EXPRESSION PROFILING OF HUANGLONGBING DISEASE IN THROUGH MOLECULAR TECHNIQUES

By

Rozina Aslam

M. Phil. Biochemistry (UAF)

A thesis submitted in partial fulfillment of the requirements for the degree of

Doctor of Philosophy

In

Biochemistry

Department of Biochemistry

Faculty of Sciences

University of Agriculture

Faisalabad, Pakistan

2016

Declaration

I, Rozina Aslam, Regd. No. 92-ag-1367, hereby declare that the contents of the thesis “EXPRESSION PROFILING OF HUANGLONGBING DISEASE IN CITRUS THROUGH MOLECULAR TECHNIQUES” are product of my own research and no part has been copied from any published source (except the references, standard mathematical and genetic models/equations/formulas/protocols etc.). I further declare that this work has not been submitted for award of any other diploma / degree. The university may take action if the information provided is found inaccurate at any stage. In case of any default, the scholar will be proceeded against as per HEC plagiarism policy.

ROZINA ASLAM

92-ag-1367

To

The Controller of Examination

University of Agriculture,

Faisalabad.

"We, the Supervisory Committee, certify that the contents and form of the thesis submitted by Rozina Aslam have been found satisfactory and recommend that it be processed for evaluation by the external Examiner(s) for the award of degree."

SUPERVISORY COMMITTEE:

CHAIRMAN ------

(Prof. Dr. Khalil-ur-Rahman)

MEMBER ------

(Prof. Dr. Muhammad Asghar Bajwa)

MEMBER ------

(Prof. Dr. Iqrar Ahmad Khan)

Dedicated to

Late Father and Mother

May Allah rest their souls in peace

ACKNOWLEDGEMENTS

First of all, immeasurable thanks to Almighty Allah for his mercy and guidance in giving me full strength to complete this task. May Allah’s peace and blessing be upon our beloved prophet Muhammad (PBUH).

I am highly grateful to my supervisor, Prof. Dr. Khalil-ur-Rahman whose expertise, generous guidance and support made it possible for me to complete this task. Without his guidance and persistent help this dissertation would not have been possible.

I would like to thank my committee member Prof. Dr. Muhammad Asghar for his precious guidance and support which was extremely valuable for my study both theoretically and practically.

My deepest gratitude, special thanks and best regards goes to my most respectable committee member, Prof. Dr. Iqrar Ahmad Khan (Vice Chancellor, University of Agriculture, Faisalabad, Pakistan) for his precious supervision and kind guidance, keen interest, prompt inspiration, timely suggestion with kindness, arrangement of all research facilities, scholarly advice and scientific approach which have helped me and made it possible to complete my research project.

I would like to express my gratitude to all my teachers for their advices, sharing knowledge and who put their faith in me and urged me to do better.

I thank profusely to all the faculty and staff, Department of Biochemistry, Centre for Agricultural Biochemistry and Biotechnology (CABB) and Greenhouse, Institute of Horticultural Sciences for their support and cooperation.

I would like to thank my parents and all other family members especially my brother Sher Afgan, sisters and in laws, for their kind help, support, motivation and prayers, all the time to achieve this difficult task. It is my privilege to thank especially my husband for his encouragement, support and help in the lab and field throughout my research period.

I express my deepest thanks to Prof. Dr. Mikeal L. Roose (Chairman, Department of Botany and Sciences, University of California, Riverside (UCR), USA.) and Dr. Claire Thomas Federici (UCR) for their kind support and help for completing molecular studies of my research. I also appreciate the kind support and help of Dr. Glen Hicks, Neerja katiar, John weiger and Clay Clark (IIGB, UCR).

I also appreciate the kind cooperation and support of Makbule koksal and Kimbely Gentile (UCR).

I acknowledge the financial support of HEC (Higher Education Commission), Government of Pakistan, and Pak-US project “Management of citrus greening by producing healthy , monitoring vectors and identification of tolerance” due to which this study became possible. I also appreciate and acknowledge Dr. Richard F. Lee, Dr. Robert Kruger and all staff members of USDA National Clonal Germplasm Repository for Citrus and Dates, Riverside, USA for their help and providing citrus germplasm seed.

Rozina Aslam

TABLE OF CONTENTS

Chapter No. TITLE Page No. ACKNOWLEDGEMENTS i LIST OF ABBREVIATIONS iv TABLE OF CONTENTS v LIST OF TABLES ix LIST OF FIGURES xii ABSTRACT xiv 1 INTRODUCTION 1 1.1 NEED FOR THE PROJECT 3 1.2 OBJECTIVES 5 2 REVIEW OF LITERATURE 6 2.1 Huanglongbing 6

2.2 Huanglongbing occurance in Pakistan 7

2.3 Liberibacter transmission by Asian citrus psyllid 7 2.4 8 Huanglongbing diagnosis 2.4.1 9 Huanglongbing diagnosis by the presence of natural vector 2.4.2 10 Huanglongbing diagnosis on the basis of symptoms 2.4.3 10 Huanglongbing diagnosis by biochemical test 2.4.4 11 Huanglongbing diagnosis by microscopy 2.4.5 11 Huanglongbing diagnosis by molecular techniques 2.5 13 Expression profiling of huanglongbing 3 MATERIALS AND METHODS 17 3.1 EXPERIMENT-1. Rearing of citrus germplasm in greenhouse 17 3.2 EXPERIMENT-2. Asian citrus psyllid rearing 22 3.3 EXPERIMENT-3. Huanglongbing diagnosis in ACP infested 25 plants and inoculation of citrus germplasm 3.3.1 Huanglongbing diagnosis 25 3.3.1.1 DNA extraction 25 3.3.1.2 DNA quatification by agarose gel electrophoresis 26 3.3.1.3 Polymerase chain reactions for HLB diagnosis 27 3.3.1.4 PCR product analysis 28 3.3.2 Inoculation of citrus germplasm 29 3.4 EXPERIMENT-4 Detection of Candidatus Liberibacter 33 asiaticus in citrus germplasm 3.4.1 Sample collection 33 3.4.2 Shipment of DNA to USA and Zymopurification 34 3.4.3 Citrus germplasm DNA quantification by spectrophotometry 35

