The relationship of Drosophila nigrospiracula and Ervinia carnegieana to the bacterial necrosis of Carnegiea gigantea Item Type text; Thesis-Reproduction (electronic) Authors Graf, Penelope Ann, 1941- Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 26/09/2021 16:49:18 Link to Item http://hdl.handle.net/10150/318458 THE* RELATIONSHIP OF Drosophila rdgrospiracula AND'Erwinia. carnegieana TO THE BACTERIAL NECROSIS OF Carnegiea gigantea. by Penelope Ann Graf A Thesis Submitted to the Faculty of the DEPARTMENT OF ZOOLOGY In Partial Fulfillment of the Requirements For the Degree of MASTER OF SCIENCE In the Graduate College THE UNIVERSITY OF ARIZONA 19 6 5 STATEMENT BY AUTHOR This thesis has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library. Brief quotations from this thesis are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author. SIGNED: APPROVAL BY THESIS DIRECTOR This thesis has been approved on the date shown below: & / c/ /(/& WILLIAM B. HEED Date ' ^ Associate Professor of Zoology ACKNOWLEDGMENTS I wish to express my sincere gratitude to Dr. William B. Heed, my major professor under whom this work was done, for his constant encouragement, suggestions and criticisms during the research and writing of this paper. I also wish to thank Dr. Stanley Alcorn for his helpful criticisms and suggestions concerning the taxonomy of Erwinia carnegieana, and Dorothy Prest and Dr. Adelaide Even son for their help in the identification of the yeasts. The general encouragement, laboratory facilities and financial aid made available in the form of a graduate research assistantship through Dr s. Hugh H. Smith and Robert J. Janssen is greatly appreciated. Special thanks also go to Paul A. Judge, Superintendent of the Saguaro National Monu­ ment, for granting approval to use the study area within the monument. I also extend my thanks to Mr. and Mrs. Fredrick Cooper for their encouragement and technical assistance, to Dorothy McKenrick, Estella Woods and Ida Williams for their help in media and glassware preparation; and to the many other persons who have contributed so graciously of their time. TABLE OF CONTENTS Page LIST OF TABLES.............................................................. vi LIST OF ILLUSTRATIONS ........................ viii ABSTRACT. ........................................................................... ix INTRODUCTION............................................................................................. 1 ECOLOGY ......... ................................................................... 5 Drosophila nigrospiracula ........................ 5 Erwinia carnegiean.a. 6 Carnegiea gigantea .................. 13 Pachycereus pringlei .................. 16 Ferocactus wislizenii ....................... 17 MATERIALS AND METHODS . ........................ 18 Collection and Analysis of Cactus Samples ........ 18 Collection of Drosophila nigr ospiracula ......... 21 Techniques for Collecting and Sterilizing Drosophila nigr ospiracula Eggs ....................... 23 Bacteriological Techniques Involved in Testing the External and Internal Microflora of Drosophila nigr ospiracula for Erwinia carnegieana ....... 27 Nutrition Tests on Drosophila nigr ospiracula. .. 28 SAGUARO AND CARDON SLIME FLUX ANALYSES...................... 30 Bacterial Analyses . 30 Yeast Analyses .................... 45 EXTERNAL AND INTERNAL MICROFLORA ANALYSES OF Drosophila nigr ospiracula ............................ 49 Laboratory Stocks ............................ 49 Field Populations ............................ 50 iv V TABLE OF CONTENTS- -Continued Page NUTRITIONAL TESTS ON Drosophila nigrospiracula ..... 53 Erwinia carnegieana. 53 Candida mycoder ma .... .............. 56 Sterols ............................ 58 SUMMARY AND CONCLUSIONS. ................................ 62 LITERATURE CITED .................................. 65 APPENDIX A ................................................ ................................................ 69 APPENDIX B ........................ 70 APPENDIX C ....................... 71 APPENDIX D . .. 73 LIST OF TABLES Table, Page 1. Some differences between Erwinia carnegieana and soft rOt collfor m 7 Z. Host-range of Erwinia carnegieana (from Alcorn, 1961). 11 3. Host-range of culturally similar bacilli isolated from other cacti or soil (from Alcorn, 1961) ............................ 