Innovative sustainable bioreactor-in-a-granule formulation of Trichoderma asperelloides.

The advancement of fungal biocontrol agents depends on replacing cereal grains with low-cost agro-industrial byproducts for their economical mass production and development of stable formulations. We propose an innovative approach to develop a rice flour-based formulation of the beneficial biocontrol agent Trichoderma asperelloides CMAA1584 designed to simulate a micro-bioreactor within the concept of full biorefinery process, affording in situ conidiation, extended shelf-life, and effective control of Sclerotinia sclerotiorum, a devastating pathogen of several dicot agricultural crops worldwide. Rice flour is an inexpensive and underexplored byproduct derived from broken rice after milling, capable of sustaining high yields of conidial production through our optimized fermentation-formulation route. Conidial yield was mainly influenced by nitrogen content (0.1% w/w) added to the rice meal coupled with the fermentor type. Hydrolyzed yeast was the best nitrogen source yielding 2.6 × 109 colony-forming units (CFU)/g within 14 days. Subsequently, GControl, GLecithin, GBreak-Thru, GBentonite, and GOrganic compost+Break-Thru formulations were obtained by extrusion followed by air-drying and further assessed for their potential to induce secondary sporulation in situ, storage stability, and efficacy against Sclerotinia. GControl, GBreak-Thru, GBentonite, and GOrganic compost+Break-Thru stood out with the highest number of CFU after sporulation upon re-hydration on water-agar medium. Shelf-life of formulations GControl and GBentonite remained consistent for > 3 months at ambient temperature, while in GBentonite and GOrganic compost+Break-Thru formulations remained viable for 24 months during refrigerated storage. Formulations exhibited similar efficacy in suppressing the myceliogenic germination of Sclerotinia irrespective of their concentration tested (5 × 104 to 5 × 106 CFU/g of soil), resulting in 79.2 to 93.7% relative inhibition. Noteworthily, all 24-month-old formulations kept under cold storage successfully suppressed sclerotia. This work provides an environmentally friendly bioprocess method using rice flour as the main feedstock to develop waste-free granular formulations of Trichoderma conidia that are effective in suppressing Sclerotinia while also improving biopesticide shelf-life. KEY POINTS: • Innovative "bioreactor-in-a-granule" system for T. asperelloides is devised. • Dry granules of aerial conidia remain highly viable for 24 months at 4 °C. • Effective control of white-mold sclerotia via soil application of Trichoderma-based granules.

Integrative workflows for the characterization of hydrophobin and cerato-platanin in the marine fungus Paradendryphiella salina.

Hydrophobins (HFBs) and cerato-platanins (CPs) are surface-active extracellular proteins produced by filamentous fungi. This study identified two HFB genes (pshyd1 and pshyd2) and one CP gene (pscp) in the marine fungus Paradendryphiella salina. The proteins PsCP, PsHYD2, and PsHYD1 had molecular weights of 12.70, 6.62, and 5.98 kDa, respectively, with isoelectric points below 7. PsHYD1 and PsHYD2 showed hydrophobicity (GRAVY score 0.462), while PsCP was hydrophilic (GRAVY score - 0.202). Stability indices indicated in-solution stability. Mass spectrometry identified 2,922 proteins, including CP but not HFB proteins. qPCR revealed differential gene expression influenced by developmental stage and substrate, with pshyd1 consistently expressed. These findings suggest P. salina's adaptation to marine ecosystems with fewer hydrophobin genes than other fungi but capable of producing surface-active proteins from seaweed carbohydrates. These proteins have potential applications in medical biocoatings, food industry foam stabilizers, and environmental bioremediation.

Application of Stemphylium lycopersici Extracts Immobilized on MCM-48type Mesoporous Materials as Biocatalysts for Monoacylglycerol Production.

