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

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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, q RT-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.

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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. The 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.

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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)

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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

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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

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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 Recombinant plasmid DNA isolation ………………. 87 3.1.3.7 Sequence data analysis of fungal ITS fragments …… 88 3.1.4 Diagnostic of slow decline disease causal fungus ……………... 88 3.1.4.1 Pathogenicity assays of PnSA roots ………………... 88 3.1.4.2 Colonization assays of fungal cultures on PnSA roots 89 3.2 Elucidation of Defence-Related Mechanism Pathways in Pepper Plants .. 90 3.2.1 Plant materials …………………………………………………. 90 3.2.2 Fungal infection assays on pepper plant tissues ……………….. 90 3.2.2.1 Pathogenic agents …………………………………... 90 3.2.2.2 Leaves infections …………………………………… 91 3.2.2.3 Roots infections …………………………………….. 91 3.2.2.4 Fungal colonization on pepper plant tissues ………... 92 3.2.3 Total RNA isolation …………………………………………… 92 3.2.3.1 Purification of isolated total RNA ………………….. 93 3.2.3.2 Quality assessment of the isolated total RNA ……… 94 3.2.4 Transcriptomics analysis of pepper defence-related genes ……. 94 3.2.4.1 RNA sequencing ……………………………………. 94 3.2.4.2 De novo assembly of unigenes ……………………... 95 3.2.4.3 Functional annotation and KEGG classification …… 95 3.2.5 Detection of microsatellites ……………………………………. 96 3.2.6 Identification of reference genes ………………………………. 96 3.2.6.1 First-strand cDNA synthesis ………………………... 96 3.2.6.2 Quantitative real-time polymerase chain reaction ….. 97

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3.3 Identification of potential biocontrol agents for slow decline …………... 98 3.3.1 Isolation of rhizobacteria ………………………………………. 98 3.3.1.1 Field sampling ……………………………………… 98 3.3.1.2 Isolation of rhizobacteria from soils ………………... 99 3.3.2 Molecular identification of isolated rhizobacteria …………….. 100 3.3.2.1 Isolation of bacterial DNA ………………………….. 100 3.3.2.2 Quality assessment of isolated bacterial DNA ……... 100 3.3.2.3 PCR amplification of bacterial 16S rRNA genes …... 101 3.3.2.4 Purification of PCR amplified 16S rRNA genes …… 101 3.3.2.5 Cloning of bacterial 16S rRNA genes ……………… 102 3.3.2.6 Recombinant plasmid DNA isolation ………………. 102 3.3.2.7 Sequence data analysis of bacterial 16S rRNA genes 103 3.3.3 Biocontrol assays of isolated rhizobacteria ……………………. 103 3.3.3.1 Siderophore production …………………………….. 103 3.3.3.2 Chitinase production ………………………………... 104 3.3.3.3 Antagonistic effects to F. solani isolate FS010 …….. 104 3.3.4 Plant growth promotion traits of isolated rhizobacteria ……….. 105 3.3.4.1 IAA production ……………………………………... 105 3.3.4.2 Root growth assays …………………………………. 105 3.3.4.3 Phosphate solubilisation ……………………………. 106 3.3.4.4 Ammonia production ……………………………….. 106 3.4 Assessment on Pepper Defence-Related Genes …………………………. 107 3.4.1 Infection of pepper plant tissues with soil-microbes …………... 108 3.4.2 Expression study of targeted pepper defence-related genes …… 108

Chapter 4: Results 4.1 Identification of Indigenous Causal Fungal Strains of Slow Decline …… 109 4.1.1 Isolation of soil-borne fungi …………………………………… 109 4.1.2 Morphological characterization of isolated fungal strains …….. 110 4.1.3 Molecular identification of isolated fungal strains …………….. 114 4.1.3.1 Isolation and quality assessment of fungal DNA …... 114 4.1.3.2 PCR and sequence data analysis of ITS regions ……. 115 4.1.4 Diagnostic of slow decline disease causal fungus ……………... 117

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4.1.4.1 Pathogenicity assays of PnSA roots ………………... 117 4.1.4.2 Colonization assays of fungal cultures on PnSA roots 118 4.2 Elucidation of Defence-Related Mechanism Pathways in Pepper Plants .. 119 4.2.1 Fungal infection assays on pepper plant tissues ……………….. 119 4.2.2 Isolation and quality assessment of pepper total RNA ………... 120 4.2.3 Transcriptomics analysis of pepper defence-related genes ……. 121 4.2.3.1 RNA-Sequencing and de novo assembly …………… 121 4.2.3.2 Gene annotation and KEGG classification …………. 121 4.2.3.3 Species distribution analysis ………………………... 126 4.2.3.4 Differentially expressed genes (DEGs) …………….. 126 4.2.3.5 Characterization of pepper resistance gene analogues 129 4.2.4 Detection of microsatellites ……………………………………. 133 4.2.5 Identification of reference genes ………………………………. 137 4.3 Identification of potential biocontrol agents for slow decline …………... 140 4.3.1 Isolation of antagonistic rhizobacteria ………………………… 140 4.3.2 Molecular identification of isolated rhizobacteria …………….. 140 4.3.2.1 Isolation and quality assessment of bacterial DNA … 143 4.3.2.2 PCR and sequence data analysis of 16S rRNA genes 144 4.3.3 Biocontrol assays of isolated rhizobacteria ……………………. 147 4.3.3.1 Antagonistic effects to F. solani isolate FS010 …….. 147 4.3.3.2 Siderophore production …………………………….. 148 4.3.3.3 Chitinase production ………………………………... 149 4.3.4 Plant growth promotion traits of isolated rhizobacteria ……….. 152 4.3.4.1 IAA measurement …………………………………... 152 4.3.4.2 Root growth stimulation ……………………………. 154 4.3.4.3 Phosphate solubilisation ……………………………. 159 4.3.4.4 Ammonia production ……………………………….. 160 4.4 Assessment on Pepper Defence-Related Genes …………………………. 161 4.4.1 Expression study of targeted pepper defence-related genes …… 161

Chapter 5: Discussion 5.1 Identification of Indigenous Causal Fungal Strains of Slow Decline …… 165 5.1.1 Characterization of isolated soil-borne fungal strains …………. 165

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5.1.2 Diagnostic of slow decline disease causal fungus ……………... 166 5.2 Elucidation of Defence-Related Mechanism Pathways in Pepper Plants .. 168 5.2.1 Total RNA isolation …………………………………………… 168 5.2.2 Data assembly and gene sequence annotation …………………. 169 5.3 Identification of potential biocontrol agents for slow decline …………... 172 5.3.1 Isolation and molecular identification of rhizobacteria ………... 172 5.3.2 Biocontrol assays of isolated rhizobacteria ……………………. 172 5.3.3 Plant growth promotion traits of isolated rhizobacteria ……….. 174 5.4 Assessment on Pepper Defence-Related Genes …………………………. 176 5.4.1 Expression study of targeted pepper defence-related genes …… 176

Chapter 6: Conclusion ……………………………………………………………. 178

References ………………………………………………………………………... 179

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PUBLICATION AND PRESENTATION

Referred Journals 1) Lau ET, Khew CY, Hwang SS. Elucidation of biological pathways in black pepper defence mechanism through RNA-Sequencing. IPC Journal of Focus on Pepper 2018; 9(1): 26-40. 2) Lau ET, Angela T, Lai PS, Khew CY, Hwang SS. Identification of soil-borne fungi isolated from disease black pepper farms in Serikin and Serian areas of Sarawak, Malaysia. IPC Journal of Focus on Pepper 2018; 9(2): 23-35.

Conference Proceedings 1) Lau ET, Khew CY, Hwang SS. De novo transcriptome sequencing of black pepper (Piper nigrum L.). Proceeding of the 12th Malaysia International Genetics Congress. Bangi-Putrajaya Hotel, Kuala Lumpur, 25-27 September 2017. pp 68. 2) Lau ET, Khew CY, Hwang SS. De novo assembly of leaf transcriptome in black pepper (Piper nigrum L.). Proceeding of the Monash Science Symposium 2016. Monash University Malaysia, Kuala Lumpur, 21-23 November 2016. pp 159.

Presentation 1) Lau ET, Khew CY, Hwang SS. Research on black pepper resistance genes. MPB Officer Conference 2018. Sultan Iskandar Building, Kuching, 08 February 2018. 2) Lau ET, Khew CY, Hwang SS. Inhibitory effects of rhizobacteria in against soil- borne pathogens. Group Discussion Seminar. Okayama University, Japan, 15 September 2017 3) Lau ET, Khew CY, Hwang SS. Development of black pepper (Piper nigrum L.) genetic database. MPB Officer Conference 2016. Sultan Iskandar Building, Kuching, 08 November 2016.

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LIST OF FIGURES

Figure Description Page

1-1 The pie chart shows the world total export of black pepper from year 29 2008 to year 2017. The data were retrieved from the statistic figures of International Black Pepper Community (IPC) and Malaysian Black Pepper Industry.

2-1 Black pepper (Piper nigrum L.) plants of cultivated variety “Semengok 35 Aman” with 6 months old that cling on the supporting poles in field. The photo was taken from a healthy black pepper farm located at Sri Aman Division of Sarawak State.

2-2 Black pepper fruits, (a) Green pepper berries; (b) Black peppercorns of 38 commerce and (c) White peppercorns of commerce. The photo (a) was taken from a healthy black pepper farm located at Serian Division of Sarawak State. Meanwhile, photos (b) and (c) were retrieved from Malaysian Black Pepper Production Technology Manual (Lai and Sim, 2011).

2-3 The morphological appearances of cultivar “Kuching”, (a) Leaves and 46 flower spikes on the lateral branches and (b) Mature fruit spikes on the lateral branches. The photos were taken from a healthy black pepper farm located at Betong Division of Sarawak State.

2-4 The morphological appearances of cultivar “Semengok Emas”, (a) 47 Leaves on the lateral branches and (b) Flower and fruit spikes on the lateral branches. The photos were taken from a healthy black pepper farm located at Betong Division of Sarawak State.

2-5 The morphological appearances of cultivar “Semengok Aman”, (a) 49 Leaves on the lateral branches, (b) Flower spikes on the lateral branches and (c) Mature fruit spikes on the lateral branches. The photos were taken from a healthy black pepper farm located at Sri Aman Division of Sarawak State.

2-6 Resistant wild pepper plant, P. colubrinum Link, (a) Potted young 53 cuttings (3 months old); (b) Potted young cuttings (6 months old); (c) Mature leaves on the lateral branches and (d) flower spikes on the lateral branches. The photos were taken from the wild pepper germplasm maintained at Malaysian Pepper Board plant house.

2-7 Symptoms of slow decline found in Malaysia black pepper farms, (a) 56 Foliage yellowing of black pepper plant with slow decline; (b) Browning of vascular tissues due to Fusarium infection; (c) Formation of root galls due to infestation by root-rot nematode. The photos were taken from a disease black pepper farm located at Sri Aman Division of Sarawak State.

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2-8 Incidences of slow decline reported in black pepper farms situated at 57 Julau and Betong divisions of the State of Sarawak in the year 2018, (a) Farm damage due to slow decline; (b) Black pepper leaves with foliar yellowing symptom due to F. solani invasion; (c) Damage of black pepper plant collar region and loss of feeder roots due to F. solani invasion; (d) Black pepper root necrosis due to formation of gall-like lesions caused by parasitic nematodes and (e) Rotten of black pepper roots due to F. solani infection. The photos were taken from the disease black pepper farms located at Julau and Betong Divisions of Sarawak State.

2-9 Symptoms of Phytophthora root rot found in Malaysia black pepper 59 farms, (a) Formation of wet slimy dark patch at collar region of infected black pepper plant; (b) P. capsici infected black pepper plant with wilted leaves and heavy defoliation; (c) Foot rot-infected leaf with fimbriate-edge lesion; (d) Foot rot-infected fruit spike with brownish- black lesion and (e) Rotten of the foot rot-infected basal stem. The photos were taken from a disease black pepper farm located at Julau Division of Sarawak State.

2-10 The characteristic symptom of black berries disease (appearance of 61 black spots on the black pepper berries), a major disease found in Malaysia black pepper farms that caused by Colletotrichum spp. The photo was taken from a disease black pepper farm located at Betong Division of Sarawak State.

2-11 Developing of white fungal mycelial mats on black pepper roots, the 62 symptom of white root rot disease found in Malaysia black pepper farms. The photo was taken from a disease black pepper located at Bintulu Division of Sarawak State.

2-12 Formation of purplish-grey velvet-like incrustation on black pepper 63 fruit spike due to velvet blight disease, a major disease found in Malaysia black pepper farms. The photo was taken from a disease black pepper farm located at Serian Division of Sarawak State.

2-13 Mosaic pattern with light and dark green regions on black pepper leaves 64 due to stunted disease, a major disease found in Malaysia black pepper farms. The photo was taken from a disease black pepper farm located at Sri Aman Division of Sarawak State.

2-14 Horse hair-like mycelial rhizomorphs entangling black pepper leaves, 65 the symptom of horse-hair blight disease found in Malaysia black pepper farms. The photo was retrieved from Malaysian Black Pepper Production Technology Manual (Lai and Sim, 2011).

2-15 Formation of pink incrustations on black pepper plant stems, the 65 symptom of pink disease found in Malaysia black pepper farms. The photo was retrieved from Malaysian Black Pepper Production Technology Manual (Lai and Sim, 2011).

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2-16 Formation of distinctive white thread-like fungal mycelia on black 66 pepper leaves, the symptom of white thread blight disease found in Malaysia black pepper farms. The photo was retrieved from Malaysian Black Pepper Production Technology Manual (Lai and Sim, 2011).

2-17 Major insect pests found in Malaysia black pepper farms, (a) Black 67 pepper weevil; (b) Green black pepper bug and (c) Tingid bug. The photos were retrieved from Malaysian Black Pepper Production Technology Manual (Lai and Sim, 2011).

2-18 Minor insect pests found in Malaysia black pepper farms, (a) Flea 67 beetle; (b) Leaf hopper; (c) Aphids; (d) Mealy bugs and (e) Scale insects. The photos (a) – (d) were retrieved from Malaysian Black Pepper Production Technology Manual (Lai and Sim, 2011). Meanwhile, photo (e) was taken from a disease black pepper farm located at Serian Division of Sarawak State.

3-1 Sampling of slow decline disease causal fungal strains from black 83 pepper farms situated at Mongkos and Mujat villages of Serian Division and Jagoi Duyuh village of Serikin town, (a) Collection of soils and root samples from the rhizosphere; (b) Loss of feeder roots and damage at collar region of infected black pepper plant; (c) Browning symptom in vascular xylem tissues of infected plant.

3-2 Sampling of beneficial soil rhizobacteria from black pepper farms 99 located at Jagoi Duyuh and Jagoi Sebubok villages of Serikin town, Mongkos and Mujat villages of Serian Division and Karu village of Padawan district, (a) Healthy black pepper plants with good and vigorous growth; (b) Collection of soils samples from rhizosphere.

4-1 Disease symptoms of the black pepper plants with slow decline, (a) 109 Foliar yellowing, wilting at the shoot tips, heavy defoliation and die- back of the infected plant; (b) Loss of feeder roots and damage at the collar region; (c) Death of the infected plants; (d) Formation of root galls due to infestation by root-rot nematode; (e) Rotten of the infected black pepper roots; (f) Browning of vascular tissues due to Fusarium infection.

4-2 Microscopic images of the assessed fungi, (a) Microconidia of members 112 in Group FU-01; (b) Macroconidia of members in Group FU-01; (c) Hyphae of members in Group FU-02; (d) Septate hyphae of members in Group FU-02; (e) Sporangium of members in Group PH-01; (f) Oogonium of members in Group PH-01; (g) Thin walled hyphae of member in Group RI-01 and (h) Chlamydospore of member in Group RI-01.

4-3 Morphological appearances of the four fungal groups cultured on PDA 113 medium, (a) Group FU-01 (genus Fusarium); (b) Group FU-02 (genus Fusarium); (c) Group PH-01 (genus Phytophthora) and (d) Group RI- 01 (genus Rigidoporus).

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4-4 1.0% (w/v) agarose gel electrophoresis image of the isolated fungal 114 DNA samples, Lane L1: isolate RM001; Lane L2: isolate FS002; Lane L3: isolate FS006; Lane L4: isolate FS009; Lane L5: isolate FS008; Lane L6: isolate PN001; Lane L7: isolate PN002; Lane L8: isolate FS001; Lane L9: isolate FS003; Lane L10: isolate FS004; Lane L11: isolate FS005; Lane L12: isolate FS007; Lane L13: isolate FS010.

4-5 1.5% (w/v) agarose gel electrophoresis images of fungal ITS fragments, 116 (a) Member isolates in Group FU-01; (b) Member isolates in Group FU-02; (c) Member isolates in Group PH-01 and (d) Isolate RM001, the only member isolate in Group RI-01. M: Promega 100 bp DNA ladder.

4-6 The symptoms of slow decline showed by infected black pepper 117 cuttings in diagnostic assays, (a) Control treatment; (b) Foliar yellowing after a month of infection, (c) Healthy roots and (d) Feeder roots loss and damage at collar region after three months of infection.

4-7 Electrophoresis image of the isolated total RNA evaluated by using 121 Agilent 2100 Bioanalyzer System, Lane L: RNA ladder; Lane S1: Pc- N1; Lane S2: Pc-T1; Lane S3: Pc-N2; Lane S4: Pc-T2; Lane S5: PnSA- N1; Lane S6: PnSA-T1; Lane S7: PnSA-N2; Lane S8: PnSA-T2; Lane S9: PnKch-N1; Lane S10: PnKch-T1; Lane S11: PnKch-N2 and Lane S12: PnKch-T2.

4-8 Summary of sequence homology search against NCBI nr and EBI 123 InterPro protein databases. The Venn diagram described the number of unigenes that have been annotated and aligned to the known proteins in both databases.

4-9 GO classifications of the annotated unigenes into three major groups 124 and 63 subgroups. The x-axis shows the percentage of unigenes in specific subgroups. The y-axis shows the subgroups in GO annotation.

4-10 Species distribution analysis based on blast top-hits of unigene 126 sequence homology searches against the known proteins in NCBI nr protein database.

4-11 The pie chart shows the majority of the DEGs were associated to plant- 127 pathogen interactions pathway (Path: ko04626) and plant MAPK signalling pathway (Path: ko04016).

4-12 Black pepper RGA aligned by using ClustalW algorithm. Conservative 132 substitutions are shaded using online Boxshade server. Sequence comparison shows the presence of conserved motifs Kinase-1/P-loop (GMGGVGK), Kinase-2 (VLDDVW) and Kinase-3/GLPL, indicated by letters on top of the alignments.

4-13 The summary of SSR loci has been detected in transcriptome data. In 134 total, 5,012 SSRs were detected in 4,693 unigenes. The x-axis indicates the repeat type. The y-axis indicates the number of different repeats.

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4-14 Distribution of the defined SSR motifs in different groups of black 134 pepper defence-related genes. The x-axis indicates the different groups of defence-related unigenes. The y-axis indicates the number of SSR loci.

4-15 The cycle threshold (Ct) values of six assessed internal control genes, 138 (a) i.e. elongation factor 1-α (ef1α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-tubulin, histone 3 (H3), actin and ubiquitin 7 (UBQ7) in pepper roots tissues.

4-15 The cycle threshold (Ct) values of six assessed internal control genes, 139 (b) i.e. elongation factor 1-α (ef1α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-tubulin, histone 3 (H3), actin and ubiquitin 7 (UBQ7) in pepper leaves tissues.

4-16 The average expression stability values of six assessed internal control 139 genes, i.e. elongation factor 1-α (ef1α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-tubulin, histone 3 (H3), actin and ubiquitin 7 (UBQ7). The lowest M value of Histone 3 (H3) indicated that this gene has exhibited the most stable expression in pepper plant tissues.

4-17 Phylogenetic relationship of 46 isolated rhizobacteria based on their 142 16S rRNA gene sequences. The phylogenetic tree was constructed by using Molecular Evolutionary Genetics Analysis 6.0 (MEGA6). The isolates have been grouped into three main clusters, i.e. Cluster 1: genus Burkholderia with 25 isolates, Cluster 2: genus Bacillus with 13 isolates and Cluster 3: genus Pseudomonas with 8 isolates. Numbers at each node represent bootstrap values as percentage of 100 based on 1,000 replications. Bootstrap values greater than 70% are shown. The isolates selected for plant growth-promotion analysis were bolded and highlighted.

4-18 1.0% (w/v) agarose gel electrophoresis image of the isolated bacterial 143 DNA, Lane L1: isolate JD04; Lane L2: isolate JS02; Lane L3: isolate JS05; Lane L4: isolate MO02; Lane L5: isolate MO09; Lane L6: isolate MU03; Lane L7: isolate MU07.

4-19 1.5% (w/v) agarose gel electrophoresis of PCR amplified bacterial 16S 145 rRNA gene fragments. Lane M: Promega 100 bp DNA ladder.

4-20 The antagonistic effects to F. solani isolate FS010 mycelium radial 147 growth that exhibited by the assessed rhizobacterial isolates, (a) Br. gelatini isolate JD04; (b) B. ubonensis isolate MO02; (c) Ba. subtilis isolate MU03 and (d) Control plate.

4-21 Production of siderophores exhibited by the assessed rhizobacteria on 149 CAS agar plates, (a) A colour change of CAS medium from blue to orange exhibited by the Gram-negative P. geniculata isolate JS02 on common CAS agar plate indicated siderophores production; (b) Gram- positive Br. gelatini isolate JD04 on Overlaid-CAS agar plate at 0 hour

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of incubation and (c) A colour change of CAS medium from blue to orange exhibited by the Gram-positive Br. gelatini isolate JD04 on Overlaid-CAS agar plate after 24 hours of incubation at 30°C indicated siderophores production.

4-22 Chitinolytic activities showed by two bacterial isolates of genus 150 Pseudomonas on the chitin agar plates after 16 hours of incubation at 30°C, (a) P. geniculata isolate JS02 and (b) P. beteli isolate JS05. The presence of clear zones surrounding bacteria colonies on agar plates indicated chitinase production.

4-23 The presence of pink colour in Salkowski reagents produced by all the 152 assessed rhizobacteria indicated IAA production, (a) Br. gelatini isolate JD04; (b) Ba. subtilis isolate MU03; (c) Ba. siamensis isolate MU07; (d) P. geniculata isolate JS02; (e) P. beteli isolate JS05; (f) B. ubonensis isolate MO02 and (g) B. territorii isolate MO09.

4-24 Root germination of black pepper cuttings stimulated by the two 155 greatest IAA producing rhizobacteria in the current study at the end of the trials, (a) PnSA treated by Br. gelatini isolate JD04; (b) PnSA treated by P. geniculata isolate JS02; (c) PnSA in control treatment; (d) PnKch treated by Br. gelatini isolate JD04; (e) PnKch treated by P. geniculata isolate JS02 and (f) PnKch in control treatment.

4-25 Effects of IAA producing rhizobacteria on the increment of chlorophyll 158 content in black pepper leaves at 3rd, 5th, 10th and 15th day of the trials. Experiments were conducted in triplicates with 10 cuttings per treatment. The x-axis shows the rhizobacteria isolates. The y-axis shows the percentage of chlorophyll content. Data means with same letter on the top of each column are not significantly different. However, means with different letters within each column are differs significantly at p≤0.05.

4-26 Phosphate solubilizing activities exhibited by the two members of 159 rhizobacteria in genus Burkholderia on Pikovskayas agar plates after 16 hours of incubation at 30°C, (a) B. ubonensis isolate MO02 and (b) B. territorii isolate MO09. The presence of clear zone surrounding bacterial colonies on Pikovskayas agar medium indicates phosphate solubilizing acid production.

4-27 The colour change of bacterial cell free supernatants from light yellow 160 to deep yellow in nesslerization assays after two days of incubation at 30°C, (a) P. geniculate isolate JS02; (b) B. ubonensis isolate MO03 and (c) Ba. siamensis isolate MU07. The sterile un-inoculated peptone water (light yellow solution) was served as reference in nesslerization assays. The colour change of peptone water from light to deep yellow indicates ammonia production.

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4-28 The expression level of pepper defence-related genes, i.e. Cf-9, FLS2, 162 MEKK1, PR1 and RGA2 in roots of susceptible PnSA and PnKch as well as resistant Pc infected by pathogenic F. solani isolate FS010 (Trial 1, Fs-treated), beneficial Br. gelatini isolate JD04 (Trial 2, Br- treated) and a combination of the two soil microbes infection (Trial 3, Br-Fs-treated). The y-axis represents Log2 values of gene expression fold change. Error bars denote the standard error of gene expression level.

4-29 The expression level of pepper defence-related genes, i.e. Cf-9, FLS2, 163 MEKK1, PR1 and RGA2 in leaves of susceptible PnSA and PnKch as well as resistant Pc infected by pathogenic F. solani isolate FS010 (Trial 1, Fs-treated), beneficial Br. gelatini isolate JD04 (Trial 2, Br- treated) and a combination of the two soil microbes infection (Trial 3, Br-Fs-treated). The y-axis represents Log2 values of gene expression fold change. Error bars denote the standard error of gene expression level.

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LIST OF TABLES

Table Description Page

1-1 Quantity of Malaysian black pepper rejected by importer countries in 32 metric tonnes (MT) due to high residues level.

2-1 List of black pepper (P. nigrum L.) accessions maintained in Semengok 45 Agriculture Research Centre, Department of Agriculture, Sarawak.

2-2 Recommended pesticides for soil-borne diseases found in black pepper 71 farms. The information was retrieved from Malaysian Black Pepper Production Technology Manual (Lai and Sim, 2011).

4-1 Soil-borne fungi isolated from disease farms DF1, DF2 and DF3 located 111 at Serikin and Serian areas of Sarawak.

4-2 The yield and purity range of the isolated fungal genomic DNA assessed 115 at spectrum wavelengths of 230 nm, 260 nm and 280 nm with a dilution factor of 100.

4-3 Colonization of F. solani isolate FS010 in pepper leaves and root tissues 120 at the end of pathogenicity assays.

4-4 The overview results of pepper transcriptome sequencing and assembly. 122

4-5 List of 125 KEGG pathways mapped by the annotated unigenes. 125

4-6 Number of DEGs that showed higher expression levels with log2 fold 128 change ≥ 1 in resistant P. colubrinum Link.

4-7 List of 232 black pepper RGA that shared similarities with the resistance 130 proteins published in NCBI nr protein database.

4-8 Summary of SSR searching results by screening transcriptome data. 133

4-9 Defined SSR repeat units in the black pepper defence-related unigenes. 135

4-10 Summary of primer sequences for six assessed reference genes, melting 138 temperature, amplicon length and efficiency of PCR runs.

4-11 Morphology, gram nature and antifungal effects of the isolated bacteria. 141

4-12 The yield and purity range of the isolated bacterial genomic DNA 144 assessed at spectrum wavelengths of 230 nm, 260 nm and 280 nm with a dilution factor of 100.

4-13 Molecular identification of selected bacterial isolates through sequence 146 homology analysis of 16S rRNA genes.

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4-14 Siderophore production, chitinase production and antifungal activity 151 exhibited by the selected bacterial isolates.

4-15 Phosphate solubilisation, ammonia production, IAA production and root 153 growth stimulation exhibited by the selected bacterial isolates.

4-16 Chlorophyll concentration (μmol per m2) of black pepper leaves 156 measured during 0th, 3rd, 5th, 10th and 15th day of the trials.

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LIST OF ABBREVIATIONS

AI Active Ingredient AMV Avian Myeloblastosis Virus ARC Agriculture Research Centre ATP Adenosine triphosphate BioloMICS Biological Data Management, Identification, Classification and Statistics Blastx Basic Local Alignment Search Toolx BP Biological Processes Ca Calcium

CaCl2 Calcium chloride

Ca3O8P2 Tricalcium phosphate CAS Chrome Azurol S CC Cellular Components cDNA complementary DNA Cf-9 Receptor-like protein

CH3COOH Glacial acetic acid

CH3CO2K Potassium acetate

CH6ClN3 Guanidine hydrochloride CI Chitinolytic Index C: I Chloroform: Isoamyl alcohol CMV-Pn Piper nigrum L. strain of Cucumber Mosaic Virus

Ct Cycle threshold CTAB Cetyltrimethylammonium bromide Cultivars Cultivated varieties PnKch Piper nigrum L. cultivar Kuching PnSA Piper nigrum L. cultivar Semengok Aman ddH2O double distilled water DEG Differentially expressed gene DEPC Diethyl pyrocarbonate DF 1-3 Disease farms 1-3 DNA Deoxyribonucleic acid dNTP Nucleoside triphosphate

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DoA Department of Agriculture DTT Dithiothreitol EBI European Bioinformatics Institute EDTA (Ethylenedinitrilo)tetraacetic acid ef1α elongation factor 1-α EGTA Ethylene glycol bis(2-aminoethyl ether)-N, N, N ‘N’-tetraacetic acid EST Expressed Sequence Tag ETI Effector-Triggered Immunity

FeCl3 Iron (III) chloride

FeSO4 Iron (II) sulphate Flg Flagellin FLS2 LRR receptor-like serine threonine-kinase protein GAP Good Agricultural Practices GAPDH Glyceraldehyde-3-phosphate dehydrogenase GDP Gross Domestic Product GI GenInfo GO Gene Ontology H3 Histone 3 HCl Hydrochloric acid HDTMA Hexadecyltrimethylammonium bromide HF1-5 Healthy farms 1-5 HR Hypersensitive response IAA Auxin/Indole-3-acetic acid IDM Integrated Disease Management IPC International Black pepper Community IPTG Isopropyl-β-D-thiogalactopyranoside ISR Induced Systemic Resistance (ISR) ITS Internal Transcribed Spacer K Potassium KCl Potassium chloride KEGG Kyoto Encyclopedia of Genes and Genomes

KH2PO4 Monopotassium phosphate

K2HPO4 Dipotassium phosphate

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LB Luria-Bertani LiCl Lithium chloride LRR Leucine-Rich Repeat M Reference gene stability factor MAPK Mitogen-Activated Protein Kinase MAS Marker assisted selection MEKK1 Mitogen-activated protein kinase kinase kinase 1 MF Molecular Functions Mg Magnesium

MgCl2 Magnesium chloride

MgSO4 Magnesium sulphate MISA MIcroSAtellite MM9 Minimal Media 9 MPB Malaysian Pepper Board MRLs Maximum Residue Levels mRNA Messenger RNA MSL Mean Sea Level MT Metric tonnes N Nitrogen NaCl Sodium chloride NaOAc Sodium acetate NaOCl Sodium hypochlorite NaOH Sodium hydroxide NBS Nucleotide-Triphosphate Binding Site NCBI National Center for Biotechnology Information NGS Next-Generation Sequencing

NH4Cl Ammonium chloride

(NH4)2SO4 Ammonium sulphate NPK Nitrogen-Phosphorus-Potassium Nr Non-redundant O-CAS Overlaid CAS P Phophorus P&D Pests and Diseases

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Pc Piper colubrinum Link PDA Potato Dextrose Agar PAMP Pathogen-Associated Molecular Pattern P: C: I Phenol: Chloroform: Isoamyl alcohol PCR Polymerase Chain Reaction PGPR Plant Growth Promoting Rhizobacteria PIRG Percentage of Inhibition of Radial Growth PIPES Piperazine-N,N′-bis(2-ethanesulfonic acid) PR1 Pathogenesis-related protein 1 PRRs Pattern Recognition Receptors PTI PAMP-Triggered Immunity PVP Polyvinyl pyrolidone qRT-PCR Quantitative real-time polymerase chain reaction rDNA Ribosomal DNA RGA Resistance Gene Analogues RGA2 Disease resistance protein RGA2 R-gene Resistance gene RH Relative Humidity RIN RNA Integrity Number RNA Ribonucleic acid RNA-Seq RNA-Sequencing RPKM Reads per kilo base per million rRNA Ribosomal RNA SAR Systemic Acquired Resistance SDS Sodium dodecyl sulfate SI Solubilizing Index SSR Simple sequence repeat TBE Tris-borate-EDTA TE Trace elements TIR Toll/Interleukin-1 receptor

Tm Melting temperatures Tris-HCl Tris hydrochloride UBQ7 Ubiquitin 7

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Unigene Unique gene WEGO Web Gene Ontology Annotation Plot X-Gal 5-bromo-4-chloro-3-indoyl-ß-D-galactopyranoside YEM Yeast-Extract-Mannitol

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ABBREVIATIONS OF SCIENTIFIC NAME

A. pintoi Arachis pintoi Ba. siamensis Bacillus siamensis Ba. subtilis Bacillus subtilis Br. gelatini Brevibacillus gelatini B. orientale Blechnum orientale B. territorii Burkholderia territorii B. ubonensis Burkholderia ubonensis C. capsici Colletotrichum capsici C. gloeosporioides Colletotrichum gloeosporioides C. piperis Colletotrichum piperis C. pubescens Centrosema pubescens C. salmonicolor Corticium salmonicolor C. sinensis Citrus sinensis D. curanii Dicranopteris curanii D. lineari Dicranopteris lineari D. suffruticosa Dillenia suffruticosa E. guineensis Elaeis guineensis E. zwageri Eusideroxylon zwageri F. solani Fusarium solani G. sepium Gliricidia sepium G. truncate Gleichenia truncate H. ammodendron Haloxylon ammodendron I. cylindrica Imperata cylindrica M. scandens Marasmiellus scandens M. equicrinis Marasmius equicrinis M. incognita Meloidogyne incognita N. nucifera Nelumbo nucifera P. beteli Pseudomonas beteli P. capsici Phytophthora capsici P. colubrinum Link Piper colubrinum Link P. dactylifera Phoenix dactylifera P. geniculata Pseudomonas geniculata

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P. nigrum L. Piper nigrum L. R. lignosus Rigidoporus lignosus R. microporus Rigidoporus microporus R. similes Radopholus similes R. soongorica Reaumuria soongorica V. vinifera Vitis vinifera

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CHAPTER 1

INTRODUCTION

1.1 Research Background

Black pepper, also known as “King of Spices”, is the oldest spice crop in the world. It is a perennial climbing woody plant in family Piperaceae. Black pepper is cultivated for its fruit, known as peppercorn which is very popular among spices since ancient times. It is the most important spice traded internationally, accounting for almost one-third of the total volume and total value of global spice trade (Weiss, 2002; Parthasarthy, 2008). Peppercorn is usually dried and used as seasoning in food flavouring (Nisha et al., 2007). Black pepper is known to have human health promotion and diseases prevention properties. It is used in traditional medicine since centuries for their anti-inflammatory and anti-flatulent properties. Peppercorn, the source of vitamin K and B-complex group of vitamins is rich in antioxidants that protect human body from cancers and diseases by removing harmful radicals (Srinivasan, 2007; Dawid et al., 2012; Dudhatra et al., 2012). Peppercorn is also rich in nutrient minerals such as manganese, magnesium, potassium, iron, copper, zinc, calcium, chromium, selenium and phosphorus according to Meghwal and Goswami (2012). Piperine, the volatile-oils that contributes to strong aroma and pungency of peppercorns also has been widely studied for its nutritional and medicinal properties (Jin and Han, 2010; Islam et al., 2015; Gorgani et al., 2017; Zihan et al., 2017). With its various uses and valuable properties, black pepper has secured a pivotal position in food and non-food industries, especially in pharmaceutical, cosmeceutical, perfumery and health care products development. It is expected to have better prospect in the years to come.

Apart of its nutritional and medicinal values, black pepper is also socially importance because of its high economic properties, particularly in sustaining livelihoods of rural dwellers in developing nations. Black pepper is a small farmer’s crop. It is widely cultivated in Vietnam, Indonesia, Brazil, India, Malaysia, Sri Lanka, China, Thailand and Madagascar. Other countries such as Fiji, Brunei, Zimbabwe, Malawi, Cambodia,

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Zambia and Kenya cultivate black pepper in a small scale. Black pepper is commonly grown as monocrop in most of the black pepper producing countries. However, it also has been grown as mixed crop in coconut, coffee and areca nut plantations mainly in India. Currently, Malaysia is the fifth largest black pepper producer in the world, after Vietnam, Indonesia, Brazil and India.

Black pepper is cultivated in Malaysia for a very long time (Kamarulzaman et al., 2013) and this is focused mainly in the state of Sarawak. Black pepper cultivation in Sarawak can date back to the year 1875. However, systematic and more extensive planting of black pepper in Sarawak was initiated in the year 1900 and within two decades, black pepper planting had spread over a wide area in Sarawak. It has become an important and popular cash crop in Sarawak for smallholder farmers with an average farm size of 0.2 hectare. Black pepper cultivation was concentrated in certain regions of Kuching, Kota Samarahan, Serian, Sri Aman, Betong, Sarikei, Sibu, Bintulu, Julau and Miri Divisions. Sarawak state, with a total black pepper cultivated area of 16,700 hectares has contributes to 98.2% of the country total black pepper production. The crop has provides stable income for 33,000 dwellers in the rural areas of Sarawak, and some in Johor and Sabah. Black pepper was initially planted by Chinese farmers in Malaysia in the early years. However, native farmers mostly the Iban and Bidayuh have made-up about 90% of the amount of total black pepper farmers in recent years due to assistance schemes provided by Malaysian Government.

Today, black pepper has become an important commodity crop in Malaysia. There is about 95% of the Malaysian black pepper is traded in the form of black peppercorns and white peppercorns. Meanwhile, the remaining 5% of Malaysia traded black pepper is made-up of ground black pepper and white pepper powders. Sarawak pepper, the trade name commercially used to promote Malaysian black pepper is a renowned brand worldwide with its quality ranked among the best of all black pepper producing countries. With a total export of 128,844 metric tonnes as shown in Figure 1-1, black pepper has produced RM3.5 billion of revenue for the country in the past ten years. This volume had contributed 4.5% of the world total export (2,861,739 metric tonnes). Black pepper industry is expected to contribute 0.1% of the country total GDP in the coming years and to generate RM4.5 billion worth of exports by the year 2020.

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Others Sri Lanka 145,732 MT 107,790 MT (5.09%) Malaysia (3.77%) Vietnam 128,844 MT 1,399,338 MT (4.50%) (48.90%)

Indonesia 501,245 MT (17.52%) Brazil India 358,348 MT 220,442 MT (12.52%) (7.70%)

Figure 1-1. The pie chart shows the world total export of black pepper from year 2008 to year 2017. The data were retrieved from the statistic figures of International Black Pepper Community (IPC) and Malaysian Black Pepper Industry.

However, black pepper is a very challenging crop to grow as compared to other world commodities. This is because black pepper is high nutrient demanding crop, sensitive to soil pH and moisture, susceptible to insect pests and diseases besides requiring intensive labour for cultivation. Currently, black pepper production is steadily declining in most of the producing countries. There are a number of constraints faced by farmers in black pepper cultivation. One of the major constraints is yield loss due to farm damage caused by insect pests and diseases (P&D). Diseases are more severe in contribution of farm loss than insect pests and are vary across different countries. Soil-borne diseases due to invasion of several soil-borne fungi have been reported as the most critical disease in black pepper cultivation. The diseases were reported to cause 20% to 30% annual yield loss in Vietnam, Indonesia, India and Malaysia in the past decades. Based on the figures of Malaysian Black Pepper Industry Statistic, yield loss due to invasion of soil-borne fungi in Malaysia black pepper farms is about 25% of the country total black pepper production in the past five years. Therefore, the effort to overcome soil-borne diseases is an effective strategy to improve black pepper farms production. A better knowledge

29 | P age and understanding about diverse diseases, invasion mechanisms, disease symptoms, timely diagnosis, crop resistance and interactions with pathogens would contribute to successful management of soil-borne diseases in black pepper cultivation.

With the aims to promote sustainable development of country economy, while to reduce the number of poor farmers, particularly in the State of Sarawak, Malaysian government has planned to increase black pepper annual production from the current 31,000 metric tonnes per year to 35,500 metric tonnes in the year 2020. This means that there is a need of 14.5% annual yield increment . With the implementation of current Good Agricultural Practices (GAP), farmers are expecting a minimum yield of 2 kg dried berries per plant of black pepper. However, the recent yield per plant of black pepper in Malaysia is 1.85 kg dried berries on average because of the farms damages which mainly caused by soil- borne pathogens.

With the planting density of 2,000 plants per hectare on average, it is forecasted that a minimum yield of 0.3 metric tonnes per hectare of black pepper farm was loss because of the soil-borne diseases. This figure has brought to an estimation of about RM84.6 million loss of country revenue in the year 2018 as the total black pepper production area in Malaysia for the particular year is 16,787 hectares. Therefore, improvement of soil-borne diseases management in black pepper cultivation is essential to minimize the yield loss and ensure high outputs as well as quality production in black pepper farms. Implementation and adoption of Integrated Disease Management (IDM) Program in black pepper cultivation by combining crop diseases resistance, cultural and biological control strategies is recommended as rational and holistic approach to manage soil- borne diseases and ensure high output and quality black pepper production in Malaysia.

