University of Wollongong Research Online University of Wollongong Thesis Collection University of Wollongong Thesis Collections 1991 Morphological and serological characterization of bacterial isolates from greening/dieback diseased citrus Daniel Carlos Bock University of Wollongong Recommended Citation Bock, Daniel Carlos, Morphological and serological characterization of bacterial isolates from greening/dieback diseased citrus, Doctor of Philosophy thesis, Department of Biology, University of Wollongong, 1991. http://ro.uow.edu.au/theses/1068 Research Online is the open access institutional repository for the University of Wollongong. For further information contact the UOW Library: [email protected] MORPHOLOGICAL AND SEROLOGICAL CHARACTERIZATION OF BACTERIAL ISOLATES FROM GREENING/DIEBACK DISEASED CITRUS A thesis submitted in fulfilment of the requirements for the award of the degree of Doctor of Philosophy from THE UNIVERSITY OF WOLLONGONG by Daniel Carlos Bock B.Sc.C Honours) University of the Witwatersrand, Johannesburg, R.S.A. Department of Biology 1991 ii ABSTRACT The morphological and serological characteristics of bacterial isolates from plants infected with African greening, Reunion greening and Taiwan likubin were investigated. Isolates from Australian citrus dieback affected trees and resembling the putative greening isolates, were also included. Although predominantly long thin rods, the bacterial cells were morphologically variabile in both liquid and plate cultures at 25°C and 35°C. "Round forms" developed with age and nutrient limitations. Based on colony morphology and pigmentation, the isolates were categorized into two groups - Groups 1 and 2. Whole cell protein patterns were obtained by SDS-PAGE. Patterns of the Group 1 isolates, which were conserved with growth, were similar to one another and different from the patterns of the Group 2 isolates. In an attempt to establish a bacterial detection probe, antisera were raised against the Group 1 isolates. These sera specifically reacted with all the members of this group in slot-blot immunoassays. Using the sera in western blots, characteristic serological reaction patterns were associated with the Group 1 and Group 2 isolates. Both greening-affected and dieback-affected field samples reacted specifically with the antisera in slot-blot immunoassays. A monospecific polyclonal antiserum was also raised against a 38K - 40K protein band in western blots of the Group 1 isolates. The reaction of antigenic bands in western blots of preparations of tissue from affected trees can be achieved although the techniques involved need to be refined. Metabolically, the Group 1 isolates are somewhat related to Clavibacter michioanense subsp. michiganense. However, the protein pattern and western blot results did not support this. A Clavibacter sp. was isolated from Australian citrus dieback affected trees. A Group 1 isolate was also obtained from Australian citrus dieback affected trees. Declaration This thesis is submitted in accordance with the regulations of the University of Wollongong in fulfilment of the requirements for the degree of Doctor of Philosophy. The material presented is the result of my own unaided work and has not previously been submitted at another university or institution, Daniel C. Bock 9 March, 1991 IV ACKNOWLEDGEMENTS How does one express one's appreciation for the support so freely given by several people during the course of a research project such as this? Two words spring to mind... Thank you I am ever grateful to my supervisor, Professor Helen Garnett. Professor Garnett was at all times readily available to discuss, not only the significance of latest results, but also the problems associated with those experiments that just did not work. Helen's unceasing support and encouragement throughout this study is something to be envied. The help and advice so kindly and freely provided from Venezuela by my family, in the Department of Biology by Dr. Glenda Sullivan-Tailyour and Judy Gordon, and at the Halls of Residence by Cynthia and Tom Halloran, cannot be appreciated and thanked enough. The support from my colleagues and friends: Christine McComb, Greg McKay, Ashlee Moses, Patrick Roach, Paul van Rijn, Pilar Roig-Faran and Gonzalo Hortelano Hap, will always be remembered. The University of Wollongong Scholarship is also gratefully acknowledged. V TE SALUDO, UNA AVENTURA NUEVA CON TODA Ml ANSI A, POR SATISFACER Ml DESEO MAS PROF UNDO... VIVIR Y ESTAR CONTENTO! PUEDA SEP PROSPERO, YASI REAL I ZAP Ml FELICIDAD. PUEDA SEP DIFICIL, PARA QUE Ml LUCHA POR EL DESAFIO PUEDA SURGIR A LA SUPERFICE. TAL ES LA VIDA, PARA VIVIR, ESTAR CONTENTO Y SATISFECHO; PARA QUE MARANA PUEDA DECIR QUE HOY HE PODIDO REAL I ZAP ME. Laura D. M. Bock 1988 CONTENTS ABSTRACT... DECLARATION... ACKNOWLEDGEMENTS... INDEX... ABBREVIATIONS... LIST OF FIGURES- LIST OF TABLES... 