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Endophytic fungi associated with Ficus carica L. (Moraceae) from Argentina: antimicrobial and biotransforming ability Melisa Isabel Barolo, María Victoria Castelli, Silvia Noelí López* Farmacognosia, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario-CONICET, Santa Fe, Argentina *Silvia Noelí López, [email protected] Endophytic fungi associated with Ficus carica L. (Moraceae) from Argentina: antimicrobial and biotransforming ability The endophytic fungal community associated with leaves of Ficus carica L. (Moraceae) from Argentina was investigated. Fifteen fungal isolates were isolated and identified by molecular methods into the genera Alternaria, Cladosporium, Curvularia, Diaporthe, Epicoccum, Myrothecium, Neofusicoccum, Nigrospora, Preussia and Ustilago. Cladosporium cladosporioides and Curvularia lunata were the most frequently isolated species. The fungal metabolic profiles were obtained by automated TLC and NMR and analysed by PC Analysis. Antifungal and antibacterial activity was assessed by bioautographic assays. In addition, the biotransforming ability of the fungal isolates was tested on F. carica extracts. Five isolates (33.3%) exhibited inhibitory activity against at least one of the microorganisms tested. Most of the fungal endophytes were able to metabolize the flavonoid rutin 1, and the coumarin psoralen 3 present in F. carica extracts. Further investigations of the psoralen biotransforming ability performed by the selected endophyte Alternaria alternata F8 showed the accumulation of the 6,7-furan-hydrocoumaric acid derivative 4 as the main biotransformation product. Our results corroborate that F. carica can live symbiotically with rich and diverse endophytic communities adding insights about their ecological interactions. Keywords: Endophytic fungi, Ficus carica, chemical profiling, antimicrobial activity, biotransformation Figure S1. Adult specimen of F. carica included in this study. Figure S2. Endophytic fungi isolated from F. carica on Petri dishes containing PDA and incubated at 28 ºC for 7 d. Figure S3. 1H NMR spectrum of psoralen 3 in CDCl3 as a solvent. Bruker 300 MHz (1H) / 50 MHz (13C) spectrometer. Figure S4. 6.00 to 9.00 ppm magnification of 1H NMR spectrum of psoralen 3. Solvent: CDCl3. Bruker 300 MHz (1H) / 50 MHz (13C) spectrometer. Figure S5. 13C NMR spectrum of psoralen 3 in CDCl3 as a solvent. Bruker 300 MHz (1H) / 50 MHz (13C) spectrometer. Figure S6. 70 to 170 ppm magnification of 13C NMR spectrum of psoralen 3. CDCl3. Bruker 300 MHz (1H) / 50 MHz (13C) spectrometer. Figure S7. HH COSY experiment of psoralen 3. Figure S8. Amplification of HH COSY experiment of psoralen 3 at region amongst 6.00 to 7.90 ppm. Figure S9. HSQC experiment of psoralen 3. Figure S10. Amplification of HSQC experiment of psoralen 3 amongst 6.00 to 8.00 ppm (1H) and 80 to 160 ppm (13C). Figure S11. HMBC experiment of psoralen 3. Figure S12. Amplification of HMBC experiment of psoralen 3 amongst 5.80 to 8.40 ppm (1H) and 70 to 180 ppm (13C). Figure S13. Quantitation of the psoralen 3 content in the F. carica 7.5 % culture medium. Polynomial regression based on the height of the peaks. Software Vision CATS v4.0. Figure S14. 1H NMR (section 2.60-7.60 ppm, CDCl3, 300 MHz) of compound 4. Figure S15. HRMS of psoralen 3. LC-MS ES-API positive mode. Figure S16. Comparison of the HRMS spectra obtained by direct infusion in negative (left) and positive (wright) mode of A. alternata F8 extracts and the corresponding controls at 21 d incubation. The ions m/z = 274.26; 381.29 and 383.11 (positive mode) and their correspondent ions in the negative mode are contaminants reported by the equipment operator. T21C: control medium at 21 d; T21P: control medium + 46 μg/mL psoralen 3 incubated by 21 d; T21F8: F8 grown on Control medium by 21 d; T21F8+P: F8 grown on the control medium + 46 μg/mL psoralen 3. Figure S1 Figure S2 Figure S3 Figure S4 Figure S5 Figure S6 Figure S7 Figure S8 Figure S9 Figure S10 Figure S11 Figure S12 Figure S13 Figure S14 Figure S15 Negative mode Positive mode Figure S16