Showing papers on "Trichoderma longibrachiatum published in 2021"

Trichoderma longibrachiatum and Trichoderma asperellum Confer Growth Promotion and Protection against Late Wilt Disease in the Field.

Abstract: Late wilt disease (LWD) of maize, caused by Magnaporthiopsis maydis, is considered a major threat to commercial fields in Israel, Egypt, Spain, and India. Today’s control methods include chemical and agronomical intervention but rely almost solely on resistant maize cultivars. In recent years, LWD research focused on eco-friendly biological approaches to restrain the pathogen. The current study conducted during two growing seasons explores the potential of three Trichoderma species as bioprotective treatments against LWD. These species excelled in preliminary assays performed previously under controlled conditions and were applied here in the field by directly adding them to each seed with the sowing. In the first field experiment, Trichoderma longibrachiatum successfully rescued the plants’ growth indices (weight and height) compared to T. asperelloides and the non-treated control. However, it had no positive effect on yield and disease progression. In the subsequent season, this Trichoderma species was tested against T. asperellum, an endophyte isolated from susceptible maize cultivar. This experiment was conducted during a rainy autumn season, which probably led to a weak disease burst. Under these conditions, the plants in all treatment groups were vivid and had similar growth progression and yields. Nevertheless, a close symptoms inspection revealed that the T. longibrachiatum treatment resulted in a two-fold reduction in the lower stem symptoms and a 1.4-fold reduction in the cob symptoms at the end of the seasons. T. asperellum achieved 1.6- and 1.3-fold improvement in these parameters, respectively. Quantitative Real-time PCR tracking of the pathogen in the host plants’ first internode supported the symptoms’ evaluation, with 3.1- and 4.9-fold lower M. maydis DNA levels in the two Trichoderma treatments. In order to induce LWD under the autumn’s less favorable conditions, some of the plots in each treatment were inoculated additionally, 20 days after sowing, by stabbing the lower stem section near the ground with a wooden toothpick dipped in M. maydis mycelia. This infection method overrides the Trichoderma roots protection and almost abolishes the biocontrol treatments’ protective achievements. This study suggests a biological Trichoderma-based protective layer that may have significant value in mild cases of LWD.

In Vitro and In Vivo Antifungal Activity of Sorbicillinoids Produced by Trichoderma longibrachiatum.

Abstract: In the search for antifungal agents from marine resources, we recently found that the culture filtrate of Trichoderma longibrachiatum SFC100166 effectively suppressed the development of tomato gray mold, rice blast, and tomato late blight. The culture filtrate was then successively extracted with ethyl acetate and n-butanol to identify the fungicidal metabolites. Consequently, a new compound, spirosorbicillinol D (1), and a new natural compound, 2',3'-dihydro-epoxysorbicillinol (2), together with 11 known compounds (3-13), were obtained from the solvent extracts. The chemical structures were determined by spectroscopic analyses and comparison with literature values. The results of the in vitro antifungal assay showed that of the tested fungal pathogens, Phytophthora infestans was the fungus most sensitive to the isolated compounds, with MIC values ranging from 6.3 to 400 µg/mL, except for trichotetronine (9) and trichodimerol (10). When tomato plants were treated with the representative compounds (4, 6, 7, and 11), bisvertinolone (6) strongly reduced the development of tomato late blight disease compared to the untreated control. Taken together, our results revealed that the culture filtrate of T. longibrachiatum SFC100166 and its metabolites could be useful sources for the development of new natural agents to control late blight caused by P. infestans.

Trichoderma longibrachiatum (TG1) Enhances Wheat Seedlings Tolerance to Salt Stress and Resistance to Fusarium pseudograminearum

