RACES OF FUSARHJM OXYSPORUM F. LINI IN ARIZONA by Robert B. Marlatt A Thesis submitted to the faculty of the Department of Plant Pathology in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in the Graduate College, University of Arizona 1952 Approved: UXzAsJ>L\J) t Director of Thesis ACKNOWLEDGMENT Grateful acknowledgment is made to Dr. R.B. Streets who directed the thesis. The author is indebted to Dr. J"»G. Brown, Dr. Alice M. Boyle, $nd Dr* P.D. Keener for critical reading of the manu script and for their helpful suggestions. Greenhouse space was generously furnished by Professor W.E, Bryan, Head of the Department of Plant Breeding. Acknowledgment is made also to Dr. W.S. Phillips, Head of the Department of Botany and Range Ecology, and to Edward L. Breazeale, Assistant Agricultural Chemist, for valuable aid. ii TABLE OF CONTENTS Section Page INTRODUCTION 1 Review of the Literature 2 History 2 Symptoms 3 Control 4 The Pathogen . 5 Environment 10 Resistance 14 METHODS AND RESULTS 16 Collection of Cultures 16 Single-Spore Culture Methods 18 Cultural Studies .24 Growth on Potato-Dextrose Agar 26 Growth on Malt Agar 26 Growth on Cornmeal Agar 26 Growth on Rice 27 Anastomosis and Antagonism 33 Germination Tests .... 40 Greenhouse Procedures 45 Investigation of a Bacterial Pathogen 51 Toxin Investigations ... 52 Re-isolation of the Fusarium from Stem Tips ... 68 Pathogenicity Experiment 70 Pathogenicity of Isolates 73 Comparison of Varieties 99 Arizona Isolates Compared with Those from Other Regions ........ 107 DISCUSSION 109 SUMMARY . . . 116 BIBLIOGRAPHT . . 118 APPENDIX 125 iii LIST OF TABLES Number Page 1« Sources of Flax Yielding Fusarium oxysporum f. lini • 17 2. Approximate Percentages of Macroconidia on Various Media .... ...... 20 3. Cultures on Potato-Dextrose Agar 28 4. Cultures on Malt Agar 29 5. Cultures on Cornmeal Agar 31 6. Cultures on Rice 32 7o Diameters of Colonies after Six Days Growth. on Potato-Dextrose Agar . 34 8. Diameters of Colonies after Six Days Growth on Malt Agar 35 9. Diameters of Colonies after Six Days Growth. on Cornmeal Agar . 36 10. Diameters of Colonies after Eight Days Growth on Rice 37 11. Percentages of Flax Seeds Germinated Disin fected with New Improved Ceresan 41 12. Fungi from Flax Seeds Disinfected with New Improved Ceresan . 43 13. Percentages of Flax Seeds Germinated Disinfected with. Alcoholic Mercuric Chloride 44 14. Fungi from Flax Seeds Disinfected with Alcoholic Mercuric Chloride . 45 15. Effect of Soil Mixtures on Flax Emergence . . 47 16. Change in Soil during One Month in the Greenhouse . 48 iv Number Page 17. Effect of Toxic Filtrate on Punjab Flax Seeds 55 18. Effeot of Toxic Filtrate on Punjab Flax Seeds 57 19. Effect of Toxic Filtrate on B 5128 Flax Seeds 58 20. Effeot of Toxic Filtrate on Seeds of Ten Flax Varieties 60 21o Flax Varieties Listed in Increasing Order of-Susceptibility to Filtrate . 66 22. Reaction of Punjab to Isolates. ....... 74 23. Reaction of Bison to Isolates 76 24. Reaction of Linota to Isolates 78 25. Reaction of Pergamino to Isolates ...... 80 26. Reaction of Redwing to Isolates ....... 82 27. Reaction of NDR 114 to Isolates 84 28. Reaction of C.I. 1164 to Isolates 86 29. Reaction of B 5128 to Isolates 88 30. Reaction of No. 21 to Isolates 90 31. Reaction of No. 22 to Isolates. ....... 92 32. Isolates in Order of Decreasing Pathogenicity to Ten "Varieties of Flax 95 33. Isolates in Order of Decreasing Pathogenicity to Ten Varieties of Flax Determined by Wilt after Emergence 96 34* Isolates in Order of Decreasing Pathogenicity to Ten Varieties of Flax Determined by Amount of Emergence 100 35. Isolates in Order of Decreasing Pathogenicity to Ten Varieties of Flax Determined by Number of Survivors after.5 0 Days 101 36. Varieties in Order of Decreasing Resistance Determined by Average Per Cent Wilt with all Isolates 103 Number Page 37. Varieties in Order of Decreasing Resistance Determined by Susceptibility and Resistance to Isolates 104 38. Varieties in Order of Decreasing Resistance Determined by Susceptibility and Resistance to Isolates • . . 105 39. Varieties in Order of Decreasing Resistance Determined by Susceptibility and Resistance to Isolates ..... 106 40. Comparable Isolates of Minnesota and Arizona Tests . . 