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1.2 Statements of Research Problems

Black pepper cultivation in Sarawak is restricted by soil-borne diseases that have caused significance yield loss and affect the quality of farm production. Although the precise loss estimation due to soil-borne diseases is still lacking, the diseases were reported to cause 20% to 30% annual yield loss in most of the black pepper producing countries such as Vietnam, India, Indonesia and Malaysia (Samraj and Jose, 1966; Nambiar and Sarma, 1977; Sastry, 1982; Dutta, 1984; Thangaselvabal et al., 2008; Nguyen and Bui, 2011). Therefore, effective management of soil-borne diseases is essential to minimize black pepper farm loss and improve the quality of farm production. A better knowledge and understanding about the causal fungi of soil-borne diseases is crucial for effective management of the diseases. However, the genetic studies of soil-borne diseases causal fungi in Malaysia are still in developmental stage. There is little information is known about the genome of these pathogenic fungal strains and their mechanisms of invasion. Therefore, research study need to be carried out to isolate and identify the indigenous soil-borne diseases causal fungi in Malaysia black pepper farms.

Traditional black pepper cultivation practices in Malaysia are still relies on chemical products to sustain yield and control insect pests and diseases (Pal and McSpadden, 2006). Hazard chemical products such as pesticides, fungicides and nematicides appear to be the best solution to overcome insect pests and diseases in black pepper cultivation for spectacular improvements in the productivity. However, the persistence of hazard chemical residues in peppercorns can affect the quality of exports and consumptions. Table 1-1 has shown the quantity of exported Malaysian black peppers that has been rejected by importer countries in the past five years due to high Maximum Residue Levels (MRLs). Besides that, excessive use or misuse of hazard chemical products also contribute to the environmental pollution and affects our ecological systems.

Apart from that, application of chemical fertilizers to sustain the yield of black pepper farms can cause soil acidification in the long run and subsequently restrict the nutrient uptake by black pepper roots. Moreover, with the high rainfall distribution in Sarawak throughout the year, nutrient leaching has become a major environmental issue in black pepper farms as majority of the black pepper farms in Sarawak are located at hilly areas. Nutrient leaching not only increases the cost and quantity of fertilizer inputs required 31 | P age for farm maintenance, it also brings to the pollution of ground and surface water due to nitrate runoff. Consequently, this has led to considerable of change in using hazard chemical products to control insect pests and diseases as well as to sustain yield of black pepper farms.

Disease resistance has been widely explored in many plant species (Ribeiro do Vale et al., 2001; Gururani et al., 2012; Andersen et al., 2018). However, global genomic study on black pepper is still in developmental stage. Lack of genetic information has become a major constraint in production of black pepper cultivars (cultivated varieties) with desired agronomic traits such as diseases resistance. Currently, few black pepper cultivars are recommended for planting in Malaysia. Each cultivar has its own prevalent characteristics and drawback. The cultivars “Semengok Aman” and “Kuching” are the most widely planted varieties in Malaysia. Peppercorns of these two cultivars are highly suitable to be used for production of premium black pepper and creamy white pepper products. However, these two cultivars are highly susceptible to various soil-borne diseases that have caused significance farm loss every year.

Table 1-1. Quantity of Malaysian black pepper rejected by importer countries in metric tonnes (MT) due to high residues level. Year Quantity Residues Level Standard (MT) MRLs; in ppm 2014 15 Thiamethoxam: 0.230 ppm 0.050

2015 0.5 Carbendazim 0.300 ppm 0.100

2016 33 Carbendazim: 0.200 ppm 0.100 Fipronil: 0.005 ppm 0.002 Difenoconazole: 0.080 ppm 0.050

2017 51.5 Carbendazim: 0.200 ppm 0.100 Fipronil: 0.012 ppm 0.002 Difenoconazole: 0.060 ppm to 0.110 ppm 0.050 Thiamethoxam: 0.090 ppm to 0.100 ppm 0.050

2018 41 Thiamethoxam: 0.060 ppm 0.050 Difenoconazole: 0.100 ppm 0.050

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1.3 Research Aims and Objectives

The current research was designed by focusing on three main aims. The primary aim of this study is to elucidate the molecular pathways and genes involved in pepper defence- related mechanisms through RNA-Sequencing (RNA-Seq). In order to obtain an overall and comprehensive transcriptome dataset of pepper plants, the experimental works were designed to fulfil the objectives:

i. RNA-Sequencing of disease susceptible cultivars “Semengok Aman” and “Kuching” as well as disease resistant wild pepper plant, P. colubrinum Link. ii. De novo assembly, functional annotation and detection of microsatellite in pepper transcriptome dataset. iii. Identification of mechanism pathways and expression analysis of genes related to pepper defence response.

The secondary aim of this study is to identify indigenous causal fungal strains of slow decline, which is the most critical soil-borne disease in Malaysia black pepper farms. In order to achieve the aim, experimental works were conducted to fulfil the objectives:

i. Isolation of soil-borne fungal strains from slow decline infected black pepper farms. ii. Molecular identification of isolated fungi through sequences homology analysis of fungal internal transcribed spacer (ITS) regions. iii. Diagnostic of slow decline causal fungal strains through fungal virulence tests and colonization assays.

The tertiary aim of this study is to propose alternative solution for slow decline through application of biocontrol strategies instead of the current practices which are relied on the utilization of hazard chemical products in soil-borne disease managements. In order to achieve the aim, experimental works were conducted to fulfil the objectives:

i. Isolation and identification of plant growth promoting rhizobacteria (PGPR). ii. Evaluation of plant growth promoting trait and antagonistic effect in isolated PGPR. iii. Analysis of the effects of soil microbe’s exposure on the expression levels of R- genes in pepper defence mechanism. 33 | P age

1.4 Research Contributions and Impact to Society

The outcomes of the current study were expected to: a) Establish first transcriptome dataset for local cultivated black pepper varieties that could serve as the fundamental reference for future functional genomics studies of other black pepper cultivars in Malaysia. b) Provide new perspectives to manage soil-borne diseases in Malaysia and hints on potential agronomic traits for future crop improvement program in black pepper. c) Identify indigenous slow decline causal organism to aid in future genomics analysis of this pathogenic fungus and study on their mechanisms of invasion. d) Identify antagonistic plant growth promoting rhizobacteria which are potent to be developed as biocontrol agent and biofertilizer. e) Promote bio-farming in black pepper cultivation to sustain soil health and prevent eco-system from the harmful effects of hazard chemical products. f) Ensure high output and quality farm production in order to promote sustainable development of black pepper industry in Malaysia.

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CHAPTER 2

LITERATURE REVIEWS

2.1 Black Pepper Plant

Black pepper plant as shown in Figure 2-1 with its botanical name Piper nigrum L. is native to the Western Ghats of the Kerala State in India (Damanhouri and Ahmad, 2014). This crop has subsequently spreaded to other countries through secondary introduction where it is extensively planted. Black pepper plant thrives best in tropical countries with moist and warm climate as well as Relative Humidity (RH) over 70% (Sen and Colleen, 2004; Hajeski and Nancy, 2016). The plant grows well at elevations from 50 m up to 1,500 m above the Mean Sea Level (MSL).

Figure 2-1. Black pepper (Piper nigrum L.) plants of cultivated variety “Semengok Aman” with 6 months old that cling on the supporting poles in field. The photo was taken from a healthy black pepper farm located at Sri Aman Division of Sarawak State.

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The robust and luxurious growth of black pepper plant is seen on fertile, flat or gently sloping land with not exceeding 10° which is rich in humus, with good drainage and proper light shade as well as soil pH in the range from 5.5 to 6.5. The preferable temperature range for black pepper plant is from 23°C to 32°C with 28°C is the optimum temperature for its growth. Land initially planted with cocoa and rubber trees should be avoided or used with precautions for black pepper cultivation as these areas might have been infected by soil-borne diseases. As a rainfed crop, black pepper plant requires adequate and evenly distributed annual rainfall of about 2,000 mm to 3,000 mm. Nevertheless, a dry season in a year is required for fruit setting. Excess wetness due to prolonged rainfall during the period of spike induction would result in continuation of the vegetative phase in black pepper plant growth rather than reproductive phase. On the other hands, effective moisture management during the prolonged dry periods would significantly enhance the crop productivity.

2.1.1 Leaf

The leaves of black pepper plants are in alternate attachment with one leaf per plant node at alternate sides of the branches (Lai and Sim, 2011; Sim et al., 2011; Chen et al., 2017) . The leaves are simple with palmate venation and with a grooved petiole on the leaves upper surface. The sizes, shapes, margins and relief of the leaves surface are varying in different black pepper cultivars. The leaves margins are usually entire, repand or wavy and the relief of the leaves surface can be either flat, mildly or strongly raised between the main veins. The leaves shapes are basically lanceolate to ovate and the leaves tips are either acute or acuminate. The shapes of the leaves base are usually acute, oblique, truncate, round or cordate.

2.1.2 Inflorescence

Black pepper flowers are form on a pendulous spike developed from apical buds of the plagiotropic branch derived from leaves axils of the terminal shoots (Lai and Sim, 2011; Sim et al., 2011; Chen et al., 2017). It arises at the nodes opposite the leaves on a branch. It is about 15 cm to 20 cm long and bears about 100 flowers to 120 flowers at 36 | P age maturity stage. The flowers are small, sessile, either green, light green or golden yellow in colour. Black pepper inflorescences are protected by a linear leaf-like structure called prophyll. The prophylls shrivel, turn black and fall off with the emergence of the inflorescences. Black pepper plant is either bisexual or unisexual with the staminate and pistillate flowers are on the same or different individual plants. Most of the black pepper cultivars have bisexual flowers although there are some black pepper cultivars with high percentage of pistillate flowers. Black pepper plants are usually self-pollinated. After the process of pollination, the flower spikes will develop into fruit spikes which also known as infructescences.

2.1.3 Infructescence

Most of the black pepper cultivars consist of fruit spikes which are straight. However, there is some black pepper cultivars consist of curved and twisted fruit spikes. Peppercorns or usually also known as pepper berries are the fruits of black pepper plant. Black pepper berries consist of a thin exocarp, fleshy mesocarp and hard endocarp (Lai and Sim, 2011; Sim et al., 2011; Chen et al., 2017). It is a one-seeded indehiscent fruit with 4 mm to 6 mm in diameter and enclosed by a pulpy mesocarp. Black pepper berries are pale green in colour and soft at the early stage of development. However, the berries turn dark green in colour and get harder during the mature stage. The outer skin or exocarp of black pepper berries turns yellow and is bright red in colour as well as get softer as the berries are ripe. Black peppercorns of commerce are dried fruits of black pepper plant whereas white peppercorns of commerce are dried seeds of the same black pepper plant as shown in Figure 2-2. Black pepper fruits ripe in 6 months to 9 months. The process of harvesting usually takes about 2 months to 3 months due to the continuous flushing of black pepper flowers and uneven fruits ripening. Black pepper seed is about 3 mm to 4 mm in diameter. The seed contains a minute embryo, little endosperm and copious perisperm made up of large cells containing starch. Peppercorns are rich in alkaloid piperine and volatile essential oils which give rise to their pungency and typical aroma. However, the spiciness, pungency and aroma of peppercorns are influenced by different cultivars of black pepper plants and also the growing habitat.

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(b)

(a) (c) Figure 2-2. Black pepper fruits, (a) Green pepper berries; (b) Black peppercorns of commerce and (c) White peppercorns of commerce. The photo (a) was taken from a healthy black pepper farm located at Serian Division of Sarawak State. Meanwhile, photos (b) and (c) were retrieved from Malaysian Black Pepper Production Technology Manual (Lai and Sim, 2011).

2.1.4 Stem and branch

Black pepper plant can climb up to more than 10 ft in height on supporting pole. The plant produces two different types of branches. One is straight, grows upward, monopodial with adventitious roots at each node clinging on the support. This type of branch is known as orthotropic branch or terminal shoot (Lai and Sim, 2011; Sim et al., 2011; Chen et al., 2017). The branch has indefinite growth and thickens as it grows. It is usually referred as the stem of black pepper plant. The tip of the orthotropic branch is either green, brownish green, brownish purple or purple in colour due to the presence of anthocyanin in varying intensities. Black pepper leaves are produced at each node of orthotropic branch as the branch grows upward with the help of adventitious roots that cling to the supporting pole. The other type of black pepper branch was developed from axillary buds at each node of orthotropic branch which grows in lateral, sympodial with

38 | P age flowers (inflorescence) and fruit spikes (infructescence) at the node. This type of branch is known as plagiotropic branch or lateral branch which eventually bears the flower and fruit spikes.

2.1.5 Root

Black pepper root system is developed from adventitious roots of the orthotropic branch that buried in the soils (Lai and Sim, 2011; Sim et al., 2011; Chen et al., 2017). There are prominent root initials at each node of orthotropic branch. These root initials develop into adventitious roots when moisture is available and they cling onto the supporting poles. The adventitious roots can grow into normal underground roots in soils. Therefore, cuttings taken from the orthotropic branch are usually used as planting materials for black pepper farm establishment. The number of main roots developed from the adventitious roots is highly depends on growth environment and the number of branch nodes buried in soils. The bulk of secondary and feeder roots developed from the main roots are located just below the soil surface and generally penetrate down to the soils about 50 cm in depth. The secondary and feeder roots of black pepper plant are usually found extended beyond the edge of plant canopy. However, the spread of black pepper root system is highly variable and depends mainly on planting density and growth conditions.

2.1.6 Stolon and hanging shoot

Black pepper plant produces adventitious runner shoots that known as stolon at the base of a plant (Lai and Sim, 2011; Sim et al., 2011; Chen et al., 2017). Stolon has longer internodes than orthotropic branch and also could produce adventitious roots at each node. It is usually trails on the ground and strike roots easily or climbs up when come into contact with supporting poles. In India and Sri Lanka, stolons are also used as planting materials for establishment of black pepper farm apart of orthotropic branches. Black pepper plant also could produces hanging shoots from the terminal. Different with runner shoots, hanging shoots do not undergo branching. This type of shoots is usually grows and hangs downward. Hanging shoots also can produce adventitious 39 | P age roots when climb up to a supporting pole and strike roots when come into contact with soils or when moisture is available. Hanging shoots are also used as planting materials for establishment of black pepper farms in India.

2.2 Establishment of Black Pepper Farms

2.2.1 Planting materials

Black pepper is propagated through cuttings taken from the orthotropic branches with varying number of nodes rather than seeds. Runner shoots and hanging shoots that grow from the orthotropic branches are also used as planting materials in Sri Lanka and India. The traditional agriculture practice in Malaysia recommends five-node cuttings as planting materials for establishment of black pepper farms (Lai and Sim, 2011; Sim et al., 2011; Chen et al., 2017). However, during the time of planting materials shortage, two-node cuttings or even one-node cuttings is also proposed for establishment of black pepper farms. However, field planting of such shorter cuttings is slower at initial stage as compared to five-node cuttings. Therefore, it is necessary to do rooting for such shorter cuttings in nursery for a period of 2 months to 3 months before being transplanted to the field. Besides, high quality planting materials should be used for establishment of black pepper farms as the marginal quality cuttings would affect the growth and yield. The quality control of black pepper planting materials begins with stock plant s. The cuttings used for establishment of stock plant nursery must be obtained from vigorously growing and healthy young black pepper plants of the recommended cultivars with 1 year to 3 years old. The cuttings should not be taken from insect pests and diseases affecte d plant or the plant with symptoms of nutritional disorders.

2.2.2 Land preparation

Land preparation is usually carried out during the dry season which is from May to August of the year. Clear felling, uprooting, stacking and burning of jungle tree stumps are the common standard practices that usually carried out by black pepper growers. 40 | P age

Heavy machinery is required in establishment of large black pepper farm. However, this type of activities needs to be carried out carefully to prevent removal of the top soils. Tree stumps, especially rubber tree roots must be removed thoroughly to avoid the incidence of soil-borne diseases, particularly white root disease (Lai and Sim, 2011; Sim et al., 2011; Chen et al., 2017). Glyphosate is the commonly used herbicide to reclaim the land from sheet lallang and other weeds.

2.2.3 Drainage

Black pepper plants grow well in soil with good drainage and rich in organic humus. The plants are not recommended to be planted in the areas that are either too dry or susceptible to flood (Lai and Sim, 2011; Sim et al., 2011; Chen et al., 2017). Therefore, for black pepper farms located in low-lying areas with poor drainage conditions, peripheral drains are made to get rid of excess water in farms. Field drains with 60 cm in depth and 60 cm in width are made in between every five rows to six rows of black pepper plants. It is used to connect the peripheral drains and lead the excess water away from the black pepper farms especially after rain. Besides, field drains also play a significant role in preventing soil-borne fungi from spread over in a black pepper farm particularly during the rainy season.

2.2.4 Field planting

Field planting is carried out during wet season, usually from October to December of the year. All pre-rooted cuttings, polybag-nurseried cuttings or unrooted fresh cuttings can be used as planting materials for establishment of black pepper farms. Black pepper plants are planted on planting mounds of about 20 cm in height and 50 cm in width which are set up a month before field planting (Lai and Sim, 2011; Sim et al., 2011; Chen et al., 2017). The soils are neutralized with dolomite. Field planting is usually carried out after one month of soil neutralization. Black pepper cuttings are tied up to the supporting poles that positioned at the central of planting mounds and all weeds surrounding the farms are cleared away. However, the practice of clean weeding is often

41 | P age reported to result in serious soil erosion especially in black pepper farms located at steep slopes.

Therefore, black pepper farmers are advised to adopt the practice of clean weeding by planting ground cover crops. Instead of clean weeding, circle weeding is recommended by removing weeds surrounding the planting mounds only rather than entire area in a farm. The newly planted cuttings are usually shaded with fern fronds (Dicranopteris lineari; Dicranopteris curanii; Gleichenia truncate; Blechnum orientale), lallang leaves (Imperata cylindrica), coconut fronds and other type of shades that permit free ventilation to protect black pepper cuttings from direct sunlight until they are well established. The young plants require watering every other day during the dry season in the first two years of planting. The shoots are trimmed twice a year. The plants start to bear fruit in the second year of field planting.

2.2.5 Spacing

Lining is carried out with nylon strings and measuring tape for spacing in between two planting points. Black pepper is planted at a planting distance of 1.8 m x 2.4 m or 2.1 m x 2.1 m apart in rectangular pattern (Lai and Sim, 2011; Sim et al., 2011; Chen et al., 2017). Leguminous cover crop is recommended for black pepper farms at sloping land to avoid soil erosion and to maintain soil fertility. There are two species of leguminous plants, centro (Centrosema pubescens) and pintoi peanut (Arachis pintoi) is widely recommended as cover crops for black pepper farms in Malaysia. For black pepper farms with cover crop planted at inter-rows, the planting distance is adjusted to 2.0 m x 3.0 m (for centro) and 2.0 m x 2.4 m or 2.4 m x 2.4 m (for pintoi peanut). The average planting density of a black pepper farm in Malaysia is about 2,000 plants per hectare.

2.2.6 Supporting pole

Black pepper is a woody climber that has been trained to climb up onto a dead wood or live wood supports (Lai and Sim, 2011; Sim et al., 2011; Chen et al., 2017). In traditional, dead wood supports such as Belian posts that also known as Borneo 42 | P age ironwood (Eusideroxylon zwageri) and Selangan Batu or Shorea species are widely used supporting posts in black pepper farms. However, the recent research findings have shown that numerous live woods supports of certain leguminous trees such as Gliricidia (Gliricidia sepium) and Simpuh (Dillenia suffruticosa) are suitable to be used as alternative to the dead wood supports as they are more economical (Lai and Sim, 2011; Sim et al., 2011; Chen et al., 2017). Hence, live wood supports have recently been widely recommended for black pepper cultivation in Malaysia. In black pepper cultivation, the recommended dead wood supports are usually 4 cm to 6 cm in diameter and 12 ft in length. Prior to planting, the posts are positioned at the central of planting mounds by inserting bottom end of the posts, about 2 ft into the soil with the remaining length of the posts on ground is 10 ft. Whereas, the live wood supports are maintained at a height of 3 m by pruning and training to form a single main stem with a leafy crown.

2.2.7 Pruning

Black pepper plant needs to be pruned to encourage the formation of desired canopy. Three rounds of pruning for the growing black pepper plant should be carried out during the immature phase of black pepper plant growth (Lai and Sim, 2011; Sim et al., 2011; Chen et al., 2017). The first pruning of terminal shoot is done at 6 months to 8 months after planting to allow three leader shoots to develop. The second pruning is done when the plants are about one year old or when the plant has reached a height of half post. The third pruning is carried out when the terminal shoots of a black pepper plant have reached the top of the supporting poles. The third cycle of pruning is usually coincides with the first round of berries production. Apart the pruning of black pepper plants, the live wood supports are also required to be pruned in often to provide adequate sunlight for black pepper plants. It is recommended to conduct the pruning of live wood supports prior fertilizer application to maximize the nutrients uptake by black pepper plants.

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2.2.8 Fertilizing

Application of fertilizer is important to sustain high yield in black pepper cultivation as black pepper plants have high demand on nutrients supply. Granulated chemical compound fertilizers are widely applied for black pepper cultivation in Malaysia. The recommended major nutrient compositions (Nitrogen: Phosphorus: Potassium or NPK) of fertilizers are 12:12:17:2 + trace elements (TE), 15:15:15 or 9:9:9 (Lai and Sim, 2011; Sim et al., 2011; Chen et al., 2017). During fertilizing, the fertilizers are placed in two shallow trenches made on the planting mound at both sides of the plant, which below the plant canopy. Fertilizing is carried out at 80 g per plant after one month of planting and 100 g per plant after three months of planting. The fertilizer is usually covered with a thin layer of soil after application to avoid run- off by rain water. Chicken manure in the same manner as for compound fertilizers is used after half year of field planting. It is applied at 300 g per plant at one month interval. Besides that, liming with dolomite is also carried out in black pepper farms as this practice is required to neutralize the soil pH and to improve the Calcium (Ca) and Magnesium (Mg) nutrition in black pepper plants.

2.2.9 Harvesting and processing

Black pepper plants start to bear fruits in the second year of field planting. As a production cycle of black pepper plants take about 6 months to 9 months, the first round of fruit harvesting is carried out in the third year of field planting. The process of fruit harvesting is usually started in March and completed in August of the year (Lai and Sim, 2011; Sim et al., 2011; Chen et al., 2017). Black peppercorns of commerce are processed by direct sun-dry the freshly harvested black pepper berries which show early signs of maturity. Meanwhile, white peppercorns of commerce are produced by soaking the ripe black pepper berries in clean running water to remove their pericarps prior the process of sun- dry.

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2.3 Cultivated Black Pepper Va rieties in Malaysia

In total, there are seven black pepper cultivars or cultivated varieties have been planted in Malaysia, known as “Kuching”, “Semongok Emas”, “Semongok Aman”, “Semengok Perak”, “Semengok 1”, “Nyerigai” and “Uthirancotta”. Among these, three cultivars that have been recommended to farmers for extensive planting are “Kuching”, “Semengok Emas” and “Semengok Aman” (Lai and Sim, 2011; Sim et al., 2011; Chen et al., 2017). These three cultivars are recommended to farmers due to their better characteristics in fruit setting and higher insect pests and diseases tolerance level than the other cultivars. Nevertheless, these three cultivars are varying in morphological appearances, chemical contents and fruit qualities. Apart from the seven cultivated varieties, Malaysia also possess a collection of black pepper germplasm which made up of 43 black pepper (P. nigrum L.) accessions as shown in Table 2-1 and a few wild pepper species such as P. colubrinum Link that are well maintained in Semengok Agriculture Research Centre (ARC), DoA Sarawak. These collections of black pepper germplasm are important and valuable genetic resources for future Malaysian black pepper improvement program.

Table 2-1. List of black pepper (P. nigrum L.) accessions maintained in Semengok Agriculture Research Centre, Department of Agriculture, Sarawak. Place of collection Accession No.

Sarawak K62, KCH, PN118, PN125, PN126, PN127, PN129, PN131, PN135, PN136, PN137, PN138, PN139, PN140

Indonesia PN36, PN37, PN74

India PN28, PN30, PN79, PN81, PN94, PN99, PN100, PN103, PN113, PN114, PN115, PN116, PN119, PN123, PN124, PN132, PN133, PN134

Central America PN107, PN108, PN109, PN110, PN111, PN112, PN106, PN117

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2.3.1 Cultivar “Kuching”

Cultivar “Kuching” as shown in Figure 2-3 is the traditional and most widely planted black pepper variety in Malaysia (Lai and Sim, 2011; Sim et al., 2011; Chen et al., 2017). The terminal shoot tip of this cultivar has intermediate anthocyanin coloration and is light purple in colour. The leaves of cultivar “Kuching” are lanceolate-ovate in shape and the relief of the leaves upper surface is smooth. The flowers of cultivar “Kuching” are pale yellow in colour and in contrast to those of cultivars “Semongok Emas” and “Semongok Aman”. Cultivar “Kuching” has vigorous growth and is high yielding. Generally, the fruit spikes of this cultivar have good fruit setting and the pericarps of the berries are the thinnest among the three recommended varieties. Therefore, cultivar “Kuching” is the preferred black pepper variety for creamy white pepper production. The average yield of cultivar “Kuching” is 1.7 kg to 3.4 kg per plant. However, this cultivar is susceptible to various diseases such as Phytophthora foot rot, slow decline due to Fusarium infection, black berries disease, nematode root-knot and wrinkle leaves caused by P. nigrum L. strain of Cucumber Mosaic Virus (CMV-Pn).

(a) (b) Figure 2-3. The morphological appearances of cultivar “Kuching”, (a) Leaves and flower spikes on the lateral branches and (b) Mature fruit spikes on the lateral branches. The photos were taken from a healthy black pepper farm located at Betong Division of Sarawak State. 46 | P age

2.3.2 Cultivar “Semengok Emas”

Cultivar “Semengok Emas” as shown in Figure 2-4 was derived from the breeding programme through crossing of an Indian cultivar known as “Balancotta” with cultivar “Kuching” (Lai and Sim, 2011; Sim et al., 2011; Chen et al., 2017). This cultivar was released to farmers by DoA Sarawak in the year 1991. Same as cultivar “Kuching”, the terminal shoot tip of cultivar “Semengok Emas” has intermediate anthocyanin coloration. It is light purple in colour. The leaves of cultivar “Semengok Emas” are lanceolate-ovate in shape and the relief of the leaves upper surface is mildly raised between the main veins. The flowers of cultivar “Semengok Emas” are yellow to golden yellow in colour. This cultivar has good fruit setting which is comparable to cultivar “Kuching”. However, the canopy size of cultivar “Semengok Emas” is not as dense as cultivar “Kuching”. Nevertheless, this character facilitates the picking of fruit spikes during the process of harvesting. The berries ripening of cultivar “Semengok Emas” is also more even if compared to cultivar “Kuching”. This character allows the harvesting cycles to be reduced to 2-3 rounds instead of 5-6 rounds as in cultivar “Kuching”. Apart from that, cultivar “Semengok Emas” is more tolerant to black pepper weevils and black berries disease that caused by soil-borne Collectotrichum species. However, this cultivar has no advantages over cultivar “Kuching” in nematode root-knot and wrinkled leaves disease.

(a) (b) Figure 2-4. The morphological appearances of cultivar “Semengok Emas”, (a) Leaves on the lateral branches and (b) Flower and fruit spikes on the lateral branches. The

47 | P age photos were taken from a healthy black pepper farm located at Betong Division of Sarawak State.

2.3.3 Cultivar “Semengok Aman”

Cultivar “Semengok Aman” as shown in Figure 2-5 was derived from the clonal propagation of a black pepper cultivar that originally from Costa Rica with Germplasm Accession No. PN106. This cultivar was released to farmers by DoA Sarawak in the year 2006 (Lai and Sim, 2011; Sim et al., 2011; Chen et al., 2017). Same with the other two recommended black pepper cultivars, the terminal shoot tip of cultivar “Semengok Aman” has intermediate anthocyanin coloration and is light purple in colour. The leaves are ovate in shape and the relief of the leaves upper surface is strongly raised between the main veins. The flowers of cultivar “Semengok Aman” are light green in colour. This cultivar has very good fruit setting and produces bigger berries and longer fruit spikes than cultivars “Kuching” and “Semengok Emas”. Cultivar “Semengok Aman” has exhibited good characteristic of cultivar “Semongok Emas” in that it is more uniform in berries ripening and allowing crop yield to be harvested in a shorter span of time.

In terms of its resistancy to insect pests and diseases, cultivar “Semongok Aman” is more tolerant to Phytophthora foot rot and black berries diseases as compared to cultivar “Kuching”. It is also reported to be more tolerant to black pepper weevils. In terms of chemical quality, the berries of cultivar “Semongok Aman” have higher contents of piperine, oleoresin, volatile and non-volatile oils than the berries of cultivars “Kuching” and “Semongok Emas”. The green pepper products produced from the berries of cultivar “Semongok Aman” is more pungent if compared to those produced from the berries of cultivars “Kuching”. However, the pericarps of “Semongok Aman” berries are thicker than the pericarps of “Kuching” berries. This character has caused the conversion rate to produce white peppercorns of commerce is lower in cultivar “Semengok Aman” (22%) than cultivar “Kuching” (24%). However, there is no variation as in cultivar “Semongok Emas”. Therefore, the berries of cultivar “Semongok Aman” are highly suitable to be used for production of premium black pepper products and green pepper products.

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(a) (b) (c)

Figure 2-5. The morphological appearances of cultivar “Semengok Aman”, (a) Leaves on the lateral branches, (b) Flower spikes on the lateral branches and (c) Mature fruit spikes on the lateral branches. The photos were taken from a healthy black pepper farm located at Sri Aman Division of Sarawak State.

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2.4 Resistant Wild Pepper Plant

Crop wild relatives are an important germplasm resource in crop improvement program. This is because the wild species are possible progenitors of the crops and therefore can be used as gene donors to the crops (Ford-Lloyd et al., 2011). A wild species of pepper plants known as P. colubrinum Link as shown in Figure 2-6 is valuable and important because of its resistance to a number of plant soil-borne pathogens. This woody shrub is belongs to family Piperaceae. It is an exotic Piper species native to the Northern part of South America (Krishnan et al., 2015). P. colubrinum Link is a distant relative of the cultivated black pepper species, P. nigrum L. Different with P. nigrum L., this species is grows well in marshy habitat that provided with proper light shade. P. colubrinum Link is propagates either through cuttings or seeds. Nevertheless, the seeds must be fresh during the sowing process as the viability of the seeds is low. Seeds sown in sand could germinate readily. The one-month old seedlings with approximately 5 cm to 8 cm in height can subsequently be maintained in nurseries by transplanting into ceramic vases or polybags that filled with suitable potting mixtures.

P. colubrinum Link has gained biotechnological significance. This species is of great importance because of its unique genetic characters that render it resistant to soil-borne pathogens those causing root diseases in widely cultivated P. nigrum L. P. colubrinum Link has been extensively reported as a resistant plant in against pathogenic fungi such as Phytophthora capsici and Fusarium solani. These two fungi species are the main causal organisms of black pepper foot rot and slow decline diseases (Devasahayam, 2000; Weiss, 2002; Malik and Kokkat, 2017; Malik and George, 2018). Apart from that, P. colubrinum Link also has been reported as a resistant plant in against several species of root-rot parasitic nematode such as Meloidogyne incognita and Radopholus similes that have caused significance yield loss in black pepper farms (Ramana and Mohandas, 1987).

As reported in the study of Sandeep Varma et al. (2009), P. colubrinum Link response to the infection of P. capsici through chitinase production. Molecular assessment of the chitinase genes in P. colubrinum Link was proposed as an effective solution in against a wide range of soil-borne fungal pathogens from genera Phytophthora, Collectotrichum, Fusarium as well as root-rot nematodes such as M. incognita and R. similes. Therefore, 50 | P age with its multiple resistance properties, P. colubrinum Link has immense potential to be used as genes donor plant in Marker Assisted Selection (MAS) breeding programs for transferring of desirable resistance genes to the cultivated black pepper plant, P. nigrum L. in against specific plant disease pathogens. However, due to the limitation of genetic information in P. colubrinum Link and the other Piper species, this plant is still remains biotechnologically under utilized and the probability of success for transferring of desirable resistance genes from P. colubrinum Link to the susceptible P. nigrum L. is very scanty.

Even though genomic exploration in Piper species is still in infancy, however, several efforts have been carried out by scientists to discover defence-related genes and proteins in P. colubrinum Link and P. nigrum L. For examples, Dicto and Manjula (2005) has reported successful identification of Class-V group member s of Pathogenesis-Related (PR) genes in P. colubrinum Link through PCR based suppression subtractive hybridization (SSH) technique. These genes were found to be differentially expressed in P. colubrinum Link in response to salicylic acid (SA) signalling molecule. Girija et al (2005a) has reported the role of hydroxyl methyl glutaryl CoA reductase (hmgr) genes in P. colubrinum Link in resistant to fungal pathogens besides isoprenoid biosynthesis. Apart from that, successful cloning of β-1,3-glucanase cDNA fragments, the genes that play an important role in withstanding abiotic stresses of P. nigrum L. has been carried out by Girija et al (2005b). In addition, Stephen et al. (2001) has reported successful induction of defence-related phenylalanine ammonia lyase (PAL), β-1,3- glucanase and PR proteins in P. nigrum L. in against P. capsici.

Interspecific hybridization of P. colubrinum Link and P. nigrum L. has been proposed as a potential and fast solution in order to transfer desirable resistance genes from P. colubrinum Link to P. nigrum L. This effort has been attempted in Malaysia and India. However, the results obtained are inconsistent and the efforts are being unsuccessful due to the problem of incompatibility among the two pepper species. This is mainly due to the different ploidy level of P. colubrinum Link (diploid; 2n=2x=26) and P. nigrum L. (tetraploid; 2n=4x=52). As P. colubrinum Link is a diploid whereas P. nigrum L. is a tetraploid, the two species are cross incompatible. This is because the progenies gained from the cross is predictable to be a triploid and sterile (Purseglove et al., 1981; Sim and Rosmah, 2011; Jagtap et al., 2016). 51 | P age

Vanaja et al. (2008) has reported a partly fertile black pepper hybrid was developed through interspecific hybridization between the cultivated P. nigrum L. and wild species P. colubrinum Link. Although the developed new hybrid (triploid; 2n=3x=39) has a large number of long spikes per unit area of plant canopy, the fruit setting of the hybrid is very low. Moreover, the germinated hybrids have exhibited low survival rate in field planting. Hence, the possibility to impart resistance in P. nigrum L. through application of interspecific hybridization is meagre. Besides that, it was also felt by many scientists that grafting of P. nigrum L. shoots onto the P. colubrinum rootstocks would solve the problem of soil-borne diseases in black pepper farms. In addition, the grafting technique could improve the adaptability of P. nigrum L. to marshy situations.

The related research trials were carried out in Malaysia, India and Brazil (Ravindran and Remashree, 1998; Vanaja et al., 2008). However, the grafted plants have showed high initial success but poor survival rate in the long run due to delayed graft incompatibility. As the results of field assessments, an anomalous secondary growth has been observed among two pepper species (De Waard et al., 1969). Anatomical analysis of the grafts has shown the formation of an anomalous thickening layer at graft union due to the over growth of P. nigrum L. As the grafts grow further, the stems were splited and a dark layer was observed at cambial contact region of the two pepper species wherever grafts failed. Therefore, application of P. colubrinum Link as disease-resistant rootstock is not recommended for black pepper cultivation. This is because the grafts are reported to deteriorate gradually.

Since P. colubrinum Link is resistant to numerous soil-borne diseases in black pepper cultivation, it is a rich repository of desirable defence-related genes. This species is an ideal candidate to be used as genes donor for resistance attributes in susceptible P. nigrum L. through biotechnological approaches. Due to current research findings and knowledge about defence-related mechanisms in P. nigrum L. is inadequate; therefore identification of key defence-related genes in P. colubrinum Link will be of immense use in elucidation of defence-related response and mechanism pathways in P. nigrum L. These novel research findings are valuable resource and can be applied to enhance the level of disease resistancy in susceptible P. nigrum L. and to improve the programs of disease management in black pepper cultivation as well.

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(a) (b)

(c) (d) Figure 2-6. Resistant wild pepper plant, P. colubrinum Link, (a) Potted young cuttings (3 months old); (b) Potted young cuttings (6 months old); (c) Mature leaves on the lateral branches and (d) flower spikes on the lateral branches. The photos were taken from the wild pepper germplasm maintained at Malaysian Pepper Board plant house.

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2.5 Pathogenic Organisms in Black Pepper Cultivation

Black pepper is susceptible to various pathogenic organisms such as soil-borne fungi, plant parasitic nematodes, viruses and insect pests. Yield loss due to invasion of insect pests and diseases has been identified as a major constraint in black pepper cultivation of all producing countries and Malaysia is no exception. In Malaysia, black pepper farms are affected by a myriad of diseases. Some of the diseases are devastated and usually cause heavy yield loss as well as black pepper plant death. Meanwhile, some of the diseases are less severe and only with minor economic affects. There are numerous major diseases have been found in Malaysia black pepper farms. Most of these major diseases are caused by soil-borne fungi from genera Phytophthora, Colletotrichum, Fusarium and Rigidoporus (Lai and Sim, 2011; Sim et al., 2011).

2.5.1 Major disease in Malaysia black pepper farms

2.5.1.1 Slow decline

Slow decline as shown in Figure 2-7 is the most serious debilitating disease of black pepper plants in Malaysia. It is a disease complex caused by soil-borne fungus known as F. solani and associated with a species of plant parasitic root-knot nematode, M. incognita (Albuquerque, 1961; Hamada et al., 1988; Lai and Sim, 2011; Sim et al., 2011 ). As the infected black pepper plants show distinctive symptom of foliage yellowing, therefore it is also known as yellowing disease (Kueh et al., 1993; Sitepu and Mustika, 2000). Rehman et al. (2012) reported that F. solani could produces toxin anhydrofusarubin to cause decline in host plants during invasion. This pathogenic fungus invades internal tissues of black pepper plant roots and leads to vascular wilt in black pepper plants. The fungus proliferates in water-conducting xylem vessels of host plants. It causes blockage in the xylem vessels and interferes water as well as nutrients uptake by host plants. The black pepper plants with vascular wilt usually were showed discolouration of the vascular system at main stems, trunks and branches.

Root-knot due to invasion of plant parasitic nematode (M. incognita), a microscopic and unsegmented worm is associated with F. solani invasion in causing slow decline in 54 | P age black pepper plants. Similar with F. solani, root-knot nematodes invade black pepper plant roots and form gall-like lesions that restrict water and nutrients uptake by host plants. These parasitic worms proliferate and feeding on nutrient-rich cells in the plant roots and lead wilting to the infected plants (Lai and Sim, 2011; Sim et al., 2011). Foliar yellowing, wilting at the shoot tips, browning of plant vascular tissues, leaves drop-off, dieback and sudden death are the symptoms of slow decline disease.

Slow decline causes significance yield loss in majority of the black pepper producing countries such as Malaysia, Indonesia, Brazil, India and Thailand (Ramana and Eapen, 2000). The disease is also reported to reduce economic lifespan of black pepper farms from 20 years down to 6 years or 8 years according to Duarte and Albuquerque (1991). The infected black pepper plants show a progressive decline, flaccidity and finally died after a period of time. Black pepper plant death due to slow decline is commonly occurs gradually after one year to two years of infection (Anandaraj, 2000). Slow decline was found in most of the Malaysia black pepper farms, particularly in Serian, Sri Aman, Betong and Julau divisions of the State of Sarawak.

Black pepper plants with slow decline do not show any symptoms of disease at the early stage of infection (Rehman et al., 2012; Shahnazi et al., 2012; Gogoi et al., 2017). The infected plants show varying degrees of feeder root loss and the expression of disease symptoms on the aerial parts of infected black pepper plants only detected after a considerable portion of feeder roots are lost. Infected roots show varying degrees of necrosis and presence of root galls due to root-knot nematode invasion. The fungal infection subsequently spreads to mature roots. The root damage impairs the translocation of water and mineral salts in the infected black pepper plants and leads to symptoms similar to nutritional disorders such as foliar yellowing, interveinal chlorosis, wilting of the shoot tips and die back of aerial stems. Browning of vascular tissues is usually observed at roots and collar regions of black pepper plants with slow decline.

The canopy size of the infected plants is reduced if compare with healthy plant. There is a gradual reduction in vigour and productivity of black pepper plants and ultimately leads to plant death over a period of time. Therefore, slow decline is also well known as slow wilt disease. Although it does not cause sudden death of black pepper plants such as Phytophthora foot rot, but it is more common and often outbreaks in Malaysia black 55 | P age pepper farms and cause serious damage to black pepper farms. This is mainly due to the infected plants does not show any clear disease symptoms at the early stage of infection. This has caused the necessary steps of precaution and treatment not being applied properly or not being applied at all at the first stage of infection.

Root galls

(a) (b) (c) Figure 2-7. Symptoms of slow decline found in Malaysia black pepper farms, (a) Foliage yellowing of black pepper plant with slow decline; (b) Browning of vascular tissues due to Fusarium infection; (c) Formation of root galls due to infestation by root- rot nematode. The photos were taken from a disease black pepper farm located at Sri Aman Division of Sarawak State.