Abstract: Salinity is abiotic stress that inhibits seed germination and suppresses plant growth and root development in a dose-dependent manner. Fusarium pseudograminearum (Fg) is a plant pathogen that causes wheat crown rot. Chemical control methods against Fg are toxic to the environment and resistance has been observed in wheat crops. Therefore, an alternative approach is needed to manage this devastating disease and the effects of salinity. Our research focused on the mycoparasitic mechanisms of Trichoderma longibrachiatum (TG1) on Fg and the induction of defenses in wheat seedlings under salt and Fg stress at physiological, biochemical and molecular levels. The average inhibition rate of TG1 against Fg at day 4 was 23%, 32%, 39%, and 38% respectively in the four NaCl treatments (0, 50, 100, and 150 mM). The mycoparasitic mechanisms of TG1 against Fg were coiling penetration, and wrapping of Fg hyphae. In response to inoculation of TG1 with Fg, significant upregulation of cell wall degrading enzymes (CWDEs) was observed. The expression of β-1, 6-glucan synthase (PP4), endochitinase precursor (PH -1), and chitinase (chi18-15) increased by 1.7-fold, 1.9-fold, and 1.3-fold on day 14 compared with day 3. Wheat seedlings with combined TG1+Fg treatments under different NaCl stress levels decreased disease incidence by an average of 52%; increased the superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activity by an average of 29%, 35%, and 20% respectively; and decreased malondialdehyde (MDA) and hydrogen peroxide (H2O2) content by an average of 43% and 44% respectively compared with Fg treated seedlings. The combined TG1+Fg treatment induced the transcription level of plant defense-related genes resulting in an increase in tyrosin-protein kinase (PR2), chitinase class I (CHIA1), and pathogenesis-related protein (PR1-2) by an average of 1.5-fold, 1.7-fold, and 2-fold respectively. However, the expression levels of phenylalanine ammonia-lyase (PAL) decreased 3.8-fold under various NaCl stresses. Our results suggest that TG1 enhances wheat seedling growth and controls wheat crown rot disease by strengthening the plant defense system and upregulating the expression of pathogenesis-related genes under both Fg and salt stress.

Endophytic fungi associated with soybean plants and their antagonistic activity against Rhizoctonia solani

Abstract: Fungal endophytes produce many secondary metabolites that can reduce root rot diseases Soybean is a particularly important crop worldwide Endophytic fungi can be isolated, identified, and incorporated into sustainable agriculture for the biological control of many diseases The aim of this study was to isolate some endophytic fungi for controlling the most important diseases of soybean plants and to study the mechanisms underlying this biocontrol regarding the suppression of pathogens Ten endophytic fungi were isolated from soybean plants Among them, the 3 fungi isolates that exhibited a high percentage of antagonistic activity against Rhizoctonia solani, the causal pathogen of root rot disease of soybean plants, were identified as Trichoderma longibrachiatum S12, T asperellum S11, and T atroviride PHYTAT7 The 3 fungi isolates had the ability to produce pectinase and chitinase and to solubilize phosphors Moreover, they produced siderophores and indole acetic acid (IAA), which have a strong effect on the growth of the plants The 3 isolates reduced disease severity by 64, 60, and 55%, respectively than the infected control The results suggest that certain endophytic fungi associated with soybean plants have potential for the management of root rot diseases in soybean Moreover, these isolates can be considered as having a growth-promoting effect in soybean plants

Prospection of Fungal Lignocellulolytic Enzymes Produced from Jatoba (Hymenaea courbaril) and Tamarind (Tamarindus indica) Seeds: Scaling for Bioreactor and Saccharification Profile of Sugarcane Bagasse

Abstract: The lignocellulosic biomass comprises three main components: cellulose, hemicellulose, and lignin. Degradation and conversion of these three components are attractive to biotechnology. This study aimed to prospect fungal lignocellulolytic enzymes with potential industrial applications, produced through a temporal analysis using Hymenaea courbaril and Tamarindus indica seeds as carbon sources. α-L-arabinofuranosidase, acetyl xylan esterase, endo-1,5-α-L-arabinanase, β-D-galactosidase, β-D-glucosidase, β-glucanase, β-D-xylosidase, cellobiohydrolase, endoglucanase, lichenase, mannanase, polygalacturonase, endo-1,4-β-xylanase, and xyloglucanase activities were determined. The enzymes were produced for eight filamentous fungi: Aspergillus fumigatus, Trametes hirsuta, Lasiodiplodia sp., two strains of Trichoderma longibrachiatum, Neocosmospora perseae, Fusarium sp. and Thermothelomyces thermophilus. The best producers concerning enzymatic activity were T. thermophilus and T. longibrachiatum. The optimal conditions for enzyme production were the media supplemented with tamarind seeds, under agitation, for 72 h. This analysis was essential to demonstrate that cultivation conditions, static and under agitation, exert strong influences on the production of several enzymes produced by different fungi. The kind of sugarcane, pretreatment used, microorganisms, and carbon sources proved limiting sugar profile factors.

