Discovery of mosquitocides from fungal extracts through a high-throughput cytotoxicity-screening approach

Background Mosquitoes transmit a variety of diseases. Due to widespread insecticide resistance, new effective pesticides are urgently needed. Entomopathogenic fungi are widely utilized to control pest insects in agriculture. We hypothesized that certain fungal metabolites may be effective insecticides against mosquitoes. Methods A high-throughput cytotoxicity-based screening approach was developed to search for insecticidal compounds in our newly established global fungal extract library. We first determined cell survival rates after adding various fungal extracts. Candidate insecticides were further analyzed using traditional larval and adult survival bioassays. Results Twelve ethyl acetate extracts from a total of 192 fungal extracts displayed > 85% inhibition of cabbage looper ovary cell proliferation. Ten of these 12 candidates were confirmed to be toxic to Anopheles gambiae Sua5B cell line, and six showed > 85% inhibition of Anopheles mosquito cell growth. Further bioassays determined a LC50, the lethal concentration that kills 50% of larval or adult mosquitoes, of 122 µg/mL and 1.7 µg/mosquito, respectively, after 24 h for extract 76F6 from Penicillium toxicarium. Conclusions We established a high-throughput MTT-based cytotoxicity screening approach for the discovery of new mosquitocides from fungal extracts. We discovered a candidate extract from P. toxicarium that exhibited high toxicity to mosquito larvae and adults, and thus were able to demonstrate the value of our recently developed approach. The active fungal extracts discovered here are ideal candidates for further development as mosquitocides. Graphical abstract

Feature-Based Molecular Networking—An Exciting Tool to Spot Species of the Genus Cortinarius with Hidden Photosensitizers

Fungi have developed a wide array of defense strategies to overcome mechanical injuries and pathogen infections. Recently, photoactivity has been discovered by showing that pigments isolated from Cortinarius uliginosus produce singlet oxygen under irradiation. To test if this phenomenon is limited to dermocyboid Cortinarii, six colourful Cortinarius species belonging to different classical subgenera (i.e., Dermocybe, Leprocybe, Myxacium, Phlegmacium, and Telamonia) were investigated. Fungal extracts were explored by the combination of in vitro photobiological methods, UHPLC coupled to high-resolution tandem mass spectrometry (UHPLC-HRMS2), feature-based molecular networking (FBMN), and metabolite dereplication techniques. The fungi C. rubrophyllus (Dermocybe) and C. xanthophyllus (Phlegmacium) exhibited promising photobiological activity in a low concentration range (1–7 µg/mL). Using UHPLC-HRMS2-based metabolomic tools, the underlying photoactive principle was investigated. Several monomeric and dimeric anthraquinones were annotated as compounds responsible for the photoactivity. Furthermore, the results showed that light-induced activity is not restricted to a single subgenus, but rather is a trait of Cortinarius species of different phylogenetic lineages and is linked to the presence of fungal anthraquinones. This study highlights the genus Cortinarius as a promising source for novel photopharmaceuticals. Additionally, we showed that putative dereplication of natural photosensitizers can be done by FBMN.

Isolation of Taxol and Flavin-like fluorochrome from Endophytic Fungi of Mangifera indica

Scouting for novel and plant-derived biomolecules from endophytic microbial sources draws greater focus on the discovery of novel bioactive metabolites. With this rationale, we scouted the endophytic fungi for taxol, an anticancer diterpenoid and fluorescent biomolecules. In the present study, about 31 endophytic fungal isolates recovered from the Mangifera indica leaves were screened for taxol production in M1D medium. About five isolates were shortlisted based on the thin layer chromatographic analysis of the fungal extracts. Among them Colletotrichum sp. MIP-5 has been identified as a producer of fungal taxol based on UV, FTIR, TLC and HPLC analysis. The partially purified fungal taxol showed similar spectral and chromatographic features of commercially available paclitaxel. In addition to this, we also report the production of a fluorescent compound by Penicillium sp. MIP-3. The Flavin-like compound exhibited a bright greenish-yellow fluorescence with an emission maximum in the range of 505 – 545nm. GC-MS analysis showed the occurrence of Latia luciferin, primarily associated with the bioluminescence of freshwater limpet Latia neritoides. This is the first report of this compound from Penicillium sp. In addition, therapeutically active steroid (β-Sitosterol, Stigmasterol, Campesterol), quinones (Benzo[h]quinoline, 2,4-dimethyl-) and phloroglucinol (Aspidinol) derivatives were also identified from Penicillium sp. MIP-3 based on GC-MS analysis. These molecules could potentially be used in biological and pharmaceutical applications in future.

