Control of Guignardia citricarpa by Bacillus subtilis and Trichoderma spp.

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Productivity, biological efficiency, and number of Agaricus blazei mushrooms grown in compost in the presence of Trichoderma sp. and Chaetomium olivacearum contaminants

Foi avaliado o efeito dos fungos contaminantes Trichoderma sp. e C. olivacearum na produtividade, eficiência biológica e número de cogumelos da produção do A. blazei em composto (mistura de cana-de-açúcar, palha de capim coast-cross, farelo de soja, gesso e calcário calcítico). O delineanamento foi inteiramente casualizado com três tratamentos (Trichoderma sp., C. olivacearum e testemunha) e oito repetições (caixa com 12 kg de composto colonizado com A. blazei). Após a colonização do composto pelo A. blazei, adicionou-se 150g de inóculo à base de triticale de cada um dos fungos contaminantes na superfície do composto seguido da camada de cobertura. O experimento foi conduzido em estufa com cobertura plástica, umidade relativa entre 60-90% e temperatura de 20-34ºC. A produtividade foi determinada pela relação entre a massa fresca de basidiomas e a massa úmida do composto. A eficiência biológica foi determinada pela relação entre a massa fresca de basidiomas e a massa seca do composto ao final do período de colheita. de acordo com os resultados obtidos, os fungos contaminantes C. olivacearum e Trichoderma sp. não afetaram a produtividade, eficiência biológica e número de cogumelos da produção do A. blazei em compostos previamente colonizados.

Enzymatic hydrolysis of botryosphaeran and laminarin by beta-1,3-glucanases produced by Botryosphaeria rhodina and Trichoderma harzianum Rifai

Botryosphaeran, a (1 -> 3; 1 -> 6)-beta-D-glucan produced by Botryosphaeria rhodina, and laminarin were hydrolysed by two fungal beta-glucanases predominantly of the 1,3-type produced by B. rhodina and Trichoderma harzianum Rifai grown on botryosphaeran as sole carbon source. Both beta-glucanase preparations presented different modes of attack on botryosphaeran and laminarin. Laminarin was hydrolysed to the extent of similar to 50% in 1 hand 100% within 24 h, and its hydrolysis products were mainly glucose and gentiobiose, and lesser amounts of laminaribiose and oligosaccharides of DP 3-4 during the early stages of hydrolysis, while botryosphaeran 'yielded mainly glucose and gentiobiose with some trisaccharide, but no laminaribiose or tetrasaccharide when hydrolysed by the T. harzianum enzyme. By contrast, B. rhodina beta-1,3-glucanases produced predominantly glucose during all stages of botryosphaeran hydrolysis. Some physicochemical properties of the 1,3- and 1,6-beta-glucanases, and beta-glucosidases contained in the two fungal P-glucanase preparations are also described for the first time. (c) 2006 Elsevier Ltd. All rights reserved.

Botryosphaeran, a new substrate for the production of beta-1,3-glucanases by Botryosphaeria rhodina and Trichoderma harzianum Rifai

Botryosphaeria rhodina and Trichoderma harzianum Rifai were grown on botryosphaeran (an exopolysaccharide (EPS) of the beta-1,3; 1,6-D-Glucan type produced by B. rhodina) as sole carbon source with the objective of producing beta-glucanases of the beta-type. Conditions for beta-1,3-glucanase production by T harzianum were examined by a statistical response surface method, and showed maximal enzyme production at 5 days growth in media containing 1.5 g/1 of EPS. Good agreement was obtained between the experimental values of beta-1, 3-glucanase activity and the corresponding values predicted by the mathernatical model. The crude beta-1,3-glucanase preparations were active towards a number of different beta-1,3-glucans and beta-glucosides. The mycelium of B. rhodina also proved to be a good substrate for beta-1,3-glucanase production by both fungal species. (c) 2005 Published by Elsevier Ltd.

Comparative growth of trichoderma strains in different nutritional sources, using bioscreen c automated system

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Production and characterization of cellulase-free xylanase from Trichoderma inhamatum

The production of extracellular cellulase-free xylanase from Trichoderma inhamatum was evaluated in liquid Vogel medium with different carbon sources as natural substrates and agricultural or agro-industrial wastes. Optimal production of 244.02 U/mL was obtained with xylan as carbon source, pH 6.0 at 25 degrees C, 120 rpm, and 60-h time culture. Optimal conditions for enzyme activity were 50 degrees C and pH 5.5. Thermal stability of T. inhamatum xylanolytic complex expressed as T(1/2) was 2.2 h at 40 degrees C and 2 min at 50 degrees C. The pH stability was high from 4.0 to 11.0.These results indicate possible employment of such enzymatic complex in some industrial processes which require activity in acid pH, wide-ranging pH stability, and cellulase activity absence.

