lrv4r'f ii I rri;Tl'l UTE ta -3 '1Q ' Ltu,R,\HY STUDIES ON SILVER LEAF DISEASE OF STONE AND POME FRUIT TREES by GEOFFREY CHARLES BISHOP B.Ag. Sc. (Hons.) Adelaide Department of Plant Pathology, lVaite Agricultural Research lnstitute, University of Adelaide, South Australia. A dissertation submitted to the University of Adelaide in partial fulfilment of the requirements for the degree of Doctor of Philosophy. May 1978 TABLE OF CONTENTS Page SI'JMMARY iii STATEMENT v ACKNOWLEDGEMENTS vi I GENERAL INTRODUCTION 2 The Disease 2 Distribution 4 Hosts of C/¡o ndrostereum purpureum 9 Economic Importance t7 Control Measnres t9 Objectives ofthc studies presented in this thesis 22 il SILVER LEAF DISEASE IN SOUTH AUSTRALIA 23 Incidence 23 Hosts of Cåondrostereum purpureum 27 Economic Importance 29 ilI COLLECTION AND CULTURE OF Chondrostereunt purpureunx 32 Collection of basidiocarps and basidiospores 32 Storage of basidíospores 32 Growth on artifìcial media 35 Maintenance of cultures 37 ry STUDIES ON THE INFECTION OF FRIIIT TREES 38 By Cho n dr o s t er eum p urpureum Quantity of inoculum reqnired to induce infection by 38 Chondrostereum purpureun in freshly pruned cherry sapwood Materials and Methods 39 Results and Discussion 39 Suiceptibility of cherry trees to different isolates 4l of Chondrostereum puryureum Materials and Methods 43 Results and Discussion 44 Phytolysin production by isolates of Chondrostereum purpureum 46 Materials and Methods 46 Results and Discussion 48 Page V STUDIES ON BIOLOGICAL CONTROL OF' Chondrostereum purpureum 5l A. Studies on Fusarùtm lateritium Nees 51 Fusarium lateritium as a plant pathogen 5l In vitro studies of the interaction between C. purpureum 52 andF.lateritium Detection of a fungitoxin by a bioassay technique 52 Inhibition of F. lateitiuntby C. purpureum 56 Inhibition of C. puryureumby enniatin s6 Production of enniatin by isolates of Fusarium lateritium 57 Materials and Methods 57 Results and Discussion 60 Sportrlation of F. lateritium on pruned cherry sapwood 65 surfaces in the field Study of a natural population of Fusariutn laterítium 67 Materials and Methods 69 Results 69 Toxicity of orchard fungicides Io F. lateitium 77 Discussion 80 Protection of cherry sapwood by F. lateritium against invasion by 80 Chondrostereum purpureun in the field Materials and Methods 80 Results and Discussion 82 B. Selection of a biological control agent for Chondrostereum purpureltm B6 Introduction 86 Sporulation of Fusøium isolates 88 Crossplating tests with the selected fungi 89 G¡owth of C. puryureurz isolates and test fungi in cherty wood blocks 89 Protection of cherry sapwood in vitro 92 VI GENERAL DISCUSSION 95 VII REFERENCES 98 VIII APPENDICES lt7 Appendix I. Publication 118 Appendix II. Media 119 Appendix III. Rainfall data for'Ferndale', Basket Range from 122 July 1975 to January 1978. Appendix IV. Orchard sprays 125 Appendix V. Correlations of spore number per g and rainfall 126 prior to sampling Appendix VI. Failule of an attempt to artificially infect apple 127 trees with Chondrostereum purpureum ERRATA page 4 line 9 BrenchJ-eY rr Iast l-ine sapnophyte 13 Tab1e Pvwmts institia 14il EseaLLonía 20 line 9 C. put'ptæewn It last line PeníopLnra gigøt'tea 21 line 3 Poz,ia ca.rboníea ll ll 11 Ethenidge 22 ll 24 infection 23 It 15 Leishrnan 27 ll 18 C. purpurewn rranll tlonll 30 It 9 for rrdischargerr trdepositionrr 32 ll t9E2I substitute for 34 It 3 Gnosclaude It il 4 C. pllrpureum il il 25 successive 38 n t2 individual 46 il 10 Albersheirn tr It 48 il 36 should nead . not only due. ' rr. a 52 ll 9 should nead . t^las found associated with twig dieback of mulbenrY...rt 60 rr 10 Canter t Ilsley (not Canten t Pnice) 63 rBl C. purpuYewn not inhibited I 65 l-ine 10 pathogen s 69 t, 28 Figr:::e 14a 7t figure Figune 14a 87 Irrje '2 I PottIe 94 il3 Bunkard 96 rt2 possess 99 nef. 15 sanbuciram 104 lt Insent: DrAeth, H.R.X. (1939) A survey of inte::action between fungi. Biological Reviews 14, 105-131' tt3 il I physiological rt6 It 6 Wood c.C. BishoP 1-4.!1.78 ul SUMMARY of Chondrostereum purpureunx has been compiled from A conrprehensive list of the hosts Australia. Thirty-nine plant species were the literature and from field observations in south previously bcen listed as hosts ,,orded as hosts in South Australia and of these 11 had not ,rclm any other countrY' and willow were found to be as effec- Basidiospores of c. purpureum from apple, brooln cherry sapwood. The isolates did, however, ,ive as those from cherry in invadilg frestrly prunecl caused' It was found that those differ in the amount of silYering of the foliage that they relatively little phytolysin' the substance isolates which causecl little foliar silvering produced that disrupts the leaf tissues and causes silvering symptoms' putpureum were initiated' Fus- Studies seeking a potential biological control agent for c. protection against another vascular ttrium