Trichoderma longibrachiatum derived metabolite as a potential source of anti‐breast‐cancer agent

Abstract: Breast cancer (BC) is one of the most common malignancies and the second leading cause of cancer deaths among women worldwide. This high rate of morbidity and mortality requires research on the antioxidant and anticancer agents that can specifically kill or inhibit cancer cells without affecting normal cells. Trichoderma fungus is a rich source of such agents and secondary metabolites. Therefore, in the current study, we aimed to examine the effect of Trichoderma longibrachiatum metabolite in BC. The findings of our study illustrated that this fungus metabolite could limit the ability of breast cancer cells to proliferate and stop their cell cycle. On the other hand, it induces apoptosis in these cells. Together, the results of the present study for the first time demonstrate that T. longibrachiatum metabolite can play an anticancer role in the treatment of breast cancer, and it may be used as a clinical drug for BC treatment.

Longibramides A–E, Peptaibols Isolated from a Mushroom Derived Fungus Trichoderma longibrachiatum Rifai DMG-3-1-1

Abstract: Five new peptaibols, longibramides A-E (1-5) with 11 amino acid residues, were isolated from a fungus Trichoderma longibrachiatum Rifai DMG-3-1-1, which was isolated from a mushroom Clitocybe nebularis (Batsch) P. Kumm collected from coniferous forest in the subboreal area of northeast China. The structures of longibramides A-E were determined by their spectroscopic data (NMR and MS-MS spectra), their absolute configurations were determined by X-ray diffractions and Marfey's analyses. The X-ray diffractions of longibramides A, B and the similar CD spectra of A-E showed that they all had α-helix conformations. Longibramides B and E showed moderate cytotoxicities against BV2 and MCF-7 cells, and also showed some inhibitory effects against methicillin-resistant Staphylococcus aureus MRSA T144. L-trans-Hyp was not commonly found in natural peptaibols, which was the 6th or 10th amino acid residue in longibramides C-E . The X-ray diffractions of longibramides A and B afforded the accuracy conformations of their secondary structures, which maybe help to interpret the structure-activity relationships of the family of peptaibols in the future.

Mycogenic Nano-Complex for Plant Growth Promotion and Bio-Control of Pythium aphanidermatum

Abstract: (1) Background: biological way is one of the most ecofriendly and safe strategies for nanomaterials synthesis. So, biosynthesis-green method was used for the preparation of Zn(II) complex (in the Nano scale) from the reaction of the schiff base ligand 2,2′-((1E,1′E)-(1,2-phenylenebis (azanylylidene)), bis(methanylylidene))bis(4-bromophenol), and Zn(II)sulphate. The biogenic ZnNP-T was characterized by different methods. Our purpose was to evaluate the ability of biosynthesis-green method for the preparation of Zn(II) complex as an antifungal agent against diseases from fungal species. (2) Methods: in this work, isolates of Pythium aphanidermatum and Trichderma longibrachiatum were obtained, and Trichderma longibrachiatum was used to prepare nano metal complex. We tested the pathogenicity of nano metal complex against seedling and germination of seeds, and we evaluated the effectiveness of ZnNP-T for growth promotion of Vicia feba in early stage and inhibitory activity against Pythium aphanidermatum. (3) Results: antagonistic activity of ZnNP-T was tested in vitro against Pythium aphanidermatum, and then the growth rates of Vicia faba were determined. The obtained data revealed that mycelial growth of pathogenic fungus was inhibited about 73.8% at 20 ppm. In addition, improved the total biomass of Vicia faba in the presence of P. aphanidermatum. All concentration of ZnNP-T positively affected root weight of Vicia faba seedlings, and positively affected shoot weight. Root and shoot lengths were affected by using 20 ppm of ZnNP-T with up to 180 and 96.5 mm of shoot and root length compared to that of the control, while germination percentage was significantly enhanced with up to 100% increase after 72 h of germination. (4) Conclusion: one of the modern challenges in vegetable or fruit production is to enhance seed germination and to grow healthy plants with strong root system. In future, there should be a focus on using of biogenic Zinc nano-complex as plant growth promoter agents.

