Bioconversion of antifungal viridin to phytotoxin viridiol by environmental non-viridin producing microorganisms.

Biotransformation of viridin, an antifungal produced by biocontrol agent, with non-viridin producing microorganisms is studied. The results show that some environmental non-targeted microorganisms are able to reduce it in the known phytotoxin viridiol, and its 3-epimer. Consequently, this reduction, which happens in some cases by detoxification mechanism, could be disastrous for the plant in a biocontrol of plant disease. However, a process fermentation/biotransformation could be an efficient approach for the preparation of this phytotoxin.

Efecto de la combinación de Metarhizium anisopliae y Gliocladium virens sobre la oviposición, eclosión y emergencia de Aedes aegypti / Effect of the combination of Metarhizium anisopliae and Gliocladium virens on the oviposition, hatching and emergency of Aedes aegypti

Resumen: Objetivo: Evaluar el efecto de la combinación de Metarhizium anisopliae y Gliocladium virens, ambos con Aqua Reslin Super, sobre oviposición, eclosión y emergencia de Aedes aegypti. Material y métodos: Se realizaron evaluaciones para determinar el efecto de los tratamientos impregnados en papel filtro y expuestos dentro de recipientes de plástico sobre la oviposición, eclosión y emergencia de Aedes aegypti. Resultados: Los resultados indicaron que las combinaciones hongo e insecticida no afectaron el comportamiento de oviposición, pero sí la eclosión de los huevos y la emergencia del adulto. Conclusión: Con los resultados se puede concluir que la combinación de hongos + insecticida puede ser una buena opción para aplicarse en sitios de oviposición con miras al desarrollo de una ovitrampa letal.

Abstract: Objective: To evaluate the effect of the combination of Metarhizium anisopliae and Gliocladium virens, both with Aqua Reslin Super, on the oviposition, hatching and emergence of Aedes aegypti. Materials and methods: Evaluations were carried out to determine the effect of treatments impregnated on filter paper and exposed within plastic containers on the oviposition, hatching and emergency of Aedes aegypti. Results: The results indicated that the fungus and insecticide combinations did not affect the oviposition behavior, but if the hatching of the eggs and the adult's emergency. Conclusion: With the results it can be concluded that the combination of fungi + insecticide can be a good option to be applied in oviposition sites with a view to the development of a lethal ovitrap.

[Effect of the combination of Metarhizium anisopliae and Gliocladium virens on the oviposition, hatching and emergency of Aedes aegypti]. / Efecto de la combinación de Metarhizium anisopliae y Gliocladium virens sobre la oviposición, eclosión y emergencia de Aedes aegypti.

OBJECTIVE: To evaluate the effect of the combination of Metarhizium anisopliae and Gliocladium virens, both with Aqua Reslin Super, on the oviposition, hatching and emergence of Aedes aegypti. MATERIALS AND METHODS: Evaluations were carried out to determine the effect of treatments impregnated on filter paper and exposed within plastic containers on the oviposition, hatching and emergency of Aedes aegypti. RESULTS: The results indicated that the fungus and insecticide combinations did not affect the oviposition behavior, but if the hatching of the eggs and the adult's emergency. CONCLUSIONS: With the results it can be concluded that the combination of fungi + insecticide can be a good option to be applied in oviposition sites with a view to the development of a lethal ovitrap.

OBJETIVO: Evaluar el efecto de la combinación de Metarhizium anisopliae y Gliocladium virens, ambos con Aqua Reslin Super, sobre oviposición, eclosión y emergencia de Aedes aegypti. MATERIAL Y MÉTODOS: Se realizaron evaluaciones para determinar el efecto de los tratamientos impregnados en papel filtro y expuestos dentro de recipientes de plástico sobre la oviposición, eclosión y emergencia de Aedes aegypti. RESULTADOS: Los resultados indicaron que las combinaciones hongo e insecticida no afectaron el comportamiento de oviposición, pero sí la eclosión de los huevos y la emergencia del adulto. CONCLUSIONES: Con los resultados se puede concluir que la combinación de hongos + insecticida puede ser una buena opción para aplicarse en sitios de oviposición con miras al desarrollo de una ovitrampa letal.

Antimicrobial activity and active compounds of a Rhus verniciflua Stokes extract.

