Epidemiology of blackleg disease of canola, caused by Leptosphaeria maculans

Transcription

1 Epidemiology of blackleg disease of canola, caused by Leptosphaeria maculans Bita Naseri Thesis submitted for the degree of Doctor of Philosophy in The University of Adelaide School of Agriculture, food & Wine AUSTRALIA 2006

2 Table of contents Abstract... I Abbreviations... III Acknowledgements...IV Publications...VI Chapter 1. Introduction and Literature review Introduction Background Canola cultivation in Australia History of blackleg disease Disease symptoms Epidemiology of blackleg Life cycle of Leptosphaeria maculans Inoculum Sources of inoculum Production of inoculum Dissemination Infection Disease control Disease resistance Stubble management Crop rotation Disease escape Chemical control Biological control Forecasting of disease Conclusion Research objectives...23 A

3 Chapter 2. General material and methods Plant materials Canola cultivars Plant growth Potting soil Plant growth stage Greenhouse conditions Canola stubble Controlled environments Controlled environment cabinet Incubators Fungi L. maculans on canola stubble Isolation and purification of fungi from canola stubble Storage of fungal isolates Statistical analysis...29 Chapter 3. Formation of pseudothecia and ascospores of L. maculans Introduction Materials and Methods Effect of temperature on pseudothecium formation under continuous wetness Effect of darkness on pseudothecium formation under continuous wetness Effect of discontinuous wetness on pseudothecium formation Development of a method to study the effect of temperature on ascospore release in a controlled environment Assessment of ascospore release inside a plastic tray Assessment of ascospore release inside a Petri dish Statistical analysis Results Effect of temperature on pseudothecium formation under continuous wetness Effect of darkness on pseudothecium formation under continuous wetness Effect of discontinuous wetness on pseudothecium formation Development of a method to study the effect of temperature on ascospore release in a controlled environment Assessment of ascospore release inside a plastic tray Assessment of ascospore release inside a Petri dish Discussion...47 B

4 Chapter 4. Germination and hyphal growth from ascospores of L. maculans in a controlled environment Introduction Materials and Methods Experimental design Effects of temperature on germination and germ tube elongation of L. maculans ascospores on agar-coated slides Preparation of ascospore suspension Preparation and inoculation of agar-coated slides Assessment of ascospore germination and germ tube elongation Effects of temperature and cultivar on germination and germ tube elongation of L. maculans ascospores on plant tissue Inoculation of leaf and cotyledon Assessment of ascospore germination and elongation of germ tubes Effect of duration of incubation and cultivar on hyphal growth Statistical analysis Results Effect of temperature and cultivar on ascospore germination Effect of temperature and cultivar on germ tube elongation On agar-coated slides On plant tissue Effect of duration of incubation and cultivar on hyphal growth Discussion...72 Chapter 5. Incubation period of L. maculans on canola in a controlled environment Introduction Materials and Methods Experimental design Plant material Inoculation Effect of rubbing the leaf surface Lesion development and recovery of L. maculans from inoculated plants Statistical analysis Results Effect of temperature, wetness duration, cultivar and leaf position Effect of rubbing the leaf surface Lesion development and recovery of L. maculans from inoculated plants...89 C

5 5.4 Discussion...90 Chapter 6. Survival of L. maculans and associated mycobiota on buried canola stubble and changes in soil microbiota Introduction Materials and Methods Burial of canola stubble in sand and field soil L. maculans and associated mycobiota on buried canola stubble Effect of environmental factors and soil temperature Pseudothecium formation on buried canola stubble Effect of storage Effect of burial Pathogenicity of L. maculans Soil microbiota Statistical analysis Results Burial of canola stubble in sand and field soil L. maculans and associated mycobiota on buried canola stubble Effect of environmental factors Pseudothecium formation on buried canola stubble Effect of storage Effect of burial Pathogenicity of L. maculans Soil microbiota Discussion Chapter 7. Interactions between L. maculans and associated fungi on canola stubble in vitro Introduction Materials and Methods Dual culture on agar plates Dual culture on agar-coated slides Inoculation of blackleg-affected stubble with fungi Effect of inoculant fungi on germination of L. maculans ascospores Effect of spores of potential antagonists on germination of L. maculans ascospores Effect of fungi on decay of canola stubble Statistical analysis D

6 7.3 Results Dual culture on agar plates Dual culture on agar-coated slides Inoculation of blackleg-affected stubble with fungi Effect of inoculant fungi on germination of L. maculans ascospores Effect of spores of potential antagonists on germination of L. maculans ascospores Effect of fungi on decay of canola stubble Discussion Chapter 8. General discussion Appendix Appendix Appendix Appendix References E

