ENVIRONMENTAL FACTORS AFFECTlNG

PREDATOR-PREY RELATIONSHIPS AMONG YEASTS

Ankica Pupovac-Velikonja

Department of Plant Sciences

Submitted in partial fulfilment of the requirements for the degree of

Master of Science

Faculty of Graduate Studies The University of Western Ontario

London, Ontario November 1998

O Ankica Pupovac-Velikonja 1999

National library 1+1 of,, Bibliothèque nationale du Canada

Acquisitions and Acquisitions et Bibliograp hic Services services bibliographiques

395 Wellington Street 395, me Welhgton OttawaON K1AON4 OtGawaON K1AON4 canada canada

The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Libmy of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sell reproduire, prêter, distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/fïlm, de

reproduction sur papier ou sur format électronique.

The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor subsbntial extracts from it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation.

ABSTRACT

A novel type of contact necrotrophic relationship among yeasts, characterized

as yeast predation, has been studied. Predacious yeasts, the teleornorphs of which

were recently classified into a single genus (Saccharomycopsis), and their potentiai

prey from several yeast genera were grown separately and in CO-culture on vanous

media. Haustorium formation and cell penetration were obseived using Iight and

electron microscopy. Known predacious yeasts are sulfur auxotrophs. Therefore,

their predation dynamics was studied on synthetic media in which the content and

type of organosulfur source were varied. Contrary to what was expected, even very

low organosulfur concentrations (e.g., 2 1 pprn methionine) stimolated, rather than

inhibited predation with an eventual elimination of the prey. Endo-kglucanase, but

not a-mannanase or chitinase activity, increased during predation. Preliminary

experirnents with inorganic salts indicated that the presence of ammonium sulfate

inhibited predation and suppressed endo-blucanase activity.

Kevwords: A~thmascus, auxotrop h, beta-g lucanase, Candida, haustorium,

interfungal, methionine, organosulfur, necrotrophic, parasitic,

penetration, predacious yeast, predation, predator, predatory yeast,

prey, su lfu r, Saccharomycopsis, yeast.

iii

ACKNOWLEDGEMENTS

Herewith I express my most sincere gratitude to Professor Marc André

Lachance, my principal advisor. 1 consider myself honoured and very forninate for

having been chosen by him to work on a hitherto unexplored topic and ... well, boldly

to go where no one has gone before. It was a pleasure to work with him and under

his guidance (...well, most of the time), for he is a combination of a thoroughbred

scientist, an artist. a philosopher, a tnie gentleman, and a fnend - a rara avis,

indeed.

I am very grateful to rny other advisors, Professor Charles G. Trick and

Professor Priti Krishna. for their help, adviœ and encouragement. They indebted

me with generously offering their fime and showing interest in my work. They also

taught me that you c m cal1 your supervisor simply André (instead of sehr geehrter

Herr Professor Doktor Doktor honoris causa, as we fiorn Europe usually do).

My special thanks to Professor Jane Bowles for her guidance during our

yeast-sampling field trip in Southwestem Ontario; to Professor Carlos Augusto

Rosa, post-doctoral fellow from Bello Horizonte, for his optimism and the soond of

his enchanting Brazilian CDS in the lab; Mrs. Birgit Schlag-Edler Nienna, Austria)

for excellent electron microphotography; Mr. Tom Haffie and my fellow TAS for

making my teaching assistantship in PS 290 (Genetics) a tnily enjoyable

experience.

Also many thanks to the secretanal and technical staff (Donna Cheshuk,

Stefani Tichboume, Elizabeth Myscich) for their help and patience.

Finally, my love to Ogo and Joran. They know what for.

And to Nikka, who I brought into this world only two and a half days after

defending this thesis, 1 10 days ahead of tirne. She was patiently waiting for me to

finish and is now fighting her own silent battle to grow, breathe on her own and join

US.

TABLE OF CONTENTS

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Certificate of Examination ii

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Abstract and Keywords iii

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgernents iv

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Table of contents v

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . List of TabIes viii

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . List of Figures ix

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . List of Plates xiv

. . . . . . . . . . . . . . . . . . . . . . . . . . Introduction the Scope of the Thesis 1

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fungi - A Unique Phylum 2

. . . . . . . . . . . . . . . . . . . . The Evidence of Fungi in the Fossil Record 4

. . . . . . . . . . . . . . Taxonomie and Phylogenetic Position of the Fungi 6

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fungal Cell Walls 9

. . . . . . . . . . . . . . . . . . . . . lnterfungal Relationships Mycoparasitism 15

. . . . . . . . . . . . . . . . . . . . Yeasts as Fungal Parasites and Predators 18

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Killer Yeast Phenornenon 19

YeastPredation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Thesis Objectives 24

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Materials and Methods 25

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Microorganisms 25

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Culture Media 26

. . . . . . . . . . . Selection of Most Acüve Predator-Prey Combinations 27

Search for Media Components That Affect Predation . . . . . . . . . . . . 28

.............................. Light Microswpy of Predation 28

Light Microscopy of Post-Predation Viability ................... 29

Electron Microscopy of Interactions in Liquid Media ............. 29

Predation Monitoring on Selected Media (Colony Counting) . . . . . . 30 . . . . . . . . . . . . . . . . . . . . . . . . Effect of inorganic Salts on Predation 31

. . . . . . . . . . . . . . . . . . . . . . Predation-Associated Enzyme Activities 32

PrelirninaryTests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

EnzymeAssays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 . . . . . . . . . . Effect of Inorganic Ions on P(1+3)-Glucanase Activity 34

Thin-Layer Chromatography of Mono- and Oligosaccharides . . . . . 34

ExoglucanaseAssay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results and Discussion 36

. . . . . . . . . . . Selection of Most Active Predator-Prey Combinations 36

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Light Microscopy of Predation 39

. . . . . . . . . . . . . . . . . . . . . . Interactions on Solid Media . Predation 39

. . . . . . . . . . Interactions on Solid Media . Post-Predational Viability 42

Scanning Electron Microscopy of Interactions in Liquid Media . . . . . 45

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . General Effect of Nutrients 53

. . . . . . . . . . . . . . . . . . . . . . . . . . Time Course (Continuous Growth) 53

. . . . . . . . . . . . . . . . . . Predaüon Dynamics (Discontinuous Growth) 67

. . . . . . . . . . . . Search for Media Components That Affect Predaüon 89

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Role of Organic SuMir 90

The Role of L-Methionine . Predation of Saccharomycopsis

. . . . . . . . . . . . . . . . . . . . . ja vanensis on Saccharomyces cerevisiae 92

The Role of L-Methionine . Predation of Saccharomycopsis

. . . javanensis and Candida strain 'W1 " on Metschnikowia hibisci 107

The Role of Other Organosulfur Sources . Predation of

.... Saccharomycopsis javanensis on Saccharomyces cerevisiae 124

Effect of Inorganic Salts on Predation of Saccharomycopsis

.................... ja vanensis on Saccharomyces cerevisiae 1 37

..................... Predation-Associated Enzyme Activities 138

....................................... Preliminary Tests 139

Total P(1-+3)-Glucanase and a-(l-+4>Mannanase Activities . . ; . 140

. . . . . . . . . Effect of Inorganic Ions on P(1-3)-Glucanase Acüvity 147

Hydrolytic Cleavage of P(1+3>Glucan to Glucose and

........................................ Oligoglucosides 148

Exo-Po-GlucanaseAssay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155

........................................... Conclusions 158

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Literature 160

CumkulumVitae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171

vii

LIST OF TABLES

1 2.1 Sugar Distribution in Fungal Cell Walls . . . . . . . . . . . . . . . . . . . . . . . 12

1.2.2 Polysaccharide Chernotypes of Fungal Cell Walls . . . . . . . . . . . . . . 13

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2.3 Different PGlucans from Fungi 15

. 1 3.1 .1.1 Mycocinogenic Yeast Genera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

1 .3.1 .2.1 Yeast Teleomorphç with Demonstrated Predacious Behaviour . . . . 23

1.3.1.2.2 Yeast Anamorphs with Dernonstrated Predacious Behaviour . . . . . 24

Yeast Strains Studied As Potenüal Predators and Preys . . . . . . . . . 25

Basic Nutrient Media . composition and Properties . . . . . . . . . . . . . 26

Selective Media . Composition and Properties . . . . . . . . . . . . . . . . . 27

Potential Predator-Prey Combinations . . . . . . . . . . . . . . . . . . . . . . . 38

Effect of Various Complex lngredients of Nutrient Media on the

Predation of Saccharomycopsis javanensis Grown in Co-Culture

. . . . . . . . . . . . . . . . . . . . . . . . . . . . with Saccharomyces cerevisiae 89

Predator-Prey Pairs and LNB (Basal) Medium Supplements in

. . . . Experiments on the Role of Organic Sulfur in Yeast Predation 91

Effects of lnorganic Salts on Predation of Saccharomycopsis

javanensis on Saccharomyces cere visiae . . . . . . . . . . . . . . . . . . . . 1 37

viii

LIST OF FIGURES

Modern Classification of Organisms From the Former Phylum

Mycota . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Polyphyly of the 'Traditional Fungi", Mostly According to SSU rDNA

Sequencing Data, and Their Position Among Other Taxa . . . . . . . . . 8

Types of Fungal Interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 6

Mechanisrns of Fungal Combat . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Interface Types in Mycoparasitism . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Growth of Saccharomycopsisjavanensis and Saccharomyces

cerevisiae on GY Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - 55

Growth of Saccharomycopsis javanensis and Saccharomyces

cerevisiae on GY Agar - Staüsticç . . . . . . . . . . . . . . . . . . . . . . . . 57

Growth of Saccharomycopsis javanensis and Saccharomyces

cerevisiae on Basal Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

Growth of Saccharomycopsisjavanensis and Saccharomyces

cerevisiae on Basal Medium - Statistics . . . . . . . . . . . . . . . . . . . 61

Growth of Saccharomycopsis javanensis and Saccharomyces

cerevisiae on Rich Medium (YM Agar) . . . . . . . . . . . . . . . . . . . . . . . 63

Growth of Saccharomycopsisjavanensis and Saccharomyces

cerevisiae on Rich Medium (YM Agar) - Staüstics . . . . . . . . . . . 65 Predation Dynamics of Saccharomycopsis javanensis and

Saccharomyces cerevisiae on GY Agar with 0.1 gfL Yeast Extract . 69

Predation Dynamics of Saccharomycopsis javanensis and

Saccharomyces cerevisiae on GY Agar with 0.1 gl L Yeast Extract

.......................................... - Statistics 71

P redation Dynamics of Sacchammycopsis javanensis and

Saccharomyces cerevisiae on GY Agar with 1 glL Yeast Extract . . 73

Predation Dynamics of Saccharomycopsis javanensis and

Saccharomyces cerevisiae on GY Agar with 1 glL Yeast Extract

........................................... - Statistics 75

Predation Dynamics of Saccharomycopsis javanensis and

Saccharomyces cerevisiae on GY Agar with 10 glL Yeast Extract 77

Predation Dynamics of Saccharomycopsis javanensis and

Saccharomyces cerevisiae on GY Agar with 10 glL Yeast Extract

- Statistics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

Predation Dynamics of Saccharomycopsis javanensis and

Saccharomyces cerevisiae on Low Nitrogen Basal (LNB) Agar:

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . O. 1 glL (NH4),S04 8 1

Pred ation Dynamics of Saccharomycopsis javanensis and

Saccharomyces cerevisiae on Low N itrogen Basal (LN B) Agar:

. . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1 gR (NH4),S04 - Statistics 83

Predatio n Dynamics of Saccharomycopsis javanensis and

. . . . . Saccharomyces cerevisiae on Basal Agar: 5 g1L (NH,),SO, 85

Predation Dynamics of Saccharomycopsisjavanensis and

Saccharomyces cerevisiae on Basal Agar: 5 glL (N H4),S04

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - Statistics 87

Influence of Organic Sulfùr on the Predation Dynarnics of

Saccharomycopsis ja vanensis and Saccharomyces cerevisiae on

. . . . . . . LNB Medium with Varying Concentrations of m me thionine 93

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.1.1 - No Methionine (Control) 94

3.5.1 -1 -a Influence of Organic Sulfur on the Predaiion Dynamics of

to Saccharomycopsis javanensis and Saccharomyces cerevisiae on

3.5.1 -6-a LNB Medium with Varying Concentrations of L-Methionine

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - Statistics 100

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.1.1 -a - No Methionine (Control) 101

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.1 -2-a - 0.0001 g1L L-Methionine 102

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.1.3.a - 0.001 glL m me thionine 103

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.1.4.a - 0.1 g/L m me thionine 104

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.1 -5-a - 1 g / l m me thionine 105

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.1 -6-a - 10 glL L-Methionine 106

3.5.2.1 Influence-of Organic Sulf'ur on the Predation Dynamics of

to Saccharomycopsis javanensis and Metschnikowia hibisci on LN B

3.5.2.3 Medium with Varying Concentrations of L-Methionine . . . . . . . . . . 108

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.2.1 - No Methionine (Control) 109

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.2.2 - 0.001 g R L-Methionine 110

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.2.3 - 10 glL m me thionine 111

3.5.2.1-a Influence of Organic Sulfur on the Predation Dynamics of

to Saccharomycopsis ja vanensis and Metschniko wia hibisci o n LN B

3.5.2.3-a Medium with Varying Concentrations of L-Methionine

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - Statistics 112

3.5.2.1 -a - No Methionine (Control) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 13 3.5.2.2-a - 0.001 glL L-Methionine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114

3.5.2.3-a - 10 glL m me thionine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

3.5.2.4 Influence of Organic Sulfur on the Predation Dynamics of Candida

to strain 'WI " and Metschnikowia hibisci on LN B Medium with Varying

3.5.2.6 Concentrations of L-Methionine . . . . . . . . . . . . . . . . . . . - . . . . . . . 116

3.5.2.4 - No Methionine (Control) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 17

3.5.2.5 - 0.001 g1L L-Methionine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118

3.5.2.6 - 10 g/L L-Methionine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 19

3.5.2.4-a Inff uence of Organic Sulfur on the Predation Dynamics of Candida

to strain '7N1 " and Metschnikowia hibisci on LNB Medium with Varying

3.5.2.6-a Concentrations of L-Methionine

- Statistics . . ........................................ 120

3.5.2.4-a - No Methionine (Control) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

3.5.2.5-a - 0.001 g/L m me thionine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122

3.5.2.6-a - 10 glL m me thionine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

3.5.3.1 Role of Organic Sulfur in the Predation Dynamics of Saccharomycop-

sis javanensis and Saccharomyces cerevisiae on LN8 Agar with

1 glL DL-Methionine (Racemate) . . . . . . . . . . . . . . . . . . . . . . . - . . 125

3.5.3.1-a Role of Organic Suhr in the Predation Dynamics of Saccharomycop-

sisjavanensis and Saccharomyces cerevisiae on LNB Agar with

1 glL DL-Methionine (Racemate) - Statistics . . . . . . . . . . . . . . . 127

3.5.3.2 Role of Organic Sulfur in the Predation Dynamics of Saccharomycop-

sis javanensis and Saccharomyces cerevisiae on LN B Agar with

1 glL Sodium Thioglycollate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129

xii

3.5.3.2-a Role of Organic Sulf'ur in the Predation Dynamics of Saccharomycop-

sis javanensis and Saccharomyces cerevisiae on LN B Agar with

1 g R Sodium Thioglycollate - Statistics ................... 131

3.5.3.3 Role of Organic Sulfur in the Predation Dynamics of Saccharomycop-

sis javanensis and Saccharomyces cerevisiae on LN B Agar with

1 g R L-Cysteine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133

3.5.3.3-a Role of Organic SuIfUr in the Predation Dynamics of Saccharomycop-

sis javanensis and Saccharomyces cemvisiae on LN B Agar with

1 g/L L-Cysteine - Statistics . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135

3.7.2.1 Beta-(1 -+J>Glucanase Assay 48 Hours After Co-Culture of Saccharo-

mycopsis javanensis and Saccharomyces cerevisiae on Methio n i ne-

. . . . . . . . . . . . . . . . . . . . . Supplemented Predation Medium (GY) 143

3.7.2.2 Beta-(1+3>Glucanase Assay 6 Days After Co-Culture of Saccharo-

mycopsis javanensis and Saccharomyces cerevisiae and in Pure

Culture of Saccharomycopsis javanensis on Methionine-

..................... Supplemented Predation Medium (GY) 145

3.7.3.1 Effect of lnorganic Salts on j glu cana se Activity from Co-Culture of

Saccharomycopsis javanensis with Saccharomyces cerevisiae on

~redatiofl Medium (GY) with and without Methionine

. . . . . . . . . . . . . . (Washed Cells from Co-Culture after 48 hours) 149

3.7.3.2 Effect of lnorganic Salts on P-Giucanase Activity from Co-Culture of

Saccharomycopsis ja vanensis with Saccharomyces cerevisiae on

Predation Medium (GY) with and without Methionine

. . . . . . . . . . . . . . . . (Supematant from Co-Culture alter 48 hours) 151

3.7.5.1 Exo-P~Glucanase Activity Assay in Pure Cultures and Co-Cultures

of Saccharomycopsis javanensis with Saccharomyces cerevisiae on

Predation Medium (GY)

. . . . . . . . . . . . . (Washed Cells and Supernatants affer 48 hours) 156

xiii

P redation of Saccharomycopsis fermentans on Saccharomyces

cerevisiae in Slide Culture on GY Agar . . . . . . . . . . . . . . . . . . . . . . 40

Post-Predational Recovery in Slide Culture of Saccharomycopsis

javanensis and Saccharomyces cerevisiae onYM agar . . . . . . . . . 43

Predation of Sacchamrnycopsis javanensis on Saccharomyces

cerevisiae in Liquid Predation Medium (GY): Scanning Electron

Microscopy of Predator-Prey Cell Contacts . . . . . . . . . . . . . . . . . . . 47

Details of Haustoria Formation and Penetration During Co-Culture of

Saccharomycopsis javanensis and Saccharomyces cerevisiae in

Liquid GY Medium (Scanning Electron Microscopy) . . . . . . . . . . . . 49

Predation of Saccharomycopsis javanensis on Schirosaccharomyces

pombe in Liquid Predation Medium (GY): Scanning Electron

. . . . . . . . . . . . . . . . . . . Microscopy of Predator-Prey Cell Contacts 51

Thin Layer Chromatogram of Laminarin Hydrolyzate Obtained with

Washed Cells Rom Co-Culture of Saccharomycopsis javanensis and

Saccharomyces cerevisiae on GY agar . . . . . . . . . . . . . . . . . . . . . 153

xiv

INTRODUCTION - THE SCOPE OF THE THESIS

The present thesis constitutes a continuation and an integral part of

studies of a new antagonistic interfungal phenomenon (LACHANCE and PANG, 1997)

exhibited by certain yeasts grown in co-culture with other yeasts. A relationship of

necrotrophic parasiüsrn, or predation. of those novel strains upon others, chosen as

hosts, became manifest under certain culturing conditions. This predatory

behaviour came as a surprise. as such a relationship, although known and well

documented for many other fungi, was hitherto unknown among yeast genera. In

fact, this phenomenon had been seen at least once in the past (KREGER-VAN RIJ and

VEENHUIS, 1973), but its true nature and significance remained unrecognized.

