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Epidemiology of Blackleg Disease of Canola, Caused by Leptosphaeria

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Epidemiology of Blackleg Disease of Canola, Caused by Leptosphaeria Epidemiology of blackleg disease of canola, caused by Lepto sph ueríu maculuns Bita l{aseri Thesis submitted for the degree of Doctor of Philosophy in The University of Adelaide Schoot of Agriculture, food & Wine AUSTRALIA 2006 Table of contents 4bStfaCt.............................................................o...............................""tttt I AbbreViatiOnS..........................o............................................................. III ACknOwIedgemellts................................................................................IV PUbtiCatiOnS..........................................o......................................o..........VI Chapter 1. Introduction and Literature review................................... L 1.1 Introduction......... 1 t 1.2 Background......... 7.2.1 Canola cultivation in Australia.... 2 l.2.2History of blackleg disease ......... J 4 1 .2.3 Disease syrnptoms ....................... 1.3 Epidemiology of blackleg .6 1.3.1 Life cycle of Leptosphaeria maculans ,6 1.3.2Inoculum.. .8 1.3.2.1 Sources of inoculum 8 1.3.2.2 Production of inoculum...... 10 72 1 .3.2.3 Dissemination..................... 1.3.3 Infection t4 1.4 Disease control """"""16 1.4.1 Disease resistance............'... """""""""""16 1.4.2 Stubble management............ """"""""""'I7 1.4.3 Crop rotation """"'18 1.4.4 Disease escape... """"""""""" 19 1.4.5 Chemical control """"""""""'19 1.4.6 Biological contro1................ """""""""""20 1.5 Forecasting of disease...... """"""'21 1..6 Conclusion........... """"22 1.7 Research objectives.............. """"22 A Chapter 2. General material and methods 24 2.1 Plant materials.. 24 2.1.I Canola cultivars 24 2.l.2Plant growth 25 2.1.2.1 Potting soil.................., 25 2.1.2.2 Plant growth stage....... 25 2.1.2.3 Greenhouse conditions 25 2.1.3 Canola stubble 26 2.2 Contr olled environments ...................26 2.2.7 Controlled environment cabinet ....26 2.2.2 Inctbators .......... ....27 2.3 Fungi.. 27 2.3.1 L. maculans on canola stubble 27 2.3.2lsoLation and purifîcation of fungi from canola stubble... 28 2.3.3 Storage of fungal isolates 28 2.4 Statistical analysis................ ........29 Chapter 3. Formation of pseudothecia and ascospores of L. müculüns 30 3.1 Introduction ......... 30 3.2 Materials and Methods ................33 3.2.1Effect of temperature on pseudothecium formation under continuous wetness 34 3.2.2Bffect of darkness on pseudothecium formation under continuous wetness 35 3.23 Effect of discontinuous wetness on pseudothecium formation.. 35 3.2.4 Development of a method to study the effect of temperature on ascospore release in a controlled environment 36 3.2.4.t Assessment of ascospore release inside a plastic tray 36 3.2.4.2 Assessment of ascospore release inside a Petri dish .. 3/ 3.2.5 Statistical analysis .............. 3/ 3.3 Results .......39 3.3.1 Effect of temperature on pseudothecium formation under continuous wetness ...............39 3.3.2Bffect of darkness on pseudothecium formation under continuous wetness 42 3.3.3 Effect of discontinuous wetness on pseudothecium formation........ 43 3 .3.4 Development of a method to study the effect of temperature on ascospore release in a controlled environment ...,,,..46 3.3.4.I Assessment of ascospore release inside a plastic tray ........46 3.3.4.2 Assessment of ascospore release inside a Petri dish.. ........46 B Chapter 4. Germination and hyphal growth from ascospores of Z. maculøns in a controlled environment........................................... ......52 4.1 Introduction......... """'52 4.2Waterials and Methods .."""""""54 4.2.7 Expenmental design....... 54 4.2.2Bffects of temperature on germination and germ tube elongation of Z. maculans ascospores on agar-coated slides.... 55 4.2.2.1 Preparation of ascospore suspenslon................ 55 4.2.2.2 Preparation and inoculation of agar-coated slides.'...".. 56 4.2.2.3 Assessment of ascospore germination and germ tube elongation. 57 4.2.3 Effects of temperatüe and cultivar on germination and germ tube elongation of Z. maculans ascospores on plant tissue 4.2.3.1Inoculation of leaf and cotyledon.. 4.2.3.2 Assessment of ascospore germination and elongation of germ tubes............ 4.2.4Bffect of duration of incubation and cultivar on hyphal growth...... 4.2.5 Statistical analysis ........ 4.3 Results 60 4.3.1 Effect of temperature and cultivar on ascospore germination.............. 60 4.3.2Effect of temperature and cultivar on geÍn tube elongation.............'.. 64 4.3.2.1 On agar-coated slides 64 4.3.2.2 On plant tissue........ 67 4.3.3 Effect of duration of incubation and cultivar on hyphal growth.'.. 72 5.2. 