Monoacylglycerols are eco-friendly and inexpensive emulsifiers with a range of applications. The traditional synthetic route is not eco-friendly, while enzymatic catalysis offers milder reaction conditions and higher selectivity. However, its application still is limited due to the costs. In this context, endophytic fungi can be source to new biocatalysts with enhanced catalytic activity. Based on this perspective, the aim of this study was perform the synthesis of MAG's through transesterification reactions of solketal and different vinyl esters, using crude and immobilized lipolytic extracts from the endophytic fungi Stemphylium lycopersici, isolated from Humiria balsamifera. The reactions were conducted using 100 mg of biocatalyst, 1 mmol of substrates, 9 : 1 n-heptane/acetone, at 40 °C, 200 rpm for 96 h. In the reactions using the ILE and stearate, laureate and decanoate vinyl esters it was possible to obtain the correspondent products with conversion rates of 52-75 %. Also, according to the structure drivers used in MCM-48 synthesis, different morphologies and conversions rates were observed. Employing [C16MI] Cl, [C14MI] Cl and [C4MI] Cl, the 1-lauroyl- glycerol conversion was 36 %, 79 % and 44 %, respectively. This is the first work involving the immobilization of an endophytic fungi and its utilization as a biocatalyst in the production of MAG's.

Conservation of the Pal/Rim Pathway in Ustilaginomycetes.

The ability of fungi to effectively sense and internalize signals related to extracellular changing environments is essential for survival. This adaptability is particularly important for fungal pathogens of humans and plants that must sense and respond to drastic environmental changes when colonizing their hosts. One of the most important physicochemical factors affecting fungal growth and development is the pH. Ascomycota fungal species possess mechanisms such as the Pal/Rim pathway for external pH sensing and adaptation. However, the conservation of this mechanism in other fungi, such as Ustilaginomycetes is still little studied. To overcome this knowledge gap, we used a comparative genomic approach to explore the conservation of the Pal/Rim pathway in the 13 best sequenced and annotated Ustilaginomycetes. Our findings reveal that the Rim proteins and the Endosomal Sorting Complex Required for Transport (ESCRT) proteins are conserved in Ustilaginomycetes. They conserve the canonical domains present in Pal/Rim and ESCRT proteins of Ascomycota. This study sheds light on the molecular mechanisms used by these fungi for responding to extracellular stresses such as the pH, and open the door to further experimentations for understanding the molecular bases of the signaling in Ustilaginomycetes.

Antarctic marine sediment as a source of filamentous fungi-derived antimicrobial and antitumor compounds of pharmaceutical interest.

Antarctica harbors a microbial diversity still poorly explored and of inestimable biotechnological value. Cold-adapted microorganisms can produce a diverse range of metabolites stable at low temperatures, making these compounds industrially interesting for biotechnological use. The present work investigated the biotechnological potential for antimicrobial and antitumor activity of filamentous fungi and bacteria isolated from marine sediment samples collected at Deception Island, Antarctica. A total of 89 microbial isolates were recovered from marine sediments and submitted to an initial screening for L-glutaminase with antitumoral activity and for antimicrobial metabolites. The isolates Pseudogymnoascus sp. FDG01, Pseudogymnoascus sp. FDG02, and Penicillium sp. FAD33 showed potential antiproliferative action against human pancreatic carcinoma cells while showing no toxic effect on non-tumor cells. The microbial extracts from unidentified three bacteria and four filamentous fungi showed antibacterial activity against at least one tested pathogenic bacterial strain. The isolate FDG01 inhibited four bacterial species, while the isolate FDG01 was active against Micrococcus luteus in the minimal inhibitory concentration of 0.015625 µg mL -1. The results pave the way for further optimization of enzyme production and characterization of enzymes and metabolites found and reaffirm Antarctic marine environments as a wealthy source of compounds potentially applicable in the healthcare and pharmaceutical industry.

Silencing of the Laccase (lacc2) Gene from Pleurotus ostreatus Causes Important Effects on the Formation of Toxocyst-like Structures and Fruiting Body.

A wide variety of biological functions, including those involved in the morphogenesis process of basidiomycete fungi, have been attributed to laccase enzymes. In this work, RNA interference (RNAi) was used to evaluate the role of the laccase (lacc2) gene of Pleurotus ostreatus PoB. Previously, transformant strains of P. ostreatus were obtained and according to their level of silencing they were classified as light (T7), medium (T21) or severe (T26 and T27). The attenuation of the lacc2 gene in these transformants was determined by RT-PCR. Silencing of lacc2 resulted in a decrease in laccase activity between 30 and 55%, which depended on the level of laccase expression achieved. The silenced strains (T21, T26, and T27) displayed a delay in the development of mycelium on potato dextrose agar (PDA) medium, whereas in the cultures grown on wheat straw, we found that these strains were incapable of producing aerial mycelium, primordia, and fruiting bodies. Scanning electron microscopy (SEM) showed the presence of toxocyst-like structures. The highest abundance of these structures was observed in the wild-type (PoB) and T7 strains. However, the abundance of toxocysts decreased in the T21 and T26 strains, and in T27 they were not detected. These results suggest that the presence and abundance of toxocyst-like structures are directly related to the development of fruiting bodies. Furthermore, our data confirm that lacc2 is involved in the morphogenesis process of P. ostreatus.