3.4.4 Real-time PCR for Candidatus Liberibacter asiaticus detection 36 in citrus germplasm 3.4.4.1 Taqman based qPCR protocol for Las detection in citrus 37 germplasm 3.5 EXPERIMENT-5 Expression profiling of huanglongbing 39 disease in citrus 3.5.1 RNA extraction and shipment to USA 39 3.5.2 Synthesis of cDNA using NEB MMLV enzyme 41 3.5.3 Conventional PCR for the detection of internal control gene 42 malate dehydrogenase 3.5.4 Treatment of RNA samples with DNase 43 3.5.5 Qubit quantification of RNA samples 44 3.5.6 Qubit assay for RNA 45 3.5.7 Qubit assay for DNA 46 3.5.8 Purification of citrus germplasm RNA 48 3.5.9 Expression profiling of huanglongbing disease in citrus by real 53 time quantitative PCR 3.5.9.1 SYBR green based quantitative real time PCR 53 3.5.10 RNA-Seq library preparation for Illumina 59

4 RESULTS AND DISCUSSION 61 4.1 EXPERIMENT-1 Rearing of citrus germplasm in greenhouse 61 4.2 EXPERIMENT- 2 Asian citrus psyllid rearing 65 4.3 EXPERIMENT- 3 Huanglongbing diagnosis in ACP infested 68 plants and inoculation of citrus germplasm 4.3.1 Huanglongbing diagnosis 68 4.3.2 Inoculation of citrus germplasm 68 4.4 Experiment 4. Detection of Candidatus Liberibacter asiaticus 74 in citrus germplasm 4.4.1 Correlation between HLB symptoms and Ct values 75 4.5 Experiment 5. Expression Profiling of Huanglongbing Disease 81 in Citrus Germplasm by real time quantitative PCR 4.6 Assessment of RNA-Seq library quality on bioanalyzer 96 5 SUMMARY 98 6 RECOMMENDATIONS 100 REFERENCES 101

LIST OF TABLES

Table No. Title Page No. 3.1 Citrus germplasm sown for the detection and expression 18 profiling of huanglongbing. 3.2 Nutrients composition applied to the germplasm 22 3.3 Composition of CTAB buffer for DNA extraction 26 3.4 The primers sequence for HLB diagnosis by conventional 27 PCR studies 3.5 Composition of reaction mixture for conventional PCR 28 3.6 Thermal profile for conventional PCR 28 3.7 Composition of 5 X TBE buffer for one liter solution 28 3.8 Composition of 50X TAE buffer for one liter solution 29 3.9 DNA clean and concentrator contents 34 3.10 The primer/probe sequence for Las detection by qPCR studies 37 3.11 Reaction mixture for Taqman based qPCR 38 3.12 Composition of RNA extraction buffer 40 3.13 Nucleic acid concentrations for initial evaluation of RNA by 41 nanodrop 3.14 Primers and dNTPs cocktail for cDNA 41 3.15 Reverse transcription (RT) reaction cocktail 42 3.16 Composition of PCR reaction mix for MDH gene 42 3.17 PCR Conditions for MDH gene 43 3.18 Specification of Qubit assay for RNA and DNA 45 3.19 Qubit assay values for RNA and DNA 46 3.20 Comparison of RNA concentrations checked by nanodrop and 48 bioanalyzer along with RIN values 3.21 RNA clean and concentrator contents 48 3.22 Nanodrop concentrations of RNA after purification 49 3.23 Composition of TE buffer for primer dissolving 54 3.24 Thermal conditions for SYBR green based PCR reactions 55 3.25 List of primers used for expression profiling of HLB in citrus 55 by qPCR. 3.26 Citrus germplasm tested for expression profiling of HLB 57 4.1 Survived citrus germplasm from 97sown genotypes of citrus 62 4.2 Citrus genotypes expressing huanglongbing symptoms 70 4.3 Mean cycle threshold values in citrus germplasm DNA for the 79 detection of Candidatus Liberibacter asiaticus 4.4 Comparison of expression of 7 genes in 45 genotypes of HLB 82 infected citrus using qRT PCR analysis 4.5 Sample information for cDNA library concentrations by 96 nanodrop and index primer sequences

LIST OF FIGURES

Fig. No. Title Page No. 3.1 Greyish black polythene bags having a size of 15x9 inch 18 used for citrus germplasm seed sowing 3.2 Sweet plants in field with a huge population of ACP 23 adults 3.3 Sweet orange leaf samples with HLB symptoms from field 24 trees for molecular detection of Candidatus Liberibacter asiaticus 3.4 Sweet orange and plants in growth room 25 3.5 Sweet orange leaf sample with blotchy mottle and vein 26 yellowing symptom after infestation of ACP in growth room conditions for HLB pathogen detection 3.6 Healthy control plants of citrus (A to F) in greenhouse 31 3.7 Citrus germplasm replicates for inoculation of HLB 32 bacterium (A through D) 3.8 Few genotypes of citrus out of 51 showing graft/midrib 33 inoculation 3.9 DNA precipitated in isopropanol for storage and shipment to 40 USA 3.10 Thermal profile for real-time PCR detection of HLB in 40 plants using iQ5 3.11 Total RNA samples extracted from Carrizo, Corsican, Indian 45 and Troyer, with and without DNase treatment 3.12 Total RNA electrophoresis file run summary produced on 47 Agilent 2100 bioanalyzer of already DNase treated and not treated samples 4.1 Groups of citrus presenting total number of sown 64 and survived 4.2 64 Germination of citrus germplasm seedlings 4.3 Colonies of ACP on Sweet orange and Kinnow plants in 66 growthroom conditions 4.4 Appearance of blotchy mottle, vein yellowing and leaf 67 curling symptoms 4.5 Successful rearing of ACP resulting in establishment of ACP 67 colonies 4.6 DNA extracted from healthy controls and HLB infected 69 genotypes of citrus 4.7 Amplification of16S&23S intergenic region showing 69 800bpamplicon specific for Candidatus Liberibacter asiaticus 4.8 Candidatus Liberibacter asiaticus detection using OI1/OI2C 70 primer in sweet orange plants infested by ACP 4.9 Candidatus Liberibacter asiaticus detection using A2/J5 70 primer in sweet orange plants infested by ACP 71 4.10 Bars showing huanglongbing symptoms expression in citrus germplasm 4.11 Inoculated citrus plants in screenhouse showing blotchy 72 mottle and vein yellowing symptoms of HLB 4.12 HLB positive citrus genotypes with a range of Ct values 76 from 36.9-20 and their number in that range presenting the titer of Candidatus Liberibacter asiaticus on the basis of Ct values 4.13 Ct values of 51 genotypes of graft inoculated citrus 76 germplasm 4.14 Candidatus Liberibacter asiaticus amplification plot showing 78 Las detection in citrus plants 4.15 Plant’s cytochrome oxidase gene amplification plot in citrus 78 plants 4.16 Correlation between HLB symptoms and Ct values of citrus 80 germplasm 4.17 Relative expression of sulfate transferase (CsSULF) in 84 healthy and inoculated citrus germplasm 4.18 Relative expression of glusose 1 phosphate adenyl 86 transferase (CsSB1) in healthy and inoculated citrus germplasm 4.19 Relative expression of granule bound starch synthase 87 (CsSB2) in healthy and inoculated citrus germplasm 4.20 Relative expression of alpha amylase-1(CsSD1) in healthy 88 and inoculated citrus germplasm

