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Aspects of the biology of entomogenous fungi and their associations with arthropods 2002

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ASPECTS OF THE BIOLOGY OF ENTOMOGENOUS FUNGI AND THEIR ASSOCIATIONS WITH ARTHROPODS By E D U A R D O M E L I T O N J O V E L A Y A L A B . S c . Agronomy, Nat ional Schoo l of Agricul ture, E l Sa lvador 1980 B . S c . Botany, Cal i fornia State Poly technic University, P o m o n a , Cal i forn ia, U S A 1993 M . S c . Ethnobotany, Universi ty of Brit ish Co lumb ia , Vancouve r , C a n a d a 1996 A T H E S I S S U B M I T T E D IN P A R T I A L F U L F I L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F D O C T O R IN P H I L O S O P H Y In T H E F A C U L T Y O F G R A D U A T E S T U D I E S Department of Botany W e accept this thesis as conforming to the required s tandards T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A Augus t 2002 © Eduardo M. Jove l A y a l a , 2002 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada II ABSTRACT I investigated several aspects of the biology of entomogenous fungi (mostly Clavicipitaceae with few species of Hypocreaceae). My primary motive in this research was to gain an understanding of the interactions between entomogenous fungi and arthropods. My study included field collections and identification of entomogenous fungi from BC and a few collections from Peru and Idaho. I addressed some aspects of the interactions among arthropods and fungi, life histories of fungi under laboratory conditions, and observations of chemical changes of fungi growing in the presence of heavy metals. About fifty entomogenous fungi were collected in the province, mainly as anamorphs, but this permitted isolation and cultivation of many species. Of special interest is a small group of fungi parasitic on spiders some of which may be new records for western Canada. Interactions of entomogenous fungi and heavy metals yielded a cerebroside not detected, or known to be produced, in the absence of heavy metals. This compound showed antibiotic activity against Staphylococcus aureus. The induction of this cerebroside by exposure to copper also is a promising approach to obtaining new drugs, or to increase the yield of selected compounds, from these organisms. The biological activities of other extracts were assessed, demonstrating additional compounds of interest (e.g., antiviral, antibacterial, phototoxic and antifungal substances). Cultures grown on substances rich in oils and proteins (nuts and seeds) appeared to induce development beyond the anamorph stage to early teleomorph form. No perithecia developed although large synnemata and relatively bright pigmentation were observed. The ability to induce complete development of ascocarps would be of laboratory interest in the possible production of substances from wild ascocarps. Cultures obtained in this study will be deposited in the Canadian National Culture Collection, Ottawa, in the Canadian Culture Collection (UBC). Further research remains necessary to fully understand the relationship between teleomorph and anamorph stages of entomogenous fungi, their nutritional requirements, for the production of teleomorph stages under laboratory conditions, and particularly to establish systems that may allow a chemical exploration for new drugs. The preliminary studies of anti-arthropod activity by entomogenous fungi were partially successful in controlling a variety of laboratory-reared and naturally growing arthropods. The production of bio-pesticides is currently of great interest because of the problems with chemical pesticides and environmental pollution. Gaining a better understanding of the biology of these organisms will allow us to develop new genetic strains of species for both biopesticides and biosynthetic applications. Both kinds of substances can contribute to maintain the health and equilibrium of the coastal temperate rainforest of the Pacific Northwest. iii TABLE OF CONTENTS Abstract '. ii Table of Contents iii List of Tables .• vi List of Figures vii List of Abbreviations xii Acknowledgements xiv CHAPTER ONE: General Introduction 1 Historical Significance of Entomogenous Fungi 6 Cordyceps sinensis (Berk.) Sacc. and Traditional Chinese Medicine 7 Biologically Active Substances from the Hypocreales .....8 Potential of Biological Control and Mycopesticides 8 Heavy Metals and Entomogenous Fungi 9 Objectives 10 Study Area 11 CHAPTER TWO: Collection, Isolation, and Cultivation of Entomogenous Fungi 13 INTRODUCTION 13 OBJECTIVES 15 STUDY AREAS 16 METHODS 16 Field Collections 16 Voucher Specimens 17 Maintenance of Fungal Isolates and Stock Cultures 18 Attempts to Produce Ascomata in Culture 19 Entomogenous Fungi Inoculated on Experimental Arthropods 19 iv Arthropod Maintenance 20 Free Fatty Acid Analysis of Selected Entomogenous Fungi 21 RESULTS 21 Survey of Entomogenous Fungi in British Columbia 21 Field Collections 22 Microscopic Documentation of Field Collections 27 Culture Collections 29 Attempts to Produce Ascomata using Seeds and Nuts 30 Arthropod Inoculation and Synnemata Production 34 Free Fatty Acid Analysis 39 DISCUSSION 40 CHAPTER THREE: Screening of Entomogenous Fungi for Bioactivity 45 INTRODUCTION 45 OBJECTIVES 46 METHODS 46 Fungal Extract Preparation 46 Antibacterial Bioassay 46 Photoactivated Antibiotics and Antifungal Substances 48 Antiviral Bioassays 48 Screening for Cyclosporine A 49 Amino Acid Analysis of Cordyceps sinensis 50 RESULTS 51 Biological Activity Assays: Antifungal, Antibiotic, and Phototoxic 51 Photoactivated Antibiotics and Antifungal Substances 53 Antiviral Tests 54 V Amino Acid Analysis of Cordyceps sinensis 56 Screening for Cyclosporine A 57 D I S C U S S I O N 57 C H A P T E R F O U R : Evaluation of E F Tolerance to Heavy Metals 60 I N T R O D U C T I O N 60 M E T H O D S 62 Preparation of Fungal Cultures 62 Comparison of Fungal Extracts by TLC and HPLC 63 Fractionation of Crude Extracts 63 R E S U L T S 65 Effects of Heavy Metals on Entomogenous Fungi 65 Fungal Growth and Morphology 65 Fungal Tolerance to Heavy Metals 69 Bioactive Compounds Produced by EF Growing under HM Conditions 71 Isolation of a Cerebroside 71 Antimicrobial Activity of a Cerebroside 72 D I S C U S S I O N 73 Effects of HM on EF Growth, Morphology, and Tolerance 73 Bioactive Compounds Produced by EF under HM Conditions 75 L I T E R A T U R E C ITED 78 A P P E N D I C E S 99 Appendix A 100 Appendix B 104 vi LIST OF TABLES Table 1.1 Number of strains, location, and substrate of species of entomogenous fungi collected in Canada . Isolates deposited at the National Culture Collection in Ottawa, Canada 3 Table 2.1 Spec ies of laboratory-reared arthropods and wild arthropods, and their stages used in experiments 20 Table 2.2 Habitats where entomogenous fungi were collected, hosts on which fungi were obtained, number of collections, and percentage of total collection in British Columbia 22 Table 2.3 Production of synnemata using different seeds and nuts. Inoculated plates were kept at room temperature (25°C ± 4°C), an equal number in the dark, and half exposed to light (Sylvania Grow-light), on a cycle of 12/12 hr UD. X = synnemata; - = no synnemata, but small amounts of mycelial growth present; N/T = not tested. Synnemata developed under both treatments 31 Table 2.4 Synnemata production from arthropods inoculated with selected entomogenous fungi. X = synnemata produced 35 Table 2.5 Free fatty acids present in selected entomogenous fungal strains. Co lumns A and B indicate two free fatty acids that were present but not identified 39 Table 3.1 Biological activity of entomogenous fungal extracts. Symbols : + = biological activity observed, ++ = biological activity only when irradiated with U V light, - = no biological activity observed. Escherichia coli, Bacillus subtilis, Staphylococcus aureus, and Pseudomonas aeruginosa were used for anti-bacterial b ioassays. Only Paecilomyces sp. (80-14a) showed moderate antifungal activity against Candida albicans 52 Table 3.2 Antiviral bioassay. E t O a C extracts from entomogenous fungi were tested against herpes simplex virus (HSV-1) . Toxicity test results: T = toxicity, PI = partial inhibition, C l = complete inhibition, and V = virus alive 55 Table 3.3 R f values for standard amino acids used in 2 D - T L C analysis of Cordyceps sinensis. Solvent system A : Butanol-Water-Acetic Ac id (BWA) in a ratio of 45:20:20; solvent system B: 1-Propanol- N H 4 O H in a ratio of 55:45 56 Table 3.4 Free fatty acids present in selected entomogenous fungal strains. Co lumns A and B indicate two free fatty acids that were present but not identified 58 Table 4.1 Selected entomogenous fungi growing in media containing heavy metals. Concentration was 500 pg/L of M Y P medium. Y = growth under the particular metal 69 Table A.1 List of entomogenous fungi collected in British Columbia 100 Table A .2 Media used in different experiments for culturing entomogenous fungi 101 Table A . 3 Reported biological activities and other characteristics of Paecilomyces marquandii 103 vii LIST OF FIGURES Figure 1.1 Taxonomic relationships of entomogenous fungi. Higher-level taxonomic relationships are shown for the groups as a whole, and some of the major genera and species of E F within the Clavicipitaceae. Anamorphs are included in the mitosporic Trichocomataceae. Diagram created with information from the National Centre for Biotechnology Information/US and the National Library of Medicine /National Institute of Health, 2001 5 Figure 1.2 Collection areas for entomogenous fungi within the coastal western hemlock and sub-alpine mountain hemlock biogeoclimatic zones in British Columbia (shaded green) 11 Figure 1.3 Coast Mountains, British Columbia. Study areas: a) Squamish River Valley, looking north towards Elaho River Valley, and b) Old-growth Douglas-fir forest in the upper Elaho Valley 12 Figure 1.4 Locations of field collections areas for entomogenous fungi in a) Idaho and b) Amazonian Peru 12 Figure 2.1 Entomogenous fungi collection sites in south coastal British Columbia 16 Figure 2.2 Torrubiella arachnophila. Voucher 96-21. Growing on a spider. Anamorph. Collected in the Squamish Valley, B C . June 8, 1998 22 Figure 2.3 Beauveria bassiana. Voucher 84-2. Growing on a beetle. Anamorph. Collected in the Mamquam Watershed, Squamish area, B C . October 9, 1998 23 Figure 2.4 Gibellula pulchra. Voucher 98-3. Growing on a spider. Teleomorph. Collected in Pacif ic Spirit Park, U B C , B C . July 17, 1999 23 Figure 2.5 Cordyceps nigrella. Voucher 100-3. Growing on beetle larvae. Teleomorph. Collected in Tofino, Vancouver Island, B C . June 20, 2001 23 Figure 2.6 Paecilomyces inflatus. Voucher 98-2. Growing on beetle larva. Anamorph. Collected in Pacific Spirit Park, U B C , B C . July 17, 1999 23 Figure 2.7 Cordyceps capitata. Voucher 82-25. Growing on Elaphomyces granulatum. Teleomorph. Collected in the Squamish Valley, B C . August 22, 1998 23 Figure 2.8 Cordyceps tuberculata. Voucher 01-04. Growing on a butterfly. Teleomorph. Collected in the Lower Amazon Basin, along the Yarapa River, northeastern Peru. July 5, 2001 24 Figure 2.9 Torrubiella sp. Voucher 01-02. Growing on a spider. Teleomorph. Collected in the Lower Amazon Basin, along the Yarapa River, northeastern Peru. July 7, 2001 24 Figure 2.10 Torrubiella mirabilis. Voucher 01-08. a) Growing on a spider. Teleomorph. Collected in the Lower Amazon Basin, along the Yarapa River, northeastern Peru. July 7, 2001. b) aspergilloid terminal head, c) attachment to the host 24 Vlll Figure 2.11 Paecilomyces sp. Voucher 94-1. Growing on a moth cocoon. Anamorph. Collected along Marine Drive, UBC campus, BC. October 11, 1998 24 Figure 2.12 Torrubiella raticaudata. Voucher 01-09. a) Growing on a spider. Teleomorph. Collected in the Lower Amazon Basin, along the Yarapa River, northeastern Peru. July 3, 2001. b) Whip-like structure protruding from host 25 Figure 2.13 Paecilomyces sp. Voucher 97-4. a) Growing on wasps. Anamorph. Collected in Ladner, BC. June 9, 1999. b) Rose gall with wasps 25 Figure 2.14 Gibellula sp. Voucher 01-06. Growing on a spider. Anamorph. Collected in the Lower Amazon Basin, along the Yarapa River, northeastern Peru. Jul 7, 02 25 Figure 2.15 Gibellula sp. Voucher 98-3. Growing on a spider. Anamorph. Collected in Pacific Spirit Park, UBC, BC. August 29, 1999 25 Figure 2.16 Paecilomyces sp. 85-14. Synnemata. Growing on a mummified caterpillar. Collected in the Capilano Watershed (GVRD), BC. October 10, 1998 26 Figure 2.17 Gibellula sp. Voucher 80-15. Growing on a spider. Anamorph. Collected in Cypress Bowl, BC. June 19, 1998 26 Figure 2.18 Gibellula sp. Voucher 100-1. a) Growing on a spider. Anamorph. Collected in Nanaimo, Vancouver Island, BC. October 22, 1998. b) Synnemata protruding from host, showing conidiophores 26 Figure 2.19 Whorled conidiophores in Verticillium sp 27 Figure 2.20 Sympodial conidiogenous cells arising from short swollen denticulated rachis of Beauveria sp. Teleomorph not observed or reported. Type species: Beauveria bassiana (Bals.) Vuill 27 Figure 2.21 Paecilomyces sp. Penicillate with terminal whorls of phialides (cells with a swollen basal portion, tapering at end). Teleomorphs: Byssochlamys Westling, Talaromyces C.R. Benjamin, Thermoascus Miehe. Type species: Paecilomyces variotii Bainier 27 Figure 2.22 Paecilomyces sp. conideogenous cells bearing conidia 28 Figure 2.23 Gibellula sp. Compact terminal conidiophore. a) Electron Scanning Microscope, and b) oil immersion 100x 28 Figure 2.24 Cordyceps loydii. a) sporocarps, and b) fragmented spores. Oil immersion 100x 28 Figure 2.25 Anamorph isolated from Cordyceps militaris (01-07, Peru collection). Short phialides bearing spores 29 Figure 2.26 Beauveria bassiana a) colony growing on MYP medium, b) conidiophore showing phialides, and c) sketch of conidiophore and conidia 29 Figure 2.27 Paecilomyces inflatus a) colony growing on MYP medium, b) conidiophore showing phialides, and c) sketch of conidiophore and conidia 30 ix Figure 2.28 Verticillium sp. a) colony growing on M Y P medium, b) conidiophore showing phialides, and c) sketch of conidiophore and conidia 30 Figure 2.29 Paecilomyces spp. synnemata produced on walnut mesocarp: a) single, no branching; b) furcated. Photographs under 10x dissecting microscope with Nikon 5x Coolpix digital camera 31 Figure 2.30 Paecilomyces sp. a) synnemata produced following a step down in nutrients, b) magnified to emphasize the intensity of the orange pigment. No ascocarps developed. M Y P broth containing % of nutrients 32 Figure 2.31 Synnemata of Paecilomyces sp. growing on peanuts, a) and b) illustrate two different strains of Paecilomyces sp. Colonies were six weeks old 32 Figure 2.32 Paecilomyces marquandii was initially grown at room temperature (25°C ± 4°C) for one week, and then transferred to a cold room (4°C), under dark conditions. Culture was grown in M Y P liquid medium 33 Figure 2.33 A three-month-old culture of Paecilomyces tenuipes producing abundant aerial mycel ia and synnemata. Culture was grown in M Y P liquid medium 33 Figure 2.34 Synnemata of a) Paecilomyces marquandii (73-21) growing on pecans, b) Cordyceps militaris (5298) growing on walnuts, c) P. marquandii growing on walnuts 34 Figure 2.35 Synnemata growing on the cocoon of Spodoptera litoralis. Caterpillar was inoculated before metamorphosis occurred. The cocoons were kept in moist sterile chambers at room temperature (24°C) until synnemata developed, a) Paecilomyces marquandii, b) Paecilomyces inflatus(Bums\de) J .W. Carmich. , and c) Paecilomyces tunuipes 35 Figure 2.36 Percent survival of crickets, Acheta domestica, inoculated with four strains of entomogenous fungi collected in British Columbia. Total number of crickets on Day 0 was 30. The experiment duration was 10 days. Control is represented by pale blue line 36 Figure 2.37 Percent survival of arthropods inoculated with entomogenous fungi. Fungi used to inoculate each arthropod species are identified in bars 'Day 0'. Total number of arthropods on Day 0 was 30. The experiment duration was 10 days. Control is represented by pale blue line 36 Figure 2.38 a) Adult beetles Tenebrion molitor parasitized by Beauveria bassiana. b) Fungal growth is more abundant at the joints and mouths of T. molitor. The isolate was obtained from an unidentified beetle collected at Priest Lake, Idaho, U S A 37 Figure 2.39 Paecilomyces marquandii (strain 85-15a) effectively colonized Trichoplusia ni, an agricultural pest 38 Figure 2.40 Synnemata developed from an adult beetle Tenebrion mollitor inoculated with spores of Paecilomyces sp. (95-10). The beetle was immobil ized after three days of inoculation. Synnemata did not develop until two months after inoculation....38 Figure 2.41 The artificial inoculation of Paecilomyces inflatus on carpenter ants did not result in production of synnemata, but mycel ia grew on and inside the ant 39 X Figure 3.1 Diagrammatic sketch of Disk diffusion b ioassay used to evaluate antibacterial, antifungal, and phototoxicity activities in E F . Extracts were used both with and without exposure to U V light to determine if photoactive antibiotic substances were present. A) A n aliquot of each extract impregnated in a sterile paper disk and allow to dry; B) the disk was then placed on the agar surface of a plate inoculated with the appropriate test organism; and C) to assess phototoxicity a duplicate plate was prepared and exposed to U V light and incubated. Gentamicin was the control for antibacterial b ioassays, nystatin was used as a control for antifungal b ioassays, and 8-methoxypsoralen (MOP) was used for phototoxicity b ioassays 47 Figure 3.2 a) Chromatogram of fraction from an extract of Paecilomyces marquandii, T L C sil ica gel; b) duplicate plate used to bioassay for photoactivated antibiotics, positive test is indicated by the yellow area. The solvent system used to develop the T L C plate was: Chloroform: M e O H (9:1); the spray reagent was Vanill in 53 Figure 3.3 a) Two-dimensional chromatography of a crude extract of Paecilomyces sp. (strain 84-16a); spray reagent: vanill in, heating at 110°C until colors developed; b) duplicate T L C plate, without spraying, overlaid with HM+PR inoculated with Staphylococcus aureus. After 18-hr incubation at 26°C, plate was spayed with reagent MTT to visualize the area of inhibition (yellow area = antibacterial activity) 54 Figure 3.4 a) Healthy monkey kidney cel ls; b) monkey kidney cells showing HSV-1 viral infection, notice the shape of the cel ls, round and smaller than those in the control; c) monkey kidney cells showing the effects of a cytotoxic crude fungal extract, many kidney cells have col lapsed or burst. Readings of the b ioassay and photographs were taken after 72 hours of incubation 55 Figure 3.5 Amino acid analysis of Cordyceps sinensis using 2-Dimensional Thin Layer Chromatography. Points indicate position of amino acids on T L C plate. Solvent systems: A) Butanol:Water:Acidic Ac id (45:20:20), and B) Propanol : N H 4 O H (55:45) 57 Figure 4.1 Fractionation scheme of the crude extract Paecilomyces marquandii. T L C , U V light, and color spot reactions using vanillin, molybdenic acid, and ninhydrin reagents detected different c lasses of compounds 64 Figure 4.2 Colonies of Paecilomyces sp. (strain 91-4) growing on M Y P medium containing heavy metals. A = copper; B = copper and iron 66 Figure 4.3 Paecilomyces marquandii growing in media containing iron (B) and copper (D). A and C are controls 66 Figure 4.4 Influence of iron and copper at different pH values on Cordyceps militaris growth rate. Growth was measured in centimeters. Error bars indicate 2 standard errors of the mean 67 Figure 4.5 Growth of Cordyceps militaris 01-07 on media containing copper at A) pH 9.0, B) iron at pH 7.0, and C) pH 9.0. Heavy metal concentration: 500 pg/L of medium (MYP) . Cultures were 6 days old 67 xi Figure 4.6 T L C showing the accumulation of intermediate or complex metabolites produced by E F growing in an iron enriched medium, 7-7, 7-9, and 7-C correspond to Cordyceps militaris (01-07) growing on iron media at pH 7.0 and pH 9.0; Cordyceps japonica 9647; and M corresponds to the medium (MYP) ; and 8 corresponds to an isolate of Torrubiella sp. Spray reagent: Vanil l in-sulphuric acid and heating. Note the blue spot at the bottom of chromatogram; sample 7-7 growing under iron conditions at pH 7.0. The spot is absent at pH 9.0 68 Figure 4.7 Thin layer chromatogram of E t O a C extracts of selected entomogenous fungi. Lanosterol (A) and ergosterol (B) standards are shown 68 Figure 4.8 Entomogenous fungi growing on M Y P medium containing heavy metals. A) Paecilomyces tunuipes, B) Paecilomyces sp., C) A member of the Clavicipi taceae, EpichloS festuca, a relative of Cordyceps sp., is symbiotic in temperate grasses 70 Figure 4.9 Chemica l structure of compound 1: (4E,8E)-N-2-hydroxyhexadecanoyl-1-0-p- glucanopyranosyl-9-methyl-C 1 8 -sphinga-4,8-diene, a cerebroside 71 Figure 4.10 Thin Layer Chromatography illustrating Compound 1: (4E,8E)-N-2- hydroxyhexadecanoyl-1-0-p-glucanopyranosyl-9-methyl-C 1 8 -sphinga-4,8-diene. Solvent system: Cyclohexane: acetone 50:50 (v/v). A) . Compound 1 spotted twice on plates and gave an intense blue color reaction with molybdenic acid. B) Repl icate plate, overlaid antibacterial bioassay; organism: Staphylococcus aureus 73 Figure B.1 D N A sequences of entomogenous fungi producing significant alignments using GenBank: a) Verticillium sp. 100-1, b) Paecilomyces tenuipes 24-2b, c) Beauveria bassiana Bet-1, d) Cordyceps militaris 01-07, e) Paecilomyces sp. 85- 15, f) Paecilomyces sp. 85-2, g) Paecilomyces marquandii 73-21. The sequences were compared using blast analysis against Neurospora crassa genome (Whitehead Institute) 105 Figure B.2 Chromatograms of free fatty acids from selected entomogenous fungi analyzed by gas chromatography. Vial number as indicated in chromatogram report: 1 = Beauvaria bassiana Bet-3; 2 = Paecilomyces sp. 85-14; 3 = Verticillium sp . 