The faculty of being predacious appears to be associated with some other

characteristics, pertinent to the strains known and studied thus far:

al1 known predacious yeasts were collected from specific habitats, namely

wounded trees (Fagaceae) or biocoenoses of insects/flowers (Malvaceae); predation is not an obliaatory behaviour, sinœ the yeasts grow well when

cultured on suitable media in the absence of a host;

predation is manifested by the appearance of elonaated protuberances

(haustoria or penetration peas), with which the predator penetrates its host;

haustorial penetration is lethal to the host;

with only three anamorphic exceptions, the known strains are

ascos~oroaenic teleomomhs;

host s~ecificitv varies with each predacious species, and appears to be

confined to the fungi;

medium comoosition is of crucial importance, with certain standard nutrients

effectively inhibiting predaüon (e.g., yeast extract) and others having the

opposite effect (e-g.. trace amounts of L-methionine);

phvsical conditions of cultivation - temperature and agitation - have a

marked influence on the outcome and extent of predation;

metabolic deficiencies in the utilization of sulfate appear to be a characteris-

tic of al! recognized predacious strains;

phvlogenetic relatedness among the examined strains is high, allowing the

inclusion of al1 the teleomorphs into a single genus (KURTZMAN and R O B N ~ ,

1995);

The significance of the findings on predatory yeasts is twofold:

they open up a new cha~ter in the study of interfungal relations, with the

possibility of shedding new light on the ecology of yeast comrnunities;

they might eventually offer new insights and lead to new methods in the

biocontrol of plant pathoaens and the mycoflora involved in food s~oilaoe

(e.g., DEAK and BEUCHAT, 1996).

1 .l FUNGl - A UNIQUE PHYLUM

The systematic position of organisrns which customarily were called

"fungi" has been a fiuctuating one in the many past and modern attempts at

correctly positioning them in relation to other living creatures. This fluctuation is

refiected in historical attempts at their description, classification and the explanation

of their ongin (AINSWORTH, 1965).

Fungi have accornpanied humans from the very beginnings of individual and

collective consciousness. Most intuitively, one thinks of the edible or poisonous

fruiting bodies of macroscopic fungi. Some of those structures had different uses,

like the dried bracket fungi used as tinder, or the hallucinogen-containing fungi in

sharnanic rites. On the other hand, whether or not the organisms thernselves were

visible and perceived as such, the human population had benefitted or suffered from

thern. Examples of harm done by certain fungi are the destruction of crops, food

spoilage, diseases of plants, animals and man, rnycotoxicoses and deterioration of

many materials. Examples of benefits to man are the leavening of bread, the

production of various fermented and fungus-processed foods and alcoholic

fermentations. In spite of this large body of experience with fungi, the awareness

of the existence of those not visible to the unaided eye had to wait for the

microscope to be constnided by Antonie van Leeuwenhoek in the 17th century.

Traditionally, fungi were regarded and classified as plants rather than

animals. probably because mushrooms and toadstools were generally found in

associations with plants, did not run away and, as such, had been studied by

botanists. After the Gutenbergian revolution they were included in conternporane-

ous herbals and one such description, in "The Great Herbal" from 1526, portrays

them in Paracelsian and Aristotelian ternis, showhg a preoccupation with the

poisonous nature of some of them (in AINSWORTH, 1965):

Fungi ben musherons. They be colde and moyst in the thyrde degre and that is shewed by theyr vyolent moysture. There be two maners of them, one maner is deedly and sleeth hem that eateth of them and be called tode stooles, and the other dooth not.

Over the course of centuries fascination with fungi had been growing steadily,

inspiring the lives and works of outstanding nahiralists, who helped to promote the

study of fungi from the spheres of black rnagic and gastronomy to the rank of a

science. A systematic study of fungi started some two and a half centuries ago

with PietAntonio Micheli's [1679-1737 "Nova genera plantarum" (ALEXOPOULOS et

al., 1996). An introductory account of this development with further references is

given by AINSWORTH (1 965).

It is now estabfished that fungi are truly cosmopolitan organisms, a paradigm

of ubiquity in the biosphere. There is probably no ecological niche that could not be

colonized by. and no other organism that could not be host to a suitably equipped

fungus. In cornparison with other taxa (e.g., plants and animals), fungi were not

particularly innovative in the acquisition of new biochemical/physiological traits

(CARLILE, 7980). Moreover, their development often followed parallel, convergent

and retrograde evolutionary paths (SAVILE. 1968). Their recipe for success seems

to consist primarily in their formidable reproductive and survival capacities, their

propensity for associations with other organisms and the ability to adapt to highly

variable nutrient sources.

From an estirnated 1.5 million fungal species, only about 69,000 are

described (CS%), making the fun@ one of the least well known taxa of the living

world (HAWKSWORTH, 1997 ), comparable to the estimated known-to-unknown ratio

of 1 :30 for the postulateci 30 million arthropod species (ERWIN, 1982).

1.1.1 THE EVIOENCE OF FUNGI IN THE FOSStL RECORD

Fungi had developed presurnably in marine environments in the

Precambrian (>570 Ma b-p.), and rnany of them must have lived in close

associations with otherforms of life, particularly cyanobacteria and algae. A gradua!

colonization of aquatic habitats with decreasing salinity must have been a

prerequisite for terrestrialization (HALLBAUER and VAN WARMELO, 1 974; PIRONNSKI ,

1976). This transition is assumed to have taken place at the latest in the Silurian

(438-408 Ma b-p.) (SHERWOOD-PIKE and GRAY, 1985). within the protective interior

of early land colonizers from the plant kingdom (parasitic or symbiontic

associations). Some of these earliest known terrestrial filamentous fungal fossils

rnay already represent members of the Ascomycota.

In the course of evolution some fungi ihen developed strategies to wunter

desiccation, leave their hosts and start exploiting new ecoiogical niches, created by

the spreading of vegetaüon. Indeed, fossil evidence of what appeared to represent

vesicular-arbuscular mycorrhizal (VAM) fungi associated with Devonian (408-360

Ma b.p.) vascular plants would support argumentations that such fungus-plant

associations were the cause rather than a consequence of land colonization

(PIROZYNSKI and MALLOCH, 1975; PIROZYNSKI, 1976, 1981 ; MALLOCH et al., 1980;

LEWIS, 1987). However, later reevaluations of these fossils (e.g., PIROZYNSKI and

DALPC, 1989) make them more likely candidates of saprobic groups. Be that as it

may, fungi were early land colonizers and continued gradually to conquer the

diversifying ecological niches.

All fungi are heterotrophs, completely lacking photosynthetic pigments and

chloroplasts and feeding by way of uptake of dissolved nutrients. In this single

fundamental respect they differ from almost al1 plants. In fact, when considering the

Uiree phylogenetically most recent and most closely related kingdoms of the

taxonomie superkingdom Eukaryofae - the Plantae, Animalia and Fungi - the

crucial determinant for the assignrnent of organisms into one of these three crown

taxa is their mode of nutrition (MOORE. 1996):

Plan tae: autotrophic or closely related to autotrophs

Animalia: phagotrophic heterotrophs or closely related to phagotrophs

Fungk al1 lysotrophic heterotrophs

The ecological roles that these three kingdoms have played are those of

producers, consumers and degraders. Howewer, the thickness of coal deposits from

the Carboniferous (360-286 Ma b.p.), derived from the recalcitrant remains of

pteridophytes, bears witness to a world in which fungi could not yet fiourish

(CORNER, 1964; cited in MOORE, 1996). Only the advent of easily rotting flowering

plants enabled an explosion of the fungal biota, lasting up to the present time.

TAXONOMIC AND PHYLOGENETIC POStTfON OF THE FUNGl

Profound differences between fungi and other "tnie" plants have been

recognized early on and many mycologists in the past were unwrnfortable with fungi

being classifieci among plants- Their generaf lack of motility, absence of

photosynthesis. ingestion of dissolved nutrients, presence of ceIl walls and a

fonerly assumed (and later largely abandoned) descent from algae were sufficient

to regard them more closely related to plants than to animals (HICKMAN, 1965).

Consequently, fungi ended up in the taxonornic kingdom of plants at a time when

only two kingdoms were established. Atternpts to increase the number of these

largest taxa to three (HAECKEL, 1866) or even five (COPELAND, 1956), in order to

arrive at a natural, phylogenetic system of classification and to accomodate protists,

did not alter the taxonornic position of the fungi as a whole.

As late as three decades ago fungi still cunstituted the second phylum (or

division) - Mycota or Mycobionta - of the kingdorn Plantae. But a change was

imminent due to the following developments in biology (ALEXOPOULOS et al.. 1996):

realization that the hitherto employed kingdom classification is unnatural;

acknowledgment of fungal polyphyly;

acceptance of phylogenetic systematics;

application of methods molecular systematics;

discovery of new taxa;

increasing and re-evaluated fossil records.

Decisions about phylogenetic relatedness are complicated by the inherent

diffÏculty in differentiating between primitive and acquired characters. Nowadays,

fungi are categorized in a separate kingdom (WHITTAKER. 1969), not only because

of their differences from "true" plants (and their similariaes to animals), but also

because of their problematic and still incompletely resolved evolutionary origin.

Phylum Chytndiornycota Phylum Zygomycota Phyf um Ascomycota Ph y lum Basidiomycota

Phylum Oomycota P hy lurn Hyphohytnomycota Phylum Labynnthulomycota

Protists

Phylum Plasmodiophoromycota Ph y l urn Dictyosteliornycofa Phylum Acrasiomycota Phylum Myxomycota

Figure 1.1.2.1 Modem classification of organisms from the former phylum Mycota (according to HAWKSWORTH et al.. 1994).

The long overdue and finally formal dissociation of fungi from plants paved

the way for further separation of fungal taxa. The classical phylum of "fungi" is

definitely polyphyletic, which is most conclusively demonstrated by cornparison of

the small subunit ribosornal DNA (SSU rDNA) fragments (e-g., BRUNS et al., 1991 ;

1993). In order to account for this poiyphyly, HAWKSWORTH (1 991 ) proposed the

terni fungi (with lower case 9, to denote "organisms studied by mycologists". What

used to be the lowest subclass of the primitive, and abandoned taxon of

Phycomycetes - the Oomyceüdae, was promoted to the level of the newly

proposed kingdom Chrornista (FORSTER et al., 1990). This designation was not

8

accepted, however. and the previously proposed kingdom of Stramenopiia

(PATERSON, 1989) now includes oomycetes as well as other former fungi

(hyphochytrids and labyrinthulids) and some taxa traditionally belonging to algology.

The most recently accepted classification places "traditional fungi" into two separate

kingdoms and four protist phyla (Fig. 1 .1.2.1).

Plants

rl - Amoebofiagellates

"--- Euglenoids

Figure 1.1.2.2 Polyphyiy of the "traditional fungi" (boldface), mostly according to SSU rDNA sequencing data, and their position among other taxa (compiled frorn various authors by ALEXOPOULOS et al., 1996, q.v.).

9

The first two of the former sub-phyla of the Mycota, the cellular slime molds

(Acrasiomycota) and aie true slime molds (Myxomycota) have been split into four

phyla, but common ancestors are still elusive (PATTERSON and SOGIN,-1992).

Phylogenetic relationships of the Fungi with other organisms are shown in Figure

1 -1.2.2.

The taxonomy of the fungi is süll changing on al1 levels as new

molecular data become available. Therefore, it should not be assumed that the use

of the terni fungi in the continuation of this text automatically excludes those

organisms sensu HAWKSWORTH (1 991 ) which phylogenetically do not belong into the

kingdom Fungi, particularly since many quotations from literature pre-date or simply

disregard this still unresolved matter.

1.2 FUNGAL CELL WALLS

Any functional living ceIl is equipped with a cell membrane

(cytoplasmic membrane or plasmalemma) - a complex partition between self and

non-self, to separate and protect its metabolic machinery from instabilities and

hazards of the outside world and to exploit resources fully. lntracellular functions

and processes are compartmentalized by similar structures. Most cellular or

subcellular membranes are topologically stnictured, Ruid double layers of

amphiphiiic molecules, and structures enclosed within such membranes could be

compared to micelles of water-in-oil-in-water emulsions.

This combination of hydrophilicity on the intemal and extemal surfaces

with hydrophobicity in the interior of the membrane entails cnicially important

functionalities to the cell membrane. Some of these functionalities are:

maintenance of an aqueous cell interior in aqueous environments,

prevention of simple diffusion into aie ceIl of rnany low-molecular weight

compunds, including nutrients and potentially hazardous substances,

prevention of simple diffusion out of the cell of many products of cell

rnetabolism,

establishment of a physical barrier for macromolecules, including enzymes,

maintaining of appropriate concentrations and gradients of precursors,

products and electrolytes,

maintaining electric fields by ionic charge separation and thereby enabling

bioenergetic processes,

providing a matrix for the incorporation of membrane-bound and

transmembrane proteins, enzymes and enzyme complexes,

providing a matrix for structures of intercellular recognition and signal

transduction.

The most important functionality which the membrane cannot provide

is structural stability and mechanical resistance. These are the main properîies of

cell walls - physically n'gid outer envelopes separated from cell membranes by a

periplasmic space. Without such reinforcements. fungal protoplasts would lyse in

media of lower osmotic pressure. Other, l e s obvious funcüonalities associated with

cell walls may be defined as mu l t i f~n~ona l organelles of protection, shape, cell

interaction, signal reception, attachment and specialized enzymic activity (FLEET,

1991 ).

Not al1 cells are equipped with walls. Protobacteria are assumed to

have posessed cell walls as early as about 3.5 billion years ago (BARTNICKI-GARCIA,

1984), and al1 extant eubacteria and archaebacteria have them. Among the

eukaryotic taxa, cell walls are missing in animal and protist cells. With a

phagotrophic mode of nutrition characterizing the latter two taxa. cell walls would be

an impediment, whereas the photoautotrophism of plants and the lysotrophism of

fungi (both accompanied by the buildup of high osmotic gradients against the

exterior) mandate the presence of an external reinforcement (RUIZ-HERRERA, 1 992).

Prokaryoüc cell walls are very different from their eukaryotic analogues and their

further description lies beyond the scope and purpose of this chapter.

The main components of al1 fungal cell walls are polysaccharides

(typically -80%) and proteins (typically 3% to 20%). Polysaccharides may be linear

or branched, cross-linked or not via sugar moieties or other small molecules (e-g.

amino-acids). In addition to simple sugar monomers as building blocks (rnainly

glucose), sugar derivatives (e.g., amino-sugars and uronic acids), and substituent

groups (such as acetyl) may be present. Types and amounts of sugars found in cell

walls of individual taxa are given in Table 1.2.1.

f he principal component of plant ceIl walls is cellulose, Po-(1+4)-

glucan. Except for one known species from the phylum Chytndomycota (FULLER

and CLAY, 1992), cellulose is absent in Fungi, but is the main cell wall element in

Stramenopila (oomycetes and hyphochwds). Pectins, which are partially

rnethoxylated poly[l,4a-~-galacturonides] with varying degree of este rification, and

are present as a reinforcing cernent in the central larnellae between plant cells, have

not been found in fungi.

Fungal wall polysaccharides are a broad class of homo- and

heteropolymers, some of them in the fom of glycoproteins, with very different

funcüons, ranging from structural rigidity in al1 species, to virulence in pathogenic

fungi (e.g . . SAN-BLAS et ai., 1 977), to protedive capsule formation (e.g . , GQLUBEV,

1991).

BARTNICKI-GARCIA (1 968) identified eight chernotypes of fungal cell

walls according to their main polysaccharides. Only five of them characterize the

monophyletic fungal kingdom of today (Table 1.2.2).

-- 1 Chitosan-chitin 1 Zygomycota (except Tnchornycetes) - 1 Chytridiomycota, "Euascomycetes" (i .e., filamentous Ascomycetes). "Homobasidiomycetes" (i.e., Hymenomycetes and Gasteromyce ts) , Deuteromyce tes

Mannan-glucan

Mannan-chitin

Table 1.2.2 Polysaccharide chernotypes of fungal cell walls (after BARTNICKI- GARCIA, 1 968).

"Hemiascomycefes" (Le., Archiascomycetes and Saccharomycetales)

"Heterobasidiomyce fes" (i. e., Urediniomycetes, Usfilaginomycetes)

Polygaiactosamine- galactan

Fungal walls are ever-changing cornplex composites of different

materials, held together by non-covalent interactions and possibly covalent bonds

consisting of (a) polysaccharide fibrïls (chitin and glucans); and (b) a matn'x in which

Tkhomycetes

the fibrils are embedded, consisting of kand a-glucans, chitosan, polyuronides,

glycoproteins, Iipids, salts and pigments (RUIZ-HERRERA, 1992; p. 9).

One of the main constituents of fungal cell walls is chitin, poly[1,4-P-2-

acetamido-2deoxy-D-glucopyranoside], determined to contain about 2,000 sugar

monomers boa when obtained from crustaceans (MU~ARELLI, 1984) and when

enzymatically synthesized in vitro (CALVO-MENDES and RUIZ-HERRERA, 1 987). C hitin

is present in fungal cell walls in amounts of about 2% to 20% wall dry rnass. Low

values are generally found in yeasts, whereas the content in sorne fungi may be

considerably higher (-60%) (RUIZ-HERRERA, 1978). In the cell wall of S. cerevisiae

chitin accounts only for about 1.2% of the total dry mass (CABIB and BOWERS, 1971 ;

BERAN et al., 1972), but most of it is localized in bud scars (up to -70%; HOLAN et

aL, 1981).

Characteristic for the Zygomycetes is chitosan, poly[l,4-PZ-amino-2-

deoxy-o-g lucopyranoside], which is synüiesized by chitin deacetylation (DAVIS and

BARTNICKI-GARCIA, 1984) and does not appear to have any direct structural role.