Evaluation of the beta-glucanolytic enzyme complex of Trichoderma harzianum Rifai for the production of gluco-oligosaccharide fragments by enzymatic hydrolysis of 1,3;1,6-beta-D-glucans

Botryosphaeran, a new exopolysaccharide from the endophytic fungus Botryosphaeria rhodina MAMB-05, and algal laminarin were hydrolyzed by partially-fractionated enzymes of the beta-glucanolytic complex from Trichoderma harzianum Rifai. beta-Glucanase fractions (F-I and F-II) separated by gel permeation chromatography presented different modes of attack on botryosphaeran and laminarin. Botryosphaeran was hydrolyzed to the extent of 66% (F-I) and 98% (F-II) within 30 min, and its main hydrolysis products were gluco-oligosaccharides of DP >= 4, with lesser amounts of glucose, di- and tri-saccharides. The action of enzyme fractions I and II on laminarin resulted in 15% conversion to glucose, while the percentage of saccharification was radically different (70% for F-I and 25% for F-II). The different product arrays within the polysaccharide hydrolysates can be explained by the difference in the enzymes' specificities within each enzyme fraction, and the molecular structures of the polysaccharides and their complexity.

Impacto de herbicidas em isolados de Trichoderma spp.

O uso de microrganismos é uma alternativa para o controle de doenças em plantas. Todavia, é prudente verificar a interação desse com os demais métodos de controle empregados em determinada cultura. Dessa forma, objetivou-se avaliar a fungitoxicidade dos herbicidas sobre o crescimento e desenvolvimento dos isolados de Trichoderma spp. Utilizou-se o delineamento inteiramente casualizado, em esquema fatorial 6 x 6 x 4, com quatro repetições. O fator A correspondeu aos herbicidas pendimethalin, clomazone, carfentrazone-ethyl, oxadiazon, thiobencarb + propanil e byspiribac-sodium; o fator B, às doses dos herbicidas - 0, 25, 50, 75, 100 e 200% da dose recomendada; e o fator C, aos isolados de Trichoderma spp. AJAM 18, CE 66, TRI 01 e TRI 02. O ensaio foi realizado em condições in vitro; avaliaram-se o crescimento micelial radial (CMR) e a esporulação dos isolados após aplicação dos herbicidas. Observaram-se diferenças de sensibilidade dos isolados para o mesmo produto testado. O oxadiazon reduziu o CMR dos isolados AJAM 18 e TRI 01 em 66 e 35%, respectivamente. No entanto, reduziu apenas 16% do CMR do isolado TRI 02 e não alterou o CMR do isolado CE 66 mesmo em 200% da dose recomendada. Verificaram-se diferentes efeitos dos produtos em cada isolado. A mistura comercial de thiobencarb+propanil foi altamente tóxica aos isolados de Trichoderma spp., com reduções em torno de 85% no CMR e no número de esporos. Por outro lado, o byspiribac-sodium pouco afetou os isolados, apresentando reduções inferiores a 10% no CMR e na esporulação. O carfentrazone-ethyl e byspiribac-sodium demonstraram ser compatíveis com os isolados de Trichoderma spp. estudados.

Filtration enrichment method for isolation of auxotrophic mutants of Trichoderma harzianum rifai

A obtenção de marcas genéticas, quer sejam para resistência a drogas, quer para auxotrofia, é uma etapa trabalhosa mas importante em pesquisa genética. Esse trabalho visou a obtenção de mutantes auxotróficos de Trichoderma harzianum utilizando-se a técnica de enriquecimento por filtração. A técnica mostrou-se superior à técnica convencional de isolamento total. Doze mutantes auxotróficos obtidos foram testados quanto a estabilidade, crescimento e resistência ao fungicida benomil. Eles apresentaram taxas de crescimento e esporulação comparáveis à linhagem parental e dois mutantes foram resistentes a benomil em uma concentração de 500µg/ml.

Genetic recombinants in Trichoderma pseudokoningii (Rifai) without typical parasexuality

Heterokaryons were obtained by hyphal anastomosis in Trichoderma pseudokoningii using strains with morphologic and double auxotrophic genetic markers. Stable haploid and unstable hyperhaploid recombinants were detected in monosporic colonies derived from heterokaryons and selected in appropriate media or by an enrichment filtration technique. Diploids were not observed and the recombinant frequencies were higher than those found in fungi with a typical parasexual cycle. The mechanism of recombinant emergence may involve a transient diploid formation followed by chromosomal losses at the hyphal stages, resulting in haploid and hyperhaploid recombinants. The possibility of intrachromosomal rearrangement with the involvement of cytoplasmatic DNA as a heterokaryotic transformation system cannot be excluded.

Atividade antagônica in vitro de isolados de Trichoderma spp. ao fungo Phytophthora citrophthora

Gummosis is among the main fungal diseases of the citrus. It is caused by Phytophthora sp. and usually shows up in the lap of the plant, provoking rottenness and gum exudation, and expands causing the plant death for constrictions in the cambium or phloem which interrupts the descending flow of sap. The objective of this work was to evaluate the antagonistic in vitro activity of Trichoderma spp. to the fungi Phytophthora citrophthora. Phytophthora citrophthora was exposed to five environments of antagonism (without antagonist and with four strains of Trichoderma viride, T. virens, T. harzianu and T stromaticum), The in vitro essay was accomplished through the method of paired cultures. A completely randomized desing was used with five treatments and three replications, and each plot was represented by three petri dishes. The isolates of Trichoderma demonstrated significant effect in the inhibition of the mycelial growth of the fungi Phytophthora citrophthora, and the fungi Trichoderma stromaticum presented larger antagonism to the fungi P. citrophthora while the T harzianum presented antagonism smaller.

