Trichoderma biodiversity in China

In the present study, we made further investigation into the diversity of Trichoderma in China than previous ones utilizing comprehensive approaches of morphological microscopic observation and phylogenetic analysis by detecting molecular markers. One thousand nine hundred ten Trichoderma strains were isolated from soil or other materials in China: East (Anhui, Fujian, Jiangsu, Jiangxi, Shandong, Zhejiang province and Shanghai municipality), South-West (Guizhou, Qinghai, Shanxi, Sichuan and Yunnan province, Tibet Autonomous Region and Chongqing municipality), South-East (Guangdong, Guangxi, Hainan province), and Middle China (Henan, Hubei and Hunan province). Representative isolates were verified at the species level by morphological characters and the oligonucleotide barcode program TrichoOKey v.10 and the custom BLAST server TrichoBLAST, using sequence of the ITS 1 and 2 region of the rDNA cluster and partial sequences of translation elongation factor 1-alpha(tef1-α). A total of 23 Trichoderma species were identified : T.asperellum, T.atrioviride, T.aureovriride, T.brevicompactum, T.citrioviride, T.erinaceum, T.gamsii, T.hamatum, T.harzianum (H.1ixii), T.intricatum, T.koningii (H.koningii), T.koningiopsis, T.longibranchiatum, T.pleuroticola, T.reeseii (H.jecorina), T.sinensis, T.spirale, T.stromaticum, T.tomentosum, T.velutinum, T.vermipilum, T.virens (H.virens), T.viride. Among them, 3 species: T.intricatum, T.stromaticum, T.vermipilum were first reported in China; T.harzianum (H,1ixii) was the most widely distributed species in China. This study further shows that, the highest biodiversity of Trichoderma population appeared in South-West China.     

Chitinous Material in Trichoderma reesei

Trichoderma reesei was grown for 180h in batch culture in an 8 liter stirred fermenter using a glucose-rich medium. Concentrations of glucose, ammonia, cell dry weight, debris and lipid are presented for two runs. Cell dry weights reached 26.9g/L and 19.6g/L in these runs. The debris from solvent-extracted cells was chitin which accumulated to greater than 75% of the final cell dry weights.     

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.     

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.     

Enhanced Expression of Endochitinase in Trichoderma harzianum with the cbh1 Promoter of Trichoderma reesei

Production of extracellular endochitinase could be increased 5-fold in the mycoparasite fungus Trichoderma harzianum by using the cellulase promoter cbh1 of Trichoderma reesei, whereas the total endochitinase activity increased 10-fold. The cbh1 promoter was not expressed on glucose and sucrose in T. harzianum and was induced by sophorose and on cellulase-inducing medium. The endogenous endochitinase gene was expressed at a low basal level on glucose and sucrose. No specific induction by crab shell chitin or sophorose was observed.     

Lipid accumulation in Trichoderma species

Abstract Two filamentous fungi, Trichoderma harzianum and Trichoderma viride , were compared for their ability to synthesize lipids on different carbon and nitrogen sources. Three culture media were selected for each strain after preliminary screening. All the test media were nitrogen-deficient (C/N = 60) so as to stimulate lipid accumulation. For both microorganisms the glucose-ammonium sulphate medium was the most conducive to lipid production: a lipid accumulation of 17% (w/w) of biomass dry weight was obtained for T. harzianum and of 32% (w/w) of biomass dry weight for T. viride . In sucrose-sodium nitrate medium T. harzianum was able to accumulate almost 25% (w/w) of its biomass in lipid form. However the small quantity of biomass produced (2 g dry weight/l) limited the quantity of lipid obtained. Neutral lipids, free fatty acids and phospholipids were monitored during 8 days of cultivation of the two fungi.     

Protoplasts of Trichoderma viride

High yields of protoplasts from the 18-hr old mycelium of Trichoderma viride were obtained by using the lytic system, produced by Streptomyces venezuelae RA and Micromonospora chalcea grown on a synthetic medium containing laminarin and chitin, when 0.7 M MgSO₄ or (NH₄)₂SO₄ were used as osmotic stabilizers. Regeneration of these protoplasts occurred through the production of an abortive tube and direct germination of the protoplasts. Regeneration could also take place in the medium used to produce protoplasts, but the process was different in many details.     