Potential of biocontrol agents against Ganoderma lucidum causing basal stem rot in mesquite (Prosopis cineraria) in arid regions of India

Abstract: This study investigates the potential of native biocontrol agents (BCAs) as controls against Ganoderma lucidum causing root rot mortality in Indian mesquite. The disease is prevalent in sandy soils where trees grow under rainfed conditions. In addition, a beetle namely Acanthophorus serraticornis damages the roots, resulting in increasing vulnerability of the host thereby allowing easy of the pathogen. In dual culture tests, Ganoderma infected cowpea root bit experiment and compatibility with insecticides revealed that the three BCAs (Trichoderma longibrachiatum, T. harzianum, and Aspergillus nidulans) significantly inhibited G. lucidum mycelial growth. The highest mycelial growth inhibition (47.6%) was recorded after 96 h followed by 39.8% and 29.3% at 72 and 48 h, respectively, by T. longibrachium. Cell free filtrates of T. longibrachiatum, T. harzianum, and A. nidulans were superior in inhibiting mycelium growth. A low concentration (3 ml) of T. longibrachiatum was more effective in inhibiting mycelium growth compared to other BCAs. Both Prosopis juliflora compost and onion residue compost amendments as food substrates favored the growth of these BCAs, which ultimately reduced the viability of Ganoderma-colonized root bits of cowpea. Studies on compatibility between insecticides and BCAs suggests that T. longibrachiatum, harzianum and A. nidulans can be combined with phorate or chloropyriphos (both organophosphates) at variable concentrations if amended together for partially infected trees, or as a prophylactic measure in healthy trees. These studies demonstrate that there is considerable opportunity for using native BCAs against G. lucidum in managing root rot disease.

Transcriptomic and Ultrastructural Analyses of Pyricularia Oryzae Treated With Fungicidal Peptaibol Analogs of Trichoderma Trichogin.

Abstract: Eco-friendly analogs of Trichogin GA IV, a short peptaibol produced by Trichoderma longibrachiatum, were assayed against Pyricularia oryzae, the causal agent of rice blast disease. In vitro and in vivo screenings allowed us to identify six peptides able to reduce by about 70% rice blast symptoms. One of the most active peptides was selected for further studies. Microscopy analyses highlighted that the treated fungal spores could not germinate and the fluorescein-labeled peptide localized on the spore cell wall and in the agglutinated cytoplasm. Transcriptomic analysis was carried out on P. oryzae mycelium 3 h after the peptide treatment. We identified 1,410 differentially expressed genes, two-thirds of which upregulated. Among these, we found genes involved in oxidative stress response, detoxification, autophagic cell death, cell wall biogenesis, degradation and remodeling, melanin and fatty acid biosynthesis, and ion efflux transporters. Molecular data suggest that the trichogin analogs cause cell wall and membrane damages and induce autophagic cell death. Ultrastructure observations on treated conidia and hyphae confirmed the molecular data. In conclusion, these selected peptides seem to be promising alternative molecules for developing effective bio-pesticides able to control rice blast disease.

Effects of mixed culture fermentation of Bacillus amyloliquefaciens and Trichoderma longibrachiatum on its constituent strains and the biocontrol of tomato Fusarium wilt.

Abstract: AIMS To study the effects of mixed culture fermentation (MCF) of Bacillus amyloliquefaciens and Trichoderma longibrachiatum on its constituent strains and the application values for agricultural production, with the intention of developing efficient and environmentally friendly biocontrol agents. METHODS AND RESULTS In this study, an in vitro antifungal growth experiment showed that the inhibitory rate of the MCF broth on pathogenic fungi (Fusarium oxysporum f. sp. lycopersici, Botrytis cinerea, Trichothecium roseum and Colletotrichum gloeosporioides) was less than that of B. amyloliquefaciens culture fermentation (BCF). Moreover, the content and gene expression of lipopeptide antibiotics were also lower than that in the BCF group. However, the pot experiments based on irrigation with appropriately diluted fermentation broth showed that the biocontrol effect of MCF on tomato Fusarium wilt was significantly higher than that of TCF (T. longibrachiatum culture fermentation) and BCF, and was approximately 15.79% higher than that of the BTF group which made by mixing equivalent amounts of BCF and TCF. In MCF broth, two micro-organisms antagonized and coexisted, and the growth of T. longibrachiatum was inhibited. Using transcriptomic analysis, we speculated that MCF can upregulate the expression of genes related to carbon and nitrogen metabolism, oxidation-reduction activity, sporulation, environmental information response and chemotaxis, and biosynthesis of secondary metabolites of B. amyloliquefaciens, which might enhance the nutrient substances metabolism and competitiveness, survival ability, colonization and adaptability to the environment to increase its biocontrol potential. CONCLUSIONS Mixed culture fermentation could promote the more reasonable and effective utilization of biocontrol micro-organisms though improving biocontrol effect, enhancing strains survival and competitiveness, increasing beneficial metabolites, combined with resistance induction or synergistic control. SIGNIFICANCE AND IMPACT OF THE STUDY Using MCF agronomically utilizes biocontrol agents in an efficient way, which has a good potential for commercial implementation and could reduce production costs.