Diversity of endophytic fungi from the root bark of Syzygium zeylanicum, and the antibacterial activity of fungal extracts, and secondary metabolite

Syarifah, Elfita, Widjajanti H, Setiawan A, Kurniawati AR. 2021. Diversity of endophytic fungi from the root bark of Syzygium zeylanicum, and the antibacterial activity of fungal extracts, and secondary metabolite. Biodiversitas 22: 4572-4582. The decoction of the root bark of Syzygium zeylanicum has been used as traditional medicine, such as for treating pathogenic bacterial infections. Endophytic fungi that live in medicinal plant tissues have a high species diversity and biological activities correlate with their host. Therefore, this study aimed to explore the diversity of endophytic fungi from the root bark of S. zeylanicum and to determine the antibacterial activity of endophytic fungi and their secondary metabolites. In this study, we isolate and identify the endophytic fungi from the root bark of S. zeylanicum, continued by screening their antibacterial activity against two Gram-negative bacteria (Escherichia coli InaCCB5 and Salmonella thypi ATCC1048 and two Gram-positive bacteria (Staphylococcus aureus InaCCB4 and Bacillus subtilis InaCCB1204) by the Kirby-Bauer method. The fungal extract with the highest antibacterial activity proceeded with the isolation and determination of the structure of their bioactive compounds. The isolates were morphologically identified. Isolates that showed strong antibacterial activity were identified by molecular identification. Isolation of bioactive compounds was carried out by chromatographic techniques and the determination of the structure of pure chemical compounds was performed by the spectroscopic analysis. In total, there were 8 isolates of endophytic fungi were obtained from the root bark of S. zeylanicum, namely SZR1 – SZR8. SZR2 isolate has the highest antibacterial activity. Molecular identification through phylogenetic analysis showed that SZR2 isolate had high similarity with Penicillium brefeldianum. Isolation of bioactive compounds from SZR2 produced compound 1 in the form of light yellow crystals which showed strong antibacterial activity against S. typhi, E. coli, and B. subtilis with MIC values of 64 g/mL. Compound 1 was identified as p-hydroxybenzaldehyde, which was also obtained in its host. In conclusion, the endophytic fungus Penicillium brefeldianum produces similar secondary metabolites and antibacterial activity as its host plant.

Utilizing a novel fungal enzymatic cocktail as an eco-friendly alternative for cellulose pulp biobleaching

Enzyme cocktails can alter the lignin and hemicellulose content in wood cell walls, improving the bleaching process during pulp production and offsetting the need for toxic chemicals. In this study, brown pulp was biobleached with a mixture of crude fungal extracts rich in xylanase and laccase, respectively produced from Aspergillus tamarii Kita and Trametes versicolor on waste materials. The optimal conditions for biobleaching were a mixture of xylanase and laccase crude extracts (1 to 2 v/v), at a temperature of 36 °C and a pH of 5.5. The treated brown cellulose pulp showed a reduction in the Kappa number by 1.83 points, representing an efficiency of 20.3%. In addition, the brightness increased by 4.65 points in comparison to the control. Hence, studies involving the application of the standardized cocktail during the hydrolysis of lignocellulosic residues, e.g., barley residue and sugarcane bagasse, led to the formation of 85 g/L and 25 g/L of reducing sugars, respectively. Moreover, the standardized cocktail caused greater deinking of the recycled paper pulp.

Media and strain studies for the scaled production of cis-enone resorcylic acid lactones as feedstocks for semisynthesis

