Trichoderma koningiopsis (Hypocreaceae) has the smallest mitogenome of the genus Trichoderma.

Introduction: Fungal mitogenomes exhibit remarkable variation in conformation, size, gene content, arrangement and expression, including their intergenic spacers and introns. Methods: The complete mitochondrial genome sequence of the mycoparasitic fungus Trichoderma koningiopsis was determined using the Illumina next-generation sequencing technology. We used data from our recent Illumina NGS-based project of T. koningiopsis genome sequencing to study its mitochondrial genome. The mitogenome was assembled, annotated, and compared with other fungal mitogenomes. Results: T. koningiopsis strain POS7 mitogenome is a circular molecule of 27,560 bp long with a GC content of 27.80%. It harbors the whole complement of the 14 conserved mitochondrial protein-coding genes (PCG) such as atp6, atp8, atp9, cox1, cox2, cox3, cob, nad1, nad2, nad3, nad4, nad4L, nad5, and nad6, also found in the same gene order to other Hypocreales. The mitogenome also contains 26 transfer RNA genes (tRNAs), 5 of them with more than one copy. Other genes also present in the assembled mitochondrial genome are a small rRNA subunit and a large rRNA subunit containing ribosomal protein S3 gene. Despite the small genome size, two introns were detected in the T. koningiopsis POS7 mitogenome, one of them in cox3 gene and the other in rnl gene, accounting 7.34% of this mitogenome with a total size of 2,024 bp. A phylogenetic analysis was done using the 14 PCGs genes of T. koningiopsis strain POS7 mitogenome to compare them with those from other fungi of the Subphyla Pezizomycotina and Saccharomycotina. T. koningiopsis strain POS7 was clustered together with other representatives of Trichoderma lineage, within the Hypocreales group, which is also supported by previous phylogenetic studies based on nuclear markers. Discussion: The mitochondrial genome of T. koningiopsis POS7 will allow further investigations into the taxonomy, phylogenetics, conservation genetics, and evolutionary biology of this important genus as well as other closely related species.

Low-cost homemade cocktails for enzymatic conversion of sugarcane and cassava bagasses.

The agricultural industries generate lignocellulosic wastes that can be modified by fungi to generate high value-added products. This work aimed to analyze the efficiency and the cost-effectiveness of the bioconversion of sugarcane and cassava bagasses using low-cost homemade enzymatic cocktails from Aspergillus niger LBM 134. Both bagasses were pretreated with a soft alkaline solution without any loss of polysaccharides. After the hydrolysis, a 28% of conversion to glucose and 42% to xylose were reached in the hydrolysis of sugarcane bagasse while an 80% of saccharification yield, in the hydrolysis of cassava bagasse using the homemade enzymes. Furthermore, a more disorganised surface and no starch granules were observed in the sugarcane and cassava bagasses, respectively. The bioethanol yield from sugarcane and casava bagasses was predicted to be 4.16 mg mL-1 and 2.57 mg mL-1, respectively. A comparison of the cost of the homemade and the commercial enzymes was carried out. Similar hydrolysis percentages were achieved employing any enzyme; however, it was 1000-2000 times less expensive using the homemade cocktails than using the commercial enzymes. Therefore, the cost of obtaining glucose from bagasses was most expensive when applying the commercial enzymes. Moreover, the hydrolysis of the cassava bagasse was most efficient with the homemade cocktails. The importance and novelty of this work lie in the similar performance and the lower cost of the homemade cocktails from the fungus A. niger LBM 134 compared with the commercial enzymes on the hydrolysis of the sugarcane and cassava bagasses.

Aspergillus niger LBM 134 isolated from rotten wood and its potential cellulolytic ability.

Aspergillus is a genus of filamentous and cosmopolitan fungi that includes important species for medical mycology, food, basic research and agro-industry areas. Aspergillus section Nigri are efficient producers of hydrolytic enzymes such as cellulases that are employed in the cellulose conversion. Hence, the search of new cellulolytic isolates and their correct identification is important for carrying out safe biotechnological processes. This study aimed to characterise the cellulolytic potential of Aspergillus sp. LBM 134, isolated from the Paranaense rainforest (Argentina) and to identify the isolate through a polyphasic approach. The fungus was identified as Aspergillus niger and its cellulolytic potential was evaluated by using Congo red technique and fluorescence plate assays for carboxymethyl cellulase, ß-glucosidase and cellobiohydrolase, respectively. All three cellulase activities were positive; this bio-prospective positioned A. niger LBM 134 as a promising alternative for industries that require organisms capable of carrying out cellulosic biomass processing.

