Cellular damage during drying and storage of Trichoderma harzianum spores

Factors that cause cellular damage during the drying and storage of Trichoderma harzianum conidia were independently studied to determine their effects on spore viability. Specifically, thermal stress and dehydration levels (water activity, a_w = 0.1–0.7) were assessed for their effect on spore survival. In addition, environmental conditions, such as water activity and temperature, were evaluated during storage of the spores. T. harzianum spores produced in liquid culture are highly sensitive to thermal stress, but dehydration does not seem to be a factor that influences spore death during desiccation. An inverse correlation between spore survival and the specific concentration of malondialdehyde (MDA) was observed during storage, especially when the conidia moisture levels were lower than the monolayer moisture levels. We prepared spore suspensions without additives and spray-dried the samples. Our data showed that reduced sample viability was mainly caused by the temperature of the drying process, an effect that appears to be independent of water activity.     

Induction of trehalose in spores of the biocontrol agent Trichoderma harzianum

Spores of the biocontrol agent Trichoderma harzianum P1 produced in liquid media and harvested in the stationary sporulation stage SSS (after 60?h), had higher viability after slow (>4×) and fast drying (>12×) than their counterparts harvested in the exponential sporulation stage, ESS (after 30?h). The trehalose content of SSS spores was almost 20× higher than that of ESS spores (0.16 vs. 3.4?mg/100?mg, respectively). Heat shock (40?°C?×?90?min) effectively increased the trehalose content 2.5× with respect to untreated SSS spores. The trehalose content achieved in heat-treated SSS spores was almost 60% higher than the maximum reached by holding the spores under water-stress at 97% relative humidity prior to drying.     

Trichoderma harzianum metabolites pre-adapt mushrooms to Trichoderma aggressivum antagonism

Trichoderma spp. is the cause of green mold, a disorder that affects cultivated mushrooms. The aims of the study were to establish whether improvement of mushroom resistance to Trichoderma aggressivum could be obtained by inducing reaction mechanisms before contact with the pathogen and whether this ability was species or strain dependent. Twenty nine isolates of Agaricus bisporus, 29 isolates of Lentinula edodes and 18 isolates of Pleurotus spp. were studied. The effect of T. harzianum metabolites on mycelial growth of these isolates was evaluated on YMEA (yeast, malt extract and agar), supplemented or not with Lysing Enzymes from T. harzianum (Sigma?, L1412). Mycelial growth generally was affected by Lysing Enzymes, but some L. edodes and Pleurotus spp. adapted to Lysing Enzymes. When mycelium was taken from a first culture with Lysing Enzymes and placed on YMEA with Lysing Enzymes for a second culture, their growth rate was not different from those of the controls. In the case of A. bisporus, only partial adaptation was obtained with a few isolates. The effect of adaptation to Lysing Enzymes on resistance to T. aggressivum was assayed for one strain of each group. Trichoderma aggressivum was exposed to the margin of 5- to 9-day-old mushroom colonies. Agaricus bisporus produced brown droplets, and T. aggressivum overgrew its mycelium. Lentinula edodes and P. ostreatus produced brown lines blocking the progression of T. harzianum, both on YMEA and YMEA plus Lysing Enzymes. The line was visible after 3 d on YMEA and after only 2 d on YMEA plus Lysing Enzymes. Improvement in the resistance to antagonists by introduction of some of their metabolites to the culture medium is a method for mushroom protection.     

Parameters optimization of chitin hydrolysis by Trichoderma harzianum chitinase under assay conditions

A kinetic analysis and optimization of reaction conditions for the enzymatic hydrolysis of chitin using chitinase produced by Trichoderma harzianum NCIM 1185 was carried out. Swollen chitin was used as the substrate for chitinase. The central composite design was followed for this optimization. The required volume ratio of the major reactants for maximum hydrolysis was determined. The pH and temperature optima were found to be 4.75 and 47 °C respectively. K _m and V _max for this enzyme were 4.643 kg/m³ and 0.1542 U respectively.     