4.21 Relative expression of alpha amylase-3 (CsSD2) 90 4.22 Relative expression of beta amylase 9 (CsSD3) 91 4.23 Relative expression of cytochrome P450 monooxygenase 92 (CsSUR2) 4.24 Gene expression of seven genes presenting fold change in 93 forty five genotypes of citrus. 4.25 Bioanalyzer results of RNA-seq libraries 97

ABSTRACT

Huanglongbing (HLB), also called greening, is a fatal disease of citrus. Candidatus Liberibacter, a Gram negative and non culturable bacterium, is the pathogen of huanglongbing. It becomes difficult to detect this bacterium because it is unevenly distributed in its citrus hosts.The bacteria are transmitted naturally to citrus by an insect vector called citrus psyllid. Hussain and Nath documented the occurrence of HLB and its related vector Diaphorina citri Kuwayama in Pakistan in 1927.In this study, 97 accessions of citrus germplasm were raised in greenhouse. of 97 accessions, 51 were survived. The seed of citrus germplasm was obtained from National Clonal Germplasm Repository for Citrus and Dates, Riverside, California, USA and University of Agriculture Faisalabad (UAF), Pakistan. For HLB diagnosis in the leaf samples of sweet orange (Citrus sinensis) Succari from field, conventional polymerase chain reaction technique was employed. Asian citrus psyllid (ACP) was captured from HLB positive sweet orange field trees and released on healthy plants of sweet orange and kinnow for rearing and infestation in the controlled conditions of growth room. Those plants were also tested for their HLB positivity by using 16s rDNA primers OI1/OI2c and Candidatus Liberibacter asiaticus specific primer A2/J5. After confirmation of HLB, sweet orange plants infested by ACP in growth room, were usedfor the inoculation of fifty one genotypes of citrus and citrus relatives. Candidatus Liberibacter asiaticus was detected in 96.07% of the inoculated citrus germplasm by Taqman based real time PCR using primers set HLBasfpr.SYBR green based real time qPCR was performed to differentiate expression of genes in HLB infected and healthy leaf samples of forty five genotypes of citrus and its relatives. Seven genes, includingsulfate transferase (CsSULF), glucose-1-phosphate adenyl transferase (CsSB1), granule bound starch synthase (CsSB2), alpha amylase (CsSD1), alpha amylase 3 (CsSD2), beta amylase9 (CsSD3) and cytochrome P450 mono oxygenase 83B1(CsSUR2) were tested for expression profiling. Gene expression data analysis for relative quantity represented the down regulation ofcarbohydrate metabolism related genes (CsSB1and CsSD3) in Clausena harmandiana while transporter gene CsSULF expressed at equal level in healthy and diseased Glycosmis pentaphylla. Remaining genes did not express in both of the varieties. These two genotypes may be placed in tolerant category against HLB as the causal bacterium was not detected in them. In Tahitian and Schuab, no gene was amplified except CsSULF gene that was down regulated indicating strong root system for plant survival. CHAPTER- 1

INTRODUCTION

Huanglongbing (HLB) is the most serious infectious disease of citrus and major threat to citrus industry (Teixeira et al., 2005a ; Hall et al., 2012; Volk and Suszkiw, 2013). It is the main cause of decrease in in major citrus growing regions of Asia and Africa. In regions, where HLB is endemic, citrus trees produce unmarketable fruit as it abscise prematurely and mostly die within 5 to 8 years (Roistacher, 1991; Baldwin et al., 2010). Due to severity of HLB in the groves of the Yang-Cun Oversea Citrus Research Institute in Guang Dong, China, 378000 trees, infected with HLB pathogen, were uprooted during 1993 and 1995 (Zhuang, 2003). It has caused a loss of about 100 million citrus plants in African and Asian countries (Timmer et al., 2003; Gottwald et al., 2007; Nageswara-Rao et al., 2013). Among the citrus growing countries of the world, only Australia and the Mediterranean basin are found to be HLB free yet (Finlay et al., 2009).

Candidatus Liberibacter, a Gram negative, non culturable and phloem limited bacterium is the causal organism ofHLB.This bacterium is placed in genus alpha-Proteobacteria(Jagoueix et al., 1994; Li et al., 2009). This bacterium was defined and named as Candidatus (non culturable bacteria) Liberibacter by Murray and Schleifer in1994. There are three types of this bacterium: Candidatus Liberibacter asiaticus (Ca. Las), Candidatus Liberibacter africanus (Ca. Laf)(Da Graca, 1991) and Candidatus Liberibacter americanus(Ca. Lam) (Teixeira et al., 2005b). Candidatus Liberibacter asiaticus is heat tolerant and express symptoms in both cool and warm (22 to 32°C) environment whereas the African form,Candidatus Liberibacter africanus is heat sensitive that develops at temperatures ranging from 22 to 25°C.

