BACTERIOPHAGES OF ERWINIA AMYLOVORA: THEIR ISOLATION, DISTRIBUTION, CHARACTERIZATION, AND POSSIBLE INVOLVEMENT IN THE ETIOLOGY AND EPIDEMIOLOGY OF FIRE BLIGHT BY David F. Ritchie A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1978 C4 \\”’> ABSTRACT BACTERIOPHAGES OF ERWINIA AMYLOVORA: THEIR ISOLATION, DISTRIBUTION, CHARACTERIZATION, AND POSSIBLE INVOLVEMENT IN THE ETIOLOGY AND EPIDEMIOLOGY OF FIRE BLIGHT BY David F. Ritchie Studies were conducted exploring the involvement of Erwinia amyZovora bacteriophages in the etiology and epidemiology of fire blight. Phages were isolated from aerial structures of apple in association with E. amyZovora. Phages and E. amylovora were monitored and isolated in a Michigan State University (MSU) orchard during 1975 and 1976 and in 10 commercial, south- western-Michigan apple orchards in 1976. Fourteen phage strains isolated from nine apple orchards were used in the characterization studies. These were divided into two major groups. Two phage types from group I and one phage type from group II were characterized using chemical, serological, ultra— centrifugal, and electron microscopic methods. Phage and E. amylovora interactions were studied in liquid and solid media and in apple seedlings. David F. Ritchie Phage and E. amyZovora populations reached 106 plaque-forming-units (pfu)/g of fresh plant tissue and 108 colony-forming-units (cfu)/g of fresh plant tissue, respectively. Maximum phage and E. amyZovora populations coincided with maximum fire blight severity. Isolation of phage was seasonal, while E. amyZovora was isolated from diseased tissue throughout the year. Two morphological and serological groups of phages were isolated. Group I phages produced two plaque types: PEal(h) plaques were 2-3 mm in diameter surrounded by an expanding, translucent halo; while PEal(nh) plaques were 1-2 mm in diameter without the halo. Group IT phages represented by PEa7, produced 0.5—1.0 mm diameter plaques with an expanding, trans— lucent halo. PEal(h) was polyhedral, ca. 60 nm in diameter with a spike—like tail structure; PEa7 had an octahedral head, ca. 75 nm in diameter with a rigid, striated tail, ca. 135 nm long. Phages PEal(h) and PEal(nh) had a buoyant density of 1.53 g/cc and a sedimentation coefficient of 566 S. The buoyant density of PEa7 was 1.44 g/cc with a sedimentation coefficient of 1037 S. The growth cycle of PEal(h) was 70 min yielding ca 50 pfu/productive cell, while PEa7 was 135 min with ca. 8 pfu/productive cell. PEal(h) and PEal(nh) required 10 min at 55 C for David F. Ritchie complete thermal inactivation; 10 min at 65 C was required for PEa7. Host range for both phage groups was limited to E. amyZovora and several Erwinia herbicola strains. Phage PEal(h) adsorbed more rapidly to capsulated than to acapsulated strains of E. amyZovora while PEa7 adsorbed more rapidly to acapsulated strains of E. amyZovora. Growth of PEal(h) with E. amyZovora 110 rifr resulted in the synthesis of a capsular degrading factor (CDF). The CDF activity was inhibited by PEal(h) antiserum but not by PEa7 nor E. amyZovora 110 rifr antisera. The CDF affected only capsulated strains of E. amyZovora and several strains of E. herbicola. Its activity was completely destroyed by heating for 10 min at 85 C. The degradation of the capsule was responsible for the expanding, trans- lucent halo surrounding the plaque; alteration of the bacterial growth from a mucoid, white character- istic to non-mucoid, vitreous growth; high frequencies of PEal(h)-resistance; a delay in symptom development; and an increased sensitivity to streptomycin sulfate. The phages produced their greatest effect on E. amylovora indirectly through the CDF degradation of the bacterial capsule. Phage and E. amyZovora monitoring and laboratory data suggested that phage may have an effect on the epidemiology of fire blight. This dissertation is dedicated to my parents ACKNOWLEDGMENTS I would like to express my deep and sincere gratitude to Dr. Edward J. Klos for his guidance, support, and the freedom which he allowed me to pursue the many facets of this research. I am also indebted to him for introducing me to the many aspects of tree fruit disease control and to many of the persons working in this area of plant pathology. I appreciate the time and guidance given by my guidance committee members Drs. Donald C. Ramsdell, Alfred W. Saettler, and Loren R. Snyder. Special thanks are due to many persons in the department, particularly Dr. Harry Murakishi, who so graciously allowed me to use the scientific equipment housed in their laboratories. I am indebted to Dr. Karen Baker for her cheerful and eager assistance with the electron microscopy and to Kassandra Keever and Diane Borgic for their assistance in preparing and performing many of the experiments. I appreciate the friendships of the many persons whom I have learned to know while at Michigan State; these friendships have made my tenure at MSU one of iii the most enjoyable and memorable periods of my life. The support of the Michigan State Agricultural Experiment Station and the Michigan Pear Research Association is gratefully acknowledged. év TABLE OF CONTENTS Page LIST OF TABLES. . . . . . . . . . . . J . . . . . . LIST OF FIGURES . . . .'. . . . . . . . . . . . . . GENERAL INTRODUCTION AND LITERATURE REVIEW. . . . . 1 LITERATURE- -CIT.ED. ' ' g‘ o o o o o o o o o o o o o o o 6 PART I ISOLATION, ’ DISTRIBUTION, AND POPULATIONS OF ERWINIA AMYLOVORA BACTERIOPHAGES ON AERIAL STRUCTURES OF APPLE INTRODUCTION 0 Q o o o o o o o o Q Q o 9 o o o 9 o o l 2 Isolation of Erwinia amyZovora Bacteriophage from Aerial Parts of Apple Trees . . . . . . . 13 MATERIALS AND METHODS . . . . . . ... . . . . . . . l3 Erwinia amyZovora isolation . . . . . . . . . l3 Bacteriophage isolation . . . . . . . . . . . l3 Phage characterization. . . . . . . . . . . . 13 RESULTS AND DISCUSSION. . . . . . . . . . . . . . . l4 LITERATURE CITED. . . . . . . . . . . . . . . . . . 15 MATERIALS AND METHODS . . . . . . . . . . . a . . . 17 Monitoring of Erwinia amyZovora and its bacteriophages. . . . . . . . . . . . . 17 Monitoring of populations in a MSU orchard. . 18 Monitoring of populations in ten growers' orchards. . . . . . . . . 18 The effect of desiccation on phage PEal(h). . 18 RESULTS 0 O O O C O O O C O O O O O O O O O O O O 0 2 o Populations in a MSU orchard. . . . . . . . . 20 Populations in ten growers' orchards. . . . . 25 Effect of desiccation on infectivity of phage PEal(h) . . . . . . . . . . . . . . . 25 ‘U DISCUSSION! I O Q I C I I I O I I Q Q Q 9 0 LITERATURE CITED. . . . . . . . . . . . . . PART II CHARACTERIZATION OF ERWINIA AMYLOVORA BACTERIOPHAGES INTRODUCTION. . . . . . . . . . . . . . . . MATERIALS AND METHODS . . . . . . . . y . . Media and cultural conditions . . . Phage isolation . . . . . . . . . . Host range and plaque morphology. . Phage growth and purification . . . O I O O 'Phage thermal inactivation and optimum~ growth temperature. . . . . . . . . 9 One—step growth and adsorption experiments. Electron microscopy . . . . . Serology. . . . . . . . . . Sedimentation coefficients. Buoyant density . . . ... . I I I ~I I I I RESULTS I I I I I I I I I I I I I I I I I I Isolation, plaque morphology, and host range . . . . . . . . . . . Phage growth and purification. . . . Thermal inactivation and optimum growt temperature . . . . . . . . . . . . One-step growth and adsorption rates. Electron microscopy . . . . . . . . . Serology. . . . . . . . . . . . . . Sedimentation coefficients. . . . . . DISCUSSION. . . . . . . . . . . . . . . . . LITERATURE CITED. . . . . . . . . . . . . . PART III INTERACTION OF ERWTNIA AMYEOVORA WITH ITS BACTERIOPHAGE INTRODUCT ION I I O O Q O O O O O O I I O O O MATERXALS AND METHODS I I Q I I t I o I I I Media...... IIII con- 13 O I O O O . Selection of Eruinia amylovora mutants. Expanding, translucent halo . . . . . vi 0 O O D O O Page 29 33 35 36 36 37 37 37 39 40 41 42 43 44 44 48 49 57 61 61 65 69 75 78 80 80 80 82 Page Effect of temperature on the development and expansion of the halo. . . . . . . . . . . 82 Diffusion of the phage from plaques. . . . . 82 Altered colony morphology and sensitivity to phage . . . . .. . . . . . . . . 83 Stability of phage resistance and pr colony characteristics . . . . . . . . . 83 Effect of temperature on the development of pr colony characteristics. . . . . . . . . 84 Interaction of PEal(h) and Erwinia amylovora 110 rifr in broth and the development of pr colony characteristics. . . . . . . . 84 Effect of PEal(h) antiserum on the develop- ment of pr colony characteristics. . . . . 84 Agar diffusible substance. . . . . . . . . . 85 ,Effect of phage lysates on mature-lawns- of Erwinia amyZovora . . . . . . . . . . 85 Effect of phage lysates on the Erminia amylovora capsyle. . . . . . . . . 86 Serological relationship between pr and wild type Erwinia amyZovora. . . . . . 86 Mutation frequencies of phage resistance 86 Fluctuation test . . . . . . . . . . . . . 87 Test for lysogeny. . . . . . . 87 Growth and effect of phage PEalCh) on . Erwinia amylovora 110 rifr . . . . . . . . 87 Symptom development by Erwinia amyZovora . . 88 Hypersensitive reaction in tobacco . . . . . 90 RESULTS. 0 I I I I I I I I I I I I I I I I I I I I 91 Expanding, translucent halo. . . . . . . . 91 Effect of temperature on the development . and expansion of the halo. . . . . . . . . 91 Diffusion of the phage from the plaques. . . 95 Altered colony morphology and sensitivity to phage . . . . . . . 95 Factors contributing to the establishment . of pr type colonies. . . . . . . . . . . . 98 Agar diffusible substance. . . . . . . . . . 102 Effect of phage lysates on Erwinia amyZos' ora. . . . . . . . . . . . . 102 Serological relationship between pr and wild type Erwinia amyZovora. . . . . . . . 105 Resistance of Erwinia amyZovora to phage strains. . . . . . . . 105 Growth and effect of phage PEal(h) on . Erwinia amylovora 110 rifr . . . . . . . 105' Symptom development by Erwinia amylovora . P 112 Hypersensitive reaction in tobacco . . . . . 118 vii Effect of phage PEal(h) purification on the titer of Erwinia amylovora capsule degrading factor. . . . . . . . . . . . . DISCUSSION I I I I I I I I I I I I I I I I I I I I LITERATURE CITED. . . . . . . . . . . . . . . . . PART IV SOME PROPERTIES OF THE BACTERIOPHAGE PEal(h)—ASSOCIATED CAPSULE DEGRADING FACTOR INTRODUCTION. . . . . . . . . . . . . . . . . . . MATERIALS AND METHODS . . . . . . . . . . . . . . Source of capsule degrading factor. . . . . ‘ capsule degrading faCtor. I I I I I I I I I Effect Of capsule degrading factor on different bacteria strains. . . . . . . . Thermal inactivation of the capsule degrading factor. . . . . . . . . . . . . Effect of antisera on capsule degrading factor activity . . . . . . . . . . . . . Effect of crude capsule degrading factor RESULTS on Erwinia amyZovora sensitivity to streptomycin sulfate. . . . . . . . . . . I I I I. I I I I I I I I I I I I I I I I I Source of capsule degrading factor. . . . . Effect of the capsule degrading factor. on different bacterial strains. . . . . . Thermal inactivation. . . . . . . . . . . . Effect of antisera on capsule degrading factor. . . . . . . . . . . . . . . . . . Effect of capsule degrading factor on Erwinia amyZovora 110 rif sensitivity to streptomycin sulfate . . . . . . . . . DISCUSSION. . . . . . . . . . . . . . . . . . . . LITERATURE CITED. . . . . . ... . . . . . . . . . APPENDICES APPENDIX A. A1. Bacterial strains and their source. . . . . viii Page 118 122 132 138 140 140 140 141 141 141 142 144 144 144 144 149 149 153 156 158 Page APPENDIX E El. Optimum concentration of polyethylene glycol (PEG), 6000 average molecular weight, for the precipitation of bacteriophages PEal(h) and PEa7. . . . . . . . . . . . . . . . . . 161 APPENDIX C Cl. Symptom development by bacteriophage PEal(h)v sensitive (Wild type and rifr) Erwinia amglovora and PEal(h)-resistant E. amylovora (p) I I I I I I I I I I I I I I I I I I I 162 C2. Symptom development by bacteriophage PEal(h)— sensitive (wild type and rifr) Ewrinia amyZovora and PEal(h)-resistant E. amylovora (Pr) I I I I I I I I I I I I I I I I I I I I 16 3 C3. Symptom development by bacteriophage PEal(h)— sensitive Cwild type) Erwinia amylovora and PEaSCh)—resistant E. amylovora (pr) . . 164 C4. Simultaneous inoculation of apple seedlings with a mixture of Erwinia amyZovora and bacteriophage PEal(h) and the effect on symptom development . . . . . . . . . . . . 165 C5. Simultaneous inoculations of apple seedlings with a mixture of Erwinia‘amylovora and bacteriophage PEal(h) and the effect on . symptom development . . . . . . . . . . p . 166 C6. Simultaneous inoculation of apple seedlings with a mixture of Erwinia amylovora and bacteriophage PEal(h) and the effect on symptom development . . . . . . . . . . . . 168 APPENDIX D D1. Diagrammatic representation of the inter— action between bacteriophage PEal(h) and capsulated Erwinia amylovora. . . . . . . . 169 Table LIST OF TABLES Page PART I The occurrence of Erwinia amylovora and E. amyZovora bacteriophages on aerial parts of Jonathan apple trees during the summer of 1975 at East Lansing, Michigan. . . . . . 14 Host range of Erwinia amyZovora bacterio— .phages PEal, PEaZ, and PEaS. . . . . . . . . 14 Erwinia amylovora and E. amylovora bacterio— phage populations in a Michigan State University orchard at East Lansing, Michigan . . . . . . . . . . . . . . . . . . 21 Erwinia amylovora and E. amylovora bacterio— phage populations in ten growers‘ orchards in southwestern Michigan . . . . . . . . . . 26 Effect of desiccation on the survival of Erwinia amyZovora bacteriophage PEal(h) on apple leaf discs . . . . . . . . . . . . . . 28 PART II Bacteriophage strains, plaque morphology, source, and date of isolation from Michigan OI‘ChardS I I I I I I I I I I I I I I I O I I 45 Propagation of group I bacteriophages and the development of halo and non—halo plaques. . . . . . . . . . . . . . . . . . . 47 Occurrence of halo and non-halo plaque— forming phages in halo plaques of-phage‘ PEal (h) I I I I I I I I I I I I I I I I I I I 4.7 Purification and concentration of bacterio— phage PEal (h) I I- I I I I I I I I I I I I 0 2 50 Purification and concentration of bacterio— Phage PEa7 I I I I I I I I ' I I I I q o I I 51 Adsorption of phages PEal(h) and PEa7 to capsulated and acapsulated strains of- Erwinia amyZovOra. . . . . . . . . . . . . . 52 Table 7' 10. ll. 12. l3. 14. Page Antiphage sera neutralization of the 14 Erwinia amylovora bacteriophage strains . . . 66 Sedimentation coefficient calculations for phages PEal(h), PEal(nh), and PEa7. . . . 68 PART III Phage - Erwinia amylovora combinations which resulted in halo formation. . . . . . . ‘93 Diffusion of phages PEal(h) and PEal(nh) from the true plaque. . . . . . . . . . . . . 96 Instability of bacteriophage PEal(h)— resistance and altered colony morphology. of Erwinéa amylovora 110 rifr . . . . . . . . 99 ,Effect of temperature on the development of thepr type of colony. . . . . . . . . . . 100 Results of the incubation of Erwinia amyZovora 110 rifr with bacteriophage PEal(h) in NBGYE and the development of pr type colonies when plated on NAG . . . . . . . . . 100 Effect of bacteriophage PEal(h) antiserum on the development of pr type colonies. . . . 101 Ability'of bacteriophage lysates to affect mature lawns of Erwinia amylovora . . . . . . 104 The ability of bacteriophage lysates to remove the capsule from Erwénia amyZovora cells . . . . . . . . . . . . . . . . . . . . 104 Mutation frequencies of Erwinia amyZovora strains to bacteriophages . . . . . . . . . . 107 Fluctuation test for Erwinia amyZovora and resistance to bacteriophage PEal(h) . . . . . 108 Ability of bacteriophages PEal(h) and PEa7 to form plaques on several capsulated and acapsulated strains of Erwinia amyZovora. . . 119 Symptom production by several capsulated and acapsulated strains of Erwinia amylovora. . . 119 Ability of wild type (W3t.), rifampin-resistant friff), and rifnrpsalchysresistant. (rifrpr) Erwénia amyZovora strains to the hypersensi— tive reaction in tobacco leaves . . . . . . . 120 Effect of bacteriophage..PEal(h) purification on the titer of the Erwinéa amyZovora , capsule degrading factor . . . . . . . , . . 121 xi Al. Bl. C1. C2. C3. C4. Page PART IV Effect of capsule degrading factor on different bacterial strains. . . . . . . . . 145 Thermal inactivation of bacteriophage PEal(h)-associated capsule degrading factor . . . . . . . . . . . . . . . . . . . 148 Effect of bacteriophage and Erwinia amyZovora antisera on the activity of phage PEal(h)-associated capsule degrading factor . . . . . . . . . . . . . . . . . . . 148 Results of disc assay of streptomycin sulfate on Erwinia amylovora 110 rifr and E. amylovora 110 rifr — associated capsule degrading factor . . . . . . . . . . . . . . . . . . . 150 Effect of bacteriophage PEal(h) — associated capsule degrading factor (CDF) on the uptake of streptomycin sulfate by Erwinia amyZovora 110 rifr . . . . . . . . . . . . . 151 APPENDIX A Bacterial strains and their source . . . . . l58 APPENDIX B Optimum concentration of polyethylene glycol (PEG), 6000 average molecular weight, for the precipitation of bacteriophages PEal(h) and PEa7 . . . . . . . . . . . . . . . . . . 161 APPENDIX C Symptom development by bacteriophage PEal(h)- sensitive (wild type and rifr) Erwinia amyZovora and PEal(h)-resistant E. amyZovora (Pr) I I I I I I I I I I I I I I I I I I I I 162 Symptom development by bacteriophage PEal(h)— sensitive (wild type and rifr ) Erwinia angovora and PEal(h)-resistant E. amyZovora (p) I I I I I I I I I I I I I I I I I I I I 163 Symptom development by bacteriophage PEaS(h)- sensitive (wild type) Erwinia amyZovora and PEa5(h)—resistant E. amyZovora (pr ) . . . . 164 Simultaneous inoculation of apple seedlings with a mixture of Erwinia amyZovora and bacteriophage PEal(h) and the effect on symptom development. . . . . . . . . . . . . 165 xii Tabla C5. C6. Page Simultaneous inoculations of apple seedlings with a mixture of Erwinia amyZovora and bacteriophage PEal(h) and the effect on symptom development. . . . . . . . . . . . . 166 Simultaneous inoculations of apple seedlings with a mixture of Erwinia amyZovora and bacteriophage PEal(h) and the effect on symptom development. . . . . . . . . . . . . 168 xiii Figure LIST OF FIGURES Page PART I A) Plaque morphology of phage PEal(halo) and PEal(non-halo), arrow, after 18 hours of incubation at 27 C with Erwinia amyZovora isolate 110 on 2.5 ml of top-agar consisting of 0.7% nutrient agar, 0.5% glucose, and 015% yeast extract. B) Plaque morphology .of phage PEal after 48 hours under the same conditions as in A. . . . . . . . . . . . . . 15 One-step growth curve of phage isolates PEal, PEaZ, and PEaS with Erwinia amyZovora 110 as host . . . . . . . . . . . . . . . . . 15 Seasonal detection of Erwinia amyZovora bacteriophages in association with aerial structures of apple in a Michigan State University orchard at East Lansing. . . . . . 24 PART II Plaque morphologies of bacteriophages after 60 hr of incubation at 23 C with Erwinia amylovora 110 rifr as host. . . . . . 46 Sedimentation profiles of bacteriophages PEal(h) and PEal(nh) in 10 - 40% sucrose gradients . . . . . . . . . . . . . . . . . . 51 Ultraviolet adsorption spectra of 2-cycle, linear 10 - 40% sucrose gradient purified bacteriophages PEal(h) and PEa7 . . . . . . . 53 Isopynic centrifugation profiles and buoyant densities of bacteriophages PEal(h) and PEal(nh) in cesium chloride gradients . . . . 54 Thermal inactivation of bacteriophages PEal(h), PEal (nh) ’ PEa7I I I I I I I I I I I I I I I I 56 Optimum growth temperatures of bacterio- phages PEalCh) and PEa7 . . . . . . . . . . . 58 One-step growth curves for bacteriophages PEal (h) and PEa7 I I I I I I I I I I I I I I I 59 xiv Figure 8. 9. 10. 