Crystallization and preliminary X-ray diffraction analysis of endoglucanase III from Trichoderma harzianum

Endoglucanases are enzymes that hydrolyze cellulose and are important components of the cellulolytic complex. In contrast to other members of the complex, they cleave internal beta-1,4-glycosidic bonds in the cellulose polymer, allowing cellulose to be used as an energy source. Since biomass is an important renewable source of energy, the structural and functional characterization of these enzymes is of interest. In this study, endoglucanase III from Trichoderma harzianum was produced in Pichia pastoris and purified. Crystals belonging to the orthorhombic space group P212121, with unit-cell parameters a = 47.54, b = 55.57, c = 157.3 angstrom, were obtained by the sitting-drop vapour-diffusion method and an X-ray diffraction data set was collected to 2.07 angstrom resolution.

RNA Interference of Endochitinases in the Sugarcane Endophyte Trichoderma virens 223 Reduces Its Fitness as a Biocontrol Agent of Pineapple Disease

The sugarcane root endophyte Trichoderma virens 223 holds enormous potential as a sustainable alternative to chemical pesticides in the control of sugarcane diseases. Its efficacy as a biocontrol agent is thought to be associated with its production of chitinase enzymes, including N-acetyl-beta-D-glucosaminidases, chitobiosidases and endochitinases. We used targeted gene deletion and RNA-dependent gene silencing strategies to disrupt N-acetyl-beta-D-glucosaminidase and endochitinase activities of the fungus, and to determine their roles in the biocontrol of soil-borne plant pathogens. The loss of N-acetyl-beta-D-glucosaminidase activities was dispensable for biocontrol of the plurivorous damping-off pathogens Rhizoctonia solani and Sclerotinia sclerotiorum, and of the sugarcane pathogen Ceratocystis paradoxa, the causal agent of pineapple disease. Similarly, suppression of endochitinase activities had no effect on R. solani and S. sclerotiorum disease control, but had a pronounced effect on the ability of T. virens 223 to control pineapple disease. Our work demonstrates a critical requirement for T. virens 223 endochitinase activity in the biocontrol of C. paradoxa sugarcane disease, but not for general antagonism of other soil pathogens. This may reflect its lifestyle as a sugarcane root endophyte.

Conformational stability of recombinant manganese superoxide dismutase from the filamentous fungus Trichoderma reesei

Superoxide dismutases (SODS; EC 1.15.1.1) are part of the antioxidant system of aerobic organisms and are used as a defense against oxidative injury caused by reactive oxygen species (ROS). The cloning and sequencing of the 788-bp genomic DNA from Trichoderma reesei strain QM9414 (anamorph of Hypocrea jecorina) revealed an open reading frame encoding a protein of 212 amino acid residues, with 65-90% similarity to manganese superoxide dismutase from other filamentous fungi. The TrMnSOD was purified and shown to be stable from 20 to 90 degrees C for 1 h at pH from 8 to 11.5, while maintaining its biological activity. (C) 2011 Elsevier B.V. All rights reserved.

Identification of differentially expressed genes from Trichoderma harzianum during growth on cell wall of Fusarium solani as a tool for biotechnological application

Background: The species of T. harzianum are well known for their biocontrol activity against many plant pathogens. However, there is a lack of studies concerning its use as a biological control agent against F. solani, a pathogen involved in several crop diseases. In this study, we have used subtractive library hybridization (SSH) and quantitative real-time PCR (RT-qPCR) techniques in order to explore changes in T. harzianum genes expression during growth on cell wall of F. solani (FSCW) or glucose. RT-qPCR was also used to examine the regulation of 18 genes, potentially involved in biocontrol, during confrontation between T. harzianum and F. solani. Results: Data obtained from two subtractive libraries were compared after annotation using the Blast2GO suite. A total of 417 and 78 readable EST sequence were annotated in the FSCW and glucose libraries, respectively. Functional annotation of these genes identified diverse biological processes and molecular functions required during T. harzianum growth on FSCW or glucose. We identified various genes of biotechnological value encoding to proteins which function such as transporters, hydrolytic activity, adherence, appressorium development and pathogenesis. Fifteen genes were up-regulated and sixteen were down-regulated at least at one-time point during growth of T. harzianum in FSCW. During the confrontation assay most of the genes were up-regulated, mainly after contact, when the interaction has been established. Conclusions: This study demonstrates that T. harzianum expressed different genes when grown on FSCW compared to glucose. It provides insights into the mechanisms of gene expression involved in mycoparasitism of T. harzianum against F. solani. The identification and evaluation of these genes may contribute to the development of an efficient biological control agent.