Isolation and identification of South African indigenous fungal strains and assessment of their ability to degrade diclofenac in synthetic wastewater

Abstract: Pharmaceutical compounds barely removed from wastewater have become a huge concern due to their adverse effects on all environments, human health and wildlife. Among the treatment techniques, white-rot fungi (WRF) has shown a high diclofenac sodium (DCF) removal efficiency. The aims of this study were to isolate and identify South African fungi, to culture and optimize their growth, and to assess their ability to remove DCF from synthetic wastewater. Simple microscopic and FEG-SEM analysis were used followed by PCR analysis performed using ITS1/ITS4, EF1/EF2 and s-tubulin primers. Five fungal species closely related (approximately 98 to 100% similarity) to Trichoderma longibrachiatum, Trametes polyzona, Aspergillus niger, Mucor circinelloides and Rhizopus microsporus, were successfully isolated and identified. The highest growth (8.7±0.07 cm) was observed with T. longibrachiatum in culture medium composed of glucose-anhydrous-agar-peptone at 30±1.5 °C. The optimum C/N ratio of growth was found at 5.5:1. After 5 days, the highest DCF removal efficiency from a concentration of 10 mg/L was recorded for R. microsporus (72.29±3.5%), followed by A. niger (61.2±4.5%), M. circinelloides (61.2±3.5%), T. polyzona (22.42±4.5%), and by T. longibrachiatum (0%). Apart from the WRF, this study showed that other fungal strains can also be used for the degradation of recalcitrant pharmaceuticals.

Bio-production of alkaline protease by Trichoderma longibrachiatum and Penicillium rubidurum using different agro-industrial products

Abstract: Alkaline protease being active in neutral to alkaline pH has huge demands in food, detergent, leather and pharmaceutical industries. Its production from agro-industrial wastes not only lowers the production costs but also reduces the environmental problems. Hence, the present study aimed to search for new potential microbes, which can produce alkaline protease enzyme, to meet the industrial demands. In this study, 13 fungal spp. were isolated on potato dextrose agar medium (PDA) from mangrove soil through serial dilution, and then were streaked on the skim milk agar medium for qualitative screening of protease production. Out of 13 fungal spp.; only 7 spp. were able to produce proteolytic zones through the proteolytic assay. The Relative enzymatic index (REI) value (Zone diameter/Colony diameter) of all the fungal isolates that produced proteolytic zones on skim milk agar medium was evaluated. Only 2 fungal isolates which showed maximum REI value were selected, and then identified morphologically and molecularly as Trichoderma longibrachiatum (Accession no. MF144551) and by Penicillium rubidurum (Accession no. MF144561). Submerged fermentation was carried out using different agro industrial substrates to quantify for protease production, where the supernatants obtained were used for alkaline protease estimation. Among the different tested substrates, soybean powder and wheat bran were the most suitable substrates for maximum protease production by T. longibrachiatum (233.78±7.12 U/ mg) and P. rubidurum (228.61±11.13 U/ mg), respectively. The partial purified enzyme from these fungi showed maximum proteolytic potentials at pH 8.0 (P. rubidurum) and pH 9.0 (T. longibrachiatum), with optima temperature of 40 °C. Among the tested heavy metals, only Mn2+ expressed marginal enhancement of the protease enzyme activity.