The Rhus verniciflua Stokes (RVS) extract is used as a traditional herbal medicine in Southeast Asian countries such as Korea and China. In the present study, one phenolic acid and six flavonoids were isolated from an 80% ethanol RVS extract to examine their antimicrobial activities. These compounds were identified as 3',4',7-trihydroxyflavone (1), methyl gallate (2), gallic acid (3), fusti (4), fisetin (5), butin (6), and sulfuretin (7) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nuclear magnetic resonance spectroscopy. The antimicrobial activities of compounds 5 and 6 (at a dose of 16 µg/mL each) were superior to that of the control, cycloheximide (at a dose of 25 µg/mL), against Hypocrea nigricans; additionally, the activities of compounds 1 and 2 (at a dose of 8 µg/mL each) were superior to the control against Penicillium oxalicum. Also, chemical compounds 1 and 5 (at a dose of 16 µg/mL each) had higher activities than the control (25 µg/mL) against Trichoderma virens. Chemical compound 1 (at a dose of 8 µg/mL) had a similar activity to that of the control against Bacillus subtilis. The obtained results suggest that the RVS extract could be a promising food and nutraceutical source because of the antimicrobial properties of its phenolic compounds.

Inactivation kinetics and conformation change of Hypocrea orientalis ß-glucosidase with guanidine hydrochloride.

The relationship between unfolding and inactivation of Hypocrea orientalis ß-glucosidase has been investigated for the first time. The secretion of ß-glucosidase from H. orientalis is induced by raw cassava residues. The enzyme was 75 kD without glycosylation. Guanidine hydrochloride (GuHCl) could reversibly inactivate the enzyme with an estimated IC50 value of 0.4 M. The inactivation kinetics model by GuHCl has been established and the microscopic inactivation rate constants are determined. The values of forward inactivation rate constants of free enzyme are found to be larger than that of substrate-enzyme complex suggesting the enzyme could be protected by substrate during denaturation. Conformational change of the enzyme during denaturation is observed as the intrinsic fluorescence emission peaks appeared red-shift (334-354 nm) with intensity decreased following increase of GuHCl concentrations. Inactivation extent is found to be greater than conformation change of the whole enzyme, indicating that the active site of H. orientalis ß-glucosidase might be a more flexible region than the whole enzyme.

[Bacterium Arthrobacter agilis UMCV2 and diverse amines inhibit in vitro growth of wood-decay fungi]. / La bacteria Arthrobacter agilis UMCV2 y diversas aminas inhiben el crecimiento in vitro de hongos destructores de madera.

The kingdom Fungi is represented by a large number of organisms, including pathogens that deteriorate the main structural components of wood, such as cellulose, hemicellulose and lignin. The aim of our work was to characterize the antifungal activity in Arthrobacter agilis UMCV2 and diverse amines against wood-decaying fungi. Four fungal organisms (designated as UMTM) were isolated from decaying wood samples obtained from a forest in Cuanajo-Michoacán, México. Two of them showed a clear enzymatic activity of cellulases, xylanases and oxido-reducing enzymes and were identified as Hypocrea (UMTM3 isolate) and Fusarium (UMTM13 isolate). In vitro, the amines showed inhibitory effect against UMTM growth and one of the amines, dimethylhexadecylamine (DMA16), exhibited strong potential as wood preventive treatment, against the attack of decaying fungi.

Heat inactivation kinetics of Hypocrea orientalis ß-glucosidase with enhanced thermal stability by glucose.

Thermal inactivation kinetics of Hypocrea orientalis ß-glucosidase and effect of glucose on thermostability of the enzyme have been determined in this paper. Kinetic studies showed that the thermal inactivation was irreversible and first-order reaction. The microscopic rate constants for inactivation of free enzyme and substrate-enzyme complex were both determined, which suggested that substrates can protect ß-glucosidase against thermal deactivation effectively. On the other hand, glucose was found to protect ß-glucosidase from heat inactivation to remain almost whole activity below 70°C at 20mM concentration, whereas the apparent inactivation rate of BG decreased to be 0.3×10(-3)s(-1) in the presence of 5mM glucose, smaller than that of sugar-free enzyme (1.91×10(-3)s(-1)). The intrinsic fluorescence spectra results showed that glucose also had stabilizing effect on the conformation of BG against thermal denaturation. Docking simulation depicted the interaction mode between glucose and active residues of the enzyme to produce stabilizing effect.