Control biológico de Fusarium graminearum : utilización de Trichoderma spp. y biofumigación con parte aérea de Brassica juncea

Los objetivos de este trabajo fueron: determinar la factibilidad de la utilización combinada de dos métodos de control biológico: la aplicación del hongo antagonista Trichoderma spp. y la biofumigación con la parte aérea de Brassica juncea en el estadio de fin de fructificación; evaluar su efecto sobre el crecimiento del patógeno Fusarium graminearum. Se trituraron plantas de B. juncea y se colocaron en recipientes de plástico en dosis de 5 y 10 g. Sobre el material triturado se apoyó una caja de Petri con agar papa glucosado al 2%, que contenía un disco con micelio de F. graminearum o Trichoderma spp. o ambos hongos. Los recipientes de plástico se cerraron e incubaron a 25±2°C en oscuridad durante 7 días. Finalizado este período, se midió el diámetro de las colonias. Se obtuvieron los siguientes resultados: i) cuando se biofumigaron por separado, no se observó efecto fungistático de B. juncea sobre Trichoderma spp. ni sobre F. graminearum; ii) en ausencia del biofumigante, Trichoderma spp. inhibió significativamente el crecimiento de las colonias de F. graminearum, iii) la combinación de Trichoderma spp. y la biofumigación con B. juncea mostró un efecto sinérgico sobre el control del crecimiento miceliar de F. graminearum. Los resultados in vitro sugieren que el crecimiento de Trichoderma spp. y su potencial efecto de biocontrol sobre F. graminearum, no son afectados por la biofumigación con B. juncea. La utilización combinada de Trichoderma spp. y la biofumigación con B. juncea, tendría un efecto sinérgico sobre el control del crecimiento de F. graminearum.

Mycoparasitic interaction relieves binding of the Cre1 carbon catabolite repressor protein to promoter sequences of the ech42 (endochitinase-encoding) gene in Trichoderma harzianum

The fungus Trichoderma harzianum is a potent mycoparasite of various plant pathogenic fungi. We have studied the molecular regulation of mycoparasitism in the host/mycoparasite system Botrytis cinerea/T. harzianum. Protein extracts, prepared from various stages of mycoparasitism, were used in electrophoretic mobility-shift assays (EMSAs) with two promoter fragments of the ech-42 (42-kDa endochitinase-encoding) gene of T. harzianum. This gene was chosen as a model because its expression is triggered during mycoparasitic interaction [Carsolio, C., Gutierrez, A., Jimenez, B., van Montagu, M. & Herrera-Estrella, A. (1994) Proc. Natl. Acad. Sci. USA 91, 10903–10907]. All cell-free extracts formed high-molecular weight protein–DNA complexes, but those obtained from mycelia activated for mycoparasitic attack formed a complex with greater mobility. Competition experiments, using oligonucleotides containing functional and nonfunctional consensus sites for binding of the carbon catabolite repressor Cre1, provided evidence that the complex from nonmycoparasitic mycelia involves the binding of Cre1 to both fragments of the ech-42 promoter. The presence of two and three consensus sites for binding of Cre1 in the two ech-42 promoter fragments used is consistent with these findings. In contrast, the formation of the protein–DNA complex from mycoparasitic mycelia is unaffected by the addition of the competing oligonucleotides and hence does not involve Cre1. Addition of equal amounts of protein of cell-free extracts from nonmycoparasitic mycelia converted the mycoparasitic DNA–protein complex into the nonmycoparasitic complex. The addition of the purified Cre1::glutathione S-transferase protein to mycoparasitic cell-free extracts produced the same effect. These findings suggest that ech-42 expression in T. harzianum is regulated by (i) binding of Cre1 to two single sites in the ech-42 promoter, (ii) binding of a “mycoparasitic” protein–protein complex to the ech-42 promoter in vicinity of the Cre1 binding sites, and (iii) functional inactivation of Cre1 upon mycoparasitic interaction to enable the formation of the mycoparasitic protein–DNA complex.