The natural vector of the pathogen is citrus psyllid. There are two species of psyllid vector:Diaphorina citri Kuwayama(Hemiptera: Sternorryncha: Lividae) and Trioza erytreae (del Guercio)(Hemiptera: Sternorrynca: Triozidae) reported for citrus greening(Aubert, 1987).Two more psyllid vectors: Diaphorina communis Mathur (Donovan et al., 2012) and Cacopsylla (Psylla) citrisuga have been confirmed as a carrier of Candidatus Liberibacter asiaticus‟ by conventional and real-time PCR (Cen et al., 2012). However, transmission studies are underway to determine whether Las-positive psyllids can transmit Las to healthy citrus. The Diaphorina

1 citri Kuwayama (D. citri) is a natural vector of both Candidatus Liberibacter asiaticus (Las) and Candidatus Liberibacter americanus (Lam), while Trioza erytreae(T. erytreae) is the vector of Candidatus Liberibacter africanus (Laf). Among huanglonbing infected trees, Las is the most abundant species (Jagoueix et al., 1994; Teixeira et al., 2005b; Bove, 2006). HLB can also be transmitted through budding/grafting of infected budwood (Lin, 1956; Lopes et al., 2009b; Zhang et al., 2012).

HLB symptoms on leaves are just like zinc deficiency and may cause confusion. Disease symptoms do not appear in the host plant immediately after infestation. It can take years the time between infection and symptom appearance (Timmer et al., 2003; Chin et al., 2014). The leaf symptoms similarity with zinc and other nutrients create problem in HLB detection and demands molecular techniques for analysis. The most common symptoms of HLB are blotchy mottling and vein yellowing along with vein corking. Sometimes, at initial stages of infection, the above mentioned symptoms are observed only on one part of the canopy, therefore the name of the disease in China is huang-long-bing means yellow shoot disease (Garnier and Bove, 1993). Fruit yield reduces as a result of HLB infection. Fruit of small size with lopside grows on the diseased plant. HLB affected fruit do not color properly and therefore the name greening is given to the disease in South Africa. Quality deteriorated fruit falls at premature stage due to ripening from the peduncular end, and often aborted seeds are seen in the diseased fruit. Infected leaves become small and upright, followed by leaf drop and dieback. Early flowering in HLB affected plants has also been observed (Albrecht and Bowman, 2008; Martinelli et al., 2012).

Huanglongbing was found to be present in north-eastern and north-western India in the 1800s and early 1900s. A severe damage caused by populations of Diaphorina citri at Sargodha from 1915 to 1920 has been reported in detail (Husain and Nath, 1927; Gottwald et al., 2007). Thus, the natural vector of huanglongbing pathogen, Diaphorina citri Kuwayama has been present in Pakistan (Indo-Pak subcontinent) for 100 years. HLB has been known in China for more than 100 years but reported first time in 1919 by Reinking (Reinking, 1919). Interviews with technicians and farmers of China from 1947 to 1955 revealed the presence of huanglongbing in China in the 1800s (Lin, 1956) but very likely originated in Taiwan in 1870 (Bove, 2006). Later, in South Africa, HLB symptoms were reported on citrus in 1937 and called as greening disease (Van de Merwe and Andersen, 1937). Huanglongbing is widely present

2 throughout the Southern and Eastern Africa, Southwestern part of the Saudi Arabian Peninsula and from Pakistan to China in Asia (Garnier and Bove,1993). For successful management of HLB, a powerful observation of disease epidemic and rapid and accurate detection of pathogen is very important but, the detection of the pathogen is very difficult because of its low titer and uneven distribution in its citrus hosts (Planet et al., 1995; Garnier et al., 2000; Bove, 2006; Li et al., 2007). The invention of prize winning improvements to the polymerase chain reaction (PCR) technique of Kary Banks Mullis in 1985 (Saiki et al., 1985; Mullis et al., 1986; Mullis and Faloona, 1987) has made the detection of HLB bacterium possible and easy at the early stages of infection. Different methods are being used to control the vector and pathogen of HLB disease. Control of psyllid vector by the application of systemic and topical insecticides have found to be ineffective (Ichinose et al., 2010). All varieties of commercially cultivated citrus are susceptible to HLB. The tolerant varieties against huanglongbing are not yet commonly available (Grafton et al., 2013).

It is very important to understand the citrus host response to pathogen for the development of HLB management strategies. Various studies have been conducted to identify genes and proteins in response of HLB pathogen in leaves, juice vesicles and fruit peel (Albrecht and Bowman, 2008; Kim et al., 2009; Martinelli et al., 2012; Martinelli et al., 2015). Studies on gene expression changes revealed a number of different processes like, photosynthesis, carbohydrate metabolism, cell defense and transport. The first study of transcriptome profiling in leaves in response to Ca. L. asiaticus using microarray technique was reported in 2008 by Albrecht and Bowman. For gene expression/transcriptome profiling of HLB in citrus, along with microarray and qRTPCR, a high throughput sequencing technique known as RNA- Seq is also being used (Martinelli et al., 2012; Martinelli et al., 2015).

1.1 NEED FOR THE PROJECT

Citrus is very important fruit of Pakistan that remains in the market throughout the year. Citrus fruits are used in fresh as well as juiceform around the world. Citrus juice industry is the number one among juice industries of the world. Source of income for millions of people depends upon citrus industry (Khan, 2007). Citrus fruits contain a vareity of vitamins, minerals and bioactive compounds beneficial for human beings. Vitamin Cprevents coronary heart diseases, cancers and cataract(Weber et al., 1996; Kaur and Kaur, 2015).

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Citrus production of the world is 122976 thousand tones. Pakistan ranks at the 13th position in the world with 2150 thousand tones production from an area of 198 thousand hectare. Average yield in Pakistan is approximately 11 tons per hectare; about 36% of per hectare production in the USA (Anonymous, 2013). Per hectare yield of citrus in Pakistan is lower as compared to the majority of countries of the world due to many reasons, HLB is one of them. In Florida, USA, HLB was reported in 2005. Up to 2014, approximately 33% revenue of the total production of the state has been wasted due to HLB and more than 8000 persons associated with citrus industry lost their jobs (Chin et al., 2014). About 100 million citrus trees have been destroyed in African and Asian countries due to HLB (Gottwald et al., 2007; Nageswara-Roa et al., 2013). It is suggested to use enhanced nutritional program (ENP) for the maintenance of citrus tree health, fruit yield and quality of Candidatus L. asiaticus infected trees, but, in commercial trials conducted in Florida, consisting of application of micronutrients and metallic ions on HLB affected trees resulted in uncontrolled disease dynamics and pathogen titer. It has also been observed that the existing ENP methods resulted in increased inoculum of HLB bacterium and disease spread in the citrus orchards (Gottwald et al., 2012; Johnson et al., 2014).