La bacteria Arthrobacter agilis UMCV2 y diversas aminas inhiben el crecimiento in vitro de hongos destructores de madera / Bacterium Arthrobacter agilis UMCV2 and diverse amines inhibit in vitro growth of wood-decay fungi

El reino Fungi está representado por innumerable cantidad de organismos entre los cuales se encuentran hongos patógenos que deterioran los principales componentes estructurales de la madera, como celulosa, hemicelulosa y lignina. El objetivo de nuestro trabajo fue caracterizar la actividad antifúngica y la producción de diversas aminas de Arthrobacter agilis UMCV2 con acción antagónica sobre hongos xilófagos. Para ello, se aislaron 4 organismos fúngicos (designados en conjunto UMTM) a partir de madera en descomposición en un bosque de pino encino de la comunidad de Cuanajo, Michoacán, México. Dos de ellos presentaron una clara actividad enzimática de celulasas, xilanasas y enzimas accesorias óxido-reductoras, y fueron identificados como pertenecientes a 2 géneros agresivos para la madera: Hypocrea (aislado UMTM3) y Fusarium (aislado UMTM13). In vitro, las aminas evaluadas mostraron tener efecto inhibitorio sobre el crecimiento de los UMTM y la dimetilhexadecilamina; uno de estos compuestos mostró un fuerte potencial para ser utilizado como tratamiento preventivo contra el ataque de hongos destructores de madera.

The kingdom Fungi is represented by a large number of organisms, including pathogens that deteriorate the main structural components of wood, such as cellulose, hemicellulose and lignin. The aim of our work was to characterize the antifungal activity in Arthrobacter agilis UMCV2 and diverse amines against wood-decaying fungi. Four fungal organisms (designated as UMTM) were isolated from decaying wood samples obtained from a forest in Cuanajo-Michoacán, México. Two of them showed a clear enzymatic activity of cellulases, xylanases and oxido-reducing enzymes and were identified as Hypocrea (UMTM3 isolate) and Fusarium (UMTM13 isolate). In vitro, the amines showed inhibitory effect against UMTM growth and one of the amines, dimethylhexadecylamine (DMA16), exhibited strong potential as wood preventive treatment, against the attack of decaying fungi.

Identification of a genomic region containing a novel promoter resistant to glucose repression and over-expression of ß-glucosidase gene in Hypocrea orientalis EU7-22.

A high concentration of glucose in the medium could greatly inhibit the expression of cellulase in filamentous fungi. The aspartic protease from fungus Hypocrea orientalis EU7-22 could efficiently express under both induction condition and glucose repression condition. Based on the sequence of structure gene of aspartic protease, the upstream sequence harboring the putative promoter proA for driving the expression of aspartic protease was obtained by genome walking. The upstream sequence contained the typical promoter motifs "TATA" and "CAAT". The ß-glucosidase gene (Bgl1) from H. orientalis was cloned and recombined with promoter proA and terminator trpC. The expression cassette was ligated to the binary vector to form pUR5750-Bgl1, and then transferred into the host strain EU7-22 via Agrobacterium tumefaciens mediated transformation (ATMT), using hygromycin B resistance gene as the screening marker. Four transformants Bgl-1, Bgl-2, Bgl-3 and Bgl-4 were screened. Compared with the host strain EU7-22, the enzyme activities of filter paper (FPA) and ß-glucosidase (BG) of transformant Bgl-2 increased by 10.6% and 19.1% under induction condition, respectively. The FPA and BG activities were enhanced by 22.2% and 700% under 2% glucose repression condition, respectively, compared with the host strain. The results showed that the putative promoter proA has successfully driven the over-expression of Bgl1 gene in H. orientalis under glucose repression condition.

From an electrophoretic mobility shift assay to isolated transcription factors: a fast genomic-proteomic approach.