Cellulolytic activity of wild type and mutant Trichoderma pseudokoningii

Strains of Trichoderma pseudokoningii are promising objects for genetic studies and cellulase production. Auxotrophic mutants with deficiencies in the biosynthesis of aminoacids, nucleotides and vitamins (up to five markers) in addition to morphological aspects like conidial colour were obtained from two strains of double auxotrophic mutants using UV radiation. In order to compare the cellulolytic capabilities of the T. pseudokoningii (wild type strain), some of its mutants and T. reesei QM9414 we performed semiquantitative cellulase assays and quantitative determination of the enzymes exoglucanase and endoglucanase. The semiquantitative test showed that the strains with minimal mycelial growth rate were better producers. Both tests revealed that two of the studied mutants, TG3 and TG4 presented a yield higher than the wild type, reaching 30% more exoglucanase and 70% more endoglucanase. These results indicate that the wild type was improved for cellulase production. Highly significant values of correlation were found for exoglucanase and endoglucanase activities, suggesting that these enzymes may be co-regulated in T. pseudokoningii.

Comparison of β-1,3-glucanase production by Botryosphaeria rhodina MAMB-05 and Trichoderma harzianum Rifai and its optimization using a statistical mixture-design

Botryosphaeria rhodina MAMB-05 produced β-1,3-glucanases and botryosphaeran when grown on glucose, while Trichoderma harzianum Rifai only produced the enzyme. A comparison of long-term cultivation (300h) by B. rhodina demonstrated a correlation between the formation of botryosphaeran (48h) and its consumption (after 108h), and de-repression of β-1,3-glucanase synthesis when glucose was depleted from the nutrient medium, whereas for T. harzianum enzyme production commenced during exponential growth. Growth profiles and levels of β-1,3-glucanases produced by both fungi on botryosphaeran also differed, as well as the production of β-1,3-glucanases and β-1,6-glucanases on glucose, lactose, laminarin, botryosphaeran, lasiodiplodan, curdlan, Brewer's yeast powder and lyophilized fungal mycelium, which were dependent upon the carbon source used. A statistical mixture-design used to optimize β-1,3-glucanase production by both fungi evaluated botryosphaeran, glucose and lactose concentrations as variables. For B. rhodina, glucose and lactose promoted enzyme production at the same levels (2.30UmL -1), whereas botryosphaeran added to these substrates exerted a synergic effect favorable for β-glucanase production by T. harzianum (4.25UmL -1). © 2010 Elsevier B.V.

Agaricus blazei as a Substrate for the Production of β-1,3-Glucanase by Trichoderma harzianum Rifai

Extracellular β-1,3-glucanase was produced by Trichoderma harzianum Rifai cultivated in the Agaricus blazei (Agaricus brasiliensis) extract as a substrate in submerged fermentation. A 22-central composite factorial design was developed using the time of culture (x1/day) and Agaricus blazei extract concentration (x2/(g/L)) as variables, and the results were analyzed using response surface methodology (RSM). The results showed that the Agaricus blazei extract concentration was the most important variable in the production of β-1,3-glucanase, and the maximum β-1,3-glucanase activity (0.77 U/mL) was obtained in one day of cultivation. The β-glucan present in the cell wall of Agaricus blazei mushroom proved to be a good substrate for inducing the production of specific β-1,3-glucanase by Trichoderma harzianum Rifai.

Comparing submerged and solid-state fermentation of agro-industrial residues for the production and characterization of lipase by Trichoderma harzianum

Lipase production by Trichoderma harzianum was evaluated in submerged fermentation (SF) and solid-state fermentation (SSF) using a variety of agro-industrial residues. Cultures in SF showed the highest activity (1.4 U/mL) in medium containing 0.5 % (w/v) yeast extract, 1 % (v/v) olive oil and 2.5 C:N ratio. This paper is the first to report lipase production by T. harzianum in SSF. A 1:2 mixture of castor oil cake and sugarcane bagasse supplemented with 1 % (v/w) olive oil showed the best results among the cultures in SSF (4 U/g ds). Lipolytic activity was stable in a slightly acidic to neutral pH, maintaining 50 % activity after 30 min at 50 C. Eighty percent of the activity remained after 1 h in 25 % (v/v) methanol, ethanol, isopropanol or acetone. Activity was observed with vegetable oils (olive, soybean, corn and sunflower) and long-chain triacylglycerols (triolein), confirming the presence of a true lipase. The results of this study are promising because they demonstrate an enzyme with interesting properties for application in catalysis produced by fermentation at low cost. © 2012 Springer-Verlag and the University of Milan.