In Pakistan, HLB has been ignored unintentionally. Due to ignorance, uprooting of citrus trees has been a major practice in Pakistan without knowing the actual cause of decline. In these situations, a detailed study of HLB and its natural vector was truly needed so as to assess the citrus germplasm for gene expression changes in response of HLB to manage the disease. Further, the present research was optimistically designed to seek knowledge of modern and sophisticated techniques for HLB diagnosis. Moreover, ACP rearing in controlled conditions of growthroom was aimed for HLB transmission in sweet orange and kinnow plants and use of those plants for inoculation of citrus germplasm. The fundamental goal of this research is to help protect citrus plants. Use of latest technology for disease detection is required to manage HLB and save the citrus in Pakistan.

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1.2 OBJECTIVES

This study was aimed to analyse citrus germplasm for HLB pathogen detection and gene expression changes in response of HLB infection. The main objectives of this research are summarized as under:

1. Citrus germplasmrearing and inoculation in greenhouse conditions

2. Molecular detection of citrus greening (Huanglongbing)

3. Expression profiling of huanglongbing in citrus through molecular techniques

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CHAPTER 2

REVIEW OF LITERATURE

2.1. Huanglongbing

Huanglongbing (HLB), also called citrus greening, is a fatalillness of citrus. The disease is transmitted by jumping lice called citrus psyllid. There are two types of psyllid vectors confirmed for huanglongbing.One of the two psyllid vectors is D. citri Kuwayama(Hemiptera: Sternorryncha: Lividae) and the other is T. erytreae (del Guercio)(Hemiptera: Sternorrynca: Triozidae) (Aubert, 1987). Candidatus Liberibacter, a gram negative, non culturable, phloem resident bacterium is the pathogen of the disease. It has 25 nm thick cell wall. Three species of this bacterium have found to be the cause of huanglongbing.One is Ca. L. asiaticus found in Asia and Americas, second isCa. L. africanus found in Africa (Da Graca, 1991; Koizumi, 1995) and third is Candidatus Liberibacter americanus found only in America. Candidatus Liberibacter asiaticus can tolerate heat and express symptoms in cool as well as warm (22 to 35°C) ambience while Candidatus Liberibacter africanus is sensitive for heat anddo not show symptoms above 25°C (Garnier et al., 2000; Teixeira et al., 2005a; Bove, 2006; Li et al., 2009). Diaphorina citrialso called Asian citrus psyllid (ACP) can transmit Liberibacter asiaticus (Las) and Liberibacter americanus (Lam) in citrus and citrus relatives while, Trioza erytreae is the carrier and transmitter of Candidatus Liberibacter africanus (Laf) only. Among huanglonbing infected trees, Las is the most abundant species(Teixeira et al., 2005b; Bove, 2006;Lin et al., 2015). It has been proposed by Beattie et al. (2006) that HLB may have emerged from Africa in a plant, like Vepris lanceolata. The citrus may be infected by an insect after sucking sap from Vepris and then shifted to India in the form of budwood or whole plant 300 to 500 years before. Later, it would be shifted to China by some means. Perhaps this may be the reason, that HLB was reported in China later than India despite the cultivation of citrus in China for thousands of year (Gottwald et al., 2007). In a study to find out phylogenetic link between Indian and Asian strains of HLB bacterium, it was detected in samples collected from Northeast India, East Timor and Papua. Phylogenetic examination with 16S ribosomal DNA and SNP of outer membrane protein gene disclosed more genetic closeness of Northeast Indian strain with Asian strains from Taiwan,

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Japan and Vietnam apart from Indian strains. It was further concluded in this study that Poona strain was not suitable for genetic reference for Liberibacter asiaticus (Miyata et al., 2011).

The complete genomes of all three bacterial species responsible for HLB have been sequenced those are: 1.23Mbp for Ca. L. asiaticus (Duan et al., 2009), 1.195201 Mbp for Ca.L. americanus (Wulff et al., 2014) and 1.192232 Mbp for Ca. L. africanus (Lin et al., 2015).

2.2 Huanglongbing occurrence in Pakistan

During 18th century, citrus decline was a severe trouble in Indo Pak landmass (Asana, 1958; Capoor, 1963). HLB disease was known as citrus decline in the early 1900s. In 1909, presence of large number of psyllid vector D.citri on citrus was reported in India (Crawford, 1919). Anintense destruction caused byDiaphorina citriin citrus groves of Sargodha between 1915 and 1920 was reported by Husain and Nath (1927). In Pakistan, suspected presence of greening (HLB) has been reported in many publications on the basis of visual symptoms (Cochran, 1976; Akhtar and Ahmad, 1999; Abbas et al., 2005). Chohan et al. (2007)first time confirmed the presence of HLB associated bacterium in psyllid and plant host at molecular level in Peshawar province of Pakistan. Later, inPunjab, HLB was first time diagnosed in Faisalabad district in 2011 at the University of Agriculture Faisalabad,Pakistan by polymerase chain reaction for the management of this disease (unpublished). For population dynamic studies of Asian citrus psyllid (D. citri Kuwayama), from Punjab province of Pakistan, molecular detection of HLB associated bacterium in citrus and natural vector D. citri.has also been reported. In that study, presence of HLB pathogen was confirmed by real time PCR at USDA-ARS, Riverside, California, USA (Razi et al., 2014).

2.3. Liberibacter transmission by Asian citrus psyllid For testing the feeding behaviour and the transmission of Liberibacter by D. citri, a technique was used that is known as electrical penetration graphing (EPG).In this technique, an electricity connection was established between D. citri and the plant which was to be tested.Circuit closing took place when D.citri inserted the stylet in the leaf tissue of citrus and the amplified signal changes which take place, were saved by the recorder.The waveforms of signals were in response to the stylet contact, penetration, and ingestion of xylem and phloem sap by D.citri. The stylet reached to the phloem in average of 158 minutes time. When for 8 hour, data was

