THE AXKNIC CULTURE OF WHEAT AND FLAX RUST FUNGI by AMETAVA BOSE 3.Sc.(Agr.), Bihar Agricultural College M.Sc.(Agr.), Bhagalpur University M.Sc., University of British Columbia A Thesis Submitted in Partial Fulfilment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY in the Department of Plant Science We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October, 1973 In presenting this thesis in partial fulfilment of the re• quirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Depart• ment or by his representatives. It is understood that copy• ing or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Plant Science, -University of British Columbia, Vancouver 8, British Columbia. 1 Date im-M- ^ ABSTRACT Rust fungi belonging to the order Uredinales have usually been considered the classical examples of obligate parasites amongst plant pathogenic fungi. The development of our knowledge of the metabolism, nutrition and physiology of the rust fungi has been restricted because of our inability to grow these fungi in axenic culture. In the past the physiological and biochemical investigations on rust fungi per se have been limited to studies of the ger• mination and development of germ tubes. The cultivation in vitro of Puccinia graminis tritici (Erikss. and .Henri. ) , Australian race ANZ 126-6,7, by Williams et al. (1966, 1967), together with the report of Turel (1969) on the axenic culture of Melampsora lini (Ehrenb) Lev, race 3 promoted research on culturing rust fungi on artificial medium. An artificial medium containing 3% glucose, Czapek's mineral salts, 0.1% Evan's peptone, plus defatted BSA support• ed growth and sporulation of Puccinia graminis tritici race ANZ 126-6,7. Typical pigmented uredospores and teliospores were formed after 6-3 weeks growth. The uredospores were capable of infecting the mesophyll of wheat leaves exposed by stripping back the lower epidermis. Scanning electron microscopy revealed the presence of a coating of unknown chem• ical composition around the uredospores developed in vitro and not observed on uredospores grown on wheat leaves. iv Two different strains of Melampsora lini (Ehrenb) Lev were grown on solid media containing I+% sucrose, modified Knop's tissue culture macronutrients, Berthelot's micronut- rients, yeast extract and peptone. The mycelium was generally binucleate. Spore-like structures were recorded in the stroma which resembled uredospores and teliospores. Addition of 1% defatted BSA to the medium described above greatly increased the frequency of establishment of flax rust colonies. A de• fined liquid medium, containing Czapek's mineralsy Ca++, glu• cose, aspartic acid, glutathione and cysteine, and inoculated with uncontaminated uredospores, supported good vegetative growth and sporulation of wheat stem rust (Puccinia graminis f. sp. tritici race ANZ 126-6,7). Of six North American races of wheat stem rust fungus tested, only three grew vegetatively on artificial medium. Finally a chemically defined liquid medium contain• ing sucrose, Knop's mineral salts, micronutrients, aspartic (or glutamic) acid and cysteine supported the growth of vege• tative colonies of Melampsora lini race 3 from uncontaminated uredospores In a highly reproducible manner. The formation of uredospores and teliospores of these colonies in the liquid medium was controlled by the level of Ca++ (as CatNO-^)^ and the number of colonies per flask. With 60-70 colonies per flask, uredospore formation occurred on 60 to 70% of the colonies at a Ca++ level 8.5 mM. A decrease in the Ca++ level to 4.5 mM or colony frequency to 10 per flask resulted V in only infrequent sporulation. The uredospores produced in vitro infected intact cotyledons In a normal manner. This result with flax rust represents a substantial advance in our ability to control the growth of this important 'obligate* parasite In axenic culture. vi TABLE OF CONTENTS Page FRONTISPIECE Xiv ABSTRACT iii • TABLE OF CONTENTS vi LIST OF TABLES ix LIST OF FIGURES x ACKNOWLEDGEMENTS xiii GENERAL INTRODUCTION 1 REFERENCES 16 CHAPTER I - Sporuiation and Pathogenicity of an Australian Isolate of-Wheat Rust grown in vitro. ABSTRACT..; 20 INTRODUCTION 21 MATERIALS AND METHODS 22 RESULTS 24 1. Behaviour of Germinating Uredospores on Solid Medium 21+ 2. Development of Colonies on Liquid Medium 24 3. Pathogenicity of Uredospores produced in Axenic Culture 30 4. Scanning Electron Microscopy,... 30 Page .CHAPTER II. In vitro Culture of the Flax Rust, Melampsora lini. ABSTRACT 36 INTRODUCTION 36 MATERIALS AND METHODS 37 1. Melampsora lini, Strain 1 37 2. Melampsora lini, Strain 2.... .- 40 3. Cytological Examination of the Cultures 41 RESULTS 41 1 • Melampsora lini , Strain 1. .. 