Exploring Tropical and Temperate Fungi for Biocontrol Agents

ICVCAE: 26 November 2025 Sherif S. Ebada, PhD Microbial Drugs Dept., HZI Braunschweig, Germany; [email protected]; [email protected] Exploring Tropical and Temperate Fungi for Biocontrol Agents Blackwellomyces roseostromatus Samsoniella aurantia Cordyceps javanica Laburnicola nematophila Beauveria neobassiana Polyphilus sieberi Penicillium sp. Epicoccum nigrum Agenda 1. Overview on BSMs. 2. Background on BCAs. 3. Biofilms: What, Why, When and Where? 4. Biofilm Inhibitors from Saprotrophic/Entomopathogens: R. Toshe, K. Phutthacharoen, S. S. Ebada, M. Stadler, et al. 5. Biofilm Inhibitors and Biocontrol Agents from Nematode Pathogens: J.-P. Wennrich, C. Holzenkamp, E. Sepanian, S. S. Ebada et al. 6. Cytotoxic metabolites and AMCs from Plant-Associated Fungi: M. S. Elnaggar, A. M. Elissawy, S. S. Ebada, et al. 1. Background in BSMs 1. Background in BSMs 2. Background on BCAs https://link.springer.com/article/10.1007/s42452-025-06500-9 •Up to 70% increase needed, for the population of 9.1 billion by 2050. •Pests, pathogens (100K), weeds (30K), invertebrates (1K), and insects (10K). •3 Billion tons of synthetic pesticides though crop losses about 40%(>500 billion USD). •Soil microbiome, ecological imbalance, pollution, health threats, impact on biodiversity, and pest resistance. •Bio-control market expected to rise from 6.51 to 18.15 billion USD over 2022 to 2029. •Around 300 bio-pesticide active ingredients were registered. •Microbial bio-pesticides are slowly picking up steam. 2. Background on BCAs •Microbial bio-pesticides. •Lytic enzymes, AMCs or compete for nutrients, colonization, and attachment with phytopathogens on plants. •Decomposability, no compromise on groundwater, soil, and air qualities, and less residuals in end products. •In New Zealand, 317 registered insecticides under the ACVM, 23 from microbial sources. From 1996 to 2000,10 MBCAs registered in the EU, while in the last 15 years, 27 approved. •Challegnes: Increase residual action, stability, and market presence of biopesticides. •Also, the high production costs, susceptibility to environmental influences, efficacy challenges, inadequate, and storage stability. Blackwellomyces roseostromatus Samsoniella aurantia Cordyceps javanica Beauveria neobassiana https://www.pureline.com/biofilm-threats-and-the-chlorine-dioxide-solution/ Adhesion EPS matrix Biofilm Maturation Balance 3. Biofilm: What, Why, When and Where? https://www.researchgate.net/publication/239735127 3. Biofilm: What, Why, When and Where? Surface Biofilm – Close up of a Streambed Biofilm Forming Microorganism – Electron Micrograph PDB Beauveria neobassiana Compounds 2-5: anti S. aureus biofilm of ca. 80% at 31 µg/mL Bioprospection of Tenellins Produced by the Entomopathogenic Fungus Beauveria neobassiana b. Biofilm Inhibitors from Entomopathogenic fungi b. Biofilm Inhibitors from Entomopathogenic Fungi Bioprospection of Tenellins Produced by the Entomopathogenic Fungus Beauveria neobassiana Cytotoxicity criteria: •15-Hydroxylation. •N-Hydroxylation •15-Hydroxylation and N-Hydroxylation Only tenellin exhibited slight antibacterial activity against Bacillus subtilis and Staphylococcus aureus. Halo et al. (2008) Journal of the American Chemical Society Cytotoxic