Raffinose-induced mutanase production from Trichoderma harzianum

Abstract The enzyme α (1 → 3),3-glucanohydrolase (referred to as mutanase) from the filamentous fungus Trichoderma harzianum OMZ 779 is capable of degrading the water-insoluble glucan in dental plaque. Previously, it was necessary to produce the glucan (referred to as mutan) in vitro for use as the sole carbon source and inducer of mutanase synthesis in fungal cultures. We report here that raffinose also induces the production of mutanase. The metabolism of raffinose differed from that of other sugars in metabolic end products and secreted protein profile. In addition to mutanase, we observed an approximately 15 000 M _r protein that was also regulated by carbon source and by illumination conditions.     

Cytostatic effect of L-lysine-alpha-oxidase from Trichoderma harzianum Rifai and Trichoderma viride

L-lysine-alpha-oxidase, a new fungal enzyme catalyzing oxidative L-lysine deamination, was shown to have an inhibitory effect on the in vitro synthesis of DNA, RNA and proteins in human carcinoma ovarian (CaOv) cells.     

Xylanase production by Trichoderma harzianum E58

Summary Growth of Trichoderma harzianum E58 on hemicellulose-rich media, both in batch and fermentor cultures, resulted in independent profiles for the production of xylanase and endoglucanase enzymes. Dramatic differences in the ratio of xylanase to endoglucanase activities were observed among cultures grown on cellulose-rich Solka Floc and xylan. These results indicated that the induction of xylanases and cellulases was likely to be under separate regulatory control. The specific activity and amount of xylanases produced were found to be dependent on the concentration of xylan in the growth media. Growth on oat spelts xylan or the hemicellulose-rich, water-soluble fraction from steam-treated aspenwood (SEA-WS) greatly enhanced the production of xylanases and xylosidase in the culture filtrates. Constitutive levels of xylanase and endoglucanase enzymes were detected during growth of the fungus on glucose.Offprint requests to: D. J. Senior     

Trichoderma harzianum cerato-platanin enhances hydrolysis of lignocellulosic materials

Considering its worldwide abundance, cellulose can be a suitable candidate to replace the fossil oil-based materials, even if its potential is still untapped, due to some scientific and technical gaps. This work offers new possibilities demonstrating for the first time the ability of a cerato-platanin, a small fungal protein, to valorize lignocellulosic Agri-food Wastes. Indeed, cerato-platanins can loosen cellulose rendering it more accessible to hydrolytic attack. The cerato-platanin ThCP from a marine strain of Trichoderma harzianum, characterized as an efficient biosurfactant protein, has proven able to efficiently pre-treat apple pomace, obtaining a sugar conversion yield of 65%. Moreover, when used in combination with a laccase enzyme, a notable increase in the sugar conversion yield was measured. Similar results were also obtained when other wastes, coffee silverskin and potato peel, were pre-treated. With respect to the widespread laccase pre-treatments, this new pre-treatment approach minimizes process time, increasing energy efficiency.     

Continuous production of enzymes under carbon-limited conditions by Trichoderma harzianum P49P11

Carbon-limited chemostat cultures were performed using different carbon sources (glucose, 10 and 20 g/L; sucrose, 10 g/L; fructose/glucose, 5.26/5.26 g/L; carboxymethyl cellulose, 10 g/L; and carboxymethyl cellulose/glucose, 5/5 g/L) to verify the capability of the wild type strain Trichoderma harzianum to produce extracellular enzymes. All chemostat cultures were carried out at a fixed dilution rate of 0.05 h^?1. Experiments using glucose, fructose/glucose and sucrose were performed in duplicate. Glucose condition was found to induce the production of enzymes that can catalyse the hydrolysis of p-nitrophenyl-β-d-glucopyranoside (PNPGase). A concentration of 20 g/L of glucose in the feed provided the highest productivity (1048 ± 16 U/mol h). Extracellular polysaccharides were considered the source of inducers. Based on the obtained results, a new PNPGase production process was developed using mainly glucose. This process raises interesting possibilities of synthesizing the inducer substrate and the induced enzymes in a single step using an easily assimilated carbon source under carbon-limited conditions.     

Regulation of chitinase synthesis in Trichoderma harzianum.

