellulase producers selected in comparison with nine known reference strains. Although 13 isolates showed good filter paper distintegration within 14 days, only eight isolates exhibited clearing zones around their colonies on carboxymethylcellulose (CMC) agar medium. Quantitative cellulase activity measurements, using CMC as carbon source, selected three of the eight isolates as potential cellulase producers. Using dried palm oil mill condensate as carbon source, only one of the isolates (F 11) showed similar results on both carbon sources. During media optimization for CMCase production, a four-fold increase from 0.058 to 0.275 U/ml was obtained using a medium, containing 0.1% (v/v) Tween 80 0.02% (w/v) NH₄NO₃, 0.025% (w/v) proteose-peptone and 0.1% (w/v) CMC dissolved in undiluted condensate from the sterilization of oil palm bunches, with an initial pH of 5.5.     

Desorption of zinc by extracellularly produced metabolites of Trichoderma harzianum, Trichoderma reesei and Coriolus versicolor

AIMS: To determine the role of fungal metabolites in the desorption of metals. METHODS AND RESULTS: Desorption of Zn from charcoal by three different fungi was compared against metal desorption with reverse osmosis water, a 0.1% Tween 80 solution and a 0.1 mol l(-1) CaCl(2) solution. All three fungal filtrates desorbed three times more Zn than either 0.1% Tween 80 or 0.1 mol l(-1) CaCl(2). Metal chelator production in Trichoderma harzianum and Coriolus versicolor was constitutively expressed while chelator production in Trichoderma reesei was induced by Zn. The presence of Zn inhibited the production of metal chelators by C. versicolor. Only C. versicolor was found to produce oxalic acid (a strong metal chelator). All fungi caused a marked decrease in pH, although this was not enough to explain the increased desorption of the metals by the different fungal filtrates. CONCLUSIONS: Metal chelation via organic acids and proteins are the main mechanisms by which the fungal filtrates increase zinc desorption. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study explain why plants inoculated with T. harzianum T22 take up more metal from soil, than noninoculated plants while metabolites produced by fungi could be used for metal leaching from contaminated soils.     

Trichoderma harzianum antagonistic activity and competition for seed colonization against seedborne pathogenic fungi of sunflower

This study investigated the antagonistic effects of Trichoderma harzianum isolate (TRIC8) on mycelial growth, hyphal alteration, conidial germination, germ tube length and seed colonization by the seedborne fungal pathogens Alternaria alternata, Bipolaris cynodontis, Fusarium culmorum and F. oxysporum, the causes of seedling rot in over 30% of sunflowers. The antagonistic effect of TRIC8 on mycelial growth of pathogens was evaluated on dual culture that included two inoculation assays: inoculation of antagonist at 48 h before pathogen (deferred inoculation) and inoculation at the same time with pathogen (simultaneous inoculation). TRIC8 inhibited mycelial growth of the fungal pathogens between 70·67 and 76·87% with the strongest inhibition seen with deferred inoculation. Alterations in hyphae were observed in all pathogens. Conidial germination of F. culmorum was inhibited by most of the fungal pathogens (38·28%) by TRIC8. Inhibition of germ tube length by the antagonist varied from 31·83 to 37·67%. In seed colonization experiments, TRIC8 was applied in combination with each pathogen to seeds of a sunflower genotype that is highly tolerant to downy mildew. Seed death was inhibited by TRIC8 and the antagonist did not allow growth of A. alternata, B. cynodontis and F. culmorum on seeds and inhibited the growth of F. oxysporum at the rate of 58·32%.     

哈茨木霉的培养及其对烟草疫霉生长的抑制研究

哈茨木霉是一类重要的植病生防因子。哈茨木霉TH-1分别在PDA培养基、麦芽糖培养基、查氏培养基和琼脂培养基上培养均能产孢,其中PDA培养基为最适培养基。PDA培养基上,菌丝生长适宜温度27．5℃～35℃,最适温度32．5℃,产孢最适温度27．5℃。菌丝生长适宜pH值为3～7,产孢适宜pH值为5-9,生长与产孢最适pH值为5。光照对菌丝生长影响不大但明显影响菌株的产孢数量,光照时间越长产孢量越大。对峙培养试验表明TH-1明显抑制疫霉菌的生长速率,其无菌滤液明显抑制烟草疫霉菌游动孢子的萌发,并抑制游动孢子芽管的伸长,TH-1对游动孢子萌发的相对抑制率为12．7％,对芽管生长长度的相对抑制率为63．1％。水解酶平板活性测定显示,TH-1产生β-1,3葡聚糖酶与纤维素酶,从而使烟草疫霉菌细胞壁的消解,产生非挥发性抗生素抑制烟草疫霉菌孢子萌发,但对菌丝生长影响不大。     

