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Fotential of .F'seudomonas CEPACIA AS A -fOTENTIAL OF .f'SEUDOMONAS- CEPACIA AS A BIOLOGICAL CONTROL AGENT FOR SELECTED SOILBORI\IE PATHOGENS By MANSOUR »ALIGH Bachelor of Science Arak College of Sciences Tehran, Iran 1975 Master of Science Eastern New Mexico University Portales, New Mexico 1978 Submitted to the Faculty of the Graduate College of the Oklahoma State University in partial fulfillment of the requirements for the Degree of MASTER OF SCIENCE December, 1991 ~\~J~_;.j") V=t-"\ \ V-.J\~to\) POTENTIAL OF PSEUDOMONAS CEPACIA AS A BIOLOGICAL CONTROL AGENT FOR SELECTED SOILBORNE PATHOGENS Thesis Approved: Dean of the Graduate College ii Dedicated to my lovely wife Elaheh Amouzadeh iii : IF~- J: ~· cout.P . L.~~N: , - 1"i·11Ntc=--:. oN'·'. MY FEer rP.-BE A GeNJU.,S" r - .1 . • ~ · . • iv ACKNOWLEDGMENTS I wish to express my sincere gratitude to Dr. Kenneth E. Conway, my major professor, without whose continuous support, encouragement and concern this work would have not been possible. I have benefited from his insight and knowledge greatly. I am also indebted to him for his trust, guidance and friendship. I am thankful to Dr. Carol Bender and Dr. Sharon von Broembsen, members of my committee, for their constructive suggestions throughout this investigation. I also like to express my special thank to Dr. Larry Littlefield, head of plant pathology department for his sincere advice and to give my regards to the faculty of the plant pathology department. I would like to thank Dr. Martin A. Delgado for his supervision and Dr. Margaret Essenberg for her technical advice. My gratitude also goes to Dr. Paul W. Geno, Dr. George Odell, and Mr. Daryl Larson for their help conducting mass spectra and gas chromatography techniques. My sincere gratitude also goes to Dr. Larry Claypool for his excellent advice in statistical analysis of this study. I am gratefully indepted to Miss Carol Foor for her assistance to provide the laboratory facilities during this research,. Lastly, but certainly not least, I want to express my deepest appreciation to my brother-in-law, Hamid, for his sustained help and encouragement. My profound gratitude goes to my beloved wife, Elaheh, for her patience, support, understanding, and sacrificies in achieving this goal. This thesis is dedicated to my wife. v TABLE OF CONTENTS Chapter Page I. INTRODUCTION ........... .. .................. .. .. ............ .. ...... .. ...... ...... ....... .......... .. .. 1 II. LITERATURE REVIEW ......................................... ·....................................... 3 Morphology ........................................................................................ 3 Major Synonyms ......................................................................... ...... 4 Importance of P. cepacia as Human Pathogen ... .. ....... ..... .. .. .. .. 5 Importance of P. cepacia as Plant Pathogen . ..... .. ...... ...... .. .. .. ..... 6 Importance of P. cepacia as an Industrial Bacterium ................. 7 Potential of P. cepacia as Biological Control Agent .................. 8 P. cepacia As Biocontrol Agent of Foliar Diseases .......... 8 P. cepacia as Biocontrol Agent of Soilborne Diseases ... 1 0 Possible Mechanisms of P. cepacia . .. ... .. .. .. .. .. .. .. .. .. ... .. .. .. .. .. .. 1 0 Root Colonization ............................................... .................... 11 Concept of Rhizospher~ Competence ... ... ...... ................ ... ... ........ 11 Definitions: "Rhizosphere Competence (RC)" ................... 11 Problems Involved with Selection of P. cepacia as Biocontrol Agent ...................................................................... 1 3 Production of Antibiotics by P. cepacia . .. .. ... .. .. .. .. .. ... .. .. .. .. .. .. 1 6 Pyrrolnitrin ................................................................................ 1 6 Pyoluteorin ............................................................................... 1 7 Pyrrole and the Derivatives .................................................. 17 Altercidins ................................................................................ 17 Xylocandin ............................................................................... 18 Production of Bacteriocin ...................................................... 18 Production of Siderophores by P. cepacia .................................. 18 Cepabacti n ..... .... .. .. .............. .. .. .. ...... .. .. .. .. .. ..... .. .. .. .. .. .. .. .. .. .. .. .. 1 9 Pyochelin ................................................................................. 20 Possible Production of Volatile(s) by P. cepacia ........................ 20 Genetic Investigation on Phytopathogenic P. cepacia .............. 23 P. cepacia as a Promoter of Plant Growth .. ....... ... ... ...... .... ... ... ..... 