Endophytic, extremophilic and entomophilic fungi strains biodegrade anthracene showing potential for bioremediation.

Anthropogenic activities have been increasing Polycyclic Aromatic Hydrocarbons (PAHs) release, promoting an urgent need for decontamination methods. Therefore, anthracene biodegradation by endophytic, extremophilic, and entomophilic fungi was studied. Moreover, a salting-out extraction methodology with the renewable solvent ethanol and the innocuous salt K2HPO4 was employed. Nine of the ten employed strains biodegraded anthracene in liquid medium (19-56% biodegradation) after 14 days at 30 °C, 130 rpm, and 100 mg L-1. The most efficient strain Didymellaceae sp. LaBioMMi 155, an entomophilic strain, was employed for optimized biodegradation, aiming at a better understanding of how factors like pollutant initial concentration, pH, and temperature affected this process. Biodegradation reached 90 ± 11% at 22 °C, pH 9.0, and 50 mg L-1. Futhermore, 8 different PAHs were biodegraded and metabolites were identified. Then, experiments with anthracene in soil ex situ were performed and bioaugmentation with Didymellaceae sp. LaBioMMi 155 presented better results than natural attenuation by the native microbiome and biostimulation by the addition of liquid nutrient medium into soil. Therefore, an expanded knowledge about PAHs biodegradation processes was achieved with emphasis to the action of Didymellaceae sp. LaBioMMi 155, which can be further employed for in situ biodegradation (after strain security test), or for enzyme identification and isolation aiming at oxygenases with optimal activity under alkaline conditions.

Enhanced mercury phytoremediation by Pseudomonodictys pantanalensis sp. nov. A73 and Westerdykella aquatica P71.

Mercury is a non-essential and toxic metal that induces toxicity in most organisms, but endophytic fungi can develop survival strategies to tolerate and respond to metal contaminants and other environmental stressors. The present study demonstrated the potential of mercury-resistant endophytic fungi in phytoremediation. We examined the functional traits involved in plant growth promotion, phytotoxicity mitigation, and mercury phytoremediation in seven fungi strains. The endophytic isolates synthesized the phytohormone indole-3-acetic acid, secreted siderophores, and solubilized phosphate in vitro. Inoculation of maize (Zea mays) plants with endophytes increased plant growth attributes by up to 76.25%. The endophytic fungi stimulated mercury uptake from the substrate and promoted its accumulation in plant tissues (t test, p < 0.05), preferentially in the roots, which thereby mitigated the impacts of metal phytotoxicity. Westerdykella aquatica P71 and the newly identified species Pseudomonodictys pantanalensis nov. A73 were the isolates that presented the best phytoremediation potential. Assembling and annotation of P. pantanalensis A73 and W. aquatica P71 genomes resulted in genome sizes of 45.7 and 31.8 Mb that encoded 17,774 and 11,240 protein-coding genes, respectively. Some clusters of genes detected were involved in the synthesis of secondary metabolites such as dimethylcoprogen (NRPS) and melanin (T1PKS), which are metal chelators with antioxidant activity; mercury resistance (merA and merR1); oxidative stress (PRX1 and TRX1); and plant growth promotion (trpS and iscU). Therefore, both fungi species are potential tools for the bioremediation of mercury-contaminated soils due to their ability to reduce phytotoxicity and assist phytoremediation.

Optimizing the Culture Medium of Lasiodiplodia sp. to Improve the Yield of Ethyl Acetate Extract as an Antimicrobial Source.

