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Studies on Canker in European Filbert Caused by Anisogramma Anomala

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Studies on Canker in European Filbert Caused by Anisogramma Anomala AN ABSTRACT OF THE THESIS OF Joseph D. Postman for the degree of Master of Science in Botany and Plant Pathology presented onApril 15, 1986 Title: Studies on Canker in European Filbert Caused by Anisogramma anomala Redacted for Privacy Abstract approved: . H. Ronald Cameron Anisogramma anomala causes a stem canker of filberts, Corylus avellana. This disease became a threat to the main U.S. filbert growing region when it was introduced into the Pacific Northwest around 1968. Various methods of artificially inoculating filbert trees and seeds were explored. Less than 1% of potted filbert trees inoculated with ascospores produced symptoms after 4 years. The disease was not transmitted following graft inoculation of orchard trees with infected tissue. Ascospores had no effect on germination or growth of Corylus seedlings. Spread of existing cankers was compared on the susceptible cultivar 'Daviana' and the more resistant cultivar 'Barcelona'. There was no significant difference (t=.05) in canker elongation between cultivars, or between spread up and down the stems. Vertical elongation of perennial cankers averaged 26.2 cm/year. Methods for storing ascospores prior to germination, and conditions for germinating ascospores were explored. Spores exhibited no loss of viability after 2 months when cankered twigs were stored below 0° C. Ascospores remained viable for less than 14 days following discharge in water. Drying was lethal to hydrated spores. Ascospores commenced germination 24 hours after hydration when placed on a suitable medium in a proper atmosphere. Germination occurred between 5° and 25° C, with an optimum of 20°. Optimum pH for germination was between 4.2 and 5.7. A direct relationship was detected between ascospore concentration and percent germination. Eight antibiotics were screened in an attempt to prevent bacterial contamination. 10 ppm Rifampicin or 100 ppm Kanamycin effectively inhibited bacterial contaminants without affecting ascospore germination. Germination was compared on many different complex and defined media, and with different amendments added to the media. Various Corylus extracts had no effect on spore germination. 1% ethanol and 5% sucrose increased germination. Casamino acids, cornmeal or peptone promoted growth beyond the initial germ tube. Several stages of ascospore germination and early hyphal growth are described. Studies on Canker in European Filbert Caused by Anisogramma anomala by Joseph D. Postman A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Master of Science Completed April 15, 1986 Commencement June 1986 Approved: Redacted for Privacy Professor of Botany and Plant Pathology in charge of major Redacted for Privacy Head of Department of Botany and Plant Pathology Redacted for Privacy D a f Graduate School Date thesis is presented April 15, 1986 Typed by Joseph D. Postman ACKNOWLEDGEMENT I wish to thank my major professor, Dr. H. Ronald Cameron for giving me the opportunity to work on this project, and to acknowledge funding provided by the Oregon Filbert Commission. Thanks to Dr. William Denison, Dr. Charles M. Leach, and Dr. Porter Lombard for serving on my graduate committee. I also wish to thank my parents for their encouragement throughout the years of my education. TABLE OF CONTENTS INTRODUCTION AND LITERATURE REVIEW Early Attempts to Grow Filberts. 1 Filberts in the Pacific Northwest. 3 Anisogramma anomala in the Pacific Northwest. 3 Disease Symptoms. 4 Susceptibility of Corylus Species and Cultivars. 5 The Causal Organism. 6 Lifecycle of Anisogramma anomala in the Northwest. 9 Methods of Control. 10 Quarantine 10 Sanitation 11 Chemical Control 11 Resistance Breeding 12 Research Objectives 13 CHAPTER 1 Inoculation of Corylus Cultivars and Species with Anisogramma anomala and Measurements of Annual Canker Growth. Introduction 13 Materials and Methods 14 Hypodermic inoculation of seedlings I. 14 Drench inoculation of seedlings. 14 Hypodermic inoculation of seedlings II. 15 Spray inoculation of 2 year old Corylus trees. 15 Hypodermic inoculation of C. cornuta 16 var. californica. Inoculation of germinating Corylus seeds. 16 Graft inoculations in the orchard. 16 Canker measurements. 17 Results and Discussion 17 Literature Cited 23 CHAPTER 2 Ascospore Germination Studies in Anisogramma anomala Introduction 24 Materials and Methods 25 Inoculum preparation and spore germination 25 techniques. Time required to germinate. 26 Storage of inoculum. 26 Effect of temperature. 27 Effect of light. 27 Effect of pH. 27 Effect of ascospore concentration. 28 Preventing bacterial contamination. 29 Effect of host extracts. 29 Effect of media and atmosphere. 30 Results 31 Time to germinate. 31 Optimum temperature. 31 Viability of stored inoculum. 31 Effect of light. 32 Optimum pH and range. 32 Effect of spore concentration. 42 Preventing bacterial contamination. 42 Effect of host extracts. 49 Effect of media. 49 Description of germinating ascospore. 