Resorcylic acid lactones (RALs) with a cis-enone moiety, represented by hypothemycin (1) and (5Z)-7-oxozeaenol (2), are fungal secondary metabolites with irreversible inhibitory activity against protein kinases, with particularly selective activity for inhibition of TAK1 (transforming growth factor beta-activated kinase 1). Gram-scale quantities of these compounds were needed as feedstock for semi-synthesizing RAL-analogues in a step-economical fashion. To do so, this study had three primary goals: identifying fungi that biosynthesized 1 and 2, enhancing their production by optimizing the fermentation conditions on the lab scale, and developing straight forward purification processes. After evaluating 536 fungal extracts via an in-house dereplication protocol, three strains were identified as producing cis-enone RALs (i.e., MSX78495, MSX63935, MSX45109). Screening these fungal strains on three grain-based media revealed enhanced production of 1 by strain MSX78495 on oatmeal medium, while rice medium increased the biosynthesis of 2 by strain MSX63935. Furthermore, the purification processes were improved, moving away from HPLC purification to utilizing two to four cycles of resuspension and centrifugation in small volumes of organic solvents, generating gram-scale quantities of these metabolites readily. In addition, studying the chemistry profiles of strains MSX78495 and MSX63935 resulted in the isolation of ten other RALs (3-12), two radicinin analogues (13-14), and six benzopyranones (15-20), with 19 and 20 being newly described chlorinated benzopyranones.

Extracts of endophytic fungi from leaves of selected Nigerian ethnomedicinal plants exhibited antioxidant activity

Background Plants with an ethnobotanical history are known to harbor diverse group of endophytic fungi, which constitute major natural sources of bioactive compounds. In the present study, we evaluated the antioxidant activity of endophytic fungi from eight Nigerian ethnomedicinal plants. Endophytic fungi were isolated from the leaves of Acalypha ornata, Albizia zygia, Alchornea cordifolia, Chrysophyllum albidum, Ficus exasperata, Gomphrena celosioides, Millettia thonningii, and Newbouldia laevis. Methods Endophytic fungi were isolated from the leaves of selected plants via surface sterilization. Isolated fungi were identified by internal transcribed spacer (ITS-rDNA) sequence analysis. Pure fungal strains were subjected to fermentation process on solid rice medium and metabolites extracted using ethyl-acetate. Fungal crude extracts were screened for antioxidant activity using 2, 2- diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and reduction of ferric ion assays. Gas chromatography/mass spectrometry (GC/MS) analysis was used to identify the major chemical constituents in active fungal extracts. Results A total of eighteen fungal endophytes with fungal codes CU (061 and 062); ZA (161, 162, 163, and 164); LO (261); CA (041, 042, and 043); FE (081, 082, and 084); GE (091); MO (211 and 212); and NA (021 and 022) were isolated from the eight ethnomedicinal plants A. ornata, A. zygia, A. cordifolia, C. albidum, F. exasperata, G. celosioides, M. thonningii, and N. laevis respectively. ZA 163 and MO 211 fungal extracts showed significant (p < 0.05) radical scavenging activity with IC50 values of 50.53 ± 0.01 and 86.69 ± 0.02 μg/ml respectively. Fungal extract CA 041 demonstrated significantly (p < 0.01) higher iron chelating activity than standard gallic acid with absorbance values of 0.803 and 1.107 at 250 and 500 μg/ml concentrations respectively. Pyrogallol, phenol, 2,6-dimethoxy-, phytol, dl-alpha-tocopherol, alpha-tocospiro, oleamide, methyl stearate, oleic acid, palmitic acid, campesterol, stigmasterol, β-sitosterol, urs-12-en-24-oic acid, 3-oxo-, methyl ester, lup-20(29)-en-3-one, and lupeol were detected in the selected active extracts. Conclusion These results showed that leaves of the selected Nigerian plants harbor diverse group of endophytic fungi, which can be potential antioxidant resource. Graphical abstract

Metabolites from Induratia spp. Modulating Key Enzymes in Human Hemostasis

Fungi Induratia have been poorly evaluated for their non-volatile secondary metabolites. In the present work, we evaluated the effects of non-volatile secondary metabolites released into the culture medium by Induratia spp. upon toxic alterations induced by Bothrops spp. venoms. B. atrox venom phospholipase was inhibited by Induratia sp. and I. yucatanensis around 12 and 16%. The extracts of two strains inhibited 12 to 25% of the hemolysis induced by B.moojeni venom. Thrombolysis was inhibited by 30 to 60% by the compounds present in two extracts. The coagulation induced by B. moojeni venom was prolonged by 26 to 41 s by the action of the extracts of I. yucatanensis and I. coffeana. The fungal extracts did not exerted any cytotoxic effect, nor did they induce any alteration in the other evaluated substrates show the potential use of non-volatile metabolites produced by the fungi evaluated as enzyme modulators, especially for proteases with a fundamental role in human hemostasis.