The cellulose-binding domain of the major cellobiohydrolase of Trichoderma reesei exhibits true reversibility and a high exchange rate on crystalline cellulose.

Cellulose-binding domains (CBDs) bind specifically to cellulose, and form distinct domains of most cellulose degrading enzymes. The CBD-mediated binding of the enzyme has a fundamental role in the hydrolysis of the solid cellulose substrate. In this work we have investigated the reversibility and kinetics of the binding of the CBD from Trichoderma reesei cellobiohydrolase I on microcrystalline cellulose. The CBD was produced in Escherichia coli, purified, and radioactively labeled by reductive alkylation with 3H. Sensitive detection of the labeled CBD allowed more detailed analysis of its behavior than has been possible before, and important novel features were resolved. Binding of the CBD was found to be temperature sensitive, with an increased affinity at lower temperatures. The interaction of the CBD with cellulose was shown to be fully reversible and the CBD could be eluted from cellulose by simple dilution. The rate of exchange measured for the CBD-cellulose interaction compares well with the hydrolysis rate of cellobiohydrolase I, which is consistent with its proposed mode of action as a processive exoglucanase.

Molecular evidence that the asexual industrial fungus Trichoderma reesei is a clonal derivative of the ascomycete Hypocrea jecorina.

The relationship of the important cellulase producing asexual fungus Trichoderma reesei to its putative teleomorphic (sexual) ancestor Hypocrea jecorina and other species of the Trichoderma sect. Longibrachiatum was studied by PCR-fingerprinting and sequence analyses of the nuclear ribosomal DNA region containing the internal transcribed spacers (ITS-1 and ITS-2) and the 5.8S rRNA gene. The differences in the corresponding ITS sequences allowed a grouping of anamorphic (asexual) species of Trichoderma sect. Longibrachiatum into Trichoderma longibrachiatum, Trichoderma pseudokoningii, and Trichoderma reesei. The sexual species Hypocrea schweinitzii and H. jecorina were also clearly separated from each other. H. jecorina and T. reesei exhibited identical sequences, suggesting close relatedness or even species identity. Intraspecific and interspecific variation in the PCR-fingerprinting patterns supported the differentiation of species based on ITS sequences, the grouping of the strains, and the assignment of these strains to individual species. The variations between T. reesei and H. jecorina were at the same order of magnitude as found between all strains of H. jecorina, but much lower than the observed interspecific variations. Identical ITS sequences and the high similarity of PCR-fingerprinting patterns indicate a very close relationship between T. reesei and H. jecorina, whereas differences of the ITS sequences and the PCR-fingerprinting patterns show a clear phylogenetic distance between T. reesei/H. jecorina and T. longibrachiatum. T. reesei is considered to be an asexual, clonal line derived from a population of the tropical ascomycete H. jecorina.