Complementation in Nonconidiating Mutants of Trichoderma

Nonconidiating (Con(-)) mutants were isolated from wild-type and color mutants of the fungus Trichoderma viride Pers. ex Fries. Heterokaryons were easily produced and maintained, and the complementation relationships among the Con(-) mutants were established. Most Con(-) mutants could complement one or more of the other Con(-) mutants. When marked Con(-) mutants were mixed with marked Con(+) testers, conidiating heterokaryons were formed. The conidia thus obtained produced only the parental type colonies after replating, indicating that nonconidiation is a nuclear characteristic. Allowing two Con(-) colonies to meet and produce a heterokaryon, it was found that the migration of nuclei reached a rate of 5 mm per hr, which is several times greater than the rate of hyphal elongation; it was also found that heterokaryosis of a mycelial region preceded its ability to conidiate.     

Cellobiohydrolase II is the main conidial-bound cellulase in Trichoderma reesei and other Trichoderma strains

Monoclonal antibodies have been used to determine the presence of cellobiohydrolases I and II (CBH I and II), and endoglucanase I (EG I) on the surface of conidia from Trichoderma reesei QM 9414 and RUT C-30, and 8 other Trichoderma species. For this purpose, proteins were released from the conidial surface by treatment with a non-ionic detergent (Triton X-100 and -octylglucoside), followed by SDS-PAGE/Western blotting and immunostaining. Both CBH I and II were clearly present, but — unlike in extracellular culture fluids from Trichoderma — CBH II was the predominant cellulase. In T. reesei EG I could not be detected. The higher producer strain T. reesei RUT C-30 exhibited a higher conidial level of CBH II than T. reesei QM 9414. In order to assess the importance of the conidial CBH II level for cellulase induction by cellulose, multiple copies of the chb2 gene were introduced into the T. reesei genome by cotransformation using PyrG as a marker. Stable multicopy transformants secreted the 2- to 4-fold level of CBH II into the culture medium when grown on lactose as a carbon source, but their CBH I secretion was unaltered. Upon growth on cellulose, both CBH I and CBH II secretion was enhanced. Those strain showing highest cellulase activity on cellulose also appeared to contain the highest level of conidial bound CBH II. CBH II was also the predominant conidial cellulase in various other Trichoderma sp. However, roughly the same amount of conidial bound CBH II was detected in all strains, although their cellulase production differed considerably.     

木霉属中国新记录种Trichoderma koningiopsis记述

在华东地区木霉菌资源调查中,利用内转录间隔区(ITS)序列分析和形态学鉴定方法,对从土壤中分离到的木霉菌进行鉴定,发现一个中国新纪录种,即拟康宁木霉。Trichoderma koningiopsis/Hypocrea koningiopsis Samuels,C.SuarezH.C.Evans sp. nov.。该种典型的形态特征是在PDA以及CMD(玉米粉琼脂)上有瓶梗层出现象,而在MA(麦芽提取物)培养基上没有此特征。     

Truncation of a mannanase from Trichoderma harzianum improves its enzymatic properties and expression efficiency in Trichoderma reesei

To obtain high expression efficiency of a mannanase gene, ThMan5A, cloned from Trichoderma harzianum MGQ2, both the full-length gene and a truncated gene (ThMan5A△CBM) that contains only the catalytic domain, were expressed in Trichoderma reesei QM9414 using the strong constitutive promoter of the gene encoding pyruvate decarboxylase (pdc), and purified to homogeneity, respectively. We found that truncation of the gene improved its expression efficiency as well as the enzymatic properties of the encoded protein. The recombinant strain expressing ThMan5A△CBM produced 2,460 ± 45.1 U/ml of mannanase activity in the culture supernatant; 2.3-fold higher than when expressing the full-length ThMan5A gene. In addition, the truncated mannanase had superior thermostability compared with the full-length enzyme and retained 100 % of its activity after incubation at 60 °C for 48 h. Our results clearly show that the truncated ThMan5A enzyme exhibited improved characteristics both in expression efficiency and in its thermal stability. These characteristics suggest that ThMan5A△CBM has potential applications in the food, feed, paper, and pulp industries.     