100- 1; 4 = Paecilomyces sp. 95-2; 5 = Cordyceps militaris 01-07; 6 = Paecilomyces tenuipes 24-2b; 7 = Paecilomyces marquandii 73-21; 11 = solvent control; 12 = fatty acid standards 106 Figure B.3 Chromatograms of Amino acid analysis from sporocarps of Cordyceps cyanensis. Included are: Chromatogram Report, Mol Percent Report, and Typical Amino Ac id Analys is Results (Hydrolysis Test Peptide) 107 Figure B.4 Physicochemical data used to determine the structure of the cerebroside (4E,8E)-N-2-hydroxyhexadecanoyl-1-0-p-glucanopyranosyl-9-methyl-C 1 8 - sphinga-4,8-diene. Included are: a) low resolution F A B Mass Spectrum, b) 1 3 C - N M R Spectrum, c) 1 H - 1 H C O S Y Spectrum, d) 1 H - N M R Spectrum 108 LIST OF ABBREVIATIONS "C-NMR Nuclear magnetic resonance, carbon 13 1 H- 1 3 COSY Correlated spectroscopy, carbon 13 1H-1H COSY Correlated spectroscopy, hydrogen 1H-NMR Nuclear magnetic resonance, hydrogen AFLP Amplif ied fragment length polymorphism BWA Butanol-water-acetic acid CC Column chromatography C H 2 C I 2 Dichloromethane C H C I 3 Chloroform COSY Correlated spectroscopy Y CWD Coarse woody debris CZA Czapeck media DNA Deoxyribonucleic acid EF Entomogenous fungi EtOAc Ethyl acetate EtOH Ethanol FBS Fast bombardments scan HCL Hydrochloric acid HMBC Heteronuclear multiple bond correlation HPLC High pressure liquid chromatography HSV-I Herpes simple virus, type one MeCN Acetonitrile MeOH Methanol MH Muller Hinton media m m Millimetre MS Mass spectrometry MYP Malt yeast peptone NH4OH Ammonium hydroxide PDA Potato dextrose agar PDA1 Photodiode array PTLC Preparative thin layer chromatography Rf Relative mobility SAB Sabouraud dextrose Agar SEM Scanning electron microscope TFA Trifluoroacetic acid TLC Thin layer chromatography Tween-20 Polyoxyethylene sorbitan monolaurate UV Ultraviolet light V/V Volume by volume W/V Weight by volume xiv ACKNOWLEDGEMENTS I would like to thank the many people who shared their knowledge, wisdom, and wealth so that I could complete this thesis. My warmest and sincerest gratitude and recognition are extended to my committee members, Drs. Neil Towers, Robert Bandoni, and Gary Bradfield. Neil, thank you for providing me with intellectual stimulation and scientific guidance, and for sharing wonderful adventures and stories. Bob, thank you for showing me the fascinating world of fungi and for sharing your enthusiasm and life experience in many realms. Gary, thank you for your academic guidance. There are many people who shared their scientific interests, friendship, and ideas during my years at UBC. Thanks to Dr. Humphrey for all the help in the electron microscope laboratory, Dr. Bohm for sharing his laboratory, Dr. Kuhn for assisting with fatty acid analysis, Dr. Isman for supplying laboratory-reared insects for my experiments, Dr. Brueil for helping with DNA sequences, and Dr. Hudson for his collaboration in antiviral bioassays. Thanks to Jeff Chilton from North American Reishi for providing Cordyceps sinensis material for chemical analyses. My appreciation goes out to all those who generously gave me their time, expertise and advice in my investigations. Special thanks to Dr. Rodriguez, Cornell University, for collaboration on the collection of fungi from the Amazon, and for helpful discussions and guidance with my writing. I am very grateful to people in the Towers' Laboratory, past and present. Thanks to Dr. Page for introducing me to HPLC techniques and for stimulating discussions. Thanks to friends Dr. Saxeena, Dr. Balza, Dr. Orozco, and especially Dr. Ming for his collaboration in the cerebrosides identification. Thanks to Zyta Abramowsky, for her help with some experiments and answering my questions around the laboratory. I would like to extend my great appreciation to my lab mates Kevin Usher, Fiona Cochrane, Andres Lopez, Brian Delahouse, and Salverani for their support and camaraderie over the years. Special thanks to my good friends Nick Page and Paul Kroegar for their enthusiastic interest in entomogenous fungi and for their contributions of precious local collections. Pacific Northwest Key Council members helped with collections during our forays. The Osaka Institute for Fermentation provided me with entomogenous fungi strains used in some experiments. Special thanks to the First Nations House of Learning and all my relatives for their unconditional support, understanding, and encouragement. I am particularly grateful to my elders Vince Stogan, Bob George, John O'Leary, and N'kixw'stn James, and Shirley Bear, for their advice and words of wisdom. I especially extend my appreciation to Shirley Sterling, Rosalyn Ing, Jo-ann Archibald, Madeleine Maclvior, Richard Vadan, Lee Crowchild, Alannah Young, James Andrew, and Tim Mitchel for their support, good words, thoughts, and teachings. Greater Vancouver Regional District (GVRD) provided access to the Capilano Watershed and provided logistical support. Financial support was provided by the California State University System, National Research Council of Canada, and First Nations Fellowships. I give special thanks to my mentor at California State Polytechnic University Pomona, Dr. Keith Arnold for supporting my goals. I would like to extend my warmest gratitude and admiration to Dr. Jonathan and Nancy Barker for believing in my dreams and encouraging me to realize them. Thanks to Alice Ann Bandoni for her friendship, great meals, and help with collections. My deepest acknowledgement goes to my son Edward, for his understanding of my commitments and his sacrifice all these years. I thank my parents Alejandro and Zoila, for supporting my scientific curiosity at an early age. Their love and wisdom helped me to set and reach high goals in life, and provided me with strength along my path. My brothers Alejandro and Marco Antonio, and my sister Xomaira provided me with their love, support, understanding, and courage, for which I am grateful. My acknowledgements are extended to my relatives, Badih, Samia, Randa, Mae, and Waleed Wahbe. Badih and Samia, thank you for providing me with good advice, support, and love. Randa and Tim, thanks for your encouragement. Mae and Waleed, thank you for the great memories of hiking through the Sequoias with Mojo and Candy. To my partner in life, Tanya Wahbe, I extend my deepest admiration for her love, wisdom, commitment to support my dreams, sharing great fun, for help with my field collections, and especially for the long hours dedicated to editing and reviewing this thesis. Thanks to all of you who I have failed to mention but have greatly contributed to my endeavors. This thesis is dedicated to all my relatives and friends who lost their lives while I have been away from my homeland. To all my relations. Chapter One: General Introduction 1 CHAPTER ONE: General Introduction Temperate rainforest ecosystems of the Pacif ic Northwest of North Amer ica have tremendous trophic structure and biomass. Before the end of the 19th century, these forests covered a multitude of environments and landforms of approximately 11.3 x 106 ha (Harris 1984) from northern California to A laska . In British Columbia (BC), temperate rainforests support high species diversity from micro to macro organisms. The gradient of species r ichness and abundance is associated with complex patterns and processes on different temporal and spatial sca les (Wiens 1989; Huston 1994). Historically, fire has been one of the main forces shaping these forests, but during the 20th century suppression of wildfires led to different changes in the landscape. Windstorms (Lynott and Cramer 1966), pathogens (Childs 1970), and anthropogenic activities (forest harvesting; Franklin and Forman 1987) have also played a major role in the changes and sometimes destruction of these forests. Fungi have been systematically studied for the last 300 years. They have been associated with degradation and recycling of decaying organic matter, and the maintenance of healthy ecosystems. They are a source of food for vertebrates (e.g., humans, squirrels, deer) and invertebrates (e.g., mycetophilodes, slugs, snails). They are also sources of toxins in animal food and produce many secondary metabolites useful to humans. Of an estimated 1.5 million fungi (Hawksworth 1991), only about 80,000 have been described in the literature. Entomogenous fungi (EF) are fungi that parasitize arthropods, specifically insects and spiders. The total number of E F species has been estimated to be around 15,000 or 1% of the total estimated number of fungal species today (Samson et al. 1988; Glare and Milner 1991; Hawksworth 1991; Roberts and Hajek 1992; Hajek and Saint Leger 1994). Groups with large numbers of E F are Hypocreales (520 sp), Entomophthorales (240 spp.), Septobasidiales (175 spp.), and Laboulbenieales (1730 spp.; Hawksworth etal. 1983; Weir and Hammond 1997). In the last 100 years, 4,000 scientific papers have been published on E F belonging to the Clavicipitaceae (Hywel-Jones 1997c/). This is only a fraction of the total number of mycological Chapter One: General Introduction 2 papers and only a few genera and species of E F have been selected for most experimental research (i.e., the Hypocreales and anamorphs thereof or of other groups that attack insects). Among the groups of fungi attacking arthropods are the Entomophtorales. Members of this group produce zygospores in sexual reproduction and their mycelia grow inside the hosts; the Septobasidiales and Laboulbenieales do not form hyphae within the hosts and they produce haustoria that penetrate in the host systems (digestive tract in Septobasidiales and circulatory system in Laboulbeniales). Among E F are Cordyceps sinensis (Berk.) Sacc , C. militaris (L. ex Fr.) Link.), Beauveria bassiana (Bals.) Vuil l . , Metarhizium anisopliae (Met.) Sorokin), Hirsutella sp., Verticillium lecanii (Zimm.) V iegas , Paecilomyces vanotii Bainier, and P. carneus Duche & R. Heim. Most research has been conducted in laboratories, thus little is known about their ecology or biology. Entomogenous fungi are parasites of arthropods. Canada ' s total invertebrate inventories report the presence of more than 155,000 spec ies of insects and over 4,000 species of spiders (Pojar 1991). In B C alone, more than 35,000 species of insects and 600 species of spiders have been reported, with the most representative insect orders being Diptera (true flies), Coleoptera (beetles), and Hymenoptera (wasps, bees, ants; Pojar 1991). It seems reasonable to assume that this abundance of arthropods supports significant populations of E F . However, forest pathologists, entomologists, and mycologists have only occasional ly collected E F in B C (Table 1.1). Among these collections are members of the genus Cordyceps (Fr.) Link and the anamorph genera Paecilomyces Bainer, Beauveria Vuill., Verticillium Nees and Hirsutella Pat. The life histories of most E F probably include both anamorphic and teleomorphic stages. The teleomorph is the sexual morph or state of the fungus and is characterized by ascomata (Hennebert and Weresub 1977). It is often more demanding of appropriate conditions and nutrition for its development than the anamorph. The anamorph is the mitospore form of the fungus, characterized by the production of conidia (Hennebert and Weresub 1977) and/or sclerotia (Kendrick 1992). Although, the life histories of E F have not been elucidated in cultural studies, it is known that one or more types of anamorph spores can be produced by a spec ies. Chapter One: General Introduction 3 Haploid mycelia can convert the body of an insect host into a sclerotium-like mummy. This mummy may overwinter in many temperate species. Stalked stromata are characteristic of the teleomorph states of Cordyceps species, and these arise from the mummified host (or from a host ascocarp in the case of C. capitata Holmsk. (Ex Fr.) Link.). Table 1.1 Number of strains, location, and substrate of species of entomogenous fungi collected in Canada. Isolates deposited at the National Culture Collection in Ottawa, Canada. Species Number of strains Location Substrate Paecilomyces veriotii 10 New Brunswick Plcea and Acer Otawa Pea Seeds Quebec Lipstick Calgary Radish seeds Ontario House wals Alberta Rapeseeds Alberta Plnus contorta British Columbia Brassica sp. P. tunulpes 1 Quebec Insect pupae P. carneus 4 Quebec Soil Alberta Soil Alberta Soil Alberta Soil P. farinosus 14 Ontario Laboratory contaminant Bafin Island Mites Alberta Alpine soil Quebec Amanita w'rosa Quebec Spruce budworm Manitoba Larval Megachile rotunda New Bruinswick Piceaglauca seeds P. fumosoroseus 2 Quebec Soil Ontario Gypsy moth egg masses P. Inflatus 3 British Columbia Garden soil British Columbia Farm soil British Columbia Vancouver Aquarium, Tropical Valey P. Illlaclnua 6 Quebec Soil Alberta Soil Ontario Winter wheat cv. Lennox British Columbia Soil, Simon Fraser University Ontario Laboratory contaminant P. stratlaporus 1 Quebec Soil Cordyceps hesteri 1 Ontario Soil insect C. ophlogloslodes 1 Quebec Flies? Beauveria bassiana 14 Alberta Soil Chapter One: General Introduction 4 The term synamorph applies to two or more anamorphs that have the same teleomorph (Gams 1982). The holomorph refers to the whole manifestation of the genotype including all its morphs and phases (Hennebert and Weresub 1977). The presence of teleomorphs and corresponding anamorphs existing at the same latitudes may only be substantiated in the field. Establishing the link between the sexual and asexual forms of E F remains problematic. Of more than 30,000 known Ascomycetes , only about 4,000 anamorphs have been matched to their respective teleomorphs. Molecular fingerprinting has become a complementary tool to develop the molecular taxonomy of anamorphic fungi such as Paecilomyces (Tigano et al. 1995a; 1995b) and other E F . Figure 1.1 illustrates the taxonomic relationships of E F with other major groups of Ascomycetes . Although the use of molecular tools has become helpful in determining the identity and taxonomic relationships of the anamorph stages of some E F at the genus level (Sugiyama 1994; Fukatso et al. 1997), these techniques have not been very successful in identifying individual species. For example, through the use of r D N A analysis it has been establ ished that Cordyceps sinensis (Berk.) Sacc . is closely related to Hirsutella sinensis and clearly divergent from Paecilomyces sinensis, Stachybotrys sp., and Tolypocladium sp . (Chen et al. 2001; Liu et al. 2001). Additionally, Metarhizium anisopliae var. majus has been identified as the anamorph of Cordyceps brittlebankisoides (Liu et al. 2001). Chapter One: General Introduction 5 Eukaryo ta Fungi Basidiomycota ] Ascomycota E urotiomycetes Pezizomycotina Sordariomycetes Eurotiales I Trichocomaceae Hypocreales Mitosporic Trichocomaceae Clavicipitaceae Verticillium Torrubiella ]/ A ciaviceps Paecilomyces Cordyceps Torrubiella (anamorph) Epichl&e Cordyceps brongniartii (anamorph) Cordyceps bassiana (anamorph) Cordyceps militaris Beauveria bassiana var. bassiana (anamorph) Figure 1.1. Taxonomic relationships of entomogenous fungi. Higher-level taxonomic relationships are shown for the groups as a whole, and some of the major genera and species of E F within the Clavicipi taceae. Anamorphs are included in the mitosporic Tr ichocomataceae. Diagram created with information from the National Centre for Biotechnology Information/US and the National Library of Medicine /National Institute of Health, 2001. Fungi remain a vast untapped resource of novel metabolites. Numerous fungal metabolites have been reported as antibacterial or antifungal agents. Additionally, some of these metabolites may have antitumor, antiviral, or antiprotozoan activities. Today, the occurrence, distribution, and diversity of E F are directly affected by anthropogenic activities Chapter One: General Introduction 6 such as agriculture, forestry, and industrial practices (Heliovaara and Vaisanen 1991; Puterka er al. 1994). The use of carbamide herbicides and increasing environmental pollution may negatively affect the occurrence, composition, and distribution of EF and other organisms. Historical Significance of Entomogenous Fungi Observations of diseased silkworms were made in China as early as 2700 BC; Aristotle mentioned diseased honeybees, circa 335 B.C. (Steinhaus 1956). In 1834, the Italian mycologist, Bassi discovered and associated the fungus Beauveria bassiana with the muscardin disease, a fungal infection of the silk worm, Bombix mori L. (Lepidoptera: Bombycidae). The name "Cordyceps" was first used by Link in 1833, to describe fungi growing on insects (Hawksworth et al. 1983). Fries and other mycologists referred to this fungus as Isaria HilLFr., a hyphomycete genus (Hawksworth et al. 1983). The term "Cordyceps and allies," was coined by Kobayasi in 1941, and is still used to describe members of the Clavicipitaceae growing mainly on insects (e.g., beetles, ants, butterflies, caterpillars) and spiders (e.g., Salticidae). Mitchelli, Tillet, and Bassi's contributions to science were fundamental in the formulation of "the germ theory of disease," which remains a milestone in the biological sciences (Hawksworth et al., 1983; Kendrick 1992). Fifty years after the discovery of B. bassiana, Metchnikoff reported the interaction of Metarhizium anisopliae (Metch.) and the beetle Anisoplia austriaca (Hrbst.). Three decades later, Louis Pasteur's work in pathology began with a study of parasitized silkworms. Both Bassi and Pasteur suggested that microorganisms could be used in biological control (Stainhaus 1956). Other major contributions to our understanding of the fundamental biological processes of EF are those of Thaxter (1888), Petch (1923,1931,1932, 1937,1944), Mains (1950,1951,1954), Evans and Samson (1982, 1984), Samson and Evans (1973, 1977, 1992), Kobayasi (1941), and Koyabasi and Shimizo (1963a, 1963b, 1976, 1981, 1982). Chapter One: General Introduction 7 Cordyceps sinensis (Berk.) Sacc. and Traditional Chinese Medicine Chinese medicine has a long history of use of E F . Most medicinal c laims have been attributed to Cordyceps sinensis, the "cure-all-fungus." C. sinensis is a parasitic fungus that grows on larvae of lepidopterans (Kobayasi 1941). This well-known fungus has been used in Chinese traditional medicine for thousands of years (Zhu et al. 1998a; 1998b. C. sinensis is known as tong tschong sha tso which means, "worm in winter" referring to the fungus-infected insect, and "plant in summer" referring to the fungal fruiting body (Molitoris 1994). In traditional Ch inese medicine, many i l lnesses and medical conditions are treated with C. sinensis, including hepatic conditions, cardiovascular disorders, renal failure, immunological d iseases, inflammatory conditions, and cancer (Jia-shi et al. 1998). A s with many other traditional remedies, C. sinensis has been intensively studied. However, no unique active ingredient has been identified as being responsible for its medicinal effectiveness. The pharmacological properties of C. sinensis preparations have been suggested to be related primarily to bioactive polysaccharides, modified nucleotides, and cyclosporin-l ike compounds produced by this fungus. In recent years in China, Cordyceps sinensis has been intensively collected from the wild (Yao et al. 2001). In addition, intentional fires, overgrazing, and shifting cultivation continue to destroy the alpine butterfly Leptrqptehs sp. whose larvae are the host for C. sinensis, ultimately threatening the existence of the fungus (Bhattarai and Croucher 1996). The new constitution of the Kingdom of Nepal 1991 recognizes the need to preserve the environment and to use natural resources wisely. At present, six non-timber forest products (NTFPs) , which are recognized as threatened with over-exploitation, are banned from export in unprocessed medicinal products including C. sinensis (Bhattarai and Croucher 1996). However, the socio-economic and market demands may supercede the protection of wild Cordyceps sinensis populations. Cultivation of C. sinensis and other Cordyceps spp. has long been sought to reduce exploitation of natural populations (Liu et al. 2001) and to find new sources of natural products with biological activities (e.g., ergosterol peroxide, an antitumor agent from C. sinensis Bok et al. 1999). Therapeutic Chapter One: General Introduction 8 preparations of E F from Ch inese pharmacopoeiae have been introduced into western markets, but neither quality control nor regulations exist for the commercial ization of most products. Biologically Active Substances from the Hypocreales Many species in the Hypocreales have unique interactions with a range of organisms including insects, spiders, grasses, and other fungi. Claviceps purpurea (Fr.) Tul (Clavicipitaceae) has a worldwide distribution, and several closely related spec ies are parasitic on many temperate pasture grasses and cereals (e.g., rye, wheat, barley, and hybrids). In addition, members of the genus Cordyceps (Clavicipitaceae) have attracted the attention of western researchers in recent years, not because of their high chemical diversity but because unique bioactive compounds with pharmaceutical potential (i.e. immunosupressor and anti- cholesterol drugs) have been isolated from members of this genus. For example, Cyclospor ine A (CysA) was discovered as an antifungal agent produced by the fungus Tolypocladium inflatum Gams . C y s A is widely used as an immunosupressor agent in human organ transplantation. Unlike other immunosupressants, it has inhibitory effects primarily on the activation of T lymphocytes (Borel et al. 1977; Larsson 1980; Shevach 1985; Manger er al. 1986; Noble 1995). It is also effective in treating autoimmune disorders such as psoriasis and rheumatoid arthritis (Phillips 1991). Tolypocladium inflatum was previously known Trichoderma inflatum (Dreyfuss etal. 1976) and as Cylyndrocarpon luciderm Booth (Borel 1986). Tolypocladium inflatus has been considered the anamorph of Cordyceps subsessilis (Hodge et al. 1996). Other members or relatives of these E F may be potential sources of immunosupressants and other drugs. Potential of Biological Control and Mycopesticides The demands to reduce of quantities of toxic substances released into the environment (i.e. pesticides, heavy metals, gases) have increased, and many areas of the world are experiencing the compounding effects of pollution and environmental and biological Chapter One: General Introduction 9 degradation. Integrated pest management, in which natural enemies and pest arthropods interact, plays a significant role in plant and animal protection (Hoy and Herzog 1985). Different organisms have been used as agents of biological control, including bacteria, viruses, arthropods, and fungi. Mycopesticides are fungi or fungal preparations (e.g., spore suspensions) used as biological agents to control and kill arthropods (Robert and Sweeney 1982). In general, mycopesticides have a lower kill rate than chemical insecticides, but this does not necessarily mean a reduction in crop protection (Jeffs et al. 1997). The reliance on chemical insecticides for control of mosquitoes and other insects led to a number of adverse environmental consequences (e.g., the use of DDT and its effect on wild and beneficial animals; Lawrence et al. 1994, 1995). There was little research on biological control by microorganisms in the first half of the 20 t h century (Lacey and Goettel 1995). The discovery of the insecticidal properties of Bacillus thurengensis Berliner (Tanada and Kaya 1993) continues to have a major impact. The commercial development and use of EF for controlling arthropods has lagged far behind that of Bacillus thurengensis. Inoculation of test arthropods with EF has sometimes been successful. The most common and useful method of inoculation has been the use of spore suspensions (Lacey and Goettel 1995). Positive results from pest management programs require the survival of non- targeted insects and other organisms in contact with the control agent (e.g., strains of P. farinosus capable of killing the cockroach Blattella germanica L.; Zukowski and Bajan2001). Heavy Metals and Entomogenous Fungi The increasing awareness and risks of accumulating heavy metals in the environment has led to a quest for new and improved "clean" technologies (Bakkaloglu et al. 1998). An understanding of how microorganisms tolerate heavy metals can provide insight into strategies Chapter One: General Introduction 10 for their detoxification or removal from the environment (Smith 1975; Briuns et al. 2000; Bakas 2001; Gesse l 2001). Fungi are extremely efficient scavengers for mineral nutrients, and valuable as symbionts of trees and other plants. Fungi are sensit ive to temperature, oxygen, carbon dioxide, pH and ammonia fluctuations in their environment. The availabilities of many minerals ions (e.g., copper and iron) are pH dependent; mechanisms for obtaining and sequestering such elements have evolved in many fungi. Any changes in normal biological pathways cause modification in the production of metabolites. Sterol synthesis seems to be altered by the presence of C u + + in the media, leading to disturbances in the membrane function and K+ efflux (Tarhanen et al. 1996,1998). The levels of ergosterol may depend on the metal concentration in the media, indicating that heavy metals play an important role in fungal decline (Seitz ef al. 1979), which may be compounded by other environmental factors such as acid rain, use of pesticides (Urs 1967; Storey 1986), and other anthropogenic disturbances. Objectives In this thesis, I describe the results of an investigation into the biological activity of entomogenous fungi, the potential for their use in biological