Evaluation of bioremediation strategies for treating recalcitrant halo-organic pollutants in soil environments.

The aim of this study was to investigate the bioremediation potential of polychlorinated biphenyls (PCBs) in soil, mimicking three strategies: (a) mycoaugmentation: by the addition of Trametes sanguinea and Pleurotus sajor-caju co-cultures immobilized on sugarcane bagasse; (b) biostimulation: by supplementation of sugarcane bagasse; and (c) natural attenuation: no amendments. The experiments were done in microcosms using Ultisol soil. Remediation effectiveness was assessed based on pollutants content, soil characteristics, and ecotoxicological tests. Biostimulation and mycoaugmentation demonstrated the highest PCBs-removal (approx. 90%) with a significant toxicity reduction at 90 d. The studied strains were able to survive during the incubation period in non-sterilized soil. Laccase, manganese-peroxidase and endoxylanase activities increased significantly in co-cultures after 60 d. Sugarcane bagasse demonstrated to be not only a suitable support for fungal immobilization but also an efficient substrate for fungal colonization of PCBs-contaminated soils. Mycoaugmentation and biostimulation with sugarcane bagasse improved oxidable organic matter and phosphorous contents as well as dehydrogenase activity in soil. Therefore, biostimulation with sugarcane bagasse and mycoaugmentation applying dual white-rot fungal cultures constitute two efficient bioremediation alternatives to restore PCBs-contaminated soils.

Secretomic analysis of cheap enzymatic cocktails of Aspergillus niger LBM 134 grown on cassava bagasse and sugarcane bagasse.

Currently, agroindustrial wastes are little used for generating value-added products; hence, their use of these waste to produce enzymatic cocktails for the conversion of lignocellulosic biomass to fermentable sugars is a very interesting alternative in the second-generation bioethanol process. The Ascomycota fungus Aspergillus niger LBM 134 produces hydrolytic enzymes in large proportions. In this work, A. niger LBM 134 was grown on sugarcane and cassava bagasses under optimized conditions. To identify the extracellular enzymes involved in the degradation of these agroindustrial wastes, the secretomes of the culture supernatants of the fungus were analyzed and validated by biochemical assays of the enzymatic activities. A. niger LBM 134 secreted higher quantities of xylanases and accessory hemicellulases when it grew on sugarcane bagasse, whereas more cellulases, amylases, and pectinases were secreted when it grew on cassava bagasse. These findings suggest two promising enzyme cocktails for the hydrolysis of lignocellulose carbohydrate polymers to fermentable sugars. These bioinformatic analysis were functional validates through enzymatic biochemical assays that confirm the biotechnological potential of A. niger LBM 134 for the bioconversion of hemicellulosic substrates such as sugarcane and cassava bagasses.

Trichoderma spp. from Misiones, Argentina: effective fungi to promote plant growth of the regional crop Ilex paraguariensis St. Hil.

Ilex paraguariensis St. Hil (yerba mate) is an important crop in the north of Argentina, mainly in Misiones province. The application of Trichoderma as a biocontroller and biofertilizer can replace or reduce the use of agrochemicals, decreasing the negative ecological impact. In this research, we evaluated in vitro and in vivo antagonistic and plant growth promoting (PGP) properties of Trichoderma species isolated from different regions of Misiones province. Dual culture assays of Trichoderma against phytopathogenic fungi associated with yerba mate showed that T. stilbohypoxyli LBM 120 was the most effective antagonist, inhibiting in more than 75% all phytopathogen growth. Trichoderma atroviride LBM 112 and T. stilbohypoxyli LBM 120 were positive on endoglucanase, protease, chitinase, siderophore production, and phosphate solubilisation showed the best biological control agents and PGP properties. The PGP properties of Trichoderma spp. evaluated in vivo on yerba mate seedlings showed that T. atroviride LBM 112, T. stilbohypoxyli LBM 120, and T. koningiopsis LBM 219 enhanced plant dry weight over 47% in total and 24% in the aerial part. Moreover, T. koningiopsis LBM 219 increased root dry weight 25% in contrast with in vitro controls. In conclusion, native Trichoderma strains could be a sustainable solution to improve yerba mate yield.