11. D1. Adsorption curves of bacteriophages PEal(h) and PEa7 to capsulated and acapsulated strains of Erwinia amylovora. . . . . . . . Electron micrographsof Erwinia amylovora bacteriophages. . . . . . . . . . . . . . . Antiphage sera neutralization curves for bacteriophages PEal(h), PEal(nh), PEa7. . . Sedimentation curves used to calculate the rate of sedimentation for sedimentation coefficient determinations. . . . . . . . . PART III Expanding, translucent halo associated with .the plaque formed by bacteriophage PEal(h) grown on Erwinia amyZovora llO'rifr . . . . Effect of temperature on expansion of the translucent halo surrounding plaques produced by bacteriophage PEal(h) . . . . . Bacterial growth of Erwinia amyZovora after 48 hr incubation at 23 C. . . . . . . . . . Diffusion of capsular degrading factor across bacterial-free 2.0% nutrient agar and 0.5% glucose. . . . . . . . . . . . . . . . Agar gel serological tests with steamed suspensions of Erwinia amyZovora strain 110 rifrI I I I I I I I I I I I I I 9 0 I . Growth of bacteriophage PEal(h) and its effect on Erwinia amylovora 110 rifr in NBGYE culture . . . . . . . . . . . . . . . Rate of symptom development of wild type (w.t.),.rifampin-resistant (rifr),.and bacteriophage PEal(h)-resistant (rifrpr) strains of Erwinia amyZovora. . . . . . . . Rate of symptom develOpment following inoculation of seedlings with Erwinia amyZovora and a mixture of E. amyZovora and bacteriophage PEal(h) . . . . . . . . . . . Growth and disease development of Erwinia amyZovora 105 in apple seedlings. . . . . . APPENDIX D Diagrammatic representation of the inter— action between bacteriophage PEal(h) and capsulated Erwinia amyZoqora. . . . . . . . xv Page 60 63 64 67 92 94 97 103 106 111 113 114 115 169 GENERAL INTRODUCTION AND LITERATURE REVIEW Fire blight, proven to be caused by Erwinia amyZovora (Burrill) Winslowy at aZ., almost 100 years ago,(4,10) remains an erratic but serious disease on pome fruits. In recent years new methods of apple culture have utilized dwarf rootstocks resulting in high density plantings of old and new fire blight- susceptible varieties. These factors have increased the importance of fire blight. The erratic and usually devastating occurrence of fire blight cannot be fully explained and has normally been attributed to optimum climatic conditions and host factors. Even though these factors are important, the effects of other factors on the genetic potential of E. amylovora have received little research attention. With the discovery of E. amyZovora bacteriophages on aerial structures of apple trees in 1975 (42) and 'awareness of Erskine's hypothesis (1?) that phage may be involVed in the epidemiology of fire blight, it became apparent that further investigations were warranted. The major objective of this study was to explore the possible role which phage may have upon the etiology and epidemiology of the fire blight bacterium. This was done in three ways: 1) monitoring the distribution and populations of phages on the aerial structures of apple, 2) characterization of these phages, and 3) determination of the effects of these phages on E. amyZovora. Two excellent reviews on the history, epidemiol- ogy, and control of fire blight have recently been written (5,45). A voluminous amount of literature has been published starting in 1794 with Denning's (13) description of the disease, followed by a steady flow of research reports beginning with Burrill in 1877 (10). However, fire blight is still very difficult to control, continues to spread (5,45,51), and remains a constant concern in many countries where pome fruits are grown. In the United States, fire blight has at different times and places threatened the survival of the pome fruit industry (5). It continues to limit pear production and is becoming increasingly important to apple culture in the northeastern United States (7), especially where the growth of certain susceptible varieties is desirable (1,2). The literature contains numerous unsubstantiated claims, contradictory reports, and working hypotheses which, with time and repeated citations, have become accepted as facts (45). The usual taxonomic in— consistencies of E. amylovora have been reported (8,15,33) as well as variations in virulence (3,45, 47) and morphology (3,28,53) among strains, including the instability of colony types and virulence when continuously transferred (3,25). Factors contributing to pathogenicity are not understood. Host cell wall degradation does not appear to be involved (39,46) while a bacterial produced toxin has been implicated (22,26,39). Much fire blight research has been descriptive and the study of possible factors which may affect disease development have been mostly climatic (9,32, 35,40). Efforts have been made to study the effect of coinhabitors, particularly Erwfinia herbicola (Lohnis) Dye, on the survival of E. amylovora and disease development, but results have been variable or inconclusive (19,21,38,4l,43,49). There is little doubt that climatic conditions are important for disease development. Even so, in certain years and locations, blight infections are insignificant (45); furthermore, it has been observed in California that seldom more than 100 flower strikes occurred per tree during an epiphytotic even though every flower was infested with E. amyZovora and climatic conditions appeared optimal for infection (34). The physiological condition of the host plant is important for fire blight development; any factor which stimulates the development of succulent tissue enhances fire blight susceptibility (24,30,36,48). Host defense mechanisms have been examined but their role in pathogenesis, particularly under field conditions, has not been defined (11,21,44). Little is known concerning genetic variability in E. amyZovora and the factors which may affect it in nature and how this may relate to fire blight epidemiology (45). Viruses which specifically infect bacteria, bacteriophages, are capable of altering the genetic expression of their bacterial host via two major mechanisms: transduction (55) and lysogenic conversion (6). Bacteriophages of plant pathogenic bacteria are common (37,52) and often have received attention in and of themselves because of unusual characteristics (52) or have been used in bacterial identification and detection (37). Attempts to use phages for disease control have met with only limited success (12,37,52). Phages are involved in the pathogenicity of several animal bacteria (16,23, 27) as well as in the induction of extensive pheno— typical alterations in their bacterial hosts (29,31, 50). The effect of phage and their possible role in the virulence of several phytopathogenic bacteria has been reported (14,20,54). Even though E. amyZovora phages have been report- ed since the 1950's (8,37), only one strain obtained from soil has been characterized (l7). Attempts to isolate phage from aerial structures of host plants have either been negative (17) or not reported prior to 1975 (42). Two papers have implicated the possible involvement of phage in the epidemiology of fire blight (17,18). LITERATURE CITED ALDWTNCKLE, H. S. 1974. Field susceptibility of 46 apple cultivars to fire blight. Plant Dis. Reptr. 58:819—821. ALDWINCKLE, H. 5., and J. L. PRECZEWSKI. 1976. Reaction of terminal shoots of apple cultivars to invasion by Erwinia amyZovora. Phytopath- ology 66:1439—1444. ARK, P. A. 1937. Variability in the fire—blight organism, Erwinia amyZovora. PhytOpathology ARTHUR, J. C. 1885. Proof that the disease of trees known as pear blight is directly due to bacteria. N.Y. (Geneva) Agr. Exp. Sta. Bull. 2 n.s.:1n4. BAKER, K. F. 1971. Fire blight of pome fruit: the genesis of the concept that bacteria can be pathogenic to plants. Hilgradia 40:603— 633. BARKSDALE, L. 1959. Lysogenic conversions in bacteria. Bacteriol. Rev. 23:202—212. BEER, S. V., and H. S. ALDWINCKLE. 1974. Fire blight in New York State. N.Y. Food Life Sci. Quart. 7(1):16-19. BILLING, E., L.A.E. BAKER, J. E. CROSSE, and C.M.E. GARRETT. 1961. Characteristics of English isolated of Erwinia amylovora (Burrill) Winslow et al. J. Appl. Bacteriol. 24:195—21L BROOKS, A. N. 1926. Studies of the epidemiology and control of fire blight of apple. Phyto- pathology 16:665-696. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. BURRILL, T. J. 1877. Hort. Dep..Rept., Sept.13, 1876, to Dr. J. M. Gregory, Regent. Dept. Board Trustees Ill. Ind. Univ. 8:199-200. CHATTERJEE, A. K., L. N. GIBBONS, and J. A. CARPENTER. 1969. Some observations on the physiology of Erwinia herbicola and its possible implication as a factor antagonistic to Erwinia amyZovora in the "fire-blight" syndrome. Can. J. Microbiol. 15:640-642. CIVEROLO, E. L. 1972. Interaction between bacteria and bacteriophage on plant surfaces and.in plant tissues. Pages 25-37.;g H. P. Mass Geesteranus, ed. Third Int. Conf. Plant Pathogenic Bacteria. Proc., Centre Agric. Publ. Doc. (PUDOC), Wageningen, The Netherlands. 365 p. DENNING, W. 1794. On the decay of apple trees. Trans. Soc. Promot. Agr. Arts Mfg. 2:219-22 (2nd ed., 1801. 2:185—87). DRLICA, K. A., and C. I. KADO. 1975. Crown gall tumors: are bacterial nucleic acids involved? Bacteriol. Rev. 39:186-196. DYE, D. W. 1968. A taxonomic study of the genus Erwinia. I. The "Amylovora" group. N. Z. J. Sci. 11:590-607. EKLUND, M. W., and F. T. POYSKY. 1974. Inter- conversion of type C and D strains of Clostridium botulinum by specific bacterio- phages. Appl. Microbiol. 27:251—258. ERSKINE, J. M. 1973. Characteristics of Erwinia amyZovora bacteriophage and its possible role in the epidemiology of fire blight. Can. J. Microbiol. 19:837-845. ERSKINE, J. M. 1973. Association of virulence characteristics of Erwinia amyZovora with toxigenicity of its phage lysates to rabbit. Can. J. Microbiol. 19:875-877. FARABEE, C. J., and J. L. LOCKWOOD. 1958. Inhibition of Erwinia amylovora by Bacterium sp. isolated from fire blight cankers. Phytopathology 48:209—211. 20. 21. 22. 23.. 24. 25. 26. 27. 28. 29. GARRETT, CONSTANCE M. E., J. E. CROSSE, and A. SLETTEN. 1974. Relations between phage sensitivity and virulence in Pseudomonas morsprunorum. J. Gen. Microbiol. 80:475— 483. GOODMAN, R. N. 1967. Protection of apple stem tissue against Erwénéa amylovora infection by avirulent strains and three other bacterial species. Phytopathology 57:22—24. GOODMAN, R. N., J. S. HUANG, and PI-YU HUANG. 1974. Host-specific phytotoxic polysacchar- ide from apple tissue infected by Erwinia amylovora. Science 183:1081-1082. GROMAN, N. B. 1955. Evidence for the active role of bacteriophage in the conversion of nontoxigenic Uorynebacteréum diptheriae to toxin production. J. Bacteriol. 69:9—15. HILDEBRAND, E. M., and A. J. HEINICKE. 1937. Incidence of fire blight in young apple trees in relation to orchard practives. N.Y. Cornell Agr. Exp. Sta. Mem. 203:1-36. HILDEBRAND, E. M. 1938. Growth rates of phyto— pathogenic bacteria. J. Bacteriol. 35:487— 492. HILDEBRAND, E. M. 1939. Studies on fire blight ooze. Phytopathology 29:142-156. HOLMES, R. K., and L. BARKSDALE. 1969. Genetic analysis of tox+ and tox‘ bacteriophages of Corynebacterium dfipteriae. J. Virol. 3: 586-598. HUANG, P.-Y., and R. N. GOODMAN. 1970. Morph— ology and ultrastructure of normal rod-shaped and filamentous forms of Erwinia amyZovora. J. Bacteriol. 102:862—866. JONES, W., and A. WHITE. 1968. Lysogeny in mycobacteria. I. Conversion of colony morphology, nitrate reductase activity, and TWeen 80 hydrolysis of Mycobacterium sp. ATCC 607 associated with lysogeny. Can. J. Microbiol. 14:551-555. 30. 31. 32. 33., 34. 35. 36. 37. 38. 39. 40. LEWIS, L. N., and A. L. KENWORTHY. 1962. Nutritional balance as related to leaf composition and fire blight susceptibility in the Barlett pear. Proc. Am. Soc. Hort. Sci. 81:108-115. LI, K., L. BARKSDALE, and L. GARMISE. 1961 Phenotypic alterations associated with the bacteriophage carrier state of Shigella dysenteriae. J. Gen. Microbiol. 24:355-367. LUEPSCHEN, N. S., K. G. PARKER, and W. D. MILLS. 1961. Five year study of fire blight blossom infection and its control in New York. N.Y. Agr. Exp. Sta. Bull. 963:1—17. MARTINEC, T., and M. KOCUR. 1964. A taxonomic study of Erwinia amylovora (Burrill 1882) Winslow et a2. 1920. Int. Bull. Bacteriol. Nomencl. Taxon 14:5-14. MILLER, T. D., and M. N. SCHROTH. 1972. Monitoring the epiphytic population of Erwinia amyZovora on pear with a selective medium. Phytopathology 62:1175-1182. MILLS, W. D. 1955. Fire blight development on apple in western New York. Plant Dis. Reptr. 39:206-207. NIGHTINGALE, A. A. 1936. Some chemical constituents of apple associated with susceptibility to fire blight. N.H. Agr. Exp. Sta. Bull. 613:1-22. OKABE, N., and M. GOTO. 1963. Bacteriophages of plant pathogens. Annu. Rev. Phytopathol. 1:397-418. PARKER, K. G. 1936. Fire blight: overwinter" ing, dissemination, and control of the pathogene. N.Y. Agric. Exp. Sta. Ithaca Mem. 193:1-42. PIERSTORFF, A. L. 1931. Studies on the fire— blight organism, Bacillus amyZovorus. Cornell Univ. Agr. Exp. Sta. Mem. 136:1-53. POWELL, D. 1965. Factors influencing the severity of fire blight infections on apple and pear. Mich. State Hort. Soc. Ann. Meet. 94:1-7. 41. 42. 43. 44. 45. 47. 48. 49. 50. 51. 10 RIGGLE, J. H., and E. J. KLOS. 1972. Relation— ship of Erwinia herbicola to E. amylovora. Can. J. Bot. 50:1077-1083. RITCHIE, D. F., and E. J. KLOS. 1977. Isolation of Erwinia amylovora bacteriophage from aerial parts of apple trees. Phytopathology 67:101-104. ROSEN, H. R. 1928. Variations within a bactern ial species. I. Morphologic variations. Mycologia 20: 251-275. SCHROTH, M. N., and D. C. HILDEBRAND. 1965. B-Glucosidase in Erwinia amylovora and Pseudomonas syringae. Phytopathology 55:31- 33. SCHROTH, M. N., S. V. THOMSON, D. C. HILDEBRAND, and W. J. MOLLER. 1974. Epidemiology and control of fire blight. Annu. Rev. Phyto- pathol. 12:389-412. SHAFFER, W. H., JR., and R. N. GOODMAN. 1962. Progression in viva, rate of growth in vitro, and resistance to streptomycin, as indices of virulence of Erwinia amylovora. Phytopathology 52:1201-1207. SHAW, L. 1934. Studies on resistance of apple and other roasceous plants to fire blight. J. Agr. Res. 49:283-313. THOMSON, S. V., M. N. SCHROTH, W. J. MOLLER, and W. O. REIL. 1976. Efficacy of bactericides and saprophytic bacteria in reducing coloni- zation and infection of pear flowers by Erwinia amylovora. Phytopathology 66:1457- 1459. UETAKE, H., S. E. LURIA, and J. W. BURROUS. 1958. Conversion of somatic antigens in Salmonella by phage infection leading to lysis or lysogeny. Virology 5:68-91. VAN DER ZWET, T. 1968. Recent spread and present distribution of fire blight in the world. Plant Dis. Reptr. 52:698—702. 52. 53. 54. 55. 11 VIDAVER, A. K. 1976. Prospects for control of phytopathogenic bacteria by bacteriophages and bacteriocins. Annu. Rev. Phytopathol. 14:451-465. VOROS, J., and R. N. GOODMAN. 1965. Filamentous forms of Erwinia amylovara. Phytopathology 55:876-879. WU, W. C. 1972. Phage-induced alterations of cell disposition, phage adsorption and sensitivity, and virulence in Xanthomonas citri. Ann. Phytopathol. Soc. Japan 38: 333-341. ZINDER, N. D., and J. LEDERBERG. 1952. Genetic exchange in Salmonella. J. Bacteriol. 64:679 -6990 PART I ISOLATION, DISTRIBUTION, AND POPULATIONS OF ERWINIA AMYLOVORA BACTERIOPHAGES ON AERIAL STRUCTURES OF APPLE INTRODUCT ION Prior to 1975 only one reported attempt to isolate Erwinia amylovora bacteriophages from aerial structures of fire blight-susceptible plants had been published, and it was negative (4). Up to this time, no information existed on phage populations, distribution, and their association with E..amylovora on aerial plant structures. With the isolation of E. amylovora phages from these structures in 1975, it seemed important to study the distribution, relative populations, and the association of phage with E. amylovora on apple. The objective of this section is to report the isolation of E. amylovora bacteriophages from aerial structures of apple and to describe the distribution and populations of these phages and E. amylovora on such structures in several Michigan orchards. 12 W hernJanuarv 1977 PHYTOPATHOLOGY. Volume 67. No. l WhyfhoAmaflcan Wow “Normand. St. Paul. Minnesota 55121 13 Society. Inc. Ecology and Epidemiology Isolation of Erwinia amylovora Bacteriophage from Aerial Parts of Apple Trees D. F. Ritchie and E. J. Kloa Graduate Assistant and Professor. respectively. Department of Botany and Plant Pathology. Michigan State University. East Lansing. Ml 48824. The authors thanlt D. W. Dye. M. N. Schroth. and T. 8. Sutton for kindly providing bacterial cultures. Journal Article No. 7566 of the Michigan State Agricultural Experiment Station. Accepted for publication 16 August 1976. ABSTRACT RITCHIE. D. F..andE.J. KLOS. I977.bohtioaofErwinhamybvombaauiophagefmmaerialpansofappletrea. Phytopathology 67:10l-los. Popuhtions of bwtru’a anylovoro bacterioplugs greater the II. plaque-forming units ( PFU) per gram of tissue were isohted without enrichment from diseased aerial parts of apph true during the summer of 1975. Three phage isolates were selected from different WWW loations. Two typesofplaqueswere prodMaclear-centered plaquwith WMWfireblight. a spreading translucent halo and a smaller plaque without a halo. Thirty-five bacterial isolates. consisting of nine genera. l8 species. and 15 strains of E. amgvlovora were typed: the phages lysed only E. omylovora. The burst size of the three isolates was40t050 PFU percell. Thephagescould bestored at4Cand‘20Cbutlosttiterwhenstoredat24C. Studies of bacteriophages of plant pathogenic bacteria have dealt primarily with their use as a diagnostic tool(5) or in characterization of phage-bacteria interactions (3. 6. ll). Phages of phytopathogenic bacteria may be isolated frontsoil in the vicinity of the diseased plant(7) and often from the diseased tissue of the plant (ll). Erskine (8) isolated a phage from soil at the base of barium outflows-infected trees. but was unable to 'Iolate it from infected or healthy aerial tissues. This phage (Sl) lysed both E. amylovom and a yellow saprophytic bacterium which could also be lysogenized. This report deals with the isolation and partial characterintion of E. amylovora phages from diseased and symptomless plant tissues of aerial parts of apple trees during the summer of I975. MATERIALS AND METHODS Erwlnla amylovora isolation.-Diseascd and symptomless aerial tissues of apple trees. Mala: svlmm's Mill. at Michigan State University (MSU) and three growers’ orchards in southwestern Michigan were sampled. No attempt was made to disinfest the tissues. Bacter'nl popuhtions were quantified on a tissue weight basis unless stated otherwise. Tissues were washed in distilled water for 30 to 60 minutes. Serial dilutions ranging from l0'I to l0" were made in 0.02 M potassium phosphate buffer. pH 6.8. and 0. lot!!! samples were spread over the surface of a differential medium (l3). Representative isolates were tested for pathogenicity us'utg the mdling technique (l2). Bacteriophage lsohtioo.—The tissues. washing Wormnnmwmasso MWM&M.MN5532LMMM procedures. and dilution series were the same as those described for-the isolation of E. amylovora. The plating procedures were as outlined by Adanu (l). Plaques. if present. were counted after 24 hours incubation at 24 C. Three phage isolates werechosen for f urthcr study. Phage PEal was isolated from blighted 'Jonathan' apple terminals from southwestern Michigan. PEaZ was isolated from soil at MSU. and PEaS was isolated from a symptomless ‘Jonathan' apple terminal at MSU. The phage isolates were purified by single-plaque isolation five times using E. amylovorc isolate lll l0 from the MSU orchard as propagating host. Phage cluneterintlon.—Phage lysates were prepared by scraping the top agar from plates with confluent lysis. centrifuged at l2.000 g for ID minutes. and filtered through Millipore filters (pore size. 0.45 pm). The lysates were stored over a drop of chloroform in 3.54—g (2.0 dram) screw-cap vials covered with aluminum foil. Vials of each isolate were placed at 24. 4. and -20 C to determine longevity of storage. The double-layer agar technique ( l) in standard 100- mm diameter plastic petri plates was used to examine plaque morphology. The bottom layer consisted of l2 to l5 ml of 2.0% nutrient agar supplemented with 0.5% glucose: the top layer consisted of 2.5 ml of 0.7% nutrient agar. 0.5% glucose. and 0.5% yeast extract. The phage titers were adjusted to approximately 50 plaque-fanning units (PFU) per plate. An l8- to 24-hour nutrient broth- glucose culture of E. amylovora isolate NO was used as host. The plates were incubated at 27 C and examined over a 48-hour period. The double-layer agar technique was used to type the host-range of the three phage isolates. One-tenth milliliter of a 24-hour nutrient broth-glucose culture of each bacterial isolate was added to the 2.5 ml of warm top agar. After the top layer had solidified. one loopt'ul of etch lOl l4 l02 phage isolate(titers ranged from IO‘ to 10’ PFU/ml) was PHYTOPATHOLOGY (Vol. 67 TABLE 2. Host range of Erwinr‘c amylovorlr bacteriophages spotted on sections of the plates. The host-range typing was done twice at different times. Thirty-five bacterial isolates representing nine genera. 18 species. and 15 strains of E. amylovora were typed. The one-step growth experiment for each of the three phage isolates was done in 0.8% nutrient broth. 0.5% glucose. 0.5% yeast extract at 24 C. using E. amt-Iowans isolate l 10 as the host. Phage were added to the bacteria at 1le ratio and allowed to adsorb for.l0 minutes then diluted to approximately 10’ bacteria per milliliter. Beginning at 20 minutes. samples were taken every IO minutes through a 90-minute period. The one-step growth experiment was repeated three times for each phase bolus. RESULTS AND DISCUSSlON During the monitoring of E. amylovom populations’on ‘Jonathan' apple trees at the MSU experimental orchard during the summer of I975. E. amt-[ovum phages were detected without enrichment. Phages could be isolated easily from three growers‘ orchards in southwestern Michigan during a fire blight epiphytotic. Phage was detected on all tissues sampled where E. amylavora was detected except on symptomless leaves (Table 1). Failure to detect phage on symptomless leaves may have been due to several factors: (i) the phage populations may have been below the detection levels. (ii) E. amides-om populations were lower on symptomless leaves than on other tissues. and (iii) there may be less protection on leaf surfaeee from factors such as ultraviolet light. effect of moisture. etc.. than on other tissues. It is not known whether the phages were located internally or externally to the plant surface. but they were present wherever E. amylovoro was found extensively. The three phage isolates could be stored for at least six . months at 4 C and -20 C without significant loss of titer. Titers dropped rapidly when stored at 24 C. Two distinct types of plaques were produced by the phage isolates (Fig. l). The first type produced a clear PEal. PEaZ. and PEaS Bacterial isolate PEal Phage isolate PEaS Erm‘nl'a anybvore Cal Eal Ill 68 M S U ”0 M S U lll Traverse City ”2 G. Rapids “3 G. Rapids “4 Paw Paw ll5 Spinks Corners "6 Cal Ea5 Cal 338 M S U Mae 7l5 N.C. EACCSIZ l20 N.C. BASIS l2! Paw Paw l22 Agroborrerr‘wn rune/m UCJ4l6 A. (tune/acres: UC 78 Connebacrrrium fascia: C. flocrumfociens Enrerobocrer uerogenes Erwl'm'a armseprr‘ca SR 8 E. rmrovoro SR I65 E. herbicolo ZP-l E. herbrcola ZP-Z E. Irerbrcolo A-E Bthfffdlfl rolt‘ Pseudomoms oerugr’rroso P. fluorescent P. Iochrynearss P. :0th P. mm erfzobr‘um sp. Serrrm'a sp. Xanrlromonus jugfandl's X. pruni PF—Z ‘Plus (+) indicates lysis. 'Minus (-) indicates no lysis. lllllllllllllllllllL +++§+++++++++t§ 1 llllllllllllllllllll+++§++++++++++gg llllllllllllllllllll +++§++++++++++§ TABLE l. The occurrence of fiwim'e amylovorc and E. anvfovore bacteriophages on aerial parts of Jonathan apple trees during the summer of I975 at East basing. Michigan Samples containing E. emylovm Samples containing bacteriophage and approximate concentration ( PF ll )" of bacteriophage per unit sampled Types and approximate and approximate concentration (CFU)‘ amounts of tissues sampled of E. amvlovom per unit sampled Symptomless terrnimls Eight of 25 terminals (~l.o gltermlnal) I to 2 x lo‘ CPU/terminal 3- to 4-week-old infected tenninals 2.50f25termiruls Four of 25 terminals lo‘ to 3 x lo‘ PFU/terminal 250(25terminals aadleavee(~2‘to4glsample) 5x lo’toox lO' CPU/terminal 3x10'to7x10‘rr-‘U/trmtntl Newly formed centers l5 of IS 'm Nine of 15 canker: (~02 to o: g/canker) >lo‘ CPU/g 10’ to >lo‘ Pro/r Symptomless leaves Four of five samples 0 of five samples Five samples. l0 leaves persample ~l0’ CPU/sample None Three blighted fruits Three of three fruit Three of three fruit (~05 gift-tilt) ~lo' CPU/g ~lo‘ PFU/g ‘CFU. colony-fanning units. 'PFU. Murfonning units. January I977] center 4-5 mm in diameter with a distinct translucent. spreading halo occurring at l8 hours: the second type produced a smaller plaque I-2 mm in diameter with an irregular margin and no halo. The halo may have resulted from lysogcny or the production of an enzyme capable of 7.4) Thesmnll non-halo plaque form was easily separated from the Iarpr. halo-forming plaques but the larger halo-forming plaques could not be isolated readily from those produced by the non-halo plaque form. 0f the 35 bacterial isolates tested. only the E. amybvora isolates were lysed (Table 2). With two isolates. Eal from California and Mac 7I5 from MSU. hazy plaques were produced. These two bacterial isolates are different from the other isolates in that they did not produce copious amounts of slime on nutrient agar- m I . All three phage isolates had a similar one-step growth fig. HA. I). A) Plaque morphology of phage PEal (halo) and PEaI (non-halo). arrow. after l8 hours incubation at 27 C with owth amylovm isolate I I0 on 2.5 ml of top-agar consisting of 0.7% nutrient agar. 0. 