1 Experimental design 5.2.2 Plant material........... 5.2.3 Inoculation............... 5.2.4Effect of rubbing the leaf surface 5.2.5 Lesion development and recovery of L. maculans frominoculated plants.............. 5.2.6 Statistical analysis ..... 5.3 Results .......83 5.3.1 Effect of temperature, wetness duration, cultivar and ieaf position......... .....'.83 5.3.2Ûffect of rubbing the leaf surface ..............88 5.3.3 Lesion development and recovery of L. maculans from inoculated plants.....'.................89 C 90 Chapter 6. Surviv tl of L. müculøns and associated mycobiota on bUried CanOlA StUbble and Changes in SOil miCrObiotâ.......................96 6.1 Introduction......... .......96 6.2 Materials and Methods 99 6.2.1 Burial of canola stubble in sand and field soil .....99 6.2.2 L. maculans and associated mycobiota on buried canola stubble ...100 6.23Effect of environmental factors and soil temperature ...r02 6.2.4 Pseudothecium formation on buried canola stubble . ...103 6.2.4.1 Effect of storage ...103 6.2.4.2 Effect of burial ...103 6.2.5 Pathogenicity of L. maculans ...104 6.2.6 Soil microbiota ...105 6.2.7 Statistical analysis ........... ...106 6.3 Results .....107 6.3.1 Burial of canola stubble in sand and field soil r07 6.3.2 L. maculans and associated mycobiota on buried canola stubble 1i0 6.3.3 Effect of environmental factors 116 6.3.4 Pseudothecium formation on buried canola stubble rt7 6.3.4.1 Effect of storage 117 6.3.4.2 Effect of burial 119 6.3.5 Pathogenicity of L. maculans. 119 6.3.6 Soil microbiota 121 Chapter T.lnteractions between L. muculans and associated fungi on canola stubble in vítro.......................................................................... 136 7.1 Introduction......... .....136 T.2Materials and Methods ..............138 7 .2.1 Dtal culture oî agar plates............. r39 7.2.2Dual culture on agar-coated slides. t39 7.2.3Inoculation of blackleg-affected stubble with frrngi ."'......'.......' 140 7.2.3.1Effect of inoculant fungi on germination of L. maculans ascospores......................I41 7 .2.3.2 Effect of spores of potential antagonists on germination of Z. maculans ascospores r41 7.2.4Effect of fungi on decay of canola stubble...... 142 7.2.5 Statistical analysis ............... 143 D 7.3 Results .....143 7.3.1 Duai culture on agar plates........ r43 7.3.2Dual culture on agar-coated slides 147 7.3.3 Inoculation of blackleg-affected stubble with fungi t47 7 .3 .3 .I Effect of inoculant fungi on germination of Z. maculans ascospores . ..149 7 .3.3.2 Effect of spores of potential antagonists on germination of L. maculdl,.s ascospores r49 7.2.4Effect of fungi on decay of canola stubble. 150 150 Chapter 8. General discussion 155 Appendix 1............................................................................................162 Appendix 2 163 Appendix 3............................................................................................ 165 Appendix 4 t67 References t70 E This work contains no material which has been accepted for the award of any other degree or diploma in any university or other tertiary institution and, to the best of my knowledge and belief, contains no material previously published or written by another person, except where due reference has been made in the text. I give consent to this copy of my thesis, when deposited in the University Library, being available for loan and photocopying. Signed: Date: 8l oø Abstract Blackleg, caused by L. maculans, is of major economic importance in the canola growing areas of Australia. The aim of this research was to gain information about factors affecting the epidemiology of blackleg in south-eastem Australia. The effect of temperature on a number of aspects of the life cycle of L. maculans was studied in a controlled environment. Germination of ascospores was greater on agar and cotyledons than on leaves, on susceptible cultivars than resistant cultivars, and at 15 and 20oC than at 5 and 10oC. Elongation of germ tubes was greater at higher than lower temperatures and generally greater on cotyledons than on leaves of the cultivars after incubation for 24 h at 10-20"C. Temperature had a greater influence than plant organ or cultivar on ascospore germination and the elongation of germ tubes. Temperature, wetness duration, cultivar and leaf position (leaves 1-3) influenced the incubation period of L. maculans on canola. Ascospores infected six canola cultivars over 10-20oC following leaf wetness duration of 76-72 h. Incubation period (from inoculation to the appearance of first lesions) was generally shorter at higher temperatures and following longer wetness periods. Incubation period decreased with increasing leaf age, rcgardless of the blackleg-resistance rating of the cultivars. Pseudothecia developed on naturally infested canola stubble incubated at 5-20'C under continuous wetness and a 12 h photoperiod.
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