lateritium, whichhas been found to give significant .armenincae, purpureunl both in the pathogerr of woody plants, Eutypa was tested against c. mycelial growth of c' pur- laboratory and in the field. F. lateritium was found to inliibit age of the F' lateritium pureum in vítro, the size ot the inhibition zone increasing with the of an antibiotic' colony. The invitro inhibition of C. purpureum was due to the production enniatin, by F. lateritium. apple orchard was A stndy of a natural population or Fusarium lateritium in a commercial tree bark was assessed carried out. The nunþer of viable propagules of F. lateritium on apple increased at weekly intervals oveÍ a period of 30 months. The population of F' lateritium summer' on from autumn onwards, reaching a peak in spring ancl falling to a low level over population could be several occasions duríng the growing season' temporary decreases in the When these attributed to the application of fungiciclal sprays' notably Difolitan and Dithane' found that ( 5 fungicides were tested in the laboratory for toxicity to F. lateritium ilwas the germina' p.p.m. active ingredient of both Difolitan and Dithane would completely inhibit growth' tion or F. løtertttutm macroconidia , and 4 p.p.m. A.I. Difolitan would inhibit hyphal The .,;t of rainfall on the population or F. hteritium appeated to be more complex than and that cf the fungicidal splays. No signihcant correlations were foutld between spole coutrt rain that fell, t¡ 24 hour intervals up to 7 days prior to sampling' sporulation When macroconidia of F. lateritium were applied to a freshly pruned surface, suspen- occurred within 7 days, and continued at a high level for at least 6 weeks' I{owever, a sion of F. lateritium nracroconidia applied to wouncls immediately after pruning afforded little protection against inocula of C. purpureum tntodttced on the pruned cherly sapwood either 1,3 or 5 days after pruning. When only the pathogen, C' purpureum, wasappliedto pruning wounds all sites were invaded; but when F. lateritium was appliecl 5 days before inoculation lv \¡vith C. purpureum only 68% of the sites became infected with the pathogen. Several other fungi that were isolated from the surface of pruning wounds on fruit trees were tested in the laboratory against C. purpureum and some showed promise as possible bio- logical control agents. Three fungi, a Fusørtum sp. and two Trichdermø spp. were found to prevent the invasion of cherry wood blocks by C. purpureum. Spores of all three genninated and started to grow well in advance of C. purpureum, and the Trichodermas sporulated readily on the pruned sapwood. v STATEMENT This thesis contains no material which has been accepted for the award of any other degree in any university and to the best of my knowledge and belief contains no material previously published or written by another person, except where due reference is made in the text. -7r¡-v, (cì'7 Z G. C. Bishop vl ACKNOWLEDGEMENTS I wish to thank my supervisor, Dr. M.V. Carter, for his encouragement and continued interest tlrroughout the course of tHs investigation. Special thanks must also go to Dr. A.H. Ilsley (Department of Anaesthesia and Intensive Care, Flinders Medical Centre, Bedford Park, South Australia) and Dr. J. Rishbeth (Botany School, University of Cambridge, England) who gave helpful advice at different stages of the work. I am grateful to Mr. D.E. Symon @epartment of Agronomy, Waite Institute) for supplying botanical names of host plants in South Australia, to Drs. P.[I.B. Talbot and J.H. Warcup @epartment of Plant Pathology, Waite lnstitute) for criticisms and suggestions during the preparation of the manuscript and to Mr. D.T. I(lpatrick for his keen observation of Silver Iæaf Diseaæ during his years with the South Australian Department of Agriculture. I would like to thank Mr. M. Bumbieris for translation of several German publications, and Mr. R'P. Cook and Mr. T. Hancock (Biometry Section, Waite lnstitute) for advice on statistical tests. My thanks also to Lindy Castree for preparation of illustrations and for technical and general assistance, Mrs. Iæa Wichman and Mr. B.A. Palk for photography and preparatio¡ of illustrations, susan campbell and Barbara Deverson for typing the text. I wish to thank Messrs.D.A., M.E. and W.N. Bishop of Bishop Orchards,Basket Range, for use of trees in their orchard and for making fungicides available for my use, and Mrs. W.J. Bishop for rainfall records. The use of the facilities in the Department of Plant Pathology, Waite Agricultu¡al Research lnstitute and finance from the Commonwealth Department of Education under the Common- wealth Postgrarluate Award scheme are also gratefully acknowledged. FIGURE I Edouard Prillieux (1829-1915) who delivered the fi¡st recorded scientific paper on Silver Leaf Disease to a meet- ing of the Sociát6 National D'Agriculture de France held on July 22 1885.