5% glucose. and 0. 5% vast extract. I) Plaque morphology of phage PEaI after 48 hours under the same conditions as in A. Note the extending. tramluaat halo of PEal (halo) while the plaque morphology of PEI (non-halo) hasretna ined the same as at I! hours (arrow) bar equals l .0 cut. RITCHIE AND KLOS: ERWlNlA AMYLOVORA PHAGE 15 l03 "- I . . . . . u. } "al 2' ' I,“ "-50 ’ d ° W 5 o 3 . E o 3 E o b 5 '5' 2‘0). I a 3 Lars-Ir mas G t—PIIIOD ' ran-co" E “.3 I'O tit tit 43 sit in 7'0 s'o vii INCUMTION TIM! (MIN) Hg. 2. Oneqtep growth curve of phage isolates PEal. PEaZ. and PBS with Erwinia amyluvora “0 as host. curve with latent periods of 30 to 40 minutes. a rise period occurring from 35 to 65 minutes from start of incubation. and a burst size of 40 to $0 PFU/ccll (Fig. 2). Whether the phage plays a role in fire blight etiology is not known. rskine investigated this possibility under laboratory conditions and hypothesized that the yellow. . .I.- L I , ilcqucutiyin' U I ‘ J serve as a reservoir ol‘ phage which under appropriate conditions may affect the severity of fire lhlight IS) A major criticism of ”“- L, , phage from aerial plant parts. Harrison and Gibbins( l0) recently reported the isolation of a temperate phage from the yellow bacterium. Erwinin herbicola. isolate Y46. after treatment with mitomyein C: however. none of the E. anylovora isolates tuted was sensitive to that phage. One of the results of phage-bacteria interaction is the killing of the phage-sensitive bacteria and the selection of phage-resistant mutants. Preliminary data from our laboratory indicate that the virulence of E. amylovora was attenuated in phageresistant bacteria. This was reported previously for E. amylovora (8) and Pseudomoms morspnmonun (9). LITERATURE CITED l. ADAMS. M. H. I959. Bacteriophages. lnterscience Ptt'b hers. New York. 592 ZADAMS. M. H. and B. H. PARK. 1956. An enzyme produced by a phage-host cell system II. The properties of polysaccharide depolymerase. Virology 2: 719- ]. BAIGENT. N. L J E DEVAY. andM. P. STARR. 7I903. Bacteno nophans of Pseudomonas syringae. Z. J. Sci. 6:73-l00. t. BILLING. E. I960. An association between camulation and phage sensitivity in Erwinia amylovora. Nature I“: 5 I9- 820. l04 PHYTOPATHOLOGY 5. BILLING. E. I963. The value of phage sensitivity tests for the identification of phytopathogenic Pseudomonas spp. J. Appl. Beet. 26393410. 6. CIVEROLO. E. L. I972. Interaction between bacteria and bacteriophage on plant surfaces and in plant tissues. Pays 25-37 in H. P. Mass Geesteranus. ed. Third Int. Conf. Plant Pathgenic Bacteria. Proc.. Centre Agric. Publ. Doe. (PUDOC), Wageningen, The Netherlands. 365 p. 7. CROSSE. J. E.. and M. K. HINGORANI. I958. A method for isohting Pseudomonas mors-prunorum phages from soil. NatureILond.) I8I:60-6I. I. ERSKINE. J. M. I973. Characteristics of Erwinia anylovora bacteriophage and its possible role in the epidemiology of tire blight. Can. J. Microbiol. I92837- “5. 16 [VOL 67 9. GARRETT. C. M. E.. J. E. CROSSE. and A. SLETTEN. I974. Relations between phage sensitiwty and virulence in Pseudomonas morsprunorurn. J. Gen. Microbiol. 80:475-483. I0. HARRISON. A. and L. N. GIBBINS. I975. 11Ie'isolation and characterintion of a temperate phage. Yul/(E2). from Erwin’u herbicola Y46. Can. J. Microbiol. 2I:937o 9“. II. OKABE. N.. and M. GOTO. I963. Bacteriophages of plant pathogens. Annu. Rev. Phytopathol. I:397-4I8. I2. RITCHIE. D. F.. and E. J. KLOS. I974. A laboratory method of testing pathogenicity of suspected Erwinta amylovora isolates. Plant Dis. Rep. 58:I8I-l83. I3. RITCHIE. D. F.. and E. J. KLOS. I976. Selective medium for isolating Erwinia smylovora from apple and pear than. Proc. Am. Phytopath. Soc. 3: (Abuse). ( In press). MATERIALS AND METHODS Monitoring of Erwinia amylovora and its bacterio— phages. The type of apple tissue selected for moni- toring was dependent on the time of year; during winter, populations in cankers were monitored, while during the growing season populations in association with current year's tissue were checked. Tissue during the growing season was collected by slipping a plastic bag over the tissue and breaking it off thus reducing the possibility of cross contamination between samples. Tissue fresh weight was determined, a known quantity of distilled water added, and the tissue was washed in the bags for 1-2 hr. Samples from cankers were collected by removal of sections with a sterile scalpel. These sections were dissect— ed into approximately 1.0 mm long pieces, fresh weight was determined, and tissues were washed for 1—2 hr in 10 ml of distilled water. Assays for phage were done using the agar overlay method (1) with E. amyZovora 110 as host and a differential medium for E. amylovora (8). The number of plaque—forming- units (pfu) and colony—forming—units (cfu) are 17 18 reported as per gram fresh weight of tissue. Monitoring of populations in a MSU orchard. Erwinia amyZovora and its phage populations were monitored on Jonathan apple trees from June 1975 to December 1976. Symptomless tissues as well as blight— ed leaves and stems, blighted fruits, newly formed cankers, and overwintering cankers were sampled. Monitoring of populations in ten;growers‘ orchards. Ten apple orchards in southwestern Michigan were monitored in 1976 with samples taken in June, July, August, and October. Samples consisted of approximately 30 g of blighted leaves and terminals taken randomly throughout the orchard and combined into a composite sample of each orchard. The effect of desiccation on phage PEal(h). Leaf discs of 1.0 cm diameter were cut from healthy, mature apple leaves (MaZus sylvestris cv. ‘Jonathan‘) using a sterile corkborer. Discs were individually inoculated on the upper surface with 0.1 ml of phage PEal(h) from a chloroformed, filtered (Millipore 0.22 pm pore size) lysate; inoculum contained ca. 109 pfu/ml in 0.02 M potassium phosphate buffer, pH 6.8. One set of discs was placed on a wire screen sus- pended over a pan of water in a clear plastic bag to maintain 95-100% relative humidity (RH). A second set was placed in a pan without water and left l9 uncovered in ambient RH of 15-25% measured with a recording hygrothermograph. The pans remained on the laboratory bench where temperatures ranged from 21-23 C. Three discs from each set were randomly chosen over a 28—day period and individually washed in 0.02 M potassium phosphate buffer, pH 6.8. Washings were diluted and assayed for phage using E. amyZovora 110 as host. RESULTS POpulations in a MSU orchard. Table 3 summarizes the monitoring results in a MSU orchard from June 1975 to December 1976. There was extensive variation in phage and E. amyZovora populations among the samples; however, phage were easily detectable during certain periods (Figure 3). These periods coincided with fire blight epiphytotics during which time E. amyZovora populations were maximal and widely distributed on blighted as well as symptomless apple tissues. During 1975, phages were detected from June through August but not during the winter of 1975-76. In 1976, phages were not detected until July, coinciding with fire blight outbreaks and were detectable through October but not in December. Even though phage populations were variable the average population for six sampling periods was approximately lO-fold greater in 1975 (2.3 x 105 pfu/g of tissue) than in 1976 (1.2 x lo4 pfu/g of tissue). The percent of samples in which both E. amylovora and phage were detected.was greater in 1975 (66.5%) than in 1976 (52.2%). In 1975, fire blight was severe from mid-May 20 21 O .O.: .O.e OOH a O i O «OH x O OH macaque Ob\HN\O O .O.: .e.: OOH a « i O «OH x O OH mumxamo OO\ON\N O .O.e .O.O OOH x H i O «OH x H OH muoxano Oe\O\HH O .O.: .O.: OOH x m t O «OH x « OH uuoxcmo On\O\OH O .O.: .O.: OOH x m i O OOH x « OH macaque OO\OH\O O .O.a O.O.: OOH x « i O OOH x O OH . .kmnoxamu Os\ON\O OO OOH x N O OOH x O OOH x OIOOH x H OOH x H OH mHmcHsuou OmurmHHm ON\O\O OO «OH x O O «OH x O «OH x O t O «OH x H O mHaeHauuu OOOOOHHO ON\OO\N OO OOH x N O OOH x N hOH x NisOH x N NOH x « OH mmuuxeuo nuance aHzmz ON\>\> me OOH x H O OOH x « «OH x O i O «OH x N Om mHmeHsuou OOuOOHHm ON\N\s OOH OOH x N-«OH x H OOH x O OOH x NiOOH x N OOH x H ON OmHneHeuou OOOOOHHO On\«N\O OH «OH x O i O mOH x N OOH x N i O «OH x H ON OeraHsuou mmmHsoudssm ON\ON\O Dam a DOW omcom com: omcom and: moamson mcwawouaoo itiitlitititittttlitt ittitittititittittitii. HospH>Hocfi coaganm pump madmanm no a osmmfiu mo m\csmo mo nonfisz manna» no oaks admanm 0 manna» mo m\aomm mbma pan mead mcwnop mcamcog umnm .............................................. ...................................... no twosome wufimuo>wcb muoum comHnowz n ca mcoOuoHomom nonsmownouonn cacaonEo .m can unencumse ogceaam .m manna 22 .Oouoouoo mums momnnmowumuonn afionostd .m can ogoaonEd cvzwssm span zofisz ca moamsnm Houou on» no ucoonom asap .omnnmofiuwuoon cacaonEo owxvasm Mo muwcatmcaanouiuowoam u mama .oaononEo Stubs.» we muHcotmcHeuowihcoHoo u Pave O .O.e .O.: OOH x N i O OOH x OH muoxeco ON\NH\NH ON «OH x H i O OH x OOH x O i O «OH x OH muoxaao ON\HN\OH OO «OH x N i O OH x OOH x « i O OOH x O mnmxamo Oh\H\O O« HOH x « i O OH x OOH x HIOOH x O OOH x O mHocHauuu eouaOHHm ON\«N\O OO NOH x H i O OH x «OH x OI«OH x O «OH x O mHmeHsumu OOOOOHHO ON\NH\O OOH «OH x OiNOH x N OH x «OH x NIHOH x O OOH x OH mHmeHsuou OOOOOHHO ON\N\O OO OOH x N i O OH x NOH x «iOOH x H NOH x O mHmcHauou OOOOOHHO ON\NH\N O .O.: .O.: .O.: .O.a OH mHmeHsuou mmoH50umssm ON\NH\O O .O.: .O.: .O.: .O.c OH mHuaHsmou OOOHaoOmssm ON\H\O O .O.: .O.: OOH x O l O OOH x « muoxeoo ON\NN\O .Hcoscquooov m manna 23 .pouomump no: u .p.:m .Hfiumd on Honsmummm Scum unoEMOp >Haowucommo ouoz poo .ouopsxo anfluouoon o: .mosmmflu wzuaoo: oucw ucwaooco>po 0: Ho OHDDHH pouwnfinxm hens “muoxcoo mowuoucw3ho>o .Aoofimmmaflk .ouopaxo Hofiuouoon Mo oocopcsno any man .0003 anuaomn ouow undemoco>po Hmscwugoo .mcwmuoa muwcwmmpcw mo mocmmoum man :H muoxgoo mcwuoucfizuo>o Scum to luommwp muoxooo when? .musmm uwsum poo maocwshou mm £05m £u3oum m.Hmo> acouuzo on» so pouusooo to: nownz mcofiuoomcw mo uaamou mnu no Hopao no name» m @003 so weaken oum3 mnoxgoo pmfiuou >H3ozm .Eoum map can mo>moa OH I o no pomomaoo £u3oum m.umo> unouuso man mo 50 mm Houmwp on» mo poumwmcoo maocfifiuou pounmwan can mmoaaoumaamfl . 80:59:08 O 033. DETECTION OF BAGTERIOPHAGES ON AERIAL STRUCTURES OF APPLE 24 "fi'IFTI'Tr'TUU—rt' a ui p. Snot-on _. H '- u a O U .— 0 u - A A A - ‘ A I: )- ' - - v - v CG 0 .- o z 1 L J _L l L L L 1L 1 l L l L J l l l '4 3 $ $ ‘5‘ u» s 150 .“e .‘e s o“ ”0“.“ out.» 05 .6.“ e .t. .s 3° ) ”“0: $3320 0399?: : ;*°:: Vb ’fi’fi" " “29“, 9.0 flat “tog 5‘ GOO HT 5 00“ TIME OF YEAR TI-IAT TISSUE WAS SAMPLED Figure 3. Seasonal detection of Erwinia amylovora bacteriophages in association with aerial structures of apple in a Michigan State University orchard at East Lansing. Note that Y-axis is qualitative not quantitative. 25 to mid-June; all of the Jonathan trees exhibited at least 50-100 strikes/tree. During 1976, fire blight was not observed until late June; some trees showed no strikes while the most severely infected trees showed fewer than 25 strikes/tree. It was the exception to detect phage in overwintering cankers; only in September and October of 1976 were they detected associated with such structures (Table 3). Populations in ten'growersl'orchards. During the June monitoring period phage were detected in 8 of 9 orchards and in 7 of 9 orchards during July (Table 4). In August phage were detected in only 1 of 10 orchards and 2 of 10 orchards in October (Table 4). Phages were readily detected as long as E. amyZovora was present but only occasionally detected when E. amyZovora was non-detectable or at low levels. No new strikes were observed in the orchards after the 18 June monitoring period. Effect of desiccation on infectivity of_phage PEal(h). Phage PEal(h) was extremely sensitive to desiccation, exhibiting a 105-fold decline in titer within 24 hr at 15-25% RH (Table.5). The titer then declined to non—detectability after 16 days. Phage titers on leaf discs maintained at 95-100% RH remained relatively stable for 16 days then declined l to ca. 1 x 10 pfu/leaf disc by the fourth week(Table53. 26 mOH x N v0H x m .p.: .O.: OH x N mOH x m 0m 1 o .m.: .m.: m .O.: .O.: OH x O .O.: OH x N NOH x N OOH i OH OOH x H NOH x « O «OH x H .O.: .O.: .O.: OH x H OOH x N OOH .mo «OH x H H.OH x N N o a: o a: o o: a a: ems: emu: Is 0 a: x U m O p m mm o w c ooH m m .o.: .p.: .m.: .p.: OH x v 00H x m cm i o mOH x N 59H x h m .©.: .p.: .p.: .p.: OH x m oOH N m 00H t o oOH N H mOH x H v .p.c .0.: .p.: .p.c .p.c N.OH x m 00H .oo o0H x H 00H x N m .©.: .©.: .pn: .0.: OH x m mOH x M 0m .mo mOH x m mOH x H m .p.: .p.: .p.c .p.: .m.: hOH x v mm t o mOH x m N.OH x H H >uHHo>om . . m m m m m a O\Omm \OOO \Omm O\OOO \OOO \Omu our HHn muHm \eomm O\o=mo umnssz - panache OO\ON\OH OO\ON\O ON\ON\O ON\OH\O manna OHmeom .comHnon cuwumoanuoom GO mononouo .mnmzoum sou cH mcoHuoHsmom omozmoHumuoon cacaoumso .m can oaononEo dermanm .e OHQOB 27 .GOHmEom no: .moc Q .pouoouop no: I: on .®h\mH\m co moms mo: ucsoo HMHuHcH may Hound pm>uomno ouoz moxHHum 30: oz .moxwuum uanHn oun to: own» >H0>o uo: unnu mOHOOHch OHON .pumnouo on» :H own» Hon mmxHHum uanHn mo Hones: 0:90 .momosmoHHouoon UAQaQNmEd .m mo muHcaimcHEHOMImsvon 0 Dana .caononEo omresam mo muHcstmcHEHOMlacOHoo u smug ’ FPF)’ ’ i i’h i7 9 i V ' Pi .U.c .n.c .v.: .6.: MOH x,m ; 00ng m. __ . OOH.t 0H med K H N.o._n x v OH .HuoscHucooO v oHnos .coHuoHsoocH prmo H: v coxou mOHQEom a .muHcsimcHEHOMIooUon u ammo 28 O HOH x O.H H HOH x O.N ._ _‘ _ ON O OOH x O.H H OOH x O.H HN O OOH x O.O H OOH x H.H OH NOH x H.H H NOH x O.N OOH x «.« H OOH x O.H OH NOH x H.O H OOH x N.H OOH x O.« H OOH x N.H O OOH x O.« H OOH x O.O OOH x H.N H OOH x H.H H OOH x O OOH x O OO .O.O OON s OH .O.O OOOH . OO HOOOO. OHHm m mew omHm\Dmm omo Honasz p . m H m Q .mome «OOH mHmmm :0 Aannmm nonsmoHHmuomn oHononEo ewrwsam mo Ho>H>Hsm on» :0 :ernUOHnoo mo yoouuu .m mHnoB DISCUSSION Erwinia amylovora bacteriophages were commonly found on aerial structures of apple in orchards infested by E. amyZovora. Not only were phages detected in an MSU orchard during consecutive summers of 1975 and 1976, but also in 10 orchards in south- western Michigan during the summer of 1976. During fire blight epiphytotics, phages were detected in association with all types of blighted tissue and occasionally in association with symptomless tissue as well. The more severe (number of strikes) fire blight was in an orchard the easier it was to detect phage. As the incidences of fire blight strikes decreased and the blighted tissue desiccated the ability to detect phage decreased. As obligate parasites bacteriOphages are entirely dependent on their bacterial host for multiplication, and are therefore affected by the metabolic condition of the bacterial host (1). In nature E. amylovora should grow optimally when climatic conditions are favorable, coupled with an abundance of susceptible 29 30 host tissue. Such conditions are usually present in Michigan orchards from mid-May through June. During this period the most rapid development of fire blight epiphytotics occur, resulting in a sufficient population of E. amyZovora to support phage growth. Even though phages cannot multiply apart from their bacterial host they can survive in the absence of their host; the length of time they remain infectious is dependent on numerous factors (1). The E. amyZovora phages were able to tolerate numerous physical conditions, e.g. high and low temperatures, survival in absence of their host (Section II of dissertation), but were very sensitive to desiccation (Table 5). Similar drying conditions are common in blighted tissue one to two months following infection by E. amylovora. During the absence of susceptible plant tissue and favorable climatic conditions E. amylovora remained detectable (Table 3), but probably existed in an hypobiotic state (5) not conducive for phage growth. Thus, drying of blighted tissues and reduction in the presence of a highly metabolically active bacterial host were probably instrumental in the seasonal detectability of the phages. This seasonal detectability of E. amyZovora phages may explain why they were previouskyunreported 31 from aerial structures of plants susceptible to E. amyZovora. Numerous studies involving the iso— . lation of E. amylovora from blighted tissue have dealt with isolations from cankers during the winter and early spring (2,3,11); phages were not detected in cankers later than October. E. amylovora populations have been monitored primarily in symptom— less rather than blighted tissue (6,9,10); phages were not commonly detected in association with blighted rather than symptomless tissue. Furthermore, dilutions are commonly used to obtain individual colonies (6) reducing the likelihood of detecting phage. Phage plaques were normally discernible for only 24-36 hr before becomdng masked by phage- resistant E. amyZovora and other phage insensitive bacteria. ' At both the MSU orchard and the 10 southwestern Michigan orchards, fire blight epiphytotics were stationary or declining at the time of maximum recovery of E. amyZovora phages. It would be tempting to suggest that this was the effect of phage. Phage does affect virulence of E. amyZovora ‘under laboratory conditions (Section III of dissertation); however, the decline in fire blight epiphytotics coincided closely with the stoppage of terminal-bud growth, thus reducing the amount of 32 susceptible tissue. Possibly a more quantitative monitoring of phages and the development of fire blight would provide a more definitive answer to the role of phage in fire blight development in Michigan orchards. Other factors such as nutritional condition and growth stage of the host plant, climatic conditions (high humidity, warm temperatures, rain, etc.), and insects are also involved in maximum development of fire blight epiphytotics. It has been suggested that saprophytic microorganisms can affect the development of fire blight (7), since these phages are parasites of E. amyZovora it is possible that they may be one of the controlling factors in the development of fire blight. It 1. 2. 3. 4. 5. 6. 7. 8. 9. LITERATURE CITED ADAMS, M. H. 1959. BacteriOphages. Interscience Publishers, New York, NY 592 p. BAKER, K. F. 1971. Fire blight of pome fruits: The genesis of the concept that bacteria can be pathogenic to plants. Hilgardia 40:603- 633. BEER, S. V., and JOHN L. NORELLI. 1977. Fire blight epidemiology: factors affecting release of Erwinia amylovora by cankers. Phytopathology 67:1119-1125. ERSKINE, J. M. 1973. Characteristics of Erwinia amyZovora bacteriophage and its role in the epidemiology of fire blight. Can. J. Microbiol. 19:837-845. LEBEN, C. 1974. Survival of plant pathogenic bacteria. Ohio Agric. Res. Dev. Cent., Spec. Circ. 100. 21 p. MILLER, T. Dq.and M. N. SCHROTH. 1972. Monitor~ ing the epiphytic pOpulation of Erwinia amylovora on pear with a selective medium. Phytopathology 62:1175-1182. RIGGLE, J. H., and E. J. KLOS. 1972. Relation- ship of Erwinia herbicola to Erwinia amyZov- ora. Can. J. Botany 50:1077-1083. RITCHIE, D. F., and E. J. KLOS. 1978. Differen- tial medium for isolation of Erwinia amylov- ora. Plant Dis. Rept. 62:167-169. SUTTON, T. B., and A. L. JONES. 1975. Monitoring Erwinia amyZovora populations on apple in relation to disease incidence. Phytopath- ology 65:1009-1012. 33 10. ll. 34 THOMSON, S. V., M. N. SCHROTH, W. J. MILLER, and VAN W. O. REIL. 1975. Occurrence of fire blight of pears in relation to weather and epiphytic population of Erwinia amyZovora. Phytopath— ology 65:353—358. DER ZWET, T. 1969. Study of fire blight cankers and associated bacteria in pear. Phytopathology 59:607—613. PART II CHARACTERIZATION OF ERWINIA AMYLOVORA BACTERIOPHAGES INTRODUCTION Erwinia amyZovora bacteriophages have been iso— lated from soil since the 1950‘s (14); however, only one phage strain, obtained from soil, has been partial- ly characterized (10). In 1975 and 1976, E. amyZovora phages were isolated from aerial structures of apple in several Michigan orchards. Some of the biological, chemical, and physical properties of these bacterio- phages are presented in this section. 35 MATERIALS AND METHODS Media and’cultural‘conditiOns. Bacteria were grown on 2.0% (w/v) nutrient agar supplemented with 0.5% (w/v) glucose, pH 6.5, (NAG medium). Phages were plated in 100 mm diameter plastic petri dishes using the soft agar overlay method (1) with 12-15 ml of NAG and 2.5 m1 of 0.7% (w/v) nutrient agar, 0.5% (w/v) glucose, and 0.25% (w/v) yeast extract, pH 6.5, composing the bottom and top layers respectively. The liquid medium consisted of 0.8% (w/v) nutrient broth, 0.5% (w/v) glucose, and 0.25% (w/v) yeast extract, pH 6.5, in distilled water (NGBYE medium). All liquid cultures, unless stated otherwise, were incubated at 22—23 C on a reciprocal shaker (80 oscillations/min, stroke length 4.0 cm). Erwinia amyZovora strain 110 rifampin resistant (110 rifr) was used for phage propagation and assay. Assay cultures were grown overnight.in 125-ml flasks containing 25 m1 of NBGYE. Prior to experiments, 5.0 m1 of inoculum were added to 25 ml of fresh NBGYE and incubated 1-2 hr; Phage assay plates were incubated at 27 C and scored 36 37 after 12—36 hr. All bacteria and phage dilutions were made in sterile 0.02 M potassium phosphate buffer (PPBL pH 6.8. Phage isolation. Bacteriophages were isolated from aerial structures of apple as described (17). Phages were purified by single-plaque isolation 3-5 times. High titer lysates for storage and preliminary characterization were obtained from plates having confluent lysis by adding 5.0 ml of PPB, scraping off the soft agar, adding chloroform to 1.0% (v/v), centrifuging for 10 min at 12,100 x g in a Sorvall RCZ-B centrifuge, and filtering the supernatant (Milli- pore 0.22 um pore size). Filtrates were stored over a drop of chloroform at 4 C. Host range and plaque morphology. Host range was determined as described (17). Plaque morphology was examined using the agar overlay method (1). Phages were diluted so that approximately 50 plaques occurred per plate. The plates were incubated at 27 C and examined during a 12-48 hr period. ‘Phage growth and purification. Erwinia amylovora 110 rifr was grown overnight in 25 ml of NBGYE, then 10 ml were added to 1000 ml of NBGYE in a 2-liter flask and grown to an optical density (0.0.) A525 of 0.06 — 0.10 (ca. 108 colony—forming—units [cfuI/ml). The appropriate amount of phage solution was added to give 38 a final multiplicity—of—infection (m.o.i.) of 0.1—1.0. The culture was incubated with shaking for 12 hr, chloroform was added to 1.0% (v/v), and shaken an additional 5-10 min. One ug/ml of bovine pancreatic DNase and 1.0 ug/ml of beef pancreatic RNase (U.S. Biochem. Corp., Cleveland, OH) were added in the presence of 10"3 M MgSO4 - 7H20 and shaken for 1 hr at 23 C. Bacterial debris was removed by centrifugation at 12,100 x g for 10 min (Sorvall RCZ-B) and the supernatant decanted and stored. Phages were precipitated with 10% (w/v) poly- ethylene glycol 6000 (PEG, J.T. Baker Chemical Co., Phillipsburg, New Jersey) in the prsence of 0.5 M NaCl with magnetic stirring for 60 min at 4 C (23).. The phages were pelleted by centrifugation at 12,100 x g for 20 min and the pellets resuspended in 5.0 ml of PPB. The phage solution was then subjected to a cycle of low speed (10 min at 3,020 x g in conical tubes) ~— high speed centrifugation (2 hr at 22K using a rotor SW30 in a Beckman Model L Ultracentrifuge). Following high speed centrifugation, supernatants were decanted and the pellets resuspended overnight in 5.0 m1 of PPB at 4 C then centrifuged for 10 min at 3,020 x g in conical tubes. The partially purified phage solution was then stored at 4 C over a drop of chloroform. 