7 recorded, average time for phloem ingestion was found 206.1 minutes.The analysis of EPG showed that up to 96 hours exposure, higher number of insects (80%) succeeded to reach the phloem on younger tissues as compare to older tissues, on which the success rate was only 20%.The ACP acquireCandidatus Liberibacter asiaticus 54% from young leaves, whereas lower number (only 10%) from older leaves.Regarding acquisition and the transmission, nymphs were found more efficient as compare to adults (McClean and Kinsey, 1964; Tjallingii, 1988). The acquisition efficiency and feeding behaviour of D.citri is changed by stage of citrus leaf development. Young leaves were preferred by all age group of insects.There are higher chances of acquisition of bacteria when D. citri complete most of its life span (from nymph to adult) on diseased branch as compare to those that ingest sap from diseased branch when become adult (Lopes et al., 2009). The Las transmission between male and female D.citri has been investigated. It was reported that male insects transmit Las sexually to females only. A 7 dayslatent period was required for detection of Las in recipient female. Las was found in both sexes genitalia and also in eggs. It was found that Las can spread within D.citri population horizontally without presence of infected plants (Mann et al., 2011). Trees infected with HLB bacterium look more attractive to adult ACP before sucking their sap. After feeding on infected trees, ACP are attracted towards healthy trees causing the dissemination of HLB bacterium. Adults need 1-25 days after acquiring Las before transmitting it to healthy trees but the adult infected with Las at nymph stage can transmit Las immediately after becoming adult (Mann et al., 2012; Hall et al., 2012). More than eighty genotypes of citrus have been studied for the colonization of ACP, only Poncirus and some of its hybrids have been found least favourite for ACP (Westbrook et al., 2011; Albrecht and Bowman, 2012; Richardson and Hall, 2013 ).

2.4. Huanglongbingdiagnosis

For huanglongbing diagnosis, different parameters have been used including:monitoring of natural vector of HLB in citrus groves, observation of huanglongbing symptoms on citrus leaves and fruits, biochemical tests for the assessment of presence of HLB pathogen, microscopic identification of HLB pathogen and molecular detection of HLB pathogen.

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2.4.1. Huanglongbingdiagnosis by the presence of natural vector Citrus psyllid is the natural vector of HLB causing bacterium. Presence of Asian citrus psyllid (ACP) in a citrus orchard may be an indication of huanglongbing suspected plants in that orchard. Monitoring of ACP is considered as an essential part of management program aimed at minimizing the occurrence and propagation of HLB. Huanglongbing occurs after a long interval of inoculation, in this way HLB bacterium can increase in number before its detection. In the presence of new leaves, females lay eggs regularly in their life. A large number of eggs may be seen on a single new flush. Generally 500-800 eggs are layed in two months reaching at maximum number of 1900 eggs. Freshly layed eggs are light yellow and oval. At maturity egg color turns orange having two red spots for eyes. ACP has five stages of nymph or instars (Husain and Nath, 1927). A population dynamics study of ACP on Kinnow mandarin, Feutrell‟s early and Musambi in Sargodha, Pakistan, reveals ACP population to be highest in the months of April and August (Ahmadet al., 2004). According to a publication in scientific notes by Halbert and Nunez (2004), D.citri is a local inhabitant of Asia. It was familiar in Brazil for many dacades. During 1990s it has grown in the countries of northern South America and the Caribbean. It was discovered in south Florida and Gaudeloupe in 1998 and since then it is spreading quickly in the Caribbean basin. It has been known that Las carrying female psyllids are not responsible for the dissemination of Las through their eggs because eggs and first and second instars were found to be free of Las (Hung et al., 2004). An egg develops in to adult from 14 days at 29 °C to 49 days at 15°C. Fresh adults become reproductive within 2-3 days and a female can lay eggs after 1-2 days of mating. ACP can increase its population in 20 to 22 days at 25 °C (Liu and Tsai, 2000). Untill 2004, no report of ACP presence heared in Oman. On Mexican ACP was found to be present at Barka in 2005. Since then, ACP presence was recorded in most of the citrus groves of Oman especially three areas including Barka, Al-Rustaq and Masirat Al Rawajeh. Haunglongbing may be soon detected in the ACP containing areas of Oman as it has already been detected in the Saudi Arabia (Al-Zadjali et al., 2008). Adult psyllids are 2.7 to 3.3 mm long. The color of wings is brown with three different colors of abdomen. The ratio of male and female psyllids is probably equal. Adults can be seen on citrus leaves or branches sitting at the angle of 45ᴼ (Wenninger et al., 2009).

9

Australia is still free from HLB pathogen and its psyllid vector but there are strong chances of spread of HLB because of its diagnosis in the neighbours of Australia including New Guinea, Indonesia and Timor Leste (Finlay et al., 2009). Temperatures between 16 and 41.6 °C are good for oviposition but 29.6 °C is ideal (Hall et al., 2012). Records of average life of adult males and females are found to be variable ranging from 32 to 51 days at 24 to 30°C for males and 90 days at 27°C for virgin females on suitable host plants (Richardson & Hall, 2013). The ACP was discovered in Brazil in 1940s, then in Florida in late 1990 and now it has been detected in a lot of citrus growing states of America, mexico, Belize, Costa Rica and California (Grafton et al., 2013)

2.4.2. Huanglongbing diagnosis on the basis of symptoms Symptoms of HLB on leaves are similar to zinc deficiency (Timmer et al., 2003). For the appearance of disease symptoms in the host plant long period is required after infestation.Different genotypes of citrus express huanglongbing symptoms at different spans but generally symptoms on new growth can be visualized after 16-20 weeks of infection. Blotchy mottle symptoms can be seen on but not clear on . express leaf curling and prominent vein corking symptoms. All symptomatic leaves ultimately defoliate after few weeks to month (Zhang et al., 2012).

2.4.3. Huanglongbing diagnosis by biochemical test

A biochemical test based on starch reaction has been successfully practiced for HLB diagnosis. It is very simple, easy and economical. The level of starch increases upto six times in HLB affected leaves than healthy ones. The results of this iodostarch test have found to be 90 to 95% correct when compared with PCR results (Taba et al., 2006; Etxeberria et al., 2007). A combination of photochemical and non photochemical quenching and chlorophyll fluorescence parameters proved to be efficient for advanced diagnosis of HLB in asymptomatic trees, (Sagaram and Burns, 2009).

