Lau Ee Tiing Thesis
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The studies of defence-related transcriptome and potential biocontrol strategies in black pepper (Piper nigrum L.) By Lau Ee Tiing A thesis submitted to the Faculty of Engineering, Computing and Science, Swinburne University of Technology Sarawak Campus, Malaysia in fulfilment of the requirements for the degree of Doctor of Philosophy 2019 0 | P a g e ABSTRACT Black pepper is an important commodity crop in Malaysia to sustain livelihoods of rural dwellers. However, the crop is susceptible to several fungal pathogens. The primary aim of this study is to elucidate molecular pathways and genes involved in black pepper defence-related system through RNA-Sequencing. Transcriptome data were assembled into 81,096 unigenes. Gene ontolgy (GO) classification divided the annotated unigenes into 63 functional subgroups with 29 biological processes (BP), 18 cellular components (CC) and 16 molecular functions (MF). In total, 2,361 differentially expressed genes (DEGs) were detected. Of which, 1,426 DEGs showed higher expression in resistant Pc compared to susceptible PnSA and PnKch. These DEGs practically demonstrated the major branches of plant-pathogen interaction pathway (Path: ko04626). These branches result in activation of receptor genes for recognition of microbial elicitors and triggering cellular signalling genes for adaptive response. In general, no studies have been reported that black pepper plants are vulnerable to diseases caused by bacteria. This is probably due to genes in the bacteria causing plant-pathogen interaction pathway are activated in order to increase plant resistance when encounter bacterial infection. Therefore, this has shed some light in the next direction of utilising plant growth promoting rhizobacteria (PGPR) as a form of inducer in triggering expression of potential R-genes to improve disease resistance in black pepper plants. In order to describe the difference of potential R-genes between susceptible and resistant plants, qRT -PCR was performed to study the effects of pathogenic fungus F. solani isolate FS010 and beneficial rhizobacteria Br. gelatini isolate JD04 exposure on the expression levels of R-genes in pepper defence mechanism. Cf-9, the gene that is responsible for recognizing fungal virulence proteins is inexpressible in susceptible PnSA and PnKch. However, this gene has displayed a promising expression level in resistant Pc. Inactivation of Cf-9 has link to the inability of PnSA and PnKch to recognise F. solani at the initial stage of invasion, consequently delay in activation of adaptive response. Furthermore, significant expression of FLS2, a gene for recognition of bacterial flagellin has been detected in both species. Expression of FLS2 gene suggested that PGPR is could play a role as biocontrol agent and act as an inducer to trigger pepper immunity. In addition to this, a number of antagonistic PGPR have been isolated. These antagonists are capable in production of siderophore and chitinase. Moreover, they are effective ammonia producer and phosphate solubiliser. 1 | P a g e ACKNOWLEDGEMENT I would like to express my sincerest gratitude and appreciation to my supervisor, Assoc. Prof. Dr. Hwang Siaw San for her patience, persistent guidance, advice and inspiration throughout the progression of my research project as well as the completion of this dissertation. It is my honourable to have her as supervisor who shows endless care of my research works. I would like to thanks her valuable advice and diligence support in respond to my doubts at times of difficulties in my research works. Besides that, I also would like to thank Prof. Eiji Matsuura, my co-supervisor from Okayama University, Japan for his advice and support in the past three years. This study forms collaboration between Swinburne University of Technology Sarawak Campus, Okayama University, Japan and Malaysian Pepper Board. My sincere appreciation also goes to management team of Malaysian Pepper Board for providing me with the opportunity to further my Ph.D. degree in Swinburne University of Technology Sarawak Campus. T he financial support (Research Collaboration Grant 2 -5172) provided by Malaysian Pepper Board is also acknowledged. I also would like to extend my sincerest gratitude and acknowledgement to School of Research, Swinburne University of Technology Sarawak Campus for granting tuition fee waiver of my study. In addition, I also would like to thank Faculty of Engineering, Computing and Science for providing me with the necessities and resources to conduct this research. Last but not least, I also would like to express my sincerest gratitude to my parent, wife and family members for their understanding, patience, encouragement and moral support at time of my study. I also