39 Further purification was obtained by layering 2.0 m1 of phage solution on liner lO-40% sucrose gradients (4, 7, 7, 7 ml), in 0.1 M potassium phosphate buffer pH 7.8, and centrifuged for 1 hr at 22 K in a Beckman SW 25.1 rotor. Phages were collected by withdrawing the band with a needle or in 1.0 ml fractions with an ISCO Model D Density Gradient Fractionator scanned with an ISCO Model UA-4 Absorbance Monitor at 254 nm. Fractions were diluted with 9.0 ml of PPB, assayed, and the fractions containing maximum phage infectivity centrifuged for 2 hr at 22K and the pellets resuspended in 2.0 ml of PPB. When greater purity was required, isopynic centrifugation was performed in 50% (w/v) cesium chloride (CsCl) for 18—24 hr at 37.5K using an SW50.1 rotor. Phage thermal inactivation and optimum growth temperature. One ml aliquots (ca. 107 plaque—forming- units (pfu)) of PEal(h), PEal(nh), and PEa7 suspended in PPB were added to thin-walled glass tubes (outside diameter 1.5 cm). The tubes were immersed in a water bath preheated and maintained (a 1.0 C) at 10 degree intervals of temperature ranging from 25965 C. After 10 min, the tubes were immediately placed in an ice bath preceeding assay for viable phage. Each phage isolate was tested twice with two replications per test. 40 Optimum temperatures for infectivity and growth of phages PEal(h) and PEa7 were determined over a range of 0-30 C. A 12 hr culture of E. amyZovora 110 rifr was 7 cfufimL. One ml aliquots were pipetted into tubes (3 tubes for diluted 1:10 in fresh NBGYE resulting in 7 x 10 each phage at each temperature) and placed in the incubators. After 30 min of acclimation, 0.1 ml of PEal(h) (12 x lO7 pfu/ml) and PEa7 (20 x 107 pfu/ml) were added to the tubes and incubated with shaking at 30-min intervals. Following 3 hr of incubation, a drop of chloroform was added to each tube and the tubes were stored at 10 C prior to assay. One-step growth and‘adSOrption'experiments. Onew step growth experiments were performed as described for phage PEal (17). The adsorption rates were determined using the chloroform method (1). One ml of phage suspension was added to 9.0 m1 of NBGYE containing a known number of cfu/ml and the number of plaque-forming—centers was determined. One—tenth m1 samples were diluted into 4.5 m1 of NBGYE containing 0.5 m1 of chloroform at 2-min intervals over a 20~min period. Electron microscopy. A drop of sucrose—gradientw purified phages PEal(h), PEal(nh), and PEa7 suspended in PPB at titers of 2 x 1010 10 10 pfu/ml, 3 x 10 pfu/ml and 1 x 10 pfu/ml, respectively, was placed on 41 parlodion, carbon—coated, copper grids (mesh 300). After 3 min the drop was removed with filter paper and a drop of 2.0% (w/v) ammonium molydate was placed on the grid for l min. The excess was removed with filter paper, the grids dried and examined in a Philips 300 transmission electron microscope operating at 60K volts. Serology. Bacteriophages PEal(h) and PEa7 were purified through two cycles of 10-40% linear sucrose gradients, suspended in PPB, and stored at 4 C. The 10 pfu/ml and 6 x 108 pfu/ml for titers were 7 x 10 PEal(h) and PEa7, respectively. Female, New Zealand white rabbits (3.2—3.6 kg) were injected at 7—day intervals with 2 ml of a 1:1 emulsified mixture of phage and Freund's complete adjuvant (Difco). The first two injections were given intramuscularly into the thighs while the third was given subcutaneously in the dorsal region. Weekly ear-bleedings (25 ml) were initiated two weeks after the final injection. Processed sera were stored frozen at -20 C. Antisera titers were determined by using 2—fold 3 dilutions in PPB containing 10' M NaCl. Homologous 7 pfu/ml, phages were added to a final titer of ca. 10 allowed to react for 10 min at 23 C then viable pfu/ml assayed. (A normal serum control was similarly treated. The reciprocal of the dilution at which no significant 42 decline in pfu could be detected was considered antiserum titer. Neutralization—rate experiments (1) were done with phages PEal(h), PEal(nh), and PEa7 using holologous and heterologous antisera. The remaining 11 phage strains were typed by diluting each to ca. 107 pfu/ml and mixing with PEal(h) antiserum (titer 100) and PEa7 antiserum (titer 40) for 10 min then assaying for viable pfu/ml. ‘Sedimentation coefficients. Sedimentation coefficients for phages PEal(h), PEal(nh), and PEa7 were determined by comparison to tobacco mosaic virus (TMV) essentially as outlined by Brakke (7). Ten-40% sucrose gradients (4, 7, 7, 7 ml), in 0.1 M potassium phosphate buffer pH 7.8, were allowed to become linear by setting at 4 C for 18 hr. One ml (O.D. A260 = 0.7) of virus suspension (in PPB) was layered on the gradients and centrifuged at 20K at 4 C in an SW25.1 rotor using a Beckman Model L Ultracentrifuge. Measurements, to i 0.5 mm, were made at 20-min intervals over a l40~min period by measuring from the meniscus of the gradient to the top of the virus band. TMV and PEal(h) were run twice while PEal(nh) and PEa7 were run once. Sedimentation distance versus time was plotted and sedimentation rates calculated for the viruses while moving from 7.5 mm to 17.5 mm in the gradients. Sedimentation coefficients were calculated 43 based on the sedimentation coefficient of TMV as 1878(12). Buoyant density. Phages PEal(h), PEal(nh), and PEa7 purified in sucrose gradients, were centrifuged to equilibrium using an SW50.1 rotor in a Beckman Model LZ-GSB Ultracentrifuge. Four-tenths ml of phage (2 x 1010 10 10 pfu/ml, 6 x 10 pfu/m1, and 2 x 10 pfu/ml, respectively) were layered on 4.5 ml of 40% (w/v) CsCl in distilled water. After 25 hr of centrifugation at 37.5K at 4 C, lO-drop fractions were collected from the bottom of the tubes. The density of each fraction at 23 C was determined by direct weighing (8) of 10 pl quantities using preweighed 10 pl disposable pipets. Fractions were dialysed for 12 hr against 0.1 M NaCl at 4 C, brought to 1.0 ml volume, assayed, and absorbance read at 260 mm. In the first experiment phages PEal(h) and PEal(nh) were found in the first fraction, therefore the experiment was repeated using 60% (w/v) CsCl. The results for phage PEa7 were from the first experiment while the results for PEal(h) and PEal(nh) were from the second experiment. RESULTS Isolation, plaque morphology, and host range. Fourteen phage strains from eight Michigan orchards were chosen because of their source of isolation and plaque morphology (Table l). The most common type of phage plaque was 2—3 mm in diameter surrounded by an expanding, translucent halo (Figure l-A). Times of initial halo development and rate of halo expansion varied among the phage-bacterial strain combinations. The second plaque type was 1-2 mm in diameter and lacked a halo (Figure lvB); it was never isolated from plant material but was commonly detected in halo type lysates. It was impossible to propagate the halo producing types in broth without the production of non—halo producing types even when the halo type phage had been single-plagued five times and sucrose gradient purified (Table 2). Non—halo producing phage were not found in halo plaques; however, when phage from halo plaques were incubated with llOrifr in NBGYE both halo and nonehalo plaques were detected (Table 3). The third plaque type, produced by phages PEa7 and PEalS, 44 4S ON\N\O .Omz _ Om». O i N HEOHOHE OO\N\O .8: mm» H .. m.O OHmmnH OO\NN\N .33 33 no» O i N EOOHOmm OQNNN .HuHHZmHOz mm» O i N EONHOOE OO\OH\O 60:23.3 on» O i N Evmomm ON\OH\O .OOOHHOO OOHHHOO mm» H u 0.0 News mu}. 5% .mo>ooH atom owunoHHm mm» m l N 2.3 comm ON\O 5m: 5263 OOOHaouaaNO me» O i N Evmmmm ON\OH\N .3: £380 Oosuom >qu2 mm» O .. N 2: «mum ON\OH\N .8: 53d OOHEOHHO me» O .. N HEOOmm EONOOE sou... OHSHHOO 0: N .. H EENOOE «QNH SO: .38 OeH O .. N EONOOE SHOE sod H.338 0: N t H Eczema OQNHO .33 3mm mm» O i N ECHOHE . mHOcHsHou _, , . . . Hasv Hoouo O mm>noH touanHm OHM: mcHocomxm Houoano osvnHm .L ouoHOmH coHHOHOmH mo mHoo can moousom >00H05QHOE movOHm mounnono somHAOHz Eonm coHUOHonH mo duct ocn .oOHSOm .mmoHoodHoE mquHm .mcHonum omnomoHHmuoom .H OHQOB Figure 1 (A to C). 46 Plaque morphologies of bacteriophages after 60 hr of incubation at 23 C with Erwinia amylovora 110 rifr as host. Phages were plated using 2.5 ml of 0.7% nutrient agar, 0.5% glucose, and 0.25% yeast extract for the top layer; the bottom layer consisted of 2.0% nutrient agar and 0.5% glucose. A) Phage PEal(h). B) Phage PEal(nh). C) Phage PEa7. Bar = 1.0 cm. 47 Table 2. Propagation of group I bacteriophages and the development of halo and non-halo plaques. Plaque—Forming—Units/ml vwv" ' r w Plaque Assay from Plague After 24 h incubation in NBGYE Isolate - H ~ - - - H -l- - ...... W- Halo .Nonehalo Halo Non-halo PEal(h) 27 x 106 n.d? 13 x 108 so x 106 PEal(nh) n.d. so x 105 n.d. 14 x 108 PEa2(h) 53 x 106 n.d. 13 x 108 70 x 106 PEa2(nh) n.d. 12 x 106 n.d. 12 x 108 PEa5(h) 70 x 106 n.d. 95 x 107 n.d. a n.d. = not detected. Table 3. Occurrence of halo and non—halo plaque-forming phages in halo plaques of phage PEal(h). Plaque—Forming-Units/ml Plaques Assay from plaques After 24 h incubation in NBGYE Halo Non-halo Halo Non-halo a 54 x 105 n.d.a 21 x 109 29 x 108 b 70 x 105 19 x 109 18 x 108 c 63 x 105 n.d. 26 x 109 80 x 108 a 87 x 105 22 x 109 75 x 108 e 68 x 105 n.d. 19 x 109 90 x 108 a n.d. 8 not detected. 48 was 0.5—1.0 mm in diameter with an expanding halo (Figure l-C). At least 12 hr of incubation at 27 C were required before this plaque type could be observed, whereas the PEal(h) plaque type was observed after 6 hr. Phages that produced plaques of the first and second types were placed in group I while phages producing plaques of the third type were placed in group II. Host range for the 14 phage strains was essentially limited to strains of E. amyZovora as was reported for PEal (l7) . Three strains (130, 150,, and 151) of the closely related, yellow saprophyte Erwinia herbicola (Lohnis) Dye (13) were lysed by PEa7 but by PEal(h) and PEal(nh) only if spotted at titers of ca. 109 pfu/m1. Efficiencies of plating for PEa7 (compared to llOrifr) were 0% for 130, 11.1% for 150, and 27.8% for 151. A weak area of lysis could be observed when a drop of 9 x 109 pfu/ml of PEa7 was spotted on strain 130 but not when lower titers were used. When group I phages were spotted on a mutant of llOrifr, resistant to PEal(h), the mutant was resistant to all group I phages but was lysed by group II phages. Phage growth and purification. NBGYE lysates of PEal(h) and PEa7 cleared only slightly even though 10 11 10 -10 pfu/m1 were produced. The purification 49 scheme yielded better recovery of group I phages than of group II phages (Tables 4, 5). .Centrifugation of phages PEal(h), PEal(nh), and PEa7 in linear 10-40% sucrose gradients for 1 hr at 22K resulted in a single, distinct band in the lower half of the gradient (Figure 2). Phages PEal(h) and PEal(nh) had maximum infec- tivity and absorbance in fraction 12, for PEa7 maximum infectivity and absorbance occurred in fraction 16. Electron microscopy showed the peaks at the top of the gradients to contain bacterial debris and empty virus particles. The ultraviolet adsorption spectra of Z-cycle, 10—40% sucrosewgradient purified preparations of phages PEal(h) and PEa7 are shown in Figure 3. Maximum infectivity and absorbance for PEal(h) and PEal(nh) were found in fraction 24 following 25 hr of isopynic centrifugation in 60% CsCl. The density of CsCl in this fraction was 1.53 g/cc (Figure 4). For phage PEa7 maximum infectivity and absorbance were found in fraction 6 following 25 hr of isopynic centrifugation in 40% (w/v) CsCl. The density of this fraction was 1.44 g/co (Figure 4). Thermal inactivation and optimum growth tempera— ture. Phages PEal(h) and PEal(nh) were completely inactivated following 10 min at 55 C while PEa7 was completely inactivated after 10 min at 65 C (Figure 5). Table 4. Purification and concentration of bacteriophage 50 PEal(h). Steps in Vol. Titer Total Recovery purification (ml) pfu/ml pfu (%) 1— Crude lysate 1000 1.6 x 1010 1.6 x 1013 100 2" After ”“3“ and 1000 1.6 x 1010 1.6 x 1013 100 RNase 3‘ “‘3’? PEG . 140 6.4 x 1010 8.9 x 1012 56 sedimentation 4" After 10‘40" .w 60 7.0 x 1010 4.2 x 1012 26 sucrose gradient 5- After 22x for 2 h 6 1.8 x 1011 1.1 x 1012 7 6' After C?“ and 2 5.0 x 1010 1.0 x 1011 0.6 dyalySis Table 5. Purification and concentration of bacteriophage PEa7. Steps in. Vol. Titer Total Recovery purification (m1) pfu/m1 pfu (%) 1- Crude lysate 1000 1.6 x 1010 1.6 x 1013 100 2.- After DNase and 1000 1.6 x 1010 1.6 X 1013 100 RNase 3" “ta”? PEG . 140 1.1 x 1010 1.4 x 1012 9 sedimentation 4" After 10'4“ . 60 7.0 x 108 4.2 x 1010 0.3 sucrose gradient 5- After 2K for2h 6 6.0 x 109 3.6 x 1010 0.2 5‘ After CSC1 and 2 5.0 x 109 1.0 x 1010 0.06 dialysis 51 HooVHomm too Aanomm momooQOHHouooo mo moHHmon coHuoucofiHtom OI x 1W nu ‘ 01 I n vs (V t~—-) In h 00— aOh 20¢“— mZO_hU<~= m—O—Q—N—O— a O Q N Ascw—cua Push-DPPbb-nbhpl-an )uuooz aauvauosav .OHQOHoOHm omOHUSO Nov 1 OH cH In 0‘ 0| x 189/an O In t-—~) I!) Is 00— tOh 20¢“. mZO_._.U<~: m—O—Q—N—O— a 0 Q N ill-b-b-h-bhp HHS—nus . D b E b D D ’ l '1 p O ' (—-)m009z aauvauosav N .n' .N ousmHm 52 L4_PE07 "00 r I er2- . V ' 775? SLO- 1 : 0 s5 3 ' ' “o H 08" 'I '- U K E» -' «505; 2 \ O a v: a. C O. < 25 2 4 6 810I2I4I6I8 FRACTIONS FROM TOP Figure 2 (cont'd). Sedimentation profile of bacteriophage PEa7 in 10 — 40% sucrose gradient. 53 ABSORBANCE Nbbin'm'sibo'too Figure 3. 1 l l .L l 1 l J L 1 220240 260 280 300 320 340 360 WAVE LENGTH (an) Ultraviolet adsorption spectra of 2-cycle, linear 10 - 40% sucrose gradient purified bacteriophages PEal(h) and PEa7. 54 onto AlISNBO (v—r) I090 momonaoHHouooo mo mmHuHmGOt ucomoon too mOHHmoHQ :oHuomDMHHucoo OHCHQOmH SOhhoo n IO to ”I In In to '9. .mucOHooum thHngo Eonoo CH Hochomm poo Aanomm .oz zo....o<¢... ON as n O. a. 2... .out O n p p - dab ow On as 00. On. CON 0 IO N con .00» ("-0) ‘OI X 1W / field [959‘ 99/0 ,AIIS'NSO (w—v) mm; on; mt; no; mug on; SOFhOm (o—o) mos: v .02 202.04”; 0. m. cm on no» l ¢:.oua H P D b D us On 0b 00. 0o. OON Oou can can .v ouomHm (“-0) ‘0) X 'IW/ nae 55 35° 'PEoT ‘ p I " L85 .. aoo - g « O I. I . _ a 250 - ii |i.o« O .9. '9 ii 5 ~9"65 3 x zoo - l: 8 .ed 3 II at ('55 >- 5; ' tt“ 7 ' :2 .g ISO-- 1: 5. m g 2 a in u. o “- IOO 7. I so ' 25 25 20 I5 IO 5 TOP FRACTION NO. BOTTOM Figure 4 (cont'd). Isopynic centrifugation profile and buoyant density of bacteriophage PEa7 in cesium chloride gradient. 56 8 I I I 1 I . PEal(h) I PEaIM‘ 7 b 0 950 7 6 .. II .J 2 5- r \ E 4 -_ ' O. 2 3 - ‘ o C \ d .J 2" \\ \ ‘ d ' i- \‘ \ \ “a L L 1 1 01‘ 25 35 45 55 65 TEMPERATURE(C°) Figure 5. Thermal inactivation of bacteriophages PEal(h), PEal(nh), PEa7. N.D. = not detected. 57 PEal(h) had an optimum growth temperature of 15- 27 C yielding approximately a loo-fold increase in pfu after 3 hr of incubation (Figure 6). At tempera— tures of 10 C or less only 3—4 pfu per pfu inoculated were produced, while at 30 C more than a 103 decrease in pfu occurred. All the other group I phage strains except PEal(nh) and PEalZ caused lysis when incubated at 30 C. Phage PEa7 grew~optima11y at 27 C yielding 9 pfu for each pfu inoculated (Figure 6). There was a net loss of pfu at temperatures of 15 C or less. One-step growth and adsorption‘rates. PEa7 had a latent period of approximately 100 min, a rise period of 35 min, and an average burst size of 8 pfu per productive cell compared to a 40 min latent period and an average burst size of 50 pfu per productive cell for PEal(h) (Figure 7). Ninety percent of PEal(h) adsorbed to E. amyZovora llOrifr within 10 min after 20 min were required to adsorb approximately 90% of PEa7 phages to the same bacterial strain (Figure 8). Contrasting results occurred when PEal(h) and PEa7 were adsorbed to a PEal(h)-resistant strain of llOrifr, E. amyZovora llOrifrpr (Figure 8). Similar results were also obtained using E. amylovora strains E8 and E9 (Figure 8). The adsorption rate constants, k, were calculated 58 '0: fi r I T r T r ‘3 II. a. Io:— /\ .- .— D Q I 5 I0 I. o “ v' s O ’.’a s» I- O ;_ 3 ’v’ 2 “o.’ 3: x" I O '0‘. ” c- .J . ’I e x >' ~2i / IO - I H II. > I O o .3 9 I0 - . " l < 0-OPEoIIhI - a: 0--PE..7 '0‘ l 1 L L i L s O 5 l0 I5 20 25 30 TEMPERATURE (0‘) Figure 6. Optimum growth temperatures of bacterio- phages PEal(h) and PEa7. 59 3 r I I t ‘l I I I T ‘0 PEal(h)-— PEa7--- J" m“ I i I 4 I 2 ,’ \ I D -‘ ~ I’ Io’- - T 1 1 '02 1 i l I 1 1 i l 1 0 I5 30 45 60 75 90 I05 IZO I35 I50 MINUTES Figure 7. One-step growth curves for bacteriophages PEal(h) and PEa7. Bars indicate range of the samples. 60 I r I 1 r r r 1 F I T. 00 . x,“ ““0 PHAGE peat») : : ”Own—HQ°:°A°~ H . ‘ .WMM: Lg '0 :' \ '2 .4 : \\\ ° 1 I : \\ 1 O- . \ 8 H O L §3 ‘°‘ _‘ .. \. m S I .. ' > :00 PHAGE 95.7 1 3* I ‘8—0—0—0‘0 \ -' I0 _ ‘°\ . . P \ \. -I In \ q I: \°-.- N1 Flo—0E8 OW—I-o‘o d ..__."o”‘;>ACAPSULATED \\6 2:2 figm> CAPSULATED ' I . 4 . O 2 ‘4 6 8 ICIIZ I4 I6 )8 20 MINUTES Figure 8. Adsorption curves of bacteriophages PEal(h) and PEa7 to capsulated and acapsulated strains of Erwinia amylovora. 61 for PEa7 with different strains of E. amyZovora, the results are summarized in Table 6. Phage PEal(h) adsorbed rapidly to capsulated but not acapsulated strains of E. amyZovora, while PEa7 adsorbed rapidly. to acapsulated but slowly to capsulated strains (Figure 8 and Table 6). Electron microscopy. Electron microscopy showed PEal(h) to be polyhedral, probably iscoahedral, approximately 60 nm in diameter with a spike-like tail structure (Figure 9—A) thus placing it in Bradley’s group C (6). Although not shown phage PEal(nh) was morphologically indistinguishable from PEal(h). PEa7 (Figure 9-B) had an octahedral head approximately 75nm in diameter with a rigid, non-contractile, striated tail approximately 135 nm long, thus fitting the criteria of Bradley's group B (6). Serology. Serological neutralization results of phages PEal(h), PEal(nh), and PEa7 are shown in Figure 10. PEal(h) antiserum, at a titer of 1000, neutralized 97% of its homologous phage within 10 min having a velocity constant K = 321 per min. Hetero— logous phages PEal(nh) and PEa7 were 94% neutralized within 5 min, K = 597 per min, and 64% neutralized 'within 30 min, approximate K = 80 per min, respectively PEa7 antiserum, titer 100, neutralized 93% of its _62 .ooHHom oaHu was» mcHHst ponuomoo no: to: woman on» no ammiom NH toHHom coHunnsocH cHEtom on» «0 coHuncHsHou on» an no commence to: woman on» no ammiom £0Hn3 an oEHu may no couoHsOHoo \o .ucoumcoo :oHumHOmoo may OHOHsOHoo ou toms moonsm.conNOmoo mo ucoouom.\a .onspoooum xcH oHocH on» >3 oouoouoo.\o OHIQH x hm.m up home + m m miOH x hv.N mm t om homo i m m miOH x NO.H mm 1 cm home i HMHH OHH x . i o HM OHIOH hm O om mm n mm + Hm. OHH mIOH x Om.H mm 1 cm Aanomm + m m OHIOH x ON.O om HovHomm t m m OHIOH x «O.N OO HSHOOO .. HOHOHH OHH miOH x mo.N am 1 cm HovHomm + HMHH OHH com o o cameo. cacao mes . 55H... 3: .a O H H t6 H .O pooumcou coHumHomo¢\o tooHOmom omozm w\e mcHouum woman can HoHHouoom .caononso cereaam mo moHoHum toeoHomooon ton UOHOHsmmoo ou homo poo HagHomm homage mo coHuanom04 .O oHnnB 63 Figure 9 (A and B). Electron micrographs of Erwinia amyZovora bacteriophages. A) Bacteriophage PEal(h), a polyhed- ral phage with a spike-like tail. B) Bacteriophage PEa7 with an octahedral head and a rigid, striated tail. IOO, -. : ' ; * ' ’ ; \ ‘uu..x::::::--w 1 M‘\'\ “.m:: '~M.: DJ '0: ‘3 L49 : :i: t Z 0. ::::3H‘ , UJ I \“h~\‘§ 1 a! MI: 4 b \. \.: > P \. 4 3 .I .- \e i 1 ’ Pia IIIsIAs I000 : ' P507 AsIOO ‘ ornnml ’ ‘ PEal(nh) 4 Pic? .OI '. . . . . 5 IO IS 20 25 30 MINUTES Figure 10. Antiphage sera neutralization curves for bacteriophages PEal(h), PEal(nh), PEa7. 65 homologous phage within 5 min, K = 56 per min. For heterologous phages PEal(h) and PEal(nh) 64% and 40%, respectively were neutralized after 30 min giving an approximate K = 8 per min for both. Serological typing of the 11 remaining strains resulted in the neutralization of all group I phages with PEal(h) antiserum but not with PEa7 antiserum; PEa7 antiserum neutralized only group II phages (Table 7). ‘Sedimentation coefficients. Estimates of sedimentation coefficients for PEal(h) and PEal(nh) were 5668 and 1037s for PEa7 (Figure 11, Table 8). 66 Table 7. Antiphage sera neutralization of the 14 Erwénia amyZovora bacteriophage strains. ...................... Percent Viable Phagea/ Phage Strains , PEal(h) antiserum .PEa? antiserum PEal(h) l 98 PEal(nh) l 97 PEa2(h) l 99 PEa2(nh) l 98 PEa3(h) l 96 PEa4(h) l 94 PEa5(h) 1 98 PEa6(h) l 92 PEa7 97 2 PEa8(h) l 88 PEa12(h) l 86 PEa13(h) l 99 PEalS 99 S PEa16(h) 1 9O a/Thage (ca. 107 pfu/ml) in 0.02 M potassium phosphate buffer plus 10‘3 M NaCl, pH 6.8, were mixed with the antiserum and allowed to react at 23 C for 10 min, diluted, and plated with bacterial host 110 rifr. The percent viable phage was determined by comparing the number of pfu propr to addition of the antiserum to the number of pfu following exposure to the antiserum, PEal(h) antiserum was used at a titer of 100 while PEa7 antiserum was used at a titer of 40. 67 T I I T f T "a? A PIC 10!) ' Flo Huh) 0 ‘0 - TMV o 9 fl I- / /q; A DEPTH(mm) or swmmunou a e f , , \a\ l \L . . l l L l L 4' CO 80 100 120 H. MINUTES OF CENTRIFUGATION Figure 11. Sedimentation curves used to calculate the rate of sedimentation for sedimentation coefficient determinations. 