Endophytes often inhabit plant tissues and cause no disease symptoms. Lasiodiplodia is generally considered a pathogenic fungus, but such a genus is capable of producing high-value bioactive molecules, such as enzymes, secondary metabolites including antimicrobials. Therefore, Lasiodiplodia sp. endophyte was cultivated in static mode for 12 days and EtOAc extracts were obtained and evaluated against pathogens afterward. Fermentation parameters (glucose, sucrose and NaNO3) were optimized by the factorial design and response surface methodology, as these are powerful tools to provide reliable information about fungal culture conditions and EtOAc extract yields were considered as response variables. Lasiodiplodia growth curve indicated that optimal production of EtOAc extract mass was achieved after 12 days of fermentation (284 mg 300 mL-1 broth), which is in agreement with values obtained from validation tests. Minimum Inhibitory Concentration (MIC) and Minimum Microbicidal Concentration (MMC) essays suggested that the endophyte produce substances presenting antimicrobial and antifungal activities against ATCC Staphylococcus aureus and Candida albicans strains at optimum point under evaluated conditions. MIC values ranged between 50 and 100 µg mL-1 for both pathogens, while MMC of C. albicans ranged from 100 to 200 µg mL-1, which evidence its fungicidal effect. Furthermore, it was found that the EtOAc extract yield can be increased by optimizing carbon and nitrogen sources in endophyte cultivation, and there was good agreement between predicted and experimental values under optimized conditions. Thus, Lasiodiplodia fungi are promising sources of antimicrobials and changes in carbon and nitrogen sources can improve the yield of secondary metabolites according to the factorial design.

Ascomycota as a source of natural colorants.

In the last few decades, there has been a great demand for natural colorants. Synthetic colorants are known to be easy to produce, are less expensive, and remain stable when subjected to chemical and physical factors. In addition, only small amounts are required to color any material, and unwanted flavors and aromas are not incorporated into the product. Natural colorants present in food, in addition to providing color, also have biological properties and effects that aid in the prevention and cure of many diseases. The main classes of colorants produced by phylum Ascomycota include polyketides and carotenoids. A promising producer of colorants should be able to assimilate a variety of sources of carbon and nitrogen and also exhibit relative stability. The strain should not be pathogenic, and its product should not be toxic. Production processes should also provide the expected color with a good yield through simple extraction methods. Research that seeks new sources of these compounds should continue to seek products of biotechnological origin in order to be competitive with products of synthetic and plant origin. In this review, we will focus on the recent studies on the main producing species, classes, and metabolic pathways of colorants produced by this phylum, historical background, impact of synthetic colorants on human health and the environment, social demand for natural colorants and also an in-depth approach to bioprocesses (influences on production, optimization of bioprocess, extraction, and identification), and limitations and perspectives for the use of fungal-based dyes.

Penicillium Ochrochloron RLS11 Secretome Containing Carbohydrate-Active Enzymes Improves Commercial Enzyme Mixtures During Sugarcane Straw Saccharification.

Filamentous fungi are prolific producers of carbohydrate-active enzymes (CAZymes) and important agents that carry out plant cell wall degradation in natural environments. The number of fungal species is frequently reported in the millions range, with a huge diversity and genetic variability, reflecting on a vast repertoire of CAZymes that these organisms can produce. In this study, we evaluated the ability of previously selected ascomycete and basidiomycete fungi to produce plant cell wall-degrading enzyme (PCWDE) activities and the potential of the culture supernatants to increase the efficiency of the Cellic® CTec2/HTec2 for steam-exploded sugarcane straw saccharification. The culture supernatant of Penicillium ochrochloron RLS11 showed a promising supplementation effect on Cellic® CTec2/HTec2, and we conducted the whole-genome sequencing and proteomic analysis for this fungus. The size of the assembled genome was 38.06 Mbp, and a total of 12,015 protein-coding genes were identified. The repertoire of PCWDE-coding genes was comparatively high among Penicillium spp. and showed an expansion in important cellulases and xylanases families, such as GH3, GH6, GH7, and GH11. The proteomic analysis indicated cellulases that probably enhanced the biomass saccharification performance of the Cellic® CTec2/HTec2, which included enzymes from GH3, GH6, and GH7 families.

Bacterial volatile organic compounds induce adverse ultrastructural changes and DNA damage to the sugarcane pathogenic fungus Thielaviopsis ethacetica.

