Culture independent analysis of greyback canegrub-associated microflora and microbial community comparision using subtractive hybridisation. Cassandra Robyn Trent BSc(Hons) School of Life Sciences Queensland University of Technology A thesis submitted to the Queensland University of Technology for the degree of Doctor of Philosophy 2010 i. ii. ABSTRACT Greyback canegrubs cost the Australian sugarcane industry around $13 million per annum in damage and control. A novel and cost effective biocontrol bacterium could play an important role in the integrated pest management program currently in place to reduce damage and control associated costs. During the course of this project, terminal restriction fragment length polymorphism (TRFLP), 16-S rDNA cloning, suppressive subtractive hybridisation (SSH) and entomopathogen-specific PCR screening were used to investigate the little studied canegrub-associated microflora in an attempt to discover novel pathogens from putatively-diseased specimens. Microflora associated with these soil-dwelling insects was found to be both highly diverse and divergent between individual specimens. Dominant members detected in live specimens were predominantly from taxa of known insect symbionts while dominant sequences amplified from dead grubs were homologous to putatively- saprophytic bacteria and bacteria able to grow during refrigeration. A number of entomopathogenic bacteria were identified such as Photorhabdus luminescens and Pseudomonas fluorescens . Dead canegrubs prior to decomposition need to be analysed if these bacteria are to be isolated. Novel strategies to enrich putative pathogen-associated sequences (SSH and PCR screening) were shown to be promising approaches for pathogen discovery and the investigation of canegrubs- associated microflora. However, due to inter- and intra-grub-associated community diversity, dead grub decomposition and PCR-specific methodological limitations (PCR bias, primer specificity, BLAST database restrictions, 16-S gene copy number and heterogeneity), recommendations have been made to improve the efficiency of such techniques. Improved specimen collection procedures and utilisation of emerging high-throughput sequencing technologies may be required to examine these complex communities in more detail. This is the first study to perform a whole-grub analysis and comparison of greyback canegrub-associated microbial communities. This work also describes the development of a novel V3-PCR based SSH technique. This was the first SSH iii. technique to use V3-PCR products as a starting material and specifically compare bacterial species present in a complex community. iv. TABLE OF CONTENTS ABSTRACT .................................................................................... iii TABLE OF CONTENTS.......................................................................v LIST OF FIGURES ..............................................................................ix LIST OF TABLES ................................................................................xi LIST OF ABBREVIATIONS............................................................ xiii STATEMENT OF ORIGINAL AUTHORSHIP................................xv ACKNOWLEDGEMENTS...............................................................xvii Chapter 1: Introduction ....................................................................1 1.1 Sugarcane industry outline....................................................................... 3 1.1.1 Sugarcane crop................................................................................. 3 1.1.2 History of the industry...................................................................... 6 1.1.3 Economic importance....................................................................... 8 1.1.4 Sugarcane pests and diseases............................................................ 9 1.2 Biological control................................................................................... 13 1.2.1 Forms of biocontrol........................................................................ 13 1.2.2 Environmental risks........................................................................ 16 1.2.3 Future of biocontrol........................................................................ 17 1.3 Canegrubs.............................................................................................. 18 1.3.1 Greyback canegrubs ....................................................................... 21 1.4 Methods for pathogen isolation.............................................................. 31 1.4.1 Traditional culturing methods......................................................... 31 1.4.2 Molecular methods used for community analysis............................ 31 1.4.3 Suppressive Subtractive Hybridisation............................................ 35 Project goals and objectives............................................................................... 40 Chapter 2: Identifying dominant bacterial members from living and dead greyback canegrubs by using 16-S rDNA cloning and sequencing. ....................................................................................41 2.1 Introduction ........................................................................................... 43 2.2 Materials and methods ........................................................................... 47 2.2.1 Canegrub specimens from the 2007 grub season............................. 47 v. 2.2.2 16S PCR.........................................................................................48 2.2.3 Cloning...........................................................................................48 2.2.4 Sequencing analysis........................................................................49 2.3 Results....................................................................................................50 2.3.1 Living Grubs...................................................................................50 2.3.2 Dead grubs..........................................................................................51 2.3.3 BLAST isolates...............................................................................54 2.4 Discussion..............................................................................................56 2.4.1 Conclusions ....................................................................................65 Chapter 3: Developing and testing a novel V3-PCR-based SSH technique for identifying pathogens from canegrubs ........................67 3.1 Introduction............................................................................................69 3.2 Materials and methods............................................................................72 3.2.1 2006 Grub Season...........................................................................72 3.2.2 Control bacteria ..............................................................................74 3.2.3 V3 PCR ..........................................................................................74 3.2.4 V3-PCR contamination...................................................................75 3.2.5 Adaptor PCR...................................................................................75 3.2.6 SSH ................................................................................................77 3.2.7 Sequencing analysis........................................................................81 3.3 Results....................................................................................................82 3.3.1 V3-PCR..........................................................................................82 3.3.2 Adaptor PCR...................................................................................87 3.3.3 P1-PCR...........................................................................................96 3.3.4 Control SSH..................................................................................100 3.3.5 Tester:driver ratio analysis............................................................102 3.4 Discussion............................................................................................103 3.4.1 V3-PCR........................................................................................104 3.4.2 Adaptor PCR.................................................................................113 3.4.3 P1-PCR.........................................................................................118 3.4.4 Control SSH..................................................................................120 3.4.5 Tester: driver ratio study...............................................................121 3.5 Conclusions..........................................................................................122 vi. Chapter 4: Evaluation of metagenomic SSH analysis for pathogen detection in canegrubs .......................................................................125 4.1 Introduction ......................................................................................... 127 4.2 Materials and methods ......................................................................... 129 4.2.1 Grub specimens............................................................................ 129 4.2.2 DNA extraction............................................................................ 129 4.2.3 Clontech kit.................................................................................