68 Table 8. Sedimentation coefficient calculations for phages PEal(h), PEal(nh), and PEa7. a/ Virion. Time Rate of Sedimentation' Ratio of Phage 820 w (min) (mm/min) Rate to TMV TMV 88.7 0.1127 ----- 187 S PEal(h) 29.3 0.3413 3.028 566 S PEal(nh) 29.3 0.3413 3.028 566 S PEa7 16.0 0.6250 5.546 1037 S a/Time for the virus bands to move from 7.5 mm to 17.5 mm in the gradients. DISCUSSION Based on the biological, chemical, and physical data these E. amylovora phages were placed in two groups. Group I was represented by PEal(h) while group II was represented by PEa7. Except for plaque morphology, the data indicated that halo and non-halo producing phages of group I were identical. Several other phage-bacterial combinations result in the production of an expanding, translucent halo which was due to the enzymatic removal of the bacterial capsule (2,4,11,22). A similar phenomenon may cause the halo surrounding the plaque produced by the E. amyZovora phages. Failure of PEal(nh)-type phages to produce a halo may be due to nonsynthesis of the enzyme or a situation similar to a Pseudomonas aeruginosa phage—2 mutant, PDPI, in which the enzyme is produced but is not enzymatically active (4) . The smaller plaque produced by PEa7 compared to that of PEal(h) may be due to the longer latent period, smaller burst size, slower adsorption rate, and differences in phage morphology, all of which affect 69 70 plaque morphology (1). Specificity of the group I phages for capsulated strains of E. amylovora was similar to that reproted for the Escherichia coli K phages (18) and KZebsieZZa phages (11,15,16). These phages were specific to capsulated strains because their attachment site was located on the bacterial capsule. Although up to 65% of PEal(h) absorbed to the acapsulated strains of E. amyZovora no increase in titer occurred. This adsorption may have been due to the presence of capsular material not detected by the India ink stain (3). A second and more likely explanation is that infection was blocked after capsule recognition. This was hypothesized to be the case with KZebsieZZa phage 7 (19). In comparison to PEal(h), phage PEa7 attached more readily to acapsulated than capsulated strains of E. amylovora suggesting that the receptors for PEa7 are located on the cell wall beneath the capsule. These data would support and explain Billing's observations that sensitivity for different phage types was related to the presence of a bacterial capsule (5). As the data indicated, group I phages have their receptors on the bacterial capsule, thus this is the first substantiated report of Kaphages infecting phytopathogenic bacteria and the presence of 71 such phages may be rather common in Enterobacteriaceae as suggested by Stirm (18). The lower percentage of recovery during purification of PEa7 compared to PEal(h) may be related to the more fragile tail structure of PEa7 which is probably necessary for attachment site recognition. It is also possible that during purification the capsular degrading factor was lost which may aid the phage particle in reaching its receptor (19,20). That reduced infectivity was the result of the physical disruption of the intact phage particles may be gained by measuring protein absorbance versus infectivity. PEal(h) had an O.D. 10 (280 nm) of 0.30 and an infectivity of 7 x 10 pfu/ml with an A 260/280 ratio of 1.51. PEa7 had an O.D. (280 nm) of 0.63 but an infectivity of only 6 x 108 pfu/ml with an A 260/280 ratio of 1.44 (Figure 3). This suggests that the protein content was greater for the PEa7 phage suspension than for PEal(h) yet the infectivity of PEa7 was approximately loo-fold less. Significant amounts of bacterial protein were eliminated since the phages had passed through 2-cycles of sucrose gradient centrifugation; furthermore, after only l-cycle no bacterial proteinaceous material such as flagella were observed in electron micrographs. Thus, PEa7 could be purified using the PEG method but 72 its infectivity was reduced. Phage PEal(h) had a wide temperature range for optimum growth compared to PEa7; however, when in- cubated at 30 C more than a 1000-fold decrease in pfu/ml occurred (Figure 6). This was not the result of physical thermal inactivation since the phage could tolerate heating for 10 min at 45 C without a significant drop in titer (Figure 5). Phages are known to adsorb to and inject their nucleic acid into their bacterial hosts. The phage is then unable to produce progeny because a protein necessary for its growth is nonfunctional at the higher temperature even though its bacterial host grows normally at that temperature (21). This situation may exist for phages PEal(h), PEal(nh), and PEa12(h). The temperature sensitivity of the non-halo form of PEal(h), PEal(nh), adds evidence that PEal(h) and PEal(nh) are very similar even though PEal(nh) does not produce a halo-plaque. Temperatures produced a different effect on the growth of PEa7 than PEal(h) (Figure 6). This indicated the the multi— plication processes of these two phage types differed. The differences intheir one—step growth (Figure 7) also supports this hypothesis. Calculation of the velocity constant, K, of antiphage sera neutralization is valid only over a 73 limited range of inactivation, usually 90-99 percent of the phage, and cannot be validly calculated outside this range (1). Thus the K values for PEal(h) antiserum with phage PEa7 and PEa7 antiserum with phages PEal(h) and PEal(nh) cannot be validly calculated; however, for purposes of comparison, estimates have been made. This results in higher K-values which suggest greater serological relatedness than actually exist. Along with the other similarities between PEal(h) and PEal(nh) was the ability of PEal(h) antiserum to neutralize homologous phage, PEal(h), K was 321 per minute; however, for heterologous phage, PEal(nh), it was 597 per minute. Antiserum prepared to other phages that produced halo plaques was shown to also neutralize both the phage and the enzyme that caused the halo (2). Lysis of host cells by PEal(nh) did not result in detectable amounts of capsule degrading factor as did lysis by PEal(h) (Section III of dissertation). The lower K for the homologous phage, PEal(h), than for heterologous phage, PEal(nh), may have resulted from competition between the virion and the capsule degrading factor for antibody. The data from the serological typing experiments (Table 7) indicated that the 14 phage strains could be placed in two groups which correlated with group I and_group 74 II plaque types. The sedimentation coefficients of PEal(h) and PEal(nh) were similar to those of the T-odd phages while that of PEa7 was similar to the T-even phages (9). This is also true for phage morphology; PEal(h) and PEal(nh) are morphologically similar to coliphage T3 while PEa7 is similar to the tailed T-even phages (6). .Group I phages were placed in Bradley's morph— ological group C, while group II phages were placed in Group B; the phages in groups B and C contain double— stranded DNA (6). Thus, these E. amyZovora phages are probably double—stranded DNA phages as was the E. amylovora phage, 81, characterized by Erskine (10). These phages are of interest for several reasons: 1) they are the first E. amylovora bacteriophages isolated from aerial structures of plants susceptible to E. amyZovora, 2) this is the first extensive characterization of more than one strain of E.amy20vora bacteriophage, 3) the phages produce an expanding, translucent halo surrounding the true plaque, and 4) the group I phages are specific for capsulated strains of E. amylovora. LITERATURE CITED ADAMS, M. H. 1959. Bacteriophages. Interscience Publishers, New~York, NY 592 p. ADAMS, M. H., and B. H. PARK. 1956. An enzyme produced by a phage-host cells system III The properties of the polysaccharide depolymerase. Virology 2:719-736. BAILEY, R. W., and E. G. Scott. 1974. Diagnostic microbiology. 4th ed. The C. V. Mosby Company, St. Louis. 414 p. BARTELL, P. F. 1977. Localization and functional role of the Pseudomonas bacteriophage 2 — associated depolymerase. In "Microbiology" (D. Schlessinger, ed.), ppT—134-137. American Society of Microbiology, Washington, D.C. BILLING, E. 1960. An association between capsulation and phage sensitivity in Erwinia amylovora. Nature 186:819—820. BRADLEY, E. D. 1967. Ultrastructure of bacterio~ phages and bacteriocins. Bacteriol. Rev. 31: 230—314. BRAKKE, M. K. 1958. Estimation of sedimentation constants of viruses by densityegradient centrifugation. Virology 6:96-1l4. BRAKKE, M. K. 1967. Density—gradient centrifugan tion. In ”Methods in virology" (K. Maramorosch and H. Koprowski, eds.), Vol. II, pp. 93w118. Academic Press, New York, NY. 692 p. CUMMINGS, D. J. 1964. Sedimentation and biological properties of T-phages of Escherichia coli. Virology 23:408-418. 75 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 76 ERSKINE, J. M. 1973. Characteristics of Erwinia amylovora bacteriophage and its possible role in the epidemiology of'fire blight. Can. J. Microbiol. 19:837-845. HUMPHRIES, J. C. 1948. Enzymic activity of bacteriophage-culture lysates I. A capsule lysin active against KZebsieZZa pneumoniae type A. J. Bacteriol. 56:683-693. LAUFFER, M. A. 1944. The influence of concentra— tion upon the sedimentation rate of tobacco mosaic virus. J. Amer. Chem. Soc. 66:1195- 1201. LELLIOTT, R. A. 1974. Bergey's manual of determinative bacteriology. R. E. Buchanan and N. E. Gibbons, eds., 8th ed. The Williams & Wilkins Co., Baltimore, MD. 1268;» OKABE, N., and GOTO, M. 1963. Bacteriophages of plant pathogens. Annu. Rev. Phytopathol. 1: 397—418. PARK, B. H. 1956. An enzyme produced by a phage- host cell system. I. The properties of a KZebsieZZa phage. Virology 2:711-718. RAKIETEN, M. L., A. H. EGGERTH, and T. L. RAKIETEN. 1940. Studies with bacteriophages active against mucoid strains of bacteria. J. Bacteriol. 40:529—545. RITCHIE, D. F., and E. J. KLOS.. 1977. Isolation of Erwinia amchvora bacteriophage from aerial parts of apple trees. Phytopathology 67:101-104. STIRM, S. 1968. Escherichia coli K bacterio~ phages I. Isolation and introductory characterization of five Escherichia coli K bacteriophages. J. Virol. 2:1107-1114. STIRM, S., and E. FREUND-MOLBERT. 1971. Escherichia coli capsule bacteriophages. II. Morphology. J. Virol. 8:330-342. STIRM, S., W. BESSLER, F. FEHMEL, and E. FREUND— MOLBERT. 1971. Bacteriophage particles with endoeglycosidase activity. J. Virol. 8:3439 346. 77 21. STENT, G. S. 1963. Molecular biology of bacterial viruses. W} H. Freeman and Company, San Francisco. 474 p. 22. SUTHERLAND, I. W., and J. F. WILKINSON. 1965. Depolymerases for bacterial expolysaccharides obtained from phage—infected bacteria. J. Gen. Microbiol. 39:373-383. 23. YAMAMOTO, K. R., B. M. ALBERTS, R. BENZINGER, L. LAWHORNE, and G. TREIBER. 1970. Rapid bacteriophage sedimentation in the presence of polyethylene glycol and its application to large-scale virus purification. Virology 40:734—744. PART III INTERACTION OF ERWINIA AMYLOVORA WITH ITS BACTERIOPHAGE INTRODUCT ION Bacteriophages of phytopathogenic bacteria have been known since the 1920‘s (11,36). Three reviews dealing with these phages have been written since 1963 (11,36,48). A number of these phages have been extensively characterized, and several show unusual morphologies or biochemical properties (29,30,33,47, 48). Because of the impressive destruction of bacteria by phage in the laboratory, the temptation has always been to test the therapeutic effect of phages. Yet, no substantiated evidence exists where a pathogen causing an epidemic disease has been controlled by a virulent phage under natural conditions. Several attempts to control phytopathogenic bacteria through the use of phages have been unsuccessful (11,36,48). It was thought that the influence of phage on a bacterial population was limited to the elimination of the sensitive cells by virulent phages. Phage have now been shown to control or influence the virulence as well as control some of the other phenotypic 78 79 characteristics of their bacterial host via lysogenic conversion (6,14,20,21,27,32,46) or transduction (53). Many of these systems have been extensively studied; however, there are few cases where similar situations with phytopathogenic bacteria and their phages have been reported and extensively studied (12,15,16,l7,52). When strains of Erwinia amyZovora were mixed with phage of the PEal(h) type, the following phenomena occurred: development of a spreading, translucent halo surrounding the true plaque; alteration of colony morphology; high frequency of phage resistant survivorS' which easily reverted to phage sensitivity; and a delay in symptom development. This section deals with the description and explanation of these phenomena and the possible involvement of phage in fire blight epidemiology. MATERIALS AND METHODS Media. For the growth of bacteria on solid medium 2.0% nutrient agar (Difco) with 0.5% glucose, pH 6.5, was used (NAG medium). Broth medium was composed of 0.8% nutrient broth (Difco), 0.5% glucose, and 0.25% yeast extract, pH 6.5 (NBGYE medium). Where the agar overlay method (1) was used the bottom layer consisted of 12—15 ml of NAG and the top layer consisted of 2.5 ml of 0.7% nutrient agar, 0.5% glucose, and 0.25% yeast extract, pH 6.5. The growth of E. amyZovora in inoculated apple seedlings was monitored with TTN medium (42) and NAG to which 50 ug/ml of rifampin (NAG/rif) had been added after autoclaving. All dilutions, resuspensions, and inoculations of phages and bacteria were done in 0.02 M potassium phosphate buffer, pH 6.8 (PPB). "Selectio “of Erwinia‘amylovcra‘mutants. Spontanea ous mutants resistant to the antibiotic, rifampin (49) were selected from nine pure, pathogenic strains of E. amylovora. Bacteria from three to five colonies of each strain were streaked on NAG plates. After 24 hr 80 81 of incubation at 27 C, 2.0 ml of sterile PPB were added to each plate and the bacteria resuspended with a sterile, glass rod to make a slurry containing lolqlflll colony-forming—units (cfu)/ml. One-tenth ml of this slurry was plated on NAG rif plates and incubated at 27 C for 48 hr. Mutation frequencies were approximate- ly l x 10-9. Two of the fastest growing colonies were selected for each strain, resuspended in PPB, streaked on NAG, incubated for 48 hr, and representative colonies tested for rifampin resistance (rifr) on NAG rif. Phage-resistant mutants (pr) of the E. amylovora rifr strains were selected to phage PEal(h). One ml (ca. 3 x 108 cfu) of a 12 hr old broth culture of E. amylovora was added to 1.0 m1 (ca. 4 x 108 pfu) of phage. Five minutes were allowed for phage adsorption, dilutions of 10-2, 10-4, and 10"6 were made, and 0.1 ml of each dilution was spread over the surface of NAG plates over which had previously been spread 0.1 ml (ca 1 x 109 pfu/ml) of a chloroform, filter—sterilized phage suspension. Following 48 hr incubation at 23 C, 3-5 colonies from each strain were resuspended in PPB and streaked on NAG. Cultures were tested for phage and rifampin resistance and stored on nutrient agar at 4 C. 82 Expandingj‘translucent halo. Plaque and halo development were studied using the agar overlay method (1). To test the number of phagevE. amyZovcra strain combinations that resulted in halo formation, 13 phage strains and 20 E. amyZovora strains were used. One— tenth m1 of ca. 2 x 109 cfu/ml of each bacterial strain was added to top agar and layered over the bottom layer, after solidification a loopful of phage suspension (ca. 104 pfu) was spotted on the plates and incubated at 27 C for 36 hr. Effect of temperature'on the development and expansion of the halo. Erwinia amyZovora 110 rifr and phage PEal(h) were plated such that ca. 25 plaques per plate developed. The plates were incubated at 27 C for 6 hr, at which time plaques were first visible, the plates were then transferred to incubators set at 0, 10, 15, 23, 27, and 35 C air temperatures. The diameters of five plaques, including the halo, per plate were measured to i 0.1 mm at specified time intervals. The effect of temperature on the expansion of established haloes was tested in a similar manner; however, haloes were allowed to develop for 18 hr at 27 C prior to transfer to the desired temperatures. Diffusion of the phage from the plaques. Three, 24 hr old plaques produced by phages PEal(h) and 83 PEal(nh) were sampled for pfu. The points of sterile toothpicks were inserted into the true plaque and 2-3 mm outside of the plaque prior to halo development then dropped into 1.0 ml of PPB and assayed for phage. Plates were incubated an additional two days during which time the halo extended through the previously sampled areas. Samples were taken again from these areas and outside of the halo as described. '.Alteredcolony morphology and'sensitivity to ‘ph§g_. Bacterial colony morphology was studied using NAG plates. Phage sensitivity was determined by the agar overlay technique; test bacteria were added to the top agar. After solidification diluted phage solutions were spotted on the agar surface. The plates were incubated at 27 C for 24 hr and observed for lysis. The ability of phage to increase in titer in a bacterial culture was also used as a test for phage sensitivity. "Stability of phage resistance‘and'pr‘colony ‘ characteristics. Ten single-colony subcultures of E. amylovcra 110 rifr were selected. These cultures were incubated for 14 hr in NBGYE then diluted and plated on NAG and NAG over which 0.1 ml of a sterile sclution of phage PEal(h) had been spread. Plates were incubated for 48 hr at 23 C. The number of colonies which grew on NAG and NAG + PEal(h) were 84 recorded. Three colonies from four of the NAG + PEalOU plates were transferred to NBGYE and incubated on a reciprocal shaker for 12 hr then plated on NAG for pr and wild type (w.t.) colonies as well as tested for phage sensitivity. Effect of temperature on the deveIOpment of p? 9 colony characteristics. One—tenth m1 (2 x 10 pfu/ml) of PEal(h) was spread over the surface of NAG plates. 6 dilution of an 18 hr NBGYE One-tenth ml of a 10— culture of E. amyZovora 110 rifr were spread over the surface and the plates incubated for a minimum of 48 hr at 10, 15, 23, 27, 30 and 35 C. ' Interaction of PEal(h)‘and'Erwinia‘amylovora llO rifrin broth‘and the development"of‘pr'colony‘char— acteristics. One ml of an 18 hr NBGYE culture of E. amyZovora 110 rifr was added to lZS-ml flasks of 25 m1 of fresh NBGYE and incubated at 1 hr. Phage PEal(h) was then added to the 110 rifr cultures at multiplicities—of—infection (m.o.i.) of 0.1, 1.0, 10.0, and 100.0. The cultures were incubated an. additional 1 hr, diluted, plated on NAG, incubated at 23 C for 48 hr, and the number of pr and w.t. colonies counted. ' Effect of PEal(h)'antiserum on the development of .Pr colony characteristics. One-tenth ml of PEal(h) antiserum, diluted 1:10, was spread over the surface 85 of NAG plates, phage PEal(h) and E. amchvcra were then spread over this surface. The plates were incubated for 48 hr at 23 C at which time pr and w.t. colonies were counted. Agar diffusible substance. A test similar to that described by Rakienten ct aZ.(40) was used to determine whether a colony morphology alteration could be caused by an agar diffusible substance. One loopful of a NBGYE culture of 110 rifr was spotted in the center of an NAG plate, approximately 10. cm from this spot and surrounding it were placed four additional spots of 110 rifr. On the center spot was placed a loopful of phage PEal(h) suspension (ca. 108 pfu/ml) and the plate incubated at 23 C and observed over a lOeday period for changes in colony morphology. "Effect of phageglysates‘on'mature‘lawns‘of Erwinia amylovora. Phage require a metabolically active host for multiplication (1) thus once the bacterial lawn of the agar overlay is more than 18-241n: old plaques will not develop nor continue to expand. If plaque—like zones do occur or plaques continue to spread, then some factor other than phage multiplica- tion is involved. To test for such effects produced by lysates of phages PEal(h), PEal(nh), and PEa7, mature lawns (lawns 2 days or older) of E. amyZovora 110 rifr were used. A spot test similar to that 86 described by Adams and Park (2) was used to test for these effects and to determine the titer of such a substance. Effect of phage lysateS'on‘the Erwinia amylpvora ‘capsule. One-m1 aliquots of a 48 hr old NBGYE culture of 110 rifr were pipetted into sterile test tubes, India ink stains were (5) made, and immediately examined under phase contrast for the presence of a capsule. One ml quantities of chloroform-sterilized lysates of phages PEal(h), PEal(nh), PEa7, and E. amyZovora 110 rifr were added to the NBGYE cultures of 110 rifr. India ink stains were made and examined at 5—min intervals. This method made it possible to directly observe the loss of bacterial capsules and to estimate the rate of capsule degradation. ‘ Serological relationship'between‘pr and wild type Erwinia amyZovora. Antiserum to E. amyZovora 110 rifr was prepared in a rabbit according to the method outlined by Allan and Kelman (3). Double diffusion tests with Ouchterlony plates (38) were done using NBGYE cultures of 110 rifr and 110 rifrpr which had 9 cfu/ml been pelleted and resuspended in PPB to ca. 10 and steamed for 1 hr. ‘ Mutation frequencies of phage resistance. The percent of E. amchvora cells resistant to phages 87 PEal(h), PEal(nh), and PEa7, after growth for 18 hr in NBGYE was determined for 20 E; amylovora strains. Ones tenth m1 of chloroform, filter—sterilized phage lysates (ca. 108 pfu/m1) were spread over the surface of NAG plates. Dilutions of 10’2, 10—4, and 10"6 of the E. amyZovora strains were made and 0.1 m1 aliquots spread over the surface of NAG + phage and NAG plates. After incubation for 48 hr at 23 C the number of colonies which grew~on NAG and NAG + phage were counted and the percent of E. amylovcra survivors calculated. Fluctuation test. The fluctuation test developed by Luria and Delbruck (35) was used to determine whether the resistance to phage PEal(h) was the result of a spontaneous mutation or an adaptation. Two strains of E. amchvora, 110 rifr and 118 w.t., were tested with phage PEal(h) as outlined by Braun (9) . ‘ TeSt'for lysogeny. Temperate phages can often be detected by one of several methods: a low percentage-may be spontaneously released, or inducing agents may be used such as ultraviolet light or the antibiotic mitomycin C (4,37). Several of the stable pr strains of E. amylovora were tested for the production of phage using these methods. Growth and'effect‘of'phage'PEal(h)'on‘Erwinia amylovora'lTO'rifr. Ten ml of an 18 hr old NBGYE 88 culture E. amylovora 100 rifr (ca. 109 cfu/ml) were added to each of two flasks of 100 m1 of fresh NBGYE and incubated an additional 30 min. Bacteria were assayed for the presence of a capsule using the India ink stain (5) and the number of cfu/ml determined by plating on NAG. To one of the flasks was added 1.0 ml of approximately 109 pfu/ml and the actual number of pfu/m1 immediately assayed. At 10 min intervals, starting at 10 min after the addition of PEal(h), the absorbance at 525 nm was read. The presence or absence of a bacterial capsule was determined, 1.0 ml of the culture was added to tubes containing 0.1 ml of chloroform and saved for capsule degrading factor assay, and the number of cfu and pfu/ml were determined by plating on NAG and using the agar overlay method, respectively. The presence of a capsule degrading factor was