activity (4) Test Cell Line IC50 (µM) Positive Control 1 2 3 4 5 6 Epothilone B (nM) L929 (fibroblast) n.a. 70.1 48.2 0.79 6.8 5.7 0.65 KB3.1 (ovary) n.a. 64.7 20.2 0.79 6.0 4.9 0.17 A549 (lung) n.d. n.d. n.d. 0.24 2.6 24.1 0.05 MCF-7 (breast) n.d. n.d. n.d. 2.0 8.1 7.3 0.07 b. Biofilm Inhibitors from Entomopathogenic Fungi Bioprospection of Tenellins Produced by the Entomopathogenic Fungus Beauveria neobassiana •Tenellin derivatives (2-4) revealed promising activities in a dose-dependent manner. •In 2-pyridone derivatives, the aliphatic side chain at C-3 potentiates antibiofilm activity. •Hydroxylation at C-15 on of the side chain as in 15hydroxytenellin (5) negatively affected the biofilm inhibitory activity. b. Biofilm Inhibitors from Entomopathogenic Fungi Blackwellomyces roseostromatus b. Biofilm Inhibitors from Entomopathogenic Fungi. IC50 (µM) Positive Control Test Cell Line 1 2 3 4 5 6 Epothilone B (nM) L 929 (murine) ** * * ** ** 26.0 0.65 KB 3.1 (cervix) 29.2 * * 36.2 2.2 8.4 0.17 PC -3 (prostate) n.t. n.t. n.t. n.t. 2.5 7.5 0.09 MCF -7 (breast) n.t. n.t. n.t. n.t. 6.9 13.9 0.07 SKOV -3 (ovary) n.t. n.t. n.t. n.t. 25.0 12.3 0.09 A 431 (skin) n.t. n.t. n.t. n.t. 4.3 8.5 0.06 A 549 (lung) n.t. n.t. n.t. n.t. 11.5 11.1 0.05 Cytotoxic (IC50 in µM) activity results of 1-6against mammalian cells. (*): Slight inhibition of cell proliferation, (**): no cytotoxic activity observed, n.t.: not tested. b. Biofilm Inhibitors from Entomopathogenic Fungi. Nematicidal assay of 1–6against Caenorhabditis elegans. Corrected mortality rate of compounds 1–6(10 µg mL-1,50 µg mL-1 and 100 µg mL-1) against C. elegans after 18 hof treatment. A solution of ivermectin (1 µg mL-1) was used as a positive control. b. Biofilm Inhibitors from Entomopathogenic Fungi. II. Biofilm Inhibitors from Entomopathogenic Fungi. Antibiofilm and Cytotoxic Metabolites from the Entomopathogenic Fungus Samsoniella aurantia b. Biofilm Inhibitors from Entomopathogenic Fungi IC50 (µM) Positive Control Test Cell Line 1 2 3 4 5 6 Epothilone B (nM) Mouse fibroblast (L929) n.a. n.a. 0.6 1.2 1.0 0.8 0.65 Human endocervival adenocarcinoma (KB3.1) 47.3 79.0 0.5 1.2 1.0 0.8 0.17 Human prostate carcinoma (PC-3) 68.6 n.d. 6.2 n.d. 2.3 2.8 0.09 Human breast adenocarcinoma (MCF-7) 9.7 n.d. 0.2 n.d. 1.2 1.1 0.07 Human ovarian cancer (SKOV-3) 26.0 n.d. 0.3 n.d. 1.5 1.5 0.09 Human epidermoid carcinoma (A431) 18.2 n.d. 0.2 n.d. 1.0 1.0 0.06 Human lung carcinoma (A549) 52.0 n.d. 0.3 n.d. 3.0 3.2 0.05 Test Microorganism MIC (µg/mL) Positive Control (µg/mL) Staphylococcus aureus (DSM 346) 66.6 33.3 16.6 16.6 8.3 n.i. 0.21G Escherichia coli (DSM 1116)n.i. n.i. 66.6 n.i. n.i. n.i. 0.42G Bacillus subtilis (DSM 10) 66.6 33.3 16.6 8.3 4.2 n.i. 16.6O Pseudomonas aeruginosa (PA14)n.i. n.i. n.i. n.i. n.i. n.i. 0.21G Wickerhamomyces anomalus (DSM 6766)n.i. n.d. n.i. n.d. n.d. n.i. 16.6N Candida albicans (DSM 1665)n.i. n.i. n.i. n.i. n.i. n.i. 8.3N Acinetobacter baumannii (DSM 30008)n.i. n.d. n.i. n.d. n.d. n.i. 0.52C Chromobacterium violaceum (DSM 30191)n.i. n.d. n.i. n.d. n.d. n.i. 1.70G Schizosaccharomyces pombe (DSM 70572)n.i. n.d. 66.6 n.d. n.d. n.i. 8.30N Mucor hiemalis (DSM 2656)n.i. n.i. n.i. n.i. n.i. n.i. 8.30N Rhodotorula glutinis (DSM 10134)n.i. n.d. n.i. n.d. n.d. n.i. 4.20N Mycolicibacterium