Besides that, the unclear and confuse disease symptoms similar to nutritional disorders (nitrogen deficiency) that showed by the infected plants at the late stage of infection has caused misleading to black pepper farmers in taking proper precaution steps to prevent the disease from spread over. Moreover, it was also too late in taking treatments to cure the disease at the late stage of infection. Similar with Phytophthora foot rot, cultural control plays a significant role in managing slow decline. Farm hygiene practices and proper maintenance could aid in prevention of slow decline incidence. However, these practices are impractical during disease outbreak. Therefore, to prevent and control the disease effectively, an integrated disease management system through biotechnological approaches should be adopted in black pepper farm agricultural practices. There were a few incidents of slow decline have been reported outbreak in several black pepper farms situated at Julau and Betong divisions of the State of Sarawak in the year 2018. The

56 | P age disease has found to cause high yield loss and serious damage to the farms within a short period of time as shown in Figure 2-8.

(c)

(a)

Root galls

(d)

(b) (e) Figure 2-8. Incidences of slow decline reported in black pepper farms situated at Julau and Betong divisions of the State of Sarawak in the year 2018, (a) Farm damage due to slow decline; (b) Black pepper leaves with foliar yellowing symptom due to F. solani invasion; (c) Damage of black pepper plant collar region and loss of feeder roots due to F. solani invasion; (d) Black pepper root necrosis due to formation of gall-like lesions caused by parasitic nematodes and (e) Rotten of black pepper roots due to F. solani infection. The photos were taken from the disease black pepper farms located at Julau and Betong Divisions of Sarawak State.

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2.5.2 Other diseases in Malaysia black pepper farms

2.5.2.1 Phytophthora foot rot

Phytophthora foot rot as shown in Figure 2-9 is deemed the most destructive soil-borne disease in black pepper cultivation. The disease can cause high mortality of black pepper plants. It is usually occurs in black pepper farms located at lowland areas or with poor water drainage. Phytophthora foot rot is caused by a soil-borne fungus known as P. capsici (Lai and Sim, 2011; Sim et al., 2011; Chen et al., 2017). This fungus is usually remains dormant in soil during the dry season and will get activated during rainy season as the soil moisture builds up. All parts of the black pepper plants are susceptible and prone to infection by this fungus. Phytophthora foot rot consists of two phases of infection, known as aerial phase and soil phase. Aerial phase consists of infection on leaves, stems and spikes while the soil phase infection usually starts at the fibrous root system, reaches the main root and ultimately the collar or foot region of the bush.

P. capsici, the causal fungus of this disease is generally known as water mold because of their behaviours to get activate, thrive, grow, generate and infect plant roots in water. During the event of invasion, this fungus produce swimming spores which also known as zoospores to attach black pepper roots and start infection. Phytophthora foot rot is often reported by black pepper farmers from Betong and Julau divisions of the State of Sarawak. This is because majority of the black pepper farms in these two divisions are located at very outskirts areas that with high humidity climate and soil water retention.

Phytophthora foot rot is usually spread over either through infected planting materials, fungal zoospores in soil water, rain splashes or contaminated farm implements. The disease can cause sudden wilting, drying and death of black pepper plants. The infection usually starts with a formation of water soaked lesions at the collar region of the plant and changes to wet slimy dark patches as the wet weather persists. The infected plants showed disease symptoms such as heavy defoliation, branches and leaves drop-off, root rotted and finally collapse leading to death within a month as the disease progresses. Therefore, Phytophthora foot rot is also well known as quick wilt disease. Although Phytophthora foot rot is the most critical disease in Malaysia black pepper farms, the 58 | P age incidence of Phytophthora foot rot is not as common as slow decline. This is because the causal fungus of this disease, P. capsici depends solely on the wet weather and high moisture condition to activate its growth. Generally, waterlogged environment in black pepper farms is conducive for the development of Phytophthora foot rot. Therefore, establishment of proper and good drainage system in black pepper farms is necessary for prevention of the disease.

(a) (b) (c)

(d) (e) Figure 2-9. Symptoms of Phytophthora root rot found in Malaysia black pepper farms, (a) Formation of wet slimy dark patch at collar region of infected black pepper plant; (b) P. capsici infected black pepper plant with wilted leaves and heavy defoliation; (c) Foot rot-infected leaf with fimbriate-edge lesion; (d) Foot rot-infected fruit spike with brownish-black lesion and (e) Rotten of the foot rot-infected basal stem. The photos were taken from a disease black pepper farm located at Julau Division of Sarawak State. 59 | P age

Application of farm hygiene practices is important to avoid the disease from spread over to a new area. As examples, sharing of farm tools between different black pepper farms is not encouraged and all the farm tools need to be disinfected before and after use. Besides that, black pepper cuttings used for establishment of new farms must be gained from sources with foot rot disease-free. Proper farms maintenance with regular pruning of lower branches which are 30 cm to 60 cm above the ground should be practiced to avoid black pepper branches come into contact with soil. The traditional management of Phytophthora foot rot is depends very much on early precaution steps and followed by prompt application of suitable fungicides (Lai and Sim, 2011; Sim et al., 2011; Chen et al., 2017).

2.5.2.2 Black berries disease

Black berries disease as shown in Figure 2-10 is caused by either one of the three soil- borne fungi from genus Colletotrichum that known as C. gloeosporioides, C. capsici or C. piperis (Lai and Sim, 2011; Sim et al., 2011; Chen et al., 2017). Black berries disease is one the serious diseases found in Malaysia black pepper farms. The characteristic symptom of this disease is the appearance of black spots on black pepper berries which may also be found on flower spikes and black pepper leaves. The berries of the infected black pepper plants turn black and dry-up as well as the whole spike detaches from the branch as the disease progresses.

The popularly planted black pepper cultivar “Kuching” in Sarawak is very susceptible to black berries disease. The practice of farm hygiene by removing of infected plant tissues such as leaves, flower spikes and fruit spikes that dropped onto the ground is believed could help to reduce the incidence of black berries disease. Apart from that, previous research studies conducted by DoA Sarawak has shown that application of live wood supports such as G. sepium and D. suffruticosa in black pepper farms also could aid in prevention of black berries disease incidence.

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Figure 2-10. The characteristic symptom of black berries disease (appearance of black spots on the black pepper berries), a major disease found in Malaysia black pepper farms that caused by Colletotrichum spp. The photo was taken from a disease black pepper farm located at Betong Division of Sarawak State.

2.5.2.3 White root rot

White root rot as shown in Figure 2-11 is a dangerous soil-borne fungal disease that can kill black pepper plants in a short period of time. The disease is either caused by Rigidoporus lignosus or Rigidoporus microporus (Lai and Sim, 2011; Sim et al., 2011; Chen et al., 2017). The characteristic symptom of white root rot is developing of white fungal mycelial mats on roots and collar regions of black pepper plants. The infection softened black pepper roots and make the infected roots crumble easily when disturbed. The infected plants stop in growth. Leaves yellowing, defoliation and die-back are the other common symptoms of the disease. Even though the disease is not as common as the other soil-borne diseases such as Phytophthora foot rot and slow decline, the white root rot disease has caused significance yield loss and plant damage particularly to the black pepper farms those are establish ed in abandon rubber estates.

White root rot disease is usually initiated when the condition of the farms is wet, mild and humid. Prevention of the causal fungi to spread over to nearby plants is important to control the white root rot disease. Therefore, the infected black pepper plants and roots must be removed and destroyed immediately after the plants are properly diagnosed for the disease. Apart from that, small trench can be constructed surrounding the infected areas to prevent rain water runoff from distributing the causal fungi to nearby areas.

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Figure 2-11. Developing of white fungal mycelial mats on black pepper roots, the symptom of white root rot disease found in Malaysia black pepper farms. The photo was taken from a disease black pepper located at Bintulu Division of Sarawak State.

2.5.2.4 Velvet blight

Velvet blight, a disease complex in black pepper plants as shown in Figure 2-12 is caused by fungi from genera Septobasidium in association with scale insects Pinnaspis spp (Lai and Sim, 2011; Sim et al., 2011; Chen et al., 2017). Septobasidium spp. forms symbiosis relationship with scale insects by conferring protection on the scale insect and derives nutrients from the latter in return. The characteristic symptom of velvet blight disease is formation of purplish-grey velvet-like incrustation on black pepper plant stems, branches, leaves and fruit-spikes. The heavily infected black pepper plant tissues are usually dry-up and drop-off as the disease progresses.

Since the scale insects provide the source of nutrients to Septobasidium spp., therefore eliminating the former through application of white oil and albolineum could reduce the incidence of velvet blight disease. Besides that, removal of the infected black pepper plant tissues such as branches, leaves, flowers and fruit spikes from the farms could also avoid the disease to spread over in black pepper farms.

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Figure 2-12. Formation of purplish-grey velvet-like incrustation on black pepper fruit spike due to velvet blight disease, a major disease found in Malaysia black pepper farms. The photo was taken from a disease black pepper farm located at Serian Division of Sarawak State.

2.5.2.5 Stunted disease

Stunted disease as shown in Figure 2- 13 is the only major disease in Malaysia black pepper farms that is caused by viruses rather than fungal pathogens. The causal organisms of the disease in Malaysia are either Piper nigrum L. strain of Cucumber Mosaic Virus (CMV-Pn) or Piper yellow mottle virus (Lai and Sim, 2011; Sim et al., 2011; Chen et al., 2017). The infection causes mosaic pattern with light and dark green regions on black pepper leaves is the characteristic symptom of stunted disease.

As the infected black pepper plants produce crinkled young leaves with small size and reduced photosynthetic area, the disease is also known as wrinkled-leaf disease. This disease is usually transmitted through infected cuttings and unsterile farm implements. Different from the other diseases caused by fungal pathogens, the stunted disease usually spreads slowly and death of the infected black pepper plants is rare been observed. However, the infected black pepper plants stunted in growth and become unproductive.

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Figure 2-13. Mosaic pattern with light and dark green regions on black pepper leaves due to stunted disease, a major disease found in Malaysia black pepper farms. The photo was taken from a disease black pepper farm located at Sri Aman Division of Sarawak State.

2.5.3 Minor diseases in Malaysia black pepper farms

Apart of the major diseases, there are also several minor diseases have been found in Malaysia black pepper farms. The minor diseases were seldom been found in black pepper farms and only with minor economic affects. However, these diseases also could cause significance yield loss and plant damages if the disease incidents do not handled in proper ways.

2.5.3.1 Horse hair blight

Horse hair blight (Figure 2-14) , a minor disease in black pepper farms that caused by an epiphytic fungus known as Marasmius equicrinis is usually associated with poorly maintained farms. The fungus produces horse hair-like mycelial strands or rhizomorphs dangling from branches to entangle black pepper leaves, fruit spikes and other branches under it in an irregular network (Lai and Sim, 2011; Sim et al., 2011). The plant parts entangled by fungal rhizomorphs are easily detached. The disease can be controlled through farm hygiene practices such as clearing and removal of infected and dead plant parts from the plant canopy. 64 | P age

Figure 2-14. Horse hair-like mycelial rhizomorphs entangling black pepper leaves,e th symptom of horse hair blight disease found in Malaysia black pepper farms. The photo was retrieved from Malaysian Black Pepper Production Technology Manual (Lai and Sim, 2011).

2.5.3.2 Pink disease

Pink disease (Figure 2-15) due to infection of the fungus known as Corticium salmonicolor usually causes balding canopy in black pepper plants where all the branches and leaves are dry up and drop-off (Lai and Sim, 2011; Sim et al., 2011). The disease observed as the formation of pink incrustations on the black pepper plant stems and branches. Since a dense humid canopy is the main factor that encourages the growth of the causal fungus, proper pruning maintenance to reduce the canopy humidity is efficient in prevention of pink disease.

Figure 2-15. Formation of pink incrustations on black pepper plant stems, the symptom of pink disease found in Malaysia black pepper farms. The photo was retrieved from Malaysian Black Pepper Production Technology Manual (Lai and Sim, 2011).

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2.5.3.3 White thread blight

White thread blight (Figure 2-16) caused by the fungus known as Marasmiellus scandens is commonly observed as the formation of distinctive white thread-like fungal mycelia along the stems and branches of black pepper plants (Lai and Sim, 2011; Sim et al., 2011). The infected black pepper branches, leaves and fruit spikes usually dry up, turn brown and suspended by a network of fungal mycelia as the disease progresses. As pink disease, white thread blight disease can be avoided through proper pruning maintenance to reduce the humidity of black pepper plant canopy.

Figure 2-16. Formation of distinctive white thread-like fungal mycelia on black pepper leaves, the symptom of white thread blight disease found in Malaysia black pepper farms. The photo was retrieved from Malaysian Black Pepper Production Technology Manual (Lai and Sim, 2011).

2.5.4 Insect pests of Malaysia black pepper cultivation

The major insect pests found in Malaysia black pepper farms are such as black pepper weevil, black pepper tingid bug and green black pepper bug as shown in Figure 2-17. Meanwhile, the minor insect pests found in Malaysia black pepper farms are such as flea beetle, scale insect, mealy bug, leaf hopper and aphid as shown in Figure 2-18 (Lai and Sim, 2011; Sim et al., 2011).

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(a) (b) (c) Figure 2-17. Major insect pests found in Malaysia black pepper farms, (a) Black pepper weevil; (b) Green black pepper bug and (c) Tingid bug. The photos were retrieved from Malaysian Black Pepper Production Technology Manual (Lai and Sim, 2011).

(a) (b) (c)

(d) (e) Figure 2-18. Minor insect pests found in Malaysia black pepper farms, (a) Flea beetle; (b) Leaf hopper; (c) Aphids; (d) Mealy bugs and (e) Scale insects. The photos (a) – (d) were retrieved from Malaysian Black Pepper Production Technology Manual (Lai and Sim, 2011). Meanwhile, photo (e) was taken from a disease black pepper farm located at Serian Division of Sarawak State. 67 | P age

2.6 Soil-borne Diseases

Soil-borne disease is the most dangerous disease that has been faced by black pepper farmers in Malaysia. It is a major constraint in many agricultural crops production, including black pepper. The disease is caused by numerous pathogens and parasites that naturally present in soil, such as fungi, bacteria, viruses and plant parasitic nematodes. Soil serves as an ecosystem for propagation of diverse microorganisms that perform various roles. Of which, some of the soil microorganisms are useful and benefit to the biological processes. However, some of them are dangerous transmitters of diseases.

In soil, there are various causal organisms of soil-borne diseases. These organisms usually remain idormant n an environment where the interaction among them and host plants as well as soil conditions is in a balanced system. However, the incidence of diseases will be initiated if the environmental balance is disrupted and the pathogenic organisms have become dominant in surrounding environment. Soil-borne disease is often difficult to control with application of conventional strategies as the disease causal organisms are native in soil. However, the diseases can be suppressed by improving resistancy level of host plants to the pathogens or by creating a hostile environment for pathogens through utilization of antagonistic microorganisms (Osbourn, 2001; Asghar and Mohammad, 2010; Tamm et al., 2011; Katan, 2017).

In general, there are two different types of soil-borne disease, known as pre-emergence damping-off and post-emergence damping-off (Azad Disfani and Zangi, 2006; Zagade et al., 2013). Pre-emergence damping-off is occurs during the stage of seed germination where the plant seedlings decay in soil due to bad environmental conditions such as poorly-drained soil, compacted soil and presence of high quantity of undecayed organic matters in soil. Whereas, post -emergence damping-off is occurs when plant roots, collar regions and stems are invaded and caused serious damage by soil-borne organisms. The infected plants subsequently fall over and die. As the cultivated black pepper plants are propagated through cuttings rather than seeds, therefore the plants are commonly suffered by latter type of soil-borne diseases such as slow decline, vascular wilt and foot rot.

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2.6.1 Management of soil-borne diseases in Malaysia

Soil-borne pathogens survive in soils and wood debris. They remain dormant during the dry season as water is essential for activation of their growth (Azad Disfani and Zangi, 2006; Zagade et al., 2013). As the soil moisture rises up during the rainy season, the population of pathogen propagules and zoospores will increase. The pathogens get activated and start their infection to the foliage and root as well as collar regions of black pepper plants and finally leading the infected plants to death. There is no way to completely kill the soil-borne pathogens as they are native in the nature soil. Instead, prevention of soil-borne pathogens from propagation and spread over is the only way to stop the incidences of soil-borne diseases.

As soil-borne fungi prefer wet and high moisture condition to propagate, therefore adequate drainage should be provided in black pepper farms to avoid water logging. Injury to black pepper root system due to cultural practices such as digging should be avoided especially during the rainy season. Over fertilization also need to be avoided as this activity could cause root injury and subsequent fungal infection. The freshly emerging runner shoots should not be allowed to trail on the ground. They must either be tied back to the standard or pruned off. When the live supports are used to train black pepper, the branches of the supporting trees must be pruned off with regular. This is to ensure better air circulation and adequate sunlight penetration in black pepper farms in order to avoid humidity rise up as this could reduce the probability of fungal infection. High humidity condition with RH more than 80% and low temperature within 22°C to 29°C in a black pepper farm is conducive for disease spread and initiation.

Since soil-borne diseases are frequently spread through infected planting materials and farm implements, hence farm hygiene must be prioritized. Black pepper cuttings used for farm establishment must come from the reliable sources without any history of soil- borne diseases. All the reused farm implements such as farming tools and supporting poles need to be disinfected before use. Destroy and remove the dead plants or infected plants which are beyond recovery from the black pepper farms is necessary. This process aims to reduce the source of inoculum and restricts the spread of pathogens.

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The diseased plant materials need to be disposed by burning or composting. These materials are strictly prohibited to be used for mulching purpose in black pepper farms before they are completely broken down. This is because soil-borne pathogens usually thrive on rotting organic matters to hunt for nutrients to support their growth. Hence, utilization of such immature organic matters or compost materials could promote the growth of soil-borne pathogens rather than to enhance the growth of black pepper plants. Besides that, such organic matters may even stunt and kill the black pepper plants through decomposition. The process of decaying organic matters could release waste products which are toxic and harmful to plants. A period of five months to six months is usually required for organic matters to well-rotted and completely broken down to produce nutrient elements which are usable by plants.

Application of soil disinfestation through solarisation during hot weather is recommended especially for black pepper farms have been infected by soil-borne diseases. In the process of soil disinfestation through solarisation, the soil is covered with a piece of thin and transparent polyethylene sheet. The edges of the polyethylene sheet are entirely enclosed by bury the edges of about 25 cm to 30 cm or even more deep into the soil to avoid air blowing or circulation under it. Solarisation process is carried out for about one month to two months. The treatment is able to kill most of the soil-borne pathogens. Whereas, the normal soil microorganisms survive in sufficient quantity to compete with pathogens and prevent them from recolonization in the treated soils. Solarisation is also able to kill weed seeds and insect pests in soil as well.

Hazard chemicals such as fungicides and nematicides appear to be the best solution to overcome soil-borne diseases for spectacular improvements in productivity. This is the most widely applied solution for management of soil-borne diseases in Malaysia. When a disease infected plant is diagnosed in a farm, all the surrounding plants including the infected plant need to be treated with the recommended chemical pesticides as listed in Table 2-2 to overcome and prevent the disease from spread over. Apart from that, it is also recommended to drench the disease prone planting points in black pepper farms with chemical pesticides and keeping those points without planting for at least one year. This process aims to completely overcome the pathogens in soils and avoid the coming new replanted black pepper plants to be infected by the same pathogens.

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2.6.2 Identification of soil-borne diseases causal fungus

Identification of causal fungus is essential for proper management of soil-borne diseases in black pepper farms (Singh et al., 2006). Morphological characteristics such as spore- producing structures, colony features and hyphal appearance are traditionally employed practices for fungal classification (Shahnazi et al., 2012). These characters are reported as key for identification and taxonomy classification of many fungal species such as Fusarium and Phytophthora (Mchau and Coffey, 1995; Shahnazi et al., 2012). Although morphological appearance are routinely used in fungal taxonomic studies, however, these approaches may not perform well especially at the level of fungal species identification as there are limited numbers of morphological characters that could be used for fungal species discrimination (Raja et al., 2017). Therefore, molecular approaches, which are effective and faster than traditional morphological identification, are widely employed to identify plant fungal pathogens (De Biazio et al., 2008).

Nuclear ribosomal internal transcribed spacer (ITS) regions have been reported as the most useful genetic markers in molecular ecology study of fungi (Peay et al., 2008). ITS regions refer to DNA sequences or corresponding transcribed regions in polycistronic rRNA precursor transcript that situated between the small and large-subunit ribosomal RNA (rRNA) genes in fungal chromosome. These regions have been widely used as universal barcode sequences for molecular identification of fungi at the species to genus level, and even within species in the studies of geographical races identification (Korabecna, 2007; Nilsson et al., 2008; Conrad et al., 2012; Schoch et al., 2012; Mahmoud and Zaher, 2015; Sanghita and Bibhas, 2015). Due to the higher degree of variation than other regions of rRNA, taxon-selective ITS amplification with universal ITS1 and ITS4 markers has become routine for identification and classification of many fungal species instead of traditional morphological identification approaches (White et al., 1990; Hibbett, 1992; Gardes and Bruns, 1993; Henson and French, 1993; Oliver, 1993; Chillali et al., 1998; Taylor et al., 2000).

PCR amplification of ITS regions has been reported as an effective method to detect pathogenic Fusarium species in agricultural crops as the defined ITS region sequences are highly variable in genus Fusarium (O’Donnell, 1992; Bowers et al., 2007; De Biazio et al., 2008). However, molecular analysis of indigenous Fusarium species in 72 | P age

Malaysia is still in developmental stage and there is little information is known about this pathogenic fungus. Therefore, isolation and identification of indigenous Fusarium species, the causal fungus to slow decline disease in Malaysia black pepper farms is essential for study the invasion mechanisms of this pathogenic fungus.

2.7 Genetic Research of Cultivated Black Pepper Plants in Malaysia

Due to the importance of black pepper in country economic, Department of Agriculture (DoA) Sarawak has intensified research on black pepper in the past decades. Many of the studies have been dedicated to explore the black pepper farming practices, insect pests and diseases management, crop breeding as well as genetic fingerprints. However, little is known about the molecular background in black pepper plants. Malaysian Pepper Board (MPB), with its mission to promote and develop Malaysian black pepper industry, has successfully isolated a number of Resistance Gene Analogues (RGA) from two pepper species known as Piper nigrum L. cv. Semengok Aman (susceptible to diseases) and P. colubrinum Link (diseases resistant wild pepper plant) by using degenerate primers designed to identify Nucleotide-Triphosphate Binding Site (NBS)- Leucine-Rich Repeat (LRR) domains of black pepper disease resistance genes or R- genes (Lau et al., 2012).

The partial sequence length of the isolated pepper RGA is range from 490 bp to 540 bp and the predicted pepper RGA protein sequences consist of Kinase-1 (P-loop), Kinase-2 (VLDDVW) and Kinase-3 (GLPL) domains. These RGA protein sequences are 42% to 47% similar to the sequences of other plant disease resistance proteins that deposited in National Center for Biotechnology Information (NCBI) GenBank database. However, several sequences of the isolated RGA proteins were found identical among susceptible P. nigrum L. cv. Semengok Aman and resistant P. colubrinum Link although the two plants are from different species. These results have showed that similar R-genes may be involved in defence-related mechanism pathways of different Piper species. This has encouraged MPB to conduct further research in studying the complexity of gene regulations in pepper plants defence mechanisms.

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One of the efforts of MPB toward this purpose is to establish Expressed Sequence Tag (EST) database for P. nigrum L. cv. Semengok Aman (Lau, 2013). EST database is generated by high-throughput single-pass sequencing of complementary DNA (cDNA) library. This technique was used for genome exploration of numerous plant species such as alfalfa (Hays and Skinner, 2001), mint (Lindqvist et al., 2006), castor oil plant (Lu et al., 2007), wheat (Lazo et al., 2004), grape (Xu et al., 2009) and cotton (Kumar et al., 2006). EST database provides direct evidence to all the sampled transcripts and therefore is a rich resource for plant genes discovery and annotation (Shivashankar et al., 2006). Although ESTs is a profile of expressed gene sequences, but it is usually does not contain full length gene sequence. This is because ESTs are relatively short DNA sequences generated from the 5’ and 3’ ends of cDNA clones. Besides that, EST approach also permits low quality bases of data to be generated due to single-pass DNA sequencing. Redundant of gene sequences is another limitation of EST technique. In order to overcome these limitations and to gain more unique gene sequences (unigenes), a new and advanced technique than EST approach is necessary for more comprehensive establishment of genetic database for pepper plants.

The new developments of sequencing technologies in the recent dedaces have facilitated the elucidation of molecular mechanisms and pathways in black pepper plants. RNA- Seq is a revolutionary tool that provides many advantages in transcriptome profiling. This advanced technique, by using Next-Generation Sequencing (NGS) platforms such as Illumina/Solexa, ABI/SOLiD and Roche/454, has offered many advantages than EST and traditional microarray-based gene expression approaches. It is significantly cheaper, faster, needs significantly less DNA and is more accurate and reliable than traditional sequencing approaches. RNA-Seq can be used to cover a wide application from simple mRNA profiling to entire transcriptome analysis in an organism. This technology also assists in understanding the molecular mechanisms and signalling pathways that control the development of an organism. RNA-Seq has been employed to built the first dataset of black pepper root transcriptome in the year 2012 (Gordo et al., 2012). It also has been employed to describe fruit transcriptome in black pepper according to Hu et al. (2015) and to discover black pepper R-genes involved in phenylpropanoid metabolism pathway in against Phytophthora foot rot as reported by Hao et al. (2016). With the advantages and wide applications of RNA-Seq technologies, the genomic sequence information of

74 | P age black pepper is within reach to aid the achievement of goals in the improvement of crop qualities and diseases management.

2.8 Biological Control

Biological control or biocontrol is an environmental friendly method of controlling and reducing plant diseases and insect pests by using other living organisms, such as natural predators, parasitoids, pathogens and competitors to plant diseases causal organisms and insect pests. It is a promising practice to replace hazard chemicals in controlling insect pests and diseases of agricultural crops. Implementation of biocontrol can be carried out through three basic practices, known as importation, augmentation and conservation as described by Unruh and Tom (1993). Importation type of biocontrol practice is refers to the introduction of natural enemies to a new locale where naturally the organisms do not occur, in the hope of achieving control of plant pathogens. Even though it is a classical approach of biocontrol, but the introduced population of natural enemies may transform and become dangerous pathogen to the crops (Mahr and Ridgway, 1993).

Augmentation biocontrol is a practice to induce existing populations of natural enemies in a locale to combat pathogens. This practice constitutes prevention rather than cure. Plant pathogens are usually suppressed and kept down to low level in a locale by gradually inoculation of biocontrol agents to the locale in order to set up a longer-term control. Augmentation is an effective and commonly applied practice to control plant pathogens. However, the success of this practice is highly depends on interactions between plant pathogens and biocontrol agents (Ridgway and Vinson, 1977). The population conservation of existing natural enemies adapted in a locale is a simple and cost-effective biocontrol practice (Bosch and Telford, 1964). It is usually conducted through habitat manipulation to favor natural enemies and ensure the survival as well as high reproduction of these organisms.

Currently, biocontrol has become a critical concern to overcome soil-borne diseases in Malaysia black pepper farms for spectacular yield improvement. It is an important component of black pepper IDM program in Malaysia for soil-borne diseases. Biocontrol agents for plant diseases are generally known as antagonists, which usually 75 | P age comprise of microorganisms such as rhizobacteria. Rhizosphere, the narrow zone of soil influenced by plant roots, is populated by diverse range of microorganisms that play an important role in enhancing plant growth (Saharan and Nehra, 2011; Hrynkiewicz and Baum, 2012). Bacteria that colonize plant roots at all stages of plant growth and able to enhance the nutrient status of host plants are named as plant growth promoting rhizobacteria or PGPR (Antoun and Kloepper, 2001). The widely reported PGPR comprise of species from genera Bacillus, Pseudomonas , Streptomyces, Burkholderia, Stenotrophomonas, Arthrobacter and Serratia (Joseph et al., 2007; Messiha et al., 2007; Vijayalakshmi et al., 2011; Ashwini and Srividya, 2014; Vanitha and Ramjegathesh, 2014; Law et al., 2017). These groups of bacteria have been reported as effective biofertilizers as well as biocontrol agents for root rot, stalk rot and damping-off diseases in various agricultural crops such as sweet pepper, maize, tomato and banana (Djordjevic et al., 2011; Farooq and Bano, 2013; Apastambh et al., 2016). Biocontrol products of these bacteria have been used in several ways to reduce diseases in seeds, seedlings and planting materials of tomato and pepper plants in the field with various degrees of success.

PGPR improve the productivity and growth of crop plants as well as to assist in sustainability of safe environment in many different ways if compare with chemical pesticides and fertilizers. These bacteria form a mutualistic relationship with host plants. They colonize at the surface of plant roots and the host plants are benefit from the nutrients provided by bacteria. Earlier studies have reported that PGPR enhance root growth by increasing the number of root hairs through auxin or indole-3 -acetic acid (IAA) production (Spaepen and Vanderleyden, 2011; Khin et al., 2012; Ahmed and Hasnain, 2014). PGPR also aid in crop nutrition as they could improve the available contents of macronutrients such as nitrogen (N), phosphorus (P) and potassium (K) in soil through events of nitrogen fixation, phosphate and potassium solubilisation. These activities could improve NPK uptake by crop plants (Oteino et al., 2015; Júnior et al., 2016; Mahendra et al., 2016). Therefore, PGPR has played a crucial role in biofertilization. The earlier research findings showed the soil treated with PGPR could significantly increase the growth and yields of agricultural crops (Lim and Kim, 2009; Asif et al., 2018).

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Apart of biofertilization, PGPR consist of numerous characters which allow them to actively colonize at the rhizosphere and prevent deleterious effects of plant pathogens (Rangajaran et al., 2003; Saikia et al., 2005). The antagonistic effects of PGPR are usually related to the production of several secondary metabolites such as antibiotics, siderophore, hydrogen cyanide and hydrolytic enzymes that cause antibiosis to plant pathogens (Zhang et al., 2001; Haas and Keel, 2003; Charlotte and Peter, 2012; Ahmed and Holmstrom, 2014). The well-known genera of PGPR such as Pseudomonas, Bacillus and Serratia are reported to have chitinolytic activity (Mabuchi et al., 2000; Someya et al., 2001; Viterbo et al., 2001; Wen et al., 2002; Huang et al., 2005). These rhizobacteria play an important role in biological control of fungal pathogens as they are capable of lysing chitin, a major constituent of the fungal cell wall (Yu et al., 2002; Zhang and Fernando, 2004; Abdullah et al., 2008). Even though chemical pesticides and fertilizers are effective to replenish nutrients in soils and to eliminate soil-borne diseases in black pepper farms, but they are not considered as long term solutions due to the concerns of exposure risks such as environmental pollution, health hazards, residue persistence and high input costs. Therefore, biological approach by using other living entities such as PGPR was proposed as an ideal alternative to chemical inputs in black pepper cultivation.

Apart to aid in black pepper plant nutrition and growth, yield improvement as well as diseases management, these root-associated bacteria also can boost the resistancy level of black pepper plants and rendering the entire black pepper plant more resistant to pathogens by activating plant defence-related response through induced resistance (Jetiyanon et al., 2003; Zhang et al., 2004; Beneduzi et al., 2012). Although a variety of biocontrols have been defined nowadays, but further development and effective adoption of these bacterial agents in black pepper crop agricultural practices will require a greater understanding of the complex interactions among black pepper plants, biocontrol agents and pathogens.

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2.9 Plant Defence Mechanisms

Plant defence mechanisms are determined by the presence of resistance genes (R-genes) that enable plants to recognize microorganisms and activate inducible defence systems to combat microbial invasion (Grant et al., 1998). Genes conferring resistance to various infections caused by soil-borne microorganisms such as bacteria, fungi and parasitic soil worms have been isolated from many plant species (Baker et al., 1997; Gebhardt, 1997; Hammond-Kosack and Jones, 1997). Earlier studies have revealed that most of the plant R-genes encodes cytoplasmic proteins that contain nucleotide-triphosphate binding site (NBS) and leucine-rich repeat (LRR) domains (Hulbert et al., 2001). The motifs of NBS -LRR domains are well conserved in R-genes of difference plant species that commonly known as resistance gene analogues or RGA (Kanazin et al., 1996; Shen et al., 1998).

The widely reported plant RGA are such as NBS-LRR repeats, receptor like kinases, pentatricopeptide repeats and apoplastic peroxidases (Sekhwal et al., 2015). The genetic analysis of plant RGA with NBS-LRR domains has confer that this group of genes is able to recognize and respond to infections caused by numerous soil-borne pathogens, nematodes and viruses in various crop species (Leister et al., 1996; Yu et al., 1996; Aarts et al., 1998; Seah et al., 1998; Speulman et al., 1998). For example, Mi and I2CI proteins in tomato are confer resistance to root-knot nematode and slow decline, respectively (Ori et al., 1997; Milligan et al., 1998). N protein in tobacco is reported resistance to mosaic virus (Whitham et al., 1994). Cre3 protein in wheat is also found to be effective in against root-knot nematodes (Lagudah et al., 1997). L6 and M proteins in flax are resistance to fungus rust diseases (Lawrence et al., 1995; Anderson et al., 1997). The other members of plant NBS-LRR resistance protein are such as Prf in tomato, Xa1 in rice, RPM1 in Arabidopsis and RGC2 in lettuce (Bent et al., 1994; Salmeron et al., 1996; Meyers et al., 1998; Yoshimura et al., 1998).

In natural environment, plant cells are exposed to various infections such as bacteria, fungi, viruses and root-knot nematodes (Faulkner and Robatzek, 2012). These organisms invade plant cells to access nutrients for their proliferation and subsequently caused damage to host cells (Giraldo and Valent, 2013). Therefore, plants have developed numerous mechanisms to defend themselves in against pathogen invasions. Different with animal cells, plant cells do not developed an acquired immune system to 78 | P age recognise non-self-invaders. Instead, plant defence response depends on recognition of invading pathogens by Pattern Recognition Receptors (PRRs) that usually found on the surface of cell plasma membranes. This mechanism is known as Pathogen-Associated Molecular Pattern (PAMP) Triggered Immunity (PTI) and is the first layer of plant defence mechanism (Bittel and Robatzek, 2007; Segonzac and Zipfel, 2011). Through PRRs, plant cells could recognize conserved microbial invader proteins and variable pathogen effectors. This recognition triggers defence response in plants that involves the functions of NBS-LRR class of proteins.

Bacterial flagellin (flg) is the best characterized invader proteins that trigger PTI in plant cell (Park et al., 2014). The peptide flg22, a conserved section of 22 amino acids in the N-terminal of bacterial flagellin has been frequently used as a synthetic inducer to trigger PTI mechanism by activating FLS2, a LRR receptor like serine/threonine protein kinase in plant cells (Felix et al., 1999). Activation of FLS2 triggers Mitogen-Activated Protein Kinase (MAPK) signalling pathway through formation of PRR-PAMP complex. The signalling initiated by FLS2 is transmitted through MAPK signalling pathway and activates plant defence genes for emission of antimicrobial compounds (Asai et al., 2002; Boudsocq et al., 2010).

Apart of FLS2, the Cf-9 disease resistance protein of plant cells is confers resistance to fungal pathogens that express the corresponding avirulence proteins, Avr9 (Rivas et al., 2002). Cf-9 is located predominatly in the plasma membrane. It is an extra cytoplasmic membrane anchored proteins consist of LRR domain and short cytoplasmic domain but without an apparent signalling domain. Cf-9 is consistent with a receptor function to recognize and respond to fungal invader proteins. But, the mechanism of this protein in signal transduction has yet to be determined (Dixon et al., 2000). Same with the FLS2, Cf-9 is also a regulator to produce reactive oxygen species and hypersensitive response/localized programmed cell death (Pieterse et al., 2009; Bart et al., 2011; Tena et al., 2011; Mazzotta and Kemmerling, 2011; Henry et al., 2013; Larroque et al., 2013).

The second layer of plant defence is known as Effector-Triggered Immunity (ETI). This mechanism defines the relationship in between plants and their pathogens through the interaction of plant resistance proteins and pathogen-derived virulence factors (Spoel and Dong, 2012). Pathogens can suppress PTI by directly inject their virulence factors 79 | P age called effectors into plant cell through secretion systems. When PTI is defeated, plant cells become truly exposed to pathogens. To carry on with invasion, pathogens release their effectors into host cell cytosol. Effectors aid in pathogenesis by assisting pathogen attachment, pathogen movement, disrupts host cellular processes, interrupt host proteins translation and eventually evade host defence response (Rajamuthiah R and Mylonakis, 2014).

Pathogenic effectors are recognised by NBS-LRR proteins presence in cytoplasm of plant cells. NBS-LRR proteins coded by plant R-genes consist of specific domains such as LRR and Toll/Interleukin-1 receptor (TIR) which permit them to direct binding with pathogenic effectors (DeYoung et al., 2006). ETI is triggered by MAPK signalling pathways (Hayden et al., 2006; Vallabhapurapu and Karin, 2009; Arthur and Ley, 2013). This mechanism response is more amplified and faster than PTI. ETI restricts pathogen invasions through several mechanisms such as to release antimicrobial molecules such as hydrolytic enzymes to lyse effectors, to deposit callus at the infection sites as physical barrier, to cause encasement of invaders and to develop hypersensitive response leading the infected host cells to apoptosis (Greenberg and Yao, 2004; Jones and Dangl, 2006). Both PTI and ETI alleviate pathogen invasion by inducing downstream defence-related responses in plants.

Induced resistance of plants comprise of two major modes of action known as induced systemic resistance (ISR) and systemic acquired resistance (SAR). These two modes of induction can be either local or systemic depends on the type of stimuli. Local resistance mechanism usually involves cell wall reinforcements through callose apposition and lignification process that mediated by plant RGA corresponding to the production of apoplastic peroxidases, an enzyme released in plant in response to wounding due to mechanical damage (Polle et al., 1994; Minibayeva et al., 2015). Apart from that, the induced local defence mechanisms in plants also include production of antimicrobial secondary metabolites and the accumulation of pathogenesis-related (PR) proteins, a group of enzyme act directly to lyse invader cells or induce localized cell death.

The systemically enhanced local resistance mechanism in plant which is activated upon root colonization by non-pathogenic rhizobacteria is known as ISR (Pieterse et al., 80 | P age

2001; Siddiqui and Shaukat, 2002; Bakker et al., 2003) Rhizobacteria-mediated ISR mode of action is reported to increase physical or chemical barrier of host plant cells to against a broad spectrum of pathogenic invaders rather than direct killing or restriction of the invaders (Choudhary et al., 2007). The ISR pathway is mediated by jasmonic acid (JA) and/or ethylene (ET) which is stimulated following applications of non-pathogenic rhizobacteria (Hartman et al., 2016). However, development of ISR in host plant cells is highly depends on specific recognition of microbial components such as flagella, signal molecules and antifungal secondary metabolites such as siderophores (Chilekampalli and Ramu, 2013). Moreover, infected plant cells are able to produce a long distance signal to induce systemic expression of defence known as SAR in distal non-colonized cells (Shah, 2009).

SAR is a resistance response occurs in entire plant that induced by earlier localized exposure to an invader. Therefore, SAR is considered analogous to the innate immune system found in animals (Ausubel, 2005). It is an important mechanism either for plant to resist diseases or to recover from diseases. Instead of ethylene and jasmonic acid, SAR is mediated by salicylic acid (SA), a compound produced following pathogen infection. Different with ISR, SAR is triggered by accumulation of PR proteins and can elicit hypersensitive response (HR) and cause encasement of invaders in a small area of the site of infection (Gautam and Stein, 2011) as well as directly to lyse invader cells or induce localized cell death.

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CHAPTER 3

MATERIALS AND METHODS

3.1 Identification of Indigenous Causal Fungal Strains of Slow Decline

3.1.1 Isolation of soil-borne fungi

3.1.1.1 Field sampling

Three black pepper farms located at Mongkos and Mujat villages of Serian Division and Jagoi Duyuh village of Serikin town that have been reported with the incidents of slow decline in the year 2016 were selected as sampling sites in the current study. The farms were designated DF1 (Jagoi Duyuh) (1°19'38.0"N 110°00'12.9"E), DF2 (Mongkos) (0°53’06.1”N 110°35’21.7”E) and DF3 (Mujat) (0°53'26.5"N 110°33'59.3"E). The average farm size of the selected disease farms are about 0.5 acre which consist of roughly 400 black pepper plants in total. Majority of the black pepper cultivars planted in these farms is variety “Semengok Aman” (45%) and variety “Kuching” (40%). Apart from that, the farms also contain 15% other black pepper cultivars such as variety “Semengok Emas” (8%) and variety “Semengok Perak” (7%).

Rhizospheric soils and root samples of infected black pepper plant s were collected from the selected disease farms. A number of three individual plants for each black pepper cultivar planted in the disease farms that have been confirmed with the symptoms of slow decline disease were randomly selected as sampling points per farm location. The infected black pepper roots together with the adherent soils were carefully removed from rhizosphere as shown in Figure 3-1. The collected rhizospheric soils and disease root samples were packed and sealed in sterile collection bags prior to delivery to the laboratory.

