“Identification, diversity and detection of Xanthomonas strains associated with pistachio dieback in Australia” Alireza Marefat Thesis submitted for the degree of Doctor of Philosophy at The University of Adelaide Discipline of Plant and Pest Science Faculty of Sciences December 2005 Dedicated to my son, ‘ Armin’ ABSTRACT.....................................................................................................................I DECLARATION ...........................................................................................................III ACKNOWLEDGEMENTS .............................................................................................. IV ABBREVIATIONS......................................................................................................... VI CHAPTER 1: REVIEW OF THE LITERATURE ...................................................................1 1.1 Introduction...........................................................................................................1 1.2 The pistachio industry...........................................................................................2 1.2.1 Origin and history.............................................................................................2 1.2.2 World production..............................................................................................3 1.2.3 Australian production........................................................................................3 1.3 Pistachio dieback ...................................................................................................5 1.3.1 History, distribution and importance .................................................................5 1.3.2 Symptoms.........................................................................................................6 1.3.3 The causal agent ...............................................................................................7 1.4 Xanthomonas Dowson 1939 .................................................................................10 1.4.1 History, distribution and importance ...............................................................10 1.4.2 Description .....................................................................................................10 1.5 Identification and characterisation of xanthomonads........................................11 1.5.1 Non DNA-based methods ...............................................................................11 1.5.1.1 Morphological and structural description..................................................12 1.5.1.2 Metabolic testing......................................................................................12 1.5.1.3 Fatty acid methyl ester (FAME) analysis..................................................13 1.5.1.4 Gel electrophoresis of whole cell proteins ................................................15 1.5.1.5 Serological techniques..............................................................................15 1.5.2 DNA-based molecular techniques ...................................................................17 1.5.2.1 PCR-independent methods .......................................................................18 1.5.2.1.1 DNA-DNA hybridisation ......................................................................18 1.5.2.1.2 Restriction endonuclease analysis..........................................................19 1.5.2.1.3 Restriction fragment length polymorphism analysis (RFLP)..................19 1.5.2.2 PCR-based techniques..............................................................................20 1.5.2.2.1 Multiple arbitrary primed PCR (MAAP) ...............................................21 1.5.2.2.2 Amplified fragment length polymorphism analysis (AFLP)...................21 1.5.2.2.3 Repetitive sequence-based PCR ............................................................22 1.5.2.3 PCR-based detection techniques...............................................................23 1.6 Summary and objectives .....................................................................................25 CHAPTER 2: GENERAL MATERIALS AND METHODS.....................................................28 2.1 Collection of xanthomonad strains associated with pistachio dieback ..............28 2.2 Collection of other phytobacteria occurring in and around pistachio orchards ....................................................................................................................................29 2.3 Collection of type and reference strains of Xanthomonas ..................................31 2.4 Propagation and maintenance of bacteria..........................................................33 2.5 Collection of seeds and other plant materials for pathogenicity tests................33 2.6 DNA extraction from bacteria ............................................................................34 CHAPTER 3: GENETIC DIVERSITY WITHIN THE PISTACHIO DIEBACK PATHOGEN ........37 3.1 Introduction.........................................................................................................37 3.2 Materials and methods ........................................................................................38 3.2.1 Experimental protocol for rep-PCR fingerprinting ..........................................38 3.3 Data analysis........................................................................................................40 3.4 Results..................................................................................................................41 3.5 Discussion.............................................................................................................42 CHAPTER 4: CHARACTERISATION OF XANTHOMONADS CAUSING PISTACHIO DIEBACK ....................................................................................................................................57 4.1 Introduction.........................................................................................................57 4.2 Materials and methods ........................................................................................58 4.2.1 Biochemical and physiological tests................................................................58 4.2.2 Biolog GN2 Microplate System ......................................................................59 4.2.3 SDS-PAGE of whole cell proteins ..................................................................59 4.2.4 Sequencing of the 16S-23S rDNA spacer region .............................................61 4.2.5 16S rRNA gene sequencing ............................................................................62 4.2.6 DNA-DNA hybridisation................................................................................65 4.2.7 Comparison with pathovars of X. translucens using rep-PCR..........................66 4.3 Results..................................................................................................................66 4.3.1 Biochemical and physiological tests................................................................66 4.3.2 Biolog GN2 Microplate System ......................................................................70 4.3.3 SDS-PAGE of whole cell proteins ..................................................................72 4.3.4 Sequencing of the 16S-23S rDNA spacer region .............................................73 4.3.5 Sequencing of the 16S rRNA gene..................................................................73 4.3.6 DNA-DNA hybridisation................................................................................74 4.3.7 Comparison with pathovars of X. translucens using rep-PCR..........................78 4.4 Discussion.............................................................................................................87 CHAPTER 5: PATHOGENICITY OF XANTHOMONADS ASSOCIATED WITH PISTACHIO ....93 5.1 Introduction.........................................................................................................93 5.2 Materials and methods ........................................................................................94 5.2.1 Pathogenicity to members of the Anacardiaceae .............................................94 5.2.2 Pathogenicity to members of the Poaceae .......................................................97 5.3 Results................................................................................................................100 5.3.1 Pathogenicity to members of the Anacardiaceae ...........................................100 5.3.2 Pathogenicity to members of the Poaceae .....................................................101 5.4 Discussion...........................................................................................................110 CHAPTER 6: DETECTION OF XANTHOMONAD PATHOGENS OF PISTACHIO IN AUSTRALIA ...............................................................................................................116 6.1 Introduction.......................................................................................................116 6.2 Materials and methods ......................................................................................117 6.2.1 Detection of bacteria with X. translucens -specific primers, T1 & T2.............118 6.2.2 PCR assay for specific detection of the pistachio pathogens..........................119 6.2.2.1 Selection and design of primers..............................................................119