Clonagem molecular, expressão, purificação e caracterização estrutural da endoglucanase de Trichoderma harzianum visando o desenvolvimento de coquetéis enzimáticos para a produção de etanol lignocelulósico

O aumento na demanda mundial por energia, a perspectiva de encolhimento dos recursos energéticos e a preocupação global com a questão ambiental, despertaram o interesse por fontes alternativas de energia. A biomassa lignocelulósica é abundante e de baixo custo, com potencial para complementar a produção em larga escala de combustíveis. A degradação das moléculas constituintes da parede celular à açúcares fermentescíveis e então à etanol, ocorre através da hidrólise enzimática da biomassa. Contudo, a utilização de enzimas para esse fim encontra-se em estágio exploratório e representa um gargalo na implementação de tecnologias de etanol 2G em escala industrial, desencadeando a busca de celulases bioquimicamente mais ativas, estáveis e economicamente viáveis. O presente trabalho visou a caracterização da endoglucanase I do fungo Trichoderma harzianum, e para isso foi realizada expressão, ensaios bioquímicos e biofísicos do domínio catalítico (ThCel7B-CCD) e da proteína inteira (ThCel7B-full). A enzima exibiu um perfil acidofílico, com atividade ótima em pH 3,0 a 55°C. A proteína também se mostrou capaz de hidrolisar uma variedade de substratos, sendo a maior atividade hidrolítica em β-glucano (75 U mg-1). Ao analisar a estabilidade térmica medida a 55°C em pH 5, a atividade residual manteve-se intacta por mais de 2 meses. Outra característica relevante foi o elevado grau de sinergismo entre ThCel7B e ThCel7A. Análises de microscopia eletrônica de flocos de aveia submetidas à hidrólise com ThCel7B evidenciaram os efeitos de degradação do substrato em relação às amostras controle. O conjunto desses resultados, além de importante para a compreensão do mecanismo molecular de ThCel7B e de outras endoglucanases da família GH7, também revelou uma enzima de interesse biotecnológicos uma vez que o comportamento ácido e sua estabilidade térmica são características relevantes para aplicações industriais sob condições extremamente ácidas.

Secretome analysis of Trichoderma atroviride T17 biocontrol of Guignardia citricarpa

The fungal species Guignardia citricarpa is an important pathogen in citriculture. Members of the fungal genus Trichoderma are recognized as biocontrol agents but studies on the interactions between both fungi are scarce. This study aimed to identify extracellular proteins secreted by Trichoderma atroviride T17 that are related to the control of G. citricarpa. Two-dimensional gel electrophoresis (2D) was used to study the patterns of proteins secreted by T. atroviride T17 in medium containing glucose (control) and in medium containing G. citricarpa GC3 inactivated mycelium. We identified 59 of the 116 spots differentially expressed (50.86%) by LC–MS/MS. Of these, we highlight the presence of glycoside hydrolases (CAZy families 3, 43, 54, 76 and 93), chitinase, mutanase, a-1,3-glucanase, a-1,2-mannosidase, carboxylic hydrolase ester, carbohydrate-binding module family 13, glucan 1,3-b-glucosidase, a-galactosidase and Neutral protease 2. These proteins are related to mycoparasitism processes, stimuli and therefore to the biological control of pathogens. The results obtained are in agreement with reports describing an increase in the secretion of proteins related to mycoparasitism and biological control and a reduction in the secretion of proteins related to the metabolism of Trichoderma species grown in the presence of the pathogen. Moreover, these results are pioneer in understanding T. atroviride interaction with G. citricarpa. For the first time, we identified potential candidate proteins that may have a role in the antagonism mechanism of G. citricarpa by T. atroviride T17. Thus our results shed a light into the molecular mechanisms that T. atroviride use to control G. citricarpa.

Caracterización del agente causal de la producción radicular del aguacate y Trichoderma sp., como potencial agente de control biológico en Nicaragua.

