Investigation of induced resistance against Fusarium circinatum in Pinus patula by Katrin Fitza Submitted in partial fulfilment of the requirements for the degree Magister Scientiae In the Faculty of Natural and Agricultural Sciences Department of Genetics University of Pretoria April 2012 Supervisor: Dr. Sanushka Naidoo Co-supervisors: Prof Alexander A. Myburg and Prof Emma Steenkamp © University of Pretoria TABLE OF CONTENTS Declaration ............................................................................................................... vi Preface .................................................................................................................... vii Acknowledgments .................................................................................................. ix Abbreviation ............................................................................................................. x List of Figures ........................................................................................................ xiii List of Tables ......................................................................................................... xvi Chapter 1 Literature Review – Induced resistance: from model plants to pine species 1.1 Introduction .................................................................................................... 2 1.2 Pinus patula ................................................................................................... 4 1.2.1 Biology of Pinus species ..................................................................................... 4 1.2.1.1 Morphological characteristic features of conifers …....……….. .....….. 6 1.2.1.2 Genomic resources available for pine …………..…..…..…................ 7 1.2.2 Ecological and economic importance of pine ...................................................... 8 1.2.2.1 The South African Forestry Industry ………………...……………….... 9 1.3. Pitch canker caused by Fusarium circinatum …...................................... 10 1.3.1 Taxonomy of the pathogen .............................................................................. 10 1.3.2 Symptoms associated with pitch canker .......................................................... 10 1.3.3 Genetic variation in resistance against F. circinatum in the pine species ........ 11 1.3.4 Reproduction of F. circinatum .......................................................................... 12 1.3.5 Pathogenicity and environmental factors causing infection ………...….…….... 13 1.3.6 Distribution of the fungus ………….…………………………….….………...... .... 14 1.4 Current status on the management of the disease .................................. 14 1.5 Induced resistance ...................................................................................... 15 1.5.1 Pattern-triggered immunity (PTI) ………………………………….………..…...... 16 1.5.2 Effector triggered immunity (ETI) ……………………………….….....……..... …. 16 1.5.2.1 The functions of R genes ……………...……………….……..….….... 17 ii © University of Pretoria 1.5.3 Systemic acquired resistance (SAR) ………..................................................... 19 1.5.3.1 The agricultural perspective on SAR …………………..……...…...... 20 1.5.4 Induced systemic resistance (ISR) ….…………................................................ 21 1.5.4.1 Function of NPR1 in the JA signalling ………………...…………...… 22 1.5.5 Systemic induced resistance (SIR) .................................................................. 23 1.5.6 Mechanism of defence against biotrophs and necrotrophs …...……..………... 23 1.5.7 What is known about defence in conifers ......................................................... 24 1.5.8 The host pathogen interaction (Pine – Fusarium) ............................................ 26 1.6 Inducer application as a means to improve resistance ........................... 27 1.6.1 Biologically and chemically derived inducers known from literature ................ 29 1.7 Conclusion ................................................................................................... 34 1.8 Aim of the study .......................................................................................... 35 1.9 References ................................................................................................... 36 Chapter 2 Elucidation of defence responses associated with inducer application in Pinus patula ..................................................................................... ................... 47 2.1 Abstract ........................................................................................................ 48 2.2 Introduction .................................................................................................. 49 2.3 Materials and methods ................................................................................ 52 2.3.1 Plant material ................................................................................................... 52 2.3.2 Inducer preparation and concentration optimization ….…….................. .......... 52 2.3.3 Screening of the effectiveness of the Inducers under nursery conditions in Pinus patula seedlings .................................................................................... 54 2.3.4 Evaluation of the top inducers under greenhouse conditions for induced resistance ............................................................................................ ............ 54 2.3.5 Artificial inoculation technique of pine seedlings with F. circinatum ................. 56 2.3.6 Scoring system categorizing disease severity ……………............................... 56 2.3.7 Statistical analysis of P. patula lesion measurements .…………………........... 57 2.3.8 Detection of putative defence gene orthologs in P. patula ............................... 57 iii © University of Pretoria 2.3.9 Primer design and sequence validation …................................................. ....... 58 2.3.10 RNA extraction and cDNA synthesis .............................................................. 61 2.3.11 Reverse transcriptase - quantitative PCR (RT-qPCR) analysis of induced plant material ................................................................................................ 62 2.4 Results .......................................................................................................... 64 2.4.1 Effectiveness of inducers triggering induced resistance in P. patula ............... 64 2.4.2 Identification of putative defence marker orthologs in P. patula using phylogenetics ................................................................................................. 70 2.4.3 Detecting reference gene orthologs in P. patula using phylogenetics. …......... 81 2.4.4 Expression of known defence related marker genes in response to chitosan application ....................................................................................................... 83 2.4.4.1 Primers designed for defence marker genes ……............................ 86 2.4.4.2 Marker genes expression analysis using RT-qPCR ………..…........ 86 2.5 Discussion ................................................................................................... 91 2.5.1 Identification of chitosan as a promising resistance inducer in Pinus …......... 91 2.5.2 Other inducers that influenced disease severity ………………………........ ...... 93 2.5.3 Comparison of field versus greenhouse conditions ......................................... 94 2.5.4 The purpose of the booster application ............................................................ 95 2.5.5 The inoculation technique employed currently ……………………………......... 96 2.5.6 Phylogenetic approach taken to identify putative defence marker genes ........ 96 2.5.7 Defence responses elicited by chitosan application in P. patula …………....... 97 2.6 Conclusion and Future prospects…......................................................... 101 2.7 References ................................................................................................. 103 3. Concluding Remarks …............................................................................... 111 iv © University of Pretoria Supplementary Data Supplementary Data A............................................................................................... 114 Contig maps created from the ESTs obtained from ConiferGDB Supplementary Data B............................................................................................... 117 Initial neighbour joining phylogenetic trees constructed with the ESTs available from Pinus taeda as well contig maps Supplementary Data C............................................................................................... 127 Products of reference and target specidic primers analyzed on a 1.5% (w/v) agarose gel Supplementary Data D............................................................................................... 128 Stability results of the putative defence gene orthologs in P. patula. Supplementary Data E............................................................................................... 130 TBLASTX results of RT-qPCR targets against TAIR and the contig sequences Supplementary Data F............................................................................................... 132 Expression data for the selected putative defence marker orthologs for chitosan 10 mg/m treated P. patula Supplementary Data G.............................................................................................