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(a) (b) (c) Figure 3-1. Sampling of slow decline disease causal fungal strains from black pepper farms situated at Mongkos and Mujat villages of Serian Division and Jagoi Duyuh village of Serikin town, (a) Collection of soils and root samples from the rhizosphere; (b) Loss of feeder roots and damage at collar region of infected black pepper plant; (c) Browning symptom in vascular xylem tissues of infected plant.

3.1.1.2 Direct isolation of fungal strains from disease roots

Direct isolation of fungal strains from infected roots was carried out by using modified method of Shahnazi et al. (2012). The disease root samples were carefully washed with adequate tap water to remove excess soils prior to surface sterilization in 1% sodium hypochlorite (NaOCl) solution for 5 min. The sterilized root samples were rinsed twice with adequate sterile double distilled water (ddH2O) and followed by air drying on the sterile filter papers. After drying process, the root samples were cut into small sections of 5 mm in length before plating on Potato Dextrose Agar (PDA) medium. The plates were incubated at 30°C for 48 hours. The individual fungal cultures grown from the root segments were further isolated by transferring them onto fresh PDA plates. The new culture plates were then incubated at 30°C for 7 days.

3.1.1.3 Isolation of fungal strains from soil samples

Isolation of fungus from rhizospheric soil samples were carried out by using modified dilution plating method of Parmeter (1970). One gram of soils was suspended in 9 ml of sterile ddH2O. The soil suspension was further diluted with a serial of 10-fold dilution for six times. An amount of 0.2 ml 1:106 diluted soil suspensions was then spread on PDA plates. The plates were incubated at 30°C for 16 hours. The individual fungal 83 | P age cultures grown on the plates were further isolated by transferring them onto a fresh PDA plate. The new culture plates were then incubated at 30°C for 7 days.

3.1.2 Morphological characterization of isolated fungal strains

The pure fungal cultures grown on PDA plates were analysed for their morphological appearances. The morphological characters such as colony colour, shape, texture, spore and hyphal appearance were assessed through microscopic observation. The lactophenol cotton blue microscope slides were prepared according to the method of Astrid (1999). Microscopic visualization was carried out by using Leica (Germany) DM4000 compound microscope with 40X and 100X magnifications. The morphological appearances of the isolated fungal cultures were compared to the works of Gallegly and Hong (2008), Fatin et al. (2017) and Gogoi et al. (2017).

3.1.3 Molecular identification of isolated fungal strains

3.1.3.1 Isolation of fungal DNA

DNA isolation was carried out on fungal mycelium by using a modified CTAB-based isolation method of Doyle and Doyle (1990). DNA isolation buffer consists of 2% (w/v) cetyltrimethylammonium bromide (CTAB), 0.1 M Tris hydrochloride (Tris-HCl), 25 mM ethylenediaminetetraacetic acid (EDTA), 1% (w/v) polyvinylpyrrolidone (PVP), 1.4 M NaCl and 1% (v/v) β-mercapto ethanol (was added prior to use). A ratio of 10 ml isolation buffer per gram of fungal mycelium was used. Fungal tissues were grinded with pre-chilled pestle and mortar in the presence of liquid nitrogen. The grinded tissues were added to pre-heated isolation buffer with 65°C. The homogenate was incubated at 65°C for 15 min. During incubation, the tube was gently inverted every 5 min to mix the homogenate.

The homogenate was then cooled to room temperature. Cell debris, protein contaminants and other impurities were separated from DNA through phenol: chloroform: isoamyl alcohol (PCI) purification. Equal volume of PCI (25: 24: 1, v/v) 84 | P age was added to the homogenate. The tube was gently inverted for 20 times and followed by centrifugation at 13,000 rpm at 4°C for 15 min. After the process of centrifugation, the upper aqueous phase was collected and re-extracted with equal volume of chloroform: isoamyl alcohol (CI) at ratio 24: 1 (v/v). The centrifugation process was repeated. Fungal DNA was then recovered through isopropanol precipitation.

The re-collected upper aqueous phase of homogenate was added with 2/3 volume of cold isopropanol with -20°C and 0.1 volume of 3 M sodium acetate (NaOAc) with pH 5.2. The mixture was incubated at -20°C for half an hour. Fungal DNA was recovered from the mixture through centrifugation at 14,000 rpm at 4°C for 15 min. The obtained DNA pellets were washed with 5 ml of 70% (v/v) pre-cold ethanol (-20°C) and then centrifuged at 13,000 rpm at 4°C for 5 min to remove excess ethanol. The washed DNA pellets were subsequently air dried at room temperature. The isolated fungal DNA was then re-suspended in 300 μl of sterile ddH2O.

RNA contamination was eliminated from the isolated fungal DNA by using Promega RNase A Solution according to the manufacturer’s instruction. Fungal DNA was treated with 1% RNase by adding 3 μl of Promega RNase A Solution to the suspended DNA. The reactions were carried out at 37°C for 30 min, followed by heat inactivating of RNase at 65°C for 10 min. The purified DNA was re-treated with equal volume of PCI (25: 24: 1, v/v), followed with equal volume of CI (24: 1, v/v) and recovered through 2/3 volume of cold isopropanol (-20°C). The precipitated DNA pellets were washed with 1 ml of 70% (v/v) pre-cold ethanol (-20°C); air dried at room temperature and then re-suspended in 100 μl of sterile ddH2O. The isolated fungal DNA was stored at -20°C prior to next experimental stage.

3.1.3.2 Quality assessment of isolated fungal DNA

The intact of purified fungal DNA was assessed by using Invitrogen (USA) SYBR® Safe DNA Gel Stain prepared 1% (w/v) agarose gel. Fungal DNA was electrophoresed at 100 volts for an hour in 1X TBE (90 mM Tris-borate, 2 mM EDTA with pH 8.0) buffer. The agarose gel was visualized by using ThermoFisher Scientific (USA) Safe Imager™ 2.0 Blue-Light Transilluminator and documented by using Bio-Rad 85 | P age

(California) Gel Documentation 2000 System. The isolated fungal DNA was then quantified by using Gene-Quant (USA) UV Spectrophotometer at spectrum wavelengths of 230 nm, 260 nm and 280 nm with a dilution factor of 100.

3.1.3.3 PCR amplification of fungal ITS regions

Universal primers ITS1 (5’-tccgtaggtgaacctgcgg-3’) and ITS4 (5’-tcctccgcttattgatatgc- 3’) were used for PCR analysis of fungal ITS regions, the defined molecular barcode for identification of fungi species as reported by Conrad et al. (2012) and Raja et al. (2017). PCR amplifications were carried out by using Applied Biosystems (USA) VeritiTM 96- Well Thermal Cycler. The total 25 μl PCR reaction volume consists of 1X PCR buffer [20 mM Tris-HCl, 50 mM potassium chloride (KCl)], 1.5 mM magnesium chloride

(MgCl2), 0.2 mM each nucleoside triphosphate (dNTP) mix, 0.4 μM each primer, 1 unit Taq polymerase (Invitrogen, USA) and 30 ng fungal template genomic DNA. PCR reaction conditions were set as 1 cycle of initial denaturation at 94°C for 2 min, followed by 35 cycles of denaturation at 94°C for 30 s; annealing at 55°C for 30 s; extension at 72°C for 30 s and ended with 1 cycle of final extension at 72°C for 5 min.

3.1.3.4 Purification of PCR amplified ITS fragments

PCR amplicons were gel-excised by using Invitrogen (USA) PureLink Quick Gel Extraction Kit according to the manufacturer’s instruction. The 1.0% (w/v) agaorse gel slices containing PCR amplicons were dissolved in 3 gel volumes of Gel Solubilisation Buffer L3 at 50°C for 10 min. During incubation, the tubes were gently inverted in every 2 min for well mixing of the mixture to permit proper gel dissolution. After gel slices appear dissolved, the tubes were further incubated for an additional 5 min to ensure complete gel dissolution. To get optimal DNA yields, 1 gel volume of isopropanol at room temperature was added to the tube containing well dissolved gel slice and mix well. PCR fragments were then recovered through membrane binding. The mixture was applied to Quick Gel Extraction Column inside a Wash Tube. The column was centrifuged at 13,000 rpm for 1 min at room temperature. The flow through was discarded. PCR fragments bond on the membrane of the extraction column were 86 | P age washed by using Wash Buffer W1 containing ethanol. The fragments were then eluted from the wash column membrane by using Elution Buffer E5. The purified PCR products were stored at -20°C prior to next experimental stage.

3.1.3.5 Cloning of fungal ITS fragments

The purified PCR products were cloned by using Promega (USA) pGEM®-T Easy Vector System II according to the manufacturer’s instruction. The total 10 μl ligation volume consists of 1X rapid ligation buffer [30 mM Tris-HCl (pH 7.8), 10 mM MgCl2, 10 mM dithiothreitol (DTT), 1 mM adenosine triphosphate (ATP), 5% polyethylene glycol], 50 ng pGEM®-T Easy vector, 3 Weiss units T4 DNA ligase and 3 μl PCR products of fungal ITS fragments. Ligation was carried out overnight at 4°C. The recombinant vectors were subsequently transformed into JM109 high efficiency competent E. coli cells through heat-shock at 42°C for 90 s without shaking. The transformants were incubated at 37°C for 16 hours on LB medium consists of 100 μg/ml ampicillin, 0.5 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) and 80 μg/ml 5- bromo-4-chloro-3-indolyl-β-D-galacto pyranoside (X-Gal) as selective agents. Blue and white colour screening of bacterial colonies as well as PCR amplification with T7 and SP6 promoter primers were carried out to identify the presence of positive clones.

3.1.3.6 Recombinant plasmid DNA isolation

The recombinant vectors were isolated from positive bacteria clones by using Promega (USA) Wizard® Plus SV Minipreps DNA Purification Kit according to the manufacturer’s instruction. One ml of overnight grown bacterial culture was centrifuged at 13,000 rpm for 2 min to pellet the cells. The supernatant was discarded and the cells were suspended in 250 µl of Cell Resuspension Solution [50 mM Tris-HCl with pH 7.5, 10 mM EDTA, 100 μg/ml RNAse A]. The suspended bacterial cells were treated with 250 µl of Cell Lysis Solution [1% sodium dodecyl sulfate (SDS), 0.2 M sodium hydroxide (NaOH)], followed by 10 µl of Alkaline Protease Solution and 350 µl of

Neutralization Solution [4.09 M guanidine hydrochloride (CH6ClN3), 0.759 M potassium acetate (CH3CO2K), 2.12 M glacial acetic acid (CH3COOH) with pH 4.2]. 87 | P age

The suspension was centrifuged at 13,000 rpm at room temperature for 10 min to discard cell debris. Recombinant plasmid DNA was then recovered through membrane binding. Cell lysate, the aqueous phase of suspension was applied to Minicolumns and centrifuged at 13,000 rpm for 1 min at room temperature. The flow through was discarded. Recombinant plasmid DNA bond on the membrane of Minicolumns was washed with Column Wash Solution (60 mM CH3CO2K, 8.3 mM Tris-HCl with pH 7.5, 0.04 mM EDTA with pH 8.0, 60% ethanol) and then eluted from the columns membrane by using Nuclease Free Water. The isolated plasmid DNA with PCR amplified fungal ITS fragments were stored at -20°C prior outsourced for single-pass DNA sequencing services.

3.1.3.7 Sequence data analysis of fungal ITS fragments

The purified recombinant plasmid DNA PCR amplified fungal ITS fragments were outsourced for single-pass DNA sequencing services. The obtained sequence data of fungal ITS fragments were searched against NCBI GenBank non-redundant (nr) database by using Biological data management, identification, classification and statistics (BioloMICS) software for similarity analysis (Robert et al., 2011).

3.1.4 Diagnostic of slow decline disease causal fungus

3.1.4.1 Pathogenicity assays of PnSA roots

In order to diagnose the causal fungal strains of reported slow decline incidents that occurred in disease farms DF1, DF2 and DF3 located at Serikin and Serian areas of Sarawak, three month-old black pepper cuttings of susceptible P. nigrum L. cv. Semengok Aman (PnSA) were used as host plants in fungal pathogenicity assays. Four fungal strains coded FS010, FS008, PN002 and RM001 isolated from the disease farms were used as pathogenic agents to infect the roots of PnSA, the main black pepper cultivar had been planted in the disease farms. Pathogenicity assays were conducted in triplicates with the presence of three biological replicates.

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Root infections were carried out by using a modified method of Edel-Hermann et al. (2012). Three month-old PnSA cuttings were carefully uprooted from the soil medium. The roots and collar regions of the uprooted PnSA were washed carefully with adequate tap water to remove excess soils prior wounded with sterile scalpels to facilitate fungal penetration. The washed PnSA roots were dipped in fungal conidial suspensions for 10 min before transplanted into polybags filled with sterile soil medium. Fungal infections were repeated at 2 days interval by drenching the soil surrounding collar regions of the infected PnSA cuttings with fresh fungal conidial suspensions. This is to prevent fungal conidial suspensions from drying out during the period of pathogenicity assays and also to ensure the continuous of fungal infections at PnSA roots. Fungal infections were continued fore thre months until the infected PnSA cuttings have shown clear symptoms of slow decline disease. In pathogenicity assays, the PnSA roots treated with sterile ddH2O through the same process were served as control treatments.

3.1.4.2 Colonization assays of fungal cultures on PnSA roots

Colonization assays were carried out to measure the percentage of PnSA roots that have been colonized by pathogenic agents. The infected PnSA roots were randomly sampled for assessments. The harvested root samples were washed with adequate tap water, surface sterilized in 1% NaOCl solution for 5 min, rinsed twice with adequate sterile ddH2O, air dried on sterile filter papers, cutted into small segments of 5 mm in length and plated on PDA medium at 30°C for 48 hours. Individual fungal cultures grown from the root segments were isolated by transferring them onto new plates with fresh PDA medium. The new culture plates were incubated at 30°C for 7 days. The number of root segments that have been colonized by fungal cultures was measured. The percentage of fungal colonization was subsequently calculated through the formula, Colonization (%)

= (Nf/Nt) x 100%, where Nf denotes number of root segments with fungal colonization and Nt denotes total number of root segments (Porras et al., 2007). Isolation of fungal DNA was carried out by using a modified CTAB-based isolation method of Doyle and Doyle (1990). The isolated fungal cultures were identified through PCR amplification and sequencing of ITS regions.

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3.2 Elucidation of Defence-Related Mechanism Pathways in Pepper Plants

3.2.1 Plant materials

In order to elucidate the molecular pathways and defence-related genes in black pepper, three month-old cuttings of P. nigrum L. cv. Semengok Aman (PnSA) and P. nigrum L. cv. Kuching (PnKch) were used as plant materials in the current study. As the two black pepper cultivars are susceptible to F. solani and other soil-borne fungi invasion, hence a resistant wild pepper plant known as P. colubrinum Link (Pc) that well maintained in MPB Germplasm Nursery was also included in the current study with the aim to obtain more comprehensive transcriptome dataset of black pepper defence-related mechanism pathways.

3.2.2 Fungal infection assays on pepper plant tissues

3.2.2.1 Pathogenic agents

F. solani isolate FS010, the diagnosed causal fungal strain of slow decline incident reported in disease farm DF3 located at Mujat village of Serian Division was used as pathogenic agent in the current study. This fungal isolate was used as pathogenic agent to infect the leaves and roots of susceptible PnSA and PnKch as well as resistant Pc cuttings. The total RNA isolated from pepper leaves was used for conducting RNA-Seq. Pepper leaves were harvested from an individual cutting at three time points of infection during pathogenicity assays, i.e. prior infection (0th day), first week after infection (7th day) and second week after infection (14th day). This aimed to assess the expression of possible pepper R-genes at different time frame of infection process. Meanwhile, pepper roots were harvested at the end of pathogenicity assays (three months after infection) when the symptoms of slow decline disease such as foliar yellowing, defoliation, plant wilt, damage of collar regions and loss of feeder roots were observed on infected pepper cuttings. The pepper root total RNA was isolated and used in expression analysis for the assessement of targeted pepper R-genes.

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3.2.2.2 Leaves infections

Leaves infections were carried out by using a modified method of Spina et al. (2008). A number of ten healthy leaves per individual of pepper plant were randomly selected for infection. The leaves were washed with adequate tap water, surface sterilized with 1%

NaOCl, rinsed twice with adequate sterile ddH2O and air dried prior fungal infections. Each leaf was infected by applying 10 μl F. solani conidial suspension to a wound near petiole that created by using sterile scalpel to facilitate F. solani penetration. The fungal infections were repeated daily by applying equal volume of fresh conidial suspension to the same infection points at leaves petioles. This is to ensure the continuous of F. solani infection at pepper leaves by preventing conidial suspension from drying out during the period of pathogenicity assays. F. solani infections on pepper leaves were continued for two weeks until the infected leaves have shown clear symptom of foliar yellowing. In pathogenicity assays, pepper leaves treated with sterile ddH2O through the same process were served as control treatments. Leaves infections were carried out in three biological replicates.

3.2.2.3 Roots infections

The infections of F. solani isolate FS010 at PnSA, PnKch and Pc roots were carried out by using a modified method of Edel-Hermann et al. (2012). The three month-old pepper cuttings were carefully uprooted from the soil medium. The roots and collar regions of pepper cuttings were washed carefully with adequate tap water to remove excess soils prior wounded by using sterile scalpels to facilitate F. solani penetration. The washed pepper roots were dipped in F. solani conidial suspensions for 10 min prior transplanted into polybags filled with sterile soil medium. Fungal infections were repeated at 2 days interval by drenching the soil surrounding collar regions of the infected pepper cuttings with fresh F. solani conidial suspensions. This is to prevent conidial suspensions from drying out during the period of pathogenicity assays and also to ensure the continuous of F. solani infections at pepper roots. The fungal infections were continued for three months until the infected pepper cuttings have shown clear symptoms of slow decline disease such as foliar yellowing, defoliation, plant wilt, damage of collar regions and

91 | P age loss of feeder roots. In pathogenicity assays, the pepper roots treated with sterile ddH2O through the same process were served as control treatments.

3.2.2.4 Fungal colonization on pepper plant tissues

Colonization assays were carried out to measure the percentage of infected pepper leaves and roots that have been colonized by pathogenic agent, F. solani isolate FS010. The infected pepper plant tissues were randomly sampled for assessments. The collected pepper plant tissues samples were washed with adequate tap water, surface sterilized in

1% NaOCl solution for 5 min, rinsed twice with adequate sterile ddH2O, air dried on sterile filter papers, cutted into small segments of 5 mm in length and plated on PDA medium at 30°C for 48 hours. Individual fungal cultures grown from the pepper plant tissues segments were isolated by transferring them onto new plates with fresh PDA medium. The new culture plates were incubated at 30°C for 7 days. The number of pepper plant tissues segments that have been colonized by F. solani isolate FS010 was measured. The percentage of F. solani colonization at the infected pepper leaves and roots was then calculated through the formula as described by Porras et al. (2007),

Colonization (%) = (Nf/Nt) x 100%, where Nf denotes number of pepper plant tissues segments with F. solani isolate FS010 and Nt denotes total number of pepper plant tissues segments. Isolation of F. solani DNA was then carried out by using a modified CTAB-based isolation method of Doyle and Doyle (1990). Identity of isolated fungal cultures (F. solani isolate FS010) was further reconfirmed through PCR amplification and sequencing of ITS regions.

3.2.3 Total RNA isolation

Total RNA isolations were carried out on pepper leaves and roots by using a modified CTAB-based extraction method. All solution prepared for RNA isolation was treated with 0.1% diethyl pyrocarbonate (DEPC). RNA isolation buffer consists of 2% CTAB, 0.1 M Tris-HCl, 25 mM EDTA, 1.4 M NaCl, 1% PVP and 1% β-mercaptoethanol (was added prior to use).

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A ratio of 10 ml isolation buffer per gram of frozen plant tissues was used. The frozen plant tissues were grinded with pre-chilled pestle and mortar in the presence of liquid nitrogen. To the grinded plant tissues, 10 ml of pre-heated (65°C) isolation buffer was added. The homogenate was incubated at 65°C for 30 min. During incubation, the tube was gently inverted in every 5 min to mix the homogenate. The homogenate was then cooled to room temperature and added with equal volume of PCI (25: 24: 1, v/v). The tube was gently inverted for 20 times and centrifuged at 13,000 rpm at 4°C for 15 min. The upper aqueous phase was collected and re-extracted with equal volume of CI (24: 1, v/v). The centrifugation process was repeated. Total RNA was recovered through LiCl precipitation. The re-collected upper aqueous phase was added with 0.2 volume of cold 10 M LiCl with -20°C. RNA precipitation was carried out at -20°C for overnight. Total RNA was then recovered from LiCl solution through centrifugation at 14,000 rpm at 4°C for 15 min. The precipitated RNA pellets were washed with 5 ml of 75% (v/v) cold ethanol (-20°C), centrifuged at 13,000 rpm at 4°C for 5 min to remove excess ethanol, air dried at room temperature and then re-suspended in 100 μl of sterile 0.1% (v/v) DEPC treated water. The isolated total RNA was stored at -80°C prior to next experimental stage.

3.2.3.1 Purification of isolated total RNA

DNA impurities were removed from the isolated total RNA by using Promega (USA) RQ1 RNase-Free DNase I Solution according to the manufacturer’s instruction. The total 10 μl DNase reaction volume consists of 1X RQ1 RNase-free DNase reaction buffer [40 mM Tris-HCl with pH 8.0, 10 mM magnesium sulfate (MgSO4), 1 mM

CaCl2] and 1 unit of RQ1 RNase-free DNase per 1 μg of total RNA. The reactions were carried out at 37°C for 30 min, followed by heat inactivating of DNase at 65°C for 10 min with 0.1 volume of RQ1 DNase stop solution [20 mM egtazic acid (EGTA) with pH 8.0]. The purified total RNA were treated with equal volume of PCI (25: 24: 1, v/v), followed with equal volume of CI (24: 1, v/v) and recovered through 2/3 volume of cold isopropanol (-20°C). The precipitated RNA pellets were washed with 1 ml of 75% (v/v) cold ethanol) (; -20°C air dried at room temperature and then re-suspended in 100 μl of sterile 0.1% (v/v) DEPC treated water. The purified total RNA was stored at -80°C prior to next experimental stage. 93 | P age

3.2.3.2 Quality assessment of the isolated total RNA

The intact of purified total RNA was assessed by using Agilent (USA) RNA 6000 Nano Kit according to the manufacturer’s instructions. RNA was electrophoresed in Agilent RNA 6000 Nano Gel-Dye Mix [65 μl RNA 6000 Nano gel matrix, 1 μl RNA 6000 Nano dye concentrate] that loaded in a sterile Agilent (USA) RNA Nano Chip. Each sample well on RNA Nano Chip consists of 5 μl Agilent RNA 6000 Nano marker and 1 μl isolated total RNA samples. The ladder well on the chip consists of 5 μl Agilent RNA 6000 Nano marker and 1 μl Agilent RNA ladder instead of isolated total RNA samples. The concentration and RNA Integrity Number (RIN) of the purified total RNA was then documented by using Agilent (USA) 2100 Bioanalyzer. The procedures of electrode decontamination of the Bioanalyzer system were carried out with RNaseZAP solution and RNase-free water prior the chip runs was started. This is mainly to avoid decomposition of isolated total RNA samples during electrophoresis process.

3.2.4 Transcriptomics analysis of pepper defence-related genes

3.2.4.1 RNA sequencing

Messenger RNA (mRNA) in leaves total RNA were poly-A enriched with oligo-dT attached magnetic beads. The molecules were fragmented into small pieces by using divalent cations under elevated temperature. The cleaved RNA fragments were reverse- transcribed into single stranded cDNA with random hexamer primers. RNA strands were cleaved from the newly synthesized first-strand cDNA through RNase H treatment. Second-strand cDNA were then synthesized as replacement strands with DNA polymerase I. The reactions were carried out by using Illumina TruSeq RNA Library Prep Kit according to the manufacturer’s instruction. A single base “A” was then added to the newly synthesized cDNA fragments during the end repairing process. The products were ligated with sequencing adapters, purified and enriched through PCR amplification. PCR reaction conditions were set as 1 cycle of initial denaturation at 98°C for 30 s, followed by 15 cycles of denaturation at 98°C for 10 s; annealing at 60°C for 30 s; extension at 72°C for 30 s and ended with 1 cycle of final extension at 72°C for

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5 min. The cDNA libraries were subsequently sequenced by using Illumina HiSeqTM 2000 platform. RNA-Seq service was outsourced to ScienceVision Sdn. Bhd.

3.2.4.2 De novo assembly of unigenes

The raw data obtained from Illumina HiSeqTM 2000 sequencer were pre-filtered with Bowtie 2 software (Langmead and Salzberg, 2012). Reads of low quality, those with unknown nucleotides larger than 5%, short reads with less than 35 base pairs and adapter for reverse transcription and sequencing were discarded during quality trimming. Clean reads were subjected to base quality with Q score of 30. This is to ensure that only good quality bases derived from the mRNA were used for de novo assembly. CLC genomics software was used to assemble reads (default kmer as 28). The sequenced reads from 12 samples were combined and the overlapping bases in the short reads were assembled into contigs with high coverage. The reads were subsequently mapped back to the contigs to obtain longest assembled sequences defined as unigenes that cannot be extended on either end. The received transcriptome sequences were analysed against a set of eukaryotic core proteins to assess for completeness by using Core Eukaryotic Genes Mapping Approach (CEGMA; Parra et al., 2007). The results of assembly were assessed on the basis of total number of unigenes, the distribution of unigene length and the N50 statistic.

3.2.4.3 Functional annotation and KEGG classification

Unigene sequences were blasted against NCBI non-redundant (nr) protein databases (E- value ≤ 10-5) to define putative functions of the unigenes by using Basic Local Alignment Search Toolx (Blastx). Furthermore, unigenes were search against the European Bioinformatics Institute (EBI) InterPro proteins by using InterProScan software (Jones et al., 2014). Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) classifications of the unigenes were subsequently conducted by using Web Gene Ontology Annotation Plot (WEGO) (Ye et al., 2018) and Blast2GO (Gotz et al., 2008) software, respectively. GenInfo (GI) identifiers of the homologous sequences were used to retrieve sequences IDs by mapping of unigenes to 95 | P age nr reference protein database (UniProt and InterPro) (Conesa and Gotz, 2008). Sequences IDs were subsequently matched to GO terms and annotation (Ashburner et al., 2007) to define reliable Enzyme Codes (EC) and KEGG pathways of the unigenes. The expression of unigenes was compared between two pepper species to investigate differentially expressed genes (DEGs) by using CLC genomics software.

3.2.5 Detection of microsatellites

The position of simple sequence repeats (SSRs) in transcriptome data were identified by using MIcroSAtellite (MISA) tool (Sebastian et al., 2017). The search was carried out to detect bi-, tri-, tetra-, penta- and hexanucleotide repeats with minimum 100 bases in between two SSRs. The MISA results were search against the annotated unigenes to assign putative functions to the defined SSR loci (Victoria et al., 2011). The SSR repeats located in the sequences of defence-related unigenes were also identified.

3.2.6 Identification of reference genes

3.2.6.1 First-strand cDNA synthesis

The first-strand cDNA were prepared for qRT-PCR analysis of the targeted R-genes by using Promega (USA) AMV Reverse Transcription System according to the manufacturer’s instruction. The total 20 μl reverse transcription volume consists of 1X reverse transcription buffer [10 mM Tris-HCl with pH 9.0 at 25°C, 50 mM potassium ® chloride (KCl), 0.1% Triton X-100], 5 mM MgCl2, 1 mM each dNTP, 1 u/μl ® Recombinant RNAin Ribonuclease Inhibitor, 0.5 μg Oligo (dT)15 primer, 15 u AMV Reverse Transcriptase and 1 μg total RNA. The reverse transcriptions were carried out with 10 min of initial RNA incubation at 70°C to remove RNA secondary structures and followed by 45 min of reverse transcription at 42°C. The reactions were terminated by heat inactivation of AMV reverse transcriptase at 95°C for 5 min. This is to prevent reverse transcriptase from binding to the newly synthesized first-strand cDNA. The reactions were then incubated on ice for 5 min. First-strand cDNA were stored at -20°C prior to next experimental stage. 96 | P age

3.2.6.2 Quantitative real-time polymerase chain reaction (qRT-PCR)

Internal control genes were used to assess transcriptome quality and to provide fundamental basis for scanning of interested DEGs in the current study. Several reference genes have been assessed on their suitability as internal control genes in qRT- PCR. Therefore, in the current study, candidate reference genes were identified by literature studies, with focus on those genes that has been frequently used in other plants. The sequence of candidate reference genes were obtained from the transcriptome data by search through gene descriptions in functional annotation. Primers amplify the candidate reference genes were designed by using Primer3 software (Koressaar and Remm, 2007; UnteRGAser et al., 2012).

The qRT-PCR was carried out by using ABI (USA) QuantStudioTM 6 Flex Real-Time PCR System in accordance with the manufacturer’s instructions. For each analysis, qRT-PCR was carried out in three sample replications. The total 20 μl reaction volume consists of 0.4 μM each primer, 2 μl c DNA, 10 μl Applied Biosystems SYBR Select Master Mix and DEPC treated water. The specificity of qRT-PCR primers was verified by conducting melting curve analysis at the end of each run. To ensure reliability of the runs, a serial 10-fold dilution from 1,000 ng/μl to 1 ng/μl of pooled cDNA of root and leaf samples was carried out to define the efficacy of qRT-PCR through equation, Efficiency = [10(-1/slope) - 1) x 100 (Radoni et al., 2004; Ridgeway and Timm, 2015). A standard curve was made by plotting log of cDNA dilutions against Ct values of the runs. To define the most stable reference gene, the reference gene stability factor (M) of the genes under investigation was determined by using geNorm (Vandesompele et al.,

2002) algorithm based on Ct values.

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3.3 Identification of potential biocontrol agents for slow decline

3.3.1 Isolation of rhizobacteria

3.3.1.1 Field sampling

Rhizospheric soil samples were collected from five healthy farms located at Jagoi Duyuh and Jagoi Sebubok villages of Serikin town, Mongkos and Mujat villages of Serian Division and Karu village of Padawan district in the current study. The selected black pepper farms were designated HF1 (Jagoi Duyuh) (1°19'38.0"N 110°00'12.9"E), HF2 (1°19'36.5"N 110°00'12.9"E) (Jagoi Sebubok), HF3 (0°53’06.1”N 110°35’21.7”E) (Mongkos), HF4 (0°53'26.5"N 110°33'59.3"E) (Mujat) and HF5 (1°17’04.4”N 110°16’47.0”E) (Karu). The location of HF1, HF3 and HF4 are adjacent to and within 1 km to 3 km distance from the selected disease farms DF1, DF2 and DF3 as mentioned earlier. These three farms were highly recommended in the current study as the farms are free of slow decline infection even though they are next to the disease farms DF1, DF2 and DF3 which have been seriously damaged by slow decline. Therefore, there is a high opportunity for the presence of beneficial microorganisms in HF1, HF3 and HF4 that are antagonistic to the causal organisms of slow decline incidents reported in disease farms DF1, DF2 and DF3. The average farm size of the selected healthy black pepper farms are about 0.62 acre which consist of approximately 500 black pepper plants in total.

Similar to the disease farms DF1, DF2 and DF3, most of the black pepper cultivars planted in these farms are varieties Semengok Aman and Kuching with 235 plants (47%) and 180 plants (36%), respectively. The remaining 17% (85 plants) of the cultivated black pepper plants consist of varieties Semengok Emas (39 plants), Semengok Perak (25 plants) and Semengok 1 (21 plants). A number of three healthy black pepper plants which have exhibited good and vigorous growth were randomly selected as sampling points for each cultivar per farm location. The healthy black pepper roots together with the adherent soils were carefully removed from rhizosphere (Figure 3-2) , packed and sealed in sterile collection bags prior to delivery to the laboratory.

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(a) (b) Figure 3-2. Sampling of beneficial soil rhizobacteria from black pepper farms located at Jagoi Duyuh and Jagoi Sebubok villages of Serikin town, Mongkos and Mujat villages of Serian Division and Karu village of Padawan district, (a) Healthy black pepper plants with good and vigorous growth; (b) Collection of soils samples from rhizosphere.

3.3.1.2 Isolation of rhizobacteria from soils

Rhizobacteria were isolated from rhizospheric soil samples by using Luria-Bertani (LB) agar medium through serial dilution plating method modified from Parmeter (1970). The 1.5% w/v LB agar plate consists of 1% sodium chloride (NaCl), 1% tryptone and

0.5% yeast extract. One gram of soil was suspended in 9 ml of sterile ddH2O. The soil suspension was then diluted with a serial 10-fold dilution for six times. An amount of 0.2 ml 1:106 diluted soil suspensions was spread on LB agar plates. The plates were incubated at 30°C for 16 hours. Individual bacterial colonies with different morphological appearances that grown on LB plate were further isolated by transferring them onto a fresh PDA plate consists of F. solani isolate FS010 mycelium plug at the centre of the plate. The fungus was isolated from the disease farm DF3 located at Mujat village of Serian Division which was confirmed as causal organism to the reported incidents of slow decline disease in that farm. The fungus was served as the selective agent in the screening of antagonistic rhizobacteria in this experiment. The new culture plates were then incubated at 30°C for 7 days.

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3.3.2 Molecular identification of isolated rhizobacteria

3.3.2.1 Isolation of bacterial DNA

DNA isolation was carried out on bacterial cells by using Promega (USA) Genomic Purification Kit according to the manufacturer’s instruction. One ml of overnight grown bacterial culture was centrifuged at 13,000 rpm for 2 min to pellet the cells. The supernatant was discarded and bacterial cells were re-suspended in 480 µl of 50 mM EDTA. The suspended bacterial cells were treated with 0.2 volume of lytic enzyme. The reaction was incubated at 30°C for 30 min. The purpose of this treatment is to weaken the bacterial cell wall so that efficient bacterial cell lysis can take place. The supernatant was then discarded through centrifugation at 13,000 rpm for 2 min. Bacterial cells were lysed with 600 µl of Nuclei Lysis Solution. Cell lysis was carried out at 80°C for 5 min. The cell lysate was cooled to room temperature and added with 3 µl of RNAse Solution. RNase treatment was carried out at 30°C for 15 min. The RNase-treated cell lysate was then added with 200 µl of Protein Precipitation Solution. The mixture was then vertexed vigorously at high speed for 20 s and incubated on ice for 5 min. After centrifugation at 13,000 rpm for 3 min, the supernatant containing the DNA was transferred to a clean tube and added with 600 µl of isopropanol at room temperature. Bacterial DNA was recovered through centrifugation at 14,000 rpm for 2 min. The precipitated DNA were washed with 600 µl of 70% (v/v) ethanol at room temperature, centrifuged at 13,000 rpm at 4°C for 5 min to discard excess ethanol, air dried at room temperature and re- suspended in 100 μl of DNA Rehydration Solution. The isolated DNA was stored at - 20°C prior to next experimental stage.

3.3.2.2 Quality assessment of isolated bacterial DNA

The intact of purified DNA was assessed by using Invitrogen (USA) SYBR® Safe DNA Gel Stain prepared 1% (w/v) agarose gel. Bacterial DNA was electrophoresed at 100 volts for an hour in 1X TBE (90 mM Tris-borate, 2 mM EDTA with pH 8.0) buffer. The agarose gel was visualized by using ThermoFisher Scientific (USA) Safe Imager™ 2.0 Blue-Light Transilluminator and documented by using Bio-Rad (California) Gel Documentation 2000 System. The isolated DNA was then quantified by using Gene- 100 | P age

Quant (USA) UV Spectrophotometer at spectrum wavelengths of 230 nm, 260 nm and 280 nm with a dilution factor of 100.

3.3.2.3 PCR amplification of bacterial 16S rRNA genes

Universal primers 8F (5’- agagtttgatcctggctcag-3’) and U1492R (5’-ggttaccttgttacgactt- 3’) were used in PCR analysis to amplify bacterial gene that code for 16S ribosomal RNA (rRNA), the component of bacterial 30S (small subunit) ribosome. PCR amplifications were carried out by using Applied Biosystems (USA) VeritiTM 96- Well Thermal Cycler. The total 25 μl PCR reaction volume consists of 1X PCR buffer [20 mM Tris-HCl, 50 mM potassium chloride (KCl)], 1.5 mM magnesium chloride

(MgCl2), 0.2 mM each nucleoside triphosphate (dNTP) mix, 0.4 μM each primer, 1 unit Taq polymerase (Invitrogen, USA) and 30 ng template bacterial genomic DNA. PCR reaction conditions were set as 1 cycle of initial denaturation at 94°C for 2 min, followed by 35 cycles of denaturation at 94°C for 30 s; annealing at 55°C for 30 s; extension at 72°C for 30 s and ended with 1 cycle of final extension at 72°C for 5 min.

3.3.2.4 Purification of PCR amplified 16S rRNA genes

PCR amplicons were gel-excised by using Invitrogen (USA) PureLink Quick Gel Extraction Kit according to the manufacturer’s instruction. The 1.0% (w/v) agaorse gel slices containing PCR amplicons were dissolved in 3 gel volumes of Gel Solubilisation Buffer L3 at 50°C for 10 min. During incubation, the tubes were gently inverted in every 2 min for well mixing of the mixture to permit proper gel dissolution. After the gel slices appear dissolved, the tubes were further incubated for an additional 5 min to ensure complete gel dissolution. To get optimal DNA yields, 1 gel volume of isopropanol at room temperature was added to the tube containing well dissolved gel slice and mix well. PCR fragments were then recovered through membrane binding. The mixture was applied to Quick Gel Extraction Column inside a Wash Tube. The column was centrifuged at 13,000 rpm for 1 min at room temperature. The flow through was discarded. PCR fragments bond on the membrane of the extraction column were washed by using Wash Buffer W1 containing ethanol. The fragments were then eluted 101 | P age from the wash column membrane by using Elution Buffer E5. The purified PCR products were stored at -20°C prior to next experimental stage.

3.3.2.5 Cloning of bacterial 16S rRNA genes

The purified PCR products were cloned by using Promega (USA) pGEM®-T Easy Vector System II according to the manufacturer’s instruction. The total 10 μl ligation volume consists of 1X rapid ligation buffer [30 mM Tris-HCl (pH 7.8), 10 mM MgCl2, 10 mM dithiothreitol (DTT), 1 mM adenosine triphosphate (ATP), 5% polyethylene glycol], 50 ng pGEM®-T Easy vector, 3 Weiss units T4 DNA ligase and 3 μl PCR products of fungal ITS fragments. Ligation was carried out overnight at 4°C. The recombinant vectors were subsequently transformed into JM109 high efficiency competent E. coli cells through heat-shock at 42°C for 90 s without shaking. The transformants were incubated at 37°C for 16 hours on LB medium consists of 100 μg/ml ampicillin, 0.5 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) and 80 μg/ml 5- bromo-4-chloro-3-indolyl-β-D-galacto pyranoside (X-Gal) as selective agents. Blue and white colour screening of bacterial colonies as well as PCR amplification with T7 and SP6 promoter primers were carried out to identify the presence of positive clones.

3.3.2.6 Recombinant plasmid DNA isolation

The recombinant vectors were isolated from positive bacteria clones by using Promega (USA) Wizard® Plus SV Minipreps DNA Purification Kit according to the manufacturer’s instruction. One ml of overnight grown bacterial culture was centrifuged at 13,000 rpm for 2 min to pellet the cells. The supernatant was discarded and the cells were suspended in 250 µl of Cell Resuspension Solution [50 mM Tris-HCl with pH 7.5, 10 mM EDTA, 100 μg/ml RNAse A]. The suspended bacterial cells were treated with 250 µl of Cell Lysis Solution [1% sodium dodecyl sulfate (SDS), 0.2 M sodium hydroxide (NaOH)], followed by 10 µl of Alkaline Protease Solution and 350 µl of

Neutralization Solution [4.09 M guanidine hydrochloride (CH6ClN3), 0.759 M potassium acetate (CH3CO2K), 2.12 M glacial acetic acid (CH3COOH) with pH 4.2]. The suspension was centrifuged at 13,000 rpm at room temperature for 10 min to 102 | P age discard cell debris. Recombinant plasmid DNA was then recovered through membrane binding. Cell lysate, the aqueous phase of suspension was applied to Minicolumns and centrifuged at 13,000 rpm for 1 min at room temperature. The flow through was discarded. Recombinant plasmid DNA bond on the membrane of Minicolumns was washed with Column Wash Solution (60 mM CH3CO2K, 8.3 mM Tris-HCl with pH 7.5, 0.04 mM EDTA with pH 8.0, 60% ethanol) and then eluted from the columns membrane by using Nuclease Free Water. The isolated plasmid DNA was stored at - 20°C prior outsourced for sequencing.

3.3.2.7 Sequence data analysis of bacterial 16S rRNA genes

The purified recombinant plasmid DNA was outsourced for sequencing. The sequence data of bacterial 16S rRNA gene fragments obtained from single-pass sequencing were searched against the EZbiocloud (EzTaxon) database, a taxonomically united database represented by quality-controlled bacterial 16S rRNA gene and consists of a complete bacterial hierarchical taxonomic system with more than 62,900 species (Yoon et al., 2017). A phylogenetic tree for classification of the isolated bacteria was further developed by using Molecular Evolutionary Genetics Analysis 6.0 (MEGA6) software (Tamura et al., 2013).