would like to thank my friends and lab assistants for their help, guidance and support in my work. 2 | P a g e DECLARATION BY CANDIDATE I, Lau Ee Tiing, higher degree research student of Doctor of Philosophy, from Faculty of Engineering, Computing and Science, Swinburne University of Technology Sarawak Campus hereby declare that the examinable outcome of this dissertation: a) Contains no material which has been accepted for the award to the candidate of any other degree or diploma, except where due reference is made in the text of the examinable outcome; b) To the best of the candidate’s knowledge contains no material previously published or written by another person except where due reference is made in the text of the examinable outcome; and c) Where the work is based on joint research or publications, discloses the relative contributions of the respective workers or authors. ……………………….. LAU EE TIING Student No.: 100080442 As the principal coordinating supervisor, I hereby acknowledge and certify that the above mentioned statements are legitimate to the best of my knowledge. ………………………………………………. (ASSOC. PROF. DR. HWANG SIAW SAN) 3 | P a g e TABLE OF CONTENTS Page Abstract ………………………………………………………………………....... 1 Acknowledgement ………………………………………………………………... 2 Declaration by Candidate ………………………………………………………… 3 Table of Contents ………………………………………………………………… 4 Publication and Presentation ……………………………………………………... 10 List of Figures ……………………………………………………………………. 11 List of Tables ……………………………………………………………………... 18 List of Abbreviations ……………………………………………………………... 20 Abbreviations of Scientific Name ………………………………………………... 25 Chapter 1: Introduction 1.1 Research Background …………………………………………………… 27 1.2 Statements of Research Problems ……………………………………….. 32 1.3 Research Aims and Objectives ………………………………………….. 33 1.4 Research Contributions and Impact to Society ………………………….. 34 Chapter 2: Literature Reviews 2.1 Black Pepper Plant ………………………………………………………. 35 2.1.1 Leaf …………………………………………………………….. 36 2.1.2 Inflorescence …………………………………………………... 37 2.1.3 Infructescence ………………………………………………….. 38 2.1.4 Stem and branch ……………………………………………….. 38 2.1.5 Root ……………………………………………………………. 39 2.1.6 Stolon and hanging shoot ……………………………………… 39 2.2 Establishment of Black Pepper Farms …………………………………... 40 2.2.1 Planting materials ……………………………………………… 40 2.2.2 Land preparation ……………………………………………..... 40 2.2.3 Drainage ……………………………………………………….. 41 2.2.4 Field planting ………………………………………………….. 41 2.2.5 Spacing ………………………………………………………… 42 4 | P a g e 2.2.6 Supporting pole ………………………………………………... 42 2.2.7 Pruning ………………………………………………………… 43 2.2.8 Fertilizing ……………………………………………………… 44 2.2.9 Harvesting and processing …………………………………….. 44 2.3 Cultivated Black Pepper Varieties in Malaysia …………………………. 45 2.3.1 Cultivar “Kuching” ……………………………………………. 46 2.3.2 Cultivar “Semengok Emas” …………………………………… 47 2.3.3 Cultivar “Semengok Aman” …………………………………… 48 2.4 Resistant Wild Pepper Plant …………………………………………….. 50 2.5 Pathogenic Organisms in Black Pepper Cultivation …………………….. 54 2.5.1 Major disease in Malaysia black pepper farms ………………... 54 2.5.1.1 Slow decline ………………………………………… 54 2.5.2 Other diseases in Malaysia black pepper farms ……………….. 58 2.5.2.1 Phytophthora foot rot ………………………………. 58 2.5.2.2 Black berries disease ………………………………... 60 2.5.2.3 White root rot ……………………………………….. 61 2.5.2.4 Velvet blight ………………………………............... 62 2.5.2.5 Stunted disease ……………………………………… 63 2.5.3 Minor diseases in Malaysia black pepper farms ………………. 64 2.5.3.1 Horse hair blight ……………………………………. 64 2.5.3.2 Pink disease ………………………………………… 65 2.5.3.3 White thread blight …………………………………. 66 2.5.4 Insect pests of Malaysia black pepper cultivation ……………... 66 2.6 Soil-borne Diseases ……………………………………………………… 68 2.6.1 Management of soil-borne diseases in Malaysia ……………… 69 2.6.2 Identification of soil-borne diseases causal fungus …………… 72 2.7 Genetic Research of cultivated black pepper plants in Malaysia ……….. 73 2.8 Biological Control ………………………………………………………. 75 2.9 Plant Defence Mechanisms ……………………………………………… 78 Chapter 3: Materials and Methods 3.1 Identification of Indigenous Causal Fungal Strains of Slow Decline …… 82 3.1.1 Isolation of soil-borne fungi …………………………………… 82 5 | P a g e 3.1.1.1 Field sampling ……………………………………… 82 3.1.1.2 Direct isolation of fungal strains from disease roots .. 83 3.1.1.3 Isolation of fungal strains from soil samples ……….. 83 3.1.2 Morphological characterization of isolated fungal strains …….. 84 3.1.3 Molecular identification of isolated fungal strains …………….. 84 3.1.3.1 Isolation of fungal DNA ……………………………. 84 3.1.3.2 Quality assessment of isolated fungal DNA ……….. 85 3.1.3.3 PCR amplification of fungal ITS regions …………... 85 3.1.3.4 Purification of PCR amplified ITS fragments ……… 86 3.1.3.5 Cloning of fungal ITS fragments ………………….... 87 3.1.3.6