Land plants lack chitin and chitosan completely, but chitin occurs in green algae,

diatoms chrysoflagellates, protozoans, coelenterates and nematodes, and iç the

major organic structural material in tf?e annelids, molluscs and arthropod

exoskeletons (RUIZ-HERRERA, 1992; p. 91).

In the context of this thesis the most important aspect is the presence

of homopolysaccharides that provide mechanical strength. These are characterized

predominantly by k(1-3) and P(1-+6) glycosidic Iinkages between the sugar

moieües. The natural conformation of a-glucans is an intramolecular spiral,

resulting in arnorphous and more readily soluble matrices (e.g., amylose in starch).

On the other hand, repeating P-glucosidic bonds irnpart to the poiymenc chains an

increased tendency to intermolecular hydrogen bonding , the consequence of which

is a higher crystallinity and a lower solubility of the polymer (e.g., cellulose). An

intertwined triple helical structure with six glucose subunits per tum has been

proposed for the unbranched P-1.3-glucan synthesized by recovering protoplasts

of Saccharomyces cerevisiae (KOPECKA and KREGER, 1986). A collection of

bonding types and properties of the main P-glucans is given in Table 1.2.3.

The differences in solubility between individual glucans shown in Table

3 may be due prirnarily to the size of the molecule. but also to the exclusion of water

from densely packed clusters of high crystallinity. In addition to that, evidence was

found of chitin-glucan covalent bonding. In the case of yeasts this was shown for

ceIl walls of Candida albicans (SURARIT, et al., 1988) and bud scars of S. cerevisiae

(MOL and WESSELS, 1987).

The most important type of branched glucans consists of chains of

el ,3 giucans wiUi significant proportÏons of pl ,ô-glycosidically linked side chains.

Among yeasts, the most thoroughly studied ones are those from S. cerevisiae.

Linkage type

p1,3 unbranched

Solubility

Alkali

pl ,3 with occasional pl ,6 branching of single glucose uni&

pl ,3 with significant Pl ,6 branching Alkali or 1 insolu bIe

Water

p1,3 m-1.4

&1,3 / ~ 1 , 4 / p-1,6

Occurrence

Synthesized by regenerating yeast proto plasts

Un known

Alkali

lntracellular mycolarni- narans, extracellular mucilage

-- -

Achlya , Anniilarfa - - - -

Most abundant celf watl glycan in yeasts

Fraction of yeast glucan

Table 1.2.3 Different Pglucans f'orn fungi (from RUIZ-HERRERA, 1992)

INTERFUNGAL RELATIONSHIPS - MYCOPARASITISM

Mycoparasitic fungal interactions (Figure 1 -3.1 ) are numerous and

many are well characterized. Both nectrotrophic and biotro phic hostlparasite

relationships between filamentous fungi exist (JEFÇRIES and YOUNG, 1994; pp. 46-

78). Combat strategies for the antagonizing fungi are outlined in Figure 1.3.2.

Contact necrotrophy can result from, or be induced by close proximity between

antagonistic mycelia with or without physical contact. Such interactions are also

known as hv~hal interference (IKEDIUGWU and WEBSTER, 1970). On the other hand,

destructive penetration of one mycelium by another is categorized as invasive

necrotrophy (LUMSDEN, 1992). The various kinds of antagonistic interactions at the

level of mycelial interfaces are represented in Figure 1.3.3. Common phenornena

in the destruction of the attacked rnycelium include marked alteration in the

16

pemeability of the plasmalemma. loss of turgor. granulation. vacuolation and

coagulation of the cytoplasm. In some of those interactions autolytic phenornena

in the host mycelium also appear to play a role. Interference can be rnediated by

hydrolases (e.g.. chitinases, ~1,3-glucanases, proteases) (RIDOUT et al., 1988). the

activity of which may increase in the presence of host cell wall, even in the forrn of

purified cell wall components (ELAD et al., 1 985). Low-molecular weig ht factors

which diffuse through cellulose membranes were also found to be responsible for

necrotrophic interactions (IKEDIUGWU and WEBSTER, 1970).

1 NEUTRALlSTlC 1 1 PRIMARY RESOURC 1 (COMM ENSALISTIC) 1 1 CAPTURE 1 - - - - -- -

Not detrimental to either, not Initial colonization of beneficial to both organisms. substrate by a fungus.

Beneficial to both I SECONDARY RESOURC CAPTURE

organisms. Cornpetifive replacement of one fungus by another within a partiwlar niche.

Figure 1.3.1 Types of fungal interaction (after COOKE and RAYNER. 1984).

1 YESYUISMS 1 o y e s o /.ROM/ OFCOMBAT Contact? a Total? MYCELiAi (ANTAGONIS INVOLVEMENT

(e.g., volatile and (e.g., parasitism and non-volatile antibiosis) hyphal interference)

Figure 1.3.2 Mechanisms of fungal combat (after RAYNER and WEBBER. 1984)

I Yes I

Speciatized interfaces

BIOTROPHS 810TROPHS

Figure 1.3.3 Interface types in mycoparasiüsm (from JEFFRIES and YOUNG, 1994)

YWSTS AS FUNGAL PARASITES AND PREDATORS

Yeasts do not occurr at random in the biosphere but form

communities of species (LACHANCE and STARMER, 1998). LACHANCE (1 990) defines

the yeast habitat as "a place or collection of places that share sufficient similarities

to result in their tendency to harbor similar yeast wmmunities". There are four

major terrestrial yeast habitat types - soil. plants, animals. and the atrnosphere (Do

CARMO-SOUSA, 1969). Soif, along with aquatic habitats, is problematic to a degree,

in that it is sometimes difficult to detemine whether a particular yeast is an

autochthonous or an allochthonous organism for a particular habitat. Yeast

communities and their natural habitats are cuvered in detail by PHAFF and STARMER

(1 987).

Yeasts are non-rnotile, stnctly chernoorganotrophic microfungi

(WALKER, 1998). They posess relatively modest physiological faculties (LACHANCE,

1990) and are consequently saprotrophic or, in a minonty of cases, parasitic on

animais and man. Members of both of these groups benefit from the ready

availability of nutrients in their respective habitats. Yeasts are not usually among the

eariy colonizers of more recalcitrant substrata.

Antagonistic interactions among yeasts have received much less

attention than those among filamentous fungi and lower fungal genera. It may be

argued that the opposite would be ramer surprishg because of their predominantly

saprotrophic way of life. One may expect cornpetition for nutrients to take place

arnong yeasts, as it normally happens when mixed microbial populations share

common resources. Other than that, only two tnily antagoniçtic phenornena among

yeasts are known as of now - the killer phenomenon and yeast predation.

THE KJLLER YEAST PHENOMENON

Certain yeast species produce extracellular, diffusible proteins

or glycoproteins (killer toxins) which are lethal to other (sensitive) yeast strains, but

to which the producers thernselves are immune. The phenornenon was first

observed with Saccharomyces cerevisiae strains 35 years ago (MAKOWER and

BEVAN, 1963), and was extensively reviewed since (e.g., YOUNG, 1987; WICKNER,

1992,1996). Similar toxins were later found to be produced by other fungi, namely

Ustilago (KOLTIN, 1988) and Polysphondylium, an acrasid slime mold (MIZUTANI et

al., 1990). Therefore, the name mycocins (coined after the bacteriocins) has been

proposed as preferable to the hitherto used term yeast killer toxins (GOLUBEV, 1998).

Mycocin production (killer activity) may be assayed (YOUNG, 1987)

by inoculating the suspected killer strain onto a n'ch nutrient agar containing

suspended cells (usually =4 x 10' ml'') of a mycocin-sensitive strain. The agar is

supplemented with methyiene blue (30 mg/L). Since the toxins are active in a

relatively narrow range of pH values (3 s pH s 6), the media are customarily

bufiered with sodium citrate (4.3 s pH s 4.7). Killer activity becornes manifest after

an incubation for 2-3 days, usually at 1 8-20°C (because of toxin thermolability). A

growth inhibition zone around the inoculurn is surrounded by a halo of blue-stained

dead cells of the sensitive strain. This zone of dead cells is indicative of a mycocin-

producing strain.

The discovery of S. cerevisiae rnycocins has ultimately led to an

intensive search for this trait in sorne existing yeast collections. The most

comprehensive of these surveys was the one in the National Collection of Yeast

Cultures (Colney Lane, Norwich, UK), done by PHILLISKIRK and YOUNG (1 975).

They found killer strains with a frequency of 6% (59 out of 964 strains from 28

genera) among commercial strains, and 31 % (27/86) among standard genetic

strains of the same collection. The study also revealed mat the killer trait was not

a peculiarity of Saccharomyces. Strains from the genera Candida, Debaryomyces,

Kluyveromyces, Pichia, Tonrlopsis (Candida), and, es pecially , Hansenula were

20

found to be kiiler toxin producers. The search for yeasts in natural habitats (STUMM

et al., 1977) gave an incidence of 17% (2611 57). Today, there are about 80 known

mycocinogenic species from about 20 di#ferent genera (Go~uew, 1998), as outlined

in Table 1.3.1.1 -1.

Yeast mycocins are protein or glycoprotein dimers or tnmers, usually

with a relative molecular weight of ICI-20 kDa. However, glycoprotein rnycocins

from Pichia and Khyveromyces are one order of magnitude larger (2 100 kDa).

Genes encoding Saccharomyces mycocins are based on Iinear

&RNA plasmids (WICKN ER. 1 996). Mycocins from KIuyveromyces laciis (STARK et

al., 1990) and Pichia acaciae (MCCRACKEN et al., 1994) are encoded on linear DNA

plasmids. A number of other killer yeasts inherits the mycocin genes

chromosomally, and for others still, the genetics of inheritance is unknown

(references in GOLUBEV, 1998, and WALKER, 1998).

Genus Genus Genus

Candida Hanseniaspora Rhodotorula

Saccharomyces

Kluyveromyces Cysto filobasidium

Trichosporon Debaryomyces

Table 1 .LI .1.1 Mycocinogenic yeast genera (after GOLUBEV. 1998)

All mycocins are divided into 12 classes (killer phenotypes, K,-KI,

and ha), according to cross-reactivity between species (YOUNG, 1 987) of which the

toxÏns Ki-K, and K, are secreted by Saccharomyces strains. One of thern (K,)

requires two viruses. MI and LA. The MI virus encodes a preprotoxh which is

hydrolyzed into the mycocin itself, an ap dimer (PALFREE and Busseut 197b), and

into a resistance factor, a giycosylated y-peptide (ZHU et al. 1993). The L-A virus

carries the gene for the capsid protein of both viruses (WICKNER, 1996).

The mode of action is diMerent for the individual mycocins. The best

studied system is the one of K, toxin (SKIPPER and BUSSEY, 1977; DE LA PENA et al.,

1981). About 1 o4 KI molecules are needed to kill one sensitive cell and the activity

reaches its maximum after 2 3 hours of exposure (SKIPPER and BUSSEY, 1977). It

starts with the attachment of the Psubunits to the j3-(1+6)-glucan chains in the cell

wall of the susceptible species. After mat, a-subunits are inserted through the cell

membrane. thereby forming channels which upset tJie transport of H' and K+ ions,

amino acids and ATP (MARTINAC et al., 1990).

No known mycocins are active against prokaryotes, plants or

anirnals. However, it has been shown that some mycocin-producing yeasts inhibit

the growth of a number of wood-rotang and phytopathogenic fungi (WALKER et al.,

1995).

YEAST PREDATION

As early as 1973 morphological feahires of anastornotic

interacüons between the filamentous yeast Saccharomycopsis (Althroascus,

Endomycopsis) javanensis and several other yeast genera had been studied

(KREGER-VAN RIJ and VEENHUIS, 1973). Electron micrographs revealed the

formation of denticles on hyphae and single cells of A. javanensis. These, in tum,

grew out into stalks which penetrated cell walls of other susceptible yeasts. It was

unclear, however, whether the process of penetration was parasitic in nature, or

whether it occurred only on dead cells.

Studies conducteci in this department (LACHANCE and PANG, 1997)

suggest that yeast penetration is, in fact, predation. The phenornenon appears to

be rare. Thus far, only eleven species have been shown to behave as predators

under certain conditions. Eight of them are teleomorphs (Table 1.3.1.2.1). The

remaining three are anamorphs belonging to the genus Candida (Table 1.3.1 -2.2).

The teleomorphs in Table 1.3.1.2.1 are phylogenetically related

(KURRMAN and R O B N E T 1995) and are currently classified as members of the

same genus Saccharomycopsis (KURTZMAN and FELL, 1998).

Interestingly, al1 these species share an unusual requirement for

organic sulfur. From an evolutionary aspect, a metabolic deficiency of SO,-uptake,

which per se would be quite a disadvantage in cornpetition with prototrophic

populations, could have becorne a seledive benefit to such auxotrophs in

environments where high levels of toxic sulphate analogues are encountered:

selenate(V1) (Seo?) and chrornate(V1) (~102') ions are both transported across the

cell membrane by Iow-specif~city S0:--permeases (BRETON and SURDIN-KERJAN,

1 977).

According to MHANCE and PANG (1997), the following general

prerequisites have to be fulfilled for predation to occur: (1 ) a nutrient medium poor

in organic sulfur, (2) growth on a solid support or, at least, in a stagnant and

convection-free layer of liquid, and (3) presence of a suitable organism as prey.

Yeast predation involves fomation of protuberances (haustoria) on

the cell surface of the predator grown in rnixed culture with its prey, subsequent

penetraüon of the tips of the haustoria into vegetative prey cells and/or spores. The

overall outcorne of this antagonistic relationship is a disniption of the cytoplasmic

integrity of the prey cells and, in sorne cases and under certain conditions. a

decimated prey population.

Predacious yeasts are not obligate parasites, as they grow

saprophytïcally on common media. In fact, n'ch, cornplex media or some simple

media with added methionine may suppress predation (LACHANCE and PANG. 1 997).

Moreover. it appears that the induction of hailstorium formation and the penetration

23

of prey cells is a function of organosulfur availability in the environment Because

predation-eliciting signais yield a positive response only when predator and prey

cells are in close proximity, sulfur-containing metabolites may act as chemotactk

signals for the induction of haustoflurn formation. The fact, however, that organisms

with radically different cell envelopes, e.g., the yeast-like alga Protofheca zopfii

(CONTE and PORE, 1973) failed to elicit a predatory response could mean that a

specific ledn-mediated dl-cel l recognition between predator and prey is needed.

Systematic name in use at time when predacious behaviour was demonstrated

Arfhroascus javanensis (Klocker) von Aix (1 972)

New classification as Saccharomycopsis (KURTZMANN and SMITH, 1998)

S. javanensis (Klocker) Kurtzmann 8 Robnett (1 995)

Arthmascus fermentans C.-F. Lee. F.- L. Lee, Hsu & Phaff (1994)

S. fermentans (C.-F. Lee, F A . Lee, Hsu & Phaff) Uurtanann & Robnett (1 995)

Afihroascus schoenii (Nadson 8 Krasil'nikov) Bab'eva, Vustin, Naumov & Vinovarova (1 985)

S. schoenii (Nadson & Krasil'nikov) Kurtzmann 8 Robnett (1 995)

Botryoascus synnaedendrus (D. B. Scott 8 van der Walt) von AIX (1972)

Saccharomycopsis crataegensis Kurtzmann 8 Wickerham (1 973)

S. synnaedendra D.B. Scott & van der Walt (van der Walt and D.B. Scott 1971)

--

Guilliermondella selenospora Nadson 8 Krasil'nikov (1 928)

1 UNCHANGED

- - -- -- -

S. synnaedendra (Nadson & Krasil'nikov) Kurtzmann & Robnett (1 995)

Sa ccharomycopsis fib uligera (Lindner) Klocker (1 924)

Table 1.3.1.2.1 Yeast teleomorphs with demonstrated predacious behaviour

Saccharomycopsis malanga (Dwidjoseputro) Kurhrnan, Vesonder & Srniley (1974)

UNCHANGED

1 Swcies name 1 1 Candida sp. [UWO-PSI 91-124.1 1 1 Candida sp. [UWO-PSI 91-127.1 1

Candida sp. [UWO-PSI 95-697.4 (Strain 'Wl")

Table 1.3.1.2.2 Yeast anamorphs with demonstrated predacious behaviour

1.4 THESIS OBJECTIVES

Based on what was said in the previous Section 1.3.1 -2, the following

study objectives were defined pnor to the beginning of experirnental work:

- To continue the study of phenomena associated with yeast predation by

selecting potential predator-prey pairs upon rnicroscopical and electron

rnicrosco pical examination of cultures.

To fmd growth conditions (environmental factors) underwhich penetration

does not take place, in order to help explain the reason for yeast

predation.

To investigate the production of hydrolases and the hydrolysis of prey cell

wall components associated with penetraoon.

MATERIALS AND METHODS

MICROORGANISMS

All microorganisms used in this study are preserved under liquid nitrogen

in the culture collection of the Department of Plant Sciences, University of Western

Ontario (UWO[PS]) and with only one exception (the alga Prototheca zopfi!' all of

them were yeasts or yeast-like fungi. They have been selected on the basis of their

predacious behaviour or their potential to serve as prey. The origin of isolates in

both groups is summarized in Table 2.1 .l.

1 Saccharomycopsis javanensis 1 82-52 ( Oak flux 1

1 PREDATOR

Saccharomycopsis ja vanensis

1 Saccharomycopsis synnaedendra 1 96-1 2.1 1 Oak frass 1 1 Saccharomycopsis selenospora 1 8 1 -1 08 1 Oak flux 1

1 UWO[PS] No.

92-247.1

1 ~accharomycopsi& fermentans 1 CBS 7830' 1 Orcahrd soi1 1

1 ORlGlN

Drosophila sp.

Candida strain "Wq" I 95-697.4

1 Aureobasidium pullolans 1 95879.1 1 Hibiscus fly 1

Hibiscus beetle

Rhodotorula M u t a

Schizosaccharomyces pombe

1 Prototheca zopW 1 95-917.2 1Opuntiarot 1

Table 2.1 -1 Yeast strains studied as potential predators and preys (* Centraalbureau voor Schimmelcultures, Julianlaan 67, 2628 BC Delft, The Nemerlands )

1 PREY 1

-

95-922.4

94-208.2

Saccharomyces cerevisiae 96.6

Me fschniko wia hibisci 1 95-747.4

Hibiscus fiy

Fermentation

Fermeniing birch sap

Hibiscus fl ower

Nutrient media were used for recovery of cultures after liquid hi, storage

(YM agar), maintenance of cultures in the course of study (GY agar and YE-fortifieci

GY agar), colony counts (selective media) and individual experiments (special

media designed for predation assessment).