哈茨木霉超氧化物歧化酶基因克隆与特性分析

构建了哈茨木霉菌丝的cDNA文库,并获得了3298条ESTs序列,对哈茨木霉(Trichoderma harzianum)ESTs序列本地数据库进行tBlastn检索,获得了哈茨木霉超氧化物歧化酶cDNA序列。cDNA序列全长751 bp,开放阅读框465bp,编码154个氨基酸组成的多肽,蛋白分子量为15.7kD。BlastP同源性分析表明该基因与麦角真菌(Claviceps purpurea)相似性最高为86%;与解脂耶氏酵母菌(Yarrowia lipolytica)相似性最低为72%。三级结构预测表明,其活性中心可能与His47,His49,His64,His72,His81,His121,D84位点有关,并构成其活性中心骨架。     

重寄生菌哈茨木霉的研究及其在植病生防中的应用

综述了国内外对哈茨木霉菌产生的抑菌活性物质的研究概况,包括几丁质酶、葡聚糖酶和蛋白酶在内的细胞壁降解酶和抗生素,以及哈茨木霉菌生防产品的应用现状,并提出了其在植病生防中的研究展望。     

Isolation and characterization of protease overproducing mutants of Trichoderma harzianum

Several Trichoderma strains have been reported to be effective in controlling plant diseases, and the action of fungal hydrolytic enzymes is considered as the main mechanism involved in the antagonistic process. Strain Trichoderma harzianum T334 is a potential biocontrol agent against plant pathogenic fungi with the ability to produce low levels of proteases constitutively. To improve its fungal antagonistic capacity, mutagenetic program was undertaken for the construction of protease overproducing derivates. The mutant strains were obtained by means of UV-irradiation and were selected for p-fluorophenyl-alanine resistance or altered colony morphology. It was revealed by means of specific chromogenic protease substrates that both trypsin-like and chymotrypsin-like protease secretion was elevated in most of the mutant strains. The profiles of isoenzymes were different between the mutants and the wild-type strain, when examined by gel filtration chromatography. Certain mutants proved to be better antagonists against plant pathogens in in vitro antagonism experiments. This study suggests the possibility of using mutants with improved constitutive extracellular protease secretion against plant pathogenic fungi.     

Comparison of aerial and submerged spore properties for Trichoderma harzianum

Abstract Spores produced by aerial mycelium of Trichoderma harzianum PI, a potential biocontrol agent, showed both higher UV-resistance and longer viability after storage than those produced within liquid media ('submerged' spores). Aerial spores were produced in clusters, had a thick outer wall, and few organelles. Trehalose content was significantly lower than in submerged spores. Conversely, submerged spores were mostly collapsed, not clustered and larger than aerial spores. They had many cytoplasmic organelles and a thinner outer wall. These spores were hydrophilic, while aerial ones were highly hydrophobic. On analysis, the latter was related with the presence of a single major low molecular mass protein (< 14 kDa). This protein was nearly absent in extracts from walls of submerged spores but was found in the extracellular medium. An involvement of the outer wall layer in the resting state of T. harzianum spores is proposed.     