24 Distinction between Phytopathogenic and Clinical Strains of P. cepacia .................................................................... 25 Production of Enzymes by P. cepacia ... .. ... .. ...... ..... .. ..... ........ ....... 25 Formulation of P. cepacia ................................................................ 27 Selective Media for Isolation of P. cepacia .................................. 30 Influence of Environmental Factors on P. cepacia ..................... 30 Statement of Thesis Problem ......................................................... 32 References .................................................................. ....................... 33 vi Chapter Page Ill. CHARACTERIZATION OF PSEUDOMONAS CEPACIA STRAINS AND EVALUATION OF RHIZOSPHERE COLONIZATION ON ROOTS OF VINCA ( CATHANTHUS ROSE US L.) .... .. .. .. .. .. .. .. .... .. .. 41 Abstract .. .... ........ .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ... .. .. 4 1 Introduction ........................................................................................ 42 Materials and Methods .................................................................... 44 Description of P. cepacia Strains ........................................ 44 Preparation of Bacterial Inoculum ....................................... 44 Pathogenicity Test of Strains of P. cepacia on Onion ...... 44 Petri Dish Assay for Fungal Inhibition ...... ................... ........ 45 Production of Siderophore(s) by P. cepacia and P. aeruginosa ..................................................................... 45 Production of Pectolytic Enzymes by P. cepacia and P. aeruginosa ................................ ..... ...... .. ...... .. .. ...... 46 Inoculation of Seed by Antagonistic Bacteria ................... 46 Directed Bioassay of P. cepacia on Seed Germination ........................................................................ 4 7 Effect of P. cepacia Concentration on Seed Germination ................................................ .... ........ ............ 4 7 Determination of Root Colinization Ability of P. cepacia ...... .. .. .... .. .... .. .. .. .... .... .. .. .. .. .. .. .. .. ... .. .. .. ... .. .. .. .. 4 8 Determination of Doubling Time of P. cepacia OK-2 ........ 48 Antagonistic Effect of P. cepacia against R. so/ani OK-330 (AG-4) ................................................... 49 Drenching Method Using Different Concentrations of P. cepacia OK-2 .... ........................................... ...... .. .. .. .. 50 Results ................................................................................................ 50 Pathogenicity Test of Strains of P. cepacia on Onion ...... 50 Petri Dish Assay for Fungal Inhibition by Strains of' P. cepacia .. ..... .... .. .. .. .. ............ ... .. .. .. .. .. .... .. .. .. .. .. .. .. .. .. .. .. 51 Production of Siderophore(s) by P. cepacia and P. aeruginosa .. .. .. ............ ................ ...... ............................. 51 Production of Pectotytic Enzymes by P. cepacia and P. aeruginosa ................................... ...... ...... .. .. .. ........ 51 Direct Bioassay of P. cepacia on Seed Germination....... 51 Determination of Root Colinization Ability of P. cepacia .............................................., ............. :. ... .. .. .. .. .. 52 Determination of Doubling Time of P. cepacia OK-2 ....... 52 Antagonistic Effect of P. cepacia against R. so/ani 330 (AG-4) ........................................................................... 53 Drenching Method Using Different Concentrations of P. cepacia OK-2 ............................................................. 53 Discussion.......................................................................................... 54 References ......................................................................................... 64 vii Chapter Page IV. PRODUCTION OF AMMONIA BY PSEUDOMONAS CEPACIA AND PSEUDOMONAS AERUG/NOSA: QUANTIFICATION AND EFFECT ON HOST AND PATHOGEN...................................... 67 Abstract ....................................... ~....................................................... 67 - Introduction ........................................................................................ 68 Materials and Methods ..................................................................... 70 Characteristics of P. cepacia Strains .................................. 70 Indirect Effect of P. cepacia on Seed Germination........... 70 Detection and Identification of Volatile Compound(s) ..... 70 Effect of Volatile Compound(s) on Seed Germination _and Root Elongation of Vinca Seeds and Growth of Selected Fungi..............................................................
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