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8 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-eastern 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 20 o C than at 5 and 10 o C. 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 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 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, regardless 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. The time taken for pseudothecia to mature after harvest, ranged from 58.3 days at 5 C to 22.2 days at 15 C. Fewer pseudothecia developed on stubble incubated in darkness at 15 C than in light. Pseudothecia took longer to mature when wetness was interrupted than continuous. More ascospores were released at 20 C than 5-15 C, although peak sporulation occurred earlier at 5-10 C. Discharge of ascospores continued for 8 h at 5-10 o C, for 10 h at 15 o C, and for 12 h at 20 o C. I

9 The effect of burial of infested canola stubble in sand and field soil (in pots) on pathogen survival, pathogenicity and stubble-associated fungi was studied over 13 months in ambient conditions. The isolation frequency of L. maculans from stubble decreased by 63.3% after 13 months of burial, regardless of soil type. Although the frequency of isolation of L. maculans and Alternaria spp. from stubble decreased over time, that of Stachybotrys chartarum, Fusarium spp. and Coprinus sp. increased substantially over the sampling period. Stubble buried in field soil underwent more decay than in sand over the 13 months. Pseudothecium formation on buried stubble decreased with increasing duration of burial and ceased after 11 months in the field soil and 13 months in sand. The time required for pseudothecia to mature was 25 days for stubble retrieved after one month and 30 days when retrieved 10 months after burial, regardless of soil type. Ascospores from the stubble recovered from burial caused typical Phoma leaf spots on canola over the first 9 months of burial. Over the year, 16 genera of fungi and 12 colony types of bacteria were isolated from the field soil on peptone-rose bengal agar (PRA) and on crystal violet agar (CVA), respectively. Populations of fungi and bacteria isolated on soil-extract agar were markedly greater than those on PRA and on CVA, respectively. Growth and sporulation of L. maculans on agar media and on infested canola stubble was affected by a variety of fungi obtained from stubble buried in soil or from field soil. Antagonistic activities observed included lysis, deformation, overgrowth and inhibition of L. maculans hyphae, and reduction of pseudothecium density on stubble to one-third or less than that on controls following inoculation with F. equiseti, Gliocladium roseum, Trichoderma aureoviride, Sordaria sp. and an unknown Coelomycete. S. chartarum and Coprinus sp. reduced the mass of canola stubble in vitro. Findings will contribute to a better understanding of blackleg and to the development of more effective control measures. II

10 Abbreviations ANOVA CFU CVA cv. cvs. DNA DV8A G _ G+ GS LSD NA NUV PDA PRA RO SEA WA Analysis of variance Colony forming units Crystal violet agar Cultivar Cultivars Deoxyribonucleic acid Dilute vegetable juice agar Gram-negative bacteria Gram-positive bacteria Growth stage Least significant difference Nutrient agar Near ultra violet Potato dextrose agar Peptone-rose Bengal agar Reverse osmosis Soil-extract agar Water agar III

11 Acknowledgements I would like to thank my supervisor, Associate Professor Eileen Scott, and my co-supervisor, Mrs Jenny Davidson, for their kind encouragement, support and guidance throughout this project. I would also like to thank the Iranian Ministries of Science, Technology and Research, and Agriculture for providing a scholarship to support this project. I would also like to thank: Mrs Kathryn Dowling for valuable statistical advice and assistance throughout the research. Dr Michael Priest for identifying and providing DAR numbers for selected fungi. Mr Colin Rivers for kind assistance and advice within soil analysis. Mr Trent Potter for assisting with field visits and supplying canola seed and stubble. Assoc. Prof. Eileen Scott s plant pathology research group for their friendship and constructive criticism during seminar practices. Mrs Jenny Davidson s crop pathology research group for their technical support, assistance and friendship. Dr Peter Crisp for assisting to set up the weather station and advice for recording data. Prof. Dani Shtienberg for instruction in analysis of data and advice for presenting results. Mr Farid Moradinezhad for helpful advice in statistical analysis. Dr Alireza Marefat for assistance and advice for grouping bacterial isolates. IV

12 To the following people for assistance and advice during this project; Ms Sharon Clapham, Mrs Heather Fraser, Dr Gary Taylor, Dr Ian Riley, Dr Amanda Able. To my family for all their encouragement, patience and support throughout this project and to my lovely little son, Armin. V

13 Publications Naseri B, Davidson J, Sowsnowski M, Scott E, Effect of temperature and cultivar on germination and hyphal growth from ascospores of Leptosphaeria maculans on canola stubble buried in soil. In: Proceedings of The XVth International Plant Protection Congress. Beijing, China: May. Naseri B, Davidson J, Scott E S, Survival of Leptosphaeria maculans on canola stubble buried in soil. In: Proceedings of the 15th Biennial Australasian Plant Pathology Society Conference. Geelong, Victoria, Australia: September. Naseri B, Davidson J, Marefat A, Scott E, Survival of Leptosphaeria maculans on buried canola stubble and changes in soil microbiota. In: Proceedings of 14th Australian Research Assembly on Brassicas. Port Lincoln, South Australia, Australia: 3-7 October. VI