The composition and properties of basic media (solidified form) are given

in Table 2.2.1. Selecüve media for wIony counts were used to detenine selectively

the cell numben of predators and preys. The composition and properties of the

latter are given in Table 2.2.2.

Composition Description

YM + 1 ppm

YM + Cyc cycloheximide

- -- - -

YM + 5 ppm YM +

cetyltrimethylammonium CTAB

bromide (CTAB)

Selective for predacious yeasts.

lnhibits growth of prey yeasts used in

this study.

Selective for Metschnikowia hibisci.

CTAB inhibits growth of predator.

Table 2.2.2 Selective media - composition and properties

2.3 SELECTION OF MOST ACTIVE PREDATOR-PREY COMBINATIONS

Nearly equal amounts of actively growing potenüal predator and prey

cultures were picked from maintenance medium (GY agar slants), combined on GY

agar plates (predation medium), and incubated at 25'C. After 24 hours, sarnples

of the mixed cultures were transfered to thin agar slabs (1.8% w/v) on microscope

slides, covered with coverslips and examined for haustorium formation and cell

penetration under phase contrast (oil immersion, magnification 1000x) with a Leitz

Orholux microscope.

W hen Schizosaccharomyces pombe was studied as the potential prey of

Saccharomycopsis javanensis. the predator was inoculated first onto GY agar,

incubated for 24 hours at 25°C. and then mixed with S. pombe and incubated for

another 24 hours before rnicroscopy.

2.4 SEARCH FOR MEDIA COMPONENTS THAT AFFECT PREDATION

To a 20-gR aqueous solution of yeast extract (Difcu) an equal volume of

97% (vEv) ethanol or acetone was added slowly and under agitation. The resulting

mixtures separated into a solid phase (precipitate) and a clear aqueous solvent

phase (supernatant). The supematants were carefully decanted and evaporated in

a water bath. These evaporates as well as the collected precipitates were

subsequently dried ovemight in a vacuum desiccator. Each of the four fractions

thus obtained was redissolved in the initial volume of water. These solutions were

supplemented with glucose (1 0 g/L) and agar (1 5 g/L), and after sterilization plates

were poured.

Another series of plates was prepared, containing the usual predation

medium (GY, see Table 2.2.1), suppiemented with one other complex nutrient

Chosen were malt extract, peptone and tryptone (20 glL each), and a vitamin

mixture (140 mgR) (al1 from Difco).

Predation on these media was assayed as describeci in Section 2.3.

2.5 LlGHT MICROSCOPY OF PREDATION

A thin slab of GY agar was placed on a sterile microscope slide,

inoculated with a mixture of Saccharomycopsis fennentans and Saccharomyces

29

cerevisiae, covered with a sten'le coverslip, and left at the microscope stage for up

to 3 days. The mixed culture was periodically inspected and photographed.

2 -6 LIGHT MICROSCOPY OF POST-PREDATION VlABILlTY

Microscopy was performed after predator and prey had grown together

for a period of tirne on predation agar (GY). Their subsequent recovery on rich

medium (YM) was studied microscopically as described in Section 2.5.

Approximately equal amounts of Saccharomycopsis javanensis and

Saccharomyces cerevisiae were transferred from GY agar maintenance slants.

mixed together on GY agar plates (predation medium) and incubated for ca. 15

hours at 25°C. The mixed culture thus obtained was suspended in sterile H,O,

transferred onto a thin slab of YM agar, placed on a sterile microscope slide. and

covered. Selected regions in the field of view were photographed ai diHerent

intervals to document changes (Plate 3.2.2.1-A).

2.7 ELECTRON MICROSCOPY OF INTERACTIONS IN LIQUID MEDIA

The following four predator-prey pairs were cultured in still and shaken

liquid predation medium (GY) and studied by scanning electron microscopy:

Saccharomycopsis javanensis - Saccharomyces cerevisiae

Saccharomycopsis javanensis + Schizosaccharomyces pombe

Saccharomycopsis synnaedendrus -r Metschnikowia hibisci

Candida strain "W 1 " -, Metschnikowia hibisci

Pure cultures were taken from maintenance slants and suspended in

small amounts of sterile water. The suspensions were vortexed at medium speed.

Predator suspensions (20-pL aliquots) were wmbined with prey suspensions (200-

pL aliquots) in 25 mL of liquid predation medium in 250-mL Erlenmeyer flasks.

Incubation was camed out at room temperature for 3 or more days.

Scanning electron microscopy was perforrned as follows:

Ceils Born liquid predation medium (204- aliquots) were placed on

Nuclepore membranes (0.45 Pm pore sire) resting on agar plates. The membranes

were floated onto 2.5% (wlv) glutaraldehyde in 0.1 M sodium cacodylate buffer.

ARer fixation for 15 min or more, the membranes were rinsed in cacodylate buffer,

dehydrated for 15 min in 1,Z-dimethoxypropane (propylene glycol dimethyl ether)

acidified with dilute HCI and critical-point dried. Sarnples were gold-coated by

plasma sputtering (5 min), and observed in a Hitachi F4500 Field Emission

Scanning Electron Microscope. Images were recorded electronically (Plates 3.3.1,

3.3.2 and 3.3.3).

2.8 PREDATION MONITORING ON SELECTED MEDIA

(COLONY COUNTING)

Cultures were grown on the surface of a piece of dialysis membrane

resting on agar medium. The membrane barrier serves as a nument-permeable

support for the growing cultures and allows the entire biomass to be removed from

the culture medium. Cell loss is thereby minimized and reliable colony counts can

be obtained.

Separate suspensions of predator and prey cells were prepared from pure

cultures grown on maintenance agar slants and vortexing in sterÎle &O. Celi

densities of these original suspensions were made to be roughly equal, and aliquots

of suspensions were mixed at a 1/10 predator-prey ratio.

Controls were prepared by diluting separate predator and prey suspen-

sions to the same respective concentrations as in the mixture.

Mixed suspensions and controls thus prepared were applied as 20-pL

aliquots into the centre of pieces of dialysis membrane (3 cm x 3 cm) lain flat on the

surface of a selected agar medium in a PetrÎ dish. Multiple (up to six) membrane

cultures of each predatodprey combination and control were initially prepared and

incubated at 25°C.

At selected intervals individual plates were withdrawn from the incubator

for counting. This consisted of lifong the membrane from the underlying agar,

submerging it into 3 mL of sterile surfactant-supplemented water (Tweeng 80. ca.

10 mgfL) and vortexing at medium speed. Without removing the membrane, a serial

dilution of the resulting suspension was made by transfemng 200 PL of the undiluted

suspension into 3 m l of surfactant-supplemented sterile H,O and repeating this

same dilution pattern 4 to 5 times as required.

Suitable dilutions were plated for colony counts. Twenty-microlitre

aliquots of each dilution were applied, in triplicate, ont0 the surface of YM agar with

or without selective agents (see below) and the liquid was alfowed to dry. A total of

12 spots per plate were thus obtained. The plates were incubated for 24 hours and

the small colonies were counted under a dissection microscope (1 0 x magnification).

Medium composition and incubation temperatures differed depending on

the yeast counted (Table 2.2.2). For Saccharomyces cerevisiae the plates (YM

agar) were incubated at 37°C. At this temperature S. javanensis did not grow.

EFFECT OF INORGANIC SALTS ON PREDATION

Predation of Saccharomycopsis javanensis on Sacharomyces cerevisiae

was studied on predation medium containing selected inorganic salts andlor L-

methionine. Predation on these media was assayed as described in Section 2.3.

A series of plates was prepared with predation medium (GY, Table 2.2.1).

supplemented with one of the following:

10 g/L ammonium sulfate, (NH&SO,

10 g R potassium sulfate, K2S04

10 g/L potassium nitrate, KNO,

10 gR ammonium sulfate, (NH,),SO, + 10 g R m me thionine

10 g/L bmethionine

no additive (control)

2.1 O PREDATION-ASSOCIATED ENZYME ACTIVITIES

2.10.1 PRELIMINARYTESTS

Preliminary tests were carrieci out to assess the presence or absence of

P-glucanase and chitinase adivities in predator-prey pairs of Saccharomycopsis

javanensis with Saccharomyces cerevisiae and Candida strain 'W1" with

Metschnikowia hibisci grown separately and together on predation agar (GY) and

on LNB agat with added DL-methionine (1 glL).

Plates with dialysis membranes were inoculated with suspensions of pure

cultures and their mixtures (cf. Section 2.8). Incubation was at 25°C. Cells were

harvested after 24 and 48 hours by washing the membranes in 0.5 mL of 50-mM

succinate buffer, pH = 5.2.

To determine extracellular enzyme activities. the ceIl suspensions were

immediately centrifuged (8 x 1 O3 min-') and the dl-free supematank were carefully

pipetted off and mixed with equal volumes of the required substrate solutions

(laminarin or chitin).

The reaction mixtures were incubated at (34 I 1)"C. Five-pL samples

were taken every W hour and applied directiy to the surface of filter paper (Whatman

No. 1) and air dried. The presence of reducing sugars was detected with the

alkaline silver nitrate reaction as described by LACHANCE and PHAFF (1975) (see

reagents in Section 2.9.3).

2.1 0.2 ENZYME ASSAYS

Cultures of Saccharomycopsis javanensis and Sacchammyces cerevisiae

grown separately or together were assayed for hydrolysis of laminarin and yeast

rnannan. Both activiües were assayed spectrophotometrically by measuring the

accumulation of reducing sugars using the Nelson-Somogyi colour reaction as

described by SPIRO (1 966).

The culture medium for this experiment was GY agar supplemented with

5 g/L me thionine (to stimulate predation). Multiple plates wSth dialysis membranes

were inoculated with suspensions of pure cultures or their mixture (cf. Section 2.8).

Incubation was at 25°C. Cells were harvested after 48 hours and 6 days by washing

the membranes in 1 .O ml of 50-mM succinate buffer, pH = 5.2.

To detemine extracellular enzyme activities, the resulting cell

suspensions were immediately œntrifuged (8 x lo3 min-') and the cell-free

supernatants were carefully pipetted off and mixed with equal volumes of the

required substrate solution.

For the assay of dl-bound acüvities, centrifugeci cell pellets were washed

3 times by resuspending thern with vortexing in fresh succinate buffer and

centrifuging. The washed cells were resuspended in 2.0 mL of the respective

su bstrate solution.

The reaction mixtures were incubated at (34 I 1 )OC (Figures 3.7.2.1,

3.7.2.2, 3.7.3.1, and 3.7.3.2). Samples were taken periodically. Those which

contained cells were œntrifuged (8 x IO3 min") and the clear supernatants were

pipetted off for further use.

The reaction in these sarnples was stopped by adding equal volumes of

the "modified copper reagenf' (SOMOGYI, 1952; as quoted in SPIRO, 1966) and

stored in a refrigerator until the end of sample taking, at which time the rest of the

procedure was applied.

Calibration graphs were prepared with varying concentrations of glucose

and mannose in 5&mM succinate buffer, pH = 5.2.

2.10.3 EFFECT OF INORGANIC IONS ON P(1+3>GLUCANASE ACTlVlN

The media described in Section 2.9 were used to grow CO-cultures of

Saccharomycopsis ja vanensis and Saccharomyces cerevisiae on dial ysis

membranes, as detailed in Sections 2.8 and 2.10.2, 2. The enzyme assays were

then camed out after 48 hours as outlined in Section 2.1 0.2, 3 sqq.

2.10.4 THIN-LAYER CHROMATOGRAPHY

OF MONO- AND OLIGOSACCHARIDES

The production dynamics of glucose and oligoglucosides from laminarin

by yeast 1 , b ~ ~ - g l u w n a s e s was monitored using thin layer chromatography.

Predator and prey were cultured and the reaction mixtures with laminarin

as substrate were prepared and incubated as outfined in Section 2.9.1. The only

difference was that the reacüons were stopped by appling samples ont0 the surface

of TLC-plates.

Prior to the enzyme assay tests were conducted to detemiine the best

chrornatographic conditions (stationary phase and solvent system, spot application

volume, single vs. multiple elution). Prefabricated Kieselgel60 TLC-plates, 20 cm

x 20 cm, 0.2 mm layer thickness (E. Merck, Darmstadt, Germany) were chosen.

Pre-drying of plates did not result in any improvement Best separations were

achieved with the temary eluent consisting of 3 parts n-butanoi + 1 part glacial

acetic acid + 1 part water (STAHL, 1969). Double development in a single direction

gave the most satisfactory results.

Individual sarnples were applied in 5-pL aliquots approx. 3 cm from the

lower plate edge, spaced 1.5 cm from each other (12 spots per plate). During

application the spots were dned with a hair drier. The duration of a single

developrnent in the above solvent system, up to about 1 cm from the upper plate

edge, was 5% houn. Pnor to the second development the plates were dried

ovemight in a vacuum oven (approx. 50°C).

The separated spots were visualized by spraying with Reagent 1 [0.5 mL

saturated aqueous Ag NO, in 100 mL acetone, solubilized by the dropwise addition

of water]. After drying, the plates were sprayed with Reagent 11 [2.3 mL 55% (wlw)

NaOH in 100 rnL 95% ethanol].

The staining procedure was repeated until the spots were dark. Spot

visibility was enhanced by heating the plates in an oven for 1 - 2 minutes at 120°C

after the final spraying.

2.1 0.5 EXOGLUCANASE ASSAY

Saccharomycopsis javanensis and Saccharomyces cere visiae were

grown separately and in co-culture on dialysis membranes resting on GY agar (cf.

Section 2.8). Cells were haniested and washed after 48 hours of incubation (cf.

2.1 0.2). The resulting centrifuged cells and supematants were assayed for exo-P

glucanase acüvity with a solution of pnitrophenyl-PD-glucoside (1 O glL). These

assays were carn'ed out in the same manner as those described in Section 2.1 0.2,

3 et sqq. The reacüon was stopped after appropriate time intervals with a solution

of sodium carbonate (40 glL Na,CO,), using 2 mL of this solution for 0.1 mL of

recation mixture. Thereafter the absorances of these solutions were measured in

a 1-cm spectrophotometer cell at A = 450 nm.

RESULTS AND DISCUSSION

SELECTION OF MOST ACTIVE PREDATOR-PREY COMBINATIONS

In order to find suitable combinations of cultures to study yeast predation,

the following criteria were applied:

presence of haustoriurn-mediated predation;

susceptibility of host to its predator;

predator and prey rnorphologically distinguishable from each other;

unicellular growth of both organisms on solid substrats (important for

counting ).

Five potentially predacious yeasts were tested against six prospective

prey species (Table 2.1.1). The prey included the hemiascomycetous yeast S.

cerevisiae, the holoascornycetous yeast M. hibisci, the heterobasidiomycetous yeast

Rh. minuta, the archiascomyœtous fission yeast Sz. pombe, the ascomycetous

yeast-like mould A. pullulans, and the achlorophyllous alga P. zopfii.

Tested were 23 predator-prey cornbinations. Observations from these

experiments are surnmarized in Table 3.1.1. Predation was most active with

Saccharomycopsis javanensis UWO[PS] 92-247.1 preying u pon S. cerevisiae.

Consequently, in the majority of later experiments this particular predator-prey

combination was used. An almost complete prey elimination on GY agar was

achieved after 24 houn. Morphological features characteristic for predators

(haustorkm formation, prey cell penetration) were clearly manifest, as well as the

progressively deteriorating status of the prey (details in Sections 3.2.1 and 3.2.2).

Similar results were obtained with Saccharomycopsisjavanensis UWO[PS] 82-52.

The othertwo tested rnembers of the genus Saccharomycopsis gave less

satisfacto ry results. W hen S. selenospora - fo merl y GuiMermondelia selenospora

Nadson et Krassilnikov- is grown in pure culture on GY agar, numerous thin lateral

branches can be seen along its hyphae, many of them bearing asci in the form of

ellipsoidal and ovoid structures. This corresponds closely to the description in the

systematic discussion of the species (KREGER-VAN RIJ, 1984). It was also reported

that S. selenospora produces lateral denticles which. when grown out to stalks, are

capable of penetrating its own mycelium and dead cells (KREGER-VAN RIJ and

VEENHUIS, 1973; KREGER-VAN RIJ, 1984). The filamentous growth of this organism

would preclude accurate counting of cell numbers.

S. synnaedendra - fonerly Bot~oascus synnaedendm (Scott et van

der Walt) von Arx - is known to produce a branched, septate mycelium with

sphencal or oval blastospores which conjugate with hyphal cells to produce

sphencal aval or elongated asci (KREGER-VAN RIJ, 1984). Morphologically similar

features were seen on GY agar in pure culture as well as in CO-culture with potential

prey and precluded a clear conclusion with regard to predation.

It should be noted that the species description of S. javanensis- formerly

Arthroascus javanensis von Am - also mentions the faculty of its myceliurn to

produce short lateral branches (denticles) which grow out to stalks and may then

penetrate dead cells (KREGER-VAN RIJ, 1984; KREGER-VAN RIJ and VEENHUIS. 1973).

However, with this species it is easy to distinguish between actual prey penetration

and other phenomena, as predation is perforrned by single cells that arise either

from budding or by hyphal fragmentation. In the conditions used in this study, true

hyphal growth was not oserved.

The alga P. zopfii, unrelated to the fungi (Huss and SOGIN, 1990) and with

a radically different cell envelope composition, did not induœ predatory behaviour.

Moreover, al1 tested predators were apparently outcompeted by the alga on GY

agar. This was particularly conspicuous with S. javanensis. growing poorly in the

f o m of sparse, very thin cells among the much more abundant P. zopfii.

Fission yeasts, including Schizosacchamyces pombe, differ from other

yeasts in many ways (SIPICZKI. 1995). Despite earlier daims that S. pombe was not

susceptible to penetration (KREGER-VAN RIJ and VEENHUIS, 1973; UCHANCE and

38

PANG, 7 997). active predation after 24 hrs was observed when S. pombe was added

to one-day old cultures of S. javanensis.

h

Saccharomycopsis ja vanensis

UWO[PS] 92-247.1

Saccharomycopsis javanensis

UWO[PS] 82-52

1 Candida strain 'W1"

LEGEND:

a predation very pronounced (n.t.) not tested a predation clearly visible (7) predation uncertain

predation modest O no predation observed

+ prey overgrew its predator

Table 3.1.1 Potential predator-prey combinations.