Due to an increasing demand for sustainable agricultural practices, the adoption of microbial volatile organic compounds (VOCs) as antagonists against phytopathogens has emerged as an eco-friendly alternative to the use of agrochemicals. Here, we identified three Pseudomonas strains that were able to inhibit, in vitro, up to 80% of mycelial growth of the phytopathogenic fungus Thielaviopsis ethacetica, the causal agent of pineapple sett rot disease in sugarcane. Using GC/MS, we found that these bacteria produced 62 different VOCs, and further functional validation revealed compounds with high antagonistic activity to T. ethacetica. Transcriptomic analysis of the fungal response to VOCs indicated that these metabolites downregulated genes related to fungal central metabolism, such as those involved in carbohydrate metabolism. Interestingly, genes related to the DNA damage response were upregulated, and micro-FTIR analysis corroborated our hypothesis that VOCs triggered DNA damage. Electron microscopy analysis showed critical morphological changes in mycelia treated with VOCs. Altogether, these results indicated that VOCs hampered fungal growth and could lead to cell death. This study represents the first demonstration of the molecular mechanisms involved in the antagonism of sugarcane phytopathogens by VOCs and reinforces that VOCs can be a sustainable alternative for use in phytopathogen biocontrol.

First report on the production of phytotoxic metabolites by Mycoleptodiscus indicus under optimized conditions of submerged fermentation.

An alternative to controlling weeds resistant to conventional herbicides is the isolation of new active principles. Fungi can produce phytotoxic metabolites that may be used in the development of new herbicides. The objectives of this study were: (1) isolate, select, and identify a fungus producer of phytotoxic metabolites and (2) optimize the culture conditions of this fungus in a low-cost culture medium, with the aim of increasing the phytotoxic effects of their metabolites in weeds and commercial plants. Fungi were isolated from the leaves of Conyza sp. with disease symptoms and selected according to the production of phytotoxic metabolites in solid and submerged fermentation in a low-cost culture medium. A Plackett-Burman Design and Central Composite Rotational Design were used to optimize the conditions of temperature, agitation, pH, and concentrations of glucose and yeast extract in submerged fermentation. The phytotoxic metabolites produced under optimal conditions were tested on 10 commercial plants and weeds that are difficult to control. Of the nine fungi isolated, Mycoleptodiscus indicus UFSM54 produced higher leaf lesions. The production of phytotoxic metabolites was optimized when the fungus was cultivated at 35°C, 50 rpm, and 1.5 g L-1 of glucose in submerged fermentation. The metabolites of M. indicus caused severe phytotoxic effects on germination and seedling growth, and enhanced lesion development on detached plant leaves. The present study is the first to report on the production of phytotoxic metabolites by M. indicus, a potential producer of bioherbicides.

Antimicrobial and antioxidant activities of secondary metabolites from endophytic fungus Botryosphaeria fabicerciana (MGN23-3) associated to Morus nigra L.

This study was to evaluate the biological activity of the extract of Botryosphaeria fabicerciana isolated from leaves of Morus nigra. The volatile compounds from the crude extract were analysed by GC-MS which demonstrate that mellein and ß-orcinaldehyde were are the major compounds. The best minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the extract was observed against Gram-positive bacteria, with a MIC of 15.6 µg/mL towards B. cereus and MIC of 62.5 µg/mL towards S. aureus and B. subtilis. MBC values of 31.25 µg/mL, 62.5 µg/mL, and 250 µg/mL were observed towards B. cereus, B. subtilis, and S. aureus, respectively. The cytotoxicity analyses showed CC50 of 115 µg/mL. The crude extract showed antioxidant activity by the DPPH, ABTS, and FRAP assays. Therefore, the extract of the endophytic fungus presented biotechnological potential as an antibacterial and antioxidant agent.

Medicinal potentialities and pathogenic profile of Lasiodiplodia genus.

Considering that current biotechnological advances have been contributing towards improving the well-being of humanity, endophytic fungi, such as Lasiodiplodia, are promising sources of new substances to be used in chemical, pharmaceutical and agrochemical processes. Bioactive secondary metabolites are examples of such substances, although it is widely known that Lasiodiplodia inflicts irreparable damage to several crops of major economic importance. They are often produced as a response against biotic and abiotic factors, thus revealing that they play different roles, such as in signaling and defense mechanisms. Therefore, this review presents a few subtle differences between pathogenicity and mutualistic endophyte-host interactions. Moreover, the main secondary metabolites produced by Lasiodiplodia endophytes have been described with respect to their relevant antimicrobial and cytotoxic activities.