control, and their resistance to heavy metals. My primary motive in this research was to gain an understanding of the interactions between E F and arthropods, but some experiments dealt with biological activity of E F products against other organisms. The tolerance of E F to high concentration of metal ions departs from the main goals of the research. My objectives in this research were to: 1) Survey B C entomogenous fungi in the field and establish a culture collection, 2) Cultivate Cordyceps species, including isolates obtained from culture collections, collected sporocarps and infected insects, 3) Inoculate experimental arthropods with E F to assess their potential role as agents of biological control, Chapter One: General Introduction 11 4) Examine the antibiotic, antifungal, phototoxic, and antiviral activities of E F , 5) Isolate and characterize the main bioactive compounds, and 6) Evaluate the growth of E F under heavy metal conditions to explore their potential use in bioremediation. Study Area E F were collected from field locations within the coastal western hemlock (CWH) and sub- alpine mountain hemlock (SAMH) biogeoclimatic zones of south coastal B C temperate rainforest (Figure 1.2). These biogeoclimatic zones include wet maritime-montane (CWHvm2), mountain hemlock (MH), and moist maritime windward (Mhmml ) sub-zones (Meidinger and Pojar 1991). C W H occurs west of the coastal mountains, from sea level to 900m on inward slopes in the south and mid-coast (up to 1050 m on leeward slopes); mean annual precipitation is 2228 mm (1000-4400 mm); mean annual temperature 8° C (5.2-10.5° C ; Meidinger and Pojar 1991). S A M H occurs above C W H , from 400 to 1000 m; annual precipitation ranges from 1700- 5000 mm (20-70% is snow), mean annual temperature ranges from 0.0-5.0° C (Meidinger and Pojar 1991). Figure 1.2. Collection areas for entomogenous fungi within the coastal western hemlock and sub-alpine mountain hemlock biogeoclimatic zones in British Columbia (shaded green). Chapter One: General Introduction 12 E F were obtained in the field from the Capi lano watershed, Cypress Provincial Park, the University of British Columbia Endowment Lands (Pacific Spirit Park), Ladner, the Squamish, Elaho, and Mamquam River Val leys (Coast Mountains; 49° N, 122°W; Figure 1.3), and the Morrell Nature Sanctuary (Nanaimo, Vancouver Island). Fourteen collections were obtained at sites outside B C as follows: Idaho, Priest Lake, (Figure 1.4a; cedar western hemlock forest) and Peru, Loreto, Ya rapa River, (Figure 1.4b; tropical rainforest). Figure 1.3. Coast Mountains, British Columbia. Study areas: a) Squamish River Val ley, looking north towards Elaho River Val ley, and b) Old-growth Douglas-fir forest in the upper Elaho Val ley. Figure 1.4. Locations of field collections areas for entomogenous fungi in a) Idaho and b) Amazon ian Peru. CHAPTER TWO: Collection, Isolation, and Cultivation of EF 1 3 CHAPTER TWO: Collection, Isolation, and Cultivation of Entomogenous Fungi INTRODUCTION The diverse habitats present in the Coast Mountains of south coastal British Columbia (BC) have ecological conditions in which various groups of fungi, including entomogenous fungi (EF) can thrive. Some of the temperate rainforest fungi probably overwinter as conidia or mycelia in the soil , in other substrates such as decaying wood, or plant parts. Spec ies of Cordyceps almost certainly survive as sclerotium-like hyphal masses in the host (mummies), the ascomata developing in the spring. For some small species, the development may occur in the spring, early summer, or fall but this is not known. It is known that some larger species produce sporocarps in the spring from buried mummies, but nothing is known about the survival of an individual conidium in the winter, and that may be rather poor. Nevertheless, hyphae survive freezing regularly in the laboratory (e.g., freezing in deep freezer, or freeze-drying are the two most common methods of maintaining cultures). A limiting factor in the study of E F has been the lack of a clear understanding of culture requirements for fungi that grow on or within living insects and spiders. Knowledge of essential growth conditions is required to understand the nature of the fungus-host relationship. A n understanding of the factors affecting inoculum persistence is needed (Omstad and Carruthers 1990) to predict the success of E F as potential biological control agents. Additionally, satisfying requirements for the production of teleomorphs in culture would permit genetic manipulation and selection to aid in the process of developing such agents. Spiders, which feed on a wide variety of insects and other soft-bodied invertebrate animals, attack and subdue their prey using fangs to inject a poison. Most spiders are ecologically beneficial, while some are very toxic and may kill large mammals including humans. A n understanding of the interactions between E F and spiders can be useful in the production of selective mycopesticides. CHAPTER TWO: Collection, Isolation, and Cultivation of EF 14 Some Cordyceps species and their all ies produce useful compounds with medical , agricultural, nutritional, and industrial uses. Most are parasites of arthropods and their traditional medical uses are well known and descr ibed in the literature (e.g., C. sinensis used in traditional Ch inese medicine; Jia-shi et al 1998). S o m e anamorphs of this group have been useful as agents of biological control (Burassa et al. 2001), and in the recovery of heavy metals (Bakkaloglu et al. 1998). Local strains of E F may be better adapted to local environmental pressures, and could be available for use as biological control agents in agriculture, forestry, and some industrial applications. A n improvement in culture techniques and an understanding of nutritional requirements of E F may lead to increased yield of natural compounds with specif ic biological activities (e.g., antibiotics). The number of existing E F living collections (or isolates existing in the Canad ian National Fungal Collections, Central Experimental Farm, Ottawa) remains very limited. Some fi lamentous fungi produce synnemata, structures composed of semi-compacted or strongly adherent groups of erect conidiophores bearing conidia on the apices, and in some cases , both on the apex and laterally. The production of synnemata in vitro may be a helpful taxonomic determinant for anamorphs of E F . Conid ia borne on these clustered conidiophores making up the synnemata are probably the same as those on single conidiophores. The production of ascocarps in vitro offers many potential uses such as the production of inoculum, and breeding of appropriate strains for specif ic purposes. With the appropriate manipulation of environmental conditions and media nutrients, these synnemata should produce perithecia rather than the conidial state. Claviceps Tul . and Epichlde (Fr.) Tul . are two genera in the Clavicipi taceae. Spec ies of Claviceps have a wide distribution and are considered parasites of grasses and sedges. Members of Epichlde are ascomacetous fungi that appear to be symbionts of grasses. They are seed transmitted and considered monophyletic (Kaldu et al. 1997). Epichlde festucae Leuchtm. Schardl & M.R. S iegel , which is a common symbiont of the grasses Festuca, Lolium, and Koeleria (Schardl 2001), produces the anti-insect alkaloids, peramine and loline, and the CHAPTER TWO: Collection, Isolation, and Cultivation of EF 15 anti-vertebrate alkaloids, lolitrem B a n d ergovaline. It also has an efficient vertical transmission via host seeds, a mildly pathogenic state associated with the E. festucae sexual cycle, and plays a role in improving the survival of host plants (Schardl 2001). Acremonium Link is a form genus, a genus created to include anamorphic spec ies of different fungi, of which members are grass symbionts with unknown links to teleomorphs. It is generally considered a highly polyphyleticform genus containing distantly related fungi (Glen etal. 1996). S o m e species of Acremonium are probably anamorphs of ascomycetes belonging to the Clavicipi taceae (Glen et al. 1996). In this chapter I describe collections from a survey of E F in B C , provide photographs of the representative groups of E F collected, and evaluate a number of substrata for possible teleomorph production. I also describe procedures for establishing a culture collection, and cultivation of Cordyceps spp. and E F anamorphs to examine the teleomorph stage in culture. Data obtained from free fatty acid analysis and rDNA sequencing of selected E F isolates were used to support morphological identification. This research contributes to enlarge the existing living culture collections of the Canad ian National Fungal Collections in Ottawa. OBJECTIVES 1. To survey B C entomogenous fungi in the field and establish a culture collection. 2. To cultivate Cordyceps spp. and E F anamorphs from field collections so that all life history stages, especial ly the teleomorph stage are formed in culture. CHAPTER TWO: Collection, Isolation, and Cultivation of EF 16 STUDY AREAS Most collections were obtained from field locations in south coastal B C (Figure 2.1). Additional collections were obtained from sites in Idaho, U.S. , and the Lower Amazon Basin, northeastern Peru. A detailed description of study areas can be found in Chapter One. Figure 2.1. Entomogenous fungi collection sites in south coastal British Columbia. METHODS Field Collections E F were collected in the field from 1998 to 2001. Monthly collecting trips were carried out from November to Apri l , and weekly collections occurred during spring (March to June) and fall (August to October). The June, July and August collections were focused on ephemeral species appearing after rainy periods. Field collections included sporocarps of Cordyceps spp. and arthropods with possible sporocarps formation, or superficial fungal growth suggesting E F anamorphs. Initially, sampling was done randomly using visual encounter surveys. Later, the searches were narrowed to specif ic habitats (i.e. decomposing logs). A knife or a hatchet was used to remove loose bark CHAPTER TWO: Collection, Isolation, and Cultivation of EF 17 from coarse woody debris for visual examination. Samples were examined in the field using a magnifying glass, then placed in 20-ml scintillation vials, or sterile bags. Cultures were isolated from field collections using MYP medium containing tetracycline to eliminate bacterial growth. If conidia were present they were transferred directly from the sample to the agar plates. Young colonies were transferred from initial isolation plates to MYP (Bandoni 1971) plates without tetracycline. Stock cultures were maintained in MYP slant stored at 4°C. Other media used included MYP with the addition of copper, SAB, PDA, and water agar plates with the addition of tetracycline (see Appendix A for media ingredients and description). Specimens were photographed using dissecting, compound, and electron scanning microscopes (SEM). Most photographs were taken using a digital camera. Other images were acquired using a 1200 DPI scanner, or using an automatic Minolta camera, model 3Xi, using 400 ASA film. Inoculated plates were kept upside down in an incubator at 26°C. Young colonies were immediately transferred to MYP agar containing copper sulphate. Finally, the clean culture was transferred to MYP agar without any metals. Slants were prepared for all stock cultures. The EF strains obtained from the Osaka Institute for Fermentation, Japan were used as controls in my experiments. Isolates were identified by microscopic examination of the sporulating structures, morphology, pigmentation, and other morphological characteristics. The rDNA of seven fungal strains were extracted, sequenced, and partial sequences were compared to gene sequences in the Genebank. Similarities to other EF are shown in Appendix B. Voucher Specimens Voucher specimens were dried and placed individually in cardboard boxes; small specimens were glued into individual boxes. These specimens were deposited in the Herbarium of the Botany Department, UBC. Each specimen was accompanied by information indicating the collection date, hosts, location, and associated habitat. Final identification of some collections were not available for inclusion in this thesis. Collection numbers and CHAPTER TWO: Collection, Isolation, and Cultivation of EF 18 provisional names have been assigned to vouchers and living collections. In many cases, the host could not be identified because of extensive decomposition (e.g., when the mycelia had covered most of the host body), or only parts of the host were found and collected. Fungi were identified using light microscopy, and some images were obtained using a Scanning Electron Microscope (SEM). Field collections were photographed using dissecting and compound microscopes with a digital camera. Maintenance of Fungal Isolates and Stock Cultures Stocks of fungal strains were maintained on MYP, PDA, and SAB (Hawksworth et al. 1983) at 4°C on Petri dishes and in slant culture tubes. Small scintillation vials (20 ml) were used for the preparation of slant cultures. Each strain was stored in triplicate on MYP medium. Water stock cultures were also prepared using Ependorff tubes containing 0.5 ml water (distilled and autoclaved). A small amount of mycelium taken from the edge of an active growing colony was placed in an Ependorff tube, sealed with parafilm, and stored at 4°C. The water culture stock method was developed mainly for basidiomycetes growing on wood, however it works well for a number of filamentous fungi. MYP slants were inoculated with selected fungal strains and incubated at 26°C. The caps were tightened once growth was visible, and slants were then stored at 4°C. MYP plates were inoculated and incubated at 26°C. Because the incubation time varied between isolates, plates were checked daily. Established colonies were removed from the incubator and transferred to a 4°C refrigerator. Cultures to be used for chemical extractions were grown at room temperature (25°C + 4°C) unless otherwise indicated. The most useful media for the cultivation of EF were malt yeast peptone (MYP), malt agar (MA), potato dextrose agar (PDA), Sabouraud dextrose agar (SAB), Czapek (Dox) Agar (CZA), and water agar (WA) (see Appendix A). CHAPTER TWO: Collection, Isolation, and Cultivation of EF 19 Attempts to Produce Ascomata in Culture To attempt the production of synnemata in vitro, I used field isolates from southern BC: Paecilomyces marquandii (Massee) S. Hughes, Paecilomyces tunuipes (Peck) Samson, Verticillium sp., and Cordyceps militaris strain 5711, obtained from the Osaka Institute for fermentation. I used different substrates including arthropods, seeds, and nuts as sources of nutrients for EF. Walnut, peanut, poppy, sesame, and flax seeds were placed in separate glass Petri dishes containing 5 drops of water each and sterilized by autoclaving at 121°C (Autoclave, Amsco 3021) for 30 min. Seeds and nuts were aseptically placed on MYP medium and inoculated with selected strains of fungi. Plates were kept at room temperature (25°C ± 4°C) under diffuse fluorescent light and reflected daylight. Entomogenous Fungi Inoculated on Experimental Arthropods To determine whether teleomorphs might be produced on experimental arthropods as substrates, MYP agar plates were inoculated with selected fungi and incubated at room temperature (25°C ± 4°C). After two weeks, conidial suspensions were obtained from these plates by adding 50 ml of a solution of 5% Tween 80R and gently rotating the plate. Conidial counts were made and the suspensions were adjusted to give a final concentration of 1x106 conidia/ml. These suspensions were used to inoculate the experimental arthropods. The inoculated pupae were placed in a deep Petri dish containing a bedding of sterile sphagnum moss or wet silica gel, or on water agar plates. Petri dishes were incubated at room temperature (25°C ± 4°C), under fluorescent light and diffuse sunlight. Other bedding materials used included sterile Whatman paper No.1, moist silica gel, or sawdust. These materials were autoclaved at 121°C for 30 min. EF strains obtained from the culture collection of the Osaka Institute for Fermentation, Japan, were also assessed using the above protocol. CHAPTER TWO: Collection, Isolation, and Cultivation of EF 20 Arthropod Maintenance Laboratory-reared arthropods used in this study included Spodoptera litoralis, Tenebrion molitor, Pseudoletta unipuncta, Christoneura rosaceana, and Trichoplusia ni (Table 2.1). These arthropods are commonly used as test organisms when looking for natural compounds or testing chemicals that can be used to control agricultural and forest pests (e.g., Trichoplusia ni). Isolates were used to inoculate arthropods of different developmental stages to determine whether ascomata production varied with developmental stage. Larval arthropods were kept at room temperature (25°C + 4°C) in sterile plastic containers. Water was added to maintain high humidity; a plastic platform was used to keep arthropods separated from water. Spodoptera larvae were fed a daily diet of premixed food, and the containers were cleaned daily. Grasshoppers and crickets were fed fresh lettuce and bran, and were maintained at 28°C, ambient humidity, and a light regime of 12:12 L:D. Beetles were fed bran and kept in wood shavings. 7. ni larvae were fed broccoli leaf, and Oncopeltus were fed an artificial mixture. Christoneura rosaceana was fed fresh spinach leaves, and Pseudotella sp. was fed a sterile sugar solution. Table 2.1. Species of laboratory-reared arthropods and wild arthropods, and their stages used in experiments. Laboratory-reared arthropods Stage of development Spodoptera litoralis Fabricius (Lepidoptera) larvae Tenebrion molitor L. (Coleoptera) adult and larval beetles Oncopeltus fasciatus (Dallas) adult Trichoplusia ni (Hubner) (Lepidoptera) larvae Pseudotella unipuncta adult Christoneura rosaceana larvae Commercially-reared arthropods Acheta domestica 3 rd instars Tenebrion molitor larvae Field collected arthropods Ground spiders (Salticidae) Pardosa sp. adult Grasshoppers - Melanoplus sp. adult CHAPTER TWO: Collection, Isolation, and Cultivation of EF 21 Free Fatty Acid Analysis of Selected Entomogenous Fungi Plates containing MYP medium and a sterile cellophane membrane were used to grow the fungal mycelia separate from the solid medium. Selected isolates of EF were used in this experiment (See Table 2.1). The membranes were placed on the agar surface, an agar disk was cut from an active-growth area of a fungal colony, and mycelia were placed upside down in the middle of each plate. These plates were kept in the dark, in an incubator set at 27°C ± 2°C for a week. The cultures were allowed to grow for another week in the light, and at room temperature 25°C ± 2°C. After two weeks, a small amount of mycelium was harvested and extracted with 2 ml acidic alcohol (1N HCI MeOH). The sample was centrifuged for 10 min at 2000 xg, and 200 pi of hexane were added to the sample and allowed to separate into two layers. The fatty acids were contained in the hexane layer, which was collected and placed in a 5 ml glass vial, one ml of 10% NaCl in MeOH were added, and the vial was tightly capped and kept in an oven at 85°C for 90 min. This hexane fraction was used to prepare the samples for gas chromatography (GC). A two-dimensional TLC method was developed for initial visualization of the lipids. R E S U L T S Survey of Entomogenous Fungi in British Columbia Fungi were often found on dead and mummified arthropods, under the bark of coarse wood debris, under logs, attached to leaves, and sticking out of leaf litter. Half of the collections came from logs different stages of decomposition (Table 2.2). Logs with soft and detachable bark were easily examined. Parasitized larvae and adult beetles were sometimes found covered with white mycelia. Some spiders were found attached to leaves. The areas of collection were selected based on accessibility and habitat. A large percentage of the fungi included in this study were isolated from specimens collected at the University of British Columbia. Several groups of EF are represented on this campus alone. CHAPTER TWO: Collection, Isolation, and Cultivation of EF 22 Table 2.2. Habitats where entomogenous fungi were collected, hosts on which fungi were obtained, number of collections, and percentage of total collection in British Columbia. Fungi Isolated Habitat Host/Substrate Number of Collections Percent of Total Collection (n=53) Paecilomyces galls wasp 2 3% Paecilomyces, Torrubiella, Verticillium, Cordyceps leaf litter insect parts, spiders, caterpillars, beetle larvae, cocoons 11 2 0 % Torrubiella, Verticillium logs spiders, beetles, ants, 26 5 0 % Paecilomyces, Beauveria branches beetles 3 5% Torrubiella, Gibellula, Cordyceps leaves spiders, insects 5 10% Paecilomyces, Cordyceps, Beauveria soil insect parts 5 10% Epichloe\ Claviceps grasses grains, inf lorescence 1 2% Field Collections My photographs of E F (Figures 2 . 2 - 2 . 