Hydrolysis of lignocellulosic forages by Trichoderma longibrachiatum isolate from bovine rumen

Abstract: Fungi of the genus Trichoderma hydrolyze lignocellulosic materials, thus promoting the production of fermentable sugars. In the present study, the production of cellulases by Trichoderma longibrachiatum from ruminal fluid was evaluated in submerged fermentation of Urochloa decumbens hay (UD) or sugarcane bagasse (SB). Moreover, the biomass of this fungus was analyzed by supplementing it with urea or ammonium sulfate and with two buffer systems for the production of cellulases by T. longibrachiatum using SB or UD. The fermentations were carried out in test tubes containing Sabouraud broth, and the carbon source was packed in nonwoven fabric sachets. The fermentations were performed at 39 °C for 10 days. The factorial design was completely randomized, and the final pH, fungal biomass, substrate dry matter degradation, total protein, and cellulolytic enzymes were evaluated after the fermentation process. The fungal development was better in the medium supplemented with ammonium sulfate than that supplemented with urea, and it showed better degradation of UB than SB. The evaluated isolate promoted better protein synthesis during fermentation of SB buffered with sodium acetate; however, the medium containing UB produced higher concentrations of total cellulase, carboxymethylcellulase, and avicelase than the medium containing SB. Thus, rapid cellulose production by this fungus may aid in the hydrolysis of lignocellulosic materials for animal feed supplementation for ruminants; furthermore, the use of these enzymes for biotechnological purposes is promising.

Mutualistic interaction of native Serratia marcescens UENF-22GI with Trichoderma longibrachiatum UENF-F476 boosting seedling growth of tomato and papaya

Abstract: A plethora of bacteria-fungal interactions occur on the extended fungal hyphae network in soil. The mycosphere of saprophytic fungi can serve as a bacterial niche boosting their survival, dispersion, and activity. Such ecological concepts can be converted to bioproducts for sustainable agriculture. Accordingly, we tested the hypothesis that the well-characterised beneficial bacterium Serratia marcescens UENF-22GI can enhance plant growth-promoting properties when combined with Trichoderma longibrachiatum UENF-F476. The cultural and cell interactions demonstrated S. marcescens and T. longibrachiatum mutual compatibility. Bacteria cells were able to attach, forming aggregates to biofilms and migrating through the fungal hyphae network. Long-distance bacterial migration through growing hyphae was confirmed using a two-compartment Petri dishes assay. Fungal inoculation increased the bacteria survival rates into the vermicompost substrate over the experimental time. Also, in vitro indolic compound, phosphorus, and zinc solubilisation bacteria activities increased in the presence of the fungus. In line with the ecophysiological bacteria fitness, the bacterium-fungal combination boosted tomato and papaya plantlet growth when applied into the plant substrate under nursery conditions. Mutualistic interaction between mycosphere-colonizing bacterium S. marcescens UENF-22GI and the saprotrophic fungi T. longibrachiatum UENF-F467 increased the ecological fitness of the bacteria alongside with beneficial potential for plant growth. A proper combination and delivery of mutual compatible beneficial bacteria-fungal represent an open avenue for microbial-based products for the biological enrichment of plant substrates in agricultural systems.

To evaluate in vitro the efficacy of fungicides, bio-control agents and plant extract against Colletotrichum capsici

Abstract: Colletotrichum capsici infection will be higher in the mature stage of chilli plant than in the early stage of plant (Krairuan et al., 2008). The fungus prefers warm humid environment for spreading the anthracnose disease uniformly and effectively. All the two contact and six systemic fungicides evaluated in vitro were found fungistatic against Colletotrichum capsici. Among all these fungicides, Tricyclazole was found to be most effective with significantly maximum mycelial growth inhibition and least mycelial growth followed by fungicides viz. Azostrobin, Tebuconazole, Difenconazole, Propiconazole, Carbendazim + Mancozeb. While comparatively minimum average radial mycelial growth inhibition was recorded with Hexaconazole followed by Propineb. All the seven fungal and one bacterial antagonists evaluated in vitro were found antifungal against Colletotrichum capsici exhibited significant mycelial growth inhibition. However, Trichoderma asperallum, Pseudomonas fluorescens, Trichoderma harzianum and Gliocladium virens recorded significantly highest mycelial growth inhibition. Rest of the bioagents, Trichoderma ligronum, Trichoderma koningii and Trichoderma longibrachiatum and Trichoderma hamatum tested also caused significant mycelial inhibition of the test pathogen.