Assessing the ability of white-rot fungi to tolerate polychlorinated biphenyls using predictive mycology.

The aim of the present study was to assess the ability of different white-rot fungi to tolerate polychlorinated biphenyls (PCBs) using predictive mycology, by relating fungal growth inhibition to ligninolityc enzyme secretion. Fungal strains were grown in the presence of PCBs in solid media and their radial growth values were modelled through the Dantigny-logistic like function in order to estimate the time required by the fungal colonies to attain half their maximum diameter. The principal component analysis (PCA) revealed an inverse correlation between strain tolerance to PCBs and the laccase secretion over time, being laccase production closely associated with fungal growth capacity. Finally, a PCA was run to regroup and split between resistant and sensitive fungi. Simultaneously, a function associated with a model predicting the tolerance to PCBs was developed. Some of the assayed isolates showed a promising capacity to be applied in PCB bioremediation. Abbreviations: Polychlorinated biphenyls (PCBs), white-rot fungi (WRF).

Cellulolytic ability of a promising Irpex lacteus (Basidiomycota: Polyporales) strain from the subtropical rainforest of Misiones province, Argentina / Capacidad celulolítica de una cepa prometedora de Irpex lacteus (Basidiomycota: Polyporales) de la selva subtropical de la provincia de Misiones, Argentina

Abstract The cellulolytic activity of fungi growing in the subtropical rainforest of Misiones (Argentina) represents a challenge in the technological development of the production of cellulosic bioethanol in the region using native sources. These fungi are promising to obtain sustainable enzyme cocktails using their enzymes. Cellulolytic ability of 22 white-rot fungi isolated from the subtropical rainforest of Misiones-Argentina in agar medium with two types of cellulosic substrates, carboxy-methylcellulose or crystalline cellulose, were comparatively analyzed, and the activity of two cellulolytic enzymes was evaluated in liquid medium. Although all isolates were able to grow and degrade both substrates in agar medium, and to produce total cellulase Filter paper (FPase) and endo-β-1,4-glucanase (EG) activities in broth, the isolate Irpex sp. LBM 034 showed the greatest enzymatic levels (FPase, 65.45 U L-1; EG, 221.21 U L-1). Therefore, the ITS sequence of this fungus was sequenced and analyzed through a phylogenetic analysis. These results indicate that the isolate LBM 034, corresponding to Irpex lacteus, has a promising cellulolytic ability and enzymes such as EG useful in sustainable saccharification of cellulosic materials in the region. Rev. Biol. Trop. 66(3): 1034-1045. Epub 2018 September 01.

Resumen La actividad celulolítica de hongos autóctonos asociados a la selva subtropical de Misiones (Argentina) representa un desafío en el desarrollo tecnológico de la producción de bioetanol celulósico en la región, mediante el uso de recursos nativos. Los sistemas enzimáticos de estos hongos tienen potencial aplicación en la obtención de cocteles enzimáticos rentables. La habilidad celulolítica de 22 hongos causantes de pudrición blanca se analizó comparativamente, que fueron aislados de la selva subtropical de Misiones-Argentina, en cultivos agarizados con dos tipos de sustratos celulósicos, carboxi-metilcelulosa o celulosa cristalina. También se evaluó la actividad de dos enzimas celulolíticas en cultivos líquidos. Aunque todos los aislamientos fueron capaces de crecer y degradar ambos sustratos en medio agarizado y revelar actividad celulolítica total y endo-β-1,4-glucanasa en cultivo líquido, el aislamiento Irpex sp. LBM 034 mostró las mayores actividades en papel de filtro con 65.45 U L-1 y endo-β-1,4-glucanasa con 221.21 U L-1, respectivamente. Por tanto, se secuenció y analizó la secuencia ITS de este hongo a través de un análisis filogenético. Estos resultados indicaron que el aislamiento LBM 034, correspondiente a Irpex lacteus, tiene una habilidad celulolítica prometedora en la producción de enzimas con actividad endo-β-1,4-glucanasa, útil en la sacarificación sustentable de materiales celulósicos de la región.

First Whole-Genome Shotgun Sequence of a Promising Cellulase Secretor, Trichoderma koningiopsis Strain POS7.

Trichoderma koningiopsis strain POS7 produces significantly large amounts of cellulase enzymes in solid-state fermentation. The Illumina-based sequence analysis reveals an approximate genome size of 36.6 Mbp, with a G+C content of 48.82% for T. koningiopsis POS7. Based on ab initio prediction, 12,661 coding genes were annotated.