Insights into the Lignocellulose-Degrading Enzyme System of Humicola grisea var. thermoidea Based on Genome and Transcriptome Analysis.

Humicola grisea var. thermoidea is a thermophilic ascomycete and important enzyme producer that has an efficient enzymatic system with a broad spectrum of thermostable carbohydrate-active (CAZy) enzymes. These enzymes can be employed in lignocellulose biomass deconstruction and other industrial applications. In this work, the genome of H. grisea var. thermoidea was sequenced. The acquired sequence reads were assembled into a total length of 28.75 Mbp. Genome features correlate with what was expected for thermophilic Sordariomycetes. The transcriptomic data showed that sugarcane bagasse significantly upregulated genes related to primary metabolism and polysaccharide deconstruction, especially hydrolases, at both pH 5 and pH 8. However, a number of exclusive and shared genes between the pH values were found, especially at pH 8. H. grisea expresses an average of 211 CAZy enzymes (CAZymes), which are capable of acting in different substrates. The top upregulated genes at both pH values represent CAZyme-encoding genes from different classes, including acetylxylan esterase, endo-1,4-ß-mannosidase, exoglucanase, and endoglucanase genes. For the first time, the arsenal that the thermophilic fungus H. grisea var. thermoidea possesses to degrade the lignocellulosic biomass is shown. Carbon source and pH are of pivotal importance in regulating gene expression in this organism, and alkaline pH is a key regulatory factor for sugarcane bagasse hydrolysis. This work paves the way for the genetic manipulation and robust biotechnological applications of this fungus. IMPORTANCE Most studies regarding the use of fungi as enzyme producers for biomass deconstruction have focused on mesophile species, whereas the potential of thermophiles has been evaluated less. This study revealed, through genome and transcriptome analyses, the genetic repertoire of the biotechnological relevant thermophile fungus Humicola grisea. Comparative genomics helped us to further understand the biology and biotechnological potential of H. grisea. The results demonstrate that this fungus possesses an arsenal of carbohydrate-active (CAZy) enzymes to degrade the lignocellulosic biomass. Indeed, it expresses more than 200 genes encoding CAZy enzymes when cultivated in sugarcane bagasse. Carbon source and pH are key factors for regulating the gene expression in this organism. This work shows, for the first time, the great potential of H. grisea as an enzyme producer and a gene donor for biotechnological applications and provides the base for the genetic manipulation and robust biotechnological applications of this fungus.

Metabolomic Analysis of Combretum lanceolatum Plants Interaction with Diaporthe phaseolorum and Trichoderma spirale Endophytic Fungi through 1 H-NMR.

Endophytic fungi are an important class of microorganisms, able to interact with a host plant via a mutualistic mechanism without visible symptoms of the fungal colonization. The synergy between endophytic fungi and their host plant can promote morphological, physiological and biochemical changes through the expression of bioactive metabolites. This work aims to correlate metabolic changes in the Combretum lanceolatum plant metabolome with its endophytic fungi Diaporthe phaseolorum (Dp) and Trichoderma spirale (Ts), and to discover corresponding metabolite-biomarkers, with the principal focus being on its primary metabolism. The 1 H-NMR metabolomic analysis of qualitative and quantitative changes was performed through multivariate statistical analysis and the identification of primary metabolites was achieved on the Madison Metabolomics Consortium Database. The presence of Dp significantly impacted the plant's metabolic pathways, improving the biosynthesis of primary metabolites such as threonine, malic acid and N-acetyl-mannosamine, which are precursors of special metabolites involved in plant self-defence. This work represents a valuable contribution to advanced studies on the metabolic profiles of the interaction of plants with endophytes.

Isolation and characterization of endophytic fungi having plant growth promotion traits that biosynthesizes bacosides and withanolides under in vitro conditions.