1.0 INTRODUCTION... 1.1 Greening - what is it?... 1.2 Symptomology... 1.2.1 Leaves... 1.2.2 Fruit... 1.2.3 Anatomical... 1.3 Aetiology... 1.4 Transmission... 1.5 Possible strains... 1.6 Isolation of the causal agent... 1.7 Serological characterization.. 1.8 Identification... 1.9 The Australian situation... 1.9.1 The Australian citrus industry. 1.9.2 Australian Citrus Dieback... 1.9.3 Symptomology.., 1.9.3.1 Leaves... 1.9.3.2 Fruit... 1.9.4 Transmission... Vll 1.9.4.1 Graft transmission... 17 1.9.4.2 Insect transmission... 17 1.9.5 A causal agent... 1 8 1.9.6 Other... 18 1.10 Research objectives... 18 2.0 MATERIALS AND METHODS... 22 2.1 Bacterial cultures... 22 2.2 Maintenance and storage... 25 2.2.1 Cultures... 25 2.2.2 Plant material... 25 2.3 Growth media... 26 2.3.1 MIG (Medium for the Isolation of Putative Greening-associated bacteria)... 26 2.3.2 Other... 26 2.4 Metabolic characterization... 27 2.5 Growth curves... 27 2.5.1 Starter cultures... 27 2.5.2 Growth cultures... 28 2.5.3 Sampling... 28 2.6 Electron microscopy... 28 2.6.1 Whole colony cross section preparation... 28 2.6.2 Negative staining... 29 2.7 Isolations... 29 2.7.1 From leaves... 30 2.7.2 From fruit... 30 2.7.3 Filter test for presumptive isolates... 31 2.8 Electrophoresis... 31 2.8.1 Culture preparation for SDS-PAGE... 31 viii 2.8.2 Gel preparation... 32 2.9 Serological applications... 33 2.9.1 Immunogen preparation... 33 2.9.1.1 Whole cells... 33 2.9.1.2 Gel piece... 34 2.9.2 Serum from blood... 36 2.9.3 IgG purification... 36 2.9.3.1 Ammonium sulphate precipitation... 36 2.9.3.2 Affinity chromatography... 37 2.9.4 Other antisera... 37 2.9.5 Cross-absorption... 38 2.9.5.1 With bacteria... 38 2.9.5.2 With plant material... 38 2.9,5.3 With LPS... 39 2.9.6 Slot-blot immunoassays... 39 2.9.6.1 Bacterial isolates... 39 2.9.6.2 Modifications to culture preparations for slot-blots... 40 2.9.6.3 Citrus tissue preparation for slot-blots... 41 2.9.6.4 Citrus tissue preparation for western blots... 42 2.9.6.5 Preparation of phloem exudates from citrus tissue for western blot analysis... 42 2.9.7 Western blots... 42 2.10 Statistical analysis... 44 2.11 Molecular weight determinations... 46 3.0 RESULTS... 47 3.1 General characteristics... 47 3.1.1 Light microscope observations... 47 3.1.2 Plate culture observations... 48 ix 3.1.2.1 Colony morphology and pigmentation... 48 3.1.2.2 Colony ultrastructure... 56 3.2 Growth curves... 56 3.3 Metabolic characterization... 74 3.4 Australian citrus dieback... 75 3.4.1 Symptomology... 75 3.4.2 Isolations... 78 3.4.3 Colony characteristics... 79 3.5 Antisera raised against whole cells... 79 3.6 Detection of the putative greening/dieback-associated isolates and laboratory cultures with the anti-SAOl, anti-SA03, anti-SA07 and anti-REOI IgG in slot-blot immunoassays... 83 3.7 SDS-PAGE of the putative greening/dieback-associated bacterial whole cell preparations... 89 3.8 Antigenic patterns of the putative greening/dieback- associated bacterial isolates reacted with anti-SAOl, anti-SA03, anti-SA07 and anti-REOI IgG... 91 3.9 Antigenic reaction patterns of SA03 and REOI with antisera 228/12, 228/13. 228/14, 228/15, 228/16 and 228/17... 98 3.10 Antisera against bacterial cell proteins... 99 3.1 0.1 Serological reaction patterns of the putative greening/dieback- associated bacterial isolates reacted with purified anti-C IgG in western blots... 1 01 3.10.2 Serological reaction patterns of B. subtilis. C. insidiosum, C. michiaanense, E. coli. P. aeruginosa. P.fluorescens and S. tvphimurium reacted with purified anti-C IgG in western blots... 104 3.1 0.3 Reaction of the putative greening/dieback-associated isolates and laboratory strains with the anti-C IgG in slot-blot immunoassays... 106 3.10.4 Reaction of SA01 culture supernate with anti-C IgG in western blots... 1 10 3.11 Variability in serological reactivity with growth... 112 3.1 1.1 Cell morphology... 1 14 3.1 1.2 Whole cell protein patterns... 1 14 3.11.3 Antigenic variation during growth... 117 3.12 Applications to affected and healthy citrus tissue... 1 1 9 3.1 2.1 Reaction of greening/dieback affected citrus preparations with anti-SAOl, anti-SA03, ant-SA07 and anti-REOI IgG in slot-blot immunoassays... 119 3,12.2 Effects of cross absorbing the antisera with healthy citrus tissue... 1 21 3.1 2.3 Evaluation of preparations from affected field samples reacted with anti-C IgG in western blots... 1 26 3.12.4 Reactions of whole cell antigens of isolate RE01 in spiked healthy grapefruit tissue preparations... 1 27 3.1 2.5 Serological reactions of antigens in healthy grapefruit tissue spiked with isolates SA01, SA03, SA05, SA06, RE01, TA01 and TA02 culture supernates with anti-C IgG in western blots..