3.2 LIGHT MICROSCOPY OF PREDATION

3.2.2 INTERACTIONS ON SOLlD MEDIA - PREDATION

Predation was studied by light rnicroscopy and microphotography of

predator-prey interactions between Saccharomycopsis fermentans and

Saccharomyces cerevisiae on predation agar (GY). Observations in slide culture

require that both predator and prey be capable of growth under reduced oxygen

tension. Therefore, S. fementans, a facultative anaerobe (Iike S. cerevisiae), was

used as predator in this experiment.

Predation of S. fermentans on S. cerevisiae growing on GY agar was

easily observed afier 72 hours. Haustona penetrating prey cells were seen at sites

of close contact (Plate 3.2.1.118. arrows). Abundant growth of S. cerevisiae was

seen only in regions away from predator mycelium or cells. Rernnants of markedly

altered prey cells could be seen trapped among the cells of S. fementans (Plate

3.2.1.118, bottom).

Plate 3.2.1 .i Predation of Saccharomycopsis fermentans on

Saccharomyces cerevisiae in slide culture on GY agar.

Phase contrast. Bar length = 5 prn

(A) t = O h

Prey cells (Sc) with adhering predator (S9. (6) t = 72 h

Growing rnycelium of S. fementans (S9 penetrates its

prey (arrows);

Unaffected region (U).

42

3.2.2 INTERACTIONS ON SOLID MEDIA - POST-PREDATIONALVIABILITY

Plate 3.2.2.1 shows a mixture of Saccharornycopsis javanensis and

Saccharomyces cerevisiae after transfer from conditions that favor predation (GY

agar) to a slide culture of YM agar, to assess the viability of penetrated cells. In A,

unpenetrated cells of S. cerevisiae developed nomally and after 20 hours overgrew

the predator. In B. a cluster of penetrated cells rernained inactive for 20 hours at

which point S. javanensis had multiplieci considerably. However, it is not clear

whether every S. cerevkiae cell in the cluster had been actually penetrated.

Without further expen'ments it is not possible to conclude with certainty whether

arrested growth of S. cerevisiae is caused by penetration, contact, or sirnply close

proximity to S. javanensis.

Plate 3.2.2.1 Post-Predational recovery in slide culture of Saccharomycopsis

ja vanensis and Saccharomyces cerevisiae on Y M agar.

Phase contrast. Bar length = 5 pm

(A) Control region (predorninantly prey cells, Sc).

Photographed at t = O h (A-1); t = 4 h (A-2); t = 20 h (A-3).

(BI Region of intense predation (growing predator cells, Sj).

Photographed at t = O h ( -1) ; t = 4 h (B-2); t = 20 h (5-3).

3.3 SCANNING ELECTRON MICROSCOPY

OF INTERACTIONS IN LIQUID MEDIA

With a suitable predator-prey combination, predatory behaviour is best

observed on solid substrats (GY agar). The occurrence of predation in liquid GY

medium is a much more uncommon phenornenon and a considerably longer time

is needed for it to take place. When the medium was agitated, no predation

whatsoever was observed. However, the best electron micrographs of predation

were obtained from samples of still Iiquid cultures and examples of these are shown

in this section.

The following four pairs were tested:

Saccharomycopsis javanensis 4 Saccharomyces cerevisiae

Saccharornycopsis javanensis -+ Schizosaccharomyces pombe

Saccharornycopsis synnaedendra -, Metschnikowia hibisci

Candida strain " W 1 " -+ Mefschniko wia hibisci

Predation was observed after 3 to 15 days, sornetimes longer. In still

cultures of S. ja vanensis with S. cerevisiae, active predation was seen after 1 5 days

(Plates 3.3.1 and 3.3.2). It appears that the contact between predator and prey

occurs exclusively via the penetration pegs (Plate 3.3.1). Areas of S. javanensis

without denticles do not appear to adhere to S. cerevisiae cells. Large clumps were

formed in the liquid medium. The latter might be the result of iectin-rnediated

agglutination (BARAK et al., 1 985; BARAK and CHET, I W O ) . The penetrated cells

ulümately collapse (Plate 3.3.2JB. arrows).

Intense predation of S. javanensis on Sz. pombe came somewhat as a

surprise, because introductory work on yeast predation did not reveal Sz. pombe as

susceptible to penetration (KREGER-VAN RIJ and VEENHUIS, 1973; LACHANCE and

FANG, 1997). The predator-prey cell contact appears much more intimate here

(Plate 3.3.3). The slender predator cells may adhere completely to their prey (Plate

3.3.3, upper right and lower left). Dense predator ceil aggregations with fdl

envelopment of the prey cell may be seen (Plate 3.3.3, lower right), reminiscent of

the entrapment of nematodes by fungal parasites. Patches of a rnucous deposit on

the surface of attacked prey cells are commun (Plate 3.3.3, lower left). The origin

and nature of these deposits are unclear.

The remaining two predator-prey pairs exhibited very lime predation in

liquid medium. This may be due to the fact that these predators are generally less

effective (Section 3.1 and Table 3.1 -1). No predation was detected in shake-flask

cultures of any of the four pain tested.

Plate 3.3.1 Predation of Saccharomycopsis javanensis on

Saccharomyces cerevisiae in Iiquid predation medium (GY):

scanning electron microscopy of predator-prey cell contacts.

Bar length = 2 Pm

Plate 3.3.2 Details of haustonum formation and penetration during CO-culture

of Saccharomycopsis javanensis and Saccharomyces cerevisiae

in liquid GY medium (scanning electron microscopy).

Bar length = 5 prn

(A) Hausto ria (a rrows)

(B) Collapsed prey cells (arrows)

Plate 3.3.3 Predation of Saccharomycopsis ja vanensis on

Schizosaccharomyces pombe in Iiquid predation medium (GY):

scanning electron microscopy of predator-prey ceIl contacts.

Bar length = 5 pm

3.4 GENERAL EFFECT OF NUTRIENTS

3 -4-1 TlME COURSE (CONTINUOUS GROWTH)

In this phase of the study experiments were done with Saccharomycopsis

javanensis and Saccharomyces cerevisiae in order to:

compare predator-prey interactions on three very different media;

find growth curves (log N vs. time) for pure and mixed cultures;

determine the interrelatedness of growth and predation;

establish a general correlation between nutnents and predation.

Experiments were carried out on nutrient-restricted medium (GY agar;

Figure 3.4.1.1 ), organosulfur-free medium (Basal agar; Figure 3.4.1.2), and

cornplete medium (YM agar; Figure 3.4.1.3).

According to the data in Figure 3.4.1.1, active predation occurred on GY

medium. When grown in pure culture, the cell yield was somewhat higher in the

predator, but in CO-culture predator cell numbers exceeded those of the prey by

almost three orden of magnitude (Figure 3.4.1.1 ).

On organosulfur-free basal medium (Figure 3.4.1 -2) S. javanensis clearly

benefited from the presence of S. cerevisiae. On the other hand, the reduction in

prey yield was much less dramaüc than on GY.

The remarkably sirnilar predator-to-prey cell density ratios of the mixed

and separated cultures on YM agar (rich medium) are shown on Figure 3.4.1 -3. In

both cases there was a 40- to 50-fold increase in ratio, due probably to a faster

growth rate of S. javanensis and possibly also to the larger cell volume of S.

cere visiae.

Maximum growth in al1 media was reached after approx. 48 hours. The

serni-logarithmic plot of the predator-to-prey ceIl ratio on GY agar (Figure 3.4.1 .l)

reveals that it is the period of exponential growth of both cultures in which predation

is most prominent

The decline of S. javanensis on medium devoid of organic sulfur was

expected, as this yeast requires sulfur in the organic fom. In the presence of prey,

the predator-prey ratio was not comparable to that observed on GY. The reason

may be that predatory activity depends on the release of organic sulfur into the

medium by growing prey cels. Whereas YM agar grown cells showed no sign of

predatory behaviour in the microscope, both GY and basal medium grown predators

formed abondant infection pegs.

Figure 3.4.1 -1 G rowth of Saccharomycopsis javanensis and Saccharomyces

cerevisiae on GY agar.

LEGEND:

Upper graph:

Lower graph:

Logarithm of wlony-foning units,

log N vs. time

Predator, grown separately

Predator, grown together with prey

Prey, grown separately

Prey, grown together with predator

Logarithm of predator-prey cell ratio,

log (NDmaw I N,,,) vs. time

Predator and prey grown separately

Predator and prey grown together

3 Time ? days 5

Figure 3.4.1 .l a Growth of Saccharomycopsis javanensis and Saccharomyces

cerevisiae on GY agar.

~ t a t i s k .

Logarithrn of colony-foming units, log N vs. time.

Error bars represent 99% confidence intewals (i2.576 O).

Each data point is a mean of 3 separate platings.

Numerical values in graphs are coefficients of variation.

Upper left graph: predator, grown separately

Upper right graph: predator, grown together wioi prey

Lower left graph: prey, grown separately

Lower right graph: prey, grown together with predator

Figure 3.4.1.2 G rowth of Sacchammycopsisjavanensis and Saccharomyces

cerevr'siae on basal medium.

LEGEND:

Upper Graph: Logarithm of colony-fomiing units,

log N vs. time

-0- Predator, grown separately

-@- Predatoi, grown together with prey ---O--- Prey, grown separately

---*--- Prey, grown together with predator

Lower Graph: Logarithm of predator-prey cell ratio,

log (Np-, I Np,,) vs. tirne

Predator and prey grown separately

Predator and prey grown together

Figure 3.4.1.2a G rowth of Saccharomycopsisjavanensis and Saccharomyces

cerevisiae on basal medium.

Statisücs.

Logarithm of colony-foming units, log N vs. time.

Error bars represent 99% confidence intervals (iî.576 O).

Each data point is a mean of 3 separate platings.

Numerical values in graphs are coefficients of variation.

Upper left graph: predator, grown separately

Upper right graph: predator, grown together with prey

Lower leff graph: prey, grown separately

Lower nght graph: prey, grown together with predator

Figure 3.4.1.3 Growth of Saccharornycopsis javanensiç and Saccharomyces

cerevisiae on rich medium (YM agar).

LEGEND:

Upper Graph:

Lower Graph:

Logarithm of colony-forming units.

log N vs. time

Predator, grown separately

Predator, grown together with prey

Prey, grown separately

Prey, grown together with predator

Logarithm of predator-prey cell ratio,

log (Np,,/ IVpmy) vs. time

Predator and prey grown separately

Predator and prey grown together

Figure 3.4.1.34 Growth of Saccharomycopsis javanensis and Saccharomyces

cerevisiae on n'ch medium (YM agar).

Statistics.

Logarithm of colony-foming units, log N vs. time.

Error bars represent 99% confidence intervals (I2.576 o).

Each data point is a mean of 3 separate platings.

Numerical values in graphs are coefficients of variation.

Upper left graph: predator, grown separately

Upper right graph: predator, grown together with prey

Lower left graph: prey, grown separately

Lower nght graph: prey, grown together with predator

3.4.2 PREDATION DYNAMICS (DISCONTINUOUS GROWTH)

Experiments in this phase were designed to clarify the predatoiy response

of Saccharomycopsis javanensis p reying u pon Saccharomyces cerevisiae in the

presence or absence of yeast extract as a predation suppressor.

Because predation appears to occur during active cell growth, and in

order to exclude effects of nutrient limitation and aging. transfers to fresh media had

to be done between the late exponential and the early stationary growth phase.

Young mycelia are more susceptible to invasive necrotrophy (LAING and DEACON,

1990) and the same may be true for yeast cultures. If predation occuw in nature,

then it must be associated with movement of yeasts to fresh substrates. Indeed,

yeasts associated with plants are consumed by insects, and periodically are

transferred to new environments (PHAFF and STARMER, 1987).

Five media were used:

GY agar with 0.1 glL yeast extract

GY agar with 1 glL yeast extract

GY agar with 10 glL yeast extract

Basal agar with 5 g/L (NH,),SO,

LNB agar with 0.1 g R (NH,),SO,

The first three media were designed to verify the suppressive effect of

yeast extract on yeast predation. Here, the amount of yeast extract was increased

1 O and 100 times.

The other two media did not contain any yeast extract at all. The nitrogen

source, ammonium sulfate, was added at two widely different concentrations to see

whether nitrogen (or sulfate) may play a regulatory role with respect to predation.

On GY agar with 0.1 g/L YE predation was so intense that after the third

day (one day after the first transfer) the predator-to-prey cell density ratios changed

alrnost 10' times, and no prey could be found in subsequent transfers (Figure

3.4.2.1). In repeated experiments the prey was eliminated af€er the first or second

transfer, depending on the initial population density (not shown).

With lglL YE, predation was still obvious and subsequent transfers

resulted in a steady decline in prey nurnbers (Figure 3.4.2.2). Predator-to-prey cell

ratio changed about Io4 times after 8 days (end of third transfer).

With 10 glL YE, predation had hardly any detectable effect on the

predator-to-prey cell ratio over an 84ay period. The ratios in mixed cells and cells

grown separately were essentially the same (Figure 3.4.2.3).

In thetwo organosulfur-free media (basal and LNB) the predator benefited

greatly and to an alrnost equal extent from the presence of prey. This can be judged

by the oscillating but elevated (one to two orders of magnitude) predator-to-prey cell

ratios (Figures 3.4.2.4 and 3.4.2.5). In controls (separate growth) the predator-to-

prey ceil ratio declined until the predator eventually disappeared. This decline was

more rapid at the higher ammonium sulfate concentration.

Figure 3.4.2.1 Pred ation dynamics of Saccharomycopsis javanensis

and Saccharomyces cerevisiae on GY agar with

0.1 gl L yeast extract.

The cultures were transferred at 2day intervals.

LEGEND:

Upper Graph: Logarithm of coiony-foming units,

log N vs. tirne

-0- Predator, grown separately

-a- Predator, grown together with prey --..O--- Prey, grown separately

---a--- Prey, grown together with predator

Lower Graph: Logarithm of predator-prey cell ratio,

log Np$ vs. tirne

Predator and prey grown separately

Predator and prey grown together

4 6

Time 1 days

Figure 3.4.2.1 a Predation dynamics of Saccharomycopsis javanensis

and Saccharomyces cerevisiae on GY agar with

0.1 gl L yeast extract.

Statistics.

Logarithm of colony-forming units, log N vs. üme.

Error bars represent 99% confidence intervals (kZ.576 O).

Each data point is a mean of 3 separate platings.

Numerical values in graphs are coefficients of variation.

Upper left graph: predator, grown separately

Upper right graph: predator, grown together with prey

Lower left graph: prey, grown separately

Lower right graph: prey, grown together with predator

Time /days T h e / days

Figure 3.4.2.2 Predation d ynamics of Saccharomycopsis javanensis

and Saccharomyces cerevisiae on GY agar wiih

1 g R yeast extract

The cultures were transferred at 2day intervals.

LEGEND:

Upper Graph: Logarithm of colony-forming uni&,

log N vs. time

-0- Predator, grown separately

-@- Predator, grown together with prey ---O--- Prey, grown separately

---a--- Prey, grown together with predator

Lower Graph: Log arithrn of predator-prey ceil ratio,

log (Alpdaw / N,,) vs. time

Predator and prey grown separately

Predator and prey grown together

4 6

Time / days

Figure 3.4.2.24 Predation dynamics of Saccharornycopsisjavanensis

and Saccharomyces cerevisiae on GY agar with

1 g/L yeast extract Statistics.

Logarithrn of wlony-forming units. log N vs. üme.

Error bars represent 99% confidence intervals (I2.576 a).

Each data point is a mean of 3 separate platings.

Numerical values in graphs are coefficients of variation.

Upper left graph: predator, grown separately

Upper right graph: predator, grown together with prey

Lower left graph: prey, grown separately

Lower right graph: prey, grown together with predator

Time f days

Figure 3.4.2.3 Predation dynamics of Saccharomycopsis javanensis

and Saccharomyces cerevisiae on GY agar with

1 0 gIL yeast extract.

The cultures were transfemd at 2-day intervals.

LEGEND:

Upper Graph:

Lower Graph:

Logarithm of colony-forming units,

log N vs. time

Predator, grown separately

Predator, grown together with prey

Prey, grown separately

Prey, grown together with predator

Logarithrn of predator-prey cell ratio,

log I N,,) vs. time

Predator and prey grown separately

Predator and prey grown together

4 6

Time / days

Figure 3.4.2.3-a Predation dynamics of Saccharomycopsis javanensis

and Saccharomyces cerevisiae on GY agar with

1 O g/L yeast extract

Statistics.

Logarithm of colony-forming units, log N vs. tirne.

Error bars represent 99% confidence intervals (12.576 a).

Each data point is a mean of 3 separate platings.

Numerical values in graphs are coefficients of variation.

Upper left graph: predator, grown separately

Upper right graph: predator, grown together with prey

Lower left graph: prey, grown separately

Lower right graph: prey, grown together with predator

Tirne / days Time ldays

Figure 3.4.2.4 Predation dynarnics of Saccharomycopsis javanensis and

Saccharomyces cerevisiae on low nitrogen basal (LNB) agar:

0.1 gIL (NH&S04.

The cultures were transferred at 2day intervals.

LEGEND:

Upper Graph: Logarithrn of colony-forrning units,

log N vs. tirne

A- Predator, grown separately

-0- Predator, grown together with prey - - - O--- Prey, grown separately

- - - a--- Prey, grown together with predator

Lower Graph: Logarithm of predator-prey cell ratio,

log (N'Ma, 1 Npy) vs. time

Predator and prey grown separately

Predator and prey grown together

4 6

Time I days

Figure 3.4.2.4-a Predation dynamics of Saccharomycopsis javanensis and

Saccharomyces cerevisiae on low nitrogen basal (LNB) agar:

0.1 g/L (N H&SO,.

Statistics.

Logarithrn of colony-forming units. log N vs. time.

Error bars represent 99% confidence intervals (I2.576 O).

Each data point is a mean of 3 separate platings.

Numerical values in graphs are coefficients of variation.

U pper left gra p h: predator, grown separately

Upper right graph: predator, grown together with prey

Lower left graph: prey, grown separately

Lower right graph: prey, grown together with predator

Time I days

Figure 3.4.2.5 Predation dynamics of Saccharomycopsis ja vanensis and

Saccharomyces cerevisiae on basal agar:

5 g/L (NH&SO,.

The cultures were transferred at 2-day intervals.

LEGEND:

Upper Graph:

Lower Graph:

Logarithm of colony-forming units,

log N vs. üme

Predator, grown separately

Predator, grown together with prey

Prey. grown separately

Prey, grown together with predator

Logarithm of predator-prey cell ratio,

log (AlpMaw / Aimy) vs. time

Predator and prey grown separately

Predator and prey grown together

4 6

Time / days

Figure 3.4.2.54 Predation dynamics of Saccharomycopsis ja vanensis and

Saccharomyces cerevisiae on low nitrogen basal (LN B) agar:

5 g/L (NHJ2SO4.