determined by the spot method (2) on mature lawns of 110 riff. Once the fractions contain- ing the factor were determined these fractions were diluted 2—fold and the reciprocal of the greatest dilution exhibiting the activity was taken as the titer. “§ymptom‘deve10pment by‘ErwiniaiamyZovcra. The effect of phage PEal(h) on symptom development of E. amyZovcra was tested using open pollinated 89 Jonathan apple seedlings (41). The bacteria were grown for 18-24 hr on NAG, resuspended in PPB, standardized to 0.30 O.D. at 525 um (ca. 108 cfu/ml), and diluted 1:10. A sterile, disposable syringe with a 25-gauge needle was used to wound and inoculate the seedlings. The needle was pushed through the hypocotyl, withdrawn, and a droplet of inoculum placed on the wound. Droplet size could be controlled by using the plunger; the bacterial concentration applied was ca. 106 cfu/droplet. Nine E. amylovora strains were used: wild types 105, 110, 112, 113, 115, 118, 121, 131, and 134 as well as rifampin and phage resistant mutants of these strains. Virulence was determined by the period of time required to produce a droplet of bacterial exudate at the point of inoculation. Several types of experi- ments were done. Seedlings were inoculated with wild type, rifampin resistant, and phage resistant strains of E. amyZovora, and the rate of symptom development was monitored. Erwinia amchvora strains and phage PEal(h) were mixed at an m.o.i. of 1.0, 10.0, and 100.0 and inoculated for 10, 15, or 30 minutes, then inoculated into the seedlings. Four to 12 seedlings per bacterial strain were used. A third experiment consisted of monitoring the symptom development and growth of three E. amyihvora strains, 105, 110, and 90 134 plus their rifampin and phage resistant mutants. The seedlings were inoculated and the bacterial growth was monitored by removing a 1.0 cm section, centered on the point of inoculation, from each seedling. Each section was crushed in PPB with a sterile glass rod, diluted, and assayed on TTN or NAG rif plates. Each sample period consisted of three seedlings per bacterial strain. «Hypersensitive reaction'in tobacco. Some plant pathogenic bacteria, including E. amyZovora, induce a hypersensitive reaction in tobacco leaves (28). To determine whether the mutation to phage resistance affected the hypersensitive response by the tobacco leaf, seven wild type, rifampin resistant, and phage 7 cfu/ml resistant strains were standardized to ca. 10 and infiltrated into tobacco leaves as described by Klement (28). RESULTS Expanding, translucent halo. One of the major characteristics of these E. amylovora phages, particularly the PEal(h) type, was the development of a continually expanding, translucent halo (Figure 1). The true plaque did not increase in diameter after 18 hr but the translucent halo surrounding the plaque' continued to expand. Halo formation was a common occurrence following the lysis of E. amyZovora by most of the phages (Table l). Exceptions were phage PEal(nh) which did not produce a halo in combination with any of the E. amylcvora strains and bacterial strains 104 and 119 which did not result in a halo with any of the phage strains. ' Effect of temperature on the development and expansion of the halo. The halo surrounding the plaque produced by PEal(h) failed to develop when incubated at 0 and 35 C (Figure Z-A). Once the halo was initiated it expanded most rapidly when incubated at 35 C (Figure 2—B). There was an increase in the rate of halo development and expansion as the incubation 91 'frfi N2hr Figure 1. 92 . . C . e. . ' C . . . 60hr nit-h . '. .-‘ \‘ Q' . 1‘! leht Expanding, translucent halo associated with the plaque formed by bacteriophage PEal(h) grown on Erwinia amylovora 110 riffi Time is hours of incubation at 23 C. Bar = 1.0 cm. 93 Table l. Phage - Erwinia amylovora combinations which resulted in halo formation. Phage strains (PEa) E- amylovora Strains 1(h) 1(nh) 2' 3, 4 s 6 7 8 12 13 15 16 104 - -----""‘“"“" 105 + — + -+ + -+ + -+ + + +- -+ + 110 + - + -+ + -+ + -+ + + 4- -+ + 112 + - + -+ + -+ + -+ + + 4- -+ + 113 + - + -+ + -+ + -+ + + +- -+ + 114 + - + 4- + 4- + +- + + +- -+ + 115 + — + -t + -+ + -+ + + +- 't + 117' + - + + + + + + + +- +’ + + 118 + - + -+ + -+ + -+ + + 4- -+ + 119 - -----""‘"""" 121 + — + -+ + -+ + -+ + + +- -+ + 122 + - + -+ + -+ + -+ + + 4- '+ + 125 + - + -t + -+ + -+ + + +- -+ + 126 + - + -+ + -+ + -+ + + +- -+ + 123 + - + -+ + -F + -+ + + +- -t + 133 + — + -+ + -+ + -+ + + +- -F + 134 + - + -+ + -+ + -+ + + +- -+ + 135 + - + -+ + -+ + -+ + + +- -t + 137 + - + -+ + -+ + -+ + + +- «t + 133 .+ - + + 1+ + + + + +- + + + All the E. amylovora strains were lysed by the 13 phage strains but only the combinations marked with "+" resulted in halo formation. 94 PLAQUE AND HALO DIAMETER (MM) muammqmma o—muemmwmmo o— L p04. ‘ o. 35' J //: "/45. ‘ /.:/o’.‘/’Io. ‘- -ég£_ o—o—é. J J a L L L l l2 24 36 48 60 72 INCUBATION TIME (HOURS) Figure 2(A and B). Effect of temperature on expansion of the translucent halo surrounding plaques produced by bacteriophage PEal(h). A) Effect on the establishment of the halo. B) Effect of temperature on expansion of the halo after its establishment. 95 temperatures were increased from 10 to 27 C. Diffusion of'thephage‘from‘the‘plaques. Phages of PEal(h) diffused from the true plaque but not beyond the halo; their diffusion coincided with or followed the development of the expanding halo that surrounded the true plaque (Table 2). .Phage PEal(nh), which did not produce a halo, were not detected beyond the margins of the true plaque (Table 2). .Altered‘colony morphology and‘sensitivity to REESE: When rifampin labeled (rifr) E. amyZovora was grown on NAG, convex, chalky white, mucoid colonies 2.0-2.5 mm in diameter developed (Figure 3—A). The rifr colonies were indistinguishable from wild type E. amyZovcra except for their ability to grow on media containing rifampin. Colonies which developed on NAG in the presence of phage PEal(h) were flat, vitreous, nondmucoid, and 1.0-1.5 mm in diameter (Figure 3-B). These pr colonies were also resistant to rifampin. When pr type colonies were streaked with NAG, two different types of bacterial growth occurred. The ‘growth which developed from the initial portion of the streak—process had pr characteristics, but near the end of the streak-process wild type (w.t.) bacterial _growth occurred. PEal(h) phages could be detected in 96 .tmuowumt uoc mes macaw away mmumofio:« :I: «omuomumm was among umnu mmumowtcfi :+: \e .oaen m mosmoum uo: mace Ancvammm mews: armada many may wraparouusm Oat: assurances» .mcwmcmmxm cm mcw>ms madman e menarche “Seaman amend a IN I + I + + m I + I + + N I + I + + H armada mo oamn.mo armada msUmHm mpwmuso EB mIN «swede mo mtfimcH mtflmuso SE mIm mo meamcH mo mtflmcH - . .oz msvam inclines . Assumes uswEmon>mQ onm kuwm I + I + m I + I + m I + I + \s H madman mo wswmam mo emwmuso BE MIN armada mo otamcH otwmuso 85 MIN osvmam mo opancH . .oz msvam inaeummm \eincflmma UCOEQOH 0>m0 OHGE GHOMGQ Q .msvam can» we» Scum Ancvflmmm pct Aavammm mmmmnm mo scamsMMHa .N manta 97 Figure 3 (A and B). Bacterial growth of Erwinia amyZovora after 48 hr incubation at 23 C. A) Capsulated, mucoid, wild type growth. B) Bacteriophage PEal(h)-resistant, acapsulated, non-mucoid. Bar = 1.0 cm. 98 the pr type of bacterial growth but no phages could be detected in the w.t. bacterial portion of growth, the w.t. bacteria were also sensitive to PEal(h) phages. Table 3 shows the results of an experiment to illus- trate the instability of the pr type E. amylovora colonies. The 10 subcultures of 110 rifr were all of w.t. characteristics when grown on NAG, but when grown on NAG + PEal(h) all of the colonies were of pr type. The growth of pr type colonies in NBGYE for 12 hr resulted in the failure to detect any pr type colonies when plated on NAG, the colonies which developed were also phate PEal(h) sensitive. ‘ Facto’r‘s‘ic’ontrib'ut'i'ng't‘o'th'eestablishment‘ofpr type colonies. Growth of E. amyZovcra 110 rifr at 10, 15, 23, and 27 C on NAG plates in the presence of phage PEal(h) resulted in pr type colonies; however, when incubated at 30 and 35 C, pr type colonies failed to develop but retained w.t. characteristics (Table 4). Incubation of E. amchvcra 110 rifr in NBGYE with PEal(h) at m.o.i. of 0.1—100 failed to produce any pr type colonies when plated on NAG; all of the colonies were of th. (Table 5). (Incubation of 110 rifr on NAG + PEal(h) resulted in pr type colonies. Phage PEal(h) antiserum also inhibited the development of pr type colonies on NAG plates and in NBGYE medium (Table 6). 99 Table 3. Instability of bacteriophage PEal(h)-resistance and altered colony morphology of Erwinia amylovora 110 rifr. bfu on NAG :fEE::(:?G Following 12 hr of culture in (108) (108) NBGYE broth. CFU on NAG (103). Subcultures ' r or 110 rlf r r Coloniesa/ ‘ r PEal(h) w.t. p w.t. p from w.t. p .-M ‘ f NAG + PEal(h) sensitivity a ' so 0 o 23 al 29 o + b 50 0 0 28 a2 49 0 + c 60 0 0 30 a3 21 0 + d 63 0 0 26 b1 58 0 + e 22 0 0 14 b2 46 0 + f 17 0 0 13 b3 56 0 + 49 0 0 21 25 0 0 22 d1 77 0 + i ' 52 0 0 20 d2 85 0 + j 48 0 0 23 d3 90 0 + f1 49 0 + f2 44 0 + £3 31 O + cfu a colony-forming-units. w.t. = wild type of mucoid E. amylovora, pr = phage resistant and non-mucoid. ‘ a/Three pr colonies that were selected from four 110 rifr sub- cultures which were grown on NAG + Peal(h). 100 Table 4. Effect of temperature on the development of the pr type of colony. Incubation NAG (cfu) NAG + PEal(h) (cfu) Temperature - (C) w.t. , pr w.t. pr 10 56 0 0 35 15 36 0 0 37 23 41 0 0 28 27 35 0 0 32 30 34 0 22 35 13 o 5 o w.t. = wild type, phage PEal(h) sensitive, mucoid Erwinia amylovora. pr = phage PEal(h) resistant, non-mucoid E. amylovora. Table 5. Results of the incubation of Erwinia amylovora 110 rifr with bacteriophage PEal(h) in NBGYE and the development of pr type colonies when plated on NAG. Colony type that developed on NAG M.O.I.a/ wildtype I106 cfu/ml) pr (106 cfu/ml) 0.1 80 0 l 60 0 10 60 0 100 110 0 no phage 430 0 b/ 0 330 a/ b/ M.O.I. = multiplicity of infection. Phage were spread over the surface of NAG plates prior to plating 110 rifr. 101 Table 6. Effect of bacteriophage PEal(h) antiserum on the development of pr type colonies. Reaction on NAG (107 cfu/ml) Reaction in NBGYE (107 cfu/ml) Treatment w.t. pr w.t. pr 11o rifr alone 239 o 135 o 110 rirr + PEal(h) 230 o 150 o antiserum 110 rifr + PEal(h) o 160 91 o 110 rifr + PEal(h) 225 0 so 0 antiserum + PEal(h) w.t. = wild type, PEal(h) sensitive. Pr = PEal(h) resistant. 102 Agar diffusible substance. Lysis of E. amyZovora cells by phage PEal(h) resulted in an agar diffusable substance which could move across bacterial-free areas of NAG and affected the appearance of E. amyZovora growth (Figure 4). Effect of phage lysates on Erwinia amylovora. Chloroformrsterilized lysates of phages PEal(h) and PEa7 produced a substance that caused a depression or clearing in mature lawns of E. amyZovora 110 rifr. The substance had a titer of 64 in the PEal(h) lysate, was non-detectable in PEal(nh), a titer of 2 in the PEa7 lysates, and was not detected in chloroform- sterilized cultures of 110 rifr or in cultures of 110 rifrpr incubated with phage PEal(h) (Table 7). None of the lysates had any detectable effect on mature lawns of 110 rifrpr. Incubation of capsulated cells of E. amchvora 110 rifr with a sterile lysate of E. amylovora 110 rifr + PEal(h) resulted in partial degradation of the capsule within 5 min and failure to detect the capsule after 10 min (Table 8). A lysate of PEa7 produced an effect on the E. amchvcra capsule after 15 min of incubation and no capsule was detected after 30 min while PEal(nh) and a chloroform-sterilized lysate of 110 rifr culture had no detectable effect on the 103 Figure 4 (A and B). Diffusion of capsular degrading factor across bacterial-free 2.0% nutrient agar and 0.5% glucose. A) Appearance of bacterial growth 24 hr after placement of bacteriophage PEal(h) on the center colony. B) Appearance of bacterial growth 10 days later. Note vitreous appearance of inner portions of the colonies surrounding the phage-inoculated center colony. 104 Table 7. Ability of bacteriophage lysates to affect mature lawns of'Erwimia amylovora. Source of lysatea/ Titerb/ Lawn of 110 rifr Lawn of 110 rifrpr llO rifr alone 0 o 110 rifr + PEal(h) 64 'o 110 rifr + PEal(nh) 0 110 rifr + PEa7 0 rr 0 110 rif p + PEal(h) o a/ Lysates were composed of cultures grown for 24 hr then chlorforme sterilized. b/The titer is the reciprocal of the greatest dilution producing a visible depression in the bacterial lawn. Table 8. The ability of bacteriophage lysates to remove the capsule from Erwinia amyZovora cells. 110 rifr cells / Minutes after Source of lysatea addition of :édgzizntgz lysate lysate 110 rifr PEal(h) PEal(nh) PEa7 O +b/ + + + + + + r + + ‘10 + + - + + 15 + + - + i 30 + + - + - 60 + + - + - a/ Lysates were composed of cultures grown for 24 hr then chloroform- sterilized. b/ + indicates the detection of a capsule using the India ink stain, - indicates non-detection of a capsule. 105 E. amyZovcra capsule (Table 8). Serological relationship‘betweenypn‘and'wild‘type Erwinia amyZovcra. The serological relationship between phage PEal(h)-resistant, acapsulated, less virulent strain of E. amylovora 110 rifr and phage PEal(h)-sensitive, capsulated, virulent strain of E. amchvora 110 rifr is shown in Figure 5. The capsulated (w.t.) E. amylcvora had an extra band not produced by the acapsulated (pr) E. amyZovora. ‘Resistance‘of‘Erwinia‘amyicvcra‘to'p‘age‘strains. Mutation frequencies of 20 E. amylovcra strains to phages PEal(h), PEal(nh), and PEa7 were high (Table 9). With only a few exceptions, less than half the cells in a bacterial culture failed to survive exposure to phage. The results of the fluctuation test with the selective agent, phage PEal(h), and E. amylovcra strains 110 rifr and 118 w.t. are shown in Table 10. There was no significant difference in the number of PEal(h)—resistant cfu among the individual cultures compared to the number of PEal(h)—resistant cfu among the individual samples from the same culture. All tests for temperate phages in the stable pr strains of E. amyZovora were negative. ' Growth and effect of'phage'PEal(h)'on‘Erwinia ‘amylovora'llO'rifr. The results of the experiment to 106 Agar gel serological tests with steamed suspensions of Erwinia amylovora strain 110 rifr. Capsulated (C) and acapsulated (AC) suspensions were placed in the center wells while the outside wells were filled with dilutions of antiserum prepared to capsulated E. amyZovora 110 rifr. Numbers in outside wells represent the titer of the antiserum placed in the well. 107 Table 9. Mutation frequencies of Erwinia amyZovora strains to bacteriophages. Percent E. amylovora survivors E. amyZovora Phage PEal(h) Phage PEal(nh) Phage PEa7 105 rifr 28.9 26.9 62.8 110 rifr 57.4 67.3 98.2 112 w.t. 59.0 n.d.a/ n.d. 113 w.t. 37.5 n.d. n.d. 115 w.t. 47.7 64.9 82.4 116 w.t. 47.3 n.d. n.d. 117 w.t. 22.7 26.7 58.7 118 w.t. 54.2 67.1 88.2 120 w.t. 0.014 n.d. n.d. 121 w.t. 0.003 0.002 98.7 122 w.t. 18.2 n.d. n.d. 126 w.t. 30.6 16.0 73.8 128 w.t. 37.3 n.d. n.d. 131 w.t. 59.0 n.d. n.d. 132 w.t. 79.0 n.d. n.d. 133 w.t. 51.7 n.d. n.d. 134 rifr 0.005 0.001 90.0 135 w.t. 60.4 n.d. n.d. 136 w.t. 78.5 95.1 95.5 138 w.t. 50.3 88.3 96.4 a/nid. = not determined. 108 Table 10. Fluctuation test for Erwinia amyZovora and resistance to bacteriophage PEal(h). Erwinia amyZovora 110 rifr Samples from single culture Samples from individual cultures Sample No. No. resistant cfu Sample No. No. resistantciu‘ I106) (106) l 29 1 25 2 29 2 23 3 32 3 29 4 45 4 42 5 40 5 18 6 35 7 19 8 16 9 22- 10 23 Average 35 25 Variance 10.3 6.5 Chi-square 5.89 23.20 P .27 .005 109 Table 10 (continued). Erwinia amylovora 118 w.t. Samples from single culture Samples from.individua1 cultures Sample No. No. resistant cfu Sample No. No. resistant cfu (106) (105) 1 53 1 45 2 46 2 45 3 50 3 48 4 52 4 44 5 49 5 52 6 50 7 51 8 43 9 49 10 52 Average 50 48 Variance 1.5 1.2 Chi-square .438 2.19 P .96 .98 110 determine the growth and effect of PEal(h) on E. amylovora 110 rifr are shown in Figure 6. The 110 rifr culture not inoculated with PEah(h) increased from 2.8 x 108 cfu/ml to 1-x 109 cfu/ml corresponding with an increase in O.D. from 0.3 to 0.8. Bacterial capsule was detected during the entire experiment, and no capsule degrading factor was detected. The culture inoculated with PEal(h) showed and increase of 2.3 x 108 cfu/ml to 3.0 x 108 cfu/ml during the first 40 min followed by a sharp decrease to 4.0 x 107 cfu/ml during the next 10 min. Colony—forming-units remained stable for 10 more min, then increased logarithmically to 8 7.5 x 10 cfu/ml by the end of the experiment. The sharp decline in cfu corresponded to a 0.1 O.D. de« crease, the loss of the bacterial capsule, an increase in the titer of the capsular degrading factor, and a sharp rise in the number of pfu from 6.5 x 107 10 pfu/m1 to 10 pfu/m1. Capsular degrading factor became Imaximal after 60 min at a titer of 32, and remained at this level. Optical density of the 110 rifr plus PEal(h) culture declined steadily as pfu increased and continued for 60 min then gradually increased. Even though the number of cfu in the 110 rifr and 110 rifr + PEal(h) were very similar, by the termination of the experiment there was approximately a 0.5 O.D. differ« ence between the 110 rifr and the 110 rifr + PEal (h) lll co «3 L06“, PFU (0) 8. CFU (°)/ML a» "1131r111fi1t1fi AAAAA ; ldfl‘f'” ' " 'smaum o A—C—A—d—0—=—; ; ; 32 ./ norm Pam.) I6 O 11.11 IIOIH “OH! 0 PE. NM 1 l-ioin-b Unhikain O D and.) 7 4 1 L l L 1*; 1 1 1 L‘l 1 1 IO 50 IOO ISO ISO MINUTES OF INCUBATION Figure 6. Growth of bacteriophage PEal(h) and its effect on Erwinia amylovora 110 rifr in NBGYE culture. The "+" or "-" along the CPU lines indicate the presence (+) or absence (-) of a bacterial capsule. 112 cultures. Symptom development by Erwinia amylovora. Erwinia amyZovora resistant to phage PEal(h) exhibited a delay in the production of symptoms compared to wild type and rifampin resistant strains (Figure 7). Even though there was variability in rate of symptom development among the E. amyZovora strains, the phage resistant strains consistently were slower in production of symptoms (Appendices C1, C2). Three E. amylovora strains resistant to phage PEa5(h) also exhibited a delay in symptom development (Appendix C3). The inoculation of seedlings with a mixture of phage PEal(h) and strains of E. amyZovora resulted in a delay in symptom development (Figure 8). All strains mixed with PEal(h) elicited delayed symptom development as compared to strains not mixed with PEal(h) (Appendices C4-C6). Wild type and rifampin resistant strains of E. amylovora 105, 110, and 134 grew exponentially and produced symptoms within 36 hr after inoculation into apple seedlings (Figure 9A-C). Populations of phage PEal(h)-resistant strains of 105 and 110 grew similar to their wild type strains during the first 12924 hr then remained relatively stable in the plant tissue (Figure 9A & B). After 120 hr none of the seedlings ' DISEASE RATING ;.io b: b»in 6:14 h)io t) Figure O .. I V 5 5 $1 0 wt - ; p O ' ' ’ : / fi— ‘ o I“ p' ,/ . / . // //f . .. I ,a I. 'l.’ / 4 ' // /! ‘ - I, 0’ ‘ ’0 o b I" , /’ '0' II, ’/ I I ”I 'l/ " I [I ,0 M/ 1 _1 A . 24 48 72 96 IZO HOURS Rate of symptom development of wild type (w.t.), rifampin-resistant (rifr), and bacteriophage PEal(h)-resistant (rifrpr) strains of Erwinia amylovora. Results are the means of eight different strains and two separate experiments. 114 LO o'WITHrOUT trams:r I :11"- ownu PEal(h) /"' 9 -: 5:5 - —u—£xiln a- /// >< 7r' ///// U i C s 6 - u1.5 P //// - (n // ff, 4 .. ./ l, I, o . ~ 9 / I ’ o .3 p / / /// . 2 .- / 01/ . / I o/ 'I I _ / j’¢”’m‘/ 4 /’ i o . ./ .z . 93/ . . .. l2 24 36 48 60 72 84,96 IOBIZO HOURS Figure 8. Rate of symptom deve10pment following inoculation of seedlings with Erwinia amylovora and a mixture of E. amyZovora and bacteriophage PEal(h). llS .ucmumflmmu Anvaomm woman can ucmumwmwu sHmEmm«u ma Hannah tom .mocmumwmmu :«mfimmwu cufi3 waxy oaflz ma “may .mmwu an3 .omumHoQOo ma .u.3 .mmsflatmmm magma :H mod cacaonEo oarwsxm mo usmEQoHo>mo ommmmwo pom cu3ouw .¢Im muomwm manor ON. mo. mm vm Nb Om mv om ¢N N. d G o - .. u m 1. I. 0 nbAN. v fig 3 m V . S v .. 0 Us I: I o. . 0 MN . II M. m r :9 .............. M VAAu; m a a AU aux. 3438 I. n... wuuunuuun...a.:a!: .6“... Egycmhx .4 m II...— ........ Hahn...“ ”Hung”--- 6 .2... .6. S tiff-Q ”O. S . . . . . . . . . . o. n q: 116 .ucmumflmou .nvammm woman can ucmumfimmn smemmau mw HQHMMH com .mocmumflmmu :fiQEMMflH nuw3 mmwu oawz ma man .mmwu paws .omumHommmo ma .u.3 .mmcfiaommm Mammm ca OHH cacaonsc omstgm mo ucmfimoHo>mp mmmmmflp 0cm nu3ouw .mum musmflm maze... . 0N. mo. mm .3 Nb 0m 0? mm .N N. G o n u I .- Gallo-o n all .I 0 m“ m. T V m. .0 a V . S v. . --...--\........ O 3 o u n {LI-.1066...“ '9 m”.— . r MM L. w G O 3 W X o._ 10.0 to lull-III L m 0 1: mama“ m u b b m. U ( w.tittiin\\.\\\\\x A...“ u u“ b b D b D P b b b b °-. 0- n 3 ll7 .ucmumwmou .nvammm ounce can ucmumflmou CHQEMMHH ma Hannah pom .mocmumflmmn :fimfimmau saws mama pHflz ma Hmah .ombu tawz .omuoasmmmo ma .u.3 .mmcflaoomm mango cfl «ma oxooonEo omstxm mo unmamoam>wt mmwmmfib com nuzouw .Ulm whomfim m m 3 O I ON. mo. mm .vm Nb 00 av mm .N N. m 0 r a u q 4 a “\Q.‘ L S I ZJUN - v . . S v T . 3 o r O\\\\“\\\“““ 1mm l m o o \rns L N . \ . \. O m IO/ \0\ k 3 .. . \. .. X 0.. W.,.wm/ a a \ \._..\..\ . to. \ 5m. IIIIUIIIIII \AMHm x7 “wax .3: 3 .l xhumuuuuuuuo: \..\.MW. 3303 .6. Iclaouoouadu \u6uu66 a 2. c {an}--. :Hu\ . u... o .3 o 72 O. BHSSIL :JO woxnso "901 118 inoculated with the PEal(h)—resistant strains exhibited symptoms. The PEal(h)-resistant strain of 134 exhibit— ed a growth curve similar to that of wild type 134 and all of the seedlings developed symptoms within 48 hr of inoculation (Figure 9C). Acapsulated strains 104 and 119 supported growth of PEal(h) to a lesser extent then the capsulated 110 rifr and E9 strains (Table 11). They showed no reduction in rate of symptom development compared to the capsulated and acapsulated strains of 110 rifr (110 rifrpr) and 29(28) (Table 12). Phage PEa7 could infect and grow~on capsulated and acapsulated strains but approximately loo-fold less on acapsulated 104 and 119 then on 110 rifrpr and E8 (Table 11). Hypersensitive reaction in tobacco. Erwinia amylovora strains resistant to phage induced the typical hypersensitive response when infiltrated into tobacco leaves (Table 13). 'Effect of phage PEal(h) purification on'thefititer of Erwinia amylovOra'capsule‘degrading"factor. The titer of the capsule degrading factor decreased as phage PEal(h) was purified in sucrose and cesium chloride (CsCl) gradients (Table 14). The factor was still detectable after 24 hr of centrifugation in a 50% CsCl gradient. 