3.3.3 Biocontrol assays of isolated rhizobacteria

3.3.3.1 Siderophore production

Siderophore production was detected by using Chrome Azurol S (CAS) plate assay (Schwyn and Neilands, 1987). The CAS agar medium consists of 10% blue dye [60 mg

CAS; 2.7 mg iron (III) chloride (FeCl3); 73 mg hexadecyltrimethylammonium bromide (HDTMA)], 10% Minimal Media 9 (MM9) salt solution [10 g ammonium chloride

(NH4Cl); 3 g monopotassium phosphate (KH2PO4); 5 g NaCl], 0.2% glucose, 0.3% casamino acid, 3.2% piperazine-N,N′-bis(2-ethane sulfonic acid) (PIPES) and 1.5% agar in 1 L of sterile ddH2O. Quantitative estimation of siderophore was done by using CAS-shuttle assay (Christina et al., 2015). Bacteria were grown in LB broth medium for 103 | P age

2 days at 30°C with constant shaking at 150 rpm. The culture supernatants were treated with equal volume of CAS-shuttle solution [0.15 mM CAS; 0.015 mM FeCl3; 0.6 mM HDTMA; 0.5 mM Piperazine buffer (pH 5.6); 4.5 mM sulfosalicylic acid]. The colour change of the supernatants to orange was determined at absorbance 630 nm after 15 min incubation. The reference treatment consists of 50% CAS solution and 50% un- inoculated LB medium. Percentage of siderophore was calculated through formula; Siderophore (%) = [(Ar - As) / Ar] x 100%, where Ar denotes absorbance of the reference and As denotes absorbance of the samples. The siderophore-mediated antagonism of bacterial isolates against F. solani isolate FS010 was evaluated through dual culture plate assay on PDA agar medium supplemented with 0 μM, 100 μM and

200 μM FeCl3 (Kumar et al., 2002; Sasirekha and Shivakumar, 2016).

3.3.3.2 Chitinase production

Chitinolytic activity of the isolated rhizobacteria was detected by using colloidal chitin as substrate in agar medium (Murthy and Bleakley, 2012). Colloidal chitin was prepared by adding 20 g chitin powder to 200 ml 12 M hydrochloric acid (HCl). The mixture was added with 3 L of ice-cold water and incubated at 4°C for 24 hours. The colloid was washed with tap water until the pH has risen up to 7.0. The colloidal chitin agar medium consists of 2% colloidal chitin, 0.07% dipotassium phosphate (K2HPO4), 0.05% magnesium sulphate (MgSO4), 0.03% KH2PO4, 0.001% iron (II) sulphate (FeSO4) and 1.5% agar. Bacteria were spot inoculated on agar medium and incubated at 30°C. Chitinolytic activity was determined by the presence of clear zone surrounding the bacteria colonies. The Chitinolytic Index (CI) was measured through formula CI = [(Dz - Dc) / Dc] where Dz denotes diameter of the clear zone and Dc denotes diameter of the colony (Wahyudi et al., 2011).

3.3.3.3 Antagonistic effects to F. solani isolate FS010

Antagonistic effects of the isolated rhizobacteria were evaluated on PDA medium through dual culture plate assay (Fokkema, 2008). The mycelium plugs of F. solani isolate FS010 were placed at the center of agar plates. Bacteria were spot inoculated at 104 | P age one side of agar plates with 3 cm in distance from the mycelium plugs. The control plate contained a single mycelium plug without bacterial inoculation. The plates were incubated at 25°C for 7 days. The percentage of inhibition of radial growth (PIRG) was calculated through formula PIRG (%) = [(Rc - Ri) / Rc] x 100%, where Rc denotes mycelium growth on the opposite side of the colonies and Ri denotes mycelium growth towards the colonies (Djordjevic et al., 2011).

3.3.4 Plant growth promotion traits of isolated rhizobacteria

3.3.4.1 IAA production

The isolated rhizobacteria were cultured in Yeast-Extract-Mannitol (YEM) broth medium (0.1% yeast extract, 1% mannitol, 0.05% K2HPO4, 0.02% MgSO4, 0.01% NaCl) containing 0.05% tryptophan at 30°C for 2 days with constant shaking at 150 rpm (Gordon and Weber, 1951; Ghosh and Basu, 2002). Cell-free supernatants from the fermented broth were treated with 0.2 volume of Salkowski reagent [0.5 M FeCl3, 70% perchloric acid]. The sterile un-inoculated YEM medium was served as control. The colour change of supernatants to pink was determined at absorbance 530 nm after 15 min incubation. The amount of IAA produced by the bacteria was calculated through a standard curve established by determining different concentration (0-100 μg/mL) of authentic IAA at absorbance 530 nm.

3.3.4.2 Root growth assays

Field trials were conducted to study the effects of IAA producing rhizobacteria on black pepper root growth. Five-node cuttings of black pepper PnSA and PnKch were used for the trials. The cuttings were surface-sterilized with 95% ethanol and washed with sterile water. Overnight grown bacteria cultures (0.1 ml, LB) were applied to the 1st to 3rd internode of the cuttings. The untreated cuttings were used as control. All cuttings were sown into sterile sand (heat treated at 100°C, 30 min) with ten cuttings per tray at equal distance. The trials were performed in triplicates. The trays were irrigated with sterile ddH2O every day and exposed to sunlight. The chlorophyll concentration of black 105 | P age pepper leaves was measured at five days interval until 15 days (Mohite, 2013) by using Apogee MC-100 Chlorophyll Concentration Meter (USA). Black pepper roots were harvested at the end of the trials and the dry weight of the roots was assessed.

3.3.4.3 Phosphate solubilisation

The ability of the isolated rhizobacteria to solubilize inorganic phosphate was detected with Pikovskayas agar medium (Sundara and Sinha, 1963). The medium consists of 1% dextrose, 0.5% tricalcium phosphate (Ca3O8P2), 0.05% yeast extract, 0.05% ammonium sulphate [(NH4)2SO4], 0.02% potassium chloride (KCl), 0.01% MgSO4 and 1.5% agar. Bacteria were spot inoculated on the medium and incubated at 30°C. The presence of clear zone surrounding the colonies indicated the presence of solubilizing activity. The Solubilizing Index (SI) was calculated through the ratio of total diameter of clear zone to the colony diameter (Premono and Moawad, 1996; Wahyudi et al., 2011).

3.3.4.4 Ammonia production

Ammonia production was assayed with peptone water (Cappuccino and Sherman, 2008). Bacteria were grown in 1.5% w/v peptone water at 30°C for 2 days with constant shaking at 150 rpm. The culture supernatants were treated with 5% Nessler reagent. Equal volume of sterile un-inoculated peptone water was served as reference of nesslerization. The colour change of supernatants to deep yellow was determined at absorbance 425 nm after 15 min of incubation. The amount of ammonia produced was calculated through a standard curve established by determining different concentration (0-100 mM) of authentic ammonia at absorbance 425 nm.

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3.4 Assessment on Pepper Defence-Related Genes

In order to describe the functions and differences of pepper defence-related genes in between resistant Pc and susceptible PnSA and PnKch, qRT-PCR analysis was carried out to study the effects of pathogenic F. solani isolate FS010 and beneficial Br. gelatini isolate JD04 exposure on the expression of targeted pepper defence-related genes. In total, five groups of differentially expressed genes that defined in the current study, i.e. Cf-9 , FLS2, MEKK1, RGA2 and PR1 were selected for further gene expression analysis. These groups of defence-related genes were incorporated in plant- pathogen interactions pathway (Path: ko04626). The main functions of the described genes are involved in the recognition of pathogen elicitors, signal transduction and activation of hypersensitive response. The disease resistance Cf-9 receptors are responsible in recognition of fungal avirulence proteins whereas the LRR-type serine threonine FLS2 receptor kinases are responsible in recognition of bacterial flagellins. T he mitogen-activated protein kinase MEKK1 is a signal transduction protein that involved in directing cellular responses to a diverse array of stimuli. Besides, disease resistance RGA2 proteins and pathogenesis- related PR1 proteins are defence-related reaction proteins that triggered in response to the presence of foreign bodies or the occurrence of an injury in host plants.

3.4.1 Infection of pepper plant tissues with soil-microbes

In this experiment, the pathogenic fungal strain, F. solani isolate FS010 and beneficial bacteria, Br. gelatini isolate JD04 were used as infection agents to treat the three month- old pepper cuttings of PnSA, PnKch and Pc. Soil-microbe infections were respectively carried out on pepper leaves and roots by using modified methods of Spina et al. (2008) and Edel-Hermann et al. (2012) as described earlier in Chapter 3. The trials were carried out in four experimental designs. In Trial 1, the leaves and roots of pepper cuttings were infected by pathogenic F. solani isolate FS010 (Fs-infected). Whereas, pepper cuttings were infected by beneficial Br. gelatini isolate JD04 in Trial 2 ( Br-infected). However, Trial 3 consists of a combination of beneficial bacterial and pathogenic fungal infection (Br-Fs-infected). In Trial 3, pepper cuttings were initially infected continuously by Br. gelatini isolate JD04 for three days before the infection of F. solani isolate FS010 were 107 | P age carried out on the same individual pepper plants at 4th day of the experiments. Trial 4 was served as control treatment in the assays. In Trial 4, pepper cuttings were treated with sterile ddH2O through the same process instead of F. solani isolate FS010 or Br. gelatini isolate JD04. Each trial was conducted in triplicates with the presence of three biological replicates.

3.4.2 Expression study of targeted pepper defence-related genes

The qRT-PCR was carried out on five targeted pepper defence-related genes, i.e. Cf-9, FSL2, MEKK1, PR1 and RGA2. To verify the expression of these defence-related genes in different organs of pepper plants, first-strand cDNA were synthesized from the total RNA of pepper leaves and roots. The yielded cDNA template from reverse transcription of pepper leaves and roots total RNA was diluted 10-fold and used as template for qRT- PCR. Primer3 was used to design qRT-PCR primers. In total, 20 μl qRT-PCR reaction volume consists of 0.4 μM each forward and reverse primer, 2 μl cDNA template, 10 μl Applied Biosystems (USA) SYBR Select Master Mix and DEPC treated water (0.1%, v/v). The qRT-PCR reaction conditions were set as 1 cycle of initial denaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 5 s; annealing at 52 °C to 58°C for 15 s and extension at 72°C for 15 s. The reliability and reproducibility of qRT-PCR runs were ensured through three biological and three technical replicates. Quantification of relative expression levels of targeted pepper defence-related genes were calculated as fold changes in between the infected and reference samples. The qRT-PCR results were expressed as mean value ± standard deviation. ANOVA were used to conduct statistical assessment in qRT-PCR and P value was set to 0.05.

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CHAPTER 4

RESULTS

4.1 Identification of Indigenous Causal Fungal Strains of Slow Decline

4.1.1 Isolation of soil-borne fungi

The infected black pepper plants in disease farms DF1, DF2 and DF3 have shown slow decline symptoms such as foliar yellowing, wilting at the shoot tips, heavy defoliation and die-back. The basal regions of these plants also have shown varying degrees of feeder root loss and damage at the collar regions. Figure 4-1 shows the disease symptoms of the infected black pepper plants.

(a) (b) (c)

Root galls

(d) (e) (f) Figure 4-1. Disease symptoms of the black pepper plants with slow decline, (a) Foliar yellowing, wilting at the shoot tips, heavy defoliation and die-back of the infected plant; (b) Loss of feeder roots and damage at the collar region; (c) Death of the infected plants; (d) Formation of root galls due to infestation by root-rot nematode; (e) Rotten of the infected black pepper roots; (f) Browning of vascular tissues due to Fusarium invasion.

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In the current study, a total of 13 soil-borne fungal isolates were obtained from disease black pepper roots and rhizospheric soil samples that collected from farms DF1, DF2 and DF3. Of which, four of the fungal isolates coded FS001, FS004, FS008 and FS010 were obtained from disease black pepper roots. Whereas, the other nine fungal isolates coded FS002, FS003, FS005, FS006, FS007, FS009, PN001, PN002 and RM001 were obtained from soil samples that collected from rhizosphere of the slow decline infected black pepper plants as described in Table 4-1. There were eight fungal isolates (disease root: FS001, FS004; rhizospheric soil: FS002, FS003, FS006, FS009, PN001, PN002) obtained from disease farm DF1 located at Jagoi Duyuh village, Serikin. Whereas, three fungal isolates were obtained from disease farm DF2 (disease root: FS008; rhizospheric soil: FS005, FS007) located at Mongkos village, Serian and two fungal isolates were obtained from disease farm DF3 (disease root: FS010; rhizospheric soil: RM001) located at Mujat village, Serian.

4.1.2 Morphological characterization of isolated fungal strains

In general, the obtained fungal isolates were categorized into four groups designated as Group FU-01 (FS001, FS003, FS004, FS005, FS007, FS010), Group FU-02 (FS002, FS006, FS008, FS009), Group PH-01 (PN001, PN002) and Group RI-01 (RM001) based on colony morphologies as shown in Table 4-1. Group FU-01 consists of three member isolates obtained from disease roots (FS001, FS004, FS010) and three member isolates obtained from rhizospheric soil (FS003, FS005, FS007). All member isolates in Group FU-01 have exhibited white and cottony mycelium with smooth margin. These isolates produced elongated microconidia with blunt end and septate macroconidia with sickle-shape. Group FU-01 was morphologically characterized as genus Fusarium. The member isolates in Group FU-02 comprise of one member isolate from disease roots (FS008) and three member isolates from rhizospheric soil (FS002, FS006, FS009). Same as member isolates in Group FU-01, all member isolates in Group-02 also have exhibited white and cottony mycelium but with circular margin. Although these fungal isolates had produced elongated microconidia with blunt end, no macroconidia have been observed in the member isolates of Group FU-02. The microscopic analysis has also characterized Group FU-02 as genus Fusarium because most of the morphological characters of Group FU-02 were observed similar to Group FU-01. 110 | P age

Table 4-1. Soil-borne fungi isolated from disease farms DF1, DF2 and DF3 located at Serikin and Serian areas of Sarawak.

ITS region Root colonization Isolate Sample Colony morphology Size Accession no. Identity (%) (bp) (%)

Group FU-01: Fusarium solani DF1: Jagoi Duyuh Village, Serikin (1°19'38.0"N 110°00'12.9"E) FS001 Root White; cottony; 529 FJ719812.1 96.59 FS003 Soil microconidia; 530 FJ719812.1 98.32 macroconidia; FS004 Root 523 FJ719812.1 97.18 hyaline; septate DF2: Mongkos Village, Serian (0°53’06.1”N 110°35’21.7”E) FS005 Soil White; cottony; 514 FJ719812.1 97.27 FS007 Soil microconidia; 539 FJ719812.1 98.10 macroconidia; hyaline; septate DF3: Mujat Village, Serian (0°53'26.5"N 110°33'59.3"E) FS010 Root White; cottony; 541 FJ719812.1 98.78 40.51 microconidia; macroconidia; hyaline; septate

Group FU-02: Fusarium solani DF1: Jagoi Duyuh Village, Serikin (1°19'38.0"N 110°00'12.9"E) FS002 Soil White; cottony; 439 KY307804.1 100 FS006 Soil microconidia; 445 KY307804.1 100 hyaline; septate FS009 Soil 452 KY307804.1 100 DF2: Mongkos Village, Serian (0°53’06.1”N 110°35’21.7”E) FS008 Root White; cottony; 450 KY307804.1 100 38.93 microconidia; hyaline; septate

Group PH-01: Phytophthora nicotianae DF1: Jagoi Duyuh Village, Serikin (1°19'38.0"N 110°00'12.9"E) PN001 Soil White; cottony; 662 HM807371.1 100 PN002 Soil sporangium; 691 HM807371.1 100 - oogonium; hyaline; coarse; septate

Group RI-01: Rigidoporus microporus DF3: Mujat Village, Serian (0°53'26.5"N 110°33'59.3"E) RM001 Soil White; cottony; 450 MH681569.1 100 - chlamydospore; hyaline; septate

Notes: “-”, No fungal colonization has been detected. 111 | P age

Group PH-01 consists of two member isolates obtained from rhizospheric soil (PN001, PN002). None of the member isolates in Group PH-01 were obtained from disease roots. All member isolates in Group PH-01 have exhibited white, submerged and rosette pattern mycelium with irregular margin. The microscopic observation has detected the presence of hyaline, septate and coarse hyphae as well as sporangia in Group PH-01. The presence of oogonia in Group PH-01 has revealed that this group of fungus was oomycete type and belongs to genus Phytophthora. Fungal isolate RM001 is the only member in Group RI-01. This isolate was obtained from rhizospheric soil. The fungus has exhibited white, thick and dense mycelium with smooth margin. The microscopic observation has also detected the presence of hyaline, septate hyphae and chlamydospore in isolate RM001. Therefore, this fungal isolate was morphologically characterized as genus Rigidoporus. Figure 4-2 shows the microscopic images of the obtained fungal isolates.

(a) (b) (c) (d)

(e) (f) (g) (h) Figure 4-2. Microscopic images (100X magnifications) of the assessed fungi, (a) Microconidia of members in Group FU-01; (b) Macroconidia of members in Group FU- 01; (c) Hyphae of members in Group FU-02; (d) Septate hyphae of members in Group FU-02; (e) Sporangium of members in Group PH-01; (f) Oogonium of members in Group PH- 01; (g) Thin walled hyphae of member in Group RI-01 and (h) Chlamydospore of member in Group RI-01.

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Classification of the member isolates in Group FU-01 and FU-02 into genus Fusarium, Group PH-01 into genus Phytophthora and Group RI-01 into genus Rigidoporus have been supported by the works done by Gallegly and Hong (2008), Fatin et al. (2017) and Gogoi et al. (2017). Figure 4-3 shows the morphological appearances of the four fungal groups cultured on PDA medium. Although Group FU-01 and FU-02 have shown high similarity through microscopic analysis, the two Fusarium isolates have shown different morphological appearances on PDA plates. Therefore, molecular approaches, which are more effective and faster than morphological characterization, were used to discriminate the four fungal groups at species level in the current study.

(a) (b)

(c) (d) Figure 4-3. Morphological appearances of the four fungal groups cultured on PDA medium, (a) Group FU-01 (genus Fusarium); (b) Group FU-02 (genus Fusarium) ; (c) Group PH-01 (genus Phytophthora) and (d) Group RI-01 (genus Rigidoporus).

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4.1.3 Molecular identification of isolated fungal strains

4.1.3.1 Isolation and quality assessment of fungal DNA

The fungal genomic DNA was isolated by using modified ion detergent CTAB-based isolation method. The quality of the isolated DNA samples was spectrophotometrically assessed at spectrum wavelengths of 230 nm, 260 nm and 280 nm with a dilution factor of 100. Table 4-2 summaries the yield and purity range of the isolated fungal genomic

DNA. Absorbance ratios of A260/230 and A260/280 are reported to present polysaccharides and polyphenolics as well as protein contaminations in DNA samples (Logemann et al., 1987; Manning, 1990; Gehrig et al., 2000; Malnoy et al., 2001; Hu et al., 2002; Zeng and Yang, 2002). Therefore, A260/280 ratios of the isolated fungal DNA samples ranged between 1.845 and 1.933 in the current study have shown insignificant levels of protein contamination. Besides that, A260/230 ratios more than 1.911 suggest low contamination of polysaccharide and polyphenolic in the isolated fungal DNA samples. Overall, the DNA yield ranged from 113 ng to 207 ng per gram of used tissues. The presence of distinct DNA bands as shown in Figure 4-4 suggested high quality DNA samples were obtained. The intactness o f isolated DNA was further confirmed through PCR assessment of ITS fragments. PCR amplicons with sizes ranged from 439 bp to 691 bp were amplified and visualized as distinct fragments on 1.5% (w/v) agarose gels as shown in Figure 4-5. These results suggested that the isolated DNA was of high purity and free of secondary metabolites such as polysaccharide, polyphenolic and protein substances that can affect and hinder PCR amplifications (Katterman and Shattuck, 1983; Fang et al., 1992; Guillemaut and Maréchal-Drouard, 1992; Salzman et al., 1999).

L1 L2 L3 L4 L5 L6 L7 L8 L9 L10 L11 L12 L13

Figure 4-4. 1.0% (w/v) agarose gel electrophoresis image of the isolated fungal DNA samples, Lane L1: isolate RM001; Lane L2: isolate FS002; Lane L3: isolate FS006; Lane L4: isolate FS009; Lane L5: isolate FS008; Lane L6: isolate PN001; Lane L7: isolate PN002; Lane L8: isolate FS001; Lane L9: isolate FS003; Lane L10: isolate FS004; Lane L11: isolate FS005; Lane L12: isolate FS007; Lane L13: isolate FS010.

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Table 4-2. The yield and purity range of the isolated fungal genomic DNA assessed at spectrum wavelengths of 230 nm, 260 nm and 280 nm with a dilution factor of 100. 1Absorbance ratios Organism Sample Code 2Yield (ng/g) A260/230 A260/280 Fungus FS001 1.974 1.856 137 FS002 2.013 1.949 178 FS003 1.997 1.981 119 FS004 2.036 1.832 139 FS005 1.988 1.955 179 FS006 2.108 1.973 183 FS007 1.935 1.877 195 FS008 2.099 1.916 188 FS009 1.972 1.999 196 FS010 1.980 1.845 207 PN001 1.964 1.878 171 PN002 1.911 1.871 113 RM001 2.121 1.933 129

Notes: 1Results are presented as mean of 3 absorbance readings. 2Yields of isolated fungal DNA per gram of tissue samples.

4.1.3.2 PCR and sequence data analysis of ITS regions

PCR have amplified fungal ITS sequence fragments with length ranged from 514 bp to 541 bp for member isolates of Group FU-01, 439 bp to 452 bp for member isolates of Group FU-02, 662 bp to 691 bp for member isolates of Group PH-01 and 450 bp for isolate RM001 in Group RI-01 (Figure 4-5) . As described in Table 4-1, the sequence identity search against NCBI GenBank nr database has shown that ITS fragments of member isolates in Group FU-01 (genus Fusarium) are 96.6% to 98.8% similar to ITS region of Fusarium solani strain MTCC 9622 with GenBank accession no. FJ719812.1. Meanwhile, the ITS fragments of member isolates in Group FU-02 (genus Fusarium) are fully identical (100%) to ITS region of Fusarium solani strain isolate FS04 with GenBank accession no. KY307804.1. For the member isolates in Group PH-01 (genus Phytophthora), sequence homology analysis has shown the ITS fragments of isolates

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PN001 and PN002 are fully identical (100%) to ITS region of Phytophthora nicotianae isolate NRCPh-6 with GenBank accession no. HM807371.1. Isolate RM001, the only member isolate in Group RI-01 (genus Rigidoporus) was found closely related to fungus Rigidoporus microporus isolate R23 (GenBank accession no. MH681569.1) as the assessed ITS fragments of these two fungi are fully identical (100%).

The results of ITS sequences homology analysis were found in line with the outputs of morphological classification as the obtained fungal isolates were also classified into genera Fusarium, Phytophthora and Rigidoporus. Nevertheless, molecular approaches through sequence homology analysis of fungal ITS regions, the widely used molecular barcodes for fungal discrimination were found faster and more effective than traditional morphological characterization particularly at the level of fungal species identification. This is due to limitation of fungal morphological characters that can be used for species discrimination through microscopic analysis, suggesting postulates of De Biazio et al. (2008) and Raja et al. (2017).

M M

FS001 FS004 FS007 FS002 FS006 FS008 FS009 FS003 FS005 FS010

(a) (b)

M M

PN001 PN002 RM001

(c) (d) Figure 4-5. 1.5% (w/v) agarose gel electrophoresis images of fungal ITS fragments, (a) Member isolates in Group FU-01; (b) Member isolates in Group FU-02; (c) Member isolates in Group PH-01 and (d) Isolate RM001, the only member isolate in Group RI- 01. Lane M: Promega 100 bp DNA ladder.

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4.1.4 Diagnostic of slow decline disease causal fungus

4.1.4.1 Pathogenicity assays of PnSA roots

To verify the causal fungus of slow decline disease reported in disease farms DF1, DF2 and DF3 located at Serikin and Serian areas of Sarawak, a total of four fungal isolates coded FS010, FS008, PN002 and RM001 which respectively represented each group of fungal isolates, i.e. Group FU-01, Group FU-02, Group PH-01 and Group RI-01 were selected as pathogenic agents in the diagnostic assays. Foliar yellowing was observed in black pepper cuttings infected with isolates FS010 from Group FU-01 and FS008 from Group FU-02 after a month of infection. As the experiment progress, feeder roots loss and damage of collar regions were observed in black pepper cuttings infected by these two isolates at the end of the experiments (three months after infection), as shown in Figure 4-6. However, there was no disease symptoms were observed in black pepper cuttings infected by isolates PN002 and RM001 as well as the control treatments.

Damage

(a) (b) (c) (d) Figure 4-6. The symptoms of slow decline showed by infected black pepper cuttings in diagnostic assays, (a) Control treatment; (b) Foliar yellowing after a month of infection; (c) Healthy roots and (d) Feeder roots loss and damage at collar region after three months of infection.

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4.1.4.2 Colonization assays of fungal cultures on PnSA roots

The percentages of black pepper roots colonized by fungal isolates FS008 and FS010 in pathogenicity assays are 38.93% and 40.51%, respectively as shown in Table 4-1. However, the two fungal isolates of Group PH-01 (PN002) and Group RI-01 (RM001) did not shown positive results in root colonization assays. The results of colonization have revealed that fungal isolates FS010 and FS008 from Group FU-01 and Group FU- 02 were the causal organisms to the reported incidents of slow decline in disease farms DF1, DF2 and DF3. These virulent isolates were obtained from disease roots. Besides that, they also found in rhizospheric soil and have been isolated as the other members of Group FU-01 (FS001, FS003, FS004, FS005, FS007) and Group FU-02 (FS002, FS006, FS009) in the current study. However, the member isolates of Group PH-01 and Group RI-01 were found not virulence to black pepper cuttings in root colonization assays. In addition, all member isolates in these two fungal groups were isolated from rhizospheric soil samples and none of them are isolated from roots tissues of infected black pepper plants in fields.

The member isolates of Group PH-01 (PN001, PN002) and Group RI-01 (RM001) are closely related to P. nicotianae and R. microporus, the well-known fungal pathogens to soil-borne diseases in many plants. However, the current study have shown that there was no involvement of these fungal isolates to the incidence of slow decline reported in disease farms DF1, DF2 and DF3. As F. solani was diagnosed as the causal organisms to the reported incidents of slow decline, isolate FS010 which shown higher percentage of root colonization (40.51%) than isolate FS008 (38.93%) was selected further analysis in next experimental stages.

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4.2 Elucidation of Defence-Related Mechanism Pathways in Pepper Plants

4.2.1 Fungal infection assays on pepper plant tissues

Three month-old black pepper cuttings of susceptible PnSA, PnKch and resistant Pc were infected by F. solani isolate FS010, the diagnosed causal fungus of slow decline incident reported in disease farm DF3 located at Mujat village, Serian. Interveinal chlorosis was observed in black pepper leaves of susceptible PnSA and PnKch after 2 weeks of infection. As the experiment progress, feeder root loss was also observed on susceptible PnSA and PnKch cuttings. However, there is no disease symptoms were observed in resistant Pc and control plants under the same trial conditions.

Fungal colonization results have shown that F. solani isolate FS010 was present in both infected leaves and root tissues of PnSA and PnKch as well as infected Pc leaves but not in Pc roots and also the control plants. In the current study, Pc has shown resistance to F. solani isolate FS010. The fungus was only detected at the infection points of Pc leaf midribs but not at the middle and upper points of the leaf in colonization assays. These results have revealed that the invasion of F. solani isolate FS010 in Pc leaves may be restricted by Pc plant defence mechanism pathways. Detection of F. solani isolate FS010 at the infection point of Pc leaf midribs was probably due to experimental introduction of this fungus to leaves during infection process. Meanwhile, F. solani isolate FS010 was also not detected in the root segments of infected Pc cuttings.

However, in susceptible PnSA and PnKch, the colonization assays have shown that F. solani isolate FS010 had invaded the roots and leaves tissues of these two plants. The fungus was detected in the root segments of both cuttings with PnKch roots (48.31%) were more susceptible to fungal invasion than PnSA roots (40.51%). Besides that, F. solani isolate FS010 was also detected at all assessed points of PnSA and PnKch leaf midribs (infection, middle and upper). The percentage of fungal colonization in PnSA and PnKch leaves were found highest at the infection points and gradually reduced from middle points to upper points of the leaf midribs. These results have revealed that F. solani isolate FS010 was proliferated in the internal tissues of PnSA and PnKch leaves and invaded far further from the infection points. Table 4-3 shows the colonization of F. solani isolate FS010 in the infected pepper plant roots and leaves. 119 | P age

Table 4-3. Colonization of F. solani isolate FS010 in pepper leaves and root tissues at the end of pathogenicity assays. Colonization (%) Tissues Points Pc PnSA PnKch Control Leaf midribs Infection 11.8 86.3 91.8 - Middle - 60.7 66.4 - Upper - 42.7 54.2 -

Roots Fibrous - 40.5 48.3 -

Notes: “-”, No fungal colonization has been detected.

4.2.2 Isolation and quality assessment of pepper total RNA

High quality total RNA was successfully isolated from pepper plant tissues by using modified ion detergent CTAB-based extraction method. In total, 12 RNA samples of pepper leaves designated Pc-N1, Pc-N2, PnSA-N1, PnSA-N2, PnKch-N1 and PnKch- N2 for normal (non-treated) samples; and Pc-T1, Pc-T2, PnSA-T1, PnSA-T2, PnKch- T1 and PnKch-T2 for F. solani isolate FS010 infected (FS-treated) samples have been prepared for RNA-Seq. Figure 4-7 shows the electrophoresis image of the isolated total RNA evaluated by using Agilent 2100 Bioanalyzer System. The distinct 25S and 18S ribosomal RNA bands were observed.

As ribosomal RNA represents more than 90% of the total RNA, hence any degradation occurred during RNA isolation process can easily be visualized as smearing or indistinct ribosomal RNA bands. The presence of distinct ribosomal RNA bands in Figure 4-7 indicates that little or no RNA degradation has occurred during the process of isolation. The intactness of the isolated total RNA was also defined by RNA integrity number (RIN), an algorithm for assigning integrity values to electrophoretic RNA measurements. A RIN value greater than 8 is generally considered sufficient to indicate high intact of the isolated RNA as reported by Chao et al. (2011). Therefore, with the RIN values ranged between 8.3 and 8.9 as shown in Figure 4-7, the isolated RNA samples were of high integrity and suitable to be used for RNA-Seq. The RNA yield in the current study was ranged from 489 ng to 679 ng per gram of used tissues.

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L S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12

RIN: 8.4 8.6 8.3 8.9 8.8 8.5 8.7 8.5 8.8 8.4 8.6 8.7 Yield (ng/g) 679 550 661 543 532 674 489 666 523 529 546 553

Figure 4-7. Electrophoresis image of the isolated total RNA evaluated by using Agilent 2100 Bioanalyzer System, Lane L: RNA ladder; Lane S1: Pc-N1; Lane S2: Pc-T1; Lane S3: Pc-N2; Lane S4: Pc-T2; Lane S5: PnSA-N1; Lane S6: PnSA- T1; Lane S7: PnSA- N2; Lane S8: PnSA-T2; Lane S9: PnKch-N1; Lane S10: PnKch-T1; Lane S11: PnKch- N2 and Lane S12: PnKch-T2.

4.2.3 Transcriptomics analysis of pepper defence-related genes

4.2.3.1 RNA-Sequencing and de novo assembly

Illumina HiSeqTM 2000 sequencer has yielded a total of 699,498,311 raw reads. An amount of 8.26% low-quality reads were filtered from the raw data during adapters and ambiguous base call trimming. This has resulted in a total of 641,741,570 clean reads. All clean reads were assembled into 253,508 transcripts in total corresponding to 81,096 unigenes with N50 length of 1,081 bp. The length distribution of assembled unigenes was ranged from 446 bp to 15,735 bp with a mean value of 1,012 bp. Transcriptome assembly have shown that majority length of the assembled unigenes were ranged in between 0.5 kb to 1 kb (64.84%); followed by 1 kb to 2 kb (28.54%), 2 kb to 3 kb (4.89%) and 3 kb to 4 kb (1.11%). Besides that, 0.22% and 0.11% of the assembled unigenes were less than 0.5 kb in length and longer than 5 kb in length, respectively as shown in Table 4-4.

4.2.3.2 Gene annotation and KEGG classification

Functional annotation of assembled unigenes was carried out on the basis of sequence homologies. All unigenes were searched against NCBI nr protein database by using 121 | P age

Blastx. In total, 77.16% (62,577) of the assembled unigenes have shown significant similarity with sequence data in NCBI nr protein database. Of which, 86.09% (53,871) of the NCBI blasted unigenes were associated to one or more GO terms. The assembled unigenes were also searched against EBI InterPro protein database to define additional reference annotations. InterProScan has shown that 45.56% (36,950) of the assembled unigenes have been aligned to known proteins in EBI InterPro protein database. Apart from that, 61.30% (22,652) of the InterPro scanned unigenes were annotated. Overall, 67.46% (54,708) of the assembled unigenes were functionally annotated in the current study. Among these unigenes, 39.88% (21,815) of the sequences have been annotated in both NCBI and EBI protein databases as shown in Figure 4-8.

Table 4-4. The overview results of pepper transcriptome sequencing and assembly. Number Percentage (%) Total raw reads 699,498,311 Total clean reads 641,741,570 Total transcripts in Pc 86,703 Total transcripts in PnSA 83,325 Total transcripts in PnKch 83,480 Total unigenes 81,096 Unigenes length (446 bp to 500 bp) 177 0.22% Unigenes length (501 bp to 1,000 bp) 52,586 64.84% Unigenes length (1,001 bp to 2,000 bp) 23,146 28.54% Unigenes length (2,001 bp to 3,000 bp) 3,966 4.89% Unigenes length (3,001 bp to 4,000 bp) 899 1.11% Unigenes length (4,001 bp to 5,000 bp) 231 0.29% Unigenes length (˃ 5,000 bp) 91 0.11% Mean length (bp) 1,012 N50 (bp) 1,081 Unigenes with blast hits 62,577 77.16% Unigenes with InterProScan 36,950 45.56% Unigenes with functional annotation 54,708 67.46% GO functional subgroups 63 Mapped KEGG pathways 125

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NR

6,196

2,510 32,056

21,815

11,788 837 0

GO InterPro annotation

Figure 4-8. Summary of sequence homology search against NCBI nr and EBI InterPro protein databases. The Venn diagram described the number of unigenes that have been annotated and aligned to the known proteins in both databases.

GO classification has divided the annotated unigenes (21,815) into 63 functional subgroups, with 29 biological processes (BP), 16 molecular functions (MF) and 18 cellular components (CC). Metabolic process, cellular process and response to stimulus process; binding, catalytic activity and transporter activity as well as cell, cell part and membrane were the major subgroups in BP, MF and CC, respectively, as shown in Figure 4-9. KEGG mapping were conducted to profile the functions of the annotated unigenes. In total, 52.14% (11,374) of the annotated unigenes were associated to 1,239 enzymes defined in 125 KEGG pathways as shown in Table 4-5. Most of these predicted enzymes (68.28%; 846 enzymes) were involved in metabolic pathways. Meanwhile, a number of 390 enzymes (31.48%) were associated to biosynthesis of secondary metabolites such as phospholipid, cysteine, purine and others. Besides that, a number of 228 enzymes (18.40%) were linked to microbial metabolism in diverse environments. In addition, several biological pathways related to plant defence metabolisms such as biosynthesis of phenylpropanoid (Dixon et al., 2002), flavonoid (Treutter, 2005), terpenoids (Singh and Sharma, 2014) and histidine (Seo et al., 2016) as well as signal transduction mitogen-activated protein kinase (MAPK) (Frank et al., 2004) and environmental adaptation plant-pathogen interaction (Boyd et al., 2013) were also detected in the current study.

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otherother organism organism part part otherother organism organism nucleoidnucleoid virionvirion part part virionvirion extracellularextracellular region region part part supramolecularsupramolecular complex complex symplastsymplast cellcell junction junction extracellularextracelluar region region CC membrane-enclosedmembrane-enclosed lumen lumen organelleorganelle part part proteinprotein-containing-containing complex ... membranemembrane part part organelleorganelle membranemembrane cellcell part part cell cell translationtranslation regulator regulator ... obsolete signal obsoletetransducer … obsolete TFIIIB-typeobsolete TFIIIB ... obsoleteobsolete gamma- gamma ... molecularmolecular carrier carrier activity activity obsoleteobsolete transcription transcription factor factor ... nutrientnutrient reservoir reservoir activity activity molecular transducermolecular transduceractivity antioxidantantioxidant activity activity MF obsoleteobsolete signal signaltransducer ... molecularmolecular function function regulator regulator structuralstructural molecule molecule activity activity transcriptiontranscription regulator regulat ... or transportertransporter activity activity catalyticcatalytic activity activity bindingbinding obsoleteobsolete transcription transcription factor ... behaviorbehaviour cellcell killing killing carboncarbon utilization utilization nitrogennitrogen utilization utilization obsolete proteinobsolete import protein ... locomotionlocomotion pigmentationpigmentation cellcell proliferation proliferation rhythmicrhythmic process process biologicalbiological adhesion adhesion detoxificationdetoxification immuneimmune system system process process positivepositive regulation regulation ... negativenegative regulation regulation ... BP multi-organismmulti-organism process process growthgrowth reproductivereproductive process process signalingsignalling reproductionreproduction multicellularmulticellular organismal organismal process process developmentaldevelopmental process process cellularcellular component component organization organization regulationregulation of ofbiological biological process process localizationlocalization biologicalbiological regulation regulation responseresponse to stimulusto stimulus cellularcellular process process metabolicmetabolic process process 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0 65.0 70.0 75.0

Figure 4-9. GO classifications of the annotated unigenes into three major groups and 63 subgroups. The x-axis shows the percentage of unigenes in specific subgroups. The y- axis shows the subgroups in GO annotation.

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Table 4-5. List of 125 KEGG pathways mapped by the annotated unigenes.