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2.4.4. Huanglongbingdiagnosis by microscopy

Judgement of the primary changes in HLB progression is vital for its control. By the use of light microscopy, HLB bacterium has been detected in leaf midrib, small fuits and its different parts (Schneider, 1981).Detection of Las in leaf midribs, roots and inner side of bark from infected citrus plants indicate that HLB bacterium is phloem limited and move through sieve tube system (Lopes et al., 2007). In a cytopathological study, electron microscopy was performed for citrus trees inoculated by HLB positive grafts under greenhouse conditions. For characterization of the initial changes during infection, the connection between ultrastructural modifications and symptom expression was determined. During those observations, the first change that occurred after pathogen attack was found to be swelling of middle lamella in new flushes without any symptom. Transmission electron microscopy also revealed the presence of large number of HLB pathogen in sieve tubes of phloem of asymptomatic new flushes. Amazingly no bacteria were observed in strongly symptomatic leaf samples during microscopic analysis indicating the probability that, at symptomatic stage of HLB, large portion of pathogen may be dead or nonviable (Folimonova and Achor, 2010). In a first study for Las detection by hybridization technique FISH, different organs of ACP and phloem of citrus leaves were used. Microscopic observations revealed scattered individual Las in phloem and haemolymph tissues of leaf and ACP respectively. In other organs, like midgut, salivary glands, ovaries, malpighian tubules and muscle tissues, Las was present in great amount (Ammar et al., 2011).

2.4.5. Huanglongbing diagnosis by molecular techniques Use of DNA probes for the detection of asiaticus species of HLB pathogen was first reported by Villechanoux et al. in 1992. A PCR technique was used by Jagoueix et al. (1996) for the detection of Ca. L. asiaticus and Candidatus Liberibacter africanus. In this method, 1160 bp amplicon of 16S rDNA digested by the use of Xbal produce three fragments of 130 nt, 506 nt and 520 nt from Ca. L. africanus and two pieces of 520 nt and 640 nt from Ca. L. asiaticus. Cloning and sequencing results of 16S and 23S intergenic segments of rDNA from Liberibacter asiaticus species isolated from samples taken from China and India and Liberibacter africanus species from Africa were found to be ~80% homologous (Jagoueix et al., 1997).

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For the simplicity of detection by PCR, rpl/kAJL- rpoBC associated genes of HLB pathogen were amplified. The primer set A2/J5 was designed in this method that confirms direct identification of Las andLaf (Hocquellet et al., 1999). By using PCR assay, Las has been detected in single adult and third to fifth instar of psyllid (Hung et al., 2004). The primer set A2/J5 has been used vastly with different and same name for the detection of HLB pathogen round the world (Ruangwong and Akarapisan, 2006; Chohan et al., 2007). Real time PCR is highly sensitive technique that is being used frequently for HLB diagnosis all over the world. Li et al. (2006) developed fluorescent labelled primer sets for HLB pathogen detection in citrus and insect vector as well as internal control gene primer for plant cytochrome oxidase. TaqMan qPCR is hundred times responsive than LAMP and standard PCR. It can be used to detect bacterium before the symptom appearance of HLB (Crosslin et al., 2006; Li et al., 2007). In the NWF province of Pakistan, HLB has been diagnosed by PCR technique using A2/J5 set of primers in psyllids and citrus leaves (Chohan et al., 2007).

Proper sampling of infected leaves is mendatory for HLB bacterium diagnosis. Although real time PCR technique can detect even ten bacteria from one gram of leaf sample, could not detect Las in non symptomatic leaves while symptomatic leaves of the same tree revealed 107 liberibacters per gram of leaf tissue in Sao Paulo, Brazil. These results represent the unequal presence of HLB pathogen in phloem (Teixira et al., 2008).

An average of 1010 „Ca. L. asiaticus‟ genomes in tissues per gram, were found due to natural infection in aboveground part of the plants. Same levels of Las genomes were observed in few root samples also but not in all. In samples inoculated in greenhouse Las genomes were found 104 when taken from site of inoculation and 1010 genomes per gram from few leaves. The samples taken from roots of these plants contain 107 genomes per gram. Tissues from symptomatic fruits were tested and Ca. L. asiaticus‟ genomes were easily detected (Li et al.,2009). Discovery of HLB pathogen by duplex PCR is cheaper and dependable comparing singleplex PCR (Donnua et al., 2012).The most common way of HLB pathogen detection round the world is DNA extraction from leaf midribs of diseased citrus plants confirming by PCR. As the taste of juice from HLB infected fruit turns bad, a method of HLB pathogen detection from fruit juice was optimized. Sonicator was used for homogenization instead of pestle and

12 mortarduring DNA extraction from juice.To remove pectin and PCR inhibitors, pectinase and elution columns were used that resulted in good amplification of DNA (Bai et al., 2013).

The PCR technique is more responsive and dependable than electron microscopy, ELISA and DNA hybridization for the detection of HLB bacterium. A buffer containing sodium sulphite produce good quality DNA as compared to commonly used DNA extraction buffers (Mahajan et al., 2013; Jagtap et al., 2013). The DNA of HLB bacterium can be isolated rapidly by pulverization and centrifugation method from leaf midrib. Direct PCR method when used to test the efficiency of rapidly extracted DNA, its performance was near to the standard PCR. Direct PCR was found to be quite simple for quarantine test (Fujikawa et al., 2013). From fifty primer pairs designed for HLB bacterium gene amplification, thirty two primer pairs successfully amplified Las DNA isolated from plants and insect vectors of HLB pathogen (Nageswara-Rao et al., 2013).

2.5Expressionprofiling of huanglongbing

In HLB infected fruit, the abscission zone at the pedicel end becomes orange in color and fruit falls prematurely. Aborted seeds with dark color are mostly found in HLB symptomatic fruit (McClean and Schwartz, 1970; Da Graca, 1991). Genes that encode p-proteins increase in response of HLB. These proteins are important for the maintenance of sieve plate turgor pressure and callose formation that deposit in sieve tubes and blocks transport of photosynthates in HLB infected phloem tissues (Knoblauch et al., 2001). For gene expression profiling, different methods like: serial analysis of gene expression, microarrays, massive parallel signature sequencing and spotted arrays have been frequently used (Pollock, 2002).

