1. Chemical Screen and Antibacterial Testing of Fungal Endophytes Andrew Pham | Dr. Sandra Loesgen | Department of Chemistry

2. Bacteria or Fungi Literally means “in the plant” Greek Endon – Within Phyton – Plant Endophytes are involved in a mutualistic symbiosis with their plant host providing bioactive metabolites in exchange for nutrients What is an Endophyte? B. Schulz, C. Boyle, T.N. Sieber (2006) “What are Endophytes? Vol. 9 1 Algae collected from Strawberry Hill, Oregon

3. Natural Product 2 Penicillin VK “Natural products are organic compounds that are formed by living systems” Hanson JR (2003) RoyalSocietyChem. D. Newman; G. Gragg; J. Nat. Prod. 2012 75: 311–335 Natural products sources represent 50% of all new drugs on the market

4. Strains Gozo 1 Gozo 4-Y Kreta 2/23 Kreta 2/28 Project 3

5. Fungal Media 4 M2 Malt extract Yeast extract D-glucose pH = 7.0 1158 NH 4 HSO 4 Malt Extract D-glucose Yeast Extract pH = 6.0 G20 Glycerine Malt extract Yeast extract

6. Project 5 Gozo 1Gozo 4-YKreta 2/23Kreta 2/28 M2 1158 G20

7. Method – Work Flow 6 TLC 10 Plate Work Up

8. Bioassay 7

9. Diffusion Disk Assay 8 Test for Activity Looking for Zone of inhibition

10. Antibacterial testing (Diffusion Disk Assay) 9 P. aeruginosa C. albicans S. aureus

11. Diffusion Disk Assay - Results 10 Staphylococcus aureus Krete 2/23 M2 Krete 2/28 G20 Krete 2/23 G20

12. Chemical Screening

13. Chemical Screening - Thin-Layer Chromatography (TLC) 12 254 nm M2 1158 G20 Gozo 1 M2 1158 G20 http://www.chemguide.co.uk/analysis/chromatography/thinlayer.html

14. Chemical Screening - Kreta 2/23 13 M21158G20 M2 1158 G20

15. Chemical Screening - Kreta 2/28 14 M2 1158 G20 M2 1158 G20

16. Chemical Screening – MS (Kreta 2/28 G20) – 28 min 15 [M+H] [M] = 592 g/mol Δ23 [M+Na] Δ18 [M-H 2 O]

17. Chemical Screening - TLC 16 Gozo 1 Gozo 4-YKrete 2/23 Krete 2/28 Stain: p-Anisaldehyde

18. Endophytes produced different natural products under different medium Continue onto new strains Kreta 2/23 & Kreta 2/28 Large scale Further purification Bioassay & Structure Elucidation Isolate new entophyte from Oregon Algae sources Conclusion Future Work 17

19. Acknowledgement 18 Dr. Sandra Loesgen Graduate Students: Ross Overacker Donovon Adpressa Chenxi (Zoe) Zhu Department of Microbiology Oregon State University

20. Questions? 19

21. August 18, 2015 20