smegmatis (ATCC 700084)n.i. n.i. n.i. 16.6 8.3 n.i. 1.70K n.a.: No activity. n.i.: No inhibition up to 67 µg/mL. n.d.: Not determined. G: Gentamycin; O: Oxytetracycline; N: Nystatin; C: Ciprofloxacin; K: Kanamycin. Cytotoxicity (IC50)and antimicrobial activity (MIC) of 1-6. b. Biofilm Inhibitors from Entomopathogenic Fungi. Cultivation Polyphilus sieberi Heterodera filipjevi infected by: Polyphilus sieberi LCMS analysis Activity-guided fractionation Antimicrobial testing Purification & structure elucidation Assessment of Pure Compounds Bacteria B. subtilis E. coli Fungi M. tenuis C. albicans Qualitative screening (Prescreening) Collaborations Plant parasitic nematodes (JKI) Human parasitic nematodes Screening Libraries (HIPS) Inhibtion of biofilm formation (H. Zeng) Quantitative testing Effects on C. elegans populations (J. Wennrich) Minimal Inhibitory concentration (MIC) (W. Collisi) Cytotoxicity (W. Collisi) Genus Species Strain no. Isolation source Region Polyphilus frankenii V 16 Pinus sylvetris roots a Villerupt , France sieberi 17 A Eggs of Heterodera filipyevi b Yozgat, Turkey 17 C REF 052 Asclepias syriaca roots c Bugac , Hungary Fungal strains investigated in this study a Isolated by Damien Blaudez and Michel Chalot. b Isolated by Samad Ashrafi. c Isolated by Dániel G. Knapp and Gabor M. Kováks 5. Biofilm Inhibitors and Biocontrol Agents from Nematode Pathogens Organism 2 MIC [µg/mL] Control [O] MIC [µg/ mL] B. subtilis 0.52 16.6 S . aureus 66.6 0.21 5. Biofilm Inhibitors and Biocontrol Agents from Nematode Pathogens 5. Biofilm Inhibitors and Biocontrol Agents from Nematode Pathogens Cytotoxic (IC50 in µM) activity results. Antimicrobial (MIC in µg/mL) activity results. 5. Biofilm Inhibitors and Biocontrol Agents from Nematode Pathogens Page 36 | 5: R= H 6: R= Cl 5. Biofilm Inhibitors and Biocontrol Agents from Nematode Pathogens Laburnicotides A–F: Acyclic N-acetyl Oligopeptides from the NematodeCyst Associated Fungus Laburnicola nematophila 5. Biofilm Inhibitors and Biocontrol Agents from Nematode Pathogens 5. Biofilm Inhibitors and Biocontrol Agents from Nematode Pathogens Strain number IC50 (µM) Positive Control Test Cell Line 1 2 3 4 5 6 Epothilone B (nM) Mouse fibroblast L929 * * n.d. 49.02 ** * 0.65 Human endocervival adenocarcinoma KB3.1 * 33.31 n.d. 63.02 76.01 * 0.17 Test Microorganism MIC (µg/mL) Positive Control (µg/mL) Staphylococcus aureus DSM 346 - 66.6 n.d. - - - 0.21G Aspergillus fumigatus ATCC 204305 0.26 0.52 8.325 n.d. n.d. n.d. 0.312A A. fumigatus CCF 3522 n.d. 0.52 n.d. n.d. n.d. n.d. 0.312A A. fumigatus (azoleresistant) CCF 6651 - 2.08 0.52 n.d. n.d. n.d. 0.156A A. fumigatus (azoleresistant) CCF 6674 16.6 0.52 0.52 n.d. n.d. n.d. 2.5A C. albicans CCM 8215 - - 8.3 n.d. n.d. n.d. 1.25A Cryptococcus neoformans CCF 1081 - 4.15 4.15 n.d. n.d. n.d. 2.5A Mucor plumbeus CCF 2612 - - 1.04 n.d. n.d. n.d. 0.312A Rhodotorula glutinis DSM 10134 - 8.3 n.d. - - - 4.20N 5. Biofilm Inhibitors and Biocontrol Agents from Nematode Pathogens 5. Biofilm Inhibitors and Biocontrol Agents from Nematode Pathogens Cordypyridones E–J (1–6), C (7) and D (8). 6. Cytotoxic and AMCs from Plant-Associated Fungi M. S. Elnaggar, A. M. Elissawy, N. M. Sabry, S. S. Ebada, et al. Dept. Pharmacognosy, ASU, Egypt Dr. M. S. Elnaggar Dr. A. M. Elissawy