119 Table 11. Ability of bacteriophages PEal(h) and PEa7 to form plaques on several capsulated and acapsulated strains of Erwénia amylovora. Erwinia amylovora Presence of Bacteriophage strain capsule PEal(h) PEa7 (plaque-forming-units/ml) 110 rifr + 8.2 x 107 5.5 x 107 110 rifrpr - o . x 107 E9 + 3.3 x 107 5.6 x 107 E8 - o . x 107 119 - - 2.4 x 105 4.0 x 105 104 - 1.0 x 106 9.7 x 105 Table 12. Symptom production by several capsulated and acapsulated strains of Erwinia amyZovora. / Rate of symptom developmenta Erwinia amylovora Capsulated Acapsulated Strain b/24 30 36 48 60 24 30 36 48 60 110 rifr .40 ..80 .80 1.00 1.00 110 rifrpr .. .20 .20 .20 .40 1.00 E9 .40 1.00 1.00 1.00 1.00 EB .00 .20 .40 .60 .60 119 .75 .75 .80 .80 1.00 104 .40 .80 .80 .80 1.00 a/ Initial symptom was the presence of a droplet of bacterial exudate at the point of inoculation. 1.00 - exudate present; .00 = exudate not present. b/ . . . . 8 Hours after inoculation of seedlings. Bacteria (ca. 5 x 10 cfy/ ml) were suspended in 0.02 M potassium phosphate buffer, pH 6.8, and 0.1 m1 inoculated at point of injury. 120 Table 13. Ability of wild type (w.t.), rifampin- resistant (rifr), and rifr-PEalCh)-resistant (rifrpr) Erwinia amyZovora strains to the hypersensitive reaction in tobacco leaves. . . Reaction in tobacco Erwtnia amylovora -i... strain w.t- rifr rifrpr 105 +“/ + + 110 + + + 112 + + + 113 + + + 115 . + + + 118 + + + 121 + + + Sections of tobacco leaves were infiltrated with ca. 5 x 107 cfu/ml of bacterial suspension (0.02 M potassium phosphate buffer, pH 6.8). a4 indicated hypersensitive response. 121 Table 14. Effect of bacteriophage PEal(h) purification on the titer of the Erwinia amylovora capsule degrading factor. Titer at which clearing on / mature lawns of Erwinia amy- Zovora 110 rifr occurs after 24 h at 23 C. Purification stepa l- Chloroformed lysate centrifuged 10 min 32 at 10,000 rpm 2. One-cycle of linear lO-40% sucrose-density- gradient centrifugation for 1 hr at 22,000 rpm 16 3. Two-cycles of linear lO-40% sucrose-density- gradient centrifugation for 1 hr at 22,000 rpm 4, Isopynic centrifugation in 50% (w/v) cesium chloride for 24 hr at 37,500 rpm a/ Detailed procedures given in Section.II of dissertation under MATERIALS AND METHODS -— Phage growth and purification. DISCUSSION Certain strains of bacteriophages which infect mucoid, capsulated strains of bacteria produce plaques surrounded by haloes or zones in which the bacteria are not lysed but the bacterial layer has become translucent and thinner than the surrounding normal bacterial growth (1,7,25,40). This expanding, trans- lucent halo has been shown to be the result of phage-induced enzymes that hydrolyse the bacterial 'capsular expolysaccharides (7,45). A similar phenom— enon occurs with many of the E. amylovora strains and most of its bacteriophage strains (Table 1). Evidence for such an enzyme-substrate reaction is also suggested by the direct relationship between the increase in temperature and increase in the rate of halo expansion (Figure Z—B). Bacterial strains 104 and 119 did not have a detectable capsule whereas all the other strains were capsulated. Capsule degrading factor could not be detected in a PEal(nh) + 110 rifr lysate (chloroform— 122 123 sterilized) using the spot test (Table 7). Possibly, PEal(nh) cannot direct the synthesis of or produce an active form of the factor. Such a non—halo mutant infecting Pseudomonas aeruginosa has been reported (7). Thus, for the production of an expanding, translucent halo the bacterial hOst must be capsulated and the infecting phage must direct the synthesis of an active form of the capsule degrading factor. Synthesis of the factor was linked to phage multiplication. Phage PEal(h) was temperature sensitive, being unable to multiply at 30 C or greater and slowly to 10 C (Figure 6, Section II). Halo formation also did not occur at 0 and 35 C (Figure 2-A). Capsule degrading factor was not detected in chloroform -steri1ized E. amylovora cultures in which PEal(h) had not multiplied (Table 7 and Figure 6). Bacterial cells from colonies showing altered morphology (Figure 3) or grown in the presence of PEal(h) (Figure 6) were acapsulated. Thus, phage PEal(h) or a phage product (capsule degrading factor) was responsible for removal of the capsule resulting in the vitreous, nonemucoid colony. The capsule degrading factor prevented the build-up of capsule exopolysaccharides surrounding the bacterial cell but did not affect the ability of E. amylovora to multiply. This explains part of the O.D. difference 124 between 110 rifr and 110 rifr + PEal(h) cultures, yet the acapsulated cells in 110 rifr + PEal(h) grew logarithmically once the capsule was removed (Figureffl. The high mutation frequencies shown in Table 9 were not due to a spontaneous mutation since there was no significant difference in fluctuation among the individual cultures compared to the samples from the same culture (Table 10). The resistance to phage occurred following exposure to the phage thus the resistance was adaptational (35). Phage PEal(h) adsorbed to capsulated strains of E. amyZovora (Figure 8, Section II) suggesting that the adsorption site was located on the capsule. Removal of the capsule by capsule degrading factor would explain the high frequency and adaptational nature of resistance to PEal(h). Removal from the factor such as streaking on NAG plates allows the formation of the capsule resulting in rapid reversion to wild type character— istics (Table 3). A similar situation was shown to occur with ShigeZZa dysenteriae strain 136-R4 and bacteriophage T7 (32). Incubation of capsulated E. amyZovora and PEal(h) at 30 C or greater (Table 4) or in the presence of PEal(h) antiserum (Table 6) prevented the establishment of pr type colonies. These two factors prevented the infection and growth of PEal(h) (Section II). Thus, a 125 factor that prevented the infection and growth of PEal(h) also prevented the establishment of pr type colonies. Failure to obtain pr type colonies following incubation of capsulated E. amylovora and PEal(h) in NBGYE (Table 5) may have been due to the dilution and plating process removing the bacteria from the factor and phage. Even though the cells were acapsulated in the NBGYE, when plated on NAG in the absence of the factor and phage they formed capsules resulting in wild type growth. There was also a high frequency of resistance to PEal(nh) but no capsule degrading factor was detected using the spot test (Table 7) nor was it observed that a PEal(nh) lysate could remove the capsule (Table 8). Exposure of capsulated E. amylovora to PEal(nh) on NAG resulted in vitreous, non-mucoid colonies indicat- ing capsule removal. Possibly, infection by PEal(nh) resulted in synthesis of a small, non-detectable (with the spot test) quantity of the factor requiring more than 60 min to remove a detectable amount of capsule. The frequency of resistance to PEa7 was greater than for PEal(h) or PEal(nh) (Table 9). Phage PEa7, although able to adsorb to capsulated strains, adsorbed more rapidly to acapsulated strains (Figure 8, Section II) suggesting that its adsorption site lies 126 beneath the capsule. A possible explanation of the resistance to PEa7 may be its inability to penetrate the bacterial capsule or by the time penetration is accomplished the cell is unable to support phage growth. This hypothesis also may account for some of the PEal(nh) resistance. Capsule degrading factor is probably located in the spike-like tail structure of the phage as serologically shown for a similar phage (7). The data in Table 14 also indicate a close association of the factor with the PEal(h) virion. One of the functions of this factor may be to allow the virion to penetrate the capsule to the cell wall where the nucleic acid can be injected into the bacterium. Such a tunneling effect was shown for KZebsieZZa phage 29 (8). Evidence that the factor enables the virion to diffuse more easily through capsulated bacterial masses is shown by the ability of phages to diffuse from the true plaque in association with the halo expansion (Table 2). Many species of Gram-positive and -negative bacteria produce extracellular polysaccharides in the form of capsules and slime (45). For some pathogenic bacteria these non-toxic extracellular polysaccharides are important for the invasiveness of the bacteria by preventing phagocytosis (2,34,51). Extracellular 127 polysaccharides and glycopeptides are also produced by plant pathogenic bacteria. At least three species of Corynebacterium have been reported to produce glyco— peptides (39), while Xanthomonas phaseoli (31), and Pseudomonas soZanacearum (26) produce extracellular polysaccharides that may play a role in pathogenegis. All strains of P. solanacearum which failed to produce extracellular slime were avirulent (26,43). Erwinia amylpvora also produces extracellular polysaccharide in the form of a capsule as shown by the India ink stain. A host-specific toxic polysaccharide from E. amylovora- infected apple tissue was reported (13,18,22). The exact involvement of this polysaccharide in the pathogenesispf fire blight is not completely understood (18,22,44). Even though there are many pathogenic bacteria which produce extracellular polysaccharides there are also many nonpathogens which produce these compounds. The interaction between a capsulated, virulent strain of E. amyZovora (E9) and an acapsulated, avirulent strain (E8) in apple tissue has been studied (22,23,24). It was reported that these two strains were serologically similar, produced typical E. amyZovora colonies on Cross & Goodman and Miller & Schroth media, and induced a hypersensitive reaction in tobacco (24). Growth of E8 and E9 on NAG and TTN 128 media was very different; E9 produced mucoid, wild type growth while E8 produced non-mucoid, pr type growth. A serological difference between wild type and pr type bacteria was detected, indicating that resistance to PEal(h) resulted in an antigenic change in E. amylovora Goodman at al. (23,24) reported that E8 was localized and unable to multiply and become systemic while E9 could. This suggested that the capsule protected the bacterial cells from being agglutinated and bound by the plant cells. Such phenomena were described for Rhizobium (10) in the nodulation process and for Agrobacterium (50) in gall formation. Binding in these systems leads to a symbiotic relationship or gall formation; whereas, with P. solanacearum (43), P. pisi (l9), and E. amyZovora (23,24) binding leads to incom— patibility resulting in avirulence for the pathogen. Exposure of wild type E. amylovora, including E9, to phage PEal(h) resulted in the induction and selection of bacteria phenotypically similar to E8. The reduction in symptom development exhibited by the pr type bacteria may be the result of a mechanism‘ similar to that described by E8 (23,24). Evidence for localization and reduction of bacterial multiplication of pr type cells in host tissue was shown for strains 105 rifrpr and 110 rifrpr (Figure 9A & B). Use of the seedling technique (41) for virulence assays showed 129 that none of the pr type bacteria including E8 were completely avirulent (Table 12). The tissue generally used has been fruits or potted trees (18,22,23,24,44), possibly the seedling stage may have been more . susceptible. The ability of strains 134 rifrpr, 104, and 119 (acapsulated strains) to multiply and produce symptoms similar to capsulated strains cannot be completely explained with the available data. Possibly, these strains lack a binding site for the plant cell to recognize or capsular polysaccharide is present but not detectable with the India ink stain and is covering the binding site. Erskine also found that phage-resistant mutants of E. amylovora exhibited reduced virulence (15). He suggested that the mutation to phage resistance was associated with a change in cell wall permeability so that the enzymes and/or toxic substances responsible for virulence passed through the bacterial cell wall at a reduced rate. Results with strain 134 rifrpr would contradict this hypothesis. Whatever the mechanism involved, the data consistently showed that E. amylovora resistant to or in the presence of PEal(h) type phages produced a delay in symptoms. PEal(h) type phages and the capsule degrading factor may be useful in inducing and selecting 130 acapsulated, virulently reduced strains of E. amylovora similar to E8. Erwinia amyZovora and its phages were detected in close association on aerial apple structures (Section I). It is thus reasonable to assume that some of the effects observed in the laboratory could occur under natural conditions. In the presence of phage, E. amylovora was at least attenuated in symptom induction. Removal from phage resulted in capsulation and restoration of normal rate of symptom development which would correspond to outbreaks of fire blight. The existence of E. amyZovora in the acapsulated state does not, at least in the laboratory, affect the ability of the bacteria to survive, except it exists more as an attenuated pathogen than a pathogen. The acapsulated strains were still capable of inducing the hyper- sensitive response in tobacco (Table 13), indicating that these strains have not been reduced to the level of a saprophyte or nonpathogen (28). It is possible that in the presence of phage and the capsule degrading factor E. amyZovora may exist much like an epiphyte. It is not to be assumed that moisture, temperature, and the physiological condition of the host plant are not important in the development of fire blight. Yet, microbial interactions and processes at the microscopic 131 or submicroscopic level may become very important when magnified to the macroscopic level. LITERATURE CITED ADAMS, M. H. 1959. Bacteriophage. Interscience Publisheres, New York, NY. 592 p. ADAMS, M. H., and B. H. PARK. 1956. 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Mechanism of wilt induction by amylovora in cotonester shoot and its relation to wilting of shoots infected by Erwinia amylovora. Phytopathol- ogy 68:89-94. SUTHERLAND, I. W. 1972. Bacterial exopolysac- charides. In "Advances in microbial physiology“-TA. H. Rose and D. W. Tempest, eds.), pp. 143-213. UETAKE, H., S. E. LURIA, and J. W. BURROUR. 1958. Conversion of somatic antigens in Salmonella by phage infection leading to lysis or lysogeny. Virology 5:68-91. VIDAVER, A. K., R. K. KOSKI, and J. L. VANETTEN. 1973. Bacteriophage 6: a lipid-containing virus of Pseudomonas.phaseolicola. J. Virol. 11:799-805. VIDAVER, A. K. 1976. Prospects for control of phytopathogenic bacteria by bacteriophages and bacteriocins. Annu. Rev. Phytopathol. 14:451-465. WEHRLI, W., and M. STAEHELIN. Actions of the rifampcins. Bacteriol. Rev. 35:290-309. WHATLEY, M. H., J. s. BODWTN, B. B. LIPPINCOTT, and J. A. LIPPINCOTT. 1976. Role for Agrobacterium cell envelope lipopolysacchar-- ide in infection site attachment. Infection and Immunity 13:1080-1083. WILKINSON, J. F. 1958. The extra cellular poly- sacchrides of bacteria. Bacteriol. Rev. 22: 46-73. 137 52. WU, W. C. 1972. Phage-induced alterations of cell disposition, phage adsorption and sensitivity, and virulence in Xanthomonas citri. Ann. Phytopathol. Soc. Japan 38: 333-341. 53. ZINDER, N. D., and J. LEDERBERG. 1952. Genetic exchange in Salmonella. J. Bacteriol. 64: 679-6990 PART FV SOME PROPERTIES OF THE BACTERIOPHAGE PEal(h)-ASSOCIATED CAPSULE DEGRADING FACTOR INTRODUCTION Synthesis of bacteriophage directed enzymes occurs soon after the insertionh of the phage nucleic acid into the bacterial cell. The majority of these enzymes are primarily involved in the synthesis of various structural components of the progeny phage (5). Other ' enzymes also may be synthesized which become associ- ated with the mature phage particle; among these enzymes are the depolymerases that act on the bacterial capsule and slime layers (6). Phage- associated capsule degrading enzymes have been shown for phages infecting Klebsiella (11,14), Escherichia coli (17), Aztobacter (8), Pseudomonas (3), and bacteria in a few other genera (18). The exact role of these enzymes is not known, but it has been postulated that they assist the phage in reaching its underlying bacterial cell wall receptor, aid in the release of phage from infected cells, and are involved in the specific molecular interaction required for phage adsorption (5,13,15).h A capsule degrading factor (CDF) was found associated with phage PEal(h) following infection of Erwinia amylovora (Section III). 138 139 This section describes some of the properties of a crude preparation of this CDF and its effect on E. amylovora sensitivity to streptomycin. MATERIALS AND METHODS Source of capsule‘degrading'factor. Erwinia amylovora strain 110 rifampin resistant;(rifr) grown in 0.8% (w/v) nutrient broth, 0.5% (w/v) glucose, and 0.25% (w/v) yeast extract (NBGYE), was inoculated with approximately 5 x 108 plaque-forming-units (pfu)/m1 of PEal(h) during the exponential growth stage (ca. 5 x 108 colony-forming-units [cful/ml). The culture was incubated at 23 C on a reciprocal shaker (80 oscillations/min) for 12 hr, sterilized by adding chloroform to 1.0% (v/v), and after 30 min centrifuged at 12,100 g for 10 min to remove bacterial debris. The supernatant represented the crude preparation of the CDF which was stored over chloroform at 4 C. "Capsule degrading'factor. The presence and titer of the CDF was assayed by the spot method (2). The agar overlay method (1) was used to obtain the CDF substrate. Mature lawns of E. amylovora 110 rifr were obtained in incubation of the agar overlay plates at 27 C for 48 hr, the plates were stored at 4 C until required. 140 141 Effect of capsule degrading factor on different bacteria strains. Twenty-five strains of bacteria, including 11 E. amylovora strains were grown in 25 m1 of NBGYE for 18-24 hr. One-tenth ml of the culture was added to molten top-agar and layered over the bottom layer, after solidification a loopful of the crude CDF was spotted on the surface and the plates incubated at 27 C. After 24 hr, lysis or no lysis of the bacterial lawn was recorded and the plates incubated an additional 24 hr at 23 C. Following this 48 hr incubation, a loopful of the crude CDF was again spotted on the mature lawns. The presence or absence of a translucent zone or depression in the bacterial lawn was recorded after 12 hr at 23 C. Thermal inactivation of the capsule degrading factor. Five-tenths m1 of the crude CDF were pipetted into thin-walled glass tubes (1.5 cm diameter) and placed in a preheated water bath for 10 min at temperatures of 25-95 C t 1 C. After 10 min, the tubes were immediately placed in an ice bath. Two-fold dilutions were made in 0.02 M potassium phosphate buffer, pH 6.8 (PPB) and a loopful of each dilution spotted on 2-week old lawns of E. amylovora 110 rifr. Effect of antisera on capsule degrading factor. activity. Bacteriophage PEal(h) and PEa7 antisera were prepared as described in Section II while 142 E. amylovora antiserum preparation is described in Section III. The antisera were diluted 1:50 in PPB, 0.5 ml was mixed with 0.5 m1 of the crude CDF, and mixtures were incubated at 23 C for 10 min. Two-fold dilutions were made in PPB and assayed for CDF activity. Effect of crude capsule degrading factor on Erwinia amylovora sensitivity to streptomycin sulfate. Two types of experiments were done to determine the effect of using the phage PEal(h)-associated CDF for removal of the E. amylovora capsule and its sensitivity to streptomycin sulfate (streptomycin sulfate 740 mg/g, Chas. Pfizer and Co., Inc., New York, NY). The first experiment used a disc assay procedure with 12.7 mm diameter antibiotic discs (VWR Scientific, S and S No. 740E). The antibiotic discs were saturated with different concentrations of streptomy- cin sulfate dissolved in PPB. These discs were placed on the surface of the top-agar which had been seeded with 0.1 m1 of an 18 hr old NBGYE culture of 110 rifr 9 cfu/ml) or 110 rifr plus 0.1 m1 of the CDF. (ca. 10 After 24 hr incubation at 27 C, the inhibition zones were measured to i 1.0 mm. A second experiment was designed to determine if loss of the bacterial capsule would cause an increase in the rate of streptomycin uptake and thus a more 143 rapid killing of the cells. One ml (ca. 109 cfu/ml) of an 18 hr old NBGYE culture of 110 rifr was added to two flasks of 25 ml each of fresh NBGYE. To one flask was added 1.0 ml of the crude CDF preparation and the flasks incubated 4 hr with shaking. Ten ml of the cultures were added to four, sterile 125-ma flasks, two flasks for the 110 rifr culture and two flasks for the 110 rifr plus CDF. To one flask in each set was added 10 ml of 100 ug/ml of streptomycin sulfate in PPB, thus giving a final concentration of 50 ug/ml, to the two remaining flasks were added 10 m1 of PPB. The number of cfu/ml in the non-streptomycin inoculated. flasks was determined by dilution and plating on 2.0% (w/v) nutrient agar plus 0.5% (w/v) glucose and taken as the cfu at time zero. In experiment I, samples were taken over a lZO-min period while in experiment II samples were taken over a 60-min period. RESULTS Source of capsule degrading factor. The method used to obtain the crude factor preparation yielded factor having a titer of 32-64 assayed with the spot test.. The enzyme remained active when stored at 4 C. .Effect of the capsule degrading factor on differ- ent bacterial strains. Phage PEal(h) plus 110 rifr lysate and PEal(h) plus 110 riffpr lysate could lyse all the E. amylovora strains except 110 rifrpr. The two E. herbicola strains also showed areas of lysis but none of the other bacterial strains were affected (Table l). Capsule polysaccharide degradation was shown only with the PEal(h) plus 110 rifr lysate and affected only the E. amylcvora and E. herbicola strains but not E. amylovora 110 rifrpr nor any of the other strains (Table 1). The supernatant from 110 rifrpr showed no lytic or CDF activity. Thermal inactivation. The CDF could be heated for 10 min at 75 C without a decrease in titer; however, heating for 10 min at 85 C caused complete loss of CDF activity (Table 2). 144 145 I u u u I u see EREESSEEsosdtrsaN .. .. I I ... I mo.” toenmmmoseo .um> usosoeosco cwxmesm u + I + + u .ugsmMH u + u + + u .u.3 and I + s + + a .u.3 was I + n + + 1 .u.3 mas I + I + + I .u.3 HNH . + u + + u .u.3 was I + I + + u .u;amaa s + u + + n .u.3 «as n u n u n 1 segues odd I + u + + I swam one u + :x% + + [\m amen mwwoeoessc sestsm saunas ous sues oee saunas ous seas OHH + .5th + assume he? 3m +- ...:mmnvm + .5th o and. org}. camuum Hmeumuomm uommmm qswmmumma masmmmo , uommmm owuaq\s LIP, \. L I O I! L( M (L, L I /. . I . , ,. ,. . 