Enzyme Enzyme Pathways Pathways (%) (%) Metabolic pathways 68.28 Cyanoamino acid metabolism 1.13 Biosynthesis of secondary metabolites 31.48 Ubiquinone and terpenoid-quinone biosynthesis 1.13 Microbial metabolism in diverse environments 18.40 Histidine metabolism 1.13 Biosynthesis of antibiotics 16.22 Diterpenoid biosynthesis 1.05 Purine metabolism 5.73 Sphingolipid metabolism 1.05 Amino sugar and nucleotide sugar metabolism 4.76 Arachidonic acid metabolism 0.97 Cysteine and methionine metabolism 4.20 Thiamine metabolism 0.97 Glycine, serine and threonine metabolism 3.31 Steroid hormone biosynthesis 0.89 Pyrimidine metabolism 3.31 Biotin metabolism 0.89 Glyoxylate and dicarboxylate metabolism 3.23 Monoterpenoid biosynthesis 0.89 Glycerophospholipid metabolism 3.23 Selenocompound metabolism 0.89 Pyruvate metabolism 3.23 alpha-Linolenic acid metabolism 0.89 Starch and sucrose metabolism 3.07 Lipopolysaccharide biosynthesis 0.81 MAPK signaling pathway - plant 2.82 Streptomycin biosynthesis 0.81 Arginine and proline metabolism 2.74 Ether lipid metabolism 0.81 Alanine, aspartate and glutamate metabolism 2.66 Fatty acid elongation 0.81 Fructose and mannose metabolism 2.58 Flavonoid biosynthesis 0.81 Porphyrin and chlorophyll metabolism 2.58 Vitamin B6 metabolism 0.81 Glycolysis / Gluconeogenesis 2.50 Biosynthesis of terpenoids and steroids 0.73 Tryptophan metabolism 2.26 Biosynthesis of unsaturated fatty acids 0.73 Propanoate metabolism 2.26 Valine, leucine and isoleucine biosynthesis 0.73 Phenylalanine, tyrosine, tryptophan synthesis 2.26 Other glycan degradation 0.73 Tyrosine metabolism 2.18 Styrene degradation 0.65 Glycerolipid metabolism 2.18 Peptidoglycan biosynthesis 0.65 Butanoate metabolism 2.10 Phosphonate and phosphinate metabolism 0.56 Carbon fixation pathways in prokaryotes 2.10 D-Glutamine and D-glutamate metabolism 0.56 Valine, leucine and isoleucine degradation 2.10 Carotenoid biosynthesis 0.56 Nicotinate and nicotinamide metabolism 2.10 Toluene degradation 0.56 Arginine biosynthesis 2.10 Retinol metabolism 0.48 Inositol phosphate metabolism 2.02 Taurine and hypotaurine metabolism 0.48 Terpenoid backbone biosynthesis 2.02 Flavone and flavonol biosynthesis 0.48 Methane metabolism 2.02 Oxidative phosphorylation 0.48 Pentose and glucuronate interconversions 1.94 Monobactam biosynthesis 0.40 Phenylalanine metabolism 1.94 Caprolactam degradation 0.40 Pentose phosphate pathway 1.86 Novobiocin biosynthesis 0.40 Folate biosynthesis 1.78 Primary bile acid biosynthesis 0.40 Carbon fixation in photosynthetic organisms 1.78 Zeatin biosynthesis 0.40 Aminoacyl-tRNA biosynthesis 1.78 Fluorobenzoate degradation 0.40 beta-Alanine metabolism 1.78 Glycosaminoglycan degradation 0.40 Nitrogen metabolism (22) 1.78 Indole alkaloid biosynthesis 0.40 Phosphatidylinositol signaling system 1.78 C5-Branched dibasic acid metabolism 0.40 Galactose metabolism 1.69 Geraniol degradation 0.40 N-Glycan biosynthesis 1.69 Linoleic acid metabolism 0.40 Lysine degradation 1.69 Anthocyanin biosynthesis 0.32 Fatty acid degradation 1.61 mTOR signaling pathway 0.32 Pantothenate and CoA biosynthesis 1.61 Glycosphingolipid biosynthesis - ganglio series 0.32 Glutathione metabolism 1.61 Chloroalkane and chloroalkene degradation 0.32 Citrate cycle (TCA cycle) 1.53 Polyketide sugar unit biosynthesis 0.32 Benzoate degradation 1.53 Atrazine degradation 0.32 Plant-pathogen interaction 1.45 Steroid degradation 0.32 Phenylpropanoid biosynthesis 1.45 Glycosphingolipid biosynthesis 0.32 Ascorbate and aldarate metabolism 1.45 Glucosinolate biosynthesis 0.24 Sulfur metabolism 1.37 Xylene degradation 0.24 Aminobenzoate degradation 1.37 Chlorocyclohexane and benzene degradation 0.24 Plant hormone signal transduction 1.29 Sesquiterpenoid and triterpenoid biosynthesis 0.24 Lysine biosynthesis 1.29 Mannose type O-glycan biosynthesis 0.24 Fatty acid biosynthesis 1.29 Isoflavonoid biosynthesis 0.24 Various types of N-glycan biosynthesis 1.29 PI3K-Akt signaling pathway 0.16 Riboflavin metabolism 1.21 Photosynthesis 0.16 Tropane, piperidine and pyridine biosynthesis 1.21 beta-Lactam resistance 0.08 Isoquinoline alkaloid biosynthesis 1.13 Synthesis of siderophore nonribosomal peptides 0.08 One carbon pool by folate 1.13 Biosynthesis of vancomycin group antibiotics 0.08 Steroid biosynthesis 1.13

125 | P age

4.2.3.3 Species distribution analysis

The NCBI blast top-hits of unigene sequences homology searches have shown that P. nigrum L. shared highest similarity to Vitis vinifera (grape plant) with 24,142 matched hits (29.77% of the total unigenes). This figure was followed by 17,043 (21.02%), 8,313 (10.25%), 6,401 (7.89%) and 5,322 (6.56%) matched hits in Nelumbo nucifera (sacred lotus), Elaeis guineensis (oil palm), Citrus sinensis (sweet orange) and Phoenix dactylifera (date palm), respectively as shown in Figure 4-10.

16.92%

Vitis vinifera 1.67% 29.77% Nelumbo nucifera 2.34% Elaeis guineensis 3.58% Citrus sinensis Phoenix dactylifera 6.56% Gossypium raimondii Theobroma cacao Populus euphratica 7.89% Others 21.02% 10.25%

Figure 4-10. Species distribution analysis based on blast top-hits of unigene sequence homology searches against the known proteins in NCBI nr protein database.

4.2.3.4 Differentially expressed genes (DEGs)

Expression profiles of 21,815 annotated unigenes were compared to investigate DEGs among resistant P. colubrinum Link and susceptible P. nigrum L. Reads per kilo base per million of mapped reads (RPKM) was used to normalize expression of unigenes. In total, 2,361 DEGs were detected in between the two pepper species. Higher expression level with log2 fold change ≥ 1 (Yang et al., 2016) was observed in 1,426 DEGs in 126 | P age resistant P. colubrinum Link as shown in Table 4-6. Most of these DEGs (79.38%, 1,132 DEGs) was mapped into KEGG pathways that were related to plant environmental adaptation and signal transduction as shown in Figure 4-11. The most regularly represented pathways are plant-pathogen interactions (Path: ko04626) pathway and plant MAPK signalling (Path: ko04016) pathway. The detected DEGs such as disease resistance Cf-9, LRR receptor-like serine-threonine kinase FLS2, mitogen-activated protein kinase, disease resistance RGA, WRKY transcription factors and pathogenesis-related genes were found highly expressed in resistant P. colubrinum Link compared to susceptible P. nigrum L.

3.30% 4.70% 1.47% Calcium-dependent protein kinase 4.77% Calmodulin Cyclic nucleotide gated channel 5.47% 15.85% Disease resistance protein Cf-9 Glycerol kinase 4.21% Nitric-oxidase synthase

Path: ko04626 Path: Respiratory burst oxidase

4.70% 3.37% Brassinosteroid receptor kinase LRR receptor-like kinase FLS2 Mitogen activated protein kinases 4.91% 4.84% Pathogenesis-related protein 1 ath: ko04016 ath: P Senescene-induced receptor-like kinase 3.72% WRKY transcription factors NB-LRR protein SUMM2 4.63% 14.24% Transcription factor VIP 1 Disease resistance RGA 4.14% NBS-LRR disease resistance 5.05% 5.26% Probable disease resistance At 5.40% Figure 4-11. The pie chart shows the majority of the DEGs were associated to plant- pathogen interactions pathway (Path: ko04626) and plant MAPK signalling pathway (Path: ko04016).

127 | P age

Table 4-6. Number of DEGs that showed higher expression levels with log2 fold change ≥ 1 in resistant P. colubrinum Link.

Object name Number of DEGs

brassinosteroid insensitive 1-associated receptor kinase 66

calcium-dependent protein kinase 67

Calmodulin 68

cyclic nucleotide-gated ion channel 78

Disease resistance protein Cf-9 60

glycerol kinase 67

LRR receptor-like serine threonine- kinase FLS2 59

mitogen-activated protein kinase kinase kinase 1 72

NB-LRR protein SUMM2 70

nitric oxide synthase 69

pathogenesis-related protein 1 77

Respiratory burst oxidase 53

senescene-induced receptor-like serine/threonine-protein kinase 75

transcription factor VIP1 48

WRKY transcription factor 25 80

WRKY transcription factor 29 117

disease resistance RGA2 196

disease resistance RGA3 36

NBS-LRR disease resistance 21

Probable disease resistance At 47

128 | P age

4.2.3.5 Characterization of pepper resistance gene analogues

As plant RGAs confer resistance to various fungal pathogens and earlier study also has showed that RGA comprise a large resistance gene family in pepper species (Lau et al., 2012), a total of 232 RGA gene sequences were identified in this study. These sequences ranged from 551 bp to 2,919 bp and encoded proteins with size ranged from 183 amino acids to 973 amino acids (Table 4-7) . Blastx analysis showed that the predicted black pepper RGA proteins were 45.38% to 92.43% similar to NBS-type resistance proteins in NCBI nr protein database. The presence of conserved motif sequences, i.e. Kinase- 1/P-loop (GMGGVGKT), Kinase-2 (VLDDVW) and hydrophobic Kinase-3/GLPL in the defined pepper RGA proteins as shown in Figure 4-12d ha predicted the functions of these proteins.

P-loop was reported as nucleoside triphosphate binding proteins that function through direct interaction with the bound nucleotides (Saraste et al., 1990). Baker et al. (1997) has reported that mutations of main residues in P-loop motifs can cause the functional failure in plant RGA proteins. Kinase-2 motif was involved in coordination of Mg2+ ion that required in phosphor-transfer reactions (Traut, 1994). As reported by Azhar and Heslop-Harrison (2008), protein structures among the Kinase-2 and GLPL motifs are corresponding to the specificity activation of signalling response in coil-coiled domains. Therefore, with the presence of P-loop, Kinase-2 and GLPL motifs, black pepper RGA were believed to play a key role in ATP binding, hydrolysis and signal transduction of plant defence that triggered by pathogen invasion (Miller et al., 2008).

129 | P age

Table 4-7. List of 232 black pepper RGA that shared similarities with the resistance proteins published in NCBI nr protein database.

Unigenes ID Object Length Sim. Mean Unigenes ID Object Length Sim. Mean Pn : GID - 00091 RGA2 830 46.71% Pn : GID - 43247 RGA3 805 55.90% Pn : GID - 00092 RGA2 612 72.00% Pn : GID - 43490 RGA3 794 51.76% Pn : GID - 00210 RGA2 825 54.00% Pn : GID - 44308 RGA2 839 69.05% Pn : GID - 00922 RGA2 834 59.62% Pn : GID - 45500 RGA2 685 51.67% Pn : GID - 00923 RGA2 621 49.76% Pn : GID - 45758 RGA2 566 56.43% Pn : GID - 01140 RGA2 668 66.57% Pn : GID - 46863 RGA2 519 67.14% Pn : GID - 01160 RGA2 1,106 64.43% Pn : GID - 48263 RGA2 582 55.00% Pn : GID - 01230 RGA2 2,618 44.81% Pn : GID - 48437 RGA2 775 55.00% Pn : GID - 01325 RGA2 630 57.29% Pn : GID - 48438 RGA2 601 47.00% Pn : GID - 01358 RGA2 594 60.76% Pn : GID - 48601 RGA2 556 84.67% Pn : GID - 01379 RGA2 508 6.43% Pn : GID - 48730 RGA2 1,286 71.95% Pn : GID - 02382 RGA2 1,445 49.38% Pn : GID - 48992 RGA2 999 72.38% Pn : GID - 02792 RGA2 1,077 49.67% Pn : GID - 49979 RGA3 864 52.95% Pn : GID - 02968 RGA2 502 58.76% Pn : GID - 50215 RGA2 533 76.24% Pn : GID - 03175 RGA2 570 68.24% Pn : GID - 51124 RGA2 2,594 74.00% Pn : GID - 03632 RGA2 1,009 68.52% Pn : GID - 51412 RGA2 888 81.43% Pn : GID - 03843 RGA2 572 61.10% Pn : GID - 51719 RGA2 503 51.33% Pn : GID - 03865 RGA2 1,572 49.10% Pn : GID - 52684 RGA3 833 73.93% Pn : GID - 03878 RGA2 508 51.05% Pn : GID - 53045 RGA3 503 53.05% Pn : GID - 04287 RGA2 859 56.67% Pn : GID - 53600 RGA2 605 91.95% Pn : GID - 05003 RGA2 674 62.95% Pn : GID - 53646 RGA2 645 51.81% Pn : GID - 05369 RGA2 1,467 47.67% Pn : GID - 54584 RGA2 1,560 67.90% Pn : GID - 05617 RGA2 631 54.67% Pn : GID - 55513 RGA2 2,186 80.38% Pn : GID - 05619 RGA2 636 49.76% Pn : GID - 55648 RGA2 831 78.57% Pn : GID - 05637 RGA2 1,555 51.33% Pn : GID - 56390 RGA2 1,417 51.57% Pn : GID - 05936 RGA2 684 53.62% Pn : GID - 56734 RGA2 1,426 53.24% Pn : GID - 06344 RGA2 1,168 53.62% Pn : GID - 56735 RGA2 789 90.71% Pn : GID - 06935 RGA2 777 42.43% Pn : GID - 56935 RGA2 725 66.86% Pn : GID - 07571 RGA2 502 64.14% Pn : GID - 57374 RGA2 875 57.62% Pn : GID - 07726 RGA2 3,132 57.19% Pn : GID - 57523 RGA2 976 62.95% Pn : GID - 08049 RGA2 833 64.19% Pn : GID - 57924 RGA2 836 80.48% Pn : GID - 08152 RGA2 563 85.62% Pn : GID - 58949 RGA2 588 67.52% Pn : GID - 08311 RGA2 1,283 50.10% Pn : GID - 59029 RGA2 1,116 52.62% Pn : GID - 08655 RGA2 562 78.23% Pn : GID - 59655 RGA2 742 68.71% Pn : GID - 09094 RGA2 1,222 60.33% Pn : GID - 59752 RGA2 528 68.81% Pn : GID - 09099 RGA2 1,073 49.67% Pn : GID - 59794 RGA2 822 68.19% Pn : GID - 09100 RGA2 708 98.95% Pn : GID - 60519 RGA2 1,572 45.05% Pn : GID - 09852 RGA2 865 70.19% Pn : GID - 60847 RGA2 618 93.86% Pn : GID - 09886 RGA2 587 51.19% Pn : GID - 61909 RGA2 1,238 51.38% Pn : GID - 10281 RGA2 579 53.95% Pn : GID - 62038 RGA2 1,286 54.57% Pn : GID - 10705 RGA2 1,554 45.81% Pn : GID - 62167 RGA2 1,283 89.71% Pn : GID - 11003 RGA2 864 62.33% Pn : GID - 62203 RGA2 1,598 53.10% Pn : GID - 11059 RGA2 599 66.29% Pn : GID - 63298 RGA2 1,193 67.43% Pn : GID - 11060 RGA2 826 51.24% Pn : GID - 63519 RGA2 690 78.43% Pn : GID - 11815 RGA2 924 50.25% Pn : GID - 63619 RGA2 553 66.33% Pn : GID - 11868 RGA2 644 56.29% Pn : GID - 63654 RGA3 564 54.86%

Pn : GID - 12284 RGA3 554 46.00% Pn : GID - 64593 RGA2 2,221 79.67% Pn : GID - 13465 RGA2 1530 65.41% Pn : GID - 65402 RGA2 784 73.05% Pn : GID - 13466 RGA2 1,371 45.38% Pn : GID - 65634 RGA2 809 66.14% Pn : GID - 13548 RGA2 564 57.19% Pn : GID - 66089 RGA2 534 48.00% Pn : GID - 13896 RGA2 580 54.00% Pn : GID - 66844 RGA2 1,565 63.43% Pn : GID - 14062 RGA2 1,880 53.19% Pn : GID - 67563 RGA2 682 88.00% Pn : GID - 14508 RGA2 549 49.62% Pn : GID - 67568 RGA2 1,015 62.80% Pn : GID - 14841 RGA2 602 54.00% Pn : GID - 67863 RGA2 676 59.48% Pn : GID - 15487 RGA3 872 62.43% Pn : GID - 68939 RGA2 504 59.29% Pn : GID - 16608 RGA2 1,545 65.00% Pn : GID - 68984 RGA3 762 52.52% Pn : GID - 16609 RGA2 1,018 42.00% Pn : GID - 69017 RGA2 2,420 56.81% Pn : GID - 16685 RGA2 944 63.52% Pn : GID - 69816 RGA2 504 81.62% Pn : GID - 16805 RGA2 730 57.52% Pn : GID - 70240 RGA2 731 65.05% Pn : GID - 16843 RGA2 579 60.00% Pn : GID - 70246 RGA2 589 69.86% Pn : GID - 16844 RGA2 593 53.33% Pn : GID - 70612 RGA2 1,257 62.76% Pn : GID - 16907 RGA2 507 65.10% Pn : GID - 70782 RGA3 511 53.71% Pn : GID - 17205 RGA2 595 49.24% Pn : GID - 71365 RGA2 1,021 63.48% Pn : GID - 17236 RGA2 771 49.33% Pn : GID - 71549 RGA2 1,943 57.00% Pn : GID - 17629 RGA1 945 55.67% Pn : GID - 71940 RGA2 2,919 57.86% Pn : GID - 17710 RGA2 590 60.67% Pn : GID - 71943 RGA2 815 89.00% Pn : GID - 17711 RGA1 1,016 54.57% Pn : GID - 72518 RGA2 2,402 59.22% Pn : GID - 17720 RGA2 1,002 62.24% Pn : GID - 72638 RGA2 3,045 57.05% Pn : GID - 19064 RGA2 1,027 61.71% Pn : GID - 72644 RGA2 601 48.86% Pn : GID - 20147 RGA2 561 57.14% Pn : GID - 72846 RGA2 2,041 59.95% Pn : GID - 20523 RGA2 1,517 50.48% Pn : GID - 73085 RGA2 2,553 59.43% Pn : GID - 21211 RGA2 622 48.10% Pn : GID - 73113 RGA2 991 58.67% Pn : GID - 21643 RGA2 591 52.10% Pn : GID - 73185 RGA2 1,380 48.43% Pn : GID - 22257 RGA2 512 65.29% Pn : GID - 73194 RGA2 3,105 52.43% Pn : GID - 22622 RGA3 867 62.24% Pn : GID - 73275 RGA2 1,975 75.76% Pn : GID - 23568 RGA2 2,333 57.33% Pn : GID - 73306 RGA2 894 65.86% Pn : GID - 24664 RGA2 691 62.33% Pn : GID - 73310 RGA2 884 53.10% Pn : GID - 24665 RGA2 1,138 43.33% Pn : GID - 73625 RGA2 1,341 64.24% Pn : GID - 24692 RGA2 588 49.29% Pn : GID - 73680 RGA2 670 67.76% Pn : GID - 24693 RGA2 2,233 44.76% Pn : GID - 73706 RGA2 1,244 54.71% Pn : GID - 24696 RGA3 534 58.05% Pn : GID - 73784 RGA2 563 55.62% Pn : GID - 24697 RGA3 1,074 55.71% Pn : GID - 73786 RGA2 1,520 68.38% Pn : GID - 24867 RGA3 1,145 91.14% Pn : GID - 73906 RGA2 684 53.00% Pn : GID - 25200 RGA2 507 49.14% Pn : GID - 74333 RGA2 734 57.67% Pn : GID - 25215 RGA2 3,385 49.90% Pn : GID - 74384 RGA2 753 54.38% Pn : GID - 25395 RGA2 580 46.86% Pn : GID - 74792 RGA3 687 52.76% Pn : GID - 25899 RGA3 536 51.60% Pn : GID - 74891 RGA2 1,021 56.05% Pn : GID - 26327 RGA2 708 56.29% Pn : GID - 74923 RGA2 567 56.10% Pn : GID - 27540 RGA2 538 56.90% Pn : GID - 75382 RGA3 505 58.43% Pn : GID - 27937 RGA2 969 51.81% Pn : GID - 75675 RGA2 1,053 58.10% Pn : GID - 28137 RGA2 2,472 67.43% Pn : GID - 75777 RGA2 3,284 60.95% Pn : GID - 28363 RGA1 1,710 52.62% Pn : GID - 75848 RGA2 719 55.24%

130 | P age

Pn : GID - 28710 RGA2 793 56.76% Pn : GID - 76153 RGA2 901 49.62% Pn : GID - 28937 RGA2 1,313 43.10% Pn : GID - 76359 RGA2 509 49.29% Pn : GID - 29337 RGA2 1,250 56.71% Pn : GID - 76635 RGA2 3,129 51.81% Pn : GID - 29381 RGA2 777 55.90% Pn : GID - 76776 RGA2 858 78.10% Pn : GID - 29584 RGA2 751 58.24% Pn : GID - 76923 RGA3 630 54.19% Pn : GID - 29813 RGA2 532 48.76% Pn : GID - 76925 RGA2 1,856 54.05% Pn : GID - 30391 RGA2 767 63.19% Pn : GID - 77550 RGA3 612 50.29% Pn : GID - 30393 RGA2 812 42.67% Pn : GID - 77632 RGA3 550 56.38% Pn : GID - 30433 RGA2 727 56.81% Pn : GID - 77652 RGA3 823 53.29% Pn : GID - 30444 RGA2 1,167 53.24% Pn : GID - 77653 RGA3 664 49.81% Pn : GID - 30698 RGA2 541 63.52% Pn : GID - 77655 RGA2 937 82.10% Pn : GID - 30855 RGA2 732 49.29% Pn : GID - 77881 RGA3 1,323 52.29% Pn : GID - 31419 RGA2 647 46.24% Pn : GID - 78026 RGA3 1,535 50.90% Pn : GID - 35797 RGA2 908 65.71% Pn : GID - 78224 RGA3 763 62.67% Pn : GID - 36261 RGA2 851 70.05% Pn : GID - 78227 RGA3 1,784 67.33% Pn : GID - 36830 RGA2 676 62.57% Pn : GID - 78231 RGA3 521 61.95% Pn : GID - 39546 RGA3 636 51.95% Pn : GID - 78252 RGA3 1,320 45.38% Pn : GID - 40038 RGA2 735 73.33% Pn : GID - 78491 RGA3 550 46.33% Pn : GID - 41008 RGA2 682 57.71% Pn : GID - 78713 RGA3 809 52.29% Pn : GID - 41026 RGA3 706 56.71% Pn : GID - 78848 RGA1 536 60.00% Pn : GID - 41273 RGA2 773 59.29% Pn : GID - 80231 RGA2 626 78.96% Pn : GID - 41793 RGA2 1,288 57.14% Pn : GID - 80586 RGA2 3,730 59.76% Pn : GID - 42433 RGA2 882 54.00% Pn : GID - 80988 RGA2 1,816 81.33% Pn : GID - 42459 RGA2 718 53.48% Pn : GID - 81017 RGA2 507 58.05%

131 | P age

Pn : GID - 00091 GMGGVGKTTLLKR-INNFM---/---GI VLDDVWKEW---/---QGIPLA Pn : GID - 00091 GMGGVGKTTLLKR--/---GIIR--RCLNDK-KFVLLLDDVWKEW---/---QGIPLA Pn : GID - 00092 --GGVGKTTLLKR--/---GIIR--RCLNDK-KFVLLLDDVWKEW---/---AGLPLA Pn : GID - 01140 GMGGVGKTTLARI--/---ALRCCLEETVMGKNLLIVLDDIWEWK---/---GGLPLA Pn : GID - 01230 GMGGVGKTTLARI--/---ALRCCLEETVMGKNLLIVLDDIWEWK---/---RGVPLA Pn : GID - 01358 --GGVGKTTLLKR--/---GIIR--RCLNDK-KFVLLLDDVWKEW---/---AGLPLA Pn : GID - 01379 --GGVGKTTLMRK--/---GMKINDALINSKDKVALLLDDIWEAI---/---GGLPLA Pn : GID - 02382 GMGGVGKTTLARI--/---ALRCCLEETVMGKNLLIVLDDIWEWK---/---QGIPLA Pn : GID - 02792 --GGVGKTTLMRK--/---GMKINDALINSKDKVALLLDDIWEAI---/---GGLPLA Pn : GID - 02968 GMGGVGKTTLARI--/---ALRCCLEETVMGKNLLIVLDDIWEWK---/---GGLPLA Pn : GID - 03632 --GGVGKTTLMRK--/---GMKINDALINSKDKVALLLDDIWEAI---/---AGLPLA Pn : GID - 03843 --GGVGKTTLLKR--/---GIIR--RCLNDK-KFVLLLDDVWKEW---/---QGIPLA Pn : GID - 04287 GMGGVGKTTLARI--/---ALRCCLEETVMGKNLLIVLDDIWEWK---/---GGLPLA Pn : GID - 05619 GMGGVGKTTLARI--/---ALRCCLEETVMGKNLLIVLDDIWEWK---/---AGLPLA Pn : GID - 05936 GMGGVGKTTLARI--/---ALRCCLEETVMGKNLLIVLDDIWEWK---/---RGVPLA Pn : GID - 07571 --GGVGKTTLMRK--/---GMKINDALINSKDKVALLLDDIWEAI---/---QGIPLA Pn : GID - 07726 --GGVGKTTLMRK--/---GMKINDALINSKDKVALLLDDIWEAI---/---RGVPLA Pn : GID - 08152 --GGVGKTTLLKR--/---GIIR--RCLNDK-KFVLLLDDVWKEW---/---GGLPLA Pn : GID - 08655 --GGVGKTTLMRK--/---GMKINDALINSKDKVALLLDDIWEAI---/---GGLPLA Pn : GID - 09099 --GGVGKTTLMRK--/---GMKINDALINSKDKVALLLDDIWEAI---/---RGVPLA Pn : GID - 09886 GMGGVGKTTLARI--/---ALRCCLEETVMGKNLLIVLDDIWEWK---/---RGVPLA Pn : GID - 10281 GMGGVGKTTLARI--/---ALRCCLEETVMGKNLLIVLDDIWEWK---/---RGVPLA Pn : GID - 10705 GMGGVGKTTLARI--/---ALRCCLEETVMGKNLLIVLDDIWEWK---/---GGVPLA Pn : GID - 11003 GMGGVGKTTLARI--/---ALRCCLEETVMGKNLLIVLDDIWEWK---/---RGVPLA Pn : GID - 11059 GMGGVGKTTLARI--/---ALRCCLEETVMGKNLLIVLDDIWEWK---/---AGLPLA Pn : GID - 11060 --GGVGKTTLLKR--/---GIIR--RCLNDK-KFVLLLDDVWKEW---/---GGVPLA Pn : GID - 11815 --GGVGKTTLLKR--/---GIIR--RCLNDK-KFVLLLDDVWKEW---/---GGVPLA Pn : GID - 11868 GMGGVGKTTLARI--/---ALRCCLEETVMGKNLLIVLDDIWEWK---/---GGVPLA Pn : GID - 13465 --GGVGKTTLLKR--/---GIIR--RCLNDK-KFVLLLDDVWKEW---/---QGIPLA Pn : GID - 13466 --GGVGKTTLMRK--/---GMKINDALINSKDKVALLLDDIWEAI---/---GGVPLA Pn : GID - 13548 GMGGVGKTTLARI--/---ALRCCLEETVMGKNLLIVLDDIWEWK---/---GGVPLA Pn : GID - 13896 --GGVGKTTLMRK--/---GMKINDALINSKDKVALLLDDIWEAI---/---AGLPLA Pn : GID - 14062 GMGGVGKTTLARI--/---ALRCCLEETVMGKNLLIVLDDIWEWK---/---RGVPLA Pn : GID - 14508 --GGVGKTTLMRK--/---GMKINDALINSKDKVALLLDDIWEAI---/---AGLPLA Pn : GID - 14841 --GGVGKTTLLKR--/---GIIR--RCLNDK-KFVLLLDDVWKEW---/---QGIPLA Pn : GID - 16608 --GGVGKTTLMRK--/---GMKINDALINSKDKVALLLDDIWEAI---/---RGVPLA Pn : GID - 16609 --GGVGKTTLMRK--/---GMKINDALINSKDKVALLLDDIWEAI---/---GGLPLA Pn : GID - 16685 --GGVGKTTLLKR--/---GIIR--RCLNDK-KFVLLLDDVWKEW---/---RGVPLA Pn : GID - 16805 GMGGVGKTTLARI--/---ALRCCLEETVMGKNLLIVLDDIWEWK---/---RGVPLA Pn : GID - 16843 GMGGVGKTTLARI--/---ALRCCLEETVMGKNLLIVLDDIWEWK---/---AGLPLA Pn : GID - 16844 --GGVGKTTLMRK--/---GMKINDALINSKDKVALLLDDIWEAI---/---GGVPLA Pn : GID - 16907 GMGGVGKTTLARI--/---ALRCCLEETVMGKNLLIVLDDIWEWK---/---QGIPLA Pn : GID - 17205 GMGGVGKTTLARI--/---ALRCCLEETVMGKNLLIVLDDIWEWK---/---QGIPLA Pn : GID - 17236 GMGGVGKTTLARI--/---ALRCCLEETVMGKNLLIVLDDIWEWK---/---QGIPLA Pn : GID - 17710 --GGVGKTTLMRK--/---GMKINDALINSKDKVALLLDDIWEAI---/---QGIPLA Pn : GID - 17720 --GGVGKTTLMRK--/---GMKINDALINSKDKVALLLDDIWEAI---/---QGIPLA Pn : GID - 19064 GMGGVGKTTLARI--/---ALRCCLEETVMGKNLLIVLDDIWEWK---/---GGVPLA Pn : GID - 20147 GMGGVGKTTLARI--/---ALRCCLEETVMGKNLLIVLDDIWEWK---/---QGIPLA Pn : GID - 20523 GMGGVGKTTLARI--/---ALRCCLEETVMGKNLLIVLDDIWEWK---/---GGLPLA Pn : GID - 21211 GMGGVGKTTLARI--/---ALRCCLEETVMGKNLLIVLDDIWEWK---/---GGLPLA Pn : GID - 21643 --GGVGKTTLMRK--/---GMKINDALINSKDKVALLLDDIWEAI---/---GGLPLA Pn : GID - 57924 GMGGVGKTTLARI--/---ALRCCLEETVMGKNLLIVLDDIWEWK---/---QGIPLA Pn : GID - 58949 GMGGVGKTTLARI--/---ALRCCLEETVMGKNLLIVLDDIWEWK---/---GGVPLA Pn : GID - 59029 GMGGVGKTTLARI--/---ALRCCLEETVMGKNLLIVLDDIWEWK---/---AGLPLA Pn : GID - 59655 --GGVGKTTLMRK--/---GMKINDALINSKDKVALLLDDIWEAI---/---QGIPLA Pn : GID - 59752 GMGGVGKTTLARI--/---ALRCCLEETVMGKNLLIVLDDIWEWK---/---GGVPLA Pn : GID - 59794 --GGVGKTTLMRK--/---GMKINDALINSKDKVALLLDDIWEAI---/---QGIPLA Pn : GID - 60519 --GGVGKTTLMRK--/---GMKINDALINSKDKVALLLDDIWEAI---/---QGIPLA Pn : GID - 60847 --GGVGKTTLMRK--/---GMKINDALINSKDKVALLLDDIWEAI---/---AGLPLA

Figure 4-12. Black pepper RGA aligned by using ClustalW algorithm. Conservative substitutions are shaded using online Boxshade server. Sequence comparison shows the presence of conserved motifs Kinase-1/P-loop (GMGGVGK), Kinase-2 (VLDDVW) and Kinase -3/GLPL, indicated by letters on top of the alignments. 132 | P age

4.2.4 Detection of microsatellites

A total of 5,012 SSR loci with di-, tri-, tetra-, penta- and hexanucleotide repeats were detected in the current study by scanning transcriptome data. Among these SSRs, the trinucleotide repeat motifs were the most abundant, with an amount of 2,951 SSRs (58.88%), followed by 1,890 dinucleotide repeat motifs (37.71%), 133 tetranucleotide repeat motifs (2.65%), 19 pentanucleotide repeat motifs (0.38%) and 19 hexanucleotide repeat motifs (0.38%) as listed in Table 4-8. The main motifs of the defined SSRs were dinucleotide AT/AT repeat (529) and TA/TA repeat (512) as well as trinucleotide CCG/ CGG repeat (316) and GAA/TTC repeat (307) as shown in Figure 4-13.

Besides that, a total of 77 SSRs inclusive of 11 dinucleotide repeat (14.29%), 48 trinucleotide repeat (62.33%) and 18 tetranucleotide repeat (23.38%) were detected in 208 pepper defence-related unigenes as shown in Table 4-9. Figure 4-14 shows the distribution of defined SSR loci in FLS2, RGA2 , MEKK1, PR1, RPS2, CDPK, BAK1, RPM1 and other groups of defence-related unigenes in the current study. These SSR motifs represent a valuable biomarker resource for future molecular breeding program of pepper plants. However, SSR primers need to be designed in future and all putative SSR primers should be validated before use.

Table 4-8. Summary of SSR searching results by screening transcriptome data. Description Number Total number of unigenes examined 81,096 Total number of identified SSRs 5,012 Number of SSR containing sequences 4,693 Dinucleotide 1,890 Trinucleotide 2,591 Tetranucleotide 133 Pentanucleotide 19 Hexanucleotide 19

133 | P age

2,951 3,000

2,500

2,000 1,890

1,500

1,000

529 512 500 309 316 307 211 193 194 148 176 170 111 101 141 133128 133 57 78 55 89 69 80 49 68 69 63 55 86 64 79 1 4 37 45 36 10 5 42 21 19 19 0

Figure 4-13. The summary of SSR loci has been detected in transcriptome data. In total, 5,012 SSRs were detected in 4,693 unigenes. The x-axis indicates the repeat type. The y-axis indicates the number of different repeats.

16 15 14 14

12 11 11

10 9 8 8 6 6 5

4

2 1

0 FLS2 RGA2 MEKK1 PR1 RPS2 CDPK BAK1 RPM1 Others

Figure 4-14. Distribution of the defined SSR motifs in different groups of black pepper defence-related genes. The x-axis indicates the different groups of defence-related unigenes. The y-axis indicates the number of SSR loci.

134 | P age

Table 4-9. Defined SSR repeat units in the black pepper defence-related unigenes.

Unigenes ID Object name SSR repeat unit SSR size Pn : GID - 00091 disease resistance RGA2 (ACA)5 15 Pn : GID - 00159 catalase isozyme 1 (AAAT)3 12 Pn : GID - 01147 cyclic nucleotide-gated ion channel (AATT)4 16 Pn : GID - 01377 disease resistance At1g50180 (TAAT)3 12 Pn : GID - 01407 chitinase 1-like (ACTT)4 16 Pn : GID - 01521 LRR receptor-like serine threonine- kinase FLS2 (CCA)6 18 Pn : GID - 01715 F-box LRR-repeat 3 (CCAT)3 12 Pn : GID - 01716 F-box LRR-repeat 3 (CCAT)3 12 Pn : GID - 02115 mitogen-activated protein kinase kinase kinase 1 (ACT)4 12 Pn : GID - 03209 chitinase 1 (ACTT)4 16 Pn : GID - 03210 chitinase 1 (ACTT)4 16 Pn : GID - 04402 LRR receptor-like serine threonine- kinase FLS2 (CAT)4 12 Pn : GID - 06226 F-box LRR-repeat 4 isoform X1 (CCAT)3 12 Pn : GID - 06227 F-box LRR-repeat 4 (CCAT)3 12 Pn : GID - 06246 F-box LRR-repeat 2 (CCAT)3 12 Pn : GID - 06480 chitinase 6-like (ACTT)4 16 Pn : GID - 07181 cysteine-rich receptor kinase 10 (GGCA)3 12 Pn : GID - 07571 disease resistance RGA2 (GTA)6 18 Pn : GID - 07641 disease resistance RPM1-like (GC)6 12 Pn : GID - 07642 disease resistance RPM1-like (CA)6 12 Pn : GID - 08159 LRR receptor-like serine threonine- kinase FLS2 (CTG)6 18 Pn : GID - 08410 calcium-dependent protein kinase (TTCA)3 12 Pn : GID - 08920 brassinosteroid insensitive 1-associated receptor kinase (TA)6 12 Pn : GID - 09036 mitogen-activated protein kinase kinase kinase 1 (TCC)4 12 Pn : GID - 09091 disease resistance RPS2 (ATA)6 18 Pn : GID - 09958 WD-40 repeat-containing MSI1 (CCAA)4 16 Pn : GID - 10644 mitogen-activated protein kinase kinase kinase 1 (ACG)5 15 Pn : GID - 10705 disease resistance RGA2 (CTT)5 15 Pn : GID - 10918 LRR receptor-like serine threonine- kinase FLS2 (GCT)4 12 Pn : GID - 11152 WD-40 repeat-containing MSI4 (CCAA)4 16 Pn : GID - 11153 WD-40 repeat-containing MSI4-like (TCTT)5 20 Pn : GID - 11478 F-box WD-40 repeat-containing At5g21040 (CCAA)4 16 Pn : GID - 11479 F-box WD-40 repeat-containing At5g21040 (CCAA)4 16 Pn : GID - 11480 F-box WD-40 repeat-containing At5g21040 (CCAA)4 16 Pn : GID - 11494 LRR receptor-like serine threonine- kinase FLS2 (TCA)6 18 Pn : GID - 11859 probable inactive leucine-rich repeat receptor kinase At1g66830 (ACGC)3 12 Pn : GID - 12346 disease resistance RPM1-like (CG)5 10 Pn : GID - 13548 disease resistance RGA2 (TTC)5 15 Pn : GID - 13731 LRR receptor-like serine threonine- kinase FLS2 (TGA)3 12 Pn : GID - 13856 disease resistance At1g50180 (TAAT)3 12 Pn : GID - 14841 disease resistance RGA2 (TTTG)4 16 Pn : GID - 15370 LRR receptor-like serine threonine- kinase FLS2 (TCG)5 15 Pn : GID - 15908 calcium-dependent protein kinase (TTCA)3 12 Pn : GID - 16117 calcium-dependent protein kinase (AATA)4 16 Pn : GID - 16128 calcium-dependent protein kinase (AATA)4 16 Pn : GID - 16395 cyclic nucleotide-gated ion channel (AATT)4 16 Pn : GID - 16608 cyclic nucleotide-gated ion channel (AATT)4 16 Pn : GID - 16805 cyclic nucleotide-gated ion channel (AATT)4 16 Pn : GID - 16823 cyclic nucleotide-gated ion channel (AATT)4 16 Pn : GID - 16838 Calmodulin (GTAT)4 16 Pn : GID - 16954 Calmodulin (GTAT)4 16 Pn : GID - 17084 Calmodulin (GTAT)4 16 Pn : GID - 17266 Calmodulin (GTAT)4 16 Pn : GID - 17358 Calmodulin (GTAT)4 16 Pn : GID - 17457 Calmodulin (GTAT)4 16 Pn : GID - 17566 Calmodulin (GTAT)4 16 Pn : GID - 21369 calcium-dependent protein kinase (AT)6 12 Pn : GID - 21434 calcium-dependent protein kinase (AC)6 12 Pn : GID - 21443 calcium-dependent protein kinase (AT)6 12 Pn : GID - 21466 calcium-dependent protein kinase (AC)6 12 Pn : GID - 21477 calcium-dependent protein kinase (ACCA)3 12 Pn : GID - 21489 calcium-dependent protein kinase (ACCA)3 12 Pn : GID - 21521 calcium-dependent protein kinase (ACCA)3 12 Pn : GID - 21641 calcium-dependent protein kinase (AT)6 12 Pn : GID - 21643 calcium-dependent protein kinase (AT)6 12 Pn : GID - 21723 calcium-dependent protein kinase (ACCA)3 12 Pn : GID - 21755 calcium-dependent protein kinase (AATA)4 16 Pn : GID - 21762 calcium-dependent protein kinase (TTCA)3 12 Pn : GID - 21780 calcium-dependent protein kinase (AT)6 12 Pn : GID - 22881 cyclic nucleotide-gated ion channel (AATT)4 16 Pn : GID - 22991 cyclic nucleotide-gated ion channel (TCTA)5 20 Pn : GID - 23039 cyclic nucleotide-gated ion channel (TCTA)5 20 Pn : GID - 23073 cyclic nucleotide-gated ion channel (TCTA)5 20 Pn : GID - 23161 cyclic nucleotide-gated ion channel (TCTA)5 20 Pn : GID - 23230 cyclic nucleotide-gated ion channel (TCTA)5 20 Pn : GID - 23268 cyclic nucleotide-gated ion channel (TCTA)5 20 Pn : GID - 23281 cyclic nucleotide-gated ion channel (AATT)4 16 Pn : GID - 23308 cyclic nucleotide-gated ion channel (TCTA)5 20 Pn : GID - 23334 cyclic nucleotide-gated ion channel (AATT)4 16 Pn : GID - 23525 Calmodulin (GTAT)4 16 Pn : GID - 23568 Calmodulin (GTAT)4 16 Pn : GID - 23576 Calmodulin (GTAT)4 16 Pn : GID - 23609 Calmodulin (GTAT)4 16 Pn : GID - 23630 Calmodulin (GTAT)4 16 Pn : GID - 23655 Calmodulin (GTAT)4 16 Pn : GID - 23713 Calmodulin (GTAT)4 16 Pn : GID - 23735 LRR receptor-like serine threonine- kinase FLS2 (GGA)4 12 Pn : GID - 23737 LRR receptor-like serine threonine- kinase FLS2 (GTT)5 15 Pn : GID - 23784 LRR receptor-like serine threonine- kinase FLS2 (ACC)5 15 Pn : GID - 23929 LRR receptor-like serine threonine- kinase FLS2 (TCG)5 15 Pn : GID - 23985 LRR receptor-like serine threonine- kinase FLS2 (TGA)3 12 Pn : GID - 24001 LRR receptor-like serine threonine- kinase FLS2 (CAG)6 18 Pn : GID - 24011 LRR receptor-like serine threonine- kinase FLS2 (CAC)5 15 Pn : GID - 24022 LRR receptor-like serine threonine- kinase FLS2 (AAT)4 12 Pn : GID - 24125 LRR receptor-like serine threonine- kinase FLS2 (AAC)5 15 Pn : GID - 24173 LRR receptor-like serine threonine- kinase FLS2 (TCG)5 15