Preliminary studies of new strains of Trametes sp. from Argentina for laccase production ability

Abstract Oxidative enzymes secreted by white rot fungi can be applied in several technological processes within the paper industry, biofuel production and bioremediation. The discovery of native strains from the biodiverse Misiones (Argentina) forest can provide useful enzymes for biotechnological purposes. In this work, we evaluated the laccase and manganese peroxidase secretion abilities of four newly discovered strains of Trametes sp. that are native to Misiones. In addition, the copper response and optimal pH and temperature for laccase activity in culture supernatants were determined.The selected strains produced variable amounts of laccase and MnP; when Cu2+ was added, both enzymes were significantly increased. Zymograms showed that two isoenzymes were increased in all strains in the presence of Cu2+. Strain B showed the greatest response to Cu2+ addition, whereas strain A was more stable at the optimal temperature and pH. Strain A showed interesting potential for future biotechnological approaches due to the superior thermo-stability of its secreted enzymes.

Preliminary studies of new strains of Trametes sp. from Argentina for laccase production ability.

Oxidative enzymes secreted by white rot fungi can be applied in several technological processes within the paper industry, biofuel production and bioremediation. The discovery of native strains from the biodiverse Misiones (Argentina) forest can provide useful enzymes for biotechnological purposes. In this work, we evaluated the laccase and manganese peroxidase secretion abilities of four newly discovered strains of Trametes sp. that are native to Misiones. In addition, the copper response and optimal pH and temperature for laccase activity in culture supernatants were determined. The selected strains produced variable amounts of laccase and MnP; when Cu(2+) was added, both enzymes were significantly increased. Zymograms showed that two isoenzymes were increased in all strains in the presence of Cu(2+). Strain B showed the greatest response to Cu(2+) addition, whereas strain A was more stable at the optimal temperature and pH. Strain A showed interesting potential for future biotechnological approaches due to the superior thermo-stability of its secreted enzymes.

Characterizatio of the oxidative enzyme potental in wild white rot fungi fromm the subtropical forest of misiones (Argentina) / Caracterización del potencial enzimático oxidativo de cepas nativas de hongos de pudrición blanca de la selva subtropical de Misiones (Argentina)

This research aimed to evaluate the potential of several native white rot fungi (WRF) isolated from subtropical environments of Misiones (Argentina) to produce different ligninolytic enzymes. Coriolus versicolor f. antarcticus BAFC 266, Pycnoporus sanguineus BAFC 2126 and Phlebia brevispora BAFC 633 showed the highest phenoloxidase activity. Ganoderma applanatum strain E, P. sanguineus BAFC 2126 and P. brevispora BAFC 633 revealed marked laccase and peroxidase activity. C. versicolor f. antarcticus, G. applanatum (strain A) and Trametes villosa, gave high positive reactions with 2,6-dimethoxyphenol oxidation at the lowest tested pH. C. versicolor f. antarcticus, G. applanatum strains D and F, T. elegans BAFC 2127and T. villosa, showed the highest manganese peroxidase activity. C. versicolor f. antarcticus also produced the highest lignin peroxidase activity. Tyrosinase activity was mostly evident in G. applanatum strains (D and F) and Phanerochaete chrysosporium HHB 11741. Kraft liquor decolorization results were variable and depended on the fungus and the liquor concentration. Some fungi with moderate ligninolytic activity showed high decolorization rates (e.g. Pleurotus sajor-caju and Steccherinium sp. BAFC 1171) indicating the significance of additional approach to evaluate a potential biotechnological application.

