Analysis of Secondary Metabolites Biosynthesized by Pathogenic and Symbiotic Fungi using High-Resolution Tandem LC-MS and Spectral Molecular Networking

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Analysis of Secondary Metabolites Biosynthesized by Pathogenic and Symbiotic Fungi using High-Resolution Tandem LC-MS and Spectral Molecular Networking, Is A Well-Researched Topic, It Is To Be Used As A Guide Or Framework For Your Research

Abstract

Fungi from Canadian crops affect many aspects of agriculture. For example, ginseng root rot caused by Ilyonectria spp. is a major issue for farmers. Ilyonectria spp. make few reported natural products and it is not known if those products are unique to virulent species. PCA and molecular networking were applied to HRMS data to establish a distinct metabolomic profile of root rot pathogens, characterized by antifungal resorcyclic acid lactones (RALs). These likely protect Ilyonectria from other soil pathogens, which gives it the opportunity to infect ginseng root.

Molecular networking was also applied to fungal endophytes from fruit crops to identify new compounds. Endophytes are symbionts that produce beneficial compounds, however, traditional screening methods are cumbersome in finding new or novel compounds. HRMS data of 302 fungal endophytes were examined to identify nine new compounds related to known antimicrobial compounds. These compounds are targets for isolating and characterizing in further studies.

Table of Contents

Thesis abstract …………………………………………………………………………………………………………………. i
Summary for Lay Audience ……………………………………………………………………………………………… ii
Co-authorship Statement …………………………………………………………………………………………………. iv
Acknowledgments…………………………………………………………………………………………………………… v
List of Tables ………………………………………………………………………………………………………………… ix
List of Figures ………………………………………………………………………………………………………………… x
List of Symbols and Abbreviations………………………………………………………………………………….. xii
List of Appendices ………………………………………………………………………………………………………… xv
Chapter 1: Introduction ……………………………………………………………………………………………………. 1
1.1 Culturing Fungi ……………………………………………………………………………………………………… 2
1.2 Analytical Techniques …………………………………………………………………………………………….. 7
1.2.1 Ultra-High-Performance Liquid Chromatography (UHPLC) …………………………………. 7
1.2.2 Mass Spectrometry …………………………………………………………………………………………… 9
1.2.2.1 Heated Electrospray Ionization (HESI) …………………………………………………………. 9
1.2.2.2 S-Lens, Flatapoles, Quadrupole Mass Filter, and C-trap ………………………………… 13
1.2.2.3 Orbitrap Mass Spectrometer ………………………………………………………………………. 15
1.2.2.4 Higher Energy Collisional Dissociation Cell ………………………………………………… 18
1.3 Metabolomics ………………………………………………………………………………………………………. 20
1.3.1 High-resolution LC-MS in metabolomics ………………………………………………………….. 20
1.3.2 Data processing in metabolomics ……………………………………………………………………… 23
1.3.2.1 xcms ……………………………………………………………………………………………………….. 23
1.3.2.2 Statistical testing ………………………………………………………………………………………. 24
1.3.2.3 Molecular Networking ………………………………………………………………………………. 26
1.3.3 Molecular Networking …………………………………………………………………………………….. 28
1.4 Thesis Objectives …………………………………………………………………………………………………. 31
Chapter 2 – Metabolomic profiling of ginseng root rot fungi ……………………………………………… 32
2.1 Chapter 2 Objectives …………………………………………………………………………………………….. 32
2.2 Introduction to Ginseng Root Rot …………………………………………………………………………… 32
2.3 Methods ………………………………………………………………………………………………………………. 36
2.3.1 Growth and plug extraction of fungal cultures ……………………………………………………. 36

2.3.2 LC-MS and LC-MS/MS analysis of plug extracts ………………………………………………. 38
2.3.3 Principal Component Analysis …………………………………………………………………………. 39
2.3.4 Molecular Networking Parameters and Visualization ………………………………………….. 40
2.4 Results and Discussion ………………………………………………………………………………………….. 41
2.4.1 Results and Discussion of Principal Component Analysis ……………………………………. 41
2.4.2 Results and Discussion of Molecular Networking ………………………………………………. 45
2.5 Conclusions and Suggestions for Future Work …………………………………………………………. 50
Chapter 3: Non-targeted screening of natural products from 302 fungal endophytes isolated from Canadian fruit crops ………………………………………………………………………………………………………. 52
3.1 Chapter 3 Objectives …………………………………………………………………………………………….. 52
3.2 Introduction …………………………………………………………………………………………………………. 52
3.3 Methods ………………………………………………………………………………………………………………. 57
3.3.1. LC-HRMS analysis of endophyte cultures ………………………………………………………… 57
3.3.2 Data processing and analysis by PCA ……………………………………………………………….. 59
3.3.3 GNPS parameters and processing……………………………………………………………………… 60
3.4 Results and Discussion ………………………………………………………………………………………….. 60
3.4.1 Griseofulvin Cluster ……………………………………………………………………………………….. 70
3.4.2 Griseofernaneoside Cluster………………………………………………………………………………. 73
3.4.3 Hirsutatin A Cluster ………………………………………………………………………………………… 75
3.4.4 Oxysporidinone Cluster …………………………………………………………………………………… 76
3.4.5. Additional Dereplicated Compounds ……………………………………………………………….. 77
3.5 Conclusions and Suggestions for Future Work …………………………………………………………. 80
Chapter 4: Conclusions ………………………………………………………………………………………………….. 82

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YourPastQuestions Brand

Additional information

Author

Natasha DesRochers

No of Chapters

4

No of Pages

127

Reference

YES

Format

PDF

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