Statistics.

Logarithm of colony-forming units, log N vs. time.

Error bars represent 99% confidence intervals (i2.576 a).

Each data point is a mean of 3 separate platings.

Numerical values in graphs are coefficients of variation.

Upper left graph: predator, grown separately

Upper right graph: predator, grown together with prey

Lower left graph: prey, grown separately

Lower right graph: prey, grown together with predator

Time I days Time idays

3.4.3 SEARCH FOR MEDIA COMPONENTS THAT AFFECT PREDATION

As was shown in Sections 3.4.1 and 3.4.2, rich media (YM agar) and

higher concencentrations of yeast extract in otherwise poor media tend to inhibit

predation. Therefore, an atternpt was made to find the sources of assumed

inhibiting factors in complex nutient ingredients. To thateffect, media with only one

such complex ingredient were prepared (cf. Section 2.4).

Also, a simple fracüonation of yeast extract solutions with ethanol and

acetone was performed. as outlined in Section 2.4, and the fractions were tested for

their respective predation-inhibiting potential.

The obtained results (Table 3.4.3.1) are a semiquantitative assessrnent

of rnicroscopic observations of Saccharomycopsis javanensis grown in co-culture

with Saccharomyces cerevisiae.

#

Cornplex ingredient

Acetone preci pitate

Haustorium formation

- -

-

-

- - - - - - - -

Evaporated aqueous

acetone

Ethanol precipitate

Evaporated aqueous ethanol

Peptone

Tryptone

1 Vitamins I +++ I +++ 1

Penetration

- -

-

-

1 Malt extract

Table 3.4.3.1 Effect of various complex ingredients of nutrient media on the predation of Saccharomycopsis javanensis g rown in CO-culture wi th Saccharomyces cerevisiae.

The intensity of haustona formation and penetration was evaluated on a subjective scale from negative (-) to very high (+++).

+ 1 - - +

+++

- -

+++ I

3.5 THE ROLE OF ORGANIC SULFUR

The role of organic sulfur in yeast predation was studied. based on the

fact that al1 known predacious yeasts are organosulfur auxotrophs (LACHANCE and

PANG. 1997). The rationale was that a sufficient concentration of L-methionine

added to an organosulfur-free medium would cover the needs of the auxotroph and

that the predatory response to the preçence of a susceptible prey organism would

not be elicited. The following predator-prey combinations were chosen:

Saccharornycopsis javanensis -r Saccharomyces cerevisiae

Saccharomycopsis javanensis + Metschnikowia hibisci

Candida strain "Wl" 4 Metschnikowia hibisci

The apparent importance of organic sulfur in the phenornenon of yeast

predation and an attempt to demonstrate a causative link between predacious

behaviour and sulfur auxotrophy prompted for an expansion of the model system.

Therefore, the second and third pair consist of a relatively large predator with a

small-celled prey and a system with both small-celled organisms, respectively.

In addition to L-methionine, some other organosulfur compounds were

tested with S. javanensis preying on S. cerevisiae. These included L-cysteine. the

racemic DL-methionine (in which only one half of the mixture is biochemically active)

and sodium thioglycollate (or rnercaptoacetate, HSGH,-COO-Na'), the anion of

which is easily taken up by yeastç. The predator-prey pairs and their respective

media are summarized in Table 3.5.1. Selective counts of Metschnikowia hibisci

were obtained by plating on YM agar + CTAB (see Table 2.2.2).

fl Cr)

V>: C')

3.5-1 THE ROLE OF L-METHIONINE - PREDATION OF

Saccharornycopsis javanensis ON Saccharomyces cerevisiae

The following expenments were based on the hypothesis that the

predatory behaviour had developed as a means to overcome an obligate

requirement for organic sulfur, and a preliminary observation that methionine

inhibited predation in Candida sp. W1. The specific objective was to determine the

lowest amount of methionine suficient to prevent predation. Concentrations of L-

methionine in the LNB medium were varied in a broad range of five orders of

magnitude (plus methionine-free LW) . The findings as a whole were rather

unexpected.

On methionine-free LNB agar the predator was gradually lost in

successive transfers (Figure 3.5.1.1). In the CO-culture the predator grew in an

oscillatory pattern of coexistence with its prey.

Only the lowest tested methionine concentration (0.0001 g/L, Le., c l PM)

promoted coexistence rather than antagonism in CO-culture (Figure 3.5.1.2). A

slightly oscillatory pattern was still observed. Contrary to what occurred in

methionine-free medium, the growth of the predator was stable even in absence of

prey. This suggests that even traces of bmethionine (=0.1 ppm) are suffident to

sustain the predator's viability, but insufficient for significant predation to take place.

When the concentration of kmethionine equalled or exceeded 0.001 glL.

(1 ppm) the prey was no longer countable 24 hrs after the first transfer (three days

after start) (Figures 3.5.1 -3 to 3.5.1 -6). Even at a concentration four orders of

magnitude higher, methionine had no inhibitory effed

Contrary to the expected outcorne, m me thionine stimulated rather than

inhibited the predatory response. Therefore, one should conclude that sulfur

auxotrophy and predation are coincidental.

Figures 3.5.1 -1 Influence of organic sulfur on the predation dynamics of

to 3.5.1.6 Saccharomycopsis javanensis and Saccharomyces cemvisiae

on LN6 medium with varying concentrations of m me thionine.

The cultures were transferred at 2-day intervals.

LEGEND:

Upper Graph: Logarithm of colony-foming units,

log N vs. time

-0- Predator, grown separately

-a- Predator, grown together with prey

---O--- Prey, grown separately

---a--- Prey, grown together with predator

Lower Graph: Logarithrn of predator-prey cell ratio,

log (Npdaw I Npm,,) vs. time

-V- Predator and prey grown separately

-V- Predator and prey grown together

Figure 3.5.1.1

Figure 3.5.1 -2

Figure 3.5.1.3

Figure 3.5.1 -4

Figure 3.5.l -5

Figure 3.5.1.6

No methionine (Control).

0.0001 glL L-methionine.

0.001 g/L L-methionine.

0.1 g/L L-methionine.

1 glL me thionine.

10 g/L me thionine.

4 6

Time 1 days

4 6

Time 1 days

4 6

Time I days

2

Time l days

Time I days

Time l days

Figures 3.5.1.l-a influence of organic sulfur on the predation dynarnicç of

to 3.5.1.6-a Saccharomycopsis javanensis and Saccharomyces cerevisiae

on LNB medium with varying concentrations of me thionine.

Statistics.

Logarithm of colony-forming units, log N vs. time.

Error bars represent 99% confidence intervals (I2.576 O).

Each data point is a mean of 3 separate platings.

Numerical values in graphs are coefficients of variati on.

U pper lefl graph: predator, grown separately

Upper right graph: predator, grown together with prey

Lower left graph: prey. grown separately

Lower right graph: prey, grown together with predator

Figure 3.5.1 .+a No methionine (Control).

Figure 3.5.1 - 2 9 0.0001 g R L-methionine.

Figure 3.5.1 - 3 4 0.001 glL L-methionine.

Figure 3.5.1 - 4 4 0.1 glL me thionine.

Figure 3.5.1 .5-a 1 glL m me thionine.

Figure 3.5.1 .6a 10 g/L m me thionine.

Time l days

64 O Q, p g 2 - z q x m 9;-3-:g

!. 5 % g o a o o o 8 6

O 2 4 6 8 1 0

7ïme ldays

O 2 4 6 8 1 0

Tfme 1 days

Time I days

. . - -. . -- -

I I I I i

O 1 2 3 4

Time l days

O 1 2 3 4

3 9 O 1 2 3 4

Time idays

O 1 2 3 4 9 3 - l

I I I I . .-- - - - . - - . -

. . ... . . . . ... . .- . . .- - . . . . - . .. .-. . - ...F::: - .--... -.*- ....... . . . - ....

. .- --.. -- . - r.: - - 2 - 1

- .. : . -y . . . . . . .

-. 1

2- O 1 2 3 4

Time /days

O 1 2 3 4 9 1 . 1 - I . - I 1 I

Time I days

1 I 1

1 2 3

Time ldays T h e I days

3-5.2 THE ROLE OF L-METHIONINE - PREDATION OF

Saccharomycopsis javanensis AND Candida strain 'W1"

O N Metschnikowia hibisci

Only two extrerne concentrations were tested: 0.001 glL (1 ppm) and

1 O glL (1 %).

The main feature of these two systems was a lower susceptibility of the

prey to both predators. Unlike in the system described in Section 3.5.1 above,

Metschniko wia hibisci suwived and grew in association with both predators even

after three consecutive transfen on both concentration extremes (Figures 3.5.2.2,

3.5.2.3, 3.5.2.5 and 3.5.2.6). The characteristic oscillatory growai pattern in CO-

culture on methionine-free LNB medium was seen with both pairs (Figures 3.5.2.1

and 3.5.2.4). although it was more pronounced with Candida strain 'W1" as the

predator (Figure 3.5.2.4).

Saccharomycopsis javanensis gradually disappeared in pure culture on

organosulfur-free LN6 agar but survived well when grown with the prey (Figure

3.5.2.1). On the other hand, Candida str. 'W1" had only a growth reduction on the

same medium, eventually going into oscillations (Figure 3.5.2.4). This leads to the

conclusion that the latter strain is not a true suifur auxotroph.

Figures 3.5.2.1 Influence of organic sulfbr on the predation dynamics of

to 3.5.2.3 Saccharomycopsis javanensis and Metschnikowia hibisci on

LNB medium with varying concentrations of L-methionine.

The cultures were transferred at 24ay intervals.

LEGEND:

Upper Graph:

Lower Graph:

Logarithm of colony-forming units,

log N vs. time

Predator, grown separately

Predator, grown together with prey

Prey, grown separately

Prey, grown together with predator

Logarithm of predator-prey cell ratio,

log (Al,,,, I Al,,,,) vs. time

Predator and prey grown separately

Predator and prey grown together

Figure 3.5.2.1 No methionine (control).

Figure 3.5.2.2 0.001 g/L m me thionine.

Figure 3.5.2.3 10 g/L m me thionine.

4 6

Time I days

4 6

Time 1 days

4 6

Time / days

Figures 3.5.2.14 Influence of organic sulfur on the predation dynarnics of

ta 3.5.2.34 Saccharomycopsis javanensis and Metschnikowia hibisci on

LNB medium with varying concentrations of L-methionine.

Statistics.

Logarithm of colony-forming units, log N vs. time.

Error bars represent 99% confidence intervals (I2.576 a).

Each data point is a mean of 3 separate platings.

Numerical values in graphs are coefficients of variation.

U pper left graph: predator, grown separately

Upper right graph: predator, grown together with prey

Lower left graph: prey, grown separately

Lower right graph: prey, grown together with predator

Figure 3.5.2.1-a No methionine (control).

Figure 3.5.2.2-a 0.00 1 gR L-methionine.

Figure 3.5.2.3-a 10 g R m me thionine.

Time /days Tirne l days

O 2 4 6 8

lime I days

O 2 4 6 8

Time /( lays Time l days

Figure 3.5.2.4 influence of organic sulfur on the predation dynarnics of

to 3.5.2.6 Candida strain 'W1 " and Metschniko wia hibisci

on LNB medium with varying concentrations of ~~rnethionine.

The cultures were transferred at 2-day intervals.

LEGEND:

Upper Graph:

Lower Graph:

Logarithm of colony-forming units,

log N vs. time

Predator, grown separately

Predator, grown together with prey

Prey, grown separately

Prey, grown together with predator

Logarithm of predator-prey cell ratio,

log (Alpdam l NPmJ vs. time

Predator and prey grown separately

Predator and prey grown together

Figure 3.5.2.4 No methionine (control).

Figure 3.5.2.5 0.001 glL m me thionine.

Figure 3.5.2.6 10 glL L-methionine.

4 6

Time / days

4 6

Time l days

4 6

Time I days

Figure 3.59.4-a Influence of organic sulfur on the predation dynamics of

to 3.5.2.64 Candida strain 'W1" and Metschniko wia hibisci

on LN6 medium with varying concentrations of me thionine.

Statistics.

Logarithm of colony-forming units, log N vs. time.

Error bars represent 99% confidence intervals (î2.576 0).

Each data point is a mean of 3 separate platings.

Numerical values in graphs are coefficients of variation.

Upper left graph: predator, grown separately

Upper right graph: predator, grown together with prey

Lower left graph: prey, grown separately

Lower rïght graph: prey, grcwn together with predator

Figure 3.5.2.4-a No methionine (control).

Figure 3. J.2.S-a 0.001 g/L L-methionine.

Figure 3.5.2.6-a 10 g/L L-methionine.

Time I days

- -

4 I 1 I l 1 ' I I I

O 2 4 6 8

Time idays

O 2 4 6 8

0 m.. a..- h-- Q.- O- ICZ- Q) hl

ts- m- nt- m - - m w

8 f Z g s . - y g

Time l days

Time l days Time fdays

3.5.3 THE ROLE OF OTHER ORGANOSULFUR SOURCES - PREDATION OF Saccharomycopsis javanensis ON

Saccharomyces cere visiae

The effect of added DL-methionine in LNB medium (Figure 3.5.3.1) was

not difTerent from that of pure L-methionine (Figures 3.5.1 -3 to 3.5.1.6). With

sodium thioglycollate (Figure 3.5.3.2) the prey survived only one day longer in co-

culture (48 houn after the first transfer). When L-cysteine was added as the sole

organosulfur source, the predator cell density only slowly exceeded that of the prey

by a factor of about 102 after the second transfer (Figure 3.5.3.3).

It is interesting to note that the steadily decreasing predator-to-prey cell

ratio in the controls with ~~cysteine revealed the incapacity of S. javanensis to use

mis amino acid as an external source of sulfur (Figure 3.5.3.3) under the conditions

used here. The latter may be due to the low solubility of this amino acid in LNB

medium, but it is more probable that it was oxidized to L,L-cystine, its dirner, and

could no longer be taken up by the yeast.

Figure 3.5.3.1 Role of organic sulfur in the predation dynamics of

Saccharornycopsisjavanensis and Saccharomyces cerevisiae

on LNB agar with 1 glL o~~methionine (racemate).

LEGEND:

Upper Graph:

Lower Graph:

Logarithm of colony-foming units,

log N vs. time

Predator, grown separately

Predator, grown together with prey

Prey, grown separately

Prey, grown together with predator

Logarithm of predator-prey cell ratio,

log (Ai,,, I N,,) vs. time

Predator and prey grown separately

Predator and prey grown together

3 4

T h e 1 days

Figure 3.5.3.1-a Rote of organic sulfur in the predation dynamics of

Saccharomycopsis javanensis and Saccharomyces cerevisiae

on LNB agar with 1 gfL DL-methionine (racemate).

Statistics.

Logarithm of colony-forming units, log N vs. tirne.

Error bars represent 99% confidence intervals (e.576 a).

Each data point is a mean of 3 separate platings.

Numerical values in graphs are coefficients of variation.

Upper left graph: predator, grown separately

Upper right graph: predator, grown together with prey

Lower left graph: prey, grown separately

Lower right graph: prey, grown together with predator

Time / days Time Idays

Figure 3.5.3.2 Role of organic sulfur in the predation dynamics of

Saccharomycopsisjavanensis and Saccharomyces cerevisiae

on LNB agar with 1 g/L sodium thioglycoltate.

LEGEND:

Upper Graph:

Lower Graph:

Logarithm of colony-foming units,

log N vs. time

Predator, grown separately

Predator, grown together with prey

Prey, grown separately

Prey, grown together with predator

Predator and prey grown separately

Predator and prey grown together

2 3 4

Time I days

Figure 3.5.3.2a Role of organic sulfur in the predation dynamics of

Saccharomycopsk javanensis and Saccharomyces cerevisiae

on LN8 agar with 1 g/L sodium thioglycollate.

Statistics.

Logarïthm of colony-foning units, log N vs. time.

Error bars represent 99% confidence intervals (I2.576 a).

Each data point is a rnean of 3 separate platings.

Numencal values in graphs are coefficients of variation.

Upper left graph: predator, grown separately

Upper right graph: predator, grown together with prey

Lower left graph: prey, grown separately

Lower right graph: prey, grown together with predator

Time I days Time Idays

3 4

Time l days

Figure 3.5.3.3- Role of organic sulfur in the predation dynamics of

Saccharomycopsis javanensis and Saccharomyces cerevisiae

on LNB agar with 1 glL ~ q s t e i n e .

Statistics.

Logarithm of colony-foming units, log N vs. time.

Error bars represent 99% confidence intervals (f2.576 O).

Each data point is a mean of 3 separate platings.

Numerical values in graphs are coefficients of variation.

Upper left graph: predator. grown separately

Upper right graph: predator, grown together with prey

Lower left graph: prey. grown separately

Lower right graph: prey. grown together with predator

Time I days

..-. .. : . : :::l:.. 6 : - . - ..:-f-- - - . 2- - . . ... - - .... - ... . ... . . . ................. . . - -.

...+.. A- k

Time ldays

3.6 EFFECT OF INORGANIC SALTS ON PREDATION OF

Saccharomycopsis javanensis ON Saccharomyces cerevisiae

As will be explained in Section 3.7, the influence of common inorganic ions

(K+, NH,', NO, SOC) on predation was studied (Section 3.7.3). This was inspired

by a study on the influence of inorganics on the production of amylases by

Endomyces (Hnrro~i and IIDA. 1964; see below). The results appear in Table 3.6.1

below, complernented with data on P-glucosidase activities on the same culturing

media (cf. Section 3.7.3 below).

Media 1 Haustoriurn 1 Penetration 1 PGlucanase formation activitv

GY + methionine +++ +++ -+ GY + (NH4),S04 + methionine 1 - 1 - 1 -

GY + KNO,

Table 3.6.1 Effects of inorganic salts on predation of Saccharomycopsis javanen-

sis on Saccharomyces cerevisiae.

The intensity of haustorium formation and penetration was evaluated on a subjective

scale from negative (-) to veiy high (+++).

Beta-glucanase activity assessrnent is an interpretation of results in Fig . 3.7.3.1 .

