ThPTR2, a di/tri-peptide transporter gene from Trichoderma harzianum

The generation of a wide ESTs library and database from Trichoderma harzianum CECT 2413 was the base for identifying the gene ThPTR2, coding for a PTR family di/tri-peptide transporter. The deduced protein sequence of the ThPTR2 gene showed the conserved motifs and also the 12 transmembrane domains typical of the PTR transporters. The highest level of ThPTR2 expression was found when the fungus was grown in chitin as sole carbon source. We also found that ThPTR2 expression was increased when Trichoderma interacted directly in solid medium with the plant-pathogenic fungus Botrytis cinerea, showing that ThPTR2 is involved in the mycoparasitic process. Additionally, its expression was triggered by nitrogen starvation and a higher level of expression was also found when Trichoderma was grown in secondary nitrogen sources like allantoin, yeast extract, and urea. However, no difference was found when Trichoderma was grown in presence or absence of glucose as carbon source. Strain T34-15, a transformant that overexpressed the ThPTR2 gene, showed about a 2-fold increase in the uptake of the dipeptide Leu-Leu. Additionally, two transformants from the strain Trichoderma longibrachiatum T52 that overexpressed ThPTR2 were also studied, confirming the role of this gene in peptide transport. Other homologous genes to ThPTR2 were identified in other Trichoderma strains. ThPTR2 is the first experimentally confirmed PTR family transporter gene from filamentous fungi.     

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。     

绿色木霉纤维素酶CBHII基因的分子克隆

本文以噬菌体lambda EMBL3 DNA为载体,通过克隆绿色木酶(Trichoderma viride)高分子量基因组DNA的部分酶解片段,并将重组分子进行体外包装后侵染Escherichia.coli K802,由此构建了绿色木霉基因文库。以李氏木霉(Trichoderma reesei)纤维素酶CBHII基因的末端片段为探针,用轮迥噬菌斑原位杂交从文库中筛选出CBHII基因的阳性克隆5个,随机取其中3个克隆用上述探针作斑点杂交,结果进一步证明克隆了全长或近全长的绿色木霉CBHII基因,用李氏木霉CBHI基因的末端片段探针作斑点杂交,结果提示CBHI与CBHII基因的末端序列之间无同源性存在。从斑点杂交的阳性克隆中提取DNA,酶切鉴定插入片段的长度,并克隆于质粒pUC19,Southern杂交结果证明获得了含绿色木霉CBHII基因的重组质粒pCBHII-14。     

A silent antifungal protein (AFP)-like gene lacking two introns in the mould Trichoderma viride

In cells of the mould Trichoderma viride, the existence of an antifungal protein (AFP)-like gene consisting of 285 bp was confirmed by Southern analysis that genomic DNA of T. viride could hybridize with the cDNA of mature AFP of Aspergillus giganteus MDH 18894. Except for the absence of two introns, the nucleotide sequence of the AFP-like gene was identical to that of the AFP gene of A. giganteus in positions 336-479, 568-649, and 706-765. The AFP-like gene could not be transcribed into its mRNA in T. viride cells as examined by RT-PCR using total RNAs of T. viride as template. Furthermore, AFP could not be detected either directly from the culture medium of T. viride or by Western analysis. However, the AFP-like gene could be actively expressed like the cDNA of AFP in Escherichia coli cell. Recombinant AFP exhibited similar antifungal activity as native AFP.     

Tricalcium phosphate solubilizing abilities of Trichoderma spp. in relation to P uptake and growth and yield parameters of chickpea (Cicer arietinum L.)

Nine isolates of Trichoderma spp. were investigated for their ability to solubilize insoluble phosphate in Pikovskaya's broth and were compared with an efficient phosphate-solubilizing bacterium Bacillus megaterium subsp. phospaticum PB that was used as the reference strain. All 9 Trichoderma isolates were found to solubilize insoluble tricalcium phosphate to various extents. Trichoderma viride (TV 97) (9.03 microg x mL(-1)), Trichoderma virens (PDBCTVs 12) (9.0 microg x mL(-1)), and Trichoderma virens (PDBCTVs 13) (8.83 microg x mL(-1)) solubilized 70% of that solubilized by the reference strain Bacillus megaterium (12.43 microg x mL(-1)). Pot culture and field evaluations with Trichoderma harzianum (PDBCTH 10), Trichoderma viride (TV 97), and Trichoderma virens (PDBCTVs 12) using chickpea (Cicer arietinum L.) 'Annegeri-1' as the test plant and rock phosphate as the phosphorus source showed significantly increased P uptake in plants treated with Trichoderma harzianum (PDBCTH 10) followed by Trichoderma virens (PDBCTVs 12) and Trichoderma viride (TV 97). Inoculation of Trichoderma spp. also showed increased growth and yield parameters of chickpea compared with the uninoculated controls under both glasshouse and field conditions.     