1 8 ) represent field collections from 1998-2001. Two small collections are also included from Priest Lake, Idaho, U.S. , and the Lower Amazon Basin, Yarapa River, northeastern Peru. Figure 2.2. Torrubiella arachnophila. Voucher 96-21. Growing on a spider. Anamorph. Collected in the Squamish Val ley, B C . June 8, 1998. CHAPTER TWO: Collection, Isolation, and Cultivation of EF 23 Figure 2.3. Beauveria bassiana. Voucher 84-2. Growing on a beetle. Anamorph. Collected in the Mamquam Watershed, Squamish area, BC. October 9, 1998. Figure 2.4. Gibellula pulchra. Voucher 98-3. Growing on a spider. Teleomorph. Collected in Pacific Spirit Park, UBC, BC. July 17, 1999. Figure 2.5. Cordyceps nigrella. Voucher 100-3. Growing on beetle larvae. Teleomorph. Collected in Tofino, Vancouver Island, BC. June 20, 2001. Figure 2.6. Paecilomyces inflatus. Voucher 98-2. Growing on beetle larva. Anamorph. Collected in Pacific Spirit Park, UBC, BC. July 17, 1999. Figure 2.7. Cordyceps capitata. Voucher 82-25. Growing on Elaphomyces granulatum. Teleomorph. Collected in the Squamish Valley, BC. August 22,1998. CHAPTER TWO: Collection, Isolation, and Cultivation of EF 24 Figure 2.8. Cordyceps tuberculata. Voucher 01-04. Growing on a butterfly. Teleomorph. Collected in the Lower Amazon Basin, along the Yarapa River, northeastern Peru. July 5, 2001. Figure 2.9. Torrubiella sp. Voucher 01-02. Growing on a spider. Teleomorph. Collected in the Lower Amazon Basin, along the Yarapa River, northeastern Peru. July 7, 2001. Figure 2.11. Paecilomyces sp. Voucher 94-1. Growing on a moth cocoon. Anamorph. Col lected along Marine Drive, U B C campus, B C . October 11, 1998. CHAPTER TWO: Collection, Isolation, and Cultivation of EF 25 Figure 2.12. Torrubiella raticaudata. Voucher 01-09. a) Growing on a spider. Teleomorph. Col lected in the Lower Amazon Basin, along the Yarapa River, northeastern Peru. July 3, 2001. b) Whip-l ike structure protruding from host. Figure 2.13. Paecilomyces sp. Voucher 97-4. a) Growing on wasps. Anamorph. Col lected in Ladner, B C . June 9, 1999. b) Rose gall with wasps. Figure 2.14. Gibellula sp. Voucher 01-06. Growing on a spider. Anamorph. Col lected in the Lower Amazon Basin, along the Yarapa River, northeastern Peru. July 7 ,2001 . Figure 2.15. Gibellula sp. Voucher 98-3. Growing on a spider. Anamorph. Collected in Pacif ic Spirit Park, U B C , B C . August 29, 1999. CHAPTER TWO: Collection, Isolation, and Cultivation of EF 26 Figure 2.16. Paecilomyces sp. 85-14. Synnemata. Growing on a mummified caterpillar. Col lected in the Capi lano Watershed (GVRD) , B C . October 10, 1998. Figure 2.17. Gibellula sp. Voucher 80-15. Growing on a spider. Anamorph. Collected in Cypress Bowl, B C . June 19, 1998. Figure 2.18. Gibellula sp. Voucher 100-1. a) Growing on a spider. Anamorph. Col lected in Nanaimo, Vancouver Island, B C . October 22, 1998. b) Synnemata protruding from host, showing conidiophores. CHAPTER TWO: Collection, Isolation, and Cultivation of EF 27 Microscopic Documentation of Field Collections I photographed the following images (Figures 2.19- 2.25) using a Zeiss bright field microscope and a digital camera. Colonies were grown on MYP medium and were one-week old when photographed. These represent some of the most common genera of EF collected during my study. All photographs were taken under oil immersion (100x). The most distinctive anamorph features are shown and listed in the legend of each figure. Figure 2.19. Whorled conidiophores in Verticillium sp. Figure 2.20. Sympodial conidiogenous cells arising from short swollen denticulated rachis of Beauveria sp. Teleomorph not observed or reported. Type species: Beauveria bassiana (Bals.) Vuill. Figure 2.21. Paecilomyces sp. Penicillate with terminal whorls of phialides (cells with a swollen basal portion, tapering at end). Teleomorphs: Byssochlamys Westling, Talaromyces C.R. Benjamin, Thermoascus Miehe. Type species: Paecilomyces variotii Bainier. CHAPTER TWO: Collection, Isolation, and Cultivation of EF 28 Figure 2.22. Paecilomyces sp. conideogenous cells bearing conidia. Figure 2.23. Gibellula sp. Compact terminal conidiophore. a) Electron Scanning Microscope, and b) oil immersion 100x. Figure 2.24. Cordyceps loydii. a) sporocarps, and b) fragmented spores. Oil immersion 100x. CHAPTER TWO: Collection, Isolation, and Cultivation of EF 29 Figure 2.25. Anamorph isolated from Cordyceps militaris (01-07, Peru collection). Short phialides bearing spores. Culture Collections I obtained 22 isolates from E F collected in the field. These include Paecilomyces, Beauveria, Verticillium, and other anamorphs of Cordyceps and Torrubiella Boud. The isolates were tested for biological activities (see Chapter Three) and heavy metal tolerance (see Chapter Four). The following photographs (Figures 2.26 - 2.28) represent some isolates that were established from B C field collections. Cultures were two-weeks old when photographed. Figure 2.26. Beauveria bassiana a) colony growing on M Y P medium, b) conidiophore showing phialides, and c) sketch of conidiophore and conidia. CHAPTER TWO: Collection, Isolation, and Cultivation of EF 30 C Figure 2.28. Verticillium sp. a) colony growing on MYP medium, b) conidiophore showing phialides, and c) sketch of conidiophore and conidia. Attempts to Produce Ascomata using Seeds and Nuts The inoculation of seeds and nuts did not produce ascomata, but different types of synnemata were produced depending on the media supplement and growing conditions. Synnematal production occurred using walnuts, pecans, and peanuts (Table 2.3). White synnemata grew from fluffy, cottony colonies growing on MYP plates with added walnuts; yellow-pigmented synnemata were more common when using pecans. Poppy and flax seeds were also assessed; tested EF grew mycelia but did not produce synnemata. CHAPTER TWO: Collection, Isolation, and Cultivation of EF 31 Table 2.3. Production of synnemata using different seeds and nuts. Inoculated plates were kept at room temperature (25°C ± 4°C), an equal number in the dark, and half exposed to light (Sylvania Grow-light), on a cycle of 12/12 hr L/D. X = synnemata; - = no synnemata, but small amounts of mycelial growth present; N/T = not tested. Synnemata developed under both treatments. Fungal Strain Walnuts Peanuts Pecans Flax seeds Paecilomyces marquandii (73-21) X X X - Paecilomyces tunuipes (84-15) X X - - Cordyceps japonica (9647) - - - - Verticillium sp. (24-2b) X - N/T - Cordyceps militaris (5298) X - - - Paecilomyces sp. (85-16) X X - - Paecilomyces sp. (80-14) X X - - Most synnemata produced under these experimental conditions were cream colored; others were pale yellow, bright orange, and simple (Figure 2.29a), furcated (Figure 2.29b), or irregularly branched. They ranged in length from 1.0 to 3.0 cm. In other cases , pigmentation was enhanced (e.g., pecans) b Figure 2.29. Paecilomyces spp. synnemata produced on walnut mesocarp: a) single, no branching; b) furcated. Photographs under 10x dissecting microscope with Nikon 5x Coolpix digital camera. CHAPTER TWO: Collection, Isolation, and Cultivation of EF 32 Following the exhaustion of nutrients in the media, the morphology of synnemata varied in size and form as well as pigment production. A n orange pigment was only produced when Paecilomyces sp. was exposed to nutrient-poor conditions (Figure 2.30). The age of the culture may also determine pigment production. The light regime used 12/12 hr L/D, enhanced the production of carotenoids. The control treatment, kept in dark conditions, produced less pigmentation and at a later age. Figure 2.30. Paecilomyces sp. a) synnemata produced following a step down in nutrients, b) magnified to emphasize the intensity of the orange pigment. No ascocarps developed. M Y P broth containing % of nutrients. Production of ascomata was attempted by using a number of substrates. Inoculated peanuts produced yellowish-white synnemata with a ropy shape (Figure 2.31). The strands were long and the pigmentation on the underside of the colony was bright yellow. Figure 2.31. Synnemata of Paecilomyces sp. growing on peanuts, a) and b) illustrate two different strains of Paecilomyces sp. Colonies were six weeks old. CHAPTER TWO: Collection, Isolation, and Cultivation of EF 33 Synnemata also developed in liquid cultures, and different synnematal morphologies and aerial hyphae were observed. After two months, synnematal development in Paecilomyces marquandii was observed, but these structures stayed in a primordial stage for three months, when the experiment was stopped (Figure 2.32). The morphology of Paecilomyces tunuipes was affected by the shape of the container and by aeration (Figure 2.33). In both P. marquandii and P. tunuipes, no ascomata were produced, but synnemata developed above a thick, yellow mycelial mat. Figure 2.32. Paecilomyces marquandii was initially grown at room temperature (25°C ± 4°C) for one week, and then transferred to a cold room (4°C), under dark conditions. Culture was grown in M Y P liquid medium. Figure 2.33. A three-month-old culture of Paecilomyces tenuipes producing abundant aerial mycelia and synnemata. Culture was grown in M Y P liquid medium. Synnematal morphology, color, s ize, and development time varied depending on substrate, temperature, type of media (e.g., solid or liquid, M Y P or P D A ) , and age of the culture. Best results were obtained with walnuts and pecans as substrate (Figure 2.34a). The pecans enhanced the production of pigmentation. CHAPTER TWO: Collection, Isolation, and Cultivation of EF 34 Figure 2.34. Synnemata of a) Paecilomyces marquandii (73-21) growing on pecans, b) Cordyceps militaris (5298) growing on walnuts, c) P. marquandii growing on walnuts. With the exception of Paecilomyces spp, Verticillium sp., and Cordyceps japonica, no synnemata were produced on arthropods by any of the assessed strains. Four weeks after inoculation of the pupae of Spodoptera litoralis, synnemata began to appear. No perithecia formed. The moss bedding was replaced with sil ica gel, and a light regime of 16/8 hr L/D was introduced. Because sil ica gel is a hydrophilic material, relative humidity in the container dropped quickly, thus si l ica was not a good bedding material. A lso , the grains of si l ica became attached to the fungal mycelia causing further dehydration. Best results were obtained by placing the pupae (inoculated host) in a deep petri dish containing water agar. Arthropod Inoculation and Synnemata Production Caterpillars of the moth Spodoptera litoralis (Figure 2.35) and the beetle Tenebrion molitor (Figures 2.38 and 2.40) were inoculated with different isolates from field collections obtained at the U B C campus. Members of the form genus Paecilomyces were the most aggressive in colonizing both wild and laboratory-reared arthropods. Arthropods were reared in the laboratory of Dr. Murray Isman, Agriculture, U B C . Other arthropods were tested and some were infected by the E F used in these experiments (see Table 2.4). CHAPTER TWO: Collection, Isolation, and Cultivation of EF 35 Figure 2.35. Synnemata growing on the cocoon of Spodoptera litoralis. Caterpillar was inoculated before metamorphosis occurred. The cocoons were kept in moist sterile chambers at room temperature (24°C) until synnemata developed, a) Paecilomyces marquandii, b) Paecilomyces inflatus (Burnside) J.W. Carmich., and c) Paecilomyces tunuipes. Table 2.4. Synnemata production from arthropods inoculated with selected entomogenous fungi. X = synnemata produced. Host Fungi Spodoptera Tenebrion Trichoplusia Melanoplus 7. molitor litoralis molitor ni sp. (larva) Cordyceps militaris C. japonica Beauveria bassiana X Paecilomyces sp. X X X X P. marquandii X X X Verticillium sp. Host mortality was observed in some of the inoculated hosts, but no visible mycelia were observed growing on the host. Percent survival of arthropods inoculated by fungi was examined and results are presented in Figures 2.36 and 2.37. Paecilomyces marquandii showed high percent mortality against Christoneura rosaceana and Tenebrion molitor. This fungus was originally isolated from a beetle larva. CHAPTER TWO: Collection, Isolation, and Cultivation of EF 36 100 a > 3 to C a 2 o a. 80 60 f- 40 20 Paecilomyces marquandii Paecilomyces marquandii Paecilomyces inflatus Treatment Verticillium sp. Control Figure 2.36. Percent survival of crickets, Acheta domestica, inoculated with four strains of entomogenous fungi collected in British Columbia. Total number of crickets on Day 0 was 30. The experiment duration was 10 days. Control is represented by pale blue line. Figure 2.37. Percent survival of arthropods inoculated with entomogenous fungi. Fungi used to inoculate each arthropod species are identified in bars 'Day 0'. Total number of arthropods on Day 0 was 30. The experiment duration was 10 days. Control is represented by pale blue line. CHAPTER TWO: Collection, Isolation, and Cultivation of EF 37 Mycel ia developed after 3-4 days and began colonizing most of the hosts. Five days after inoculation, a red secretion was produced from the posterior ends of the larvae. Similar secretions were produced by Paecilomyces marquandii growing on pecans (Figure 2.34a). The secretions were not collected for chemical analysis because of the potential risk of introducing contaminants into the experimental system, and to avoid disturbing fungal development. Treatments were checked daily, and dead arthropods were removed and placed in sterile moisture chambers (kept at 28°C). The development of mycelia on the host was taken as an indication of death by mycosis. Percent mortality was assessed after 10 days. During the first trial of this experiment, fungal contaminants (mainly Trichoderma, Penicillium, and Aspergillus) developed on the moss used as bedding material. This was probably caused by incomplete sterilization of the moss. Of all individuals in the Spodoptera control treatment, only two did not complete metamorphosis. Among the isolates that colonized S. litoralis and Tenebrion molitor were Verticillium (strains 96-10, 98-2, and 100-1), Paecilomyces marquandii (73-21), Paecilomyces tunuipes (84-15a), and Paecilomyces sp. (80-14) Three isolated strains from infested beetles collected at Priest Lake, Idaho, U S A , were identified as Beauveria bassiana. These strains were used to inoculate adult beetles and larvae of Tenebrion molitor, one strain showed strong pathogenicity under experimental conditions. Figure 2.38. a) Adult beetles Tenebrion molitor parasitized by Beauveria bassiana. b) Fungal growth is more abundant at the joints and mouths of T. molitor. The isolate was obtained from an unidentified beetle collected at Priest Lake, Idaho, U S A . CHAPTER TWO: Collection, Isolation, and Cultivation of EF 38 Larvae of the agricultural pest Trichoplusia ni were used to assess the pathogenicity of entomogenous fungus Paecilomyces marquandii. The spores of Paecilomyces marquandii germinated on the caterpillar and the mycel ia colonized the whole body (Figure 2.39). No synnemata were produced. Some experiments could not be repeated because of the limited supply of arthropods. Figure 2.39. Paecilomyces marquandii (strain 85- 15a) effectively colonized Trichoplusia ni, an agricultural pest. Of the isolates of E F tested on beetles, only Paecilomyces sp. was capable of producing synnemata on the adult beetle, Tenebrion molitor. Synnemata were white and grew predominantly from the ventral surface of the beetle (Figure 2.40). Little mycelia were present over the rest of the body. Figure 2.40. Synnemata developed from an adult beetle Tenebrion mollitor inoculated with spores of Paecilomyces sp. (95-10). The beetle was immobil ized after three days of inoculation. Synnemata did not develop until two months after inoculation. CHAPTER TWO: Collection, Isolation, and Cultivation of EF 39 Figure 2.41. The artificial inoculation of Paecilomyces inflatus on carpenter ants did not result in production of synnemata, but mycel ia grew on and inside the ant. Free Fatty Acid Analysis The most abundant fatty acids present in selected entomogenous fungal strains were 18:1 (9-octadecanoic acid; oleic acid), 18:2 (9,12- octadecanoic acid; linoleic acid), 16:0 (hexanodecanoic; palmitic acid), and 18:0 (octadecanoic acid; steric acid) in decreasing order (Table 2.5; see Appendix B, Figure B.2). The levels of 18:3 (19,12,15- octadecanoic acid; linolenic acid) relative to 18:2 are known to depend upon many factors, including culture mass. Table 2.5. Free fatty acids present in selected entomogenous fungal strains. Columns A and B indicate two free fatty acids that were present but not identified. Fungal Strains Free Fatty Acids Present A B 12:0 14:0 16:0 16:1 O) 18:0 18:1 (9) 18:2 (9,12) 18:3 (9,12,16) 20:0 22:0 22:1 (11) 24:0 c 0 E Beauvaria bassiana (BEET3) X X X X X X X Cordyceps militaris (01-07) X X X X X X X X X X X Paecilomyces marquandii (73-21) X X X X X X Paecilomyces sp. (24-2B) X X X X X X X X X X X Paecilomyces sp. (85-14A) X X X X X X X Paecilomyces sp. (95-2) X Verticillium sp. (100-1) X X X X X X X Standard X X X X X X X X X X X X CHAPTER TWO: Collection, Isolation, and Cultivation of EF 40 In Beauveria bassiana (BEET3) , 16:0 and 16:1 were found in lower concentrations than the average found for other fungi. Smal l amounts of 10:0 and 24:0 were also present in most tested strains. Paecilomyces sp. (24-2B) had the most diverse fatty acid composit ion, and most similar composit ion to that of Cordyceps militaris (01-07). Verticillium sp. (100-1) showed the highest amount of 18:0, and B. bassiana the lowest. No consistent differences in fatty acid composit ion among the strains tested were observed (See data in Appendix B). The small differences could not be used as special characters in species identification. However, these profiles may be useful for other purposes. DISCUSSION EF have not been studied intensively in most regions of the world, and the group remains essentially unknown from the standpoint of distribution, life histories, and genetics. However, available strains have been studied increasingly from the standpoint of chemistry because of the discovery of useful medical substances in the group. My research revealed a number of taxa of entomogenous fungi, including genera restricted to spiders and mites. The majority of E F in temperate rainforests collected in this study were anamorphic stages. Fewer teleomorph collections resulted from the surveys in B C . A short collection period in tropical rainforest yielded a larger proportion of teleomorphs than in temperate forests. Photoperiod and temperature may be partially responsible for the higher proportion of teleomorphic collections in the tropics. The addition of media supplements such as walnuts, peanuts and poppy seeds may simulate the proteins and lipid content in arthropods. Walnut mesocarps contain between 63- 7 0 % oil, more than 9 0 % of which is unsaturated fatty acids; the oleic acid content ranges from 12-20% (Savage et al. 2000). A lso , phytosterol and vitamin E are part of the oil fraction, and may play a role in fungal sterol metabolism (Savage ef al. 2000). I expected that these nutrients would contribute to the development of teleomorph stages in cultivation. Despite attempts, this CHAPTER TWO: Collection, Isolation, and Cultivation of EF 41 did not occur,.but the experimentation was very limited and additional experiments involving temperature, light exposure, and variation in nutrient availability might produce successful results. Fatty acids are precursors of vital structural and metabolic molecules. They are precursors of prostaglandins and related regulators. Fatty acid composit ion has been used as a key factor in genus or species identification, especial ly in bacteria, but also in Aspergillus, Penicillium, and Mortierella. I analyzed seven strains of E F for their fatty acid composit ion to determine if particular composit ions are characteristic of a specif ic E F taxon. Free fatty acid composit ion examined by G C did not show dramatic differences among strains. Thus, free fatty acid composit ion analysis is not recommended as a taxonomic character among E F . However, rDNA sequencing support morphological identification of the selected strains (Appendix B). The cultivation of Cordyceps has always been known to be problematic. Factors such as light regime, temperature, C 0 2 and 0 2 levels, age of the culture and pH may have an effect on ascomata development, and general fungal growth and physiology. The living collection resulting from this study could be used for further laboratory studies on E F as well as a taxonomic reference for future collections. The photographic documentation will complement existing information and databases on the fungi of the Pacif ic Northwest of North Amer ica . Some species remain to be fully identified. Interesting E F identified during this research are those parasitic on spiders. Spiders parasitized by members of Torrubiella were collected in different locations in B C , including Vancouver Island, Cypress Bowl Park, and the Squamish Val ley. These collections may be the first reports in B C , but this has not been officially confirmed. A n additional new record for western Canada (though not parasitic on spiders) may be that of a spec ies of Cordyceps, however final identification has not been completed. Despite the amount of forest covering the landscape of B C (48.4 million hectares; Ministry of Forests Annual Report 2001), and the economic dependency on the forest industry (e.g., M O F Revenues 2000/01 = $1.3 billion) on these forests, arthropod-fungus interactions and their CHAPTER TWO: Collection, Isolation, and Cultivation of EF 42 biological role in maintaining balanced and healthy forests remains poorly understood in B C . The wealth of knowledge and expertise on arthropod biology is substantial, and existing collections in the province house a good representation of the local arthropods. However, entomogenous fungi remain underrepresented in herbaria. It has become evident that E F play a significant role in the natural control of some arthropod pests (Tanada and Kaya 1993; Barson et al. 1994; Inyang et al. 1999). The discovery of Beauveria bassiana in 1836, by Agosto di Bass i lead to increased interest in E F . S ince then, the search for entomogenous fungi for use in biological control has continued, but with limited success (Gil lespie and Claydon 1989). Only a few mycoinsect icides have been developed based on E F spore suspensions and some of these are commercial ly available. Among these are Boverin (Beauveria bassiana), Mycar (Hirsutella thompsonii), Metaquino, Sorokin, Green muscle (Metarhizium anisopliae), and Vertalec (Verticillium lecanii; Kendrick 1992). Beauveria bassiana and Metarhizium anisopliae have shown positive results as mycoinsect icides, preparations of E F fungi used to kill insects or for tick control (Kaaya and Hassan 2000), and control of tsetse flies Glossina spp. (Kaaya and Munyiyi 1995). Monocerin and fusarentin 6,7- di-Me ether have been isolated from the entomogenous fungus Fusarium larvaru; both compounds have low molecular weights and have shown insecticidal activity against the blowfly, Calliphora erythrocephala. Destruxins A and B, dipsipeptides toxic to wax moth, Galleria mellonella Linnaeus, have been isolated from Metarhizium anisopliae (Roberts 1969). B. bassiana produces a variety of cuticle degrading enzymes including proteases, chit inases, and esterases (Gupta etal. 1991,1992) Worldwide, fourteen species of Paecilomyces are recognized as pathogens of various arthropods and nematodes found on plants and soil (Samsom 1974). High genetic variation has been reported among species of the form genus. For example, Paecilomyces fumoroseus and P. liliacenus show morphological similarities but are genetically and pathogenically different (Tigano-Milani et al. 1995a). Assessment of strain variation is still needed to select suitable strains for particular purposes (e.g., biological control, peptide production). CHAPTER TWO: Collection, Isolation, and Cultivation of EF 43 Little work was conducted on biological control until the first half of the 20 century with the discovery of Bacillus thurengensis (Tanada and Kaya 1993). The commercial development and use of E F for controlling of arthropods has lagged far behind that of Bacillus thurengensis. Because of the potential environmental benefits presented by the use of biological control agents, in contrast to the use of chemical pesticides, there has been much focus over the last 30 years on improving the survival of conidia (Smits et al. 1996) and large-scale production of inocula (Goettel 1984). The effect of myco and/or chemical pesticides on non-targeted microorganisms has been a concern for the last fifty years. The wide use of pesticides exerts a negative influence on non-targeted organisms such as soil fungi (Popraski and Majchrowicz 1995). Many chemical pesticides have shown toxicity and, in some cases , complete growth inhibition of E F (Urs etal. 1967; Horton 1980; Gardner and Storey 1985). Local strains of E F may be good candidates for the development of biological agents to be used in pest management in B C forests. Because these fungi are already an integral part of the ecosystem they may reduce the potential negative effects on other organisms as shown by chemicals. Furthermore, E F in combination with other natural pesticides have resulted in an efficient biocontrol. The biopesticide Neemark (Azadirachtin) activity has been found to be compatible, in vitro, with the E F Beauveria brongniartii and to Metarhizium anisopliae (Vyas et al. 1992). To investigate parasitism and pathogenic mechanisms, good experimental model systems are necessary where both parasite and host are easy to rear and handle. Numerous species of entomogenous fungi have been studied as potential biological control agents of arthropod pests (e.g., Butt and Wraight 1988; Butt and Humber 1989; Butt et al. 1996; Evans 1994; Jack and Jung 1998; Ibrahim et al. 1999). The selection of fungi for biological control requires a ser ies of screening b ioassays. The inoculation of wild locusts and spiders demonstrated that some entomogenous fungi have the potential to affect a wide range of arthropods. Because of the low sample s ize, my results do not allow me to make any strong conclusions. However, preliminary observations demonstrated that tested E F were not host specific, and caution should be taken CHAPTER TWO: Collection, Isolation, and Cultivation of EF 44 when using them as biological control agents. Ground spiders, Salticidae, were very susceptible to fungal infection by isolates of Paecilomyces spp. These experiments will need to be repeated to measure the degree of host specificity, mortality rate at different instars, and to assess levels of EF pathogenicity on wild populations of arthropods. CHAPTER THREE: Screening of EF for Bioactivity 45 CHAPTER THREE: Screening of Entomogenous Fungi for Bioactivity INTRODUCTION The search for and isolation of biological compounds from fungi and bacteria has been carried out for many centuries. For a long time, the most common sources of fungal inocula were soil and plant debris. Thousands of biochemical studies focusing on the screening, isolation, and synthesis of fungal compounds have been produced (Gunde-Cinerman et al. 1993; Huang and Kaneko 1996; Hancock 1997,1998; Hancock and Chaple 1999). Many of these yielded compounds useful as foods, drugs, and in industrial applications. However, the intense uses of antibiotics have led to the development of organisms that are resistant to our present drug arsenal. The need for safe molecules to stop invasive infections of fungi, bacteria and viruses has increased over the years. The need for new sources of biologically active agents is widely acknowledged. E F are a group that remain poorly documented, although a wealth of fungal chemical diversity has been demonstrated and some bioactive compounds have been isolated. Some of these compounds have passed clinical trials and are currently used in treatments of particular d iseases (e.g., Cyclosporin A , Cordycepin; Wasner and Pfleiderer 1995; Wasner et al. 1994, 1996, 1997). S o m e currently available antifungal compounds act on targets on mammalian cells (Debono and Gordee 1994). However, some of these compounds may be cytotoxic and cause adverse drug interactions. This situation is similar for antiviral drugs, which need to be specif ic to the virus and not damage the host cell. Two general modes of action of known antiviral drugs are the prevention of viral penetration (e.g., amantadine) and the inhibition of D N A replication enzymes (e.g., acyclovir). Only a few compounds with antiviral activity have been isolated from fungi. I screened extracts of E F for antiviral activity using the enveloped virus herpes simplex virus l (Herpesvir idae), a double stranded D N A virus in the herpes simplex group. This group includes chicken pox, shingles, and infectious mononucleosis viruses. The bacteria, fungi, and CHAPTER THREE: Screening of EF for Bioactivity 46 virus used in this screening include organisms relevant to human d iseases (e.g., Staphylococcus aureus methicillin resistant, Escherichia coli, and the yeast Candida albicans. These are also important model organisms used in many medical studies. The number of b ioassays may not reflect the total potential of the tested E F , and other b ioassays should be used in future studies. OBJECTIVES 1. Examine the biological activity of E F , including antibiotic, antifungal, phototoxic, and antiviral activities. 