41 2. Melampsora lini, Strain 2 43 3. Cytological Studies with Melampsora lini 1+6 DISCUSSION 51 REFERENCES.' 59 CHAPTER III. In vitro Growth of Wheat and Flax Rust Fungi on Chemically Defined Media. ABSTRACT 60 INTRODUCTION •. 61 MATERIALS AND METHODS 68 1. Production of Uncontaminated Uredospores 68 2. Culture Media.. ' 69 3. Sources of Organic Nitrogen and Sulphur 70 4. Inoculation of Media 71 5. Assessment of Growth.. 72 RESULTS 72 Wheat Rust 72 1. Trials with Solid Media 72 VXll Page 2. Trials with Liquid Media 73 A. Growth of races 15B4, 3#, 32-113 and ANZ 126-6,7 73 B. Effect of cAMP on growth of ANZ 126-6,7 73 C. Effect of aspartic acid, cysteine, gluta• thione and calcium on the growth of ANZ 126-6,7 74 Flax Rust 79 1. Trials with Solid Media., 79 A. Effect of BSA 79 B. Reinfection of host using vegetative colonies grown on solid medium 80 2. Trials with Liquid Media 8l A. Preliminary trials and effect of BSA 81 B. Effects of mineral nutrients 85 C. Effects of aspartic acid, cysteine, gluta• thione and calcium 88 D. -Reinfection of flax using uredospores grown in vitro 91 LIST OF TABLES PAGE TABLE I Growth of obligate parasites in host tissue 5 II In vitro development of rust fungi from germinating uredospores 8 III-I Effect of cyclic AMP and theophylline on growth of Puccinia graminis tritici (race ANZ 126-6,7) 77 III-II Effect of amino acid, glutathione and Ca(N0^)2 on the growth of Puccinia graminis tritici (race ANZ 126-6,7) 78 III-III Effect of different organic nitrogen sources on the growth of Melampsora lini (race 3 ) $k III-IV Growth and development of Melampsora lini (race 3) on completely defined liquid media... X TiTS-T OF FIGURES PAGE FIGURE 1-1 Uredospores with burst germ tube on basic medium 26 1-2 Two germinating uredospores and their germ tubes have crossed each other 26 1-3 Two nuclei in cytoplasm extruded from burst germ tube 26 1-4 Germ tubes originating from two uredospores showing anastomosis 26 1-5 Vegetative juvenile colonies and sporula- ting colonies developed on liquid medium 26 1-6 Developing uredospores in colony grown on 28 liquid medium 1-7 Developing teliospores and maturing uredo• spores from colony grown on liquid medium 28 1-8 Fully developed two-celled teliospores....,,... 28 1-9 Thick mat of hyphae at periphery of colony 28 grown on liquid medium, 1-10 Pustules developed on primary leaf of Little Club wheat as a result of inoculation with uredospores grown in vitro 28 1-11 Scanning electron micrographs of mature uredospores grown on intact wheat leaves 32 1-12 Scanning electron micrograph of mature uredospores grown in vitro. '. 32 1-13 Scanning electron micrograph of spines on surface of uredospores grown on intact wheat leaf 32 1-14 Scanning electron micrograph of spines on surface of uredospores grown on basic medium... 32 1-15 Scanning electron micrograph of developing spines on surface of uredospores grown on intact wheat leaf 32 1-16 Scanning electron micrograph of a teliospore grown on basic medium 32 xi PAGE FIGURE II-l A 12-day-old colony of strain 1(fourth transfer)..,, 45 II-2 Same colony viewed from below... 45 II-3 An 8-week-old colony of strain 2 grown from aseptic uredospores 4$ II-4 An"8-week-old colony of strain.2 of small size consisting entirely of aerial mycelium... 48" II-5 Fusion of germ tubes ,.. 48" II-6 Binucleate hyphae of an initial colony.. 4$ II-7 Binucleate mycelium of a transferred colony, 50 II-3 Uninucleate and binucleate hyphal cells from an old colony 50 II-9 Anastomosis of aerial hyphae of an old colony ' 50 11-10 Thin walled spherical cells from stromata . of an initial colony 50 11-11 Thin walled spherical cells interspersed by short hyphal cells 50 11-12 "Microsorus" structure from an initial colony 53 11-13 Non-pigment ed uredospore like cells 53 11-14 Uredospore like cells joined by short hyphal cells 53 11-15 Dense spore cluster from stromata of a transferred culture. ;. 55 11-16 Spore like cells formed at terminal hyphae..... 55 II- 17 Spore like cells formed in chains. 55 III- l North American isolates of wheat stem rust showing vegetative growth on liquid medium 76 III-2 Growth and development of an Australian race of wheat stem rust on liquid medium 76 .'.'hsat s tern rust ANZ 126-6,7 developed on a chemicaliv defined medium 7o FIGURE III-4 Flax rust colony initials raised on solid medium III-5 Colonies of flax rust originating as a re• sult of gelatin spore suspension seeding on solid medium III-6 Ectoparasitic mycelium of flax rust de• veloped on excised cotyledon III-7 Flax rust colonies developed on liquid medium Ill-8 Flax rust colonies developed on liquid media surface.