Beta-glucanase and chitinase biosynthesis in a culture of a mycophilic strain of Trichoderma viride

The production of extracellular 1,3-, 1,6-beta-glucanases and chitinase was studied during submerged cultivation of a Trichoderma viride strain 3/78 on various carbon sources: glycerol, glucose, lactose, sucrose, laminaran, starch, pustulan, chitin, and Agaricus bisporus fruit bodies. The synthesis of these enzymes and cellulase was studied also under the conditions of depression at low concentrations (10(-2) and 10(-3)M) of the first five aforementioned carbon sources as well as cellobiose, gentiobiose, N-acetyl-beta-D-glucosamine and 0.1% chitooligosaccharides and A. bisporus cell walls. The experiments were conducted with the washed mycelium of this strain grown for 2 days in a medium with glycerol as a carbon source. The results indicated that 1,3- and 1,6-beta-glucanases of the strain were of the constitutive nature and were repressed by such carbon sources as glycerol and glucose. Chitinase and cellulase were shown to be inducible enzymes. Chitinase was induced by N-acetyl-beta-D-glucosamine, chitooligosaccharides and A. bisporus cell walls as well as by lactose when the fungus was grown on this carbon source. Cellulase biosynthesis was induced by lactose, cellobiose and gentiobiose.     

Secretion of cellulase and beta-glucosidase by Trichoderma viride ITCC-1433 in submerged culture on different substrates.

Trichoderma viride ITCC-1433 produces high yields of cellulase and especially beta-glucosidase when grown in submerged culture on different carbon sources. Cellulase synthesis was strongly repressed in the presence of glucose and only a low constitutive activity of beta-glucosidase and carboxymethylcellulase, but no Avicelase, could be demonstrated when culturing T. viride on glucose. With carboxymethylcellulose (CMC) as a substrate the secretion of enzyme as well as growth depended on the degree of substitution, but in general CMC cannot be regarded either as a powerful inducer or as a carbon source. With insoluble cellulose, maximum enzyme production and activities were obtained using an alkali-treated cellulose powder. On this substrate the excretion of soluble protein into the culture broth increased and the protein concentration corresponded to cellulolytic activities.     

表面活性剂对绿色木霉产纤维素酶影响

利用绿色木霉,以稻草为唯一碳源,采用液态发酵的方法,分别加入生物表面活性剂鼠李糖脂和化学表面活性剂Tween 80,重点研究了生物表面活性剂对绿色木霉产纤维素酶的影响。实验分析了加入不同浓度的表面活性剂时滤纸酶活、羧甲基纤维素酶活、微晶纤维素酶活及酶液的表面张力随时间的变化情况。结果表明,添加鼠李糖脂能够促进绿色木霉产酶,分别使滤纸酶活、羧甲基纤维素酶活、微晶纤维素酶活最大提高了1.08倍,1.6倍和1.03倍。与Tween 80相比,鼠李糖脂促进产酶的效果明显优于Tween 80。     

Cloning and expression of a novel Trichoderma viride laminarinase AI gene (lamAI)

The gene lamAI, which encodes a novel laminarinase AI of Trichoderma viride U-1, was cloned using RT-PCR in conjunction with the rapid amplification of cDNA ends (RACE) technique. The open reading frame consisted of 2,277 bp encoding a protein of 759 amino acid residues, including a 32-residue signal prepropeptide. The protein showed 91% sequence similarity to the putative Trichoderma virens beta-1,3-glucanase BGN1, but no significant similarity to fungal beta-1,6-glucanases or beta-1,3-glucanases from other organisms. On 40 h incubation with a solo carbon source, northern analysis revealed that the gene was induced by 0.5% laminaran from Eisenia bicyclis but was not by the same concentration of glucose. The lamAI cDNA was functionally expressed in the methylotrophic yeast Pichia pastoris, resulting in a recombinant enzyme with as high activity against laminaran as native LAMAI. Based on these data, the probable existence of endo-beta-1,3:1,6-glucan hydrolases as a subclass of endo-beta-1,3-glucanases in some mycoparasitic fungi is suggested.     