2. Determine the amino acid composit ion in Cordyceps sinensis. 3. Screen for Cyclospor in A in E toAc crude extracts of E F . M E T H O D S Fungal Extract Preparation Aliquots of 250 ml of M Y P broth media were inoculated with selected E F and grown as previously descr ibed. The mycelia were extracted with 300 ml of hexane followed by three washes of 100 ml E t O A c over 24 hrs. The combined extracts were filtered and the volume reduced under pressure. The dried extracts were weighed and suspended in E t O A c : M e O H (2:1) to make a 10 pl/ml solution. Antibacterial Bioassay Antibiotic b ioassays were performed by the disk diffusion method (Figure 3.1); Muller- Hinton (MH; Difco) medium was used to grow bacteria and Sabouraud dextrose ( S A B ; DIFCO) medium for fungi. Sterile paper disks (6 mm) were impregnated with 10 ml of the previously prepared crude fungal extract, and the solvent al lowed to air dry in the fume hood. CHAPTER THREE: Screening of EF for Bioactivity 47 A n inoculum of each bacterial strain was suspended in a 10 ml glass tube containing 3 ml of Muller Hinton broth medium, and incubated overnight at 27°C, on a rotary shaker at 1200 rpm. Bacteria used in the bioassays included Gram (+) Bacillus subtilis, Staphylococcus aureus and Gram (-) Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae. The yeast Candida albicans was used to assess antifungal activity. Overnight cultures were diluted in M H nutrient broth to a cell density of 10 9 /ml. The bacterial and fungal suspensions were used to inoculate M Y P or S A B plates. Plates were prepared in duplicate. Crude extract Incubation Antifungal and Antibacterial Bioassay Control: gentamicin, nystatin Figure 3.1. Diagrammatic sketch of Disk diffusion bioassay used to evaluate antibacterial, antifungal, and phototoxicity activities in E F . Extracts were used both with and without exposure to U V light to determine if photoactive antibiotic substances were present. A) A n aliquot of each extract impregnated in a sterile paper disk and allow to dry; B) the disk was then placed on the agar surface of a plate inoculated with the appropriate test organism; and C) to assess phototoxicity a duplicate plate was prepared and exposed to U V light and incubated. Gentamicin was the control for antibacterial b ioassays, nystatin was used as a control for antifungal b ioassays, and 8-methoxypsoralen (MOP) was used for phototoxicity b ioassays. CHAPTER THREE: Screening of EF for Bioactivity 48 Photoactivated Antibiotics and Antifungal Substances Prepared disks containing fungal extracts were placed on plates inoculated with selected bacteria and the yeast Candida albicans. One plate of each duplicate pair was exposed to long wave U V light for one hour at room temperature. Irradiation was provided by a set of four Sylvania F20T12-BLB lamps, which gave a measured incident energy of 5 W / m 2 a n d a maximum output of 350 nm. Duplicates of these plates were kept in the incubator without lights. After U V irradiation, all plates were incubated at 37°C and examined after 18 hrs. The diameter of the zones of inhibition around the paper disk were measured with a ruler and recorded. Samples showing zones of inhibition of microbial growth only after U V irradiation were recorded as phototoxic (+). Those samples with zones of inhibition in both light and dark were reported as antibiotic. A larger zone of inhibition in light-treated samples, compared to those maintained in the dark, was considered as enhanced activity (Wat 1980; Towers 1984, 1987). Antiviral Bioassays Crude E tOAc extracts of 18 fungal strains isolated from arthropods were assayed against the enveloped double stranded D N A Herpes Simplex Virus Type I (HSV-I). A n Afr ican green monkey kidney cell line (Vero cells) was obtained from the Amer ican Type Culture Col lect ion. Vero cel ls were grown in Dulbecco's modified Medium (DEM) with 5 % Fetal Bovine Serum ( F B S ; Gibco Life Sc ience, Ontario) and 25 pg/ml gentamicin sulphate (Sigma), in 96-well microtest trays (Falcon) (Anani et al. 2000). A n atmosphere of 5 % C 0 2 a n d 9 5 % air at 37°C was provided using an incubator. Vero cel ls were considered ready for b ioassays when a monolayer of cel ls was formed, about 5-7 days after inoculation. Fungal extracts (EtOAc) were prepared from two-week-old M Y P broth cultures. The medium and mycel ia were separated by filtration, and both extracted three times with 100 ml of E tOAc , with a final hexane:acetone (80:20) wash . The fractions were pooled and dried under reduced pressure and dried extracts were stored at 4°C. For b ioassays, the dried samples were suspended in M e O H , diluted to a concentration of 100 pg/ml, and passed through a 0.2 mm CHAPTER THREE: Screening of EF for Bioactivity 49 pore diameter filter. The extract was further diluted 1:200 in D E M and dispensed in a 96 well plate containing a monolayer of Vero cells (Hudson et al. 1993, 1994). The 96-well plates of Vero cells, treated with a serial dilution of the fungal extracts, were incubated at 37°C for 60 min, and then examined under the microscope for any cytotoxic effects caused by the extracts. Cytotoxic effects included changes in cell morphology and cell membrane damage (e.g., cytoplasm leakage). A n aliquot of 100 ul of the virus suspension was added to each well and the plates were kept in an environmental chamber at 37°C for 30 min, with continuous shaking, and under long wave U V - A light (5 W / m 2 , with an emission wavelength maximum of 350 nm, from four Sylvania F20T12-BLB lamps), or kept in the dark for 30 min. After these treatments, the plates were returned to the incubator at 37°C. Cultures were examined under a dissecting microscope after incubating for 72 hrs. The absence of viral cytopathic effects indicated 100% inhibition of the virus. Partial inhibition of the virus was considered a negative result. Posit ive results were recorded at concentrations showing minimum inhibitory effect. Acyclovir was used as a positive control against herpes simplex virus. Screening for Cyclosporine A A stock solution of Cyclosporine A (Cys A) was prepared in M e O H ( H P L C grade) and stored in an amber bottle to protect it from light. A solution of 0.10 mg/ml was used to prepare a calibration curve. A dilution series of the sample was prepared by using different amounts of C y s A . The standard C y s A and acetonitrile ( H P L C grade) were obtained from S igma C o . A calibration curve was created by using a dilution series of the standard C y s A . Chromatographic analysis was performed using a H P L C Water Instrument (Lilford, MA, U.S.A.) model 60000A H P L C Pump, model 480 variable-wavelength U V detector, model 710B sample injector, and model 730 data module. A Waters R P - 1 8 analytical column (30 cm X 4.6 mm I.D., particle s ize 5 mm), with a R P - 1 8 guard column was heated to maintain the column at 70°C. The flow-rate of the mobile phase was 1ml/min and produced a pressure of 1100 p.s.i. CHAPTER THREE: Screening of EF for Bioactivity 50 U V monitoring was conducted at 215 nm. The mobile phase was a mixture of degassed acetonitrile:water (65:35) in isocratic mode. The retention time for C y s A standard was 5.7 min. Thin layer chromatography of standards and fungal extracts was performed using Si l ica GelF254 as a stationary phase, and Chlorofornrv.MeOH (97:3) as a mobile phase. T L C developed plates were visual ized using iodine vapors and a 254 nm U V light source (Roesel and Kahan 1987). Cyclospor ine A appears as a light yellow-brown spot under visible light and has an Rf value of 0.49 under the above conditions. Amino Acid Analysis of Cordyceps sinensis Ascomata of Cordyceps sinensis were obtained from North Amer ican Reishi Inc. Vancouver, although the sporocarps were originally collected in Ch ina. The fungus, including the colonized host, was submitted for amino acid analysis (see Appendix B, Figure B.3). Thirty grams of Cordyceps sinensis were separated into two parts: fungus ascocarps (15 g) and mummified caterpillar (15 g), which was not identified. The two samples were extracted separately. Samples were ground with a mortar and a pestle, using liquid nitrogen. The pulverized material was transferred to 500-ml Erlenmeyer f lasks, 200 ml of M e O H were added to each flask, and these were kept on a shaker for 24 hrs. The material was then filtered through Whatman No.1 paper and the filtrate was dried using a rotoevaporator. The concentrated crude extract was fractionated by passage through a 8.0 cm x 1.5 cm column packed with Dowex 50W-X8 mesh 20-50 cation exchange resins (JT Baker). The resin was first washed with deionized water, followed by 50 ml of a HCI solution pH 4.0, and finally 0.1 M N a O H . There were rinses of deionized water between each step. The column was then charged with 0.1 M HCI. The crude extract was dissolved in 10 ml of 5 0 % M e O H and loaded into a resin column. The non-amino acid fraction was eluted with deionized water. The amino acids bound to the resin were eluted using 10% N H 4 O H . Fifteen fractions were eluted (20 ml per fraction). CHAPTER THREE: Screening of EF for Bioactivity 51 Samples from the collected fractions were dried to remove the N H 4 O H , resuspended in M e O H and then spotted onto T L C cel lulose plates. The plates were developed with Solvent A:Butanol-Water-Acet ic acid (BWA) in a ratio of 45:20:20, and sprayed with 0 .1% g of monohydrate ninhydrin (Sigma Co.) to determine which fractions contained similar amino acids. Amino acid containing fractions were pooled together and dried. The samples were refrigerated at 4°C. The amino acid-containing fraction (90 mg) was dissolved in 6 ml M e O H and separated by T L C using Cel lu lose F T L C Plast ic sheets (EM Sc ience 10x10) using a two-dimensional development system. Amino acids reacted with ninhydrin producing purple, violet, blue, and yellow colorations. R E S U L T S Biological Activity Assays: Antifungal, Antibiotic, and Phototoxic I assessed the biological activities of some extracts from E F or the spent culture medium. Twenty crude fungal extracts of E F were active against the gram (+) bacteria Staphylococcus aureus and Bacillus subtilis. No activity was found against the gram (-) bacteria tested; only a strain of Beauveria sp. inhibited E. coli after U V light exposure. A number of biological activities were detected from the extracts of E F (Table 3.1). The wide range of biological activity was obtained using an assortment of growing conditions (e.g., radiation with U V light). Paecilomyces sp. (85-14b) was the only strain that showed antifungal activity against the yeast Candida albicans (not shown in table). In total, seven fungal extracts were phototoxic against Staphylococcus aureus after exposure to ultraviolet light (350 nm). A total of 14 extracts showed antibacterial activity against one or more of the tested bacteria. CHAPTER THREE: Screening of EF for Bioactivity 52 Table 3.1. Biological activity of entomogenous fungal extracts. Symbols : + = biological activity observed, ++ = biological activity only when irradiated with U V light, - = no biological activity observed. Escherichia coli, Bacillus subtilis, Staphylococcus aureus, and Pseudomonas aeruginosa were used for anti-bacterial b ioassays. Only Paecilomyces sp . (80-14a) showed moderate antifungal activity against Candida albicans. B. E. S. P subtilis coli aureus aeruginosa Control Methanol - - - Tetracycline + + + Fungi Beauveria bassiana(B'\) - ++ - B. bassiana (B4) ++ ++ - B. bassiana (B6) - - + B. bassiana(B7) - - + Cordyceps capitata (84-20) + + - C.japonica (9647) + - + C. militaris (01-07) + - - C. militaris (5298) + - - C. ophioglosiodes(8992) + - - C. tuberculata (01 -04) - - - Paecilomyces (84-2) - + + Paecilomyces (95-10) + - - Paecilomyces (24-2b) - - ++ Paecilomyces (98-2) - - + P. marquandii'(80-14a) + + + Paecilomyces (80-14b) + + - P. marquandii(73-2'\) - - ++ P. marquandii (95-2) + - ++ Verticillium sp. (100-2) - - ++ Verticillium sp. (100-1) - - ++ CHAPTER THREE: Screening of EF for Bioactivity 53 Photoactivated Antibiotics and Antifungal Substances Four crude extracts of E F showed antibiotic activity against Staphylococcus aureus; one extract of Beauveria bassiana inhibited E. coli. The extracts were photoactivated against S. aureus. Paecilomyces marquandii (73-21 and 95-2), Paecilomyces spp (24-2b), and Verticillium sp. (100-1 and 100-2). The crude extract of B. bassiana (B-1) was phototoxic to E. coli after U V exposure. Two dimensional T L C (2D-TLC) overlay bioassay was used to assay for antibacterial, antifungal, and phototoxic activity. The assay was also used as a guide for the fractionation of the crude extract of Paecilomyces marquandii. b Figure 3.2. a) Chromatogram of fraction from an extract of Paecilomyces marquandii, T L C sil ica gel ; b) duplicate plate used to bioassay for photoactivated antibiotics, positive test is indicated by the yellow area. The solvent system used to develop the T L C plate was : Chloroform: M e O H (9:1); the spray reagent was Vanil l in. A duplicate T L C plate was used for overlaid assay (Figure 3.3b), M H agar containing phenol red (MH-PR) was inoculated with S. aureus, poured on top of the T L C plate, allowed to solidify and incubated for 15 min. The replicate plate was irradiated with U V at 350 nm for 30 min, and then incubated for 18 hrs at 26°C. Spray reagent: MTT used to enhance the yellow area corresponding to the zone of phototoxicity. CHAPTER THREE: Screening of EF for Bioactivity 54 The same technique is shown on a two-dimensional T L C (2D-TLC), testing a different Paecilomyces crude extract for antibacterial activity. This technique allowed a fast comparison of different extracts from related strains. Figure 3.3. a) Two-dimensional chromatography of a crude extract of Paecilomyces sp. (strain 84-16a); spray reagent: vanillin, heating at 110°C until colors developed; b) duplicate T L C plate, without spraying, overlaid with HM+PR inoculated with Staphylococcus aureus. After 18-hr incubation at 26°C, plate was spayed with reagent MTT to visual ize the area of inhibition (yellow area = antibacterial activity). Antiviral Tests Five of the tested E F extracts completely inhibited HSV-1 (Table 3.2). Two strains of Paecilomyces spp. showed antiviral activity without causing cytotoxic effects. The crude extracts of two other isolates obtained from Torrubiella raticaudata, and T. mirabilis Samson & Evans showed partial viral inhibition and were not cytotoxic. Complete cytotoxicity was exhibited by Beauveria sp. (B-7), Cordyceps militaris (01-07), Paecilomyces tunuipes (24-2b), Torrubiella sp. (01-06). In the bioassay, the control consisted of Vero cells without the virus (Figure 3.4a). The cells with HSV-1 infection became smaller, round, and aggregated (Figure 3.4b). The effects of the cytotoxicity disrupt the organization of the Vero cells and causes membrane rupture and cell death (Figure 3.4c). CHAPTER THREE: Saeening of EF for Bioactivity 55 Figure 3.4. a) Healthy monkey kidney cells; b) monkey kidney cells showing HSV-1 viral infection, notice the shape of the cells, round and smaller than those in the control; c) monkey kidney cells showing the effects of a cytotoxic crude fungal extract, many kidney cells have collapsed or burst. Readings of the bioassay and photographs were taken after 72 hours of incubation. Table 3.2. Antiviral bioassay. EtOaC extracts from entomogenous fungi were tested against herpes simplex virus (HSV-1). Toxicity test results: T = toxicity, PI = partial inhibition, Cl = complete inhibition, and V = virus alive. FUNGUS Strain 200 pg/ml 100 pg/ml 50 pg/ml 25 pg/ml Beauveria bassiana B-3 V V V V Beauveria bassiana WB V V V V Beauveria bassiana B-1 V V V V Beauveria sp. B-7 T Cl V V Cordyceps militaris 01-07 T V V V Paecilomyces sp. 95-2 Cl PI V V Paecilomyces marquandii 73-21 V V V V Paecilomyces tenuipes 80-14a T Cl Cl V Paecilomyces sp. 85-15 Cl V V V Paecilomyces tenuipes 24-2b T Cl V V Torrubiella sp. 01-01 V V V V Torrubiella sp. 01-06 T V V V Torrubiella mirabilis 01-08 PI V V V Torrubiella raticaudata 01-09 PI V V V Verticillium sp 100-1 V V V V Verticillium sp. Spi 2 V V V V Beauveria bassiana Bet 4 T T PI PI Cordyceps militaris 5298 V V V V Cordyceps japonica 9647 V V V V CHAPTER THREE: Screening of EF for Bioactivity 56 Amino Acid Analysis of Cordyceps sinensis The amino acid (AA) fraction of Cordyceps sinensis sporocarps and mummified caterpillars (extracted separately) were analyzed using an amino acid (AA) analyzer (Beck 3000r), Biotechnology, U B C (see Appendix B). The data from the A A analyzer were used to prepare a two-dimensional T L C (2D-TLC) standard mixture using commercial ly available A A (Sigma Co.) and the separated A A fraction from C. sinensis. Both chromatograms served as references suitable for preliminary comparison of the amino acid content in Cordyceps spp. and related groups. The host caterpillar fraction was slightly different from the fruiting body of Cordyceps sinensis. Al l 18 standard amino acids used were detected by the A A analyzer, those in lower quantities were below 200 pmol, and most of these were not detected by T L C . The most abundant amino acids found in both caterpillars and sporocarps (amino acid analyzer; see Appendix B) were: glutamic ac id, alanine, and proline; followed by valine, threonine, serine, phenylalanine, leucine, lysine, tyrosine, isoleucine, aspartic acid, histidine, arginine, and methionine. The purpose of this analysis was to identify potential chemo-taxonomical markers for C. sinensis. No rare or unique amino acids were detected from this analysis. Table 3.3 lists amino acids detected by T L C chromatography and comparison to true standards amino acids. Table 3.3. R f values for standard amino acids used in 2 D - T L C analysis of Cordyceps sinensis. Solvent system A : Butanol-Water-Acetic Ac id (BWA) in a ratio of 45:20:20; solvent system B: 1- Propanol- N H 4 O H in a ratio of 55:45. Amino Acid R, Value Sol A (BWA) R f Value Sol B DL- Methionine 0.23 0.50 DL-Prol ine 0.13 0.27 D-Serine 0.04 0.26 Glycine 0.20 0.63 L-Alanine 0.01 0.07 L-Arginine 0.06 0.02 L-Glutamine 0.41 0.63 L-Histidine 0.09 0.33 L-Phenylalanine 0.16 0.38 L-Valine 0.27 0.49 CHAPTER THREE: Screening of EF for Bioactivity 57 A chromatogram was created using the same AA fraction analyzed in the AA analyzer (Figure 3.5). This chromatogram is a chemical fingerprint of the AA fraction of C. sinensis and could be compared with other Cordyceps spp. The Rf values were comparable to standard AA samples. 