SOME CONTRIBUTIONS TO THE BIOLOGY OF / ARMILLARIA MELLEA (VAHL EX FR.) QUEL. "being a thesis presented "by ELIZABETH K. MITCHELL, B.Se. for the degree of Doctor of Philosophy of the University of Glasgow. June, 1950 ProQuest Number: 13850814 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 13850814 Published by ProQuest LLC(2019). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 CONTENTS Page SUMMARY 1 ACKNOWLEDGEMENT S 3 I. INTRODUCTION 4 II. REVIEW OE LITERATURE 7 III. MATERIAL AND METHOD 26 IV. EXPERIMENTAL INVESTIGATIONS A. PHYSIOLOGY a) Germination of spores 32 b) Effect of light on growth 40 e) Effect of temperature on growth 48 a.) Effect of hydrogen ion concentration on growth 55 e) Effect of various media on growth 57 f) Influence of inoculum on colony growth 74 8) Culture with other fungi 77 h) Isolation of monospore cultures 88 i) Production of fructifications 89 B. INVESTIGATION OF PATHOGENICITY $1 a) Relation of nutrient supply to the virulence of A. meliea 94 b) Spores as agents of infection 99 c) Method of fungal penetration into roots 100 V. DISCUSSION 105 VI. REFERENCES 112 1 SUMMARY 1. six strains of A. mellea, obtained from different sources, were cultured in the laboratory and various aspects of their biology were studied. 2. Formation of rhizomorphs and sclerotia, and dry weight of fungal colonies, were reduced by the incid­ ence of light on both solid and liquid media. She amount and intensity of light affected the form of the colony. 3. Maximum growth of all strains took place at 25*0., but a tropical strain developed better than a temper­ ate one at higher temperatures. 4. Greater rate of linear spread of the mycelium took place at ph 7. 5. Mycelial,rhizomorphic and sclerotial development was affected by the composition of the medium and favoured by those media containing the greatest amount of dissolved nutrients. Glucose and peptone were the most suitable carbon and nitrogen sources, respectively. 6. She inoculum influenced the development of cultures and a rhizomorph-containing portion gave a colony with the maximum formation of rhizomorphs and sclerotia. 7. a colony of one strain of A. mellea produced fructifications on a peptone-glueose-saccharose solution after eight months in daylight. 8. Double culture of A. mellea with various wood-destroying and soil fungi showed that growth of a . mellea was adversely affected in most instances. 9. Spores from field fructifications gave greatest percentage germination on substrates of acid reaction at an optimum temperature of 25°0. 10. Uormal growth of mycelium,sclerotium and rhizomorphs occurred in mono spore cultures though not all isolates formed colonies. 11.fiSpores did not act as agents of infection of bean roots. 12. In agar culture of A4 mellea and bean seedlings, roots of the latter were penetrated by rhizomorphs and penetration was influenced by the nutrients in the agar. 13.- hyphal invasion of roots took place, and hyphal penetration occurred in advance of attack by rhizomorphs. 3 ACKNOWLEDGEMENTS The work described in the following pages was carried out during two years in the Botany Department of the University of G-lasgow, and one year in the Cryptogamic Botany Department of the University of Manchester. The author wishes to express her thanks to Professor John Walton for his support in obtaining a James Fleming Scholarship and subsequently a Maintenance Allowance from the Department of Scientific and Industrial Research, and also for placing the facilities of his Department at her disposal. Gratitude is also due to Dr. S-.G. Jones who supervised the investigations undertaken in Glasgow, and to Professor C.W. Wardlaw for research facilities at Manchester and his guidance dxiring the latter phase of the work. The writer is also grateful to Mr. E. Ashby and Mr. P. Dawes (Manchester) for the majority of the photographs which illustrate the thesis, and to Air. W. Anderson (Glasgow) for the remainder. Thanks are offered to Miss Betty Legge who kindly gave suggestions during the final preparation of this account. I. INTRODUCTION. Armillaria mellea (Fr.) Quel, is a Basidiomycete,a member of the order Hymenomycetales, and of the family Agaricaceae which includes the common mushrooms and toadstool's. A. mellea is of world-wide distribution and has been recorded in varying climatic conditions. It has been observed in the continents of Europe, America and Africa, and it is common in many other countries including India and Japan. The host range of A. mellea is great; both hard and soft woods are attacked, also economic and ornamental plants if growing in ground infected with the fungus. Among plants which are severely affected by this organism are the plantation crops of tea, coffee and citrus fruits. Some trees are known to have resistant varieties, The pathogenicity of A. mellea itself is thought to be a variable property, the latter possibly being due to the occurrence of different strains of the fungus, to local environmental conditions, or to the presence of other soil fungi. Many records of the occurrence of A. mellea are available and a number of investigations have been made. Some detailed studies of the physiology, including cultural characteristics and requirements have been published, but investigations of pathogenicity have often been in the form of field trials, with subsequent detailed 5 examination of the invaded tissue in some cases, Thomas (1954) gives a description of the invasion of various plants by A. mellea. The initial penetration of the host takes place hy means of rhizomorphic branches which first attach themselves to the roots by a hardening mucilage, and by hyphae growing out from near the rhizomorphic tips. These hyphae only penetrate the dead cork cells and the actual invasion of the root takes place by the entrance of the rhizomorph as a whole. Butler and Jones (1949) give an account of the Armillaria root rot which is similar to that given by: Hiley (1919) for larch. The fungus develops under the bark of the roots and spreads upwards into the trunkf in the form of a rhizomorph network. At a more advanced stage of attack, the wood is also found to be invaded by the fungus which forms xylostromata, and the host tissue finally becomes disintegrated. The presence of the fungus, as shown by a visible effect on the host, is rarely evident until the decay has reached an advanced stage. The first symptom to be seen is a yellowing of the leaves with subsequent loss of the foliage. Formation of resin appears to be stimulated in some cases, and this oozes out through cracks in the bark. This latter effect is noted in some coniferous hosts, but not in other plants which may also become invaded. Fructifications may be produced in the final stages of the disease, and are usually at ground level (see Plate 1). The “biology, ecology and pathogenicity of A. mellea, suggest many lines of work. The author has selected for particular study, various cultural and pathogenic features of the fungus. Scrutiny of the previous investigations made, show that relatively little attention has "been paid to the initial penetration of the fungus into the host, except “by the agency of rhizomorphs. The particular studies reported here are concerned with possible methods of penetration into the host, and such matters as the effect of fungal invasion on the neighbouring tissues of the host. j?rom previous investigations and observations recorded, it was seen that although the organism is well known and widespread, no completely effective control measures are available for all areas. It was thought that further information on the pathogenic habit of the fungus, particularly its invasion method and also its interrelationship with other micro-organisms of the soil might contribute to our knowledge of measures of control. In this study, some interesting data have been obtained. It was not always possible to advance an adequate explanation of the observed phenomena, but where possible this has been attempted. Plate 1. Fructifications of A. mellea growing from the base of the stem of a young conifer. Note the typical defoliation of the branches due to the disease.