A morphological and molecular perspective of Trichoderma viride: is it one or two species?

Trichoderma (Ascomycetes, Hypocreales) strains that have warted conidia are traditionally identified as T. viride, the type species of Trichoderma. However, two morphologically distinct types of conidial warts (I and II) have been found. Because each type corresponds to a unique mitochondrial DNA pattern, it has been questioned whether T. viride comprises more than one species. Combined molecular data (sequences of the internal transcribed spacer 1 [ITS-1] and ITS-2 regions and of part of the 28S rRNA gene along with results of restriction fragment length polymorphism analysis of the endochitinase gene and PCR fingerprinting), morphology, physiology, and colony characteristics distinguish type I and type II as different species. Type I corresponds to "true" T. viride, the anamorph of Hypocrea rufa. Type II represents a new species, T. asperellum, which is, in terms of molecular characteristics, close to the neotype of T. hamatum.     

EFFECT OF CERTAIN CHLORONITROBENZENES ON GERMINATION, GROWTH AND SPORULATION OF SOME FUNGI

When grown in an atmosphere saturated with tetrachloronitrobenzene (TCNB) vapour, Botrytis cinerea showed retarded germination and colony growth and its sporulation was completely suppressed. Retardation of growth was greater on dilute or acidified media and at lower temperatures.
Similar effects were shown by Bhizoctonia solani, Phytophthora parasitica, Mucor hiemalis and Trichoderma viride but none of these was as sensitive as Botrytis cinerea , especially as regards linear growth. Of two strains of Fusarium caeruleum , one was much more sensitive than the other, both in linear growth and sporulation. Pythium deBaryanum was unaffected in its growth rate.
TCNB, as a growth repressant, is fungistatic without being fungicidal.
With fungi which are sensitive, tetrachloronitrobenzene was more effective than the penta-derivative, except for Trichoderma viride where the converse applied.     

Production of xyloglucanolytic enzymes by Trichoderma viride, Paecilomyces farinosus, Wardomyces inflatus, and Pleurotus ostreatus

Different conditions of culture medium, incubation time, concentration and surfactant were tested to determine xyloglucanase activity. Trichoderma viride, Paecilomyces farinosus, Wardomyces inflatus and Pleurotus ostreatus showed increased xyloglucanase activities when the fungi grown on microcrystalline cellulose as the sole carbon source. Endoxyloglucanase activity increased with the growth of the fungi and reached a peak on day 14 of incubation, practically 95% of the activity was associated with the extracellular fraction. Precipitation with ammonium sulfate was the best concentration method for detection of endoxyloglucanase activity of the fungi. Endoxyloglucanase activity of the fungi was increased by 4 fold with the use of the non-ionic surfactant Tween 20. Six and three bands of xyloglucanase activities were observed in T. viride and P. ostreatus, respectively, whereas both P. farinosus and W. inflatus presented only one xyloglucanase activity band. These results indicate the presence of several xyloglucanases in the saprophytic fungi examined.     

Environmental factors affecting the antagonism of Pseudomonas cepacia against Trichoderma viride.

Antagonistic activity of the bacterium Pseudomonas cepacia against Trichoderma viride was greatly influenced by nutritional and environmental conditions. Xylose and trehalose strongly enhanced the antifungal activity of P. cepacia, whereas mannitol and glucose had little effect. The carbon sources that enhanced the antagonistic activity also inhibited sporulation of T. viride. Antagonism of P. cepacia was enhanced by ammonium nitrogen; however, with nitrite or nitrate there was only a little antagonism. The antagonism of P. cepacia was optimal at pH 5.0. Although P. cepacia showed maximum antagonism against T. viride at 37 degrees C, the antagonism was fairly good at temperatures as low as 18 degrees C, indicating that there is a broad range of temperature for the antifungal activity of P. cepacia.     