3.7 PREDATION-ASSOCIATED ENZYME ACTlVlTlES

Host cell wall penetration by parasitic fungi is normally accompanied by

some elevated enzymic activity on the part of the parasite (RIDOUT et al., 1988). The

integrity of the host's cell wall has to be weakened or disrupted before penetration

structures (e.g., haustoria) can enter the cytoplasm or periplasmic space. It was

therefore reasonable to expect elevated levels of hydrolases in predator-prey

interactions where penetration plays a role, particularly an increase in

polysaccharide hydrolases (glucanases, mannanases, chitinases).

As was rnentioned in Chapter 1, manifestations of interfungal antagonism

rnay require direct contact between mycelia or they rnay be mediatecl at a distance

via difisible agents (toxins, enzymes). In some cases both strategies play a role

(JEFFRIES and YOUNG, 1994; p. 53).

After a preliminary screening for enzymic activities capable of hydrolyzing

ceil wall components (Section 3.7.1), more in-depth experiments were devised

(Section 3.7.2) so as to give an appraisal of the individual contributions of both

extracellular and cell-wall bound predator hydrolases in enzyme-assisted cell wall

penetration. Therefore, samples taken from CO-cultures of predator and prey grown

on solid media were brought into suspension, and the resulüng suspensions were

immediately separated into a biomass portion and a ceIl-free supernatant. Control

samples of pure predator cultures were treated correspondingly.

In addition to such experiments on predation media (GY) with or without

added ~œrnethionine as predation activator, the influence of common inorganic ions

(K', NH,', NO;, S0:') on predation was also studied (Section 3.7.3). The latter was

motivated by an early study on the production of amylolytic enzymes by a strain of

the former mycelial yeast genus Endomyces (HATTORI and IIDA, 1964). Amylase

formation by washed cell suspensions of Endomyces IF0 01 11 was found to be

efficient with most inorganic potassium salts except with K2S04 and KNO,. The

latter two suppressed this enzyme activity. Since some species formerly assigned

to the genus Endomyces - e.g., E. fibuliger, E. javanensis - are predacious and

now belong to the genus Saccharomycopsis (KURTZMANN and R o e ~ m , 19951, it

was inferred that predator-prey interactÎons and the production of hydrolases of the

three main groups of yeast cell-wall polysaccharides - &lucanases. a-

mannanases and chitinases - also might be affecteci by inorganic ions.

Finally, laminarin hydrolysates obtained by the action of P(1-3)-

g lucanase(s) tom predator-prey cultures were chrornatog rap hically separated into

individual sugars (glucose and ~1igoglucosides). This was done in an attempt to

estimate the contribution of endoglucanases vs. exoglucanases in the disruption of

prey cell walls.

3.7-1 PRELIMINARY TESTS

Preliminary tests were conducted with the predator-prey pain:

Candidastrain"Wi"-+Metschnikowiahibisciand

Saccharomycopsis javanensis -, Saccharomyces cerevisiae.

Both pairs did not reveal any chitinase activity. Consequently, no further

chitinase assay was wrried out. Beta-(143)-glucanase was found in cultures of

both pairs, as evidenced by the coloured spot reactions obtained with culture

samples (Section 2.8.1 ).

However, in the case of Candida strain 'W1 " -+ Metschnikowia hibisci,

this enzymic activity was expressed in both pure cultures (predator and prey) as well

as in their mixture. This pair was therefore rejected from further studies.

With S. javanensis -r S. cerevisiae P(l-3)-glucanase activity was

regularly and clearly seen only in coculture. Occasionally it appeared also in older

cultures of the predator grown alone. Pure cultures of S. cerevjsjae showed no P glucanase activity whatsoever. Based upon the results of these tests, and taking

into consideration the fact that the main ceIl wall constituents of S. cerevisiae are

P(1-.3)-glucan and a-(1 +4)-mannan, only the latter pair was accepted for further

investigation.

3.7.2 TOTAL P(1-3>GLUCANASE AND a-(1 -4)MANNANASE ACTlVlTlES

These experiments were an attempt to assess freed (dissolved) and œll-

wall bound extracellular g lycan hydrolases individually du ring predation of S.

javanensis on S. cerevisiae. Curves shown in Figures 3.7.2.1 and 3.7.2.2 represent

the concentration of reducing sugar moieties liberated from the polysaccharide

substrate as a function of reaction time. In reality, the concentration of the

accumulated reducing sugars is a cornplex funcüon of many variables.

After 48 hours of growth on methionine-sup plemented predation agar

(GY), ~(l-+3)-glucanase activity could be measured only in predator-prey CO-

cultures, both as cell-wall bound activity and in cell-fiee supernatants (Figure

3.7.2.1). The rate of reducing sugar accumulation was exponential rather than

linear. It is significant to observe the absence of any measurable activity in two-day

old predator cultures (controls).

After six days of cultivation the situation changed (Figure 3.7.2.2).

Beta-(1+3)-glucanase activity emerged now in both washed cells and cell-free

supernatant of pure predator cultures. However, in both cases the rate of laminarin

hydrolysis was lower than in the corresponding samples from mixed cultures.

No mannanase activities were found, either in pure cultures or in mixtures.

The following explanations of the above findings are offered. Beta-(l+3)-

glucanase observed in No-day old mixed cultures (but not in pure predator cultures)

is an inducible acüvity produced in response to the presence of prey. Its

appearance in pure predator cultures after a longer incubation period (6 days) may

substantiate the fact thaï Saccharomycopsis javanensis penetrates its own

myceliurn (KREGER-VAN RIJ and VEENHUIS, 1973), probably in the stationary phase

or in conditions of prey depletion.

The simultaneous occurrence of predator-cell associated and freely

soluble P(1-+3)-glucanase is by no means a proof of the existence of two different

activities. The enzyme (or, possibly, several isozymes) may be essentially

extracellular but catalytically active pn'or to its complete detachment from the cell

wall. It cauld also be predominantly locaiized on the surface of cells or haustoria,

its presence in the liquid phase being possibly an artefact due to sample

manipulation or part of normal turnover. Under the conditions used here, S.

javanensis forms asci that deliquesce due to the action of glucanases and other

hydrolases. This could also cause the release of enzymes.

Laminann degradation curves obtained after 6 days with washed cells of

predator alone and predator-prey mixtures have a sigmoidal shape and differ greatly

from the exponential shape of the con-esponding supernatant fractions (Figure

3.7.2.2). it is possible that this sigmoidal shape, a characteristic of allosteric

enzymes, is lost in cell-free samples due to an alteration of the native conformation.

In other words, the binding sites for allosteric effectors rnay become ineffective once

the enzyme molecule is detached from the cell envelope and brought into solution.

It is important to note that the predator cell densities affer 6 days of

cultivation were about five times higher than in the culture suspensions obtained

after 2 days. It is reasonable to assume that the living cells themselves consume

some of the low-molecular weight sugars initially produced, which can not be the

case in cell-free supernatants.

The exponentiai appearance of most sugar accumulation curves reveals

another important fact. Polysaccharide chains may be broken down by two diffefent

classes of enzymes. Exoglucanases cleave off terminal sugar molecules. If the

reaction rab of such an enzyme does not change with time, there would be a linear

increase in reducing sugar concentration, as only one enzyme molecule can act

upon any polysaccharide chain regardless of the chain length. In other words.

reducing sugar accumulation is a linear function of time. On the other hand,

endoglucanases hydrolyse randomly along the polysaccharide chain. A disruption

of the prey cell wall must predorninantly rely upon the action of endogiucanases.

since there are few, if any, free chah ends. If an endoglucanase is to act alone on

a polysaccharide chah in solution, the accumulation of reducing sugar ends would

also be linear in the event of simple enzyme kinetics. Only when the chain is

eventually split into fragments too small to allow formation of an enzyme-substrate

cornplex, one would see a gradua1 decrease in sugar accumulation rate. The

opposite, raising dope of the obtained curves in Figure 3.7.2.2 - if not caused by

an allosteric enhancement of enzyme activity - indicates the simultaneous action

of bath endo- and exoglucanases. The acüon of the former creates an ever

increasing number of sites for the latter, and an exponential curve profile would

result.

Figure 3.7.2.1 Beta-(l-i3)-glucanase assay 48 hours after CO-culture of

Saccharomycopsisja vanensis and Saccharomyces cerevisiae

on methionine-supplemented predation medium (GY).

The concentration of reducing sugars liberated frorn laminan'n as substrate, in

millimoles per litre, was measured spectrophotometrically at A = 520 nrn against

glucose as standard (SPIRO, 1966).

Legend: O - Washed, centrifugeci cells --- O - - - Cell-free supernatant

60 90 120

Time / min

Figure 3.7.2.2 Beta-(l+3)-glucanase assay 6 days after CO-culture of

Saccharomycopsis javanensis and Saccharomyces cerevisiae

and in pure culture of S. javanensis on methionine-supple-

mented predation medium (GY).

The concentration of reducing sugars iiberated from laminah as substrate, in

millimoles pet litre, was measured spectrophotometncally at A = 520 nm against

glucose as standard (SPIRO, 1966).

Legend : O - Co-culture: washed, centrifuged cels --- 0 --- Coculture: cell-free supernatant

-V- Predator alone: washed, centrifugeci cels ---v--- Predator alone: dl-free supernatant

3.7.3 EFFECT OF INORGANIC IONS ON P(1+3}-GLUCANASE ACTlVlTY

This series of assays, carried out after 48 hours of growth, started with

tests on ordinary predation agar (GY), containing traces of endogenous methionine,

and on L-methionine supplemented GY, followed by expenments with added salts.

Here, too, no P(1+3)-glucanase activities were seen with pure predator cultures.

Assay results obtained with washed cells are represented in Figure 3.7.3.1 , those

with celf free supernatants in Figure 3.7.3.2. Two features are striking: different

curve profiles (sugar accumulation curves were not exponential) and a total absence

of glucanase activities in celbfree supematants with al1 of the inorganic salts tested.

The rather linear appearance of curves in Figure 3.7.3.1 could indicate that only one

of the two classes of glucanases was now active.

Potassium salt additions (74 mM K#04 and 99 mM KNO,) had an almost

identical effect: a slight reduction in laminafin degradation rate with respect to

cultures grown on simple GY agar and the same, almost linear (very slightly

sigrnoidal) degradation curves. indicative of only one class of glucanases.

Obviously, NH,+ plays an important role here, narnely that it fulfills the

predator's requirement for nitrogen. The inability of Saccharomycopsis javanensis

to assimilate nitrate could explain the dramaüc difference between the curves

obtained with KNO, and (NH4),S0,, respectively.

The absence of ~(1+3)-glucanase activity in cell-free supernatants when

any of the salts was present during cultivation is probably due to electrostatic

phenornena. Cells cultured in an electrolyte-rich medium with high ionic strength

can have a different overall charge on the cell envelope. This charge may, in turn,

be responsible for an anchoring of nascent enzyme molecules, due to electrostatic

interactions (ionion, ion-dipole) between the charged andlor polar groups of the

enzyme and the ionic double layer around the ceIl.

It is also possible that these salts affected ascus maturation and therefore

the reiease of enzymes into the medium.

3.7.4 HYDROLYTIC CLEAVAGE OF P(1+3>GLUCAN

TO GLUCOSE AND OLlGOGLUCOSlDES

Thin layer chromatography of laminarin hydrolyzates produced by washed

c d ls fro rn mixed cultures of Saccharomycopsis ja vanensis and Sacharomyces

cerevisiae on simple GY agar revealed a simultaneous appearance of glucose and

oligoglucosides in the reaction mixture. Oligomers with up to about eight glucose

subunits could be effectively separated (Figure 3.7.4.1 ). The detedon of 2 and 3

unit oligomers in early samples in addition to glucose points to the presence of

endo-P(1-3)-glucanase activities in conditions of predation. The presence of

glucose indicates exo-&g lucanase activity.

Figure 3.7.3.1 Effect of inorganic salts on Pglucanase acüvity

fro m co-cu ltu re of Saccharomycopsis ja vanensis wi th

Saccharomyces cerevisiae on predation medium (GY)

with and without methionine

(washed cells from co-culture after 48 hours).

The concentration of reducing sugars iiberated from laminarin as substrate. in

rnillimoles per litre, was measured spectrophotometrically at A = 520 nrn against

gfucose as standard (SPIRO, 1966).

Legend: 4- GY agar (no added methionine)

M - GY agar + 10 glL me thionine

-AS-- GY agar + 10 g/L (NH4)2S0,

-AS+M- GY agar + 10 g/L (NH4),S04 + 10 g/L r-methionine

P S - - GY agar + 10 glL K,SO,

P N - GY agar + 10 glL KNO,

Figure 3.7.3.2 Effect of inorganic salts on pglucanase activity

from co-culture of Saccharomycopsis javanensis with

Saccharomyces cerevisiae on predation med iurn (GY)

with and without methionine

(supernatant from CO-culture after 48 houn).

The concentration of reducing sugars liberated frorn laminarin as substrate, in

millimoles per litre, was measured spectrophotometrically at A = 520 nm against

glucose as standard (SPIRO, 1966).

Legend: -O- GY agar (no added methionine)

M - GY agar + 10 gfL kmethionine

Plate 3.7.4.1 Thin layer chromatogram of lamina rin hydrolyzate obtained with

washed cells from CO-culture of Sacchammycopsis javanensis

and ~acchammyces cerevisiae on GY agar.

DP = degree of polymerization.

Stationary phase:

Eluent:

Development:

Visualization:

Kieselgel 60 plates, 20 cm x 20 cm, layer thickness 0.2 mm,

not pre-dried

3 vol. pts. n-butanol + 1 vol. pt. acetic acid + 1 vol. pt. water

Double, one-dimensional elution, 5% hours each, in closed,

paper-lined chamber at RT; drying at 50°C overnight between

etutions

Repeated spraying with alkaline silver nitrate reagent (Section

2.8.3); 1-2 minutes at 120°C after final spray

3.7.5 BO-PD-GLUCANASE ASSAY

This experiment involved cultures of Saccharomycopsis javanensis and

Saccharomyces cerevisiae. It was aimed at assessing aie wntri bution of exo-P

glucanase actMty in the overall laminarin hydrolysis measured in previous

experiments (Sections 3.7.2-3.7.4). Here, laminarin as the enzyme substrate was

replaced by the chromogenic pnitrop henyl-po-glucoside, which is hyd rol yzed to

glucose and the yellow pnitrophenol only by ~o-glucosidases and exo-PD-

glucanases.

Measurements were carrieci out with both the washed cells and the first

washings (supernatants) of harvested cells.

The resultç are represented in Figure 3.7.5.1. It follows that activities are

found in pure cultures of both predator and prey, as well as in their mixtures.

The main finding was that there was no measurable ~lucanase/glucosi-

dase activity in supernatants of either of the pure cultures. Their respective washed -

cell fractions were, however, able to utilize the substrate via one or more cell-bound

activities. When predator and prey were grown in mixed culture and harvested in

an identical manner, ~glucanaselgluwsidase acüvity was readily measurable in the

supernatant. It may be hypothesized that in co-culture one or more oher P-glucana-

se/glucosidase activities are induced.

Figure 3.7.5.1 Exo-PD-glucanase activity assay in pure cultures and co-cultures

of Saccharornycopsis ja vanensis with Saccharomyces cerevisiae

on predation medium (GY)

(washed cells and supematants after 48 hours).

The activities were assayed spectrophotornetrkally at A = 450 nm by measuring the

accumulation of pnitrophenol liberated from gnitrophenyl-P~-glucoside.

Legend: 5- Cell fraction, CO-culture

-0- CelI fraction, prey culture

-A- Cell fracion, predator culture

-- - O --- Supernatant, CO-culture

0.3 .

0.2 .

O.? .

0.0 -

CONCLUSIONS

As has been ernphasised in the introductory sections, predacious

yeasts are not obligatory predators. Rather, this type of behaviour is elicited as a

response to certain conditions in the predators' environment. Experiments in this

thesis were therefore designed to elucidate at least some environmental parameters

conducive to the predatory response.

Based on the s u l ~ r auxotrophy of al1 known predacious yeasts, it was

hypothesized that predation was a means to acquire organic sulfur and that an

addition of suitable organosulfur wmpounds into the growth media would eliminate

the need for predation. Various metabolizable organosulfur (OS) compounds were

added to the LN8 nutrient medium in order to establish the threskold at which

predation would be stopped (Section 3.5). In most of the tests L-methionine was

used and the media always contained some inorganic sulfur (1s) in the fom of

ammonium sulfate. Quite wntrary to the expected outcorne, the following was

found:

No added OS + low IS 10.1 gIL INH4)7so~l. - Predator alone was not able to grow. - Predator and prey coexisted in CO-culture without predation.

Minimal OS 10.1 ma/L methioninel + low IS 10.1 g/L (NH4)2&J.

- Predator alone grew normally.

- Predator and prey coexisted in co-culture without predation.

m h e r OS II mq1L to 10 q/L methioninel + low IS 0.1 qlL INH4),SOAl. - Predator eliminated prey in CO culture within 3 to 4 days.

It can be concluded that an addition of organic sulfur stimulates rather

than inhibits predatory behaviour of methionine-auxotrophic yeasts. Methionine at

concentrations above 1 mglL is even necessary for predation to take place.

Predation is thus obviously not a strategy used by methionine auxutrophs to satisfy

their need for organic suIfW.

Later expenments with inorganic salis (Section 3.6) showed that

ammonium sulfate played an important role in suppressing predation. High

concentrations (10 g R ) in predation medium (GY), with or without added

methionine, entirely inhibited haustorium formation, prey cell penetration and

Pglucanase activity (Table 3.6.1 ). Initially, it was assumed that high concentrations

of the sulfate ion were responsible for the observed suppression of predation. But

since K2S04 (10 glL) did not have the same effect as (NH,),SO,, it appears that the

ammonium ion is strongly correlated to the regulation of predatory behaviour.

Further experiments in that direction are necessary.

Other desirable research should be directed at studying in more detail the

enzymatic mechanism of prey ceIl wall penetration, as it became apparent that

elevated levels of Pglucanase are associated with this phenornenon.

The observed differences in the extent of predation achieved with various

predator-prey pairs indicates that some fom of recognition. possibly at the level of

cell envelope, is involved. Therefore, the search for a lectin-type recognition

between predator and prey might yield interesting resulk

As far as the potenüal application of predacious yeasts is concerned.

studies with yeasts pathogenic to man and animais should be conducted.

It would also be of interest to CO-culture predacious yeasts with yeasts

commonly associated with food spoilage. These expenments should be conducted

in media prepared from or simulating actual food.

Interactions of predacious yeasts with other fungi have not been studied.

It would be very interesting to commence such studies with filamentous fungi

responsible for food spoilage and crop deterioration.