57 Discussion 62 Significance of results in the orchard 65 Summary of Results 67 Literature Cited 69 GENERAL CONCLUSION 71 BIBLIOGRAPHY 73 APPENDIX 77 LIST OF FIGURES Figure Page INTRODUCTION 0.1 Distribution of Corylus americana. 2 Redrawn after Drumke (1964) 0.2 Lifecycle of Anisogramma anomala in the Pacific 8 Northwest. Redrawn after Barr (1978), Barss (1921a), Humphrey (1873), Krantz (1974). CHAPTER 1 1.1 Germination after 12 weeks of Corylus avellana seeds 19 inoculated with Anisogramma anomala ascospores. Means of 8 replicates per treatment. 1.2 Annual increase in Anisogramma Canker lengths during 21 1981. CHAPTER 2 2.1 Time required for Anisogramma anomala ascospores to 33 germinate on water agar at 3 incubation temperatures. Percent germination of 500 spores. 2.2 Viability of Anisogramma anomala ascospores in infected 34 twigs stored at 3 temperatures. Percent germination of 300 spores after 48 hours. Means of 2 replicates. 2.3 Viability of Anisogramma anomala ascospores stored as 35 a suspension in distilled water at two temperatures. Percent germination of 300 spores, 48 hours after plating out on water agar. 2.4 Optimum temperature for germination of Anisogramma 36 anomala ascospores. Percent germination of 300 spores after 48 hours and after 72 hours. Means of 2 replicates. 2.5 Optimum temperature for germination of Anisogramma 37 anomala ascospores. Percent germination of 300 spores after 48 hours and after 8 days. Spores stored in suspension for 4 days prior to plating out. 2.6 Effect of florescent light on germination of 38 Anisogramma anomala ascospores. Percent germination of 300 spores after 48 hours. Means of 2 replicates. Figure Page 2.7 Optimum pH for germination of Anisogramma anomala 39 ascospores. Percent germination of 300 spores after 72 hours at 15 degrees C. 2.8 Effect of spore concentration on germination of 43 Anisogramma anomala ascospores. Percent germination of 200 spores on 1/2 strength PDA. 2.9 Effect of spore concentration on germination of 44 Anisogramma anomala ascospores. Percent germination of 200 spores on either water agar or PDA. 2.10 Effect of spore concentration on germination of 45 Anisogramma anomala ascospores. Means of data from Figure 8 and Figure 9 converted to percent of highest germination for each experiment. Only replicates B and D were used from Figure 8. 2.11 Influence of ethanol in water agar on germination of 50 Anisogramma anomala ascospores. Percent germination of 200 spores. Spore concentration = 5 x 10E5 spores/ml. 2.12 A. Typical germ tube emergence. B. Germ tube has 58 swollen to form a vesicle. C. Budding of secondary vesicles and emergence of secondary germ tube. D. Cluster of swollen vesicles and a secondary hypha. LIST OF TABLES Table Page CHAPTER 1 1.1 Analysis of Variance for Corylus avellana seed 18 germination data in Figure 1.1 CHAPTER 2 2.1 Viability of Anisogramma anomala ascospores from 40 stromata stored at 3 temperatures. Percent germination of 300 spores after 48 hours at 20 degrees C. Means of 2 replicates. 2.2 Viability of Anisogramma anomala ascospores following 41 drying. Percent germination of 300 spores after 48 hours at 22 degrees C. Means of 2 replicates. 2.3 Germination of Anisogramma anomala ascospores on 46 various media. Percent germination of 200 ascospores at two spore concentrations. Media contains 10 ppm Rifampicin unless indicated. 2.4 Effect of antibiotics on Anisogramma anomala ascospore 47 germination and on bacterial contamination. Percent germination of 200 spores. Antibiotics in water agar. 2.5 Effect of antibiotics on Anisogramma anomala ascospore 48 germination and on bacterial contamination. Percent germination of 200 spores. Antibiotics in PDA. 2.6 Effect of surface sterilization for 30 seconds on 51 germination of Anisogramma anomala ascospores. Perithecia surface sterilized in 50% ethanol or 2.5% sodium hypochlorite. Percent germination of 200 spores on water agar + 3 ppm streptomycin or on water agar + 40 ppm streptomycin. 2.7 Effect of water extracts of filbert tissues on 52 germination of Anisogramma anomala ascospores. Percent germination of 200 spores after either 48 hours at 15 degrees C, or 5 days at 10 degrees C. Means of 4 replicates. Extracts in media. 2.8 Effect of filbert stem extracts on germination of 53 Anisogramma anomala ascospores. Percent germination of 200 spores. Extracts in media. Table Page 2.9 Effect of ethanol extracts of filbert tissues on 54 germination of Anisogramma anomala ascospores. Percent germination of 200 spores on either water agar or PDA. Extracts in inoculum. 2.10 Effect of sucrose on Anisogramma anomala ascospore 55 germination. Germination on Czapek media with varying concentrations of sucrose as the only carbon source. Percent germination of 200 spores. 2.11 Effect of sucrose and nitrate concentration on 56 Anisogramma anomala ascospore germination. Germination on Czapek defined medium with varying sucrose and nitrate concentrations. Percent germination of 200 spores. 2.12 Germination of Anisogramma anomala ascospores either 59 under a cover slip or exposed on the surface of the media. Percent germination of 200 spores on Czapek medium, either in a candle jar or in a normal atmosphere.
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