Co-expression of two genes, a chitinase (chit42) and proteinase (prb1), implicated in mycoparasitism by Trichoderma hamatum

Mycoparasitism of fungal plant pathogens by Trichoderma species is a complex process that involves the production and coordinated secretion of cell-wall degrading enzymes. Genes implicated in mycoparasitism by Trichoderma atroviride contain motifs in the promoter region, designated MYRE1-MYRE4, that are proposed to act as binding sites for a global inducer of the mycoparasitic response. The aim of our study was to establish whether these motifs also were present in Trichoderma hamatum and whether the presence of these motifs could predict co-expression when T. hamatum was confronted by a pathogen. Using a combination of targeted, degenerate and inverse PCR, homologues of the mycoparasitism-related genes ech42 (chit42), prb1 and lam1.3 (xbg1.3-110), which encode an endochitinase, proteinase, and β-1,3-glucanase, respectively, were cloned and sequenced from T. hamatum. Alignment of the promoter regions of the three genes revealed identical regions in the chit42 and prb1 promoters, which were 6-9 base pairs in length and conserved in position. Specifically, the regulator y motifs MYRE1-MYRE4 were fully conserved, together with a fifth motif, identified by this research. A substrate assay designed to investigate the response of these genes from T. harzianum and T. hamatum to a simple carbon source (glycerol) showed that, in contrast to chit42 and prb1, xbg1.3-110 was not expressed. Further comparison of the expression patterns of these three genes between T. harzianum and T. hamatum using the glycerol substrate assay showed that no chit42 or prb1 expression could be detected in T. harzianum when it was grown under the same conditions as T. hamatum. This showed that the response of these genes to glycerol was species specific and that a single expression pattern for these genes was not common to all Trichoderma species. Confrontation assays were used to investigate the response of the three T. hamatum genes to the more complex substrate posed by the fungal pathogen Sclerotinia sclerotiorum. Once again gene expression analysis showed that both chit42 and prb1 were co-expressed and moderately induced during confrontation against Sclerotinia sclerotiorum. Although xbg1.3-110 previously had been implicated in mycoparasitism by T. harzianum, this study detected no xbg1.3-110 expression during confrontation between T. hamatum and S. sclerotiorum. These findings show that the MYRE1-MYRE4 together with MYRE5 are present in two species of Trichoderma, T. atroviride and T. hamatum and that the presence of these motifs could predict co-expression in response to two carbon sources.     

人参(Panax ginseng)根系分泌物成分对人参致病菌的化感效应

采用室内培养结合生物学测定的试验方法,研究了不同浓度人参(Panax ginseng)根系分泌物成分苯甲酸、邻苯二甲酸二异丁酯、十六酸和2,2-二(4-羟苯基)丙烷对人参立枯丝核菌(Rhizoctonia solani)、黑斑菌(Alternaria panax)、疫病菌(Phytophthora cactorum)、菌核菌(Sclerotinia schinseng)、锈腐菌(Cylindrocarpon destructans)和绿色木霉菌(Trichoderma viride)菌落生长及孢子萌发的化感效应.结果显示,不同浓度人参根系分泌物成分对人参致病菌及绿色木霉菌的化感效应存在显著差异.苯甲酸浓度与人参立枯丝核菌、菌核菌和锈腐菌菌落生长以及人参黑斑菌、锈腐菌孢子萌发呈负相关,与人参黑斑菌、绿色木霉菌菌落生长呈正相关;对人参疫病菌菌落生长的化感效应表现为低浓度和高浓度抑制,中浓度促进.邻苯二甲酸二异丁酯浓度与人参立枯丝核菌、黑斑菌、菌核菌和绿色木霉菌菌落生长以及人参黑斑菌孢子萌发呈负相关;对人参锈腐菌菌落生长和孢子萌发表现为低浓度和高浓度抑制,中浓度促进;对人参疫病菌菌落生长表现为低浓度和中浓度抑制,高浓度促进.2,2-二(4-羟苯基)丙烷浓度与人参立枯丝核菌、黑斑菌、疫病菌、绿色木霉菌菌落生长以及人参黑斑菌、锈腐菌孢子萌发呈负相关;对人参菌核菌、锈腐菌菌落生长表现中浓度促进,高浓度抑制.十六酸浓度与人参锈腐菌、疫病菌和绿色木霉菌菌落生长呈正相关,与人参锈腐菌孢子萌发呈负相关,对黑斑菌孢子萌发表现为中浓度抑制.4种根系分泌物的等量混合物浓度与人参致病菌及拮抗木霉菌菌落生长速率呈负相关.