En el cultivo de aguacate (Persea americana Mill.) se presentan problemas fitosanitarios importantes dentro de los cuales sobresalen por su relevancia las enfermedades de la raíz. Un fitopatógeno limitante de este cultivo es el oomicete Phytophthora cinnamomi Rands, que puede causar pérdidas hasta del 90%. Por tal razón el principal objetivo del estudio fue generar información acerca de la etiología del agente causal de la pudrición radicular del aguacate utilizando marcadores morfológicos y moleculares, además de proponer alternativas de manejo de carácter biológico que estén enmarcadas dentro de un programa de manejo integrado de la enfermedad. Se realizaron colectas de muestras de suelo en cuatro localidades del departamento de Masaya. La identificación morfológica del patógeno se realizó mediante claves taxonómicas y se confirmó a través de la técnica PCR-RFLP. Se identificó a P. cinnamomi como el principal agente causal de la pudrición radicular del aguacate. Los aislados de P. cinnamomi fueron enfrentados con Trichoderma sp por el método de cultivo dual en cajas Petri con medio PDA. Se determinó el porcentaje de inhibición de crecimiento radial (PICR) a las 72 horas, así como el grado de antagonismo de cada una de las cepas de Trichoderma sp utilizadas en el estudio. Las cepas de Trichoderma al enfrentarlas a aislados del patógeno P. cinnamomi se ubicaron en las Clases 1 y 2 de la escala de evaluación, por lo tanto se consideraron altamente antagonistas. Existe la posibilidad de manejo biológico de las poblaciones de P. cinnamomi con microorganismos antagonistas del género Trichoderma no solamente en agroecosistemas de aguacate, sino también en otros sistemas agrícolas y forestales donde el patógeno esté presente.

Potential of Trichoderma harzianum for control of banana leaf fungal pathogens when applied with a food source and an organic adjuvant

Trichoderma isolates were obtained from diseased leaves and fruit collected from plantations in the main banana production area in Northern Queensland. Phylogenetic analyses identified the Trichoderma isolates as T. harzianum and T. virens. The Trichoderma spp. were found to be antagonistic against the banana leaf pathogens Mycosphaerella musicola, Cordana musae, and Deight-oniella torulosa in vitro. Several products used by the banana industry to increase production, including molasses, Fishoil and Seasol, were tested as food source for the Trichoderma isolates. The optimal food substrate was found to be molasses at a concentration of 5 %, which when used in combination with a di-1-p-menthene spreader-sticker enhanced the survivability of Trichoderma populations under natural conditions. This formulation suppressed D. torulosa development under glasshouse conditions. Furthermore, high sensitivity was observed towards the protectant fungicide Mancozeb but Biopest oil (R), a paraffinic oil, only marginally suppressed the growth of Trichoderma isolates in vitro. Thus, this protocol represents a potential to manage banana leaf pathogens as a part of an integrated disease approach.

Efeito de diferentes concentrações do óleo de nim no crescimento micelial de fungos entomopatogênicos e Trichoderma harzianum.

A associação de extratos de origem vegetal com fungos entomopatogênicos pode aumentar a eficiência do controle biológico de pragas e doenças de plantas e ainda reduzir custos e impactos ambientais. No presente trabalho foi avaliado o efeito do óleo de nim, incorporado ao meio BDA (Batata-dextrose-ágar) nas concentrações de 0, 1, 10, 100, 1.000, 10.000 e 100.000 μ/L, sobre o crescimento micelial de Metarhizium anisopliae, Beauveria bassiana, Trichoderma harzianum e Lecanicillium lecanii. O óleo de nim foi adicionado ao meio antes e após a esterilização em autoclave por 20 min. e 1 atm. Na parte central das placas de Petri contendo os meios foi transferido um disco de micélio dos agentes de biocontrole e mantidas em sala de incubação a 27 ± 2°C. O crescimento da colônia foi avaliado diariamente por seis dias, considerando dois sentidos do diâmetro. Cada placa correspondeu a uma repetição sendo cinco placas por tratamento. O óleo de nim adicionado antes e após autoclavagem em todas as concentrações testadas, estimularam o crescimento micelial de todos os agentes de biocontrole. Esta associação é de grande valia para o controle biológico, pode aumentar a eficiência destes fungos e reduzir impactos ambientais.

Trichoderma in Brazil: history, research, commercialization and perspectives.

2009