12 4. Laboratory stocks of Drosophila nigrospiracula used during this study ............................ 24 5. Biochemical, cellular and colonial morphology of bacteria isolated from Carnegiea gigantea, Pachy- cereus pringlei and Drosophila nigrospiracula . » 34 6. Frequencies of Drosophila nigrospiracula, Carnegiea gigantea and Pachycereus pringlei found to possess the following bacteria: Aerobacter aerogenes (B-7), A. cloacae (B-l) and Erwinia carnegieana (B-3) . 44 7. The yeast flora associated with the slime fluxes of saguaro (Carnegiea gigantea) and cardon (Pachy- cereus pringlei) ................. 46 8. Comparison of the yeast flora associated with the saguaro (Carnegiea gigantea) as presented by Lamb (1964) and determined in this study .................................. 47 9. Summary of the nutritional studies of Erwinia carnegieana on seven strains of Drosophila nigro- spiracula under aseptic conditions ......... 54 10. Summary of the nutritional studies of Candida myco- derma on seven strains of Drosophila nigrospiracula under aseptic conditions ................................. 57 vi vii LIST OF TABLES--Continued Table Page 11. Percentage deviation from the cactus control (considered 100% development) with the addition of Beta-sitosterol,, cholesterol and stigmasterol to normal banana food; and from the normal banana food which served as the noncactus control on eight stocks of Drosophila nigr o spir acula * . * * * o * « ° » * * » o » * * * » 60 LIST OF ILLUSTRATIONS Figure Page 1. Map of the Sonoran Desert showing the distribution of Carnegiea gigantea and Pachycereus pringlei (from Shreve and Wiggins, 1964) . ............................... 15 2* Collection apparatus ........................... 22 3c Car don (Pachycer eus pringlei) from the west side of Star Mountain, San Felipe, Baja California, Mexico showing necrotic lesions from which Erwinia carnegieana was isolated . 31 4c Car don (Pachycereus pringlei) from the south side of Star Mountain, San Felipe, Baja California, Mexico showing necrotic lesions from which Erwinia carnegieana was isolated o , • 32 viii ABSTRACT The relationship of Drosophila nigrospiracula to slime fluxes of saguaro (Carnegiea gigantea) and cardon (Pachycereus pringlei) was investigated. The bacterium Erwinia carnegieana was isolated from 32 of 34 saguaros and from five of six cardon specimens. Drosophila nigro- spiraclxla collected on areas of slime flux had Erwinia carnegieana on them in 100% of the cardon samples and 94. 2% of the saguaro samples. Young saguaros cut with a sterile scalpel and exposed to flies directly from the field showed signs of bacterial necrosis when the flies were carrying Erwinia carnegieana. An analysis was made of other microorganisms found in the slime fluxes of cardon and saguaro. One yeast (Candida mycoderma) and three bacteria (B-3 Erwinia carnegieana, B-7 Aerobacter aerogenes and B-1 Aerobacter cloacae) were found common to both. The effects of Erwinia carnegieana and Candida mycoderma were tested On aseptic eggs. The Erwinia and Candida did not harm the development of the Drosophila but it cannot be said conclusively that they in any way added to their development. Several common sterols including Beta-sitosterol, the com­ mon sterol of saguaro, were tested as to their nutritional value to D. nigrospiracula. In general the flies did as well on sterols as on cactus. INTRODUCTION Slime flux is the infected sap exudate from trees or plants which may have been injured by insects, microbes or weather (Lamb, 1964). The slime flux of the saguaro, the giant cactus Carnegiea gigantea (Engelmann) Britton & Rose, has been associated with the bacterium Erwinia carnegieana Lightle, Standring and Brown since Standring?s original studies (1940) and the later studies of Lightle, Standring and Brown (1942). In 1939 Heinrich discussed the associa­ tion of the nocturnal moth Cactobrosis with the saguaro, and Boyle (1949) indicted the larvae of Cactobrosis fernaldialis Hulst as a vector of the bacterium. Boyle also noted other insects associated with the slime flux and indicated that they may play some part in the progress of the disease. These insects included Volucella sp. and Hololepta yucateca Marseuh Santana (1961) studied immature, cyclorrhaphous Diptera associated with the bacterial necrosis of the saguaro and found the