0 0.1 0.2 0.3 0.4 0.5 Rf Value Sol A (BWA) Figure 3.5. Amino acid analysis of Cordyceps sinensis using 2-Dimensional Thin Layer Chromatography. Points indicate position of amino acids on TLC plate. Solvent system: A) Butanol:Water:AcidicAcid (45:20:20); B) Propanol: NH4OH (55:45). Screening for Cyclosporine A TLC and HPLC analyses of selected EF fungal extracts did not reveal the presence of Cyclosporine A. D I S C U S S I O N Anamorphic stages seem to enjoy a higher diversity in morphological expression and changes in basic metabolic pathways. Little information is available on metabolic changes at declining stages of EF cultures. The detection of biologically active compounds also depends on an understanding of metabolic changes occurring over the life span of the fungal culture. CHAPTER THREE: Screening of EF for Bioactivity 58 The E F extracts tested showed activity against four bacteria, one fungus, and one virus. Microorganisms used in the b ioassays were selected for their relevance to human health. There was great variability in the results obtained in the screening. Variability within the same species is a well-known characteristic of fungal strains. One of the chal lenges encountered during the search for antiviral drugs was cytotoxicity. Because viruses are obligatory parasites, the inhibition or elimination of a virus frequently interferes with the host cell chemistry and physiology. Partial inhibition of the viral infection by the E F crude extract may result from low concentrations of the antiviral compound. Cytotoxicity was exhibited by four of the E F species assayed for anti-viral activity. Cytotoxicity has also been previously correlated with antitumor activity, and cytotoxic compounds have been found to be potential anticancer agents. In future research, E F extracts exhibiting cytotoxicity should be tested against cancer cell l ines. I was unable to detect the presence of Cyclospor ine A in crude extracts of E F tested, however this may require further investigation. Other Cyclospor ines may have been present in the extracts, but no standards were available for comparison. The culture medium, growing conditions, and the stage of development at the time of extraction may require further modifications. The composition of the culture medium is perhaps the most important factor in the production of this cyclopeptide. Cyclosporine A has immunosupressor activity in humans and is a useful drug for organ transplants. It is also effective in treating autoimmune disorders such as psoriasis and rheumatoid arthritis (Phillips 1991). Cyclospor ine A was discovered as an antifungal agent produced by the fungus Tolypocladium inflatum G a m s . This fungus was also known by the names Trichoderma polysporum (Link ex Pers.) (Dreyfuss 1976), and Cylyndrocarpon lucidum Booth (Borel 1986). Tolypocladium inflatus was renamed Beauveria nivea, and is considered the anamorph of Cordyceps subsessilis (Hodge et al. 1996) and Cylindrocarpon lucidum (Joug and Gantt 1987). CHAPTER THREE: Screening of EF for Bioactivity 59 Sterol metabolism seems to be highly dependent on the quantity, quality, and diversity of lipids present in the substrate. Many fi lamentous fungi are also capable of bio-transforming endogenous compounds (Hoover 1993), a characteristic used for many years, and rapidly gaining popularity in the fields of biotechnology and industrial mycology. E F may be good candidates for the development of agents of biotransformation, and their biological activity could be improved by collection and screening of wild strains, or by manipulation of establ ished fungal collections. The biological potential of E F remains untapped in the B C coastal temperate rainforests. Future research on E F with biological activities should consider the modification of growing conditions and the determination of nutritional requirements. The production of metabolites of particular interest could be manipulated to enhance their yield. This may also allow E F to behave as agents of biotransformation. CHAPTER FOUR: Evaluation of EF Tolerance to Heavy Metals 60 CHAPTER FOUR: Evaluation of EF Tolerance to Heavy Metals INTRODUCTION Fungi are known to be efficient scavengers that sequester many organic and inorganic molecules. They are capable of survival in most environments and tolerate chemical toxicity, extreme temperature and pH, starvation, and dehydration. Heavy metals (HM) such as cobalt, copper, and nickel serve as micronutrients and are used by fungi and other organisms in redox processes, enzyme functionality, electrostatic interactions, and the regulation of osmotic pressure (Bruins et al. 2000). High levels of H M are toxic to most organisms, but some fi lamentous fungi are tolerant to moderate levels of H M toxicity (Zamani 1985). Many fi lamentous fungi are capable of bio-transforming exogenous compounds (Hoover 1993), a characteristic highly appreciated and rapidly gaining popularity in the fields of biotechnology and industrial mycology. It has been suggested that as an adaptive strategy to H M toxicity, organisms generate small molecules (i.e. phenols, acidic amino acids) in large numbers on the cell surface to limit the uptake of the metals (Suresh and Subramanyan 1998). In addition, in the presence of toxic levels of heavy metals, sequestration of the heavy metals may occur (Mullen et al. 1992). E F have been isolated from soil samples using a selective medium containing copper (Baath 1991), however this medium is not 100% selective and other fungi are recovered as well. H M tolerance mechanisms used by E F remain poorly descr ibed. Metal ion interactions occur mainly in the plasma membrane, which is made up of sterols, phospholipids, glycolipids (Weete 1973), proteins, and carbohydrates (Kendrik 1992). Tox ic concentrations of metal ions can block functional groups in enzymes and transport systems, and displace essential metal ions in molecules and structural components, which may lead to increased membrane permeability and leakage of intracellular solutes (Gadd 1986). Copper is essential for the activity of a number of physiologically important enzymes in many organisms, and is required in trace amounts. In humans, enzyme-related malfunctions may contribute to severe neurological symptoms and neurological d isease. Copper is a co- CHAPTER FOUR: Evaluation of EF Tolerance to Heavy Metals 61 factor of Cu/Zn-superperoxidase-dismutase, which plays a role in the cellular response to oxidative stress by scavenging reactive oxygen species. In excess, Cu++ inhibits sterol biosynthesis of the fungus symbiont in the lichen Bryoria fuscescens (Gyeln.) Brodo & Hawks (Tarhanen etal. 1999). This inhibition leads to potassium (K+) efflux and a reduction in ergosterol concentration (Tarhanen etal. 1999). Ergosterol is an essential lipid involved in growth, and in the regulation of permeability of cell membranes. Consequently, it is essential in the viability and health of fungal cells. Ergosterol is the main constituent and most abundant sterol in the cell membrane of fungi (Weete 1973). In the bi-layer structure of the membrane, ergosterol forms clusters within the phospholipid layers (Nagle and Tristam-Nagle 2000). Some antifungal drugs (e.g., Nystatin) act by binding to ergosterol in the membrane, or by inhibiting various enzymes along the biosynthetic mevalonate pathway leading to ergosterol (Kleinkauf and von Dohren 1988). Cerebrosides, sphingolipids (SPLs), ceramides, and phospholipids are also essential constituents of fungal cell membranes. Free fatty acids, and functional groups such as carboxyls, amines, hydroxyls, and thiols are present in the cell membrane (Gadd 1986). Lanosterol is the precursor of animal sterols and also occurs in fungi. The antifungal compound fluconazole selectively inhibits lanosterol 14 a-dimethylase, a key enzyme in the maintenance of the fungal cell membrane. In Candida albicans, fluconazole was shown to produce a profound depletion of ergosterol with a significant increase in lanosterol content (Pearce 1995). The enzyme, 3-oxidosqualene-lanosterol cyclase (OSC) is involved in the biosynthesis of ergosterol (Jolidon et al. 1997) and could be a target for the development of new antifungal drugs. A substantial amount of research has focussed on fungi as sources of compounds useful in controlling mammalian steroid chemistry (Capek etal. 1996a; Gao etal. 2001a; Nanrj etal. 2001). Some of the most potent and clinically useful compounds as cholesterol metabolism effectors have been derived from fungal products. Two examples are hydroxymethyl coenzyme A reductase (HMG-CoA reductase) from Penicillium brevicompactum, and lovastatin from CHAPTER FOUR: Evaluation of EF Tolerance to Heavy Metals 62 Aspergillus terreus (Alberts et al. 1967). Paecilomyces sp. has been used in steroid biotransformation (Capek et al. 1996) and members of this genus may be potential candidates for the biotransformation of other toxic metabolites and mixtures of toxic waste (e.g., industrial residues). Toxic metals interact with essential cellular components through covalent and ionic bonding (Bruins etal. 2000). Cadmium, lead, and mercury selectively concentrate in certain fungi (Michelot et al. 1999). Some of the metal resistance mechanisms reported in microorganisms include: a) exclusion by permeability barrier, b) intra- and extra-cellular sequestration, c) active transport efflux pumps, d) enzymatic detoxification, and e) reduction in the sensitivity of cellular targets to metal ions (Bruins et al. 2000). E F may use one or more of these mechanisms to deal with H M toxicity. I evaluated the tolerance of E F to H M , and assessed the effects of H M on fungal growth and morphology. I also isolated and characterized a cerebroside with antibacterial activity. This antibiotic was produced when Paecilomyces marquandii was grown on a medium containing copper sulphate. My main interest was to gain understanding of E F responses to heavy metals, in particular changes in biological activities due to the presence of heavy metals in the culture medium. METHODS Preparation of Fungal Cultures A conidial suspension of Paecilomyces marquandii was prepared from 10-day-old petri plate cultures (MYP) . The plates were flooded with a solution of 0 .05% (v/v) Tween 80. A 100- ml sample of the conidial suspension was taken in a dilution series to give a final concentration of 2 x 10 6 spores/ml. One half ml of the final suspension was used to inoculate each Erlenmeyer flask. F lasks contained 500 ml of M Y P broth, and were incubated at 25 + 4°C, with cycles of 12/12 hr L/D using grow lights (Sylvania). Cultures were kept under these conditions for 21 days. They were then harvested, and E toAC extracts were prepared. CHAPTER FOUR: Evaluation of EF Tolerance to Heavy Metals 63 Samples for H P L C analysis were prepared from cultures grown in petri plates. The basic medium used was % strength for all ingredients of M Y P (Bandoni et al. 1970), with the addition of C u 2 S 0 4 . For the preparation of solid media containing H M , the pH was adjusted to 8.5; an alternative was to double the amount of agar in the medium. Concentrat ions of H M were prepared at 100, 200, 300, 400, 500, and 1000 pg/L. Pb , Zn , C o , Fe , and Ni were used to assess tolerance to other H M . The effects of H M on E F growth and morphology were recorded, and some were assessed chemically. Comparison of Fungal Extracts by TLC and HPLC Ethyl acetate extracts from the liquid medium were compared using T L C . The extract was applied to a 10x10-cm sil ica Ge l ( F 2 54) plate and run in C H 2 C I 2 : M e O H (9:1) as the moving phase. Plates were air dried, examined under long and short wave UV, sprayed with vanillin, and heated to 110°C until pigmentation was visible. values were established for major compounds and compared to standard samples of cyclosporin, ergosterol, lanosterol, and ergosterol peroxide. Fractionation of Crude Extracts Twenty-five liters of liquid M Y P containing 400 mg CuS04/ l i ter of medium were inoculated with a spore suspension (prepared as above). Cultures were maintained stationary, at room temperature for 21 days: two weeks in the dark and one week of 12/12 hr L/D using grow lights (Sylvania). The mycelium was then harvested by filtering through Whatman No. 1 filter paper, gently rinsing three times with distilled water, and storing at -4°C overnight. The frozen material was thawed, mixed with sea sand, triturated in a mortar, and extracted with 500 ml of E t o A c . M e O H (2:1 v/v) for five hrs at room temperature. A n additional extraction was performed using 100% EtoAc. Extracts were combined, reduced in volume using a rotary evaporator, and then the residue was extracted three times with equal volumes of E tOAc . The E tOAc extract was CHAPTER FOUR: Evaluation of EF Tolerance to Heavy Metals 64 fractionated by gel filtration using a Si l ica Ge l column (230-400 mesh) and eluted with a gradient of Hexane:Acetone (Figure 4.1). The liquid medium control and filtered spent medium were extracted with non-polar organic solvents (e.g., Hexane and EtOAc) , reduced under pressure, as above, and stored at 4°C. Twenty-five individual fractions were separated from the extract and compared by T L C (Sil ica GF254, solvent system: Cyclohexane:Acetone 50:50). These fractions were assessed for antibacterial, antifungal, and phototoxic activity. Figure 4.1. Fractionation scheme of the crude extract Paecilomyces marquandii. TLC, UV light, and color spot reactions using vanillin, molybdenic acid, and ninhydrin reagents detected different classes of compounds. Further purification of the active fraction 7-9 (Figure 4.1) was performed by using standard 20x20 cm P T L C plates (E. Merck, GF254) coated with a 0.25-mm-thick layer of si l ica. The P T L C plates were developed using a solvent system containing cyclohexane:acetone (50:50) v/v. Fractions obtained from P T L C plates were extracted in chloroform, concentrated under vacuum, examined by T L C sil ica plates GF254, and developed using the above solvent system. Plates were air-dried, examined under short and long wave UV, sprayed with vanillin reagent, and heated in an oven at 110°C until pigmentation developed. Molybdenic acid was also used as a detection reagent. The developed plates were immersed in a 5% methanolic solution of molybdenic acid, followed by heating as above. CHAPTER FOUR: Evaluation of EF Tolerance to Heavy Metals 65 Fractions 20-23 (Figure 4.1) were pooled and further fractionated by P T L C (Sil ica GF254> with cyclohexane:acetone (50-50) as the solvent system) to yield 6.0 mg of compound 1, which gave a reddish-purple color with vanillin reagent, and deep blue with a pale greenish-yellow background with molybdenic acid. Isolated pure compounds were submitted to M S , 1 H - N M R , 1 3 C - N M R , C O S Y ( 1 H- 1 H C O S Y and 1 H - 1 3 C C O S Y ) , and H M B C for structural determination. Data are included in Appendix B, Figure B.4. Fractions 1-3 (Figure 4.1) yielded an unidentified fatty acid; fractions 5-6 contained ergosterol and lanosterol; fractions 7-9 yielded an antibiotic and several pigments; fractions 10- 15 contained a combination of unidentified compounds. R E S U L T S Effects of Heavy Metals on Entomogenous Fungi Fungal Growth and Morphology A decrease in growth and changes in pigment coloration were observed in most E F species. A Paecilomyces marquandii control plate showed yellow mycel ia, a color intensifying with age (Figure 4.2). This pigmentation changed to reddish-orange in the medium containing H M . The reddish-orange pigmentation appearing in the underside of the mycel ia mat (Figure 4.2, Lower surface B) was produced in response to H M presence. Colonies of P. marquandii growing in solid and liquid M Y P - F e medium produced an orange pigment on the lower surface of the colony. Some pigments reacted positively to the reagent 1,1-Diphenyl-2picrylhydrazine ( D P P H ; a free radical). CHAPTER FOUR: Evaluation of EF Tolerance to Heavy Metals 66 H M caused a general reduction in E F growth (Figure 4.3) and biomass (Figure 4.4). The effect of pH and the addition of copper and iron to the medium is illustrated in Figure 4.4. Growth under copper conditions was assessed for C. militaris. Growth was reduced by 5 0 % in HM treatments. Pigmentation decreased in the presence of copper and iron at pH 9.0, but not in the presence of iron pH 7.0 (Figure 4.5). B C D Figure 4.3. Paecilomyces marquandii growing in media containing iron (B) and copper (D). A and C are controls. CHAPTER FOUR: Evaluation of EF Tolerance to Heavy Metals 67 Fe++ pH 7.0 SAB Cu++ pH 9.0 SAB Fe++ pH 9.0 SAB Control pH 7.0 SAB Figure 4.4. Influence of iron and copper at different pH values on Cordyceps militaris growth rate. Growth was measured in centimeters. Error bars indicate 2 standard errors of the mean. Figure 4.5. Growth of Cordyceps militaris 01-07 on media containing copper at A) pH 9.0, B) iron at pH 7.0, and C) pH 9.0. Heavy metal concentration: 500 pg/L of medium (MYP). Cultures were 6 days old. e ro D) C 13 LL C ro <u 2 21 1 1 CHAPTER FOUR: Evaluation of EF Tolerance to Heavy Metals 68 Figure 4.6 illustrates the T L C of the chloroform fraction of crude extract samples of Cordyceps militaris (01-07- pH : 7.0; 01-07-pH:9.0; and 01-07-pH 6.0-control), Cordyceps japonica 9647, Media (MYP) , and control. The solvent system used for T L C was Ch loro fornrMeOH 9:1. Figure 4.7 illustrates a T L C of EtoAc extracts of selected E F . Figure 4.6. T L C showing the accumulation of intermediate or complex metabolites produced by E F growing in an iron enriched medium, 7-7, 7-9, and 7-C correspond to Cordyceps militaris (01-07) growing on iron media at pH 7.0 and pH 9.0; Cordyceps japonica 9647; and M corresponds to the medium (MYP) ; and 8 corresponds to an isolate of Torrubiella sp. Spray reagent: Vanil l in-sulphuric acid and heating. Note the blue spot at the bottom of chromatogram; sample 7-7 growing under iron conditions at pH 7.0. The spot is absent at pH 9.0. A 9 I B , , , , — _ H f. Al WB Spa 100,1 M47 U n Erg Figure 4.7. Thin layer chromatogram of E t O a C extracts of selected entomogenous fungi. Lanosterol (A) and ergosterol (B) standards are shown. CHAPTER FOUR: Evaluation of EF Tolerance to Heavy Metals 69 Fungal Tolerance to Heavy Metals Tolerance to H M varied among E F strains of the same fungus, and among genera (Table 4.1). Sterol content and pigmentation also varied. The most toxic H M to E F was copper, followed by iron, nickel, lead and cobalt. Many isolated strains of Paecilomyces were tolerant to copper and other H M (Table 4.1). A strain of Paecilomyces spp. 85-15a was capable of growing in a medium containing 1g Cu++/L. Table 4 .1 . Selected entomogenous fungi growing in media containing heavy metals. Concentration was 500 pg/L of M Y P medium. Y = growth under the particular metal. F u n g u s / H e a v y M e t a l C o p p e r Z i n c L e a d N i c k e l Paecilomyces marquandii (73-21) Y Y Y Paecilomyces sp. (85-14) Y Y Y Paecilomyces sp. (92-4) Y Y Paecilomyces sp. (95-2) Y Y Paecilomyces marquandii (85-15) Y Y Y Paecilomyces sp. (98-2) Y Y Beauveria bassiana (5711) Y Cordyceps ophioglosiodes (8992) Y Y Cordyceps militaris (0107) Y Y Y Cordyceps japonica (9647) Y Beauveria bassiana (8554) Y Y E F growing on M Y P medium containing H M are shown in Figure 4.8. Three strains of Paecilomyces showed similar tolerance to lead (Figure 4.8A). Notice the effect of copper in the formation of concentric rings in Paecilomyces sp. 98-2 (Figure 4 .8B, lower right plate). Epichloe' festuca was tolerant to H M toxicity, but its growth was repressed. CHAPTER FOUR: Evaluation of EF Tolerance to Heavy Metals 70 Figure 4.8. Entomogenous fungi growing on M Y P medium containing heavy metals. A) Paecilomyces tunuipes, B) Paecilomyces sp., C) A member of the Clavicipitaceae, Epichloe' festuca, a relative of Cordyceps sp., is symbiotic in temperate grasses. CHAPTER FOUR: Evaluation of EF Tolerance to Heavy Metals 71 Paecilomyces marquandii was tolerant to all H M tested. The presence of Cu++ in the media resulted in a reduction of ergosterol production. T L C showed the accumulation or overproduction of different compounds. Results of a cerebroside isolated from P. marquandii are reported in the following section. Two other antibiotic compounds were detected during the extract analysis of P. marquandii, however the amounts isolated were too small to fully characterize these compounds. Bioactive Compounds Produced by EF Growing under HM Conditions From the initial screening, the isolate Paecilomyces marquandii (73-21) was selected for its fast growth rate and resistance to copper toxicity. The biomass produced by this strain under copper conditions allowed for the characterization of a cerebroside. Isolation of a Cerebroside I isolated the cerebroside 4E-8E)-N-2-hydroxyhexadecanoyl-1-0-p-glucanopyranosyl-9- methyl-C 1 8 -sphinga-4,8-diene (Figure 4.9), which was produced by Paecilomyces marquandii when growing in M Y P broth containing copper sulphate. This cerebroside may be produced by P. marquandii in the absence of copper, but in such small quantities, it was not detected. Under copper conditions, the cerebroside became one of the most abundant compounds in the chloroform fraction of the crude extract. o Figure 4.9. Chemica l structure of compound 1: (4E,8E)-N-2-hydroxyhexadecanoyl-1-0- P-glucanopyranosyl-9-methyl-C 1 8-sphinga-4,8-diene, a cerebroside. CHAPTER FOUR: Evaluation of EF Tolerance to Heavy Metals 72 The cerebroside (Figure 4.9) is an amorphous powder. E IMS m/z: 709 ( M + - H 2 0 , 3), 576 (6), 547 (9), 531 (5), 516 (11), 500(3), 474 (3), 438 (2), 368 (20), 296 (20), 276 (30), 262 (15), 225 (8), 180 (10), 97 (60), 95 (40), 83 (60), 81 (35), 69 (85), 57 (60), 55 (100); F A B M S m/z 750 (M ++Na), 728 (M ++H), 710 ( M H - H 2 0 ) ; 1 H - N M R (400MHz, C D 3 O D ) : 8 3.70 (1H, m, H-1a), 4.11 (1H, m, H-1b), 3.97 (1H, m, H-2), 4.12 (1H, m, H-3), 5.49 (1H, m, H-4), 5.75 (1H, m, H-5), 2.05 (2H, m, H-6), 2.04 (2H, m, H-7), 5.14 (1H, m, H-8), 1.98 (2H, t, J 7.3, H-10), 1.41 (2H, m, H-11), 1.25 (12H, m, H-12 to H-17), 0.89 (3H, f, J7.0, H-18), 1.61 (3H, s), 4.01 ( 1 H , m , H-2'), 1.54 (1H, m, H-3'a), 1.69 (1H, m, H-3'b), 1.40 (2H, m, H-4'), 1.20-1.35 (22H, m, H-5' to H-15'), 0.89 (3H, f, J 7.0, H-16'), 4.26 (1H, d, J , 7.8, H-2"), 3.18 (1H, dd, J 7.8, 9.1, H-3"), 3.26 (1H, m, H-4"), 3.25 (1H, m, H-5"), 3.83 (1H, dd, J 12.0, 1.8, H-6"a), 3.63 (1H, dd, J 12.0, 4.3, H - 6 " b ) ; 1 3 C - N M R (100MHz, C D 3 O D ) : 8 69.7 (C-1), 54.6 (C-2), 72.9 (C-3), 131.1 (C-4), 134.7 (C-5), 35.9 (C-6), 28.7 (C-7), 124.8 (C-8), 136.8 (C-9), 40.8 (C-10), 29.1 (C-11), 23.7-33.1 (6C, C-12 to C-17), 14.5 (C-18), 16.1 (C-19), 177.2 (C-1'), 73.1 (C-2'), 35.9 (C-3'), 26.2 (C-4'), 30.2-30.8 (11C, C -5 ' to C-15') , 14.5 (C-16'), 104.7 (C-1") , 75.0 (C-2") , 77.9 (C-3") , 78.0 (C-4") , 71.6 (C-5") , 62.7 (C- 6"). Ergosterol and lanosterol were also isolated and their identities confirmed by M S and comparison with pure standards (Appendix B, Figure B.4). Included are: a) low resolution F A B Mass Spectrum, b) 1 3 C - N M R Spectrum, c) 1 H - 1 H C O S Y Spectrum, and d) 1 H - N M R Spectrum. Antimicrobial Activity of a Cerebroside The cerebroside (4E,8E)-N-2-hydroxyhexadecanoyl-1-0-p-glucanopyranosyl-9-methyl- C 1 8 -sphinga-4,8-d iene, was identified as one of the main components of the crude fungal extract. T L C of the cerebroside illustrates antibacterial activity against a methicillin resistant strain of Staphylococcus aureus (Figure 4.10). No activity was found in phototoxicity, antiviral, and antifungal b ioassays. CHAPTER FOUR: Evaluation of EF Tolerance to Heavy Metals 73 Figure 4.10. Thin Layer Chromatography illustrating Compound 1: (4E,8E)-N-2- hydroxyhexadecanoyl-1-0-p-glucanopyranosyl-9-methyl-C18-sphinga-4,8-diene. Solvent system: Cyclohexane: acetone 50:50 (v/v). A). Compound 1 spotted twice on plates and gave an intense blue color reaction with molybdenic acid. B) Replicate plate, overlaid antibacterial bioassay; organism: Staphylococcus aureus. DISCUSSION Effects of HM on EF Growth, Morphology, and Tolerance The production of free radical scavengers may contribute to HM tolerance. This suggests that pigments may interact with HM as antioxidants, binding to HM as chelator molecules. Pigments produced by P. marquandii may act as antioxidants or chelating agents to reduce copper toxicity. TLC and HPLC analysis suggests that copper toxicity affects ergosterol and lanosterol production. Copper may also contribute to the accumulation of intermediates and/or de novo production of sterols, sphingolipids, ceramides, phospholipids, and fatty acids. Morphological and biochemical effects of HM were demonstrated in my preliminary experiments. In general, anamorph strains were more tolerant to HM than teleomorph strains (i.e. Osaka strains). HM affected EF in different ways. Zinc may enhance pigment and conidia production, leading to restricted growth of Cordyceps spp. and Beauveria spp., more so than Paecilomyces spp. HPLC analyses of in vivo copper induction suggest that yellow pigments and ergosterol in Paecilomyces sp. and Cordyceps militaris may play a role in the formation of HM-complexes. Cordyceps spp. are less tolerant to the HM tested, but more analyses are CHAPTER FOUR: Evaluation of EF Tolerance to Heavy Metals 74 needed to provide an explanation for this response. Some spec ies of E F showed resistance or tolerance to high concentrations of heavy metals, however the mechanisms of metal resistance have not been elucidated for this group. Fi lamentous fungi may play a role in the in situ biodegradation of aliphatic pollutants in the soil (April et al. 2000). I found that many species of E F exhibited resistance or tolerance to copper, iron, nickel, iron, lead, and cobalt. In other organisms, toxic concentrations of these metal ions can block functional groups in enzymes and transport systems, and displace essential metal ions in molecules and structural cell components (Gadd 1993). This leads to increased membrane permeability and leakage of intracellular solutes. E F growth rate was affected by the presence of H M in the culture media, but