HLB symptoms have been documented in detail. Leaf symptoms in HLB infected tree include blotchy mottle, vein yellowing, vein corking, small upright leaf, yellow shoot and premature drop of symptomatic leaves at the laminar or abscission zones. Gene expression changes in HLB infected fruit include small sized lopsided fruit and fruit ripening from the peduncular end instead of styler end. Citrus tree dies from several months to years after HLB infection (Bove, 2006). HLB also destroys the juice quality and incorporate bitter elements in citrus juice (Stokstad, 2006). In the first study of transcriptional profiling in citrus in response to Ca. 13 liberibacter infection using microarray technique, gene expression changes associated with various processes including transport, photosynthesis, cell defense, cellular organization and carbohydrate metabolism have been reported (Albrecht and Bowman, 2008). Due to vascular blockage, starch accumulation occurs in HLB symptomatic leaf tissue (Etxeberria et al., 2009). A reaction of sweet orange to Ca. L. asiaticus attack with microarray analysis indicated the up-regulation of starch synthesizing genes incorporating ADP-glucose pyrophosphorylase, granule bound starch synthase and starch debranching enzyme. These genes are responsible for starch accumulation in HLB infected leaves (Kim et al., 2009). The growth of fruit also affected by HLB infection, like: size become small, fruit become lopsided, color not developed properly, seeds of fruits aborted. Generally, fruit maturation delayed and premature abscission takes place. The changes in phytohormone and carbohydrate were confirmed by PCR to describe the expression of symptoms in sweet orange cultivar Valencia. Due to poor fruit growth, carbohydrate shortage takes place. Fruit setting and development is partially regulated by hormonal signal.A comparison of healthy asymptomatic fruit with symptomatic was made. In symptomatic fruit less amount of ethylene and more indole- 3-acetic acid (IAA) and abscisic acid (ABA) was found than asymptomatic fruit flavedo, that showed an imbalance of chemical constituents which results symptom appearance (Rosales and Burns, 2011).

In a study to identify genes related to HLB tolerance, transcriptional changes were observed in tolerant poncirus trifoliate hybrid US-897and susceptible Cleopatra mandarin seedlings. Affymetrix GeneChip citrus array was exploited to know the response of Ca. L. asiaticus infection in the above mentioned citrus genotypes. Microarry analysis showed exclusive upregulation of a gene for 2-oxoglutarate and Fe+2 dependant oxygenase in US-897(Albrecht and Bowman, 2012). To study early host responses in healthy and HLB infected sweet orange leaves by identifying differential gene expression using RNA- seq. data analyses provided that 6606 genes were differentially expressed out of total of 20 million reads in infected new flushes. Quantitative RT PCR analysis of 20 genes selected at random for validation of RNA-seq. results, revealed correspondence of 19genes with the RNA-Seq results (Parra, 2012). Martinelli et al. (2012) used next generation sequencing technique for the study of citrus response towards HLB bacterium infection. For this purpose, they prepared mRNA from healthy 14 and HLB infected fruit peel. Different pathways and proteins network were studied alongwith qPCR analysis of selected genes. They organized networks after pathways discovery for understanding metabolism in diseased fruit.The results showed increased transcription of light reaction genes of photosynthesis and ATP production genes. Protein breakdown and misfolding were provoked and down regulation of heat shock protein transcripts were seen at different stages of HLB. Pathways associated with hormone and carbohydrates metabolism were severly affected. Transcripts for cytokinins and gibberellins were reduced while for ethylene induced. HLB bacterium infection increased transcripts of WRKY transcription factors via salicylate and jasmonate pathways. These findings are helpfull for understanding epidemiology of HLB affected fruit. Liao and Burns(2012) reported qRT-PCR results for differentiating gene expression in HLB affected , girdled and healthy fruit tissues. Out of 15 genes which were selected, different expression was observed in 10 genes.The genes studied were, those involved in phytohormone metabolism(CsNCED,CsSUR2), a tranporter (CsSULF),carbohydrate metabolism(CsSD1,CsSB1 and CsB2), metabolism of cell wall(CsPG), for ROS scavenging (CsATC) and for degradation of chlorophyll (CsELIP).Gene expression for ethylene biosynthesis (CsACO) was up- regulated in the flavedo of girdled fruit and down-regulated in the fruit flavedo of symptomatic fruit as compare to control. The expression of shared genes, the beta amylase 9(CsSD3) and alpha amylase 3(CsSD2) changed in the similar pattern.

Majority of citrus genotypes vary in susceptibility for HLB pathogen, are found less susceptible. A study on response of lemon towards Ca. L. asiaticus infection resulted in increased quantity of starch synthase, decrease in photosynthesis-related proteins, increase of potassium concentration in the leaves of lemon, decrease in production of defense associated proteins and increase in zinc concentration (Nwugo et al., 2013). Some citrus genomes including citrus and citrus sinensis have been sequenced. Now there is need of function annotation. For this purpose, microarray and RNA seq data is available easily. It can be used for coexpression studies. In a study for coexpression network including HLB, 230 microarrays of Citrus sinensis and 371 genes from RNA seq data were used. Eight genes related to apoptosis were found during gene ontology studies. Those were BCL-2, polyubiquitin10, RING finger E3 ubiquitin ligases, myosin heavy chain-related,

15 sphingoid base hydroxylase, LAG1 longevity assurance homolog 3 and Glutaredoxin family protein. Functions of the above mentioned genes in HLB are still unknown (Du et al., 2015). Wang et al. (2016) used RNA-Seq to evaluate expression differences between two closely related cultivars after HLB infection: HLB-tolerant “Jackson” grapefruit-like-hybrid trees and HLB susceptible “Marsh” grapefruit trees. A total of 686 genes were differentially expressed (DE) between the two cultivars. Among them, 247 genes were up-regulated and 439 were down- regulated in tolerant citrus trees.

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CHAPTER-3

MATERIALS AND METHODS

For expression profiling of HLB in citrus, fiveexperiments were conducted. First, second and third experimentwas conducted to achieve the first objective of this study. Fourth and fifth experiments were conducted for the accomplishment of second and third objective respectively.The experiments are as under:

Expt.1- Rearing of citrus germplasm in greenhouse

Expt.2-Asian citrus psyllid rearing

Expt.3- Huanglongbing diagnosis in ACP infested plants and inoculation of citrus germplasm

Expt.4- Detection of Candidatus Liberibacter asiaticus in citrus germplasm

Expt.5-Expression profiling of huanglongbing disease in citrus germplasm

3.1 EXPERIMENT-1

Rearing of citrus germplasm in greenhouse

To study the gene expression changes in citrus in response of HLB, citrus germplasm was reared in greenhouse. Citrus germplasm seed (PI numbers) was acquired from the United States Department of Agriculture (USDA) National Clonal Germplasm Repository for Citrus and Dates, Riverside, California, USA and University of Agriculture, Faisalabad, Pakistan source trees.Seeds from 97 genotypes of citrus including citrus relative comprising