BACKGROUND: Hypocrea jecorina (anamorph Trichoderma reesei) is a filamentous ascomycete of industrial importance due to its hydrolases (e.g., xylanases and cellulases). The regulation of gene expression can influence the composition of the hydrolase cocktail, and thus, transcription factors are a major target of current research. Here, we design an approach for identifying a repressor of a xylanase-encoding gene. RESULTS: We used streptavidin affinity chromatography to isolate the Xylanase promoter-binding protein 1 (Xpp1). The optimal conditions and templates for the chromatography step were chosen according to the results of an electrophoretic mobility shift assay performed under repressing conditions, which yielded a DNA-protein complex specific to the AGAA-box (the previously identified, tetranucleotide cis-acting element). After isolating AGAA-box binding proteins, the eluted proteins were identified with Nano-HPLC/tandem MS-coupled detection. We compared the identified peptides to sequences in the H. jecorina genome and predicted in silico the function and DNA-binding ability of the identified proteins. With the results from these analyses, we eliminated all but three candidate proteins. We verified the transcription of these candidates and tested their ability to specifically bind the AGAA-box. In the end, only one candidate protein remained. We generated this protein with in vitro translation and used an EMSA to demonstrate the existence of an AGAA-box-specific protein-DNA complex. We found that the expression of this gene is elevated under repressing conditions relative to de-repressing or inducing conditions. CONCLUSIONS: We identified a putative transcription factor that is potentially involved in repressing xylanase 2 expression. We also identified two additional potential regulatory proteins that bind to the xyn2 promoter. Thus, we succeeded in identifying novel, putative transcription factors for the regulation of xylanase expression in H. jecorina.

Gene targeting in a nonhomologous end joining deficient Hypocrea jecorina.

The industrially applied ascomycete Hypocrea jecorina (synonym: Trichoderma reesei) exhibits a low rate of exogenous DNA integration by homologous recombination (HR). This hinders the high-throughput generation of strains by gene replacement and is therefore impeding systematic functional gene analyses towards, e.g. strain improvement for protein or enzyme production. To increase the rate of HR events during fungal transformation we identified and deleted the orthologue of the human KU70 in H. jecorina, which is required for the nonhomologous end joining (NHEJ) pathway and responsible for ectopic DNA integration. The effect of the absence of the H. jecorina tku70 on gene targeting was tested by deletion of two so far uncharacterized genes encoding a short chain dehydrogenase and a fungal specific transcription factor. Efficiency of gene targeting for both genes was >95% in a Deltatku70 strain when 1kb homologous flanking regions were used in the deletion construct. This is a significant increase in targeting efficiency compared to the parental - non-tku70 deleted - strain TU-6 where a gene knock-out frequency of only 5-10% was observed. Together with the recently annotated genomic sequence of H. jecorina, this system provides a useful tool for a genome-wide functional gene analysis on a high-throughput scale to improve the biotechnological potential of this fungus.

Sulphur metabolism and cellulase gene expression are connected processes in the filamentous fungus Hypocrea jecorina (anamorph Trichoderma reesei).

BACKGROUND: Sulphur compounds like cysteine, methionine and S-adenosylmethionine are essential for the viability of most cells. Thus many organisms have developed a complex regulatory circuit that governs the expression of enzymes involved in sulphur assimilation and metabolism. In the filamentous fungus Hypocrea jecorina (anamorph Trichoderma reesei) little is known about the participants in this circuit. RESULTS: Analyses of proteins binding to the cellulase activating element (CAE) within the promotor of the cellobiohydrolase cbh2 gene led to the identification of a putative E3 ubiquitin ligase protein named LIMPET (LIM1), which is an orthologue of the sulphur regulators SCON-2 of Neurospora crassa and Met30p of Saccharomyces cerevisiae. Transcription of lim1 is specifically up-regulated upon sulphur limitation and responds to cellulase inducing conditions. In addition, light dependent stimulation/shut down of cellulase gene transcription by methionine in the presence of sulphate was observed. Further, lim1 transcriptionally reacts to a switch from constant darkness to constant light and is subject to regulation by the light regulatory protein ENVOY. Thus lim1, despite its function in sulphur metabolite repression, responds both to light as well as sulphur- and carbon source. Upon growth on cellulose, the uptake of sulphate is dependent on the light status and essential for growth in light. Unlike other fungi, growth of H. jecorina is not inhibited by selenate under low sulphur conditions, suggesting altered regulation of sulphur metabolism. Phylogenetic analysis of the five sulphate permeases found in the genome of H. jecorina revealed that the predominantly mycelial sulphate permease is lacking, thus supporting this hypothesis. CONCLUSION: Our data indicate that the significance of the sulphate/methionine-related signal with respect to cellulase gene expression is dependent on the light status and reaches beyond detection of sulphur availability.