this may not be what happens in nature. T L C of crude extracts suggests that copper affects ergosterol production, and possibly contributes to the accumulation of intermediates and/or to the de novo production of other lipids. Steroid transformation in vitro has been shown to be a feature of some species of Paecilomyces (Capek et al. 1976). It is reasonable to assume that one mechanism of tolerance used by Paecilomyces sp. is the sequestration of metal ions (e.g., copper). Neurospora crassa immobil izes copper by binding it to phenols present in the mycelial cell wall. The accumulation of copper in the cell wall gives the mycel ia its bluish color (Lerch 1991). Metallothioneins are a family of important metal binding proteins involved in the handling of trace metals, including H M . A gene coding metallothioneins in Neurospora crassa has been reported (Muenguer and Lerch 1987) and expressed in Escherichia coli. The recombinant E. coli producing the metallothionein protein was able to accumulate large amounts of cadmium, suggesting a potential use for metallothionein-based bioabsorbent for certain H M removal applications (Pazirandeh et al. 1995). This technique could be used in the removal of H M from aqueous media (Pazirandeh and Campbel l 1998) and some E F may be good candidates for bioremediation research and applications. CHAPTER FOUR: Evaluation of EF Tolerance to Heavy Metals 75 Nickel toxicity has been linked to carcinogenicity and multiple types of cellular damage, which ultimately results in altered gene expression, rather than indirect D N A damage (Zoroddu et al. 2001). Iron is an essential trace element, abundant in nature, and involved in many metabolic and anabolic processes. Iron participates in diverse pathological processes by catalyzing the formation of reactive oxygen free radicals (Chau 2000). In addition, microorganisms excrete a variety of high affinity, low molecular-weight (500-1500) iron sequestering agents called siderophores (Wong 1983; Jalal etal. 1984). Fungal siderophores include ferrichromes, coprogens (Wong 1989), and triacetylfusarinine C (Konetschny-Rapp et al. 1988). A new c lass of siderophores, rhizopherrin, which employs neither hydroxamate nor catecholate groups, has been isolated in the zygomycetes (Drechsel etal. 1991, 1992). Bioactive Compounds Produced by EF under HM Conditions The cerebroside, 4E-8E)-N-2-hydroxyhexadecanoyl-1-0-p-glucanopyranosyl-9-methyl- C 1 8 -sphinga-4,8-d iene, was originally discovered from the imperfect fungus, Fusicossum amygdali (Ballio et al. 1979). Other sources have been described in the last two decades, including Polyporus ellisii (Gao ef al. 2001) and Termitomycetes albuminosus (Berk.) Heim. (Qi etal. 2001). Recently two cerebrosides with antifungal activity were reported from Russula achroleuca (Gao er al. 2001). Two ceramides with C 1 8 phytosphingosides have been isolated from R. cyanoxantha (Gao ef al. 2001) and Armillaria mellea (Gao et al. 2001). A new glucocerebroside was recently described from the mushroom Polyporus ellissi Berk (Gao ef al. 2001). Conclusive evidence has been shown that the ceramide hydroxyl groups are involved in l inkages with proteins (Steward ef al. 2001). The hydroxyl groups of sugar could possibly participate in ester l inkages. The main compound isolated from P. marquandii was identified as (4E-8E)-N-2- hydroxyhexadecanoyl-1-0-p-glucanopyranosyl-9-methyl-C 1 8 -sphinga-4,8-diene. Further research is required to elucidate the changes occurring in fungal metabolism and membrane CHAPTER FOUR: Evaluation of EF Tolerance to Heavy Metals 76 rearrangement under H M stress. The increase or decrease in the production of steroids in response to H M may be a fungal defence against stress and/or a toxicity tolerance mechanism. Studies of dry mycelia have shown that certain fungi have a large sorption capacity (SC) . For example, Aspergillus terreus dry mycelium has a high S C of 224 pg/g dry wt, and the mycel ia become blue with increased copper in the medium (Gulati 1999). Large amounts of b iomass waste results from some fermentation processes using Aspergillus spp., Penicillium spp. and other fi lamentous fungi. The mycel ia by-products are not utilized and are generally d isposed of as garbage. Some species of Paecilomyces may be good candidates for dry mycelia removal of H M as they showed fast growth rate and high b iomass production under H M conditions, and they can be grown in different substrates at low cost. Detection of larger amounts of the cerebroside may indicate changes in lipid biosynthesis. Mycel ia color changed from yellow to a greenish-yellow suggesting copper deposition in the cell wall of P. marquandii. In liquid medium, P. marquandii developed an orange-brownish ring on the lower side of the colony. These pigments may be acting as chelating or antioxidant agents to counter balance H M presence. The modification of sterols by free radical scavengers is suggested as a mechanism of biotransformation or immobilization of H M by E F . Many microorganisms are known to produce compounds with surface-active characteristics generally termed "biosurfactants." These are amphil ic molecules containing both lipids and hydrophobic moiety and include glycosphingolipids and gangliosides (Isoda etal. 1997). H M may interact with a variety of l igands such as carboxyl, phosphate, and amino groups. Under different pH levels several complexes of copper, iron, nickel and other H M may form. Changes in the structural arrangement of the lipid bilayer of membranes can influence the functional properties of membrane bound and peripheral proteins. These play an important role in morphogenesis and normal t issue remodelling through their interactions with the cytoskeleton (Baka 2000). CHAPTER FOUR: Evaluation of EF Tolerance to Heavy Metals 77 Reduced fungal growth observed under copper treatment could be the result of changes in the cell membrane's lipid composition and arrangement. The characterization of mixtures and single lipids may answer some of the questions of lipid bilayers in membranes. 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Journal of Inorganic Chemistry 85:47-54. Zukowski , K., and Bajan, C . (2001). Laboratory determination of the activity of insecticidal fungus Paecilomyces farinosus in reducing the numbers of cockroaches Blattella germanica L. Rocz Pans twZak l Hig. 48(2):133-8. APPENDICES 99 A P P E N D I C E S Appendix A Table A.1 List of entomogenous fungi collected in British Columbia. Table A.2 Media used in different experiments for culturing entomogenous fungi. Table A.3 Reported biological activities and other characteristics of Paecilomyces marquandii. APPENDICES 100 Table A.1. List of entomogenous fungi collected in British Columbia. Collection No. Host/Substrate Collection Date Place Paecilomyces marquandii 73-21 beetle Feb 98 UBC Paecilomyces sp. 73-22 beetle Feb 98 UBC Paecilomyces sp. 73-23 White fly March 98 UBC Verticillium sp. 80-2 Spider June 98 Squamish Beauveria bassiana 80-3 Beetle parts June 98 Squamish Beauveria bassiana 80-4 Beetle June 98 Squamish Torrubiella sp. 80-15 Spider June 98 Cypress Bowl Paecilomyces sp. 81-13 Beetle larvae August 98 Capilano GVRD Beauveria bassiana 81-14 Adult beetle August 98 Capilano GVRD Paecilomyces sp. 84-2 Beetle October 98 Capilano GVRD Paecilomyces marquandii 85-14 Caterpillar October 98 Capilano GVRD Torrubiella sp. 85-13 Spider October 98 Capilano GVRD Beauveria bassiana 91-20 CWD smear February 99 Ladner Beauveria bassiana 91-21 CWD smear February 99 Ladner Paecilomyces inflatus 94-1 Small larvae April 99 UBC Paecilomyces marquandii 95-2 Beetle larvae May 99 Capilano GVR Paecilomyces sp. 96-10 Beetle larvae May 99 UBC EpichlGe festuca 96-12 Grass June 99 UBC Gibellula sp. 96-21 Spider June 99 Squamish Paecilomyces sp. 98-2 Beetle larvae July 99 UBC Gibellula sp. 98-3 Spider July 99 UBC Beauveria bassiana 98-15 Beetle larvae • July 99 Squamish Paecilomyces marquandii 98-18 Beetle larvae July 99 Squamish Gibellula pulchra 100-1 Spider Oct 99 Vancouver Island APPENDICES 101 Table A .2 . Media used in different experiments for culturing entomogenous fungi. Malt extract-Yeast extract- Peptone (MYP): Malt extract Yeast extract Peptone Water 14.00 g 0.25 g 0.5 g 1000 ml Dissolve the solids in water, add the agar and autoclave the medium for 15 min at 121°C. Note: 1/4 of the malt in the full recipe for M Y P (Bandoni 1972) media was used for the initial isolation. Tetracycline was added at a rate of 1pg/l to suppress bacterial growth. Dissolve the malt extract in water; add the agar and autoclave the medium for 15 min at 121°C. Potato Dextrose Agar (PDA): Dissolve 36 grams of Difco P D A (pre-mixed) in 1000 ml of water and autoclave the medium for 15 min at 121°C. Oatmeal Agar: Rolled oats 30 g Agar 1 5 g Water 1000 ml Cook oatmeal in water for 15-30 min in a container over boiling water (double boiler). Filter through three or four layers of cheesecloth and bring filtrate to one liter of water. Autoclave for 15 min. at 121°C. Czapek (Dox) Agar: Sodium nitrate (Na N 0 3 ) 20 g Potassium phosphate ( K 2 H P 0 4 ) 1.0 g Potassium chloride (KCI) 0.5 g Magnesium sulphate ( M g S 0 4 . 7 H 2 0 ) 0.5 g Ferrous sulphate ( F e S 0 4 ) 0.01 g Sucrose 30.0 g Water 1000.0 ml Autoclave for 15 min. at 121°C. Malt Agar: Malt extract Agar Water 20 g 1 5 g 1000 ml A P P E N D I C E S 102 Table A .2 . Media used in different experiments for culturing entomogenous fungi, [continued] Water Agar (Plain Agar, Non-nutrient Agar): Agar(Di fco) 15 g Distilled water 1000 ml Steril ize for 15 min at 121°C. Peanut and Walnut Media: Twenty grams of peanuts and/or walnuts were placed in g lass Petri d ishes containing 0.5 ml of water. The d ishes were autoclaved for 15 min at 121°C. Two-three pieces, 5 g, of sterile peanuts and/or walnuts were placed on the surface of previously prepared water agar plates. The plates were sealed with plastic wrap to allow exchange of gasses , placed in open plastic bags, and refrigerated at 4°C until further use. Phenol Red Media (Over-laid Plate Bioassay): The following amounts have been estimated for a petri plate: Bacterial Media Muller Hinton Broth (powder) 0.42 g Agar 0.12 g Phenol red 0.8 ml D H 2 0 19.0 ml Fungi Media S A B broth (powder) 0.72 g Agar 0.12 9 Phenol red 0.8 ml D H 2 0 19.0 ml The phenol stock solution used in the media was prepared to a concentration of 0.5 pg/ml. The amount of medium in the plate can be reduced to 10-15 ml. Stains Used: Phloxine Staining Solution Phloxine was prepared as a 2% aqueous.solut ion. KOH Staining Solution 5% aqueous solution Lactophenol Cotton Blue Phenol crystals 20 g Lactic Acid 20 ml Glycerol 40 ml Distilled water 20 ml Dissolved by heating gently under a hot water tap. Add 0.05 g. of cotton blue. APPENDICES 103 Table A . 3 . Reported biological activities and other characteristics of Paecilomyces marquandii. Reported biological activities and other characteristics Paecilomyces marquandii Literature cited Ovicidal and ovistatic Basua ldo et al. 2000 Human pathogen in patients with compromised immune systems Naldi etal. 2000 Nematicidal activity C h e et al. 1999 Nematicidal activity Esnard et al. 1999 Highly resistant to f luconazole and flucytosine Guarro 1998 Nematicidal activity Chen et al. 1996 Hyalohyphomycot ic agents Sekhon et al. 1996 Nonapept ide antibiotic, cytotoxic, and phytotoxic Dosio etal. 1994 Nematicidal activity Marban etal. 1992 Keratinolytic, keratinophilic Filipello etal. 1991 Nematicidal activity Walter etal. 1990 Resistant to amphotericin B and 5-fluorocytosine, but sensit ive to imidazoles Castro etal. 1990 Resistant to amphotericin B and 5-fluorocytosine, but sensit ive to imidazoles Maslem et al. 1988 Mycosis in fish Lightner et al. 1988 Pept ide antibiotic Radios etal. 1987 Pept ide antibiotic Ross i et al. 1987 Member of the fungal winter community during the colder periods of the year. Widden 1986 Mycoparasi te V a n Der A a 1986 Ovicidal activity Lysek 1985 APPENDICES 104 Appendix B Figure B.1 D N A sequences of entomogenous fungi producing significant alignments using GenBank: a) Verticillium sp. 100-1, b) Paecilomyces tenuipes 24-2b, c) Beauveria bassiana Bet-1, d) Cordyceps militaris 01-07, e) Paecilomyces sp. 85- 15, f) Paecilomyces sp. 85-2, g) Paecilomyces marquandii 73-21. The sequences were compared using blast analysis against Neurospora crassa genome (Whitehead Institute) other analysis still pending. Figure B.2 Chromatograms of free fatty acids from selected entomogenous fungi analyzed by gas chromatography. Vial number as indicated in chromatogram report: 1 = Beauvan'a bassiana Bet-3; 2 = Paecilomyces sp. 85-14; 3 = Verticillium sp. 100- 1; 4 = Paecilomyces sp. 95-2; 5 = Cordyceps militaris 01-07; 6 = Paecilomyces tenuipes 24-2b; 7 = Paecilomyces marquandii 73-21; 11 = solvent control; 12 = fatty acid standards Figure B.3 Chromatograms of Amino acid analysis from sporocarps of Cordyceps cyanensis. Included are: Chromatogram Report, Mol Percent Report, and Typical Amino Acid Analys is Results (Hydrolysis Test Peptide). Figure B.4 Physicochemical data used to determine the structure of the cerebroside (4E,8E)-N-2-hydroxyhexadecanoyl-1-0-p-glucanopyranosyl-9-methyl-C 1 8 - sphinga-4,8-diene. Included are: a) low resolution F A B Mass Spectrum, b) 1 3 C - N M R Spectrum, c) 1 H - 1 H C O S Y Spectrum, d) 1 H - N M R Spectrum. APPENDICES 105 Figure B.1. D N A sequences of entomogenous fungi producing significant alignments using GenBank: a) Cordyceps militaris 01-07, b)Verticillium sp. 100-1, c) Paecilomyces tenuipes 24- 2b, d) Paecilomyces marquandii 73-21, e) Paecilomyces sp. 85-15, f) Paecilomyces sp. 85-2, and g) Beauveria bassiana Beet-1. The sequences were compared using blast analysis against Neurospora crassa genome (Whitehead Institute). 106 Partial nucleotide sequence of 18s rDNA of Cordyceps militaris (01-07). GTAGTCATATGCTTGTCTCAAAGATTAAGCCATGCATGTCTAAGTATAAGCAATTATACAGC GAAACTGCGAATGGCTCATTATATAAGTTATCGTTTATTTGATAGTACCTTACTACTTGGATA ACCGTGGTAATTCTAGAGCTAATACATGCTAAAAATCCCGACTTCGGAAGGGATGTATTTAT TAGATTAAAAACCAATGCCCTCTGGGCTCCTTGGTGATTCATGATAACTCTTCGAATCGCAC GGCCTTGCGCCGGCGATGGTTCATTCAAATTTCTTCCCTATCAACTTTCGATGTTTGGGTAT T G G C C A A A C A T G G T T G C A A C G G G T A A C G G A G G G T T A G G G C T C G A C C C C G G A G A A G G A G C C T G A G A A A C G G C T A C T A C A T C C A A G G A A G G C A G C A G G C G C G C A A A T T A C C C A A T C C C G A T TCGGGGAGGTAGTGACAATAAATACTGATACAGGGCTCTTTTGGGTCTTGTAATTGGAATG AGTACAATTTAAATCCCTTAACGAGGAACAATTGGAGGGCAAGTCTGGTGCCAGCAGCCG CGGTAATTCCAGCTCCATAGCGTATATTAAAGTTGTTGTGGTTAAAAAGCTCGTAGTTGAAC C T T G G G C C T G G C T G G C C G G T C C G C C T C A C C G C G T G C A C T G G T C C G G C C G G G C C T T T C C C TCTGTGGAACCCCATGCCCTTCACTGGGCGTGGCGGGGAAACAGGACTTTTACTTTGAAA AAATTAGAGTGCTCCAGGCAGGCCTATGCTCGAATACATTAGCATGGAATAATGAAATAGG ACGCGCGGTTCTATTTTGTTGGTTTCTAGGACCGCCGTAATGATTAATAGGGACAGTCGGG GGCATCAGTATTCAACGGTCAGAGGTGAAATTCTTGAATTCCTTGAAGACTAACTACTGCG AAAGCATTCGCCAAGGATGTTTTCATTAATCAGGAACGAAAGTTAGGGGATCGAAGACGAT CAGATACCGTCGTAGTCTTAACCATAAACTATGCCGACTAGGGATCGGACGATGTTATTTTT TGACGCGTTCGGCACCTTACGAGAAATCAAAGTGCTTGGGCTCCAGGGGGAGTATGGTCG CAAGGCTGAAACTTAAAGAAATTGACGGAAGGGCACCACCAGGGGTAAACTCTATATGCA GCCGCAGTAGCTCTGCTCCGAAAAGCAGCCTGAAAGGGTTAATGGTGTTCCTGACCGCCG CCCGGCGGTCGAATAATTGGTAGTCTCTTCGGAGGCGACACCCTCAAGTTGCGGGAACGG C A C G T G C G A A C G T A C G T G T G C C T T T A G A G C T G G C G C T A C C A A G C A G G C G T G G A A A G C G C GTCTGCGGCCGGGGTAATGACCTAGGGTATGGTAAAAACCCGTCAGATTAGGCAATCCGC A T C C A A G C C C C G T C G C C G C A A G G C A C G G G G A A G G A T C A G A G A C T T G A C G G G G G T G G G T A G C G G C G C A C G C T G C C T A A G A T A A A G T C C G A C T G C T C G C G A A A G C G T G T C A G A T A G G T A A C CTATAATCGGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCGCCAGGTCCAGA CACAATGAGGATTGACAGATTGAGAGCTCCTTCTTGATTTTGTGGGTGGTGGTGCATGGCC GTTTTTAGTTGGTGGAGTGATTTGTCTGCTTAATTGCGATAACGAACGAGACCTTGACCTG CTAAATAGCCCGTATTGCTTTGGCAGTACGCTGGCTTCTTAGAGGGACCATCGGTGCAATC CGAAGGAAGTTCGAGGCAAAAACAGGTCTGTAATGCCCTTAGATGTTCTGGGCCGCACGC GCGCTACACTGACGGAGCCAGCGAGTTCTTCCTTGTCCGAAAGGTCCGGGTAATCTTGTT AAACTCCGTCGTGCTGGGGATAGAGCATTGCAATTATTGCTCTTCAACGAGGAATCCCTAG TAAGCGCAAGTCATCTGCTTGCGTTGATTACGTCCCTGCCCTTTGTACACACCGCCCGTCG C T A C T A C C G A T T G A A T G G C T C A G T G A G G C G T C C G G A C T G G C C C A G G G A G G T G G G C A A C T ACCACCCAGGGCCGGAAAGCTCTCCAAACTCGGTCATTTAGAGGAAGTAAAAGTCGTAAC AAGGTCTCCGTAGGTGAACCTGC. Table B.1a. Accession number and score for the 10 most similar EF deduced from partial sequence of Cordyceps militaris (01-07), using BLAST search of Genebank sequences. Rank Accession Number Organism Probability, Score (bits) 1 AB027334 Paecilomyces tenuipes 2192 2 AB044630 Cordyceps sp. 97005 2165 3 AB044629 Cordyceps pruinosa 2151 4 AB027335 Beauveria brongniartii 2141 5 AF280633 Paecilomyces tenuipes 2137 6 D85136 Cordyceps militaris 2135 7 AB027333 Cordyceps takaomontana 2123 8 AB044631 Claviceps africana 2111 9 AF281132 Cordyceps sp. 97009 2097 10 AB032475 Paecilomyces fumoso-roseus 2089 107 Partial nucleotide sequence of 18s rDNA of Verticillium sp. (100-1). GT AGTC ATATG CTTGTCTC AAAG ATT AAG C C ATG C ATGTCT AAGTAT AAG C AATT AT AC AG C GAAGACTGCGAATGGCTCATTATATAAGTTACCGTTTATTTGATAGTACCTTACTACTTGGA TAACCGTGGTAATTCTAGAGCTAATACATGCTAAAAATCCCGACTTCGGAAGGGATGTATTT ATTAGATTAAAAACCAATGCCCTCTGGGCTCCTTGGTGATTCATAATAACTTTTCGAATCGC ATGGCCTTGCGCCGGCGATGGTTCATTCAAATTTCTTCCCTATCAACTTTCGATGTTGGGTA T T G G C C A A A C A T G G T C G C A A C G G G T A A C G G A G G G T T A G G G C T C G A C C C C G G A G A A G G A G C C T G A G A A A C G G C T A C T A C A T C C A A G G A A G G C A G C A G G C G C G C A A A T T A C C C A A T C C C G A TTCGGGGAGGTAGTGACAATAAATACTGATACAGGGCTCTTTTGGGTCTTGTAATTGGAAT GAGTACAATTTAAATCCCTTAACGAGGAACAATTGGAGGGCAAGTCTGGTGCCAGCAGCC GCGGTAATTCCAGCTCCATAGCGTATATAAAGTTGTTGTGGTTAAAAAGCTCGTAGTTGAAC C T T G G G C C T G G C T G G C C G G T C C G C C T C A C C G C G T G T A C T G G T C C G G C C G G G C C T T T C C C TCTGTGGAACCTCATGCCCTTCACTGGGTGTGGCGGGGAAACAGGACTTTTACTTTGAAAA AATTAGAGTGCTCCAGGCAGGCCTATGCTCGAATACATTAGCATGGAATAATGAAATAGGA CGTGTGGTTCTATTTTGTTGGTTTCTAGGACCGCCGTTATGATTAATAGGGACAGTCGGGG GCATCAGTATTCAATTGTCAGAGGTGAAATTCTTGGATTTATTGAAGACTAACTACTGCGAA AGCATTTGCCAAGGATGTTTTCATTAATCAGGAACGAAAGTTAGGGGATCGAAGACGATCA GATACCGTCGTAGTCTTAACCATAAACTATGCCGACTAGGGATCGGACGATGTTATTTTTTG ACGCGTTCGGCACCTTACGAGAAATCAAAGTGCTTGGGCTCCAGGGGGAGTATGGTCGCA A G G C T G A A A C T T A A A G A A A T T G A C G G A A G G G C A C C A C C A G G G G T G G A G C C T G C G G C T T A A TTTGACTCAACACGGGGAAACTCACCAGGTCCAGACACAATGAGGATTGACAGATTGAGA GCTCTTTCTTGATTTTGTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGT CTGCTTAATTGCGATAACGAACGAGACCTTAACCTGCTAAATAGCCCGTATTGCTTTGGCA GTACGCCGGCTTCTTAGAGGGACTATCGGCTCAAGCCGATGGAAGTTTGAGGCAATAACA G G T C T G T A A T G C C C T T A G A T G T T C T G G G C C G C A C G C G C G C T A C A C T G A C G G A G C C A G C G A GTACTTCCTTGTCCGAAAGGCCCGGGTAATCTTGTTAAACTCCGTCGTGCTGGGGATAGAG CATTGCAATTATTGCTCTTCAACGAGGAATCCCTAGTAAGCGCAAGTCATCTGCTTGCGTT GATTACGTCCCTGCCCTTTGTACACACCGCCCGTCGCTACTACCGATTGAATGGCTCAGTG A G G C G T C C G G A C T G G C C C A G G G A G G T G G G C A A C T A C C A C C C A G G G C C G G A A A G C T C T C C AAACTCGGTCATTTAGAGGAAGTAAAAGTCGTAACAAGGTCTCCGTAGGTGAACCTGC Table B.1b. Accession number and score for the ten most similar fungi deduced from partial sequence of Verticillium sp. (100-1), using BLAST search of Genebank sequences. Rank Accession Organism Probability, Number Score (bits) 1 AB0273334 Paecilomyces tenuipes 2192 2 AB044630 Cordyceps sp. 97005 2165 3 AB044629 Cordyceps pruinosa 2165 4 AB027335 Beauveria brongniartii 2151 5 AF280633 Beauveria bassiana 2141 6 D85136 Paecilomyces tenuipes 2137 7 AB027333 Cordyceps militaris 2135 8 AB044631 Cordyceps takaomontana 2123 9 AF281176 Claviceps africana 2111 10 AB027332 Cordyceps sp. 97009 2097 108 Partial nucleotide sequence of 18s rDNA of Paecilomyces tunuipes 24-2B. GTAGTCATATGCTTGTCTCAAAGATTAAGCCATGCATGTCTAAGTATAAGCAATTATACAGC GAAACTGCGAATGGCTCATTATATAAGTTATCGTTTATTTGATAGTACCTTACTACTTGGATA ACCGTGGTAATTCTAGAGCTAATACATGCTAAAAATCCCGACTTCGGAAGGGATGTATTTAT TAGATTAAAAACCAATGCCCTCTGGGCTCCTTGGTGATTCATAATAACTTTTCGAATCGCAT GGCCTTGCGCCGGCGATGGTTCATTCAAATTTCTTCCCTATCAACTTTCGATGTCTGGGTA T T G G C C A A A C A T G G T C G C A A C G G G T A A C G G A G G G T T A G G G C T C G A C C C C G G A G A A G G A G C C T G A G A A A C G G C T A C T A C A T C C A A G G A A G G C A G C A G G C G C G C A A A T T A C C C A A T C C C G A TTCGGGGAGGTAGTGACAATAAATACTGATACAGGGCTCTTTTGGGTCTTGTAATTGGAAT GAGTACAATTTAAATCCCTTAACGAGGAACAATTGGAGGGCAAGTCTGGTGCCAGCAGCC GCGGTAATTCCAGCTCCATAGCGTATATTAAAGTTGTTGTGGTTAAAAAGCTCGTAGTTGAA C C T T G G G C C T G G C T G G C C G G T C C G C C T C A C C G C G T G T A C T G G T C C G G C C G G G C C T T T C C CTCTGTGGAACCTCATGCCCTTCACTGGGTGTGGCGGGGAAACAGGACTTTTACTTTGAAA AAATTAGAGTGCTCCAGGCAGGCCTATGCTCGAATACATTAGCATGGAATAATGAAATAGG ACGTGTGGTTCTATTTTGTTGGTTTCTAGGACCGCCGTAATGATTAATAGGGACAGTCGGG GGCATCAGTATTCAATTGTCAGAGGTGAAATTCTTGGATTTATTGAAGACTAACTACTGCGA AAGCATTTGCCAAGGATGTTTTCATTAATCAGGAACGAAAGTTAGGGGATCGAAGACGATC AGATACCGTCGTAGTCTTAACCATAAACTATGCCGACTAGGGATCGGACGATGTTATTTTTT GACGCGTTCGGCACCTTACGAGAAATCAAAGTGCTTGGGCTCCAGGGGGAGTATGGTCGC AAGGCTGAAACTTAAAGAAATTGACGGAAGGGCACCACCAGGGGTGGAGCCTGCGGCTTA ATTTGACTCAACACGGGGAAACTCACCAGGTCCAGACACAATGAGGATTGACAGATTGAGA GCTCTTTCTTGATTTTGTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGT CTGCTTAATTGCGATAACGAACGAGACCTTAACCTGCTAAATAGCCCGTATTGCTTTGGCA GTACGCCGGCTTCTTAGAGGGACTATCGGCTCAAGCCGATGGAAGTTTGAGGCAATAACA G G T C T G T A A T G C C C T T A G A T G T T C T G G G C C G C A C G C G C G C T A C A C T G A C G G A G C C A G C G A GTACTTCCTTGTCCGAAAGGCCCGGGTAATCTTGTTAAACTCCGTCGTGCTGGGGATAGAG CATTGCAATTATTGCTCTTCAACGAGGAATCCCTAGTAAGCGCAAGTCATCTGCTTGCGTT GATTACGTCCCTGCCCTTTGTACACACCGCCCGTCGCTACTACCGATTGAATGGCTCAGTG A G G C G T C C G G A C T G G C C C A G G G A G G T G G G C A A C T A C C A C C C A G G G C C G G A A A G C T C T C C AAACTCGGTCATTTAGAGGAAGTAAAAGTCGTAACAAGGTCTCCGTAGGTGAACCTGC Table B.1c. Accession number and score for the ten most similar fungi deduced from partial sequence of Paecilomyces tunuipes (24-2b), using BLAST search of Genebank sequences. Rank Accession Number Organism Probability, Score (bits) 1 AB003951 Tritirachium sp. 3336 2 AJ301994 Myrothecium roridum 3275 3 AJ301993 Myrothecium roridum 3275 4 AJ301995 Myrothecium roridum 3259 5 AB003949 Nectria cinnabarina 3253 6 AJ302005 Myrothecium inundatum 3251 7 AJ302000 Myrothecium leucotrich 3251 8 D85136 Paecilomyces tenuipes 3245 9 AJ302003 Myrothecium verrucaria 3243 10 AJ302002 Myrothecium atroviride 3243 109 Partial nucleotide sequence of 18s rDNA of Paecilomyces marquandii (73-21). GTAGTCATATGCTTGTCTCAAAGATTAAGCCATGCATGTCTAAGTATAAGCAATTATACAGC GAAACTGCGAATGGCTCATTATATAAGTTATCGTTTATTTGATAGTACCTTACTACTTGGATA ACCGTGGTAATTCTAGAGCTAATACATGCTAAAAATCCCGACTTCGGAAGGGATGTATTTAT TAGATTAAAAACCAATGCCCTCTGGGCTCCTTGGTGATTCATAATAACTTTTCGAATCGCAT GGCCTTGCGCCGGCGATGGTTCATTCAAATTTCTTCCCTATCAACTTTCGATGTTTGGGTAT T G G C C A A A C A T G G T C G C A A C G G G T A A C G G A G G G T T A G G G C T C G A C C C C G G A G A A G G A G C C T G A G A A A C G G C T A C T A C A T C C A A G G A A G G C A G C A G G C G C G C A A A T T A C C C A A T C C C G A T TCGGGGAGGTAGTGACAATAAATACTGATACAGGGCTCTTTTGGGTCTTGTAATTGGAATG AGTACAATTTAAATCCCTTAACGAGGAACAATTGGAGGGCAAGTCTGGTGCCAGCAGCCG CGGTAATTCCAGCTCCAATAGCGTATATTAAAGTTGTTGTGGTTAAAAAGCTCGTAGTTGAA C C T T G G G C C T G G C T G G C C G G T C C G C C T C A C C G C G T G T A C T G G T C C G G C C G G G C C T T T C C CTCTGTGGAACCTCATGCCCTTCACTGGGTGTGGCGGGGAAACAGGACTTTTACTTTGAAA AAATTAGAGTGCTCCAGGCAGGCCTATGCTCGAATACATTAGCATGGAATAATGAAATAGG ACGTGTGGTTCTATTTTGTTGGTTTCTAGGACCGCCGTAATGATTAATAGGGACAGTCGGG GGCATCAGTATTCAATTGTCAGAGGTGAAATTCTTGGATTTATTGAAGACTAACTACTGCGA AAGCATTTGCCAAGGATGTTTTCATTAATCAGGAACGAAAGTTAGGGGATCGAAGACGATC