The production of chitinase by Trichoderma species is of interest in relation to their use in biocontrol and as a source of mycolytic enzymes. Fourteen isolates of the genus were screened to identify the most effective producer of chitinase. The best strain for chitinase was Trichoderma harzianum 39.1, and this was selected for study of the regulation of enzyme synthesis. Washed mycelium of T. harzianum 39.1 was incubated with a range of carbon sources. Chitinase synthesis was induced on chitin-containing medium, but repressed by glucose and N-acetylglucosamine. Production of the enzyme was optimal at a chitin concentration of 0.5%, at 28 degrees C, pH 6.0 and was independent of the age of the mycelium. The synthesis of chitinase was blocked by both 8-hydroxyquinoline and cycloheximide, inhibitors of RNA and protein synthesis, respectively. The mode of chitinase synthesis in this fungus is discussed.     

Hypocrea lixii, the teleomorph of Trichoderma harzianum

Cultures derived from ascospores of Hypocrea lixii (= H. nigricans, H. lentiformis) produced the morphological species Trichoderma harzianum in pure culture. Trichoderma harzianum, the most commonly found species of the genus, is also one of the most species frequently used in biocontrol of plant pathogens. It has not been connected previously to a teleomorph. The connection was confirmed by DNA sequence analysis. Similar to the anamorph, the teleomorph collections have a wide geographic distribution. Described in the 19^th century, Hypocrea lixii is epitypified by a collection from Thailand.     

影响根癌农杆菌介导的木霉菌遗传转化因素分析

采用根癌农杆菌介导的转化方法,以木霉菌分生孢子为受体材料,对影响转化效率的主要因素进行了分析。结果表明,农杆菌菌株类型、初始菌液量、分生孢子浓度、共培养时间以及乙酰丁香酮的诱导等因素对转化效率都具有重要的影响。通过对这些因素的分析,基本得出了根癌农杆菌转化系统对木霉菌遗传转化的特点和规律,为将该转化系统用于其它丝状真菌的遗传转化提供了重要参考。     

A new xylanase from a Trichoderma harzianum strain

A new xylanase (XYL2) was purified from solid-state cultures of Trichoderma harzianum strain C by ultrafiltration and gel filtration. SDS-PAGE of the xylanase showed an apparent homogeneity and molecular weight of 18 kDa. It had the highest activity at pH 5.0 and 45°C and was stable at 50°C and pH 5.0 up to 4 h xylanase. XYL2 had a low K _m with insoluble oat spelt xylan as substrate. Compared to the amino acid composition of xylanases from Trichoderma spp, xylanase XYL2 presented a high content of glutamate/glutamine, phenylalanine and cysteine, and a low content of serine. Xylanase XYL2 improved the delignification and selectivity of unbleached hardwood kraft pulp. Received 02 February 1999/ Accepted in revised form 17 April 1999     

Biological effects of Trichoderma harzianum peptaibols on mammalian cells

Trichoderma species isolated from water-damaged buildings were screened for toxicity by using boar sperm cells as indicator cells. The crude methanolic cell extract from Trichoderma harzianum strain ES39 inhibited the boar sperm cell motility at a low exposure concentration (50% effective concentration, 1 to 5 microg [dry weight] ml of extended boar semen(-1)). The same exposure concentration depleted the boar sperm cells of NADH(2). Inspection of the exposed boar sperm cells by transmission electron microscopy revealed damage to the plasma membrane. By using the black lipid membrane technique, it was shown that the semipurified metabolites (eluted from a SepPak C(18) cartridge) of T. harzianum strain ES39 induced voltage-dependent conductivity. The high-performance liquid chromatography-purified metabolites of T. harzianum strain ES39 dissipated the mitochondrial membrane potential (Deltapsi(m)) of human lung epithelial carcinoma cells (cell line A549). The semipurified metabolites (eluted from a SepPak C(18) cartridge) of T. harzianum strain ES39 were analyzed by mass spectrometry (MS). Matrix-assisted laser desorption ionization and nanoflow electrospray ionization MS revealed five major peptaibols, each of which contained 18 residues and had a mass ranging from 1,719 to 1,775 Da. Their partial amino acid sequences were determined by collision-induced dissociation tandem MS.