2 ,. / . , . _. , r . / .wcacuum dcaumuomn-umunuwuwo as nausea mcflouummo Tasman» we poomum .a manna 146 Nom marmxomwxoss.ssmsmeooemxmsbu Hon mxmwomnk sxmsmnooamrmsbu How mxumomkmsxn easemescnosms mom mwzomscNmm ocrosoxuxcx men erase condensersx «AN oxuommsozbk morosbmxmmm QHN cusses mcxosbtsmmm m8 _ _ ssscmoostom mssosomxmmm mom momstmm morosbmsmmm om.” Om H Sessssme assess av... .cwscfiucoo. megaromméo ..m ..coscfiucoo. a manna 147 .uouUMM mcacmummt wasmmmo mo Tocommum may mswumoflcce ssmd Hmwuouomn enzyme one cw econ ucmosamsmuu no cowmmoumot m mmumoflcse :+: Tawnz GOAthmnmmc wasmmmo manmuoouoc o: m0u00fimcfi =I:XR .mwmba Hmfiuwuomn mo noun mmumofiosw :+: sagas memba Hmwumuomn mo noun 0: mwumoamcw :I:\m .Uouomuot on tasoo uouomm mcflcmumoc wasmmmo o: urn ucmumcummsm 0:» ca tmuowuom on caroo .:.Hmmm Tmmnm .:0wummsmwuucwo an touoHHmm magnet Hmwuouoma can tmuflflfiuoumnahomoHoHso _anvammm ou ucmumwmwu sflmuum csononEc .m. Hmumwu OHH nuaz nonmasoca .£.Hmmm muons «0 ousuHso tao.un NH m mm3 Hannah OHH + .nvammm\fi .coflusawt vmua m an cwuoouoc on tasoo sows: uouomm mcwmmuooc masmmmo m mmcflmucoo accumcummsm 0:9 .cowummsuwuucoo an cmuoaaom mwunot Hmfiumuomn on» can coueawuoum nanomouoHno :03» Hugh OHH so szoum .svammm woman no musufloo tao M: NH 0 mm3 HMHH oaa + .c.Hmmm\o . .sofiummsmwuucoo bn mmumaamm manner ameumuomn ecu can cmwwafiuoumtsuomonoHso mm: soars ousuaso Odo n: NH 0 mm: HMHH 0HH\Q .Hotao no u: mv ms3mH arduouomn so meow ucoosamcmuu no QOAmmmummm m mosmoum ou buwaenm on» was uommmo ocfltmumoc Tarmmmo nachos: .csma Howumuomn mcfizoum .mcso> on» :0 menu Hmoao m moscoum ou mummba on» no buwawnm can 003 uommmo 0Hu>q\c ..omscflucoo. H manna 148 Table 2. Thermal inactivation of bacteriophage PEal(h)e associated capsule degrading factor. ................ (”Titer of factor~ ................................. 25 64 45 64 55 64 65 64 75 64 85 0 95 I ,. .‘ 0 . .. a r v 1 fi . a/ Reciprocal of the greatest dilution at which a depression could be detected in mature lawns of Erwinia amylovora 110 rifr after 24 hr of incubation at 23 C. Table 3. Effect of bacteriOphage and Erwinia amylovora antisera on the activity of phage PEal(h)- associated capsule degrading factor. Titer of Factor Erwinia amylovora PEal(h) PEa7 antiserum (As) antiserum (As) antiserum (As) Normal Before After Normal Before After Normal Before After serum As Ss serum As As serum As As 532/64 32/64 32/64 32/64 32/64 none 32/64 32/64 32/64 a/ Reciprocal of the greatest dilution producing a translucent zone/ depression on the mature lawns of E. amylovora 110 rifr after 12 hr at 23 C. 149 Effect'of-antisera:on:capsule‘degrading‘factor V fifi firmva activity.* Phage PEal(h) antiserum completely inhibited the CDF activity of the PEal(h)-associated CDF (Table 3). Antisera to E. amylovora 110 rifr, phage PEa7, and normal sera had no effect on the CDF activity (Table 3) . Effect of capsule degrading factor on Erwinia amylovora 110 rifr sensitivity to streptomycin sulfate. Larger zones of inhibition were produced on plates to which the PEal(h)-associated CDF was added (Table 4). Streptomycin at 25 ug/ml produced larger zones of. inhibition on 110 rifr plus the CDF than did 100 ug/ml on 110 rifr alone (Table 4). The addition of 50 ug/ml of streptomycin to 110 rifr plus CDF culture showed no viable bacteria within 30 min, while the culture without the CDF showed no viable bacteria within 60 min (Table 5, Exp. I). In experiment II (Table 5) no cfu were detected within 15 min in the 110 rifr CDF streptomycin culture while 30 min was required without the CDF. In both experi- ments I and II, incubation with the PEal(h)-associated CDF alone did not reduce the viability of the number of E. amylovora cells. 150 Table 4. Results of disc assay of streptomycin sulfate on Erwinia amylavora 110 rifr and E. amylovora 110 rifr - (CDF). associated capsule degrading factor V Streptomycin sulfate Diameter of zones (mm) concentration - - - .- i (119/ml) 110 rifr llo rifr + cos 0 0 0 25 15.0 19.0 50 16.0 20.0 100 18.0 22.0 a/ Mean of two replications; no measureable variation between the two replications. 151 .cmuoouoc uos n .t.c \o .p.c mm .c.: an oma .6.: mm .c.c Hm om . .c x n \s 3 m3 a S on moa x me on mod x mm ma ma boa x ca ma boa x OH ma m boa x cm om boa x ba ba 0 afloaeoumuuum mo HE\01 om mafia .bOH. afloasoummuum .boHV moo tmuMAUCmmm I moo cou6fl00mmm I No Ha\mn om Tcoam Aces. ¢cammm made was“ OHH Anvammm mafia Mme“ OHH msam.uwfiu oaa ,Hmwu OHH :wObaoumoHum mo HE\muwcslmcfleuowlbcoHoo no umnssz coarsest nmumn mane H newsmummxm .Hmwu OHH csosonso Genesis an mummasm swo>EoummHum mo mxmums was so .moo. uouomm mcwcmumot oasmmmo omunaoommm L .n.anmm Tanzaodnmuomn no pooumu .m manna 152 .Umuoouoc no: u .o.c \o .v.c 4 mm I .c.: . .. ., ,...nm . _ om .e.n . mm .n.e am on .v.z «N .O.: cN ov .n.n . mm .t.a mm on .U.: on med x ow NN om .U.: ma med x mb me ma . o a un U \6 ON moa ma mm 0H 00H x mm ON boa x cm ma m boa x mm mm boa x Hm an o cacafioumonuw . .. ...b. .: .7 .; .. .; ...... ._ mo da\m1 om Mada Abod GAUNEOummuum .bo.z mac UTQMAUOmmc L moo cwumHoommm L No HE\m1 om ocodm Aces. .ecsnmn mean nuns odd .ecanmn mafia nuns can hand menu OHH nuns ode nauseounnnum mo - L... t-.. ..-. ti. : : .-.... :E..-EE.. -- t, t: l : Elri r ..... r- E-,EIE- :EEEE - E. ”8.33006 nouns 08.3. HE\MUHss madeuowtbcodoo mo Honssz HH.ucmeuumxm ..nnneaneooc n manna DISCUSSION Relatively few enzymes that hydrolyse the bacter- ial polysaccharide capsule material and slime layer have been isolated or characterized (18). One of the best.sources of such enzymes has been certain phage- infected bacteria (3,8,11,14,17,18). Bacterial specificity of these enzymes has ranged from strain specificity (1,11) to hydrolysis of the extracellular polysaccharides of bacteria in different genera (18). The CDF produced following the lysis of E. amylovora 110 rifr by phage PEal(h) was quite specific for E. amylovora capsular polysaccharide. Exceptions were the E. herbicola strains. Failure to detect CDF in the 110 rifrpr + PEal(h) combination was the result of 110 rifrpr resistance to PEal(h). Lysis of the young lawns by this combination was the result of the unadsorbed phage. Capsule depolymerases have been useful in fraction- ating bacterial capsular polysaccharides, thus making it possible to study their structure (18). With the implication of extracellular polysaccharides in the pathogenesiswxfsome phytopathogenic bacteria (12,16), 153 154 including E. amylovora (9,10), PEal(h)-associated CDF may be useful in not only better understanding the capsular composition but also the pathogenesis of E. amylovora. The differential in the thermal inactivation of- phage PEal(h) (Section II) and PEal(h)-associated CDF suggested a method of phage separation for the CDF similar to that described by Adams and Park (2). A preliminary experiment indicated that 10 min of 75 C did not eliminate the PEal(h) infectivity. This may have been the result of the high phage titer (ca. 1010 pfu/m1). The CDF could probably be purified and concentrated using the scheme outlined for the depoly- merase of Pseudomonas aeruginosa phage 2 (4). The specific inhibition of the CDF by phage PEalGfi antiserum suggested that the CDF was of PEal(h) origin and not a factor occurring in uninfected E. amylovora. Phage PEa7 also produced a capsular degrading factor as indicated by the translucent halo surrounding the plaque (Section II). PEa7 antiserum did not inhibit the PEal(h)-associated CDF indicating the two factors were different even though both were synthesized in the same bacterial strain. This also supports the hypothesis that synthesis of the factor was under control of the phage genome. 155 The larger zone of inhibition produced by strep- tomycin on the E. amylovora 110 rifr plus CDF plates could have resulted from better diffusion of the anit- biotic. The CDF may have enhanced the diffusion by degradation of the bacterial extracellular poly- saccharide. A The more rapid killing of the 110 rifr cells incubated with streptomycin plus the CDF than with streptomycin alone indicated a more rapid uptake by the acapsulated cells. The capsule may serve as a pro- tective barrier against the rapid uptake of some chemicals. This also suggested the CDF, if active under orchard environments, might be useful to make E. amylovopa more sensitive to chemotherapeutic agents. LITERATURE CITED ADAMS, M. H. 1959. Bacteriophages. Interscience Publishers, New York, NY. 592 p. ADAMS, M. H., and B. H. PARK. 1956. An enzyme produced by a phage-host cell system II. The properties of the polysaccharide depolymerase. Virology 2:719-736. BARTELL, P. F., T. E. ORR, and G. K. H. LAM. 1966. Polysaccharide depolymerase associated with bacteriophage infection. J. Bacteriol. 92: 56-62. BARTELL, P. F., G. K. H. LAM, and T. E. ORR. 1968. Purification and properties of polysacchar- ides depolymerase associated with phage- infected Pseudomonas aeruginosa. J. Biol. Chem. 243:2077-2090. BARTELL, P. 1977. Localization and functional role of the Pseudomonas bacteriophage 2- associated depolymerase. In "Microbiology" 1977 (D. Schlessinger, ed.)7 ASM. pp. 134- 137. COHEN, S. S. 1968. Virus-induced enzymes. Columbia University Press, New York. 315 p. COSTERTON, J. W., H. N. DAMGAARD, and K. J. CHENG. 1974. Cell envelope morphology of rumen bacteria. J. Bacteriol. 18:1132-1143. EKLUND, C., and O. WYSS. 1962. Enzyme associated with bacteriophage infection. J. Bacteriol. 84:1209-1215. GOODMAN, R. N., J.-S. HUANG, and P.-Y. HUANG. 1974. Host-specific phytotoxin polysaccharide from apple tissue infected by Erwinia amylovora. Science 183:1081-1082. 156 10. 11. 12. l3. 14. 15. 16. 17. 18. 157 HUANG, P.-H., J.-S. HUANG, and R. N. GOODMAN. 1975. Resistance mechanisms of apple shoots to an avirulent strain of Erwinia amylovora. Physiol. Plant Pathol. 6:283-287. HUMPHRIES, J. C. 1948. Enzymic activity of bacteriophage-culture lysates I. A capsule lysin active against Klebsiella pneumoniae type A. J. Bacteriol. 56:683-693. HUSAIN, A., and A. KELMAN. 1958. Relation of slime production to mechanism of wilting and pathogenicity of Pseudomonas solanacearum. Phytopathology 48:155-165. KANGEGASAKI, 5., and A. WRIGHT. 1973. Studies on the mechanism of phage adsorption; interaction between phage 315 and its cellular receptor. ‘Virology 52:711-- 718. PARK, B. H. 1956. An enzyme produced by a phage- host cell system I. The properties of a Klebséella phage. Virology 2:711-718. REESE, J. F., G. DIMITRACOPOULOS, and P. F. BARTELL. 1974. Factors influencing the adsorption of bacteriophage 2 to cells of Pseudomonas aeruglnasa. J. Virol. 13:22—27. SEQUEIRA, L., and T. L. GRAHAM. 1977. Agglutina- tion of avirulent strains of Pseudomonas solanacearum by potato lectin. Physiol. Plant Pathol. 11:43-54. SUTHERLAND, I. W., and J. F. WILKINSON. 1965. Depolymerase for bacterial exopolysaccharides obtained from phage-infected bacteria. J. Gen. Microbiol. 39:373-383. SUTHERLAND, I. W; 1972. Bacterial exopoly- saccharides. ‘In "Advances in microbial physiology" (AT H. Rose and D. wu Tempest, eds.) pp. 143.213, APPENDICES APPENDIX A Table A1. Bacterial strains and their source Laboratory I.D. Number 400 Agrobacteriwfi 'twnefac’zl‘ens. ‘ U.$‘. , Upjohn Company. 401 A. tumefhciens. at 78, Upjohn Company. 10 Bacillus cereus var. mycofldes. Pfizer Company. 501 Cbrynebacterium fasciens. ATCC 13000. 500 C. flaccumfhciens. Source unknown. 502 C. michiganense. Tomato fruit, Michigan, 1976. 12 Enterobacter aerogenes. MSU Dept. of Microbiology, 1975. 104 Erwinia amylovora. Eal, Pear blossoms, California,1374. 105 E. amylovora. III. 68, Illinois, apple. 110 E. amylovora. Jonathan canker, MSU, February 1975. 111 E. amylovora. Bartlett canker, MSU, February 1975. 112 E. amylovora. Greening apple canker. Old Mission, Michigan, March 1976. 113 E. amylovora. Bartlett canker, Tome, Grand Rapids, Michigan, February 1975. 114 E. amylovora. Idared canker, Clayton, Grand Rapids, Michigan, February 1975. 115 E. amylovora. Jonathan canker, Carpenter, Paw Paw, Michigan, February 1975. 116 E. amylovora. Bartlett canker, Spinks Corners, Michigan, March 1975. 117 E. amylovora. EaS, Pear blossom, California, 1974, streptomycin resistant. 118 E. amylovora. Ea38, Pear blossom, California, 1974, streptomycin resistant. 119 E. amylovora. Mac715, McIntosh terminal, MSU. 120 E. amylovora. EACC512, apple, N. Carolina, 1975. 121 E. amylovora. EA518, apple, N. Carolina, 1975. 122 E. amylovora. Apple canker, webb orchard, Paw Paw, Michigan, March 1976. 158 Table A1 (continued). 159 v 123 124 125 126 127 128 129 131 132 133 134 135 136 137 138 108 106 140 141 100 101 103 130 5‘3 WHEELER: . amylovora. amylovora. amylovora. amylovora. amylovora. amylovora. . amylovora. amylovora. amylovora. amylovora. . amylovora. . amylovora. amylovora. amylovora. amylovora. carotovora carotovora Jonathan blighted terminal, MSU, May 1976. Blighted Jonathan leaves, Carpenter, Paw~ Paw, Michigan, June 1976. Blighted apple terminal, McLean, Hart, Michigan, 1976. Bbl, Blackberry cane, Illinois, 1976. Jonathan terminal, MSU, June 1976. E9, Missouri, Goodman, virulent, capsulated. E8, Missouri, Goodman, avirulent, acapsulated. #1, Blighted Jonathan terminal, R.Baiers, Keeler, Michigan, June 1976, #2, Blighted Jonathan terminal, Herman, Watervliet, Michigan, June 1976. #3, Blighted Jonathan terminal, Aschroft, Lawrence, Michigan, June 1976. #4, Blighted Jonathan terminal, Webb, Paw Paw, Michigan, June 1976. #5, Blighted Greening terminal, Baiers, Keeler, Michigan, June 1976. #6, Blighted Jonathan terminal, Hasell, Keeler, Michigan, June 1976. #7, Blighted crabapple terminal, Mankey, Berrien Springs, Michigan, June 1976. #8, Blighted Jonathan terminal, Mitchum, Hartford, Michigan, June 1976. var. atroseptica. SR8, Wisconsin, potato, 1969, Kelman. var. carotovora. SRl6S, Minnesota potato, 1973, Kelman. chrysanthemi. Corn, Berrien Springs, Michigan, 1977. chrysanthemi. Corn, Three Rivers, Michigan, 1977. herbicola. herbicola. herbicola. herbicola. ZP-l, Dye, New*Zea1and. ZP-2, Dye, New Zealand. A-E, from apricot, MSU. #7, Jonathan canker, 1976. Table A1 (continued). 160 ......................... 150 151 11 206 205 214 209 200 210 215 203 216 13 14 301 307 302 303 304 fiV—v’fi.fi' ‘ V‘vw E. herbicola. Bartlett canker, MSU, 1977. E. herbicola. Jonathan canker, MSU, 1977. Escherichia coli. MSU, Microbiology Dept., 1975. Pseudbmonas aeruginosa. MSU, Microbiology Dept., 1975. P. fluorescens. MSU, Microbiology Dept., 1975. P. fluorescens. Pf-l, from.pigweed, 1977. . Zachrymans. Williams, Wisconsin, 1976. morsprunorum. C28, cherry stem canker, England, 1960. solanacearum. Tobacco, Phillipines, Sequeira, 1967. . solanacearum. Tomato, Georgia, 1977. m wi:u m n: . syringae. DH24, cherry canker, Michigan, 1976. P. tomato. Pt-l, tomato, southwest Michigan, 1977. Rhizobium sp. Source unknown. SGrratia sp. Contaminate in tissue culture, 1975. Kanthomonas campestris. Cabbage, Michigan, 1974. X. campaestris. PHW42, cabbage, Wisconsin, 1977. X. juglandis. Diseased walnut, California, 1975. X. pruni. PF-2, Michigan. X. vesicatoria. Pepper plant from Georgia, 1976. APPENDIX-B Table Bl. Optimum concentration of polyethylene glycol (PEG), 6000 average molecular weight, for the precipitation of bacteriophages PEal(h) and PEa7. Percent PEG. Total plaque-forming-units of PEal(h) (w/v) Supernatant Pellet 13 x 1011 ... 5 12 x 1011 44 x 109 10‘ 26 x1010 68 x 1010 15 60 x 109 58 x 1010 20 92 x 107 . 48 x 1010 percent pEG .Total plaque—forming—units of PEa7 (w/v) Supernatant Pellet so x 1010 ... 32 x 109 36 x 109 3 10 15 11 x 108 17 x 101° 20 27 x 108 16 x 1010 Forty ml of crude phage lysate, chloroformrsterilized and the bacterial debris pelleted by centrifugation for 10 min at 12,000 g, were used per concentration of PEG tested. The appropriate concen- tration of PEG plus 0.5 M NaCl was added to the 40 ml of lysate and stirred for 1.5 hr at 4 C. The solutions were centrifuged at 12,100 g for 20 min, the supernatants decanted, and 2 m1 of 0.02 M potassium phosphate buffer, pH 6.8, used to resuspend the pellets by setting at 4 C overnight. 161 .muacslmcwau0mt>coHoo OH 5 x m .mo mo cowmcommsm Hoauouomn sues mmcwaoomm mo :ofiumasoocfi Houmm mason .m:0aumowamou ~90m no coo: .usomoum no: oumoaxo a co. «ucomoum mumcnxo \o \e n oo.a 162 .cOMumHsoocw mo unwom on» um oumosxo Haguouomn mo uoamouo a mo monomoum on» mmz Bowmaam HmfiuH:H\o oo.H me. mm. and co. oo.H oo.H oo.H mm. as. oo.a oo.H oo.H mm. mm. com: oo.H mm. mm. 00. oo. oo.H oo.a oo.H me. On. oo.H. oo.H oo.H mm. mm. Had oo.H om. 0m. 00. oo. oo.a oo.H oo.H oo.H oo.H oo.~ oo.H oo.H me. me. mad oo.H oo.H oo.H oo.H oo. oo.H oo.H oo.H oo.H me. oo.a oo.a oo.H oo.H oo.H mad oo.H ms. mm. mm. 00. oo.a oo.H oo.H mp. mp. oo.H oo.d oo.H oo.a oo.H MHH oo.H m5. me. mm. 00. oo.H oo.H oo.H oo.a oo.H oo.H. oo.H oo.H oo.H oo.H NHH oo.H oo.H oo.H mm. 00. oo.H oo.H oo.H me. me. oo.H oo.a oo.a oo.H oo.a .oaa oo.H oo.a oo.H mp. oo. oo.H- oo.H oo.H oo.H m5. oo.H oo.a oo.H oo.H oo.J\0 moa oNH mm an mv vm oma mm as we vm oma om an mv vm\ Q camuum lame penumammuniasammm new swam Aswan. nemumamou seasomam mesa odes cacaoesee ucmsmoao>mo soumfi>m mo mama \c ., . cwrvsxmm .Aumg eaoaonEu .m ucmumflmmunanvflmmm can eaononEe excessm Auwflu can omwu Udazg o>fluamcomt~neammm mmmcmo«nouomn an ucmsaofim>wo Eoumfihm .HD manna U xHDmem4 .mcoHumowamow m>wm no com: \o .muwcsumawan0mtmcoHoo oa x m .m0 m0 scamcommsm aneuouomn sue: mmcfiaooom mo downwasoocfi Hmumm mason m \e .ucomnm muoonxo u co. aucomoum mumpsxo n oo.a .coflucdsooca mo pcwom man an mumpsxm Hoflumuomn mo umumouc a mo mocmmoum ogy mo? soumahm Hmfiuaca 163 \3 cm. me. mm. ma. oo. oo.a mm. mm. me. so. new: oo.a oo.a om. co. co. oo.H oo.a. oo.H oo.a oo. 3 and oo.a om. cm. as. so. oo.a oe.a co.a as. so. HNH co. co. co. co. co. oo.a om. om. cm. ea. add co. co. co. so. so. oo.H oo.a oo.a as. co.\6 med oma mm we me am oma om ,. me ‘me ¢~\a L r - : r .: .; cwnuum Aumv ucoumwmontAcvammm can “mam AHMfiHV ucoumammu :dQEmmflm caoseNmEc cmnmaem ucmfim0a0>wfl Eoumfihm.m0.muflm\¢.. .Aumv cacaonEu .w ucmumdmmnlanvammm can uaoao~msu cmxmsam Amman can mama oaazv 0>HufimcomtAcVHmmm ommndowumuomo an ucm5m0am>o© Eoumaam .NU manna 0 xHQmem< 164 .mcofiumoaammu woman no com: o .muwcdlucfisu0mn>coHoo 00H x m .m0 m0 coflmcommsm Hawuouomn nufi3 mmdwdcmom mo coaumazOOCfi Hound muao&2~ .ucmmoum uoc mucosxo u 00. uncomonmrmuuoaxo u oo.H. .cowumHsoocfi mo ucfiom man an oumcaxm Hafiumuoon mo umdmouo m we mocmmmum may no: Boumshm HmfiuacJ\o mm. HH. co. co. co. oo.H oo.H mm. mm. mm. coo: oo.H mm. oo. oo. oo. oo.H oo.H hm. co. co. HNH mm. oo. oo. oo. oo. oo.a oo.H oo.H he. co. mad mm. co. co. co. co. oo.a co.a oo.a oo.H oo.H\o moa ONH mm me me em CNH em up mv em}~ cflmuum Away udmumfimoHIAnvmmmm mdau oHfiz oaoaonEu emxweam ucoemon>m© EoumE>m mo mumm\c .Audv csoaonEu .m unnumfimmHIAnvmmmm pom cacaonsu umzmssm Amman oaa3v o>wuwmcmmlacvmmmm mmmndofinmuomn an ucmEmon>mo Eoumsam .mo wants 0 Xwazmmmd 165 .chMuMOfiHmoH o>amzu no one: .mmsu ed“; a .u.3\s .cflmammwn owuofinwucm on» on unnumfimmu u augumw .coflmcommsm mean no HE no.0 I Ho.o saws coumasoocd coca mums mmcfiaommm map .cHE ma How muonsosfi ou vmzoaam can .m.m mm .uoumsn oumnmmocm Edflmmmuom 2 No.0 :w and poxfls who; AHE\smm moa x my Acvammm woman can Aas\zmo moa x my Mahmuomm .mmcflaommm mo cowumanoocw Houmm 930:}N .ucomoum uo: oumcsxo n 00. «Hammond oumcsxo u oo.H .coaumHooocfl mo ucfiom may um oumosxm Hmwuouomn mo omamouo m mo oocmmoum on» mm3 Eoumawm amfiuficH \d hm. om. mm. mm. no. No. oo.H co.H mm. mm. mm. ma. :mwz oo.H mm. ha. NH. mo. mo. oo.H oo.a hm. Nv. mm. ha. Hmah «ma mm. mo. mo. mo. oo. oo. oo.H oo.H mm. om. mm. ha. wau HNH oo.H oo.H mm. mm. mm. oo. oo.H oo.H oo.H mm. mm. mo. .u.3 mHH\fi oo.H mp. om. mm. oo. oo. oo.H oo.H mm. mm. mm. mm. Hmah OHH oo.H mm. mm. mm. co. co. oo.H oo.H we. mm. oo. cocks Hme moa\o oma em mu om we mm oma em Nb om me om\n - -- -L -, -..-, L awmuvm mmmcm sue? cacaQNmEe .m moose uoosuws eaoaonEc .m csoaonEc .. - It - - - cmnmssm ucmEmon>m© Eoumewm mo oumm .\B .ucmEmoHo>mo Eoumfi>m co uomwmm ecu can Assammm mmmnmo«uouomn can cacaoumsu ewswsmm mo onsuxwfi m cuaz mmcwanomm madam no cowpmasoocfi msoocmuasafim .vU manna . o xuazmmmc 166 .zofimcommsm menu no as mo.o : do.o Sufi: oouuaaoocw cons ouo3 mmcwacmom on» .cwfi am now onmnsoca on cmBOHHu 0cm .m.m mm .nommsn opmnmmonm_asfimmmuom 2 No.0 :w and flexes who} AHE\5mm med x my Anvammm omega can AH8\:MO moa x my mwuwuomm .mmCMaooom mo cowuoasoocd uopum unso=\‘ a .ucomwum no: mucosxo u oo. upcomonm ouccsxo u oo.H .cofivmasooaw mo usaom on» no mumpnxo awauouomn mo noumonc a mo monomoum on» mmz aoumahm auwuficn - y, P , \6 me. new. ma.. cc. ..oo.., ,. ... ,A,~m... em....aw....mw. .mo....._ .dmmz om. oo._. oo. . co... co. . , ..oo.a..oc.a..oo.a..cc.a..mm. _nm«u vma co. co. oo. oo. oo. oo.a ms. om. mm. oo. yuan HNH on. em. om. co. co. oo.a oo.H mm. mm. co. .u.3 mad me. mm. mm. co. co. oo.a oo.a oo.a mp. co. .u.3 mHH ms. nu. ma. co. co. me. me. me. me. co. .u.z mad mu. om. mm. co. co. co.d oo.a oo.H oo.a mm. .u.3 NHH\m oo.a co.H oo. oo. oo. oo.a oo.d oo.H mu. co. amen cad ms. mm. mm. 00. oo. oo.a co.a oo.H mm. co.\e yuan moa\e mm:e,~»_.mwwmm-e me.-m om _ .._.. . .mm ..... mat. .em.. we mM\a cwmuum . . . usoaonEu mommotmwmw-wmemwNmse m mound usonuwz uaoaonEe m umsweam FiP-D ’ .PILD' 5”?» r! 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D t? } -Ib' ucofimodo>mo Ecumfixm mo mHMM\u .pcmsmodo>mn Eoum5>m co uommmm on» can Anvdmmm mmmcdoHHouonn can cacaonsc cwewsgm mo ououxwa a cuw3 nmcaacmmm madam mo mcowumHsoocfi msoocmuanfiflm .mo manna U xHazmmmd 167 .mcoHHMOAHmou Room mo :mo}\m .omhu OHH: n .u.3 \u .cwmfimuau Ofluofinflucm onu ou mosmumwmou n yuan \b "erF» 1' ..Aemseaueoos mo manna 168 .chaHMOfiamou “sou mo cmoz\mo Hmwu\u .cofimcmmmsm man» no as mo.o I Ho.o spas tonnaaooca coca mums mmcaacoom may .cfls OH Mom muonsocfi ou oozoaam new .m.m mm .ummmsn mumsmmosm sawmmmuom 2 «0.0 c« and cmxfia mums AHE\:mm OHOH x NV Asvammm omega can AH5\:MO moa x mv wwuouomm .mmcfiaommm mo cowumHsoocfi “ovum manom\Q .ucommum uoc oumnzxo u oo. aucomoum manpsxo u oo.H .cofluMaooocw mo udflom onu um mumpsxo Hafiuouomn mo umamouc o no monomoum on» mm3 Boumshm HmwuficH .camammwu owuofinwucm 0:» cu mocmumwmou n \6 me. mm. mm. 00. oo. oo.H em. em. mm. on. com: oo.a oo.H oo.H co. co. oo.H oo.a oo.H oo.H om. away and co. co. co. co. co. oo.a oo.a oo.H mm. mm. “new HNH oo.H me. om. oo. oo. oo.H me. mm. me. am. yuan oaa oo.H om. co. co. co. oo.H oo.H oo.H oo.H mm.\NU MMHH moa\u cad em «a we em oma em up we e~\s . mound Suez cacaowmsc .m woman usonuw3 cacaonEc .m cacaonEe cmzmesm ucmEmoHo>mo EoudE>m mo mumm .\U .ucmfimon>mp Eoudswm co uommmo on» was Asvammm mmmndoflumuomn can eaononEe cmrwesm mo musuxwe m nnfi3 mmcfiaommm madam mo coflumHooocfi mooocouaoeflm .mo manna O xmazmmmd .HOHUMM mcwomummc omumasmmmo n mow .cmoaonEe cwzweam nmumHomdmo new Anvammm mmmnm0w lumuomn cmmzumn :ofluomumpcfl may no cowumucommudmu owumfiamnmmHo .HQ musmfim O NHQmemfi mas-mac Huauouuua uo couuavnuuua usnuouuou amass and caucus-noun usualou mac aw eyzwmm msuuouuon II-||0.. 00‘ . ..u .. ... .n£‘----wu u u u 5 - -. -. ...”nw” wumn.m huu>quuonon cause I:«uouuaA\\a - . . - 6 . . use noduonaunnuou no can»? 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