AGATACCGTCGTAGTCTTAACCATAAACTATGCCGACTAGGGATCGGACGATGTTATTTTTT GACGCGTTCGGCACCTTACGAGAAATCAAAGTGCTTGGGCTCCAGGGGGAGTATGGTCGC A A G G C T G A A A C T T A A A G A A A T T G A C G G A A G G G C A C C A C C A G G G G T G G A G C C T G C G G C T T A ATTTGACTCAACACGGGGAAACTCACCAGGTCCAGACACAATGAGGATTGACAGATTGAGA GCTCTTTCTTGATTTTGTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGT CTG CTT AATTG C GAT AAC G AAC GAG AC CTTAAC CTG CT AAAT AG C C C GT ATTG CTTTG G C A GTACGCCGGCTTCTTAGAGGGACTATCGGCTCAAGCCGATGGAAGTTTGAGGCAATAACA G G T C T G T A A T G C C C T T A G A T G T T C T G G G C C G C A C G C G C G C T A C A C T G A C G G A G C C A G C G A GTACTTCCTTGTCCGAAAGGCCCGGGTAATCTTGTTAAACTCCGTCGTGCTGGGGATAGAG CATTGCAATTATTGCTCTTCAACGAGGAATCCCTAGTAAGCGCAAGTCATCTGCTTGCGTT GATTACGTCCCTGCCCTTTGTACACACCGCCCGTCGCTACTACCGATTGAATGGCTCAGTG A G G C G T C C G G A C T G G C C C A G G G A G G T G G G C A A C T A C C A C C C A G G G C C G G A A A G C T C T C C AAACTCGGTCATTTAGAGGAAGTAAAAGTCGTAACAAGGTCTCCGTAGGTGAACCTGC Table B.1d. Accession number and score for the ten most similar fungi deduced from partial sequence of Paecilomyces marquandii (73-21), using BLAST search of Genebank sequences. Rank Accession Number Organism Probability, Score (bits) 1 AB003951 Tritirachium sp. 3360 2 AJ301994 Myrothecium roridum 3299 3 AJ301993 Myrothecium roridum 3299 4 AJ301995 Myrothecium roridum 3283 5 AB003949 Nectria cinnabarina 3277 6 AJ302005 Myrothecium inundatum 3275 7 AJ302000 Myrothecium leucotrich 3275 8 D85136 Paecilomyces tenuipes 3269 9 AJ302003 Myrothecium verrucaria 3267 10 AJ302002 Myrothecium atroviride 3267 110 Partial nucleotide sequence of 18s rDNA of Paecilomyces sp. (85-15). GTAGTCATATGCTTGTCTCAAAGATTAAGCCATGCATGTCTAAGTATAAGCAATTATACAGC GAAACTGCGAATGGCTCATTATATAAGTTATCGTTTATTTGATAGTACCTTACTACTTGGATA ACCGTGGTAATTCTAGAGCTAATACATGCTAAAAATCCCGACTTCGGAAGGGATGTATTTAT TAGATTAAAAACCAATGCCCTCTGGGGTGCJTGGT GATTCATAATAACTTTTCGAATCGCAT GGCCTTGCGCCGGCGATGGTTCATTCAAATTTCTTCCCTATCAACTTTCGATGTTTGGGTAT T G G C C A A A C A T G G T C G C A A C G G G T A A C G G A G G G T T A G G G C T C G A C C C C G G A G A A G G A G C C T G A G A A A C G G C T A C T A C A T C C A A G G A A G G C A G C A G G C G C G C A A A T T A C C C A A T C C C G A T TCGGGGAGGTAGTGACAATAAATACTGATACAGGGCTCTTTTGGGTCTTGTAATTGGAATG AGTACAATTTAAATCCCTTAACGAGGAACAATTGGAGGGCAAGTCTGGTGCCAGCAGCCG CGGTAATTCCAGCTCCAATAGCGTATATTAAAGTTGTTGTGGTTAAAAAGCTCGTAGTTGAA C C T T G G G C C T G G C T G G C C G G T C C G C C T C A C C G C G T G T A C T G G T C C G G C C G G G C C T T T C C CTCTGTGGAACCTCATGCCCTTCACTGGGTGTGGCGGGGAAACAGGACTTTTACTTTGAAA AAATTAGAGTGCTCCAGGCAGGCCTATGCTCGAATACATTAGCATGGAATAATGAAATAGG ACGTGTGGTTCTATTTTGTTGGTTTCTAGGACCGCCGTAATGATTAATAGGGACAGTCGGG GGCATCAGTATTCAATTGTCAGAGGTGAAATTCTTGGATTTATTGAAGACTAACTACTGCGA AAGCATTTGCCAAGGATGTTTTCATTAATCAGGAACGAAAGTTAGGGAATCGAAGACGATC AGATACCGTCGTAGTCTTAACCATAAACTATGCCGACTAGGGATCGGACGATGTTATTTTTT GACGCGTTCGGCACCTTACGAGAAATCAAAGTGCTTGGGCTCCAGGGGGAGTATGGTCGC AAGGCTGAAACTTAAAGAAATTGACGGAAGGGCACCACCAGGGGTGGAGCCTGCGGCTTA ATTTGACTCAACACGGGGAAACTCACCAGGTCCAGACACAATGAGGATTGACAGATTGAGA GCTCTTTCTTGATTTTGTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGT CTGCTTAATTGCGATAACGAACGAGACCTTAACCTGCTAAATAGCCCGTATTGCTTTGGCA GTACGCCGGCTTCTTAGAGGGACTATCGGCTCAAGCCGATGGAAGTTTGAGGCAATAACA GGTCTGTAATGCCCTTAGATGT T CT GGGCCGCACGCGCGCT ACACT GACGGAGCCAGCGA GTACTTCCTTGTCCGAAAGGCCCGGGTAATCTTGTTAAACTCCGTCGTGCTGGGGATAGAG CATTGCAATTATTGCTCTTCAACGAGGAATCCCTAGTAAGCGCAAGTCATCTGCTTGCGTT GATTACGTCCCTGCCCTTTGTACACACCGCCCGTCGCTACTACCGATTGAATGGCTCAGTG A G G C G T C C G G A C T G G C C C A G G G A G G T G G G C A A C T A C C A C C C A G G G C C G G A A A G C T C T C C AAACTCGGTCATTTAGAGGAAGTAAAAGTCGTAACAAGGTCTCCGTAGGTGAACCTGC Table B.1e. Accession number and score for the ten most similar fungi deduced from partial sequence of Paecilomyces sp. (85-15), using BLAST search of Genebank sequences. Rank Accession Number Organism Probability, Score (bits) 1 AB003951 Tritirachium sp. 3252 2 AJ301994 Myrothecium roridum 3291 3 AJ301993 Myrothecium roridum 3291 4 AJ301995 Myrothecium roridum 3275 5 AB003949 Nectria cinnabarina 3269 6 AJ302005 Myrothecium inundatum 3267 7 AJ302000 Myrothecium leucotrich 3267 8 D85136 Paecilomyces tenuipes 3261 9 AJ302003 Myrothecium verrucaria 3259 10 AJ302002 Myrothecium atroviride 3259 111 Partial nucleotide sequence of 18s rDNA of Paecilomyces (85-2). GTAGTCATATGCTTGTCTCAAAGATTAAGCCATGCATGTCTAAGTATAAGCAATTATACAGC GAAACTGCGAATGGCTCATTATATAAGTTATCGTTTATTTGATAGTACCTTACTACTTGGATA ACCGTGGTAATTCTAGAGCTAATACATGCTAAAAATCCCGACTTCGGAAGGGATGTATTTAT TAGATTAAAAACCAATGCCCTCTGGGCTCCTTGGTGATTCATAATAACTTTTCGAATCGCAT GGCCTTGCGCCGGCGATGGTTCATTCAAATTTCTTCCCTATCAACTTTCGATGTTTGGGTAT T G G C C A A A C A T G G T C G C A A C G G G T A A C G G A G G G T T A G G G C T C G A C C C C G G A G A A G G A G C C T G A G A A A C G G C T A C T A C A T C C A A G G A A G G C A G C A G G C G C G C A A A T T A C C C A A T C C C G A T TCGGGGAGGTAGTGACAATAAATACTGATACAGGGCTCTTTTGGGTCTTGTAATTGGAATG AGTACAATTTAAATCCCTTAACGAGGAACAATTGGAGGGCAAGTCTGGTGCCAGCAGCCG CGGTAATTCCAGCTCCATAGCGTATATTAAAGTTGTTGTGGTTAAAAAGCTCGTAGTTGAAC C T T G G G C C T G G C T G G C C G G T C C G C C T C A C C G C G T G T A C T G G T C C G G C C G G G C C T T T C C C TCTGTGGAACCTCATGCCCTTCACTGGGTGTGGCGGGGAAACAGGACTTTTACTTTGAAAA AATTAGAGTGCTCCAGGCAGGCCTATGCTCGAATACATTAGCATGGAATAATGAAATAGGA CGTGTGGTTCTATTTTGTTGGTTTCTAGGACCGCCGTAATGATTAATAGGGACAGTCGGGG GCATCAGTATTCAATTGTCAGAGGTGAAATTCTTGGATTTATTGAAGACTAACTACTGCGAA AGCATTTGCCAAGGATGTTTTCATTAATCAGGAACGAAAGTTAGGGGATCGAAGACGATCA GATACCGTCGTAGTCTTAACCATAAACTATGCCGACTAGGGATCGGACGATGTTATTTTTTG ACGCGTTCGGCACCTTACGAGAAATCAAAGTGCTTGGGCTCCAGGGGGAGTATGGTCGCA A G G C T G A A A C T T A A A G A A A T T G A C G G A A G G G C A C C A C C A G G G G T G G A G C C T G C G G C T T A A TTTGACTCAACACGGGGAAACTCACCAGGTCCAGACACAATGAGGATTGACAGATTGAGA GCTCTTTCTTGATTTTGTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGT CTGCTTAATTGCGATAACGAACGAGACCTTAACCTGCTAAATAGCCCGTATTGCTTTGGCA GTACGCCGGCTTCTTAGAGGGACTATCGGCTCAAGCCGATGGAAGTTTGAGGCAATAACA G G T C T G T A A T G C C C T T A G A T G T T C T G G G C C G C A C G C G C G C T A C A C T G A C G G A G C C A G C G A GTACTTCCTTGTCCGAAAGGCCCGGGTAATCTTGTTAAACTCCGTCGTGCTGGGGATAGAG CATTGCAATTATTGCTCTTCAACGAGGAATCCCTAGTAAGCGCAAGTCATCTGCTTGCGTT GATTACGTCCCTGCCCTTTGTACACACCGCCCGTCGCTACTACCGATTGAATGGCTCAGTG A G G C G T C C G G A C T G G C C C A G G G A G G T G G G C A A C T A C C A C C C A G G G C C G G A A A G C T C T C C AAACTCGGTCATTTAGAGGAAGTAAAAGTCGTAACAAGGTCTCCGTGGGTGAACCTGC Table B.1f. Accession number and score for the ten most similar fungi deduced from partial sequence of Paecilomyces sp. (85-2), using BLAST search of GenBank sequences. Rank Accession Number Organism Probability, Score (bits) 1 AB003951 Tritirachium sp. 3244 2 AJ301994 Myrothecium roridum 3283 3 AJ301993 Myrothecium roridum 3283 4 AJ301995 Myrothecium roridum 3267 5 AB003949 Nectria cinnabarina 3261 6 AJ302005 Myrothecium inundatum 3259 7 AJ302000 Myrothecium leucotrich 3259 8 D85136 Paecilomyces tenuipes 3253 9 AJ302003 Myrothecium verrucaria 3251 10 AJ302002 Myrothecium atroviride 3251 112 Partial nucleotide sequence of 18s rDNA of beet-1. G T A G T C A T A T G C T T G T C T C A A A G A T T A A G C C A T G C A T G T C T A A G T A T A A G C A A T T A T A C A G C G A A A C T G C G A A T G G C T C A T T A T A T A A G T T A T C G T T T A T T T G A T A G T A C C T T A C T A C T T G G A T A A C C G N G G T A A T T C T A G A G C T A A T A C A T G C T G A A A A T C C C G A C T T C G G A A G G G A T G T A T T T A T T A G A T T A A A A A C C A A T G C C C T C T G G G C T C C T T G G T G A T T C A T A A T A A C T T T T C G A A T C G C A C G G C C T T G C G C C G G C G A T G G T T C A T T C A A A T T T C T T C T C T A T C A A C T T T C G A T G T T T G G G T A T T G G C C A A A C A T G G T C G C A A C G G G T A A C G G A G G G T T A G G G C T C G A C C C C G G A G A A G G A G C C T G A G A A A C G G C T A C T A C A T C C A A G G A A G G C A G C A G G C G C G C A A A T T A C C C A A T C C C G A T T C G G G G A G G T A G T G A C A A T A A A T A C T G A T A C A G G G C T C T T T T G G G T C T T G T A A T T G G A A T G A G T A C A A T T T A A A T C T C T T A A C G A G G A A C A A T T G G A G G G C A A G T C T G G T G C C A G C A G C C G C G G T A A T T C C A G C T C C A T A G C G T A T A T T A A A G T T G T T G T G G T T A A A A A G C T C G T A G T T G A A C C T T G G G C C T G G C T G G C C G G T C C G C C T C A C C G C G T G T A C T G G T C C G G C C G G G C C T T T C C C T C T G T G G A A C C T C A T G C C C T T C A C T G G G T G T G G C G G G G A A A C A G G A C T T T T A C T T T G A A A A A A T T A G A G T G C T C C A G G C A G G C C T A T G C T C G A A T A C A T T A G C A T G G A A T A A T A A A A T A G G A C G T G T G G T T C T A T T T T G T T G G T T T C T A G G A C C G C C G T A A T G A T T A A T A G G G A C A G T C G G G G G C A T C A G T A T T C A A T T G T C A G A G G T G A A A T T C T T G G A T T T A T T G A A G A C T A A C T A C T G C G A A A G C A T T T G C C A A G G A T G T T T T C A T T A A T C A G G A A C G A A A G T T A G G G G A T C G A A G A C G A T C A G A T A C C G T C G T A G T C T T A A C C A T A A A C T A T G C C G A C T A G G G A T C G G A C G A T G T T A T T T T T T G A C G C G T T C G G C A C C T T A C G A G A A A T C A A A G T G C T T G G G C T C C A G G G G G A G T A T G G T C G C A A G G C T G A A A C T T A A A G A A A T T G A C G G A A G G G C A C C A C C A G G G G T G G A G C C T G C G G C T T A A T T T G A C T C A A C A C G G G G A A A C T C A C C A G G T C C A G A C A C A A T G A G G A T T G A C A G A T T G A G A G C T C T T T C T T G A T T T T G T G G G T G G T G G T G C A T G G C C G T T C T T A G T T G G T G G A G T G A T T T G T C T G C T T A A T T G C G A T A A C G A A C G A G A C C T T A A C C T G C T A A A T A G C C T G T A T T G C T T T G G C A G T A C G C C G G C T T C T T A G A G G G A C T A T C G G C T C A A G C C G A T G G A A G T T T G A G G C A A T A A C A G G T C T G T A A T G C C C T T A G A T G T T C T G G G C C G C A C G C G C G C T A C A C T G A C G G A G C C A G C G A G T A C T T C C T T G T C C G A A A G G C C C G G G T A A T C T T G T G A A A C T C C G T C G T G C T G G G G A T A G A G C A T T G C A A T T A T T G C T C T T C A A C G A G G A A T C C C T A G T A A G C G C A A G T C A T C T G C T T G C G T T G A T T A C G T C C C T G C C C T T T G T A C A C A C C G C C C G T C G C T A C T A C C G A T T G A A T G G C T C A G T G A G G C G T C C G G A C T G G C C C A G G G A G G T G G G C A A C T A C C A C C C A G G G C C G G A A A G C T C T C C A A A C T C G G T C A T T T A G A G G A A G T A A A A G T C G T A A C A A G G T C T C C G T A G G T G A A C C T G C Table B.1g. Accession number and score for the ten most similar fungi deduced from partial sequence of beet-1, using BLAST search of GenBank sequences. Rank Accession Number Organism Probability, Score (bits) 1 AB003951 Tritirachium sp. 3214 2 AJ301994 Myrothecium roridum 3237 3 AJ301993 Myrothecium roridum 3237 4 AB003949 Nectria cinnabarina 3223 5 AJ301995 Myrothecium roridum 3221 6 AJ302005 Myrothecium inundatum 3213 7 AJ302000 Myrothecium leucotrich 3213 8 D85136 Paecilomyces tenuipes 3207 9 AJ302003 Myrothecium verrucaria 3205 10 AJ302002 Myrothecium atroviride 3205 APPENDICES 113 Figure B.2. Chromatograms of free fatty acids from selected entomogenous fungi analyzed by gas chromatography. Vial number as indicated in chromatogram report: 1 = Beauvaria bassiana Bet-3; 2 = Paecilomyces sp. 85-14; 3 = Verticillium sp. 100-1; 4 = Paecilomyces sp. 95-2; 5 = Cordyceps militaris 01-07; 6 = Paecilomyces tenuipes 24-2b; 7 = Paecilomyces marquandii 73- 21; 11 = solvent control; 12 = fatty acid standards lample Name: BEET3 CONC lampie Name, DE Injection Date : Tue, 6. Nov. 2001 . Injection time : 5:55:55 PM Sample Name BEET3CONC Acq Operator : EDUARDO Acq. Method : FAMELK3.M Seq Line Vial No. Inj. No. : 8 1 1 114 I Report style: Mark2000 i Report creation date: 11/7/01 FID1A, (ENrOMO\CO1l-0bU1.UJ counts 12000-i 10000H 8000 H 6000 H 4000H 2000 CM CD CM CO cn co tii 'UL 10 15 —r~ 20 min # Meas. Ret. T Peak Type Height A r e a Area % 1 4.292 PB 2 5.898 PP 3 6.133 PB 4 6.624 PB 5 7.309 PB 6 7.658 PB 7 13.145 PB 1535.495 345.395 1845.289 7783.839 1363.154 539.625 531.537 5667.501 1157.401 7325.844 31374.496 5721.583 3792.620 2542.914 9.842 2.010 12.722 54.486 9.936 6.586 4.416 Instrument: 5890GC Pagelof 1 Injection Date : Tue, 6. Nov. 2001 .Injection time: 3:30:53 PM I Sample Name 85-14A Acq Operator : EDUARDO |Acq. Method : FAMELK3.M Seq Une Vial No. Inj. No. : 2 1 Report style: Mark2000 Report creation date: 11/7/01 FID1 A. (EN I UMU\lWiF026T7D) counts 12000H 10000 8000- 6000 4000 2000 S co II 10 15 Meas. Ret. T Peak Type Height Area Area % 1 2 3 4 5 6 7 8 3.703 4.277 4.523 5.873 6.111 6.610 7.302 9.360 BP PV VB PB PB VB BB BB 237.066 10172.760 528.052 2105.169 16492.365 25042.893 1118.774 356.320 828.868 35813.496 2290.071 6731.438 59647.195 94151.125 4613.660 1501.665 0.403 17.421 1.114 3.274 29.014 45.798 2.244 0.730 Instrument: 5890GC Pagdof 1 axa me • " '—" ample Name: 100-1 = _ _ = _ = : Tue, 6. Nov. 2001 3:55:02 PM 100-1 Injection Date : Injection time: Sample Name 1 Seq Line Vial No. Inj. No. : 3 3 1 Acq Operator Acq. Method Report style: EDUARDO FAMELK3.M Mark2000 Report creation date: 11/ w II PII ^ counts 12000 10000 8000 6000 4O00H © CO CM (O CO IO \ 5 R 8 in ro o> 15 | # Meas. Ret. T Peak Type Height I 1 3.231 BB 2 3.700 BB 3 4.270 PV 4 4.515 VB 5 5.024 BP 6 5.862 VB 7 6.111 BB 8 6.609 VB 9 7.299 BB 10 8.013 PP 11 9.359 PB 378.486 387.968 29197.275 1172.631 252.597 6105.434 50990.031 46771.109 1213.140 291.508 338.728 Area Area % 1203.089 0.253 1226.685 0.258 97583.508 20.522 4745.554 0.998 1068.031 0.225 19689.742 4.141 177050.922 37.234 165361.484 34.776 4852.896 1.021 1313.479 0.276 1411.078 0.297 Instrument: 5890GC Pagel of 1 ata tile : u m r u n _ i v . r̂nple Name: 95-2 CONC Injection Date : Tue, 6. Nov. 2001 •Injection time : 6:20:04 PM jSample Name 95-2 CONC Seq Line Vial No. Inj. No. : 9 4 1 f eq Operator : EDUARDO cq. Method : FAMELK3.M Report style : Mark2000 jReport creation date: 11/7/01 FID1 A, (ENTOMOW4FO901.D) counts 12000 H 10000 -I 8000-1 6000 H 4000 _ J u J CO 8 _ I D f 2000- l 10 15 r i i Meas. Ret. T Peak Type Height Area Area % 1 2 3 4 5 6 4.303 6.150 6.245 6.655 6.744 17.856 PV PV VB PV VB BB 407.568 711.079 285.550 850.990 351.170 333.336 1901.951 3091.594 1174.281 3825.358 1462.798 3520.015 12.700 20.644 7.841 25.543 9.768 23.504 Instrument: 5890GC Pagel of 1 Data file : C : \ H P C H E M \ 2 \ U A I M \ C I N I ^ . V . W ^ , Sample Name: 01-07 rone____===_=„===========: Injection Date : Injection time: Sample Name Tue, 6. Nov 6:44:12 PM 01-07 cone 2001 Seq Line Vial No. No. : 10 5 1 118 • Acq Operator : EDUARDO J Acq. Method : FAMELK3.M Report style: I Report creaiion date: FID1 A, (ENTOMOVXSF1001.D) Mark2000 11/7/01 counts "1 12000 10000 H 8000- 6000 4000- 2000 co . CM CO \co i CM JX~ 10 -r - 15 20 min # Meas. Ret. T Peak Type Height Area Area % I 1 3.241 BP 2 3.709 BB 3 4.285 BV 4 4.528 VB 5 5.036 PB 6 5.278 BP 7 5.883 VB 8 6.126 PB 9 6.629 VB 10 7.319 BB 11 7.638 PB 12 9.382 BB 13 18.742 PB 233.479 639.599 20768.074 1758.581 485.157 230.892 3572.870 19652.600 45970.555 1239.181 879.950 257.007 283.002 697.862 2012.796 68988.242 6761.602 2205.850 968.801 1134B.113 70837.477 167852.141 4949.947 5064.497 1113.157 2840.726 0.202 0.582 19.959 1.956 0.638 0.280 3.283 20.495 48.563 1.432 1.465 0.322 0.822 Instrument: 5890GC P a g d of 1 >ata me : u n r ^ i n_,.,̂ — _ Sample Name: 24-2B cone Injection Date : Tue, 6. Nov. 2001 Injection time : 7:08:24 PM Sample Name 24-2B cone Acq Operator : EDUARDO j Acq. Method : FAMELK3.M Report style: Mark2000 Report creation date: 11/7/01 1 Seq Line Via] No. Inj. No. : 11 6 1 119 I I FID1A, (ENrOMO\006M1U1.D) [ counts 12000 10000-1 8000H 6CKXH 4000- 2000 10 # Meas. Ret. T Peak Type Height Area 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 3.234 3.704 4.276 4.520 5.030 5.871 6.115 6.617 7.313 7.633 8.724 8.952 11.243 11.381 11.423 18.739 BB BB PV VB PB W VP VB BB PB PB BB PV W VB PB 353.486 409.500 22699.533 1260.879 297.742 4429.290 36771.512 49954.148 589.242 680.211 238.488 218.174 275.208 476.592 448.144 220.192 1128.001 1288.838 74634.070 5178.262 1296.982 13966.947 131049.211 174673.047 2310.101 3900.904 1250.128 2085.159 1123.337 1678.311 2319.244 2181.289 ro r- .l/>r- 15 —I— 20 mm Area % 0.269 0.307 17.767 1.233 0.309 3.325 31.197 41.583 0.550 0.929 0.298 0.496 0.267 0.400 0.552 0.519 Instrument: 5890GC Pagdof 1 >aia me . \nr >!-••• ~- — temple Name: 73-21 Injection Date : Tue, 6. Nov. 2001 I Injection time Sample Name 5:31:43 PM 73-21 EDUARDO FAMELK3.M 1 Seq Line Vial No. Inj. No. 7 7 1 120 Acq Operator Acq. Method Report style: Mark2000 Report creation date: 11/7/01 counts "1 12000H 10000-1 8000 H 6000 4000- "FID1A, (ENrOMO\007f-0701.Dr 2000 \«o -̂4 i— 10 15 l— 20 I I # Meas. Ret. T Peak Type Height Area Area % 1 2 3 4 5 6 3.234 4.275 4.521 5.873 6.112 6.613 BB PB BB PB BB VB 216.494 16010.773 715.577 2896.896 29328.527 18661.885 850.762 56787.437 3358.077 9727.482 108461.594 72181.953 0.338 22.591 1.336 3.870 43.149 28.716 Instrument: 5890GC Pagelof 1 )ata file : C : \ H P C H E M \ Z \ U A I A \ C I N I UIVIUW I • r • Sample Name: BLANK 1 Injection Date : Tue, 6. Nov. 2001 Injection time : 8:44:56 PM Sample Name BLANK Acq Operator : EDUARDO Acq. Method : FAMELK3.M Report style: Mark2000 Report creation date: 11/7/01 FID1A, (ENTOMOWIHSUi.u; counts 12000 10000 8000 6000H 4000 2000 Seq Line Vial No. Inj. No. : 10 15 11 1 121 # Meas. Ret. T Peak Type Height Area 15 Area % 20 min Instrument: 5890GC Pagelof 1 ata file : C: \HKunt iv i^^n.n—. ~ ample Name: STDS Tue, 6. Nov. 2001 9:09:04 PM STDS Injection Date : Injection time: Sample Name 1 Seq Line Vial No. Inj. No. : 16 12 1 122 Acq Operator [Acq. Method EDUARDO FAMELK3.M Report style: Report creation date: Mark2000 11/7/01 counts PTDTTTIENTOMO^I 2F1601 .D) f 12000H 10000 8000 H 6000- 4000 H 2000 # Meas. Ret.T Peak Type Height 1 2 3 4 5 6 7 8 9 10 11 12 2.680 BP 3.208 PB 4.263 BV 4.494 VB 5.856 PV 6.101 VB 6.604 PB 7.270 PB 7.966 PB 10.435 PV 10.772 VB 13.065 PB 38450.629 38380.203 49080.367 20792.988 33984.629 20751.988 21109.391 18317.959 32989.437 29639.160 18022.639 20405.035 i— 10 15 ~1~ 20 min Area Area % 120311.766 121883.875 162526.078 73143.531 116429.859 72723.008 72149.984 67218.906 120433.547 120315.695 67408.570 98683.641 /^«v) (AC, dl-V ) "l .1 -2 Z. (*») Instrument: 5890GC Pagdof 1 APPENDICES 123 Figure B.3 Chromatograms of Amino acid analysis from sporocarps of Cordyceps cyanensis. Included are: Chromatogram Report, Mol Percent Report, and Typical Amino Acid Analysis Results (Hydrolysis Test Peptide). 124 CO D a i CM c.RF'0 3 l b -2 I A All I! : I I i hi !! Ij'i iii ! Ii.- i l ! i i'lK ill X2 V ZP.O T u r n t a b l e P o s i t i o n Data S t a r t Dpi a D u r a t i o n Peak HI T h r e s h o l d SoMpl Inc] Ttiter-vsl Samples In Run Oc;«t"ciior ID Int . fvtri. Pint 125 P ~ c- Co] i torai i o n r i l e Reference Time Reference Offse t 1 Re ference Offse t 2 0.80 min 3.00 min ISTD Peak ID : NOP I nt ear at i on Interval : S.S to 1 S.0 P i n PEAK RET. PMOL PnOL IL) TIME BY c o r r e c . MOL ni n HEIGHT I NT STD 2 n s p e r i i c Ac i d 6.5"; SE-1 .71 854.52 5. £5 Glutamic ficid •~ T 46B5.E3 4 183.55 ,24.94 S e r i n e 8 . 22 1129.97 1SU.6S "S.04 6 i y c i ne 8. B5 435.73 439.77 2 .52 Hi st i dine 9. 08 4511.86 4051.SS 24.12 A r a l n i ne 9. Er 2 i 4.75 1 92.88 1.15 Threonine S. 83 380.72 34 1.38 '2.04 A l a n i n e 10.23 3335.Se 2555.SE 17.93 Fro I ir.e 10.45 14S9.41 1337.46 7 . se T yros i ne 12.63 1 5 5 . 8 7 1iS.55 0.89 v e l i n e 13. 45 4S4.35 434.3-2 2 .55 Me t hi cn i ne 13.7? 20.00 : 7 . 95 Cyst e:ne 14.5? 46 . 15 4 ' . 4 i 3.25 I so leuc i ne 1 5. 35 4 3.98 39.50 0 . 2 £ Leuc i ne 1 5.55 209.45 189.05 1.12 NOR 15.85 1 1 1 3 . 5 ; S00C.00 I STD Phenyl a 1 a m ne ' £ . : " 225.54 2i?3 .43 1 .2' L •/ eine 34?.74 3?5.58 ; .82 TOTAL PfiOLC RE COVfJu.'fj '87SS.47 Miniaupi Peak Threshold: 393 ufiU • 4 C5eJ-.= b e l o u : ( 44 pea? 3 f o-j.-d '. IS Dee!- i -s-tcher S a m p l e ID: EI n {-» «3 < D a l e» 126 II Ml!;! l miM lili 1̂ ii i i i i ii Hi! ii IIII I  !! •j! jl j i m j «!(. . kip. In i i i ii n I Si ii iiii inn Hf! HH !! Jil l liiiri mi H/in im it: , 20.0 5 . 3 P I U 1 e [ 0 : ! 450 ! CG T u r n t a b l e P o s i t i o n Data S t a r t Data D u r a t i o n Peak Mt rhreahold Ca.l i b r a i ion F i l e : Referencs Time : Reference Offse t 1: Reference Of f se t 2: I n t e a r a t i o n In terva l Saf-if.il i nu I nterva.1 Sonp.lf!?, In Run Operator ID Int . StrJ. fimt ISTD Peak ID 127 PERK ID ftscertic A c i d G l u t a m i c A c i d Sen i r,e G i v e m e H i s t i d i r e ftrqinine T h r e o n i n e A l a n i n e Proline. ? 5 i n e RET. T I Mf! n 07 TIMF. mi n 9 . 9 3 V 3 . 2 7 • 0 . 4 7 P E W HEIGHT U N U 4065 5433 35320 1 236 195 95S 2 556 31 88 9 5? 72 2 92240 433G3 1 9037 !4 6G72 1524 1 651 4 1 215164 217093 7847 / t o .•' a 534 1 065 1 79800 3024 SS8E 21 335 7 2F'7 PMOL BY HEIGHT • • C 7 >7(P. ! 4 . 0 470. *. 7 P " PMOL corre.c. INT STD 203 . 1 205 , 3 C 2 . 6 8 1 i s 4 3 1 .»1 r- -7 .'. 7 r*j 1 i : J: 6 4 _ i _ W 'w _ t/ iTiC ? E T c 7 V." — — V t- H u • 128 SAT w Q. ' cu ft- CO cu CO "35 La •a co ' 3-' co cu si VJ . *35 e < ."2 < o c- S "c. >•» c-. <0 s o CA M U > t L. a* i. 3 4> -J t 3 5 4> 05 I H v u e v • 3 O* 4> (A on s . 4* Ok I cw OT < • o • GC c Cd O 6- C Cd 0. c4 ( I I • • 1 Îft I/) I/) t/) «/i «n «o *n v> 0 0 0 ^ * 0 0 0 0 0 o o o o o o It < o "8 M • =L w-i o a •a g i o e < c * J to O •c o 8-a: a 3 o e < e e o od c o o ' o c Z u >. u l-> R 5 O 1 1 -a c cQ APPENDICES 129 Figure B.4 Physicochemical data used to determine the structure of the cerebroside (4E,8E)-N-2-hydroxyhexadecanoyl-1-0-B-glucanopyranosyl-9-methyl-C 1 8- sphinga-4,8-diene. Included are: a) low resolution F A B Mass Spectrum, b) 1 3 C - N M R Spectrum, c) 1 H - 1 H C O S Y Spectrum, d) 1 H - N M R Spectrum. 8- 55- o o • o CO o • ur> X - > oo -V to n o o 8T CO V CM to CO X - CO o CO o in CM O O C J 8 X" o o CO i - s i o • o o • in co o • CD co o • o in <= I j o c o co oo 2 o o 131 O L D c CD T m o CTJ E ru <j o ' x m rn o C D 10 i n • n o» un • " • C_ O ^ > Q. ID ro ••— to CL cn LO o m o > CD un cn m un ~~ r» o o o C\J o » - > L o n n o o o o o o — J 1/1 C D C D C D X 3 O O O 3 L to I o o o o un — — O O U 3 L D O ^ o • S c o m x o o o o u I D nj U J o o CX X Q 1 I O O O O O lO 3 < X C C C J 2 L U Q . zee r a o o t n e n a : u. Q i - - o. a — a o x z o. a. v> o z a. o_ o_ CL cn O x >- • LT1 OJ o "LO o - o in -OJ o -in OJ CO a. ro Q CO O Z C LU Z C J C_ X CL O r> < x cc C_> Z U J CL aj *-> cu co e o CD CO C_ O CU CL O O C= O J N N OJ X X cn O 1 o CD o x ro co ai r̂ . > rg OJ co i—i m tn cn oj oj co ~ z m co co ro C\J o cu T 10 m 00 • OJ o rn X CD I— n a o z OC X OC LL) I— CD CL > _ CO O —I —I x _ c r 3 Q O C o c n s 1 — i Q - Q - i — cn z o cn o n ^ cn 0 0 0 0 0 0 • o OJ 0 0 CD O O m o o o C3 CD 2 LU CU <c cc a a 1— a x o " o o — X cj 132 CO X cn X •a c cu 0 0 CO in m 0 a> CO —1 0 e r~ 0 CD OJ 0 OJ C_ cn 0 cn CO m CL 0 cn 0 0 c 0 • t-i cn cn OJ CJ 0 c CL 1 O _l LL. OJ 1—1 Li- Q. cn U_ cn en O O O CM m cn co cn C J cn 1 CD Q_ E e a. CL eg CJ u CL X CL X 0 0 0 O O m cn 0 in 0 cn O 0 c_ 0 OJ cu 0 OJ *—1 0 4-» OJ «-< 0 0 O CD cu 0 1 1 e 10 c_ CO a. 0 a. cr s z CL Q- a X >- « OJ OJ CJ CJ LL. Cl- u. a. eg CL x O T O OJ C J 2: S C J Q. IVI CL X ro m LO O CO z: ro CQ UO o e ~D -a I 133 " S — s 3 o Q - o o o o o _ • l£> O O O O 0 ID u3 ca cvj m Q o o <̂  — — o o o o o - — o o o w (fl CD E 3 3 O S D Q Q u u >- is a. i z o cn u. •< n o o • > o o 2 o o o o o o o o o £ 0 0 0 0 0 0 0 0 0 " " o o o o o o o in n S S - 3 o a a a o u. « : . a tn co aa u a V V • o - to • o • « 10 ^ • UD ^ O I <—• o — j o x —. _ J a x . i a. x a. _ J CL x a r o-W ii CD cn co a x a. x o. ; o e

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