Hopefully, progress in the understanding of the mechanism of predation

will prove useful in its enhancement and commercial exploitation.

160

LITERATURE

Ainsworth, G.C. (1 965). Historical introduction to mywlogy. In: 'The Fungi. An

Advanced Treatise. Vol. 1. The Fungal Cell" (Ainsworth, G.C. and Sussman,

A.S., eds.). Academic Press, New York, pp. 3-20

Alexopoulos, C. J., Mims, C.W. and Blackwell. M. (1 996). "Introductory Mycology",

4th ed. John Wiley & Sons, New York, x+868 p.

Barak, R. and Chet, 1. (1990). Lectin of Sclerotiurn rolfsii: its purification and

possible function in fungal-fungal interaction. J. Appl. Bacteriol. 69(1):101-112

Barak, R., Elad, Y., Mirelman, D. and Chet, 1. (1 985). Lectins: a possible basis

for specific recognition in the interaction of Trichoderma and Sclerotiurn roifçii.

Phytopathology 75(4):458-462

Baruiicki-Garcia, S. (1968). Cell wall chernistry, morphogenesis, and taxonomy of

fungi. Annu. Rev. Microbiol. 22:87-108

Bartnicki-Garcia, S. (1 984). Kingdoms with walls. In: "Structure, Functions and

Biosynthesis of Plant Cell Wails" (Dugger, W.M. and BaNiicki-Garcia, S., eds.)

Waverly Press. Baltimore, MD, pp. 1

Beran, K., Holan, 2. and Baldrian, J. (1 972). The chiun-glucan cornplex in Saccha-

romvces cerevisiae. 1. IR and X-ray observations. Folia Microbiol. 1 7(5):322-

330

Breton, A. and Surdin-Ke rjan, Y. (1977). Sulfate uptake in Saccharomvces cere-

visiae: biochemical and genetic study. J. Bactend 132(1):224-232

161

Bruns, T.D., White, T.J. and Taylor, J.W. (1 991). Fungal rnolecular systematics.

Annu. Rev. Ecol. Syst 22:525-564

Bruns, T.D., Vilgalys, R., Barns, SM., Gonzales, D., Hibbett, D.S., Lane, D.J.,

Simon, L., Stickel, S., Szaro, T.M., Weisburg, W.G. and Sogin, M.L. (1993).

Evolutionary relationships within the fungi: analyses of nuclear small subunit

rRNA sequences. Mol. Phylogenet EvoL 1 :231-241

Cabib, E. and Bowers, B. (1971). Chitin and yeast budding. Localization of chitin

in yeast bud scars. J. Biol. Chem. 246(1): 1 52-1 59

Calvo-Mendes, C. and Ruiz-Herrera, J. (1 987). Biosynthesis of chitosan in

membrane fractions from Mucor rouxii by the concerted action of chitin

synthetase and a particulate deacetylase. Exp. Mycol. 1 l(2): 128-140

Carlile. M.J. (1980). From prokaryote to eukaryote: gains and losses. In: "The

Eukaryote Microbial Cell" (Gooday, G.W., Loyd D. and Trinci, A.P.J., eds.).

Symp. Soc. Gen. Microbiol. 30: 1-40

Conte, M.V. and Pore, R.S. (1 973). Taxonomic implications of Prototheca and

Chlorella cell wall polysaccharide characterization. Arch. Microbiol. 92:227-233

Cooke, R.C. and Rayner, A.D.M. (1 984). r'€cology of Saprotrophic Fungi".

Longman, London and New York, 539 p.

Copeland, H.F. (1 956). "Classification of the Lower Organisms". Pacific Books,

Palo Alto, CA, ix+302 p.

Davis, L.L. and Bartnicki-Garcia, S. (1984). Chitosan synthesis by the tandem

action of chitin synthetase and chitin deacetylase from Mucor rouxii.

Biochemistry 23(6): 10651 073

Dea k, T. and Beauchat, L. R. (1 996). "Handbook of Food Spoilage Yeasts: CRC

Press, Boca Raton, FL, 210p.

de la Pefia, P., Barros, F., Gascon, S., Lazo, P.S. and Ramos, S. (1981).

Effect of yeast killer toxin on sensitive cells of Saccharomvces cerevisiae. J.

BioL Chem. 256(20): 10420-1 0425

Do Carmo-Sousa, L. (1 969). Distribution of yeasts in nature. In: "The Yeasts. Vol.

1. Bidogy of Yeasts", 1 si ed. (Rose, A.H. and Harrison, J.S., eds.). Academic

Press, London, UK, pp. 79-105

Elad, Y., Lifshitz, R. and Baker, R. (1985). Enzymatic adivity of the mycoparasite

W i u m nunn during interaction with host and non-host fungi. Physioi. Plant

Pathol. 27(2): 131 -1 48

Erwin, T.L. (1 982). Tropical forests: their richness in Coleoptera and other arthro-

pod species. Coleopt Bull. 36(1):74-75

Fleet, G.H. (1 991). Cell walls. In: "The Yeasts. Vol. 4. Yeast Organelles", 2nd

ed. (Rose. A.H. and Harrison, J.S., eds.). Academic Press, London, UK, pp.

1 99-277

Forster, H., Coffey, MD., EIwood, H. and Sogin, ML. (1990). Sequence analysis

of the small subunit ribosomal RNAs of three zoosporic fungi and implications

for fungal evolution. Mycologia 82(3):306-312

Fuller. M.S. and Clay, R.P. (1993). Observations of Gonaoodva in pure culture:

growth, development and cell wall characten'mtion. Mycologia 85(1):3845

Golubev, W.I. (1 991). Capsules. In: "The Yeasts. Vol. 4. Yeast O~anel/esnl 2nd

ed. (Rose. A.H. and Harrison, J.S.. eds.). Academic Press, London, UK, pp.

175-1 98

Golubev, W.I. (1 998). Mywcins (killer toxins). In: "The Yeasts, A Taxonomie

Study", 4th ed. (Kurtzman, C.P. and Fell, J.W., eds.). Elsevier Science B.V..

Amsterdam, The Netheriands, pp. 55-62

Haeckel, E. (1 866). "Generelle Morphologie der Organismen. Allgemeine

Gmndzüge der organischen Fonnen-WissenschaR, mechanisch begnïndet

durch die von Charles Darwin refomierfe Descendenz-Theorie. Vol. 1:

Allgemeine Anatomie der Organismen. Vol. Il: Allgemeine

Entwicklungsgeschichte der Organismen". Georg Reimer Veriag , Berlin.

Germany.

Hallbauer, D.K. and van Warrnelo, K.T. (1974). Fossilized plants in tbucholite from

Precambrian rocks of the Witwatersrand, South Afn'ca. Precambmn Res.

1 (3):199-212

Hattori, Y. and lida, S. (1 964). Amylolyüc enzymes of Endomvces IF0 01 11.

Conditions for production of enzyme by washed cell suspensions. Kogyo

Kagaku Zasshi 67(5):737-739. Cited in Chem. Abstr. 61 :7411 b

Hawksworth, D.L. (1 991 ). The fungal dimension of biodiversity: magnitude, signifi-

cance, and conservation. Mycol. Res. 95(6):641-655

1 64

Hawksworth, D.L., Kirk, P.M., Sutton, B.C. and Pegler, D.N. (1 994). "Ainsworth

and Bisby's Dictionary of the Fungi", 8th ed. CAB International, Egham, UK,

xii+6 1 6 p.

Hickman, C.J. (1965). Fungal structure and organization. In: "The Fungi. An

Advanced Treatise. Vol. 1. The Fungal Cell" (Ainsworth, G.C. and Sussman,

AS., eds.). Academic Press, New York, pp. 2145

Holan, 2.. Pokomy, V., Beran, K., Gemperle, A., Tuzar, 2. and Baldrian, J. (1981).

The glucan-chitin complex in Saccharomvces cerevisiae. V. Precise location

of chitin and glucan in bud scar and their physiwchernical characterization.

Arch. Microbiol. 1 30(4):312-318

Huss, V.A.R. and Sogin, M.L. (1990). Phylogenetic position of some Chlorella

species within the Chlorococcales based upon complete small-subunit ribosomal

RNA sequences. J. Mol. Evol. 31 (S):432-442

Ikediugwu, F.E.0 and Webster, J. (1 970). Antagonism between Coprinus hepte-

merus and other coprophilous fungi. Trans. Bnt Mycol. Soc. !H(2): 1 81 -204

Jefies, P. and Young, T.W.K. (1 994). Necrotrophic associations. In: "Interfungal

Parasitic Reîationships". CAB International, Wallingford, Oxon, UK, xvii+296

Koltin, Y. (1 988). The killer system of Ustilaoo mavdis: secreted polypeptides en-

coded by viruses. In: "Vinrses of Fungi and Simple Eukaryotes" (Koltin, Y . and

Leibowitz, M.J., eds.), Marcel Dekker, New York, pp. 209-242

Kopecka, M. and Kreger, D.R. (1986). Assembly of microfibnls in vivo and in

vitro from (1 -+3>po-glucan synthesized &y protoplasts of Saccharomyces

cerevisiae. Arch. Microbiol. l43(4):38%395

165

Kreger-van Rij, N.J.W. and Veenhuis, M. (1 973). Electron microscopy of some

special cell contacts in yeasl. J. Bacteriol. 1 l3(1):350-356

Kurtunan, C.P. and Robnett, C.J. (1 995). Molecular relationships arnong hyphal

ascomycetous yeasts and yeastlike taxa. Can. J. Bot. 73(Suppl. 1 ):S824-S830

Kurhman, C.P. and Smith, M.Th. (1998). Saccharomp~sis Schionning. In: "The

Yeasts, A Taxonomic Study", 4th ed. (Kurtzman, C.P. and Fell, J.W., eds.).

Elsevier Science B.V., Amsterdam, The Netherlands, pp. 374-386

Lachance M.-A. (1990). Yeast selecüon in nature. In: "Yeast Strain Selection"

(Panchal. C.J., ed.). Marcel Dekker, New York, pp. 2141

Lachance, M . A . and Pang, W.-M. (1 997). Predaciouç yeasts. Yeast l3(3):225-

232

Lachance, M.A., Pupovac-Velikonja. A., Schlag, B. and Guptill, C. (1997). A new

yeast habitat: yeasts. 18th International Specialized Symposium on Yeasts

(ISSYJ, Bled, Slovenia. August 24-29

Lachance, M.-A. and Starmer, W.T. (1998). Ecology and yeasts. In: "The Yeasts,

A Taxonornic Study", 4th ed. (Kurhman, C.P. and Fell, J.W., eds.). Elsevier

Science B.V., Amsterdam, The Netherlands, pp. 21-30

Laing, S A K . and Deacon, J.W. (1 990). Aggressiveness and fungal host ranges of

mycoparasitic Pythium species. Soi1 Biol. Biochem. 22(7):905-911

Lewis, D.H. (1 987). Evolutionary aspects of mutualistic associations between fungi

and photosy nthetic O rg anisms. In: "€volutionary Biology of the Fungi" (Rayner,

A.D.M., Brasier, C.M. and Moore, O., eds.). Cambridge ~niversity Press,

Cambridge, UK, pp. 161-1 78

Lumsden, RD. (1992). Mycoparasitism of soilborne plant pathogens. In: "The

Fungal Community, lts Organization and Role in the EcosystemN, 2nd edn.

(Carroll, G.C. and Wickow, D.T., eds.). Marcel Dekker, New York, pp. 275-293

Makower, M. and Bevan, E.A. (1963). The inheritance of a killer character in yeast

(Saccha romvces cerevisiae). I n: "Genetics Today. Proceedings of the 7 7 th

international Congress on Genetics, The Hague, The Nefheriands, September

1963. Vol. 1" (Geerts, S.J., Ed.), Pergamon Press, Oxford, UK, p. 202

Malloch, D.W., Pirozynski, K.A. and Raven P.H. (1 980). Ewlogical and evolutionary

significance of mycorrhizal symbioses in vascular plants (a review). Proc. Natl.

Acad. Sci. USA 77(4):2113-2118

Martinac, B., Zhu, H., Kubalski, A., Zhou, X., Culbertson, M., Bussey, H. and Kung,

C. (1990). Yeast K I toxin forms ion channeis in sensitive spheroplasts and in

artificiai liposomes. Proc. Natl. Acad. Sci. U.S.A. 87(16):6228-ô232

Mizutani, A., Hagiwara, H. and Yanagisawa, K. (1 990). A killer factor produced by

the cellular slime mold Polysphondvlium ~aiiidum. Arch. Microbiol. l53(5):413-

416

Moore, R.T. (1996). Evolutionary trends in the fungi. In: "Evolution of Microbial

Life. Fm-Fourth Symposium of the Society of General Microbiology, University

of WaMNck, March 7996" (Roberts, D. McL., Sharp, P., Alderson, G. and

Collins, M.A., eds.), pp. 205-224

Muzarelli, R.A.A. (1 985). Chitin. In: "The Polysaccharides. Vol. 3" (Aspinall, G.O.,

ed.). Academic Press, Oriando, FL, pp. 417450

Palffee, R.G.E. and Bussey, H. (1 979). Yeast killer toxin: purification and charac-

terisation of the protein toxin from Saccharomyces cerevisiae. Eur. J. Biochem.

93(3):487-493

Patterson, D. J. (1 989). Strameno~ila: chromophytes from a protistan perspective.

In: "The Chromophyte Algae: Problems and Perspectives" (Green, J .C . , Leadbeater, B.S.C. and Diver. W.L., eds.). Systematics Association Special Vol.

No. 38. Clarendon Press, Oxford, UK. pp.357-379

Patterson, D. J. and Sogin, M .L. (1 992). Eukaryote orygins and protistan diversity.

In: "The Ongin and Evolution of Prokatyotic and Eukaryotic Cells" (Harbnan , H.

and Matsuno, K., eds.). World Scientific, Singapore, pp.1346

Phaff, H.J and Starmer, W.T. (1987). Yeasts associated with with plants, insects

and soil. In: "The Yeasts. Vol. 1. Bioiogyof Yeasts", 2nd ed. (Rose, A.H. and

Harrison, J.S., 4s. ) . Academic Press, London, UK, pp. 723-1 80

Philliskirk, G. and Young, T.W. (1975). The occurrence of killer character in

yeasts of various genera. Antonie van Leeuwenhoek 41 (2): 747-1 51

Pirozynski, K.A. (1976). Fossil fungi. Ann. Rev. Phytopathol. 14:237-246

Pirozynski, K.A. (1 981 ). Interactions between fungi and plants throug h the ages.

Can. J. Bot 59(10): 1 824-1 827

Pirozynski, K.A. and Dalpé, Y. (1 989). Geological hystory of the Glomaceae with

particular reference to rnycorrhizal symbiosis. Symbiosis 7:l-36

168

Pirozynski, K.A and Malloch, D.H. (1975). The origin of land plants: a matter of

mycotrophism. BioSystems 6(3): 1 53-1 64

Rayner, A.D.M. and Webber, J.F. (1 984). lnterspecific mycelial interacüons - an

overview. In: "The Ecology and Physiology of the Fungal Mycelium" (Jennings,

D.H. and Rayner, A.D.M., eds.). Cambridge University Press, Cambridge, UK,

pp. 383-417

Ridout, C. J.. Caley-Smith, J.R. and Lynch, J.M. (1 988). Fractionation of extracellu-

lar enzymes from a mycoparasitic strain of Trichoderma harzianum. Enzyme

Microbiol. Biotechnol. 1 0: 1 80-1 87

Ruiz-Herera, J. (1 978). The distribution and quantitative importance of chitin in the

fung i. In: "Proceedings of the First international Conference on Chitin/Chitosan "

(Muzarelli, R.A.A. and Parker, ER., eds.), MITSea Grant Program, Cambridge,

MA, pp.11-21

Ruiz-Herrera, J . (1 992). "Fungal Ce11 Wall: Stmcture, Synthesis, and Assembly ".

CRC Press, Boca Raton, FL, [viii]+248 p.

Ruiz-Herrera, J. and Sentandreu, R. (1 992). Fungal ceIl wall synthesis and assem-

bly. In: "Current Topics in Medical Mycology. Vol. 3: (McGinnis, M.R. and

Borgers, M., eds.), Spnnger Verlag, New York, pp.168-185

San-Blas, G., San-Blas, F. and Serrano, LE. (1977). Host-parasite relationships

in the yeastlike fom of Paracoccidioides brasiliensis stran lVlC Pb9. Infect.

Immun. 1 5(2):343-346

Savile, D.B.O. (1 968). Possibe interrelationships between fungal groups. In: "The

Fungi. An Advanced Treatise. Vol. 111. The Fungal population " (Ainsworth,

G.C. and Sussman, A.S., eds.). Academic Press, New York, pp. 3-20

Sherwood-Pike, M.A. and Gray, J. (1985). Silurian fungal rernains: probable re-

cords of the class Ascomvcetes. Lethaia 18(1): 1-20

Sipiczki, M. (1 995). Phylogenesis of fission yeasts. Contradictions surrounding the

origin of a century old genus. Antonie van Leeuwenhoek 68:119-149

Skipper. N. and Bussey, H. (1 977). Mode of action of yeast toxins: energy require-

ment for Saccharomyces cerevisiae killer toxin. J. Bacteriol. 129(2):668-677

Stumm, C, Herrnans, J.M.H., Middelbeek, E.J., Croes, A.F. and de Vries, G.J.M.L.

(1 977). Killer-sensitive relationships in yeasts from natural habitats. Antonie van

Leeuwenhoek 43(2): 125-1 28

Walker, G.M. (1 998). "Yeast Physiology and Biotechnology". John Wiley & Sons,

New York, ix+350 p.

Whittaker, R.H. (1969). New concepts of kingdoms of organisms. Science

l63(3863): 1 50-1 60

Wickner, R.B. (1992). Double-stranded and singlastranded RNA viruses of

Saccharomvces cerevisiae. Ann. Rev. Microbiol. 46347375

Wickner, R.B. (1 996). Double-stranded RNA viruses of Saccharomvces cerevi-

siae. Microbiol. Rev. 60(1):250-265 -

1 ?O

Young, T.W. (1987). Killer yeasts. In: "The Yeasts. Vol. 1. Biologyof Yeastsn,

2nd ed. (Rose, A.H. and Hamson, J.S.. eds.). Academic Press, London. UK, pp.

Zhu, Y.S., Kane, J., Zhang, X.-Y., Zhang, M. and Tipper, D.J. (1 993). Role of the

ycomponent of preprotoxin in expression of the yeast K, killer phenotype. Yeasf

9(3):25 1-266