135 | P age

Pn : GID - 24205 LRR receptor-like serine threonine- kinase FLS2 (GTT)5 15 Pn : GID - 24206 LRR receptor-like serine threonine- kinase FLS2 (GTT)5 15 Pn : GID - 24248 Calmodulin (GTAT)4 16 Pn : GID - 24282 Calmodulin (GTAT)4 16 Pn : GID - 24298 Calmodulin (GTAT)4 16 Pn : GID - 24359 Calmodulin (GTAT)4 16 Pn : GID - 24380 Calmodulin (GTAT)4 16 Pn : GID - 24463 Calmodulin (GTAT)4 16 Pn : GID - 24464 Calmodulin (GTAT)4 16 Pn : GID - 24487 Calmodulin (GTAT)4 16 Pn : GID - 44281 pathogenesis-related protein 1 (ATT)4 12 Pn : GID - 44699 pathogenesis-related protein 1 (TGT)5 15 Pn : GID - 44879 pathogenesis-related protein 1 (CTC)4 12 Pn : GID - 53019 pathogenesis-related protein 1 (CCG)4 12 Pn : GID - 53175 pathogenesis-related protein 1 (ATT)6 18 Pn : GID - 53292 pathogenesis-related protein 1 (TTA)6 18 Pn : GID - 53518 pathogenesis-related protein 1 (CTA)6 18 Pn : GID - 53646 pathogenesis-related protein 1 (CAT)6 18 Pn : GID - 53753 pathogenesis-related protein 1 (TGT)5 15 Pn : GID - 53832 pathogenesis-related protein 1 (TTA)6 18 Pn : GID - 54126 pathogenesis-related protein 1 (CTC)4 12 Pn : GID - 54177 pathogenesis-related protein 1 (CAT)6 18 Pn : GID - 68773 mitogen-activated protein kinase kinase kinase 1 (AGG)6 18 Pn : GID - 69017 mitogen-activated protein kinase kinase kinase 1 (ACT)4 12 Pn : GID - 69154 mitogen-activated protein kinase kinase kinase 1 (AGC)5 15 Pn : GID - 69230 mitogen-activated protein kinase kinase kinase 1 (TAG)4 12 Pn : GID - 69348 mitogen-activated protein kinase kinase kinase 1 (TAT)6 18 Pn : GID - 69574 mitogen-activated protein kinase kinase kinase 1 (TCC)4 12 Pn : GID - 69754 mitogen-activated protein kinase kinase kinase 1 (CCT)4 12 Pn : GID - 69786 mitogen-activated protein kinase kinase kinase 1 (ATT)6 18 Pn : GID - 69816 mitogen-activated protein kinase kinase kinase 1 (ACT)4 12 Pn : GID - 69946 mitogen-activated protein kinase kinase kinase 1 (ACG)5 15 Pn : GID - 69953 mitogen-activated protein kinase kinase kinase 1 (ACG)5 15 Pn : GID - 70123 brassinosteroid insensitive 1-associated receptor kinase (TC)5 10 Pn : GID - 70570 brassinosteroid insensitive 1-associated receptor kinase (TG)6 12 Pn : GID - 71078 brassinosteroid insensitive 1-associated receptor kinase (GA)6 12 Pn : GID - 71378 brassinosteroid insensitive 1-associated receptor kinase (ATG)6 18 Pn : GID - 71597 brassinosteroid insensitive 1-associated receptor kinase (CGG)5 15 Pn : GID - 71776 mitogen-activated protein kinase kinase kinase 1 (TCC)4 12 Pn : GID - 71822 mitogen-activated protein kinase kinase kinase 1 (CCT)4 12 Pn : GID - 72860 LRR receptor-like serine threonine- kinase FLS2 (CCA)6 18 Pn : GID - 73091 brassinosteroid insensitive 1-associated receptor kinase (TTG)6 18 Pn : GID - 73100 brassinosteroid insensitive 1-associated receptor kinase (TA)6 12 Pn : GID - 73185 disease resistance RGA2 (AGA)5 15 Pn : GID - 73247 catalase like (AAAT)3 12 Pn : GID - 73310 disease resistance RGA2 (CTT)5 15 Pn : GID - 73439 catalase-peroxidase (AAAT)3 12 Pn : GID - 73621 LRR receptor-like serine threonine- kinase FLS2 (TAA)3 9 Pn : GID - 73661 brassinosteroid insensitive 1-associated receptor kinase (ATC)5 15 Pn : GID - 73680 disease resistance RGA2 (CACC)4 12 Pn : GID - 73781 cyclic nucleotide-gated ion channel (AATT)4 12 Pn : GID - 73784 disease resistance RGA2 (AGA)5 15 Pn : GID - 73906 disease resistance RGA2 (CCA)6 18 Pn : GID - 74045 calcium-dependent protein kinase (TTCA)3 12 Pn : GID - 74088 brassinosteroid insensitive 1-associated receptor kinase (TA)6 12 Pn : GID - 74184 calcium-dependent protein kinase (TTCT)3 12 Pn : GID - 74384 disease resistance RGA2 (TCT)5 15 Pn : GID - 74589 F-box LRR-repeat At3g18150 (CCAA)4 16 Pn : GID - 74744 disease resistance RPS2 (ATA)6 18 Pn : GID - 74853 catalase A (AAAT)3 12 Pn : GID - 74923 disease resistance RGA2 (AGT)6 18 Pn : GID - 74996 brassinosteroid insensitive 1-associated receptor kinase (TGC)5 15 Pn : GID - 75207 brassinosteroid insensitive 1-associated receptor kinase (TTG)6 18 Pn : GID - 75234 pathogenesis-related protein 1 (ATT)4 12 Pn : GID - 75376 brassinosteroid insensitive 1-associated receptor kinase (TTG)6 18 Pn : GID - 75486 cyclic nucleotide-gated ion channel (AATT)4 16 Pn : GID - 75626 LRR receptor-like serine threonine- kinase FLS2 (TGA)3 12 Pn : GID - 75635 FBD, F-box and LRR (CCAA)4 16 Pn : GID - 75777 disease resistance RGA2 (AACA)5 20 Pn : GID - 75812 brassinosteroid insensitive 1-associated receptor kinase (TGG)6 18 Pn : GID - 75848 disease resistance RGA2 (AAC)5 15 Pn : GID - 75945 pathogenesis-related protein 1 (TGT)5 15 Pn : GID - 76023 catalase-peroxidase (AAAT)3 12 Pn : GID - 76038 brassinosteroid insensitive 1-associated receptor kinase (ATG)6 18 Pn : GID - 76153 disease resistance RGA2 (CAA)6 18 Pn : GID - 76359 disease resistance RGA2 (AAG)5 15 Pn : GID - 76635 disease resistance RGA2 (GTA)6 18 Pn : GID - 76776 disease resistance RGA2 (AGA)5 15 Pn : GID - 76915 brassinosteroid insensitive 1-associated receptor kinase (TGG)6 18 Pn : GID - 76917 cyclic nucleotide-gated ion channel (AATT)4 12 Pn : GID - 76925 disease resistance RGA2-like (AGA)5 15 Pn : GID - 76943 brassinosteroid insensitive 1-associated receptor kinase (TAC)4 12 Pn : GID - 77109 LRR receptor-like serine threonine- kinase FLS2 (CCA)6 18 Pn : GID - 77164 disease resistance At1g50180 (TAAT)3 12 Pn : GID - 77655 Disease resistance RGA2 (AGA)5 15 Pn : GID - 77724 disease resistance RPM1-like (CT)6 12 Pn : GID - 77857 F-box FBD LRR-repeat At1g13570-like (CCAA)4 16 Pn : GID - 77928 disease resistance At1g50180 (TAAT)3 12 Pn : GID - 78211 leucine rich repeat (TAAT)3 12 Pn : GID - 78224 disease resistance RGA3 (AGA)5 15 Pn : GID - 78227 disease resistance RGA3 isoform X1 (AGA)5 15 Pn : GID - 78323 brassinosteroid insensitive 1-associated receptor kinase (TA)6 12 Pn : GID - 78412 disease resistance RPM1-like (AG)6 12 Pn : GID - 78542 chitinase 2-like (ACTT)4 16 Pn : GID - 78979 pathogenesis-related protein 1 (CTC)4 12 Pn : GID - 78996 disease resistance RPM1-like (AG)6 12 Pn : GID - 79127 LRR receptor-like serine threonine- kinase At3g47570 (TAAT)3 12 Pn : GID - 79718 pathogenesis-related protein 1 (CAT)6 18 Pn : GID - 79783 disease resistance RPP13 1 (TA)6 12 Pn : GID - 79789 LRR receptor-like serine threonine- kinase GSO1 (TAAT)3 12

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Pn : GID - 80192 nitric oxide synthase (TAC)4 12 Pn : GID - 80231 disease resistance RGA2 (AGA)5 15 Pn : GID - 80371 catalase (AAAT)3 12 Pn : GID - 80383 glycerol kinase (AGT)6 18 Pn : GID - 80398 Respiratory burst oxidase (AGT)6 18 Pn : GID - 80586 disease resistance RGA2 (AGT)6 18 Pn : GID - 80621 mitogen-activated protein kinase kinase kinase 1 (CCT)4 13 Pn : GID - 80803 disease resistance RPP13 4 (AGA)5 15 Pn : GID - 80973 pathogenesis-related protein 1 (CAT)6 18 Pn : GID - 80977 disease resistance At4g10780 (CCT)4 13 Pn : GID - 80988 disease resistance RGA2 (AGT)6 18 Pn : GID - 81017 disease resistance RGA2 (AGT)6 18

4.2.5 Identification of reference genes

In the current study, a total of six commonly reported reference genes were identified from transcriptome data through key word search. These genes are elongation factor 1-α (ef1α), actin, glyceraldehyde-3- phosphate dehydrogenase (GAPDH), histone 3 (H3), β- tubulin and ubiquitin 7 (UBQ7) (Nicot et al., 2005; Chandna et al., 2012; Hu et al., 2015). In total, six primer pairs with amplification lengths of 109 bp to 148 bp and melting temperatures (Tm) of 50.1°C to 58.4°C were designed as shown in Table 4-10. All PCR reactions have shown efficiencies in between 98.67% and 106.16%. The expression stabilities of six reference genes in black pepper roots and leaves were monitored by qRT-PCR. The cycle threshold (Ct) values of these reference genes in different pepper plant tissues, i.e. roots and leaves were evaluated as shown in Figure 4-

15 (a) and Figure 4-15 (b), respectively. The Ct values of these reference genes were ranged in between 23.3 to 34.5. The highest Ct value was found in GAPDH. Meanwhile, the lowest Ct value was found in ef1α. Each individual reference gene has different Ct value in different pepper plant tissues that varied not so obviously. The expression of each reference gene was evaluated by using geNORM algorithm to identify the best candidate gene in different pepper plant tissues. Gene stability measure of the six reference genes has shown that the average expression M value of H3 was the lowest (Figure 4 -16). This result has shown that H3 is the most suitable internal control gene to be used for expression analysis of targeted pepper defence-related genes in next experimental stage of the current study.

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Table 4-10. Summary of primer sequences for six assessed reference genes, melting temperature, amplicon length and efficiency of PCR runs.

Genes Primers (5’ – 3’) Ta (°C) Amplicon Length PCR Efficiency (bp) (%) Actin cccatagacactccgact 55.8 113 103.48 cgaaggactacttgttgt ef1α cctttgcctatacgaggt 52.1 109 105.28 aatggacttgcggacagt GAPDH atcggttgaggagagagg 58.1 112 101.42 tcttcttcagtagccttc H3 agtcggggctgctgtcca 55.3 109 106.16 gctctgcgacctttccat UBQ7 ctggtcgtcgcaaattag 58.4 148 98.67 gaacatcttgacgtcctg β-tubulin acaagtccgcgttccgaa 52.7 113 98.84 acgttggcccagtaagta

35.0

30.0

Values

t C 25.0

20.0 Pc PnSA PnKch

Actin ef1α GAPDH H3 UBQ7 β-tubulin

Figure 4-15 (a). The cycle threshold (Ct) values of six assessed internal control genes, i.e. elongation factor 1-α (ef1α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-tubulin, histone 3 (H3), actin and ubiquitin 7 (UBQ7) in pepper roots tissues.

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35.0

30.0

Values

t C 25.0

20.0 Pc PnSA PnKch

Actin ef1α GAPDH H3 UBQ7 β-tubulin

(b)

Figure 4-15 (b). The cycle threshold (Ct) values of six assessed internal control genes, i.e. elongation factor 1-α (ef1α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-tubulin, histone 3 (H3), actin and ubiquitin 7 (UBQ7) in pepper leaves tissues.

0.600

0.550

0.500

0.450

0.400 Average Expression Stability (M) Stability Expression Average 0.350

0.300 UBQ7 ef1α Actin GAPDH β-tubulin H3

Figure 4-16. The average expression stability values of six assessed internal control genes, i.e. elongation factor 1-α (ef1α), glyceraldehyde- 3-phosphate dehydrogenase (GAPDH), β-tubulin, histone 3 (H3), actin and ubiquitin 7 (UBQ7). The lowest M value of Histone 3 (H3) indicated that this gene has exhibited the most stable expression in pepper plant tissues.

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4.3 Identification of potential biocontrol agents for slow decline

4.3.1 Isolation of antagonistic rhizobacteria

In total, 46 bacterial isolates were obtained from 15 rhizospheric soil samples collected from healthy black pepper farms HF1, HF2, HF3, HF4 and HF5 located at Serikin, Padawan and Serian areas of Sarawak. The selection of distinct bacterial isolates on LB medium was carried out based on bacterial gram nature, colony morphological features and antifungal properties (Modi and Patel, 2017) as shown in Table 4-11. There were respectively six and twelve bacterial isolates were obtained from healthy black pepper farms HF1 and HF2 located at Jagoi Duyuh village and Jagoi Sebubok village, Serikin. In total, 22 bacterial isolates were obtained from black pepper farms at Serian areas. Of which, twelve bacterial isolates were obtained from healthy black pepper farm HF3 located at Mongkos village. Meanwhile, the other 10 bacterial isolates were obtained from healthy black pepper farm HF4 located at Mujat village. In addition, a number of six bacterial isolates were obtained from healthy black pepper farm HF5 located at Karu village of Padawan district. Overall, 19.57% (9 isolates) of the isolated bacteria has shown effective antagonism to F. solani isolate FS010. Meanwhile, 32.61% (15 isolates) of the isolated bacteria were moderate in inhibition of F. solani isolate FS010 mycelium growth. Majority of the isolated bacteria (47.82%; 22 isolates) were found to exhibit low antagonistic effects to F. solani isolate FS010.

4.3.2 Molecular identification of isolated rhizobacteria

Sequence analysis based on 16S rRNA genes has grouped the isolated bacteria into genera Burkholderia (25 isolates), Bacillus (13 isolates) and Pseudomonas (8 isolates) as shown in Figure 4-17. Of which, seven bacterial isolates coded JD04, JS02, JS05, MO02, MO09, MU03 and MU07 were selected for further assessment on plant growth promoting traits as these bacterial isolates have shown effective antagonistic effects to F. solani isolate FS010. Although bacterial isolates JS08 and K06 isolated from black pepper farms HF1 and HF5 also have shown effective antagonism to F. solani isolate FS010, these two bacteria were morphologically similar to bacterial isolates MO09 and MO02, respectively (Table 4-11) . Besides that, the MEGA phylogenetic tree has also 140 | P age revealed that bacterial isolates JS08 and K06 are genetically closely related to bacterial isolates MO09 and MO02. Therefore, these two bacteria were represented by MO09 and MO02 in the analysis of next experimental stages.

Table 4-11. Morphology, gram nature and antifungal effects of the isolated bacteria.

Gram Code Size Shape Margin Opacity Elevation Consistency Colour Antifungal Nature HF1: Jagoi Duyuh Village, Serikin (1°19'38.0"N 110°00'12.9"E) JD01 -ve Small Circular Entire Opaque Raised Moist Creamy Moderate JD02 -ve Small Circular Entire Opaque Raised Dry White Moderate JD03 -ve Large Circular Entire Opaque Flat Dry White Low JD04 +ve Small Circular Entire Opaque Raised Moist Brownish Effective JD05 -ve Small Circular Entire Opaque Raised Dry White Low JD06 -ve Small Circular Entire Opaque Raised Moist Yellowish Moderate HF2: Jagoi Sebubok Village, Serikin (1°19'36.5"N 110°00'12.9"E) JS01 -ve Large Circular Entire Opaque Raised Moist White Low JS02 -ve Small Circular Entire Opaque Raised Moist White Effective JS03 -ve Small Circular Entire Opaque Raised Dry White Moderate JS04 -ve Small Circular Entire Opaque Raised Dry White Low JS05 -ve Small Circular Entire Opaque Raised Moist Yellowish Effective JS06 -ve Small Circular Entire Opaque Raised Dry Yellowish Low JS07 -ve Small Circular Entire Opaque Convex Moist White Low JS08 -ve Small Circular Lobate Opaque Flat Moist Yellowish Effective JS09 -ve Small Circular Entire Translucent Raised Moist Creamy Low JS10 +ve Small Circular Entire Opaque Raised Moist Yellowish Moderate JS11 +ve Small Circular Entire Opaque Raised Moist Creamy Moderate JS12 -ve Large Circular Entire Opaque Flat Dry White Low HF3: Mongkos Village, Serian (0°53’06.1”N 110°35’21.7”E) MO01 -ve Small Circular Entire Translucent Raised Moist Creamy Low MO02 -ve Small Circular Lobate Opaque Raised Moist Brownish Effective MO03 -ve Large Circular Lobate Opaque Raised Moist Brownish Moderate MO04 -ve Small Circular Entire Opaque Raised Dry Creamy Low MO05 +ve Small Circular Entire Opaque Raised Moist Creamy Moderate MO06 +ve Small Circular Entire Translucent Raised Moist Creamy Low MO07 +ve Large Irregular Lobate Opaque Flat Moist White Moderate MO08 -ve Small Circular Entire Opaque Convex Moist Yellowish Low MO09 -ve Small Circular Lobate Opaque Flat Moist Yellowish Effective MO10 -ve Small Circular Entire Opaque Convex Moist White Low MO11 -ve Large Circular Entire Opaque Raised Dry White Low MO12 -ve Large Circular Entire Opaque Raised Dry White Moderate HF4: Mujat Village, Serian (0°53'26.5"N 110°33'59.3"E) MU01 -ve Small Circular Entire Opaque Raised Moist Creamy Moderate MU02 +ve Small Circular Entire Opaque Raised Moist Yellowish Moderate MU03 +ve Large Irregular Lobate Opaque Flat Dry White Effective MU04 +ve Large Irregular Lobate Opaque Flat Moist White Moderate MU05 -ve Small Circular Entire Opaque Raised Dry White Low MU06 -ve Small Circular Entire Translucent Raised Moist Creamy Low MU07 +ve Small Circular Undulate Opaque Raised Moist Brownish Effective MU08 -ve Large Circular Entire Opaque Flat Dry White Low MU09 -ve Large Circular Entire Opaque Flat Dry Yellowish Low MU10 -ve Small Circular Entire Opaque Convex Moist White Low HF5: Karu Village, Padawan (1°17’04.4”N 110°16’47.0”E) K01 -ve Large Circular Entire Opaque Flat Dry White Low K02 +ve Small Circular Entire Opaque Raised Moist Creamy Moderate K03 -ve Large Circular Entire Opaque Raised Moist White Low K04 +ve Small Circular Entire Opaque Raised Dry White Low K05 +ve Small Circular Entire Opaque Raised Dry White Moderate K06 -ve Small Circular Lobate Opaque Raised Moist Brownish Effective

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In the current study, the five bacterial isolates out of the seven selected antagonists, i.e. JD04, MO02, MO09, MU03 and MU07 were obtained from healthy black pepper farms HF1, HF3 and HF4 which are adjacent to disease farms DF1, DF2 and DF3. Therefore, it was believed that these bacterial isolates were effective antagonists to F. solani isolate FS010 as the farms of origin of these bacterial isolates were free from F. solani invasion although the farms location were next to disease farms DF1, DF2 and DF3 that have been reported seriously damaged by slow decline.

Figure 4-17. Phylogenetic relationship of 46 isolated rhizobacteria based on their 16S rRNA gene sequences. The phylogenetic tree was constructed by using Molecular Evolutionary Genetics Analysis 6.0 (MEGA6). The isolates have been grouped into three main clusters, i.e. Cluster 1: genus Burkholderia with 25 isolates, Cluster 2: genus Bacillus with 13 isolates and Cluster 3: genus Pseudomonas with 8 isolates. Numbers at each node represent bootstrap values as percentage of 100 based on 1,000 replications. Bootstrap values greater than 70% are shown. The isolates selected for plant growth- promotion analysis were bolded and highlighted.

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4.3.2.1 Isolation and quality assessment of bacterial DNA

The bacterial genomic DNA was isolated by using Promega Genomic Purification Kit according to the manufacturer’s instruction. The quality of the isolated bacterial DNA was spectrophotometrically evaluated at spectrum wavelengths of 230 nm, 260 nm and 280 nm with a dilution factor of 100. Table 4-12 summaries the yield and purity range of the isolated bacterial DNA. According to Hu et al. (2002) and Zeng and Yang (2002), the absorbance ratios of A260/230 and A260/280 represent polysaccharide, polyphenolic and protein contaminations in DNA samples. In the current study, the A260/280 ratios of the isolated bacterial DNA ranged between 1.803 and 1.925 indicated insignificant levels of protein contamination. Meanwhile, the A260/230 ratios more than 1.901 suggest weak contamination of polysaccharides and polyphenolics in the isolated bacterial DNA.

Overall, the yield of isolated bacterial DNA was ranged from 654 ng to 786 ng per gram of used tissues. The presence of distinct DNA bands as shown in Figure 4-18 suggested high quality of bacterial DNA was obtained. The intact of isolated bacterial DNA were further confirmed through PCR analysis of 16S rRNA gene sequences. PCR amplicons with sizes ranged from 1,440 bp to 1,502 bp were successfully amplified and visualized as distinct bands on 1.5% (w/v) agarose gels as shown in Figure 4-18. These results suggested that the isolated bacterial DNA was of high purity and free of secondary metabolites such as protein substances, polysaccharides and polyphenolics compounds which can affect and hinder the PCR amplifications (Salzman et al., 1999).

L1 L2 L3 L4 L5 L6 L7

Figure 4-18. 1.0% (w/v) agarose gel electrophoresis image of the isolated bacterial DNA, Lane L1: isolate JD04; Lane L2: isolate JS02; Lane L3: isolate JS05; Lane L4: isolate MO02; Lane L5: isolate MO09; Lane L6: isolate MU03; Lane L7: isolate MU07.

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Table 4-12. The yield and purity range of the isolated bacterial genomic DNA assessed at spectrum wavelengths of 230 nm, 260 nm and 280 nm with a dilution factor of 100. 1Absorbance ratios Organism Sample Code 2Yield (ng/g) A260/230 A260/280 Rhizobacteria JD04 1.977 1.925 701 JS02 1.935 1.899 678 JS05 1.901 1.826 786 MO02 2.045 1.851 743 MO09 2.168 1.803 699 MU03 2.204 1.909 654 MU07 1.929 1.889 683

Notes: 1Results are presented as mean of 3 absorbance readings. 2Yields of isolated bacterial DNA per gram of used tissues.

4.3.2.2 PCR and sequence data analysis of 16S rRNA genes

In total, seven bacterial isolates coded JD04, JS02, JS05, MO02, MO09, MU03 and MU07 were selected for further assessment as these isolates are antagonists to F. solani isolate FS010, the causal fungus of slow decline incident reported in disease farm DF3. Table 4-13 shows the results of molecular identification of selected bacterial isolates through sequence homology analysis of 16S rRNA genes. PCR have amplified the 16S rRNA gene fragments of selected bacterial isolates with sequence length ranged from 1,440 bp to 1,502 bp (Table 4-13) . These fragments were 97.64% to 99.58% similar to 16S rRNA gene sequences data in EZBioCloud database. The query sequences coverage of homology analysis was ranged from 99.0% to 99.6%. Figure 4-19 shows the agarose gel electrophoresis of PCR amplified bacterial 16S rRNA gene fragments.

Overall, the selected seven bacterial isolates have been classified into three main genera by 16S rRNA gene sequences. They are genus Bacillus with three isolates coded JD04, MU03 and MU07, genus Pseudomonas with two isolates coded JS02 and JS05 as well as genus Burkholderia with two isolates coded MO02 and MO09. The 16S rRNA gene fragments of bacterial isolates in genus Bacillus, i.e. isolates JD04, MU03 and MU07

144 | P age are 98.52% to 99.38% similar to 16S rRNA genes of Br evibacillus gelatini (KP899808), Ba. subtilis (CP013984) and Ba. siamensis (AJVF0100043). Meanwhile, the 16S rRNA gene fragments of bacterial isolates in Pseudomonas, i.e. isolates JS02 and JS05 are 99.1% to 99.3% similar to 16S rRNA genes of P. geniculata (AB021404) and P. beteli (AB021406). The 16S rRNA gene fragments of two isolates in genus Burkholderia, i.e. MO02 and MO09 are 97.64% to 99.44% similar to 16S rRNA genes of B. ubonensis (EU024179) and B. territorii (LK023503) as shown in Table 4-12.

M JD04 MU03 MU07 JS02 JS05 MO02 MO09 1,500 bp

1,000 bp 900 bp

Figure 4-19. 1.5% (w/v) agarose gel electrophoresis image of PCR amplified bacterial 16S rRNA gene fragments. Lane M: Promega (USA) 100 bp DNA ladder.

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. CP013984 KP899808 EU024179 LK023503 AB021404 AB021406 Accession no. Accession AJVF01000043

geniculata hit name taxon - Bacillus subtilisBacillus Bacillus siamensis Bacillus Pseudomonas beteli Pseudomonas Top Brevibacillus gelatini Brevibacillus Burkholderia territoriiBurkholderia Burkholderia ubonensis Burkholderia Pseudomonas Pseudomonas

98.52 99.22 99.38 99.51 99.58 97.64 99.44 Similarity (%)

bacterial isolates through sequence isolates homology bacterial analysis genes of 16S rRNA

99.5 99.0 99.5 99.1 99.3 99.6 99.2 Query coverage (%) coverage Query

1,446 1,458 1,462 1,449 1,452 1,502 1,440 Molecular identification selected of Molecular . 13 Length of 16S rRNA (bp)Length of 16S rRNA - 4

Table JS02 JS05 JD04 MU03 MU07 MO02 MO09 Isolates

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4.3.3 Biocontrol assays of isolated rhizobacteria

4.3.3.1 Antagonistic effects to F. solani isolate FS010

The strongest antagonism to F. solani isolate FS010 has shown by Br. gelatini isolate JD04 with PIRG value of 68.5% as shown in Table 4-14. Two bacterial isolates from genus Burkholderia, B. ubonensis isolate MO02 and B. territorii isolate MO09 have lower efficacy than Br. gelatini isolate JD04 with their PIRG values are 66.7% and 65.9%, respectively. Besides that, two bacterial isolates from genus Bacillus, Ba. subtilis isolate MU03 and Ba. siamensis isolate MU07 also have shown significant inhibitory effects to F. solani isolate FS010 mycelium radial growth. The smallest PIRG values were shown by two bacterial isolates from genus Pseudomonas, P. geniculata isolate JS02 and P. beteli isolate JS05 with values of 62.5% and 61.6%, respectively. Figure 4-20 shows the antagonistic effects of evaluated bacterial isolates in restriction of F. solani isolate FS010 mycelium radial growth. In the current study, the antagonistic effects of the selected bacterial isolates were found related to siderophores (a chelating agent with high affinity for ferric iron) and chitinase ( hydrolytic enzyme that lyse chitin, a major constituent of the fungal cell wall) production.

(a) (b)

(c) (d) Figure 4-20. The antagonistic effects to F. solani isolate FS010 mycelium radial growth that exhibited by the assessed rhizobacterial isolates, (a) Br. gelatini isolate JD04; (b) B. ubonensis isolate MO02; (c) Ba. subtilis isolate MU03 and (d) Control plate.

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4.3.3.2 Siderophore production

The universal siderophore assay by using CAS and HDTMA as indicators is useful in detection of siderophore produced by Gram-negative bacteria. However, the HDTMA ingredient is toxic to Gram-positive bacteria and inhibits their growth as reported by Louden et al. (2011). Despite the thicker peptidoglycan layer, Gram-positive bacteria are more receptive to HDTMA due to the absence of outer membrane. In the current study, a colour change of CAS medium from blue to orange was observed in Gram- negative P. geniculata isolate JS02, P. beteli isolate JS05, B. ubonensis isolate MO02 and B. territorii isolate MO09; but not in Gram-positive Br. gelatini isolate JD04, Ba. subtilis isolate MU03 and Ba. siamensis isolate MU07.

Instead of common CAS method, an overlay technique modified from Perez-Miranda et al. (2007) was used to detect siderophore production in Gram-positive Br. gelatini isolate JD04, Ba. subtilis isolate MU03 and Ba. siamensis isolate MU07 as shown in Figure 4-21. A modified CAS medium is cast upon culture agar plates consists of Gram positive bacterial isolates. Thus, this technique is also known as “Overlaid CAS” or “O- CAS”. Colour change of O-CAS medium from blue to orange indicated siderophores production. The amount of siderophore produced by selected bacterial isolates was found varied from the lowest 53.4% in B. territorii isolate MO09 up to the highest 74.5% in Br. gelatini isolate JD04 as shown in Table 4-14.

The bacterial isolates of genus Bacillus were the greatest siderophores producer (64.8% to 74.5%) in the current study, and followed by bacterial isolates of genus Pseudomonas (55.5% to 61.7%) and genus Burkholderia (53.4% to 58.3%). The PIRG values of the evaluated bacterial isolates were found gradually reduced if FeCl3 was supplied to the culture medium. As shown in Table 4-14, the percentage of PIRG reduction due to the presence of 200 μM FeCl3 in culture medium was ranged from 61% (P. beteli isolate JS05) to 68% (Br. gelatini isolate JD04). These results have suggested the presence of siderophore-mediated antagonism in the evaluated bacterial isolates. Apart from that, the results also have revealed that the evaluated bacterial isolates were also produced other type of antifungal metabolites as they were able to restrict the growth of F. solani isolate FS010 either in the absence (PIRG: 61.6% to 68.5%) or presence (PIRG: 35.2%

148 | P age to 40.3%) of FeCl3. Chitinase is the other antifungal compound that has been produced by the evaluated bacterial isolates to inhibit F. solani isolate FS010 in the current study.

(a) (b) (c) Figure 4-21. Production of siderophores exhibited by the assessed rhizobacteria on CAS agar plates, (a) A colour change of CAS medium from blue to orange exhibited by the Gram-negative P. geniculata isolate JS02 on common CAS agar plate indicated siderophores production; (b) Gram-positive Br. gelatini isolate JD04 on Overlaid-CAS agar plate at 0 hour of incubation and (c) A colour change of CAS medium from blue to orange exhibited by the Gram-positive Br. gelatini isolate JD04 on Overlaid-CAS agar plate after 24 hours of incubation at 30°C indicated siderophores production.

4.3.3.3 Chitinase production

Colloidal chitin plate assay has revealed that all the evaluated bacterial isolates in the current study have secreted chitinase in the presence of chitin. The two bacterial isolates of genus Pseudomonas, P. geniculata isolate JS02 and P. beteli isolate JS05 were the greatest chitinase producers with their CI values are 1.76 and 1.67 respectively as shown in Table 4-14. Figure 4-22 shows the chitinolytic activities exhibited by these two bacterial isolates on chitin agar plates after 16 hours of incubation at 30°C. The presence of clear zones surrounding bacterial colonies on agar plates indicated chitinase production. Apart from that, the bacterial isolates of genus Bacillus have also exhibited significance chitinolytic activities with the CI values for Br. gelatini isolate JD04, Ba. subtilis isolate MU03 and Ba. siamensis isolate MU07 are 1.41, 1.35 and 1.38, respectively. Lowest chitinolytic activities were exhibited by bacterial isolates of genus

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Burkholderia with the CI values for B. ubonensis isolate MO02 and B. territorii isolate MO09 are 1.32 and 1.19, respectively.

(a) (b) Figure 4-22. Chitinolytic activities showed by two bacterial isolates of genus Pseudomonas on the chitin agar plates after 16 hours of incubation at 30°C, (a) P. geniculata isolate JS02 and (b) P. beteli isolate JS05. The presence of clear zones surrounding bacteria colonies on agar plates indicated chitinase production.

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3

c a a b d cd cd

. FeCl

μM 36.4 ± 0.43 40.3 ± 0.50 40.0 ± 0.33 38.2 ± 0.27 35.2 ± 0.45 35.7 ± 0.22 35.3 ± 0.52 200

3

a c a e b d bacterial isolates bacterial de FeCl

μM PIRG (%) 1 * 53.6 ± 0.22 48.9 ± 0.15 53.0 ± 0.24 47.0 ± 0.12 52.2 ± 0.27 47.8 ± 0.31 47.2 ± 0.26 100

3

a c e d d b bc FeCl

68.5 ± 0.36 65.3 ± 0.23 61.6 ± 0.39 63.2 ± 0.20 62.5 ± 0.32 66.7 ± 0.37 65.9 ± 0.06 Without

a b ab ab ab ab ab antifungal activity antifungal selected by the exhibited

. CI: Chitinolytic Index. CI: Chitinolytic * 1.76 ± 0.10 1.19 ± 0.20 1.41 ± 0.23 1.35 ± 0.06 1.38 ± 0.22 1.67 ± 0.07 1.32 ± 0.23 Chitinase (*CI)

f f a c e b d Production of Production 1 ± standard deviation 55.5 ± 0.40 53.4 ± 0.44 74.5 ± 0.32 64.8 ± 0.13 58.3 ± 0.30 68.9 ± 0.03 61.7 ± 0.18 Siderophore (%) Siderophore

Siderophore production, chitinase production and chitinase Siderophore production,

isolate MU07 . isolate JS02 isolate MO02

isolate MO09 isolate JD04 isolate MU03 14

- 4 isolate JS05

PIRG: Percentage of Inhibition of Radial Radial of Inhibition Growth; PIRG: Percentage Each value is mean of triplicates is mean of triplicates value Each 1 * Rhizobacteria isolates Rhizobacteria

gelatini geniculate geniculate beteli ubonensis territorii Table

. . . . Br. Ba. subtilis Ba. siamensis P P B B Notes: Notes:

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4.3.4 Plant growth promotion traits of isolated rhizobacteria

4.3.4.1 IAA measurement

The evaluated bacterial isolates produced different concentration of IAA with the presence of 0.05% tryptophan. In the current study, Br. gelatini isolate JD04 is the best IAA producer with the concentration of IAA produced was 42.6 μg/ml. Meanwhile, B. ubonensis isolate MO02 produced the smallest amount of IAA with concentration 10.5 μg/ml as shown in Table 4-15. A colour change of YEM broth from light yellow to pink after Salkowski treatments as shown in Figure 4-23 indicated IAA production by all evaluated bacterial isolates.

(a) (b) (c) (d)

(e) (f) (g) Figure 4-23. The presence of pink colour in Salkowski reagents produced by all the assessed rhizobacteria indicated IAA production, (a) Br. gelatini isolate JD04; (b) Ba. subtilis isolate MU03; (c) Ba. siamensis isolate MU07; (d) P. geniculata isolate JS02; (e) P. beteli isolate JS05; (f) B. ubonensis isolate MO02 and (g) B. territorii isolate MO09. IAA standard curve formula, y = 0.008x + 0.0673 with R² = 0.9978, where y denotes absorbance reading at 530 nm and x denotes concentration of IAA (μg/mL).

152 | P age

a c d d b d d

PnKch 3.93 ± 0.11 1.90 ± 0.21 1.19 ± 0.30 0.83 ± 0.31 2.62 ± 0.13 1.11 ± 0.29 0.56 ± 0.26

a e c d b d de

Root dry weight (g) Root dry weight PnSA 4.16 ± 0.13 1.01 ± 0.18 2.35 ± 0.15 1.63 ± 0.24 3.07 ± 0.09 1.55 ± 0.24 1.27 ± 0.18

)

f a e c d b g μg/mL

24.6 ± 0.25 42.6 ± 0.12 27.2 ± 0.45 33.8 ± 0.16 28.5 ± 0.47 38.5 ± 0.46 10.5 ± 0.45 IAA ( IAA 3

f e a c g d b . Production of Production 1 y = 0.008x + 0.0673, R² = 0.9978. 0.0673, R² = 0.008x + y = 3 61.2 ± 0.19 62.4 ± 0.30 75.3 ± 0.30 69.8 ± 0.16 60.3 ± 0.26 67.6 ± 0.07 71.9 ± 0.26 Ammonia (mM) Ammonia 2

. bacterial isolates bacterial

a a b b ab ab

- * ± standard deviation detected.

1.94 ± 0.06 2.01 ± 0.12 Solubilizing 1.61 ± 0.07 1.62 ± 0.12 1.81 ± 0.05 1.77 ± 0.11 Phosphate (*SI) : Not y = 0.034x + 0.4304, 0.034x + 0.9915; y = R² = - 2

isolate MU07 Phosphate solubilisation, ammonia production, IAA production and root growth stimulation exhibited by the selected stimulation growth root by the selected productionexhibited and IAA Phosphate solubilisation, production, ammonia isolate JS02 isolate MO02

. isolate MO09 isolate JD04 isolate MU03

15 - isolate JS05

4 SI: Solubilizing Index; * SI:Index; Solubilizing Data calculated through calculated Data Each value is mean of triplicates Each 1 * Rhizobacteria isolates Rhizobacteria

gelatini geniculate geniculate beteli ubonensis territorii

. . . . Table Br. Ba. subtilis Ba. siamensis P P B B Notes: Notes: Notes:

153 | P age

4.3.4.2 Root growth stimulation

Biological feasibility assays for the effects of IAA producing bacterial isolates on black pepper root growth have shown that all evaluated bacterial isolates were effective in stimulating root growth and increasing chlorophyll content of black pepper cuttings. Br. gelatini isolate JD04, the greatest IAA producer in the current study has stimulated highest root growth in black pepper cuttings. The dry weight of black pepper roots stimulated by this bacteria isolate is 4.2 fold (in PnSA) and 3.9 fold (in PnKch) higher than control treatment as shown in Table 4-15. However, the other two bacterial isolates of genus Bacillus were less effective in stimulation of black pepper roots growth if compared with Br. gelatini isolate JD04. These two bacterial isolates have found secreted smaller amount of IAA than Br. gelatini isolate JD04. The amount of roots stimulated by these two bacterial isolates is respectively 1.6 fold (in PnSA) and 1.2 fold (in PnKch) higher than control in Ba. subtilis isolate MU03; and 1.3 fold (in PnSA) and 0.8 fold (in PnKch) higher than control in Ba. siamensis isolate MU07.

In the current study, P. geniculata isolate JS02 has shown second higher efficacy in root stimulation after Br. gelatini isolate JD04. The amount of black pepper roots stimulated by this bacterial isolate is 3.1 fold (in PnSA) and 2.6 fold (in PnKch) higher than control. It was followed by B. territorii isolate MO09 with the amount of black pepper roots stimulated is 2.4 fold (in PnSA) and 1.9 fold (in PnKch) higher than control. Similar to Ba. subtilis isolate MU03 and Ba. siamensis isolate MU07, P. beteli isolate JS05 and B. ubonensis isolate MO02 are moderate in stimulation of black pepper roots growth in the current study. The amount of black pepper roots stimulated by P. beteli isolate JS05 is 1.6 fold (in PnSA) and 1.1 fold (in PnKch) higher than control. Whereas, the amount of black pepper roots stimulated by B. ubonensis isolate MO02 is the least with 1.0 fold (in PnSA) and 0.6 fold (in PnKch) higher than control as B. ubonensis isolate MO02 is the weakest IAA producer in the current study.

The current results have shown significance correlation of bacterial IAA concentration in stimulating black pepper roots growth. The evaluated bacterial isolates in the current study are able to stimulate black pepper roots growth through IAA production. Bacterial isolates with higher capability of IAA production is able to stimulate higher amount of root growth in black pepper plants as shown in Table 4-15. Figure 4-24 shows the root 154 | P age germination of black pepper cuttings stimulated by the two greatest IAA producing bacterial isolates in the current study, i.e. Br. gelatini isolate JD04 and P. geniculata isolate JS02 compared to control treatements at the end of the trials.

(a) (b) (c)

(d) (e) (f) Figure 4-24. Root germination of black pepper cuttings stimulated by the two greatest IAA producing rhizobacteria in the current study at the end of the trials, (a) PnSA treated by Br. gelatini isolate JD04; (b) PnSA treated by P. geniculata isolate JS02; (c) PnSA in control treatment; (d) PnKch treated by Br. gelatini isolate JD04; (e) PnKch treated by P. geniculata isolate JS02 and (f) PnKch in control treatment.