哈茨木霉发酵液中肽类物质对豇豆根瘤结构和功能的影响

通过树脂吸附、离子交换、薄层层析、高效液相色谱系统等分离、纯化方法,从哈茨木霉(Trichoderm aharzianum)T2-16菌株发酵液中分离得到一种对豆科作物生长具促进作用的物质,通过质谱等方法鉴定为肽类物质。为探明该活性物质对豆科作物的促生机制,用该活性物质对豇豆种子浸种处理后,进行盆栽实验,通过对盆栽实验中根瘤结构与固氮活性变化的研究,结果显示,该活性物质可增加根瘤侵染组织的面积和根瘤细胞中类菌体的数量,降低根瘤细胞液泡化程度,促进根瘤中公共细胞周膜较多形成,加快类菌体的发育成熟,提高根瘤豆血红蛋白的含量,从而提高根瘤的固氮活性。     

Proteins Related to the Biocontrol of Pythium Damping-off in Maize with Trichoderma harzianum Rifai

Induced resistance has been evidenced as one of mechanisms of Trichoderma to control plant diseases, however, no study showed the change of host proteomics in Trichoderma-induced resistance of maize against damping-off caused by Pythium ultimum Trow. The mechanism of Trichoderma harzianum Rifai for controlling maize seedling disease caused by Pythium ultimum Trow was investigated firstly by proteome technique and the result suggested that T. harzianum strain T22 was not only able to promote seedling growth but also protein accumulation. One-dimensional electrophoresis assay showed that more bands appeared on the gel with T22 or T22 combined with P. ultimum （T22 ＋ P. ultimum） treatment than with other treatments. Enzyme assay showed that two chitinases of the root sample were more activated in the treatments with T22 than in the other treatments without T22. Proteins in the seedling roots from the various treatments were separated through protein extraction and 2-D electrophoresis technique. In the seedlings produced from the T22-treated seeds, there were 104 up-regulated proteins and 164 down-regulated proteins relative to the control, and 97 and 150, respectively, aftel treatment with T22 ＋ P. ultimum; however, with P. ultimum alone the values were much lower than with the other two treatments. The correlation coefficient values were 0.72, 0.51 and 0.49 for the comparison of protein spot distribution on gel among control with T22, P. ultimum and T22 ＋ P. ultimum, respectively. So it seemed that P. ultimum infection was more effective than T22 in interfering with the host proteome profile. Furthermore, analysis with MALDITOF-MAS showed that some important proteins associated with defensive reactions were identified in T22 or T22 ＋ P. ultimum treatments, including endochitinase, pathogenesis-related protein PRMS （pathogenesis-related maize seed）, GTP-binding protein, isoflavone reductase and other proteins related to respiration. All those proteins are probably part of the network of resistance or development-related proteins. Interestingly, P. ultimum treatment resulted in elimination of pathogenesis-related protein PRMS on gel, and therefore damping-off could be in part attributed to inhibition of the expression of this protein by P. ultimum infection. Some unknown proteins are also related to the defensive reaction of the host.     

Regulation of 36-kDa beta-1,3-glucanase synthesis in Trichoderma harzianum

The effect of carbon sources on the level of beta-1,3-glucanases in the culture filtrates of Trichoderma harzianum (Tc) was investigated. Enzyme activity was detected in all carbon sources, but highest levels were found when laminarin and purified cell walls were used. Three isoforms of beta-1,3-glucanase were produced during growth of the fungus on purified cell walls. Two isoforms were produced on chitin, chitosan, N-acetylglucosamine and laminarin, while only one was detected when the fungus was grown on cellulose and glucose. A 36-kDa beta-1,3-glucanase (GLU36) was secreted from T. harzianum (Tc) grown on all carbon sources tested as demonstrated by Western blot analysis. We found that a significant increase in the level of GLU36 in the culture filtrate follows glucose exhaustion, suggesting that this enzyme is controlled by carbon catabolite repression.     

Seed biopriming with novel strain of Trichoderma harzianum for the control of toxigenic Fusarium verticillioides and fumonisins in maize

Fusarium verticillioides is one of the most important fungal pathogens in maize causing both pre- and post-harvest losses and also capable of producing Fumonisins. In the present study attempts have been made for screening potential T. harzianum from native rhizosphere and to study its effect on Fusarium ear rot disease, fumonisin accumulation in different maize cultivars grown in India. Eight isolates of T. harzianum were isolated and T. harzianum isolate Th-8 exhibited better antifungal activity than carbendizim. Th-8 was formulated in different solid substrates like wheat bran, paddy husk, talcum powder and cornstarch. Maize seeds of kanchan (moderately resistant), pioneer (resistant) and sweet corn (susceptible) were selected for laboratory and field studies and these seeds were treated with a conidial suspension of T. harzianum at the rate of 1 × 10⁸ spore/ml and formulation at the rate of 10 g/kg. Treated seeds were subjected to evaluate F. verticillioides incidence, seed germination, seedling vigour and field emergence, yield, thousand seed weight and fumonisin production. It was found that the pure culture of T. harzianum was more effective in reducing the F. verticillioides and fumonisin incidence followed by Talc formulation than the carbendizim treated and untreated control. Formulations of T. harzianum were effective at reducing the F. verticillioides and Fumonisin infection and also increasing the seed germination, vigour index, field emergence, yield, and thousand seed weight in comparison with the control.     