El objetivo de este trabajo fue evaluar el potencial para producir enzimas ligninolíticas de diversas cepas de hongos de pudrición blanca, nativas de la Provincia de Misiones (Argentina). Coriolus versicolor v. antarcticus BAFC 266, Pycnoporus sanguineus BAFC 2126 y Phlebia brevispora BAFC 633 mostraron un gran potencial para producir fenoloxidasas. En Ganoderma applanatum cepa E, P. sanguineus BAFC 2126 y P. brevispora BAFC 633 se observó una marcada actividad lacasa y peroxidasa. C. versicolor v. antarcticus. G. applanatum (cepa A) y Trametes villosa, mostraron una reacción oxidativa altamente positiva sobre 2,6-dimetoxifenol a pH 4. C. versicolor v. antarcticus, G. applanatum (cepas D y F), T. elegans BAFC 2127 y T. villosa, mostraron una alta actividad manganeso peroxidasa y solo C. versicolor v. antarcticus mostró una alta actividad de lignino peroxidasa. La actividad tirosinasa fue evidente en las cepas D y F de G. applanatum y en Phanerochaete chrysosporium HHB 11741. La decoloración del licor negro Kraft reveló una actividad ligninolítica variable para las diferentes cepas estudiadas dependiendo de la concentración utilizada. Algunos hongos mostraron una moderada actividad ligninolítica con grandes halos de decoloración (p.e. Pleurotus sajor-caju y Steccherinium sp. BAFC 1171) indicando un significativo potencial para el aprovechamiento biotecnológico de estas cepas.

Effect of chemical and metallic compounds on biomass, mRNA levels and laccase activity of Phlebia brevispora BAFC 633.

Nine aromatic compounds (caffeic acid, syringaldehyde, vanillic acid, guaiacol, vanillin, sinapic acid, syringol, syringic acid and ferulic acid) and four metallic compounds (CuSO4, AgNO3, MnSO4, and CaCl2) were tested for their ability to increase laccase (Lac) activity in the ligninolytic basidiomycete Phlebia brevispora BAFC 633. The addition of syringaldehyde, syringol, guaiacol, sinapic acid, vanillin, ferulic acid and CuSO4 showed a positive effect on fungal growth; however, it decreased dramatically with the addition of AgNO3 and did not undergo changes in the presence of CaCl2 or MnSO4. Lac activity increased with the addition of all the compounds tested, depending on the concentration and the day of culture. P. brevispora BAFC 633 produced two isoenzymes, a constitutively expressed of 60 kDa and another of 75 kDa expressed upon induction by sinapic acid, MnSO4 or CuSO4. Lac secretion capacity of P. brevispora BAFC 633 can be increased 27 times higher than the control with the highest levels detected in the presence of 0.3 mM CuSO4 at day 14. The action is affected at pre-transcriptional level regulating at the onset of the process, however it does not rule out the effect at the post-transcriptional and post-translational levels, for which is necessary to deepen in the knowledge of all possible regulation points of gene expression.

Estandarización de marcadores moleculares microsatélites para su uso en la industria forestal de Misiones, Argentina / Standarization of microsatellite molecular markers for Misiones (Argentina) forest industry

Provincia de Misiones posee actualmente una actividad forestal en pujante crecimiento ubicóndose entre las primeras del pais. Este marco de desarrollo productivo permite predecir un ámbito de crecimiento favorecido por las nuevas condiciones del mercado internacional. Por otro lado a pesar del avance de la tecnologi­a industrial, no se ha alcanzado el nivel de desarrollo biotecnológico óptimo que conjugue la calidad genética con caracterí­sticas fenotópicas de excelencia en las especies maderables de mayor demanda en la Provincia basó¡ndose la selección en criterios netamente fenotópicos y en la experiencia del productor, sin contarse con métodos moleculares desarrollados en la región. Este trabajo presenta los resultados del Proyecto Federal de Innovacíon Productiva (PFIP Mi09) cuyo objetivo principal fue estandarizar y transferir al sector productivo un conjunto de marcadores moleculares microsalites para ser aplicado al análisis de poblaciones y forestaciones de Araucaria angustifolia y Pinus taeda provenientes de la Provincia de Misiones (Argentina). Esto permitirá conocer el perfil genético de plantaciones y poblaciones de estas especies forestales, pudiendo aplicarse a la certificación de calidad en la producción forestal o a la selección de ejemplares de especies nativas.

Misiones Province currently has the first intensive forestry activity of Argentine. This framework of productive development allows predict an area of growth favored by the new conditions of the international market. On the other side despite the progress of industrial technology, has not been reached the optimal level of biotechnological development that combining quality with genomic and phenotypic characteristics of forest species. This work presents the results of Federal Project of Productive Innovation (PFIP Mi09) whose main objective was standardize and transfer to the productive sector a set of microsatellites molecular markers to be applied to the populations analysis of Araucaria angustifolia and Pinus taeda forestation from the Misiones (Argentine). This will reveal the plantations and forest genetic profile and may be applied to genetic certification of forest production quality.