155 | P age

th ± 0.27 ± 0.38 ± 0.29 ± 0.39 ± 0.04 ± 0.13 ± 0.16 ± 0.02 ±

15 403.9 389.1 376.5 398.2 375.2 353.5 393.5 330.4

th ± 0.06 ± 0.16 ± 0.54 ± 0.27 ± 0.11 ± 0.13 ± 0.17 ± 0.06 ±

10 day of the trials. the of day

317.5 303.0 300.1 314.5 300.3 286.9 306.2 281.7 th

th ± 0.05 ± 0.05 ± 0.05 ± 0.05 ± 0.05 ± 0.05 ± 0.05 ± 0.05 ±

and 15 and 5 th 246.1 247.9 251.1 247.6 246.8 246.2 249.6 245.4 PnKch (Day) , 10 th , 5 rd rd 0.06 ± 0.08 ± 0.14 ± 0.45 ± 0.55 ± 0.09 ± 0.07 ± 0.03 ±

3 ) 2 , 3 th 242.8 244.4 247.4 244.2 243.3 242.1 246.1 243.7

th ± 0.21 ± 0.34 ± 0.67 ± 0.58 ± 0.26 ± 0.18 ± 0.13 ± 0.07 ±

0 238.2 239.8 242.7 239.6 238.7 237.6 241.5 239.1

th ± 0.16 ± 0.24 ± 0.35 ± 0.47 ± 0.41 ± 0.34 ± 0.33 ± 0.22 ±

15 431.8 415.7 400.6 430.8 402.1 378.4 419.5 352.6 leaves measured duringleaves 0 measured

Chlorophyll concentration per (μmol m th ± 0.05 ± 0.05 ± 0.05 ± 0.05 ± 0.05 ± 0.05 ± 0.05 ± 0.05 ±

10

. 339.4 323.6 319.3 340.2 321.9 307.1 326.4 300.6

) of black pepper black ) of th ± 0.09 ± 0.12 ± 0.18 ± 0.27 ± 0.53 ± 0.26 ± 0.43 ± 0.17 ±

2 5 263.1 264.8 267.2 267.9 264.5 263.6 266.1 261.9 PnSA (Day)

rd 0.49 ± 0.52 ± 0.03 ± 0.11 ± 0.05 ± 0.25 ± 0.48 ± 0.36 ±

μmol m per ± standard deviation 3 259.5 261.0 263.2 264.2 260.7 259.2 262.3 260.0

th ± 0.01 ± 0.15 ± 0.09 ± 0.23 ± 0.46 ± 0.51 ± 0.27 ± 0.31 ±

0 254.6 256.1 258.2 259.2 255.8 254.3 257.4 255.1

JS02

Chlorophyll concentration ( concentration Chlorophyll

. isolate MU07 isolate isolate MO02 isolate

isolate MO09 isolate JD04 isolate MU03 16

- Each value is mean of triplicates Each 4 JS05 isolate

gelatini geniculate geniculate beteli ubonensis territorii Rhizobacteria isolates

. . . . Table Br. Ba. subtilis Ba. siamensis P P B B Control Notes:

156 | P age

Chlorophyll concentrations of PnSA and PnKch leaves were measured during 0th, 3rd, 5th, 10th and 15th day of the experiments as shown in Table 4-16. Significant increment of leaves chlorophyll for black pepper cuttings treated with evaluated bacterial isolates was observed during 5th, 10th and 15th day of the experiments as shown in Figure 4-25. In average, the percentage of chlorophyll increment in black pepper leaves for both cultivars was 3.34% to 3.64% during 5th day of the experiments, 20.75% to 33.30% during 10th day of the experiments and 48.79% to 69.52% during 15th day of the experiments. However, there is no significant different in chlorophyll increment for black pepper leaves measured during 3rd day of the experiments (1.909% to 1.919%) if compare to control treatments (1.906%) as shown in Figure 4-25.

This is probably due to the increased number of black pepper root hairs and root laterals stimulated by IAA producing bacterial isolates after few days of sowing process have enhanced the nitrogen uptake by black pepper cuttings. Black pepper cuttings treated with Br. gelatini isolate JD04 and P. geniculata isolate JS02, the two greatest IAA- producers in the current study have shown highest amount of chlorophyll concentration if compared with others evaluated bacterial isolates. Meanwhile, black pepper cuttings treated by B. ubonensis isolate MO02 with least IAA production have shown smallest amount of chlorophyll concentration towards the end of the experiments (10th and 15th day). The current results have proposed the involvement of evaluated bacterial isolates in enhancing black pepper growth through synthesizing IAA.

157 | P age

1.919a 1.920 1.918a 1.915a 1.915 1.913a 1.911a 1.909a 1.910a 1.910 1.906a 1.905 Percentage (%) Percentage 1.900 3rd day Br. gelatini isolate JD04 B. territorii isolate MO09 P. geniculata isolate JS02 P. beteli isolate JS05 Ba. Subtilis isolate MU03 Ba. Siamensis isolate MU07 B. ubonensis isolate MO02 Control

4.00 3.64a 3.41a 3.48a 3.50 3.34a 3.37a 3.34a 3.37a

3.00 2.66b 2.50 Percentage (%) Percentage 2.00 5th day Br. gelatini isolate JD04 B. territorii isolate MO09 P. geniculata isolate JS02 P. beteli isolate JS05 Ba. Subtilis isolate MU03 Ba. Siamensis isolate MU07 B. ubonensis isolate MO02 Control

35.00 33.30a 31.25a

30.00 26.81ab 25.83b 26.36ab 25.00 23.65bc 20.75c 20.00 17.81d Percentage (%) Percentage 15.00 10th day Br. gelatini isolate JD04 B. territorii isolate MO09 P. geniculata isolate JS02 P. beteli isolate JS05 Ba. Subtilis isolate MU03 Ba. Siamensis isolate MU07 B. ubonensis isolate MO02 Control

80.00

69.57a 66.21a 70.00 62.96ab 62.29ab 57.20b 60.00 55.15b 48.79c 50.00 38.20d

Percentage (%) Percentage 40.00 30.00 15th day Br. gelatini isolate JD04 B. territorii isolate MO09 P. geniculata isolate JS02 P. beteli isolate JS05 Ba. Subtilis isolate MU03 Ba. Siamensis isolate MU07 B. ubonensis isolate MO02 Control

Figure 4-25. Effects of IAA producing rhizobacteria on the increment of chlorophyll content in black pepper leaves at 3rd, 5th, 10th and 15th day of the trials. Experiments were conducted in triplicates with 10 cuttings per treatment. The x-axis shows the rhizobacteria isolates. The y-axis shows the percentage of chlorophyll content. Data means with same letter on the top of each column are not significantly different. However, means with different letters within each column are differs significantly at p≤0.05.

158 | P age

4.3.4.3 Phosphate solubilisation

Among evaluated bacterial isolates, six of them were positive in producing phosphate solubilizing acid except Br. gelatini isolate JD04. However, the other two bacterial isolates of genus Bacillus were able to solubilize phosphate with solubilizing index of 1.81 in Ba. subtilis isolate MU03 and 1.77 in Ba. siamensis isolate MU07 as shown in Table 4-15 . B. territorii isolate MO09 and B. ubonensis isolate MO02, the two bacterial isolates of genus Burkholderia are the best phosphate solubiliser in the current study with highest solubilizing index of 2.01 and 1.94, respectively. P. beteli isolate JS05 and P. geniculate isolate JS02, the two bacterial isolates of genus Pseudomonas have produced lowest amount of phosphate solubilizing acid with their phosphate solubilizing index were 1.62 and 1.61, respectively. Figure 4-26 shows the phosphate solubilizing activities of B. territorii isolate MO09 and B. ubonensis isolate MO02 on Pikovskayas agar plates. These two bacterial isolates produced clear zone surrounding colonies on Pikovskayas agar medium which has indicates phosphate solubilizing acid production.

(a) (b) Figure 4-26. Phosphate solubilizing activities exhibited by the two members of rhizobacteria in genus Burkholderia on Pikovskayas agar plates after 16 hours of incubation at 30°C, (a) B. ubonensis isolate MO02 and (b) B. territorii isolate MO09. The presence of clear zone surrounding bacterial colonies on Pikovskayas agar medium indicates phosphate solubilizing acid production.

159 | P age

4.3.4.4 Ammonia production

The presence of deep yellow colour in cell free supernatants after nesslerization assays has indicated that all evaluated bacterial isolates were able to release high levels of ammonia. In the current study, P. geniculata isolate JS02 has produced highest amount of ammonia with concentration of 75.3 mM after two days of incubation at 30°C. All evaluated bacterial isolates were efficient in emission of ammonia with concentration ranging from the lowest 60.3 mM in Br. gelatini isolate JD04, followed by 61.2 mM in Ba. subtilis isolate MU03, 62.4 mM in Ba. siamensis isolate MU07, 67.6 mM in P. beteli isolate JS05, 69.8 mM in B. ubonensis isolate MO02, 71.9 mM in B. territorii isolate MO09 and the highest 75.3 mM in P. geniculata isolate JS02 as shown in Table 4-15. Figure 4-27 shows the colour change of bacterial cell free supernatants from light yellow to deep yellow in nesslerization assays after two days of incubation at 30°C. The colour change of peptone water from light to deep yellow indicates ammonia production.

(a) (b) (c) Figure 4-27. The colour change of bacterial cell free supernatants from light yellow to deep yellow in nesslerization assays after two days of incubation at 30°C, (a) P. geniculate isolate JS02; (b) B. ubonensis isolate MO03 and (c) Ba. siamensis isolate MU07. The sterile un-inoculated peptone water (light yellow solution) was served as reference in nesslerization assays. The colour change of peptone water from light to deep yellow indicates ammonia production.

160 | P age

4.4 Assessment on Pepper Defence-Related Genes

4.4.1 Expression study of targeted pepper defence-related genes

The expression of targeted pepper defence-related genes, i.e. Cf-9, FLS2, MEKK1, PR1 and RGA2 were explored in roots (Figure 4-28) and leaves (Figure 4-29) of susceptible P. nigrum L. and resistant P. colubrinum Link. Although several RGAs have been identified from transcriptome data, only RGA2 confers the resistance to fungal infestant has been chosen for expression study. All assessed genes have shown higher expression level in resistant Pc if compared with susceptible PnSA and PnKch after infected by pathogenic F. solani isolate FS010 and beneficial Br. gelatini isolate JD04. Low expression level of FLS2, MEKK1, RGA2 and PR1 genes has been observed in susceptible PnSA and PnKch in Trial 1 for both roots and leaves tissues (infected by pathogenic F. solani isolate FS010). However, these genes have shown high expression in susceptible PnSA and PnKch as well as resistant Pc in Trial 2 and Trial 3 (infected by beneficial Br. gelatini isolate JD04).

There was no expression has been observed for Cf-9 genes in roots and leaves tissues of susceptible PnSA and PnKch in all research trials. Although Cf-9 genes has shown high expression in Pc cuttings that infected by pathogenic F. solani isolate FS010 in Trial 1, the activities of this gene were found dramatically dropped in Pc cuttings in Trial 3. The

Log2 expression fold change values of Cf-9 genes were reduced from 3.38 in Trial 1 to 0.59 in Trial 3 for Pc roots; and 2.31 in Trial 1 to 0.55 in Trial 3 for Pc leaves. In Trial 3, infection of beneficial Br. gelatini isolate JD04 on Pc roots and leaves tissues were initially carried out three days before the infection of pathogenic F. solani isolate FS010 was introduced to the same individual Pc cuttings during 4th day of the trials. Therefore, decrease of Cf-9 gene expression has revealed that application of beneficial Br. gelatini isolate JD04 has significantly suppressed the infection of pathogenic F. solani isolate FS010 on Pc roots and leaves in Trial 3.

Besides that, the expression level of all assessed genes was found higher in resistant Pc roots if compared to Pc leaves as shown in Figure 4-28 and Figure 4-29. However, there was no significance difference has been observed in the expression level of those genes in leaves and roots tissues of susceptible PnSA and PnKch. 161 | P age

Trial 1, Fs-treated

4.00 3.38 3.50 3.16 2.87 3.00 2.82 2.50 2.00 1.50 values of fold change foldof values 1.00 2 0.27 0.25 0.28 0.27 0.50 0.11 0.22 0.14 0.15 0.21 0.12 0.13 Log 0.00 0.09 0.00 0.00 Pc PnSA PnKch

Cf-9 FLS2 MEKK1 PR1 RGA2 Control

Trial 2, Br-treated

4.00 3.42 3.45 3.44 3.50 3.15 3.00 2.52 2.56 2.38 2.43 2.41 2.41 2.37 2.50 2.35 2.00 1.50 values of fold change foldof values 1.00 2

0.50 0.11 0.15 0.11 0.13 Log 0.00 0.00 0.00 Pc PnSA PnKch

Cf-9 FLS2 MEKK1 PR1 RGA2 Control

Trial 3, Br-Fs-treated

4.50

3.77 4.00 3.56 3.64 3.42 3.50 3.00 2.48 2.45 2.44 2.36 2.45 2.36 2.50 2.32 2.26 2.00 1.50 values of fold change foldof values

2 1.00 0.59 0.12 Log 0.50 0.00 0.08 0.00 0.07 0.00 Pc PnSA PnKch

Cf-9 FLS2 MEKK1 PR1 RGA2 Control

Figure 4-28. The expression level of pepper defence-related genes, i.e. Cf-9, FLS2, MEKK1, PR1 and RGA2 in roots of susceptible PnSA and PnKch as well as resistant Pc infected by pathogenic F. solani isolate FS010 (Trial 1, Fs-treated), beneficial Br. gelatini isolate JD04 (Trial 2, Br-treated) and a combination of the two soil microbes infection (Trial 3, Br-Fs-treated). The y-axis represents Log2 values of gene expression fold change. Error bars denote the standard error of gene expression level.

162 | P age

Trial 1, Fs-treated

2.50 2.31 2.15 2.00 2.00 1.88

1.50

1.00 values of fold change foldof values

2 0.50 0.26 0.27 0.26 0.29 0.23 0.22 0.18 0.13 0.17 0.12 0.11 0.14 Log 0.00 0.00 0.00 Pc PnSA PnKch

Cf-9 FLS2 MEKK1 PR1 RGA2 Control

Trial 2, Br-treated

3.50

2.83 2.86 3.00 2.75 2.71 2.54 2.49 2.38 2.50 2.34 2.28 2.27 2.34 2.32 2.00 1.50

values of fold change foldof values 1.00

2

0.50 0.13 0.12 Log 0.08 0.00 0.09 0.00 0.00 Pc PnSA PnKch

Cf-9 FLS2 MEKK1 PR1 RGA2 Control

Trial 3, Br-Fs-treated

3.50

2.89 2.91 3.00 2.75 2.83 2.45 2.38 2.50 2.31 2.35 2.36 2.30 2.29 2.27 2.00 1.50

values of fold change foldof values 1.00

2 0.55

0.50 0.14 0.13 Log 0.00 0.09 0.00 0.00 Pc PnSA PnKch

Cf-9 FLS2 MEKK1 PR1 RGA2 Control

Figure 4-29. The expression level of pepper defence-related genes, i.e. Cf-9, FLS2, MEKK1, PR1 and RGA2 in leaves of susceptible PnSA and PnKch as well as resistant Pc infected by pathogenic F. solani isolate FS010 (Trial 1, Fs-treated), beneficial Br. gelatini isolate JD04 (Trial 2, Br-treated) and a combination of the two soil microbes infection (Trial 3, Br-Fs-treated). The y-axis represents Log2 values of gene expression fold change. Error bars denote the standard error of gene expression level.

163 | P age

CHAPTER 5

DISCUSSION

5.1 Identification of Indigenous Causal Fungal Strains of Slow Decline

5.1.1 Characterization of isolated soil-borne fungal strains

Slow decline is a disease complex caused by soil-borne fungus, F. solani and associated with plant parasitic root-knot nematode, M. incognita (Albuquerque, 1961; Hamada et al., 1988; Lai and Sim, 2011; Sim et al., 2011). It is the most serious and dangerous soil- borne disease that frequently outbreak in Malaysia black pepper farms. In the current study, a total of 13 indigenous soil-borne fungal isolates have been obtained from slow decline infected black pepper farms.

At the initial stage of fungal discrimination, the 13 fungal isolates were classified into four groups designated Group FU-01, Group FU-02, Group PH-01 and Group RI-01 based on fungal morphological appearances on PDA plates. Microscopic analysis has further classified the four fungal groups into genera Fusarium which comprise of Group FU-01 and Group FU-02, Phytophthora (Group PH-01) and Rigidoporus (Group RI- 01). However, species of the fungal isolates could not been determined by microscopic analysis. This is due to morphological characters of the fungal isolates were insufficient for microscopic discrimination of species. Even though the two Fusarium groups in the current study have shown different morphological appearances on PDA plates (Figure 4-3), these two groups of fungus were found similar to each other through microscopic analysis, except in macroconidia production (Table 4-1).

In order to effectively discriminate the species of the four fungal groups, PCR analysis of ITS region sequences was employed in the current study. As described by Peay et al. (2008), sequences of fungal ITS regions have been widely used as universal molecular barcodes for species discrimination in many fungal species. Molecular characterization appears to be an effective approach in species discrimination of the isolated fungi in the

164 | P age current study. Sequence homology analysis of PCR amplified ITS region sequences have shown that the four fungal groups in the current study were genetically closely related to four defined fungal strains in NCBI GenBank. These four fungal strains are F. solani strain MTCC 9622 (accession no. FJ719812.1), F. solani isolate FS04 (accession no. KY307804.1), P. nicotianae isolate NRCPh-6 (accession no. HM807371.1) and R. microporus isolate R23 (accession no. MH681569.1)

The current results have revealed that molecular characterization through ITS sequences homology analysis were more effective in discrimination of the isolated fungi at species level compared to morphological observation. All fungal isolates of genus Fusarium (Group FU-01 and Group FU-02) in the current study were detected as F. solani by ITS region sequences homology analysis. Hence, these fungal isolates were observed similar to each other through microscopic analysis. Moreover, the two F. solani of Group FU- 01 and Group FU-02 were further differentiated into two different strains. These outputs have further explained the uncertainty where the two F. solani have shown different morphological appearances on PDA medium. The results of molecular characterization were supported by Bowers et al. (2007) and De Biazio et al. (2008). The literatures have reported that homology analysis of ITS region sequences are an effective approach in detect Fusarium species. This is because of the defined ITS region sequences are highly variable in genus Fusarium.

5.1.2 Diagnostic of slow decline disease causal fungus

In the current study, two indigenous F. solani strains designated F. solani isolate FS008 and F. solani isolate FS010 have been diagnosed as the causal fungus of slow decline incidents reported in black pepper disease farms DF1, DF2 and DF3 located at Serikin and Serian areas of the State of Sarawak. The other fungal species such as P. nicotianae and R. microporus also have been detected in same disease farms. However, sequence homology analysis of ITS regions has revealed that 76.92% of the soil-borne fungi isolated from the disease farms were detected as F. solani. This percentage have further shown that the disease farms DF1, DF2 and DF3 were dominated by F. solani during the period of disease incidence, rather than P. nicotianae (15.38%) and R. microporus (7.69%). 165 | P age

Fungal isolation has shown that F. solani isolate FS008 and F. solani isolate FS010 were virulent fungal strains. These two strains have been detected in both disease root samples (40%) and rhizospheric soil samples (60%) that collected from disease farms. Besides that, black pepper cuttings infected by these two fungi have shown clear disease symptoms of slow decline such as foliar yellowing, loss of feeder roots and damage of collar regions at the end of pathogenicity assays. PCR analysis of ITS region sequences also has shown that F. solani isolate FS008 and F. solani isolate FS010 were re-isolated from the infected black pepper root segments in colonization assays, suggesting Koch’s postulates (Jonathan, 2017).

P. nicotianae and R. microporus have been reported as causal organisms to foot rot and white root rot diseases in pepper plants and other agricultural crops (Andrés et al., 2003; Suwandi, 2006; Saadoun et al., 2013). However, P. nicotianae isolate PN001 and P. nicotianae isolate PN002 as well as R. microporus isolate RM001 in the current study were only detected in rhizospheric soil samples collected from disease farms DF1, DF2 and DF3. None of these fungal strains were isolated from disease root samples. This indirectly has pointed out that there is no significant involvement of these fungal strains in slow decline incidents reported in disease farms DF1, DF2 and DF3. Moreover, the isolated fungal strains of P. nicotianae and R. microporus were found not virulent to black pepper cuttings during pathogenicity assays. The cuttings infected by these fungal strains do not exhibit any disease symptoms of slow decline at the end of the assays.

166 | P age

5.2 Elucidation of Defence-Related Mechanism Pathways in Pepper Plants

5.2.1 Total RNA isolation

High quality total RNA is crucial for RNA-Sequencing analysis of plant transcriptome. However, during RNA isolation, plant cells were disrupted through homogenization. Secondary metabolite substances such as polyphenolics were released from plant cell vacuoles and react rapidly with cytoplasmic enzymes. These enzymatic by-products were oxidized to form brownish and covalently linked quinines which avidly bind to RNA molecules (Loomis, 1974; Chang et al., 1993; Gasic et al., 2004). Oxidation of polyphenolic compounds in RNA isolation causes the homogenized solutions become brown colour and eventually contributes to yellowish and water insoluble RNA pellets at the end of extraction. Polysaccharide is another tricky contaminant during plant RNA isolation (Scott and Playford, 1996). Polysaccharides released from the cytoplasmic of broken plant cells into homogenized mixtures and form viscous and gluelike solution. This secondary substance co-precipitate and interact with RNA to form tight complexes that hinder RNA from resuspension in sterile DEPC treated water (Wilkins and Smart, 1996; Fu et al., 2004).

In addition to polysaccharide and polyphenolic substances, the abundant contamination of protein substances also restricts the recovery of high quality RNA. Protein is readily oxidized to form covalently linked quinines and interact irreversibly with RNA. Protein contaminants are usually co-purified with RNA and affect the purity of isolated RNA. The gelatinous and oxidized RNA pellets were hindered from reverse transcription and PCR analysis (Fang et al., 1992; Sharma et al., 2002). Therefore, the current study has proposed an improved extraction method for isolating high quality and intact total RNA from black pepper plant leaves and roots. Black pepper plants tissues were reported rich in polysaccharide, polyphenolic and protein substances (Dhanya et al., 2007; Umadevi and Anandaraj, 2015). The described extraction protocol in the current study is of high efficiency as the method is effective to prevent the interaction of secondary substances with RNA during isolation process.

167 | P age

5.2.2 Data assembly and gene sequence annotation

High-throughput RNA-Seq has been reported by Gordo et al. (2012), Hu et al. (2015) and Hao et al. (2016) as an effective approach to gain large quantity of transcriptome data in black pepper plants. In the current study, transcriptomic data of two important local black pepper cultivars known as “Semengok Aman” (PnSA) and “Kuching” (PnKch) as well as a biotechnologically important wild pepper plant, P. colubrinum Link (Pc) were described for the first time. The transcriptome data of PnSA, PnKch and Pc obtained from Illumina HiSeqTM 2000 sequencing platform were assembled by using CLC genomics workbench. Kumar and Blaxter (2010) as well as Honaas et al. (2016) have identified CLC genomics workbench as one of the leading assemblers that produce low redundant assemblies.

The efficiency of data assembly in the current study was revealed by high value of N50 (1,081 bp) and mean length of 1,012 bp in sequence assembly. The value of N50 is defined as the number of minimum contig length required to cover 50% of the genome. This means half of the transcriptome sequences in the current study are in contigs larger than or equal to 1,081 bp. These results were comparable to the reported transcriptome data in other plant species. Shi et al. (2013) has reported a data assembly of xerophyte Reaumuria soongorica plant transcriptome with N50 value of 1,109 bp and mean length of 677 bp. Meanwhile, Long et al. (2014) has reported a transcriptome data assembly with N50 value of 1,812 bp and mean length of 1,060 bp in a species of desert tree known as Haloxylon ammodendron.

Gene annotation of the assembled transcriptome data has shown that a large number of unigenes (67.46%) in the current study were functionally annotated by searching against NCBI and EBI protein databases. This is informative for the physiology of black pepper plants. The remaining 32.54% unannotated unigenes in the current study are most probability due to insufficient reference genome for pepper plants. There is also a possibility that the unannotated unigenes are novel genes which need further elucidation in future for their roles in pepper plants. GO classification has provided further insights to the roles of annotated unigenes in pepper plants. GO functional subgroups with the highest abundance in BP, MF and CC categories of the current study were similar to the

168 | P age results of Hao et al. (2016), who investigated the phenylpropanoid metabolism of black pepper in response to soil-borne P. capsici infection.

In total, 52.14% annotated unigenes in the current study were mapped into 125 KEGG pathways. Several plant defence-related metabolisms pathways such as environmental adaptation plant-pathogen interaction (Boyd et al., 2013), signal transduction mitogen- activated protein kinase (MAPK) (Frank et al., 2004), biosynthesis of phenylpropanoid (Dixon et al., 2002), flavonoid (Treutter, 2005), terpenoids (Singh and Sharma, 2014) and histidine (Seo et al., 2016) have been detected. Annotated pepper defence-related unigenes in the current study practically demonstrated the major branches of the plant- pathogen interactions pathways (Path: ko04626). These branches were associated to plant defence-related mechanisms in recognition of microbial elicitors, cellular signal transduction in response to stimuli and activation of hypersensitive response to microbial invasion that mediated by plant R-genes such as Cf-9 , FLS2, MEKK1, PR1 and RGA2.

Moreover, the annotated pepper unigenes have shown highest homology to grape plants (V. vinifera) with percentage of matched blast hits was 29.77%. This was followed by sacred lotus (N. nucifera), oil palms (E. guineensis), sweet orange (C. sinensis) and date palms (P. dactylifera) with percentage of matched blast hits was ranged from 6.56% to 21.02%. Gordo et al. (2012) has also presented that 53.93% of the predicted proteins in black pepper root transcriptome were homology to protein sequences from databases of V. vinifera. Meanwhile, Hu et al. (2015) has also reported that V. vinifera was ranked first in species distribution analysis of black pepper fruit transcriptome with percentage of sequence homology is 39.68%. The species distribution might reveal the evolutionary relationship of black pepper with other plant species.

In total, a number of 5,012 SSR loci were defined in the current transcriptome data. Polymorphic SSRs are important molecular markers which have been widely used for plant genetic diversity, gene mapping, molecular breeding and gene-based related studies (Zietkiewicz et al., 1994). Identification of SSR loci in the current study not only illustrates the valuable of the established transcriptome data but also provides available reference data for pepper research program. In addition, an amount of 77 SSR loci were detected in the sequences of 208 black pepper defence-related genes. These SSR motifs 16 9 | P age represent a valuable biomarker resource for future marker assisted selection program of pepper plants. However, SSR primers need to be designed in future and all putative SSR primers should be validated before use.

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 the plant resistant when encounter bacterial infection. Therefore, this has shed some light in the next direction of utilising plant growth promoting rhizobacteria as a form of inducer in triggering the expression of potential R-genes in order to improve defence mechanisms in black pepper plants. Application of plant growth promoting rhizobacteria to induce disease resistance in plants has been widely employed for sustainable development of various agricultural industries (Loon, 2007; Paul and Lade, 2014; Vejan et al., 2016; Backer et al., 2018; Etesami and Maheshwari, 2018),

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5.3 Identification of potential biocontrol agents for slow decline

5.3.1 Isolation and molecular identification of rhizobacteria

Plant growth promoting rhizobacteria have gained worldwide attention in the recent decade due to their abilities in promoting plant growth and suppress plant pathogens. The current study has explored the existence of indigenous PGPR species which are potentially to be utilized as biocontrol agents for black pepper cultivation in Sarawak. Isolation of PGPR from 15 rhizospheric soil samples collected from healthy black pepper farms HF1, HF2, HF3, HF4 and HF5 has yielded 46 bacterial isolates with various morphological appearances. Of which, seven of the isolated PGPR have shown effective antagonistic effects to F. solani isolate FS010. All the seven antagonists were further studied on their antagonistic effects and plant growth promoting attributes.

Sequence data analysis of bacterial 16S rRNA genes was carried out in the current study to identify the seven antagonists. This is because the sequences of bacterial 16S rRNA gene are highly conserved in different bacterial species. It is present as multigene family or operons in almost all bacteria species. The gene has been reported by Tsukuda et al. (2017) to show similar functionalities between distantly related bacterial lineages. Therefore, PCR amplification of 16S rRNA gene sequences has been widely applied for phylogenetic studies in bacteria (Eden et al., 1991; Weisburg et al., 1991; Schmidt and Relman, 1994; Gray and Herwig, 1996; Brett et al., 1998; Kolbert and Persing, 1999; Coenye and Vandamme, 2003; Lu et al., 2009; Pereira et al., 2010). The gene is used as the standard for classification and identification of bacterial genus and species particularly for those bacterial strains that do not fit any recognized biochemical profiles (Michael and Sharon, 2007). Besides that, the size of bacterial 16S rRNA gene with the length of about 1,500 bp is large enough for informatics purposes (Patel JB, 2001).

5.3.2 Biocontrol assays of isolated rhizobacteria

The ability of the obtained antagonists in suppressing mycelium growth of F. solani isolate FS010 was associated to the production of siderophores. Siderophore is a bio- molecule with low molecular weight that produced by bacteria under iron starvation. It 171 | P age is a chelating agent with a high affinity for ferric iron. Iron is essential for organisms in the processes of respiration and DNA synthesis. However, the bioavailability of iron in nature environment is limited due to low solubility of the Fe3+ ion in soil. Siderophore is strong enough to remove iron from soils and convert it to become available for the growth of microbial cells (Pahari et al., 2016; Sasirekha and Shivakumar, 2016). By producing siderophores, antagonistic bacteria could facilitates their colonization at rhizosphere and assists host plants in iron nutrition as well as suppress the growth of soil-borne pathogens through iron competition (Chincholkar et al., 2007; Vansuyt et al., 2007).

The siderophores-mediated antagonism that showed by the selected bacterial isolates in the current study was detected through the reduction of PIRG values when FeCl3 was supplied to agar medium. In dual culture plate assay, the selected bacterial isolates suppressed the growth of F. solani isolate FS010 through ferric ion competition.

Therefore, by supplying FeCl3 to the agar medium, there is no privilege for the selected bacterial isolates to restrict F. solani isolate FS010 through siderophore production as there is sufficient amount of ferric iron in the agar medium to support the growth of both organisms.

Chitinase production could be another mechanism of biocontrol showed by the selected bacterial isolates in the current study. The colloidal chitin plate assays have shown that all selected bacterial isolates are chitinase producer. These bacteria are able to restrict the growth of F. solani isolate FS010 in dual culture plate assays with the presence

(PIRG: 35.2% to 40.3%) of FeCl3 (Table 4-13). The two isolates of genus Pseudomonas, P. geniculata isolate JS02 and P. beteli isolate JS05 have shown the highest PIRG values in the siderophores-mediated antagonism assays. Interestingly, these two isolates are the greatest chitinase producer in the current study. These observations have suggested that chitinase could be other secondary metabolites produced by these two isolates to restrict the growth of F. solani isolate FS010 when there is sufficient amount of ferric iron is presented in the agar medium.

Besides, Pseudomonas is also a well-known PGPR that exhibit antagonism in against fungal pathogens through chitinase secretion (Kavino et al., 2010; Ramyasmruthi et al., 2012). Furthermore, the three bacterial isolates of genus Bacillus, i.e. Br. gelatini isolate 172 | P age

JD04, Ba. subtilis isolate MU03 and Ba. siamensis isolate MU07 that exhibited higher CI were also more effective in inhibition of F. solani isolate FS010 than B. ubonensis isolates MO02 and B. territorii isolate MO09 of genus Burkholderia. These results have indirectly suggested that the chitinase produced by the selected bacterial isolates also took part in the inhibition of F. solani isolate FS010. As chitin is an essential component for carbohydrate skeleton in fungal cell wall (Lenardon et al., 2010), the effective chitinolytic activities that exhibited by the selected bacterial isolates is very useful for future isolation of antifungal compounds to combat soil-borne fungi in black pepper cultivation.

5.3.3 Plant growth promotion traits of isolated rhizobacteria

The capability of the selected bacterial isolates in promoting pepper root development is highly related to IAA production. IAA is a phytohormone that regulates the development of plant roots by stimulating cell proliferation and elongation (Lwin et al., 2012; Glick, 2014). This hormone helps in the production of longer roots with increased number of root hairs and root laterals which are actively involved in nutrient uptake (Datta and Basu, 2000). Bacillus and Pseudomonas have been reported effective in enhancing root elongation for various plant species such as mung bean, legume plants and cabbage (Patten and Glick, 2002; Ghosh et al., 2003) through IAA secretion.

The biological feasibility assays of bacterial IAA on pepper root growth in the current study have also pointed out that the development of pepper root system stimulated by bacterial IAA is highly influenced by the concentration of synthesized IAA. Dry weight assessments on pepper roots have showed that the bacterial isolates with greater ability in IAA secretion are more efficient in stimulation of pepper root germination. This was confirmed by the treatments of pepper cuttings with Br. gelatini isolate JD04, P. genicula te isolate JS02 and B. territorii isolate MO09, the greatest IAA producers in the current study which have stimulated the production of the highest amount of lateral roots in the treated pepper cuttings, if compared to the other bacterial isolates with lower IAA secretion.

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Nesslerization assays have revealed that all the selected bacterial isolates are effective in production of deaminases. Therefore, these bacteria are able to catalyse liberation of ammonia from the organic detritus that presence in the nature sand. Application of these bacteria in IAA-mediated root growth assays could improve the nitrogen availability in the sowing medium (nature sand) and therefore benefit those pepper cuttings with larger quantity of lateral roots. The results of chlorophyll assessments have also showed that the bacterial isolates with greater abilities in IAA and ammonia production are able to produce higher content of chlorophyll in pepper leaves as nitrogen is an important structural element of chlorophyll (Daughtry et al., 2000; Tucker, 2004).

Therefore, the selected bacterial isolates are potent to be proposed as nitrogen fertilizer in black pepper cultivation. These bacteria are also an effective phosphate solubiliser. Since phosphorus is an active element in nature, it is usually not found as a free element in nature soils. It is commonly exists as an insoluble mineral salts known as phosphate rocks (Miller et al., 2010; Nicholas et al., 2015). The unavailability of phosphorus in nature soil has been recognized as one of the major growth limiting factors in agricultural system (Daniels et al., 2009). With the ability to solubilize phosphate in vitro, these bacteria are also becoming a potential alternative to inorganic phosphate fertilizers in promoting black pepper growth and yield.

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5.4 Assessment on Pepper Defence-Related Genes

Plant disease resistance is usually mediated by gene-for-gene interaction in which plants carry specific R-genes to recognize pathogen avirulence proteins (Dangl and Jones, 2001). The R-gene mediated resistance is typically accompanied by rapid localized cell death at the site of infection called the hypersensitive response (HR). As plants cannot move to escape pathogen attacks, therefore, they have developed sophisticated mechanisms to perceive such attacks and subsequently translate that perception into an adaptive response. The current study has reviewed the recognition-dependent disease resistance response in black pepper plants. The correlation of black pepper R-genes to plant- pathogen interactions (Path: ko04626) pathway and plant MAPK signalling (Path: ko04016) pathway have revealed that the defence response of black pepper plants against invading soil-borne F. solani is highly associated to PTI mechanism. The genes identified by RNA-Seq practically demonstrated the major branches of plant-pathogen interaction pathway. These branches result in activation of cell receptor genes for recognition of microbial elicitors, followed by triggering MAPK signalling genes for cellular signal transduction in response to stimuli and activation of hypersensitive response/localized programmed cell death related genes to combat microbial invasion.

5.4.1 Expression study of targeted pepper defence-related genes

To determine the differences among defence-related genes between P. colubrinum Link and P. nigrum L. after being infected with F. solani, qRT-PCR was employed to study a subset of these genes that played roles in recognition of microbial elicitors (Cf-9 and FLS2), cellular signal transduction (MEKK1 and RGA2) and hypersensitive response or localized programmed cell death (PR1). In order to standardize quantitative expression of the targeted black pepper defence-related genes, identification of suitable reference genes has been carried out in the current study. A total of six frequently used reference genes, i.e. ef1α, actin, GAPDH, UBQ7, H3 and β-tubulin were selected for their expression analysis in black pepper roots and leaves. The sequences of these genes were obtained from transcriptome data by searching through gene descriptions in functional annotation. Specific amplification and expression analysis of these genes were carried

175 | P age out through qRT-PCR. Histone H3 was defined as the most suitable reference gene of black pepper in the current study.

To access the differences of identified R-genes in between susceptible PnSA, PnKch and resistant Pc after being infected with pathogenic F. solani isolate FS010 and beneficial Br. gelatini isolate JD04, qRT-PCR was employed to analyse a subset of defence-related genes (Cf-9, FSL2, MEKK1, PR1 and RGA2) in these two pepper species that played roles in recognition of invader proteins, signal transduction and HR to obtain relative gene expression levels. Majority of the examined genes were induced in the infected plants of both black pepper species, excluded Cf-9. Based on qRT-PCR analysis, the expression of Cf-9 was not detected in susceptible PnSA and PnKch. However, this gene has displayed a promising expression profile in resistant Pc. Furthermore, lower expression levels of MEKK1, PR1 and RGA2 were also observed in the susceptible PnSA and PnKch after F. solani infection.

As Cf-9 was reported as a key receptor in recognition of fungal avirulence proteins (Rivas et al., 2002; Rivas et al., 2004; Chakrabarti et al., 2016), therefore, inactivation of Cf-9 in PnSA and PnKch were believed to prevent the plants from recognition of F. solani at the initial stage of PTI mechanism and subsequently delayed signal transduction in the infected black pepper cells for activation of adaptive response. This was reflected by the low expression levels of examined cellular signal transduction genes (MEKK1 and RGA2) and HR related genes (PR1) were observed in F. solani infected PnSA and PnKch. However, these three genes as well as the FSL2, a LRR receptor protein kinase for recognition of bacterial flagellin in plant cells have shown high levels of expression in both species of pepper plants infected by Br. gelatini. Therefore, inactive of Cf-9 in susceptible PnSA and PnKch could be the reason why P. nigrum L. is susceptible to various species of fungal pathogens. However, no studies have been reported that P. nigrum L. is susceptible to bacterial pathogens. Therefore, in the current study, application of antagonistic plant growth promoting rhizobacteria in black pepper cultivation has been suggested. PGPR is not only play a role as biocontrol agent to suppress the growth soil-borne pathogens, it also act as a synthetic inducer to trigger black pepper PTI mechanism by activating FLS2 in black pepper plant cells during soil-borne fungi invasion (Felix et al., 1999; Asai et al., 2002; Jetiyanon et al., 2003; Zhang et al., 2004; Boudsocq et al., 2010; Beneduzi et al., 2012). 176 | P age

CHAPTER 6

CONCLUSION

The overall research design were intended to achieve three main aims, which are (1) to elucidate the molecular pathways and genes involved in pepper defence-related mechanisms through RNA-Seq; (2) to identify indigenous causal fungal strains of slow decline and (3) to propose alternative solution for slow decline through application of biocontrol strategies. The aims of the research have been achieved.

For the first time, the project had presented transcriptome dataset of two Malaysian black pepper cultivars namely P. nigrum L. cv. Semengok Aman and P. nigrum L. cv. Kuching as well as a wild pepper plant namely P. colubrinum Link generated by using Illumina HiSeqTM 2000 sequencing platform. In total, 81,096 unigenes with a N50 value of 1,081 bp and average length of 1,012 bp were yielded. Of which, 67.46% (54,708) of the assembled unigenes were functionally annotated.

The characteristic of the established transcriptome data was illustrated comprehensively through bioinformatics analysis. The works of R-genes analysis, SSR loci identification and reference genes assessment suggest the availability of the established transcriptome data. The defined DEGs practically demonstrated the major branches of plant-pathogen interaction pathway (Path: ko04626). These branches result in activation of cellular receptor genes, signal transduction genes and pathogenesis-related genes to combat microbial invasion. All these data could provide fundamental reference for further functional genomics studies on other Malaysian black pepper cultivars. In addition, the defined SSR data is a valuable resource for biomarker development in future molecular breeding program of black pepper cultivars.

Besides that, by comparing the susceptible and resistant black pepper plants, qRT-PCR of genes related in black pepper plant-pathogen interactions has led to a conclusion that disease resistance in black pepper plants is accompanied by gene-for-gene interactions. It is believable that inactive of Cf-9 gene has causes the cultivated black pepper plants

177 | P age fail in recognition of soil-borne fungal pathogens and therefore delay in activation of adaptive response. These findings could serve as an important guideline in studying other soil-borne fungal pathogens in black pepper farms.

Identification of causal fungus is essential for proper management of soil-borne diseases in black pepper farms. Two indigenous fungal strains of genus Fusarium designated F. solani isolate FS008 and F. solani isolate FS010 have been identified in the current study. These two fungal strains were diagnosed as the causal fungus of slow decline incidents reported in black pepper disease farms located at Seri kin and Serian areas of Sarawak. Molecular characterization of fungal isolates through homology analysis of ITS region sequences was more effective than morphological characterization in species discrimination of isolated fungi.

Besides that, the current study has proposed biocontrol strategies as alternative solution instead of hazard chemical products in combating slow decline. In total, seven bacterial isolates that potential in restriction of pathogenic soil-borne F. solani and to promote black pepper plant growth have been identified in vitro in the current study. These antagonists are potent to be developed as biocontrol agents due to their capabilities in the production of siderophore and chitinase. Moreover, these bacteria have great potential to be developed as bio -fertilizer as they are an effective ammonia producer and phosphate solubiliser. IAA synthesized by these bacteria was also effective in stimulating root growth and improving nutrient uptake status in black pepper plants.

In order to pursue this research, some potential areas of study that can be recommended as the ways forward of the current study are such as: 1) Genome sequencing and field assessments of identified seven antagonistic PGPR. 2) Development of biological products such as biofungicides or biofertilizers. 3) Development of b iomarkers for black pepper crop improvement program.

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