木霉对月季幼苗生长的影响

为了研制促进月季生长的木霉生防菌肥，采用本实验室分离鉴定的哈茨木霉T6施于月季盆栽土壤中，分析木霉菌对"十姐妹"月季硬枝扦插发芽、幼苗生长、防御酶活性和内源激素水平的影响。结果表明：施用哈茨木霉T6的月季硬枝出芽率和幼苗枝条长度分别是对照的1.4和1.82倍；并且枝条和叶的干重、枝条和叶的含水率均显著增加。此外显著提高了叶中3种防御酶SOD、POD、CAT的活性及6种内源激素IAA、GA₃、ZT、ABA、SA和JA的水平。说明木霉T6具有促进月季生长和提高其抗性的作用，研究结果为研制促进月季生长的生物菌肥提供理论依据。     

花青素对神经退行性疾病保护作用的研究进展

对近年来花青素在神经退行性疾病中的研究进展进行综述,并从抑制氧化应激、缓解神经炎症、缓解兴奋性毒性和抑制蛋白异常聚集等方面对花青素的神经保护作用机制进行探讨,为进一步了解和研究花青素类化合物在神经退行性疾病中的预防作用提供科学依据。     

Cloning and heterologous expression of SS10, a subtilisin-like protease displaying antifungal activity from Trichoderma harzianum

Trichoderma harzianum parasitizes a large variety of phytopathogenic fungi. Trichoderma harzianum mycoparasitic activity depends on the secretion of complex mixtures of hydrolytic enzymes able to degrade the host cell wall. A gene ( SS10 ) encoding a subtilisin-like protease was cloned from T. harzianum T88, a biocontrol agent effective against soil-borne fungal pathogens. The full-length cDNA was isolated by 5' and 3' rapid amplification of the cDNA ends. The coding region of the gene is 1302 bp long, encoding 433 amino acids of a predicted protein with a molecular mass of 45 kDa and a pI of 6.1. Analysis of the deduced amino acid sequence revealed that this protein had homology to the serine proteases of the subtilisin-like superfamily (subtilases) (EC 3.4.21.) and had a predicted active site made up of the catalytic residues Asp 187, His 218 and Ser 376. Northern experiments demonstrated that SS10 was induced in response to different fungal cell walls. Subtilisin-like protease gene SS10 was expressed in Saccharomyces cerevisiae under control of the GAL1 promoter. The enzyme activity culminates (17.8 U mL⁻¹) 60 h after induction with galactose. The optimal enzyme reaction temperature was 50 °C and the optimal pH was 8. The subtilisin-like protease exerted broad-spectrum antifungal activity against Alternaria alternata, Fusarium oxysporum, Rhizoctonia solani, Sclerotinia sclerotiorum and Cytospora chrysosperma .     

哈茨木霉发酵液中肽类物质对豇豆土著根瘤菌竞争结瘤的影响

通过树脂吸附、离子交换、薄层层析、高效液相色谱系统以及紫外、质谱等分离、纯化和鉴别的方法,从哈茨木霉T2-16菌株发酵液中分离得到一种对豆科作物生长具促进作用的肽类物质。用该肽类物质对豇豆土著根瘤菌进行处理后,用AFLP技术研究了该物质对供试根瘤菌遗传稳定性和在土壤中竞争结瘤能力的影响。结果表明,传代次数和培养温度(28℃~36℃范围)对供试根瘤菌的遗传性状无明显影响,其AFLP指纹未发生明显变化;但经木霉肽类代谢产物处理后,根瘤菌竞争结瘤能力得到提高,为对照根瘤菌的1.53倍。