Endophytes are regarded with immense potentials in terms of plant growth promoting (PGP) elicitors and mimicking secondary metabolites of medicinal importance. Here in the present study, we explored Bacopa monnieri plants to isolate, identify fungal endophytes with PGP elicitation potentials, and investigate secretion of secondary metabolites such as bacoside and withanolide content under in vitro conditions. Three fungal endophytes isolated (out of 40 saponin producing isolates) from leaves of B. monnieri were examined for in vitro biosynthesis of bacosides. On morphological, biochemical, and molecular identification (ITS gene sequencing), the isolated strains SUBL33, SUBL51, and SUBL206 were identified as Nigrospora oryzae (MH071153), Alternaria alternata (MH071155), and Aspergillus terreus (MH071154) respectively. Among these strains, SUBL33 produced highest quantity of Bacoside A3 (4093 µg mL-1), Jujubogenin isomer of Bacopasaponin C (65,339 µg mL-1), and Bacopasaponin C (1325 µg mL-1) while Bacopaside II (13,030 µg mL-1) was produced by SUBL51 maximally. Moreover, these aforementioned strains also produced detectable concentration of withanolides-Withaferrin A, Withanolide A (480 µg mL-1), and Withanolide B (1024 µg mL-1) respectively. However, Withanolide A was not detected in the secondary metabolites of strain SUBL51. To best of our knowledge, the present study is first reports of Nigrospora oryzae as an endophyte in B. monnieri with potentials of biosynthesis of economically important phytomolecules under in vitro conditions.

The Fumarprotocetraric Acid Inhibits Tau Covalently, Avoiding Cytotoxicity of Aggregates in Cells.

Neurodegenerative disorders, including Tauopathies that involve tau protein, base their pathological mechanism on forming proteinaceous aggregates, which has a deleterious effect on cells triggering an inflammatory response. Moreover, tau inhibitors can exert their mechanism of action through noncovalent and covalent interactions. Thus, Michael's addition appears as a feasible type of interaction involving an α, ß unsaturated carbonyl moiety to avoid pathological confirmation and further cytotoxicity. Moreover, we isolated three compounds from Antarctic lichens Cladonia cariosa and Himantormia lugubris: protolichesterinic acid (1), fumarprotocetraric acid (2), and lichesterinic acid (3). The maleimide cysteine labeling assay showed that compounds 1, 2, and 3 inhibit at 50 µM, but compounds 2 and 3 are statistically significant. Based on its inhibition capacity, we decided to test compound 2 further. Thus, our results suggest that compound 2 remodel soluble oligomers and diminish ß sheet content, as demonstrated through ThT experiments. Hence, we added externally treated oligomers with compound 2 to demonstrate that they are harmless in cell culture. First, the morphology of cells in the presence of aggregates does not suffer evident changes compared to the control. Additionally, the externally added aggregates do not provoke a substantial LDH release compared to the control, indicating that treated oligomers do not provoke membrane damage in cell culture compared with aggregates alone. Thus, in the present work, we demonstrated that Michael's acceptors found in lichens could serve as a scaffold to explore different mechanisms of action to turn tau aggregates into harmless species.

Fungal endophytes from leaves of Mandevilla catimbauensis (Apocynaceae): diversity and potential for L-asparaginase production.

In this study, we examined endophytic fungi in leaves of Mandevilla catimbauensis, an endemic plant species found in the Brazilian dry forest (Caatinga), and endophytic fungi's potential to produce L-asparaginase (L-ASNase). In total, 66 endophytes were isolated, and the leaf-fragment colonisation rate was 11.78%. Based on morphology, internal transcribed spacer (ITS), and partial large subunit (LSU) of ribosomal DNA sequencing, the endophytic fungi isolated belonged to six Ascomycota orders (Botryosphaeriales, Capnodiales, Diaporthales, Eurotiales, Marthamycetales, and Pleosporales). Phyllosticta species were the most frequent endophytes isolated (23 isolates [45.1%] from two species). The Shannon-Wiener and Fisher alpha index average values were 0.56 and 3.26, respectively. Twenty endophytes were randomly selected for the L-ASNase production test, of which fourteen isolates showed potential to produce the enzyme (0.48-2.22 U g-1), especially Phyllosticta catimbauensis URM 7672 (2.22 U g-1) and Cladosporium sp. G45 (2.11 U g-1). Phyllosticta catimbauensis URM 7672 was selected for the partial optimisation of L-ASNase production because of its ability to generate considerable amounts of enzyme. We obtained the highest L-ASNase activity (3.47 U g-1), representing an increase of 36.02% in enzymatic production, under the following experimental conditions: a pH of 4.2, 1.0% inoculum concentration, and 2.5% L-asparagine concentration. Our study showed that M. catimbauensis harbours an important diversity of endophytic fungi with biotechnological potential for L-ASNase production.