108 vi LIST OF FIGURES . (in Appendix) Number 1. Map of the Yuma Area 2. Cajnera Luoida Drawing of Fusarium oxysporumf . lini Macrooonidium during Germination 3. Slide Bearing a Thin Layer of Agar for Obtaining Single Spores 4. Slide Bearing a Thin Layer of Agar for Observing Anastomosis 5. Examples of Cultural Types 6. Pot Protected from Rodents and Insects 7. Rice Culture used for Soil Inoculation 8. Culture Chamber 9. View of Greenhouse Experiment 10. Effects of Toxic Filtrate on Punjab Seed Germination 11. Fusarium Growing from a Stem Tip 12. Wilt caused by a Fusarium isolate vii I N T R O D U C T I O N Recent studies of flax wilt by Streets( 1950, 1951) of the Arizona Agricultural Experiment Station have shown that the disease is becoming a limiting factor in flax culture and that the most satisfactory control measure is the sub stitution of wilt-resistant varieties for the very suscepti ble commercial variety, Punjab. Some varieties, resistant when grown int he Imperial Valley in California, are very susceptible when grown in the Yuma Valley of Arizona, suggesting the existence of physio logical races of the fungus differing in their pathogenicity to certain varieties of flax. Investigators have reported the existence of physiological strains in other areas. The value of resistant varieties of flax for Arizona depends upon a determination of what races of the wilt fungus are present, so that flax varieties having the proper resistance can be recommended. Isolation of the various races can be useful as a means of providing the University flax-wilt plot with dif ferent strains of the fungus so that flax-breeding experi ments being carried on may produce flax resistant to the wilt organism in the various fields of the state. Observance of cultural traits of the strains of the fungus may provide some means of classifying them under 2 laboratory conditions and of relating them to those races obtained in other parts of the world. Review of the Literature A review of the pertinent literature has been made in order to provide a source of information useful for this and for later work on the problem* History; It is known that flax (Linum usitatissumum L.) has been cultivated for at least six thousand years, but the first mention of the wilt disease was probably made by Pliny (translated 1856) who remarked: "It has the property of scorching the ground where it is grown and of deterio rating the quality of the very soil itself," Until the nine teenth century, no one knew any more about the cause of flax wilt disease than did Pliny in the first century. Finally in 1893, Broekema (1893) separated the disease from others and suspected its parasitic nature. He selected surviving plants from an infested field and noted that their seed gave rise to a generation of flax which was somewhat resistant to the disease. Shortly thereafter in 1897 Nypels described a disease of flax in the Flanders area which he called "la brulure du lin," which probably was flax wilt (Barker, 1923). In Japan, Hiratsuka first reported that the disease was caused by a Fusarium in 1896. Bolley in 1901 described the fungus and named it Fusarium lini. By selection methods Bolley obtained varieties of flax which were resistant to the disease, and the flax industry was no longer destinedt o 3 move from "flax-siok" regions to areas where flax had never been cropped before. The first important commercial production of flaxseed in California was the eleven thousand acres planted.in the Imperial Valley in 1934. This was the outgrowth of variety trials begun in 1927 at the Imperial Valley Experiment Sta tion near El Centro. Wilt was not as yet reported from the far western states when Dillman and G-oar (1937) wrote on flaxseed production in these areas. Flax was grown experimentally in Phoenix,A rizona, as early as 1914. Mention was made of variety tests being con ducted by the Experiment Station in the University of Arizona Agricultural Experiment Station's annual reports for 1915, 1916 and in later years. Symptoms: In reporting on the wilt disease of flax, it was stated by Bolley (1901) that most susceptible plants are killed by the fungus before emerging from heavily infested soil. The organism was isolated by Schuster (1944) from primary roots of the flax plant on the day of emergence, and was found in the apex of plants seven days after emergence. Boyle (1934) reports that the whole root stele was invaded in flax twenty days old. However, Tisdale (1917b) found no considerable clogging of the vascular system by the ffusarium, despite the occurrence of microconidia in the vessels. The wilting may be partially explained by the fact that Millikan (1949) found a toxic substance which was formed by the fungus and which caused wilt symptoms. Grossman (1934) noted that a