Peptide Synthetase Gene in Trichoderma virens

Trichoderma virens (synonym, Gliocladium virens), a deuteromycete fungus, suppresses soilborne plant diseases caused by a number of fungi and is used as a biocontrol agent. Several traits that may contribute to the antagonistic interactions of T. virens with disease-causing fungi involve the production of peptide metabolites (e.g., the antibiotic gliotoxin and siderophores used for iron acquisition). We cloned a 5,056-bp partial cDNA encoding a putative peptide synthetase (Psy1) from T. virens using conserved motifs found within the adenylate domain of peptide synthetases. Sequence similarities with conserved motifs of the adenylation domain, acyl transfer, and two condensation domains support identification of the Psy1 gene as a gene that encodes a peptide synthetase. Disruption of the native Psy1 gene through gene replacement was used to identify the function of this gene. Psy1 disruptants produced normal amounts of gliotoxin but grew poorly under low-iron conditions, suggesting that Psy1 plays a role in siderophore production. Psy1 disruptants cannot produce the major T. virens siderophore dimerum acid, a dipetide of acylated N^δ-hydroxyornithine. Biocontrol activity against damping-off diseases caused by Pythium ultimum and Rhizoctonia solani was not reduced by the Psy1 disruption, suggesting that iron competition through dimerum acid production does not contribute significantly to disease suppression activity under the conditions used.     

Biology and biotechnology of Trichoderma

Fungi of the genus Trichoderma are soilborne, green-spored ascomycetes that can be found all over the world. They have been studied with respect to various characteristics and applications and are known as successful colonizers of their habitats, efficiently fighting their competitors. Once established, they launch their potent degradative machinery for decomposition of the often heterogeneous substrate at hand. Therefore, distribution and phylogeny, defense mechanisms, beneficial as well as deleterious interaction with hosts, enzyme production and secretion, sexual development, and response to environmental conditions such as nutrients and light have been studied in great detail with many species of this genus, thus rendering Trichoderma one of the best studied fungi with the genome of three species currently available. Efficient biocontrol strains of the genus are being developed as promising biological fungicides, and their weaponry for this function also includes secondary metabolites with potential applications as novel antibiotics. The cellulases produced by Trichoderma reesei, the biotechnological workhorse of the genus, are important industrial products, especially with respect to production of second generation biofuels from cellulosic waste. Genetic engineering not only led to significant improvements in industrial processes but also to intriguing insights into the biology of these fungi and is now complemented by the availability of a sexual cycle in T. reesei/Hypocrea jecorina, which significantly facilitates both industrial and basic research. This review aims to give a broad overview on the qualities and versatility of the best studied Trichoderma species and to highlight intriguing findings as well as promising applications.     

Electrofusion of Trichoderma reesei protoplasts

Protoplasts of Trichoderma reesei were fused according to the method of Zimmermann. For optimizing the fusion parameters the central composite design was used. Genetic evidence for fusion has been obtained by segregation of the auxotrophic markers in the haploid conidia. The parameters which were optimized were: pulse voltage, pulse duration and number of pulses. The optimal parameters for the fusion of T. reesei protoplasts are 90 V pulse voltage, 37 μS pulse duration and six pulses at intervals of 1-0 s.     

The glycosulphatase of Trichoderma viride

The growth of the mould Trichoderma viride on a defined medium containing either potassium d-glucose 6-O-sulphate or potassium d-galactose 6-O-sulphate as sole sources of both carbon and sulphur is marked by the production of an enzyme system capable of liberating inorganic SO(4) (2-) ions from either of the sulphate esters. The enzyme is not produced when the organism is grown with glucose (or galactose) and potassium sulphate or with glucose and methionine as sole sources of carbon and sulphur. Experimental conditions are described whereby inorganic SO(4) (2-) ions liberated from potassium glucose 6-O-sulphate by the growing mould appear in the culture medium after a constant lag period of 21-24hr. The enzyme has been shown to be a simple glycosulphatase that is active towards the 6-O-sulphate esters of d-glucose and d-galactose but not towards potassium glucose 3-O-sulphate. The properties of the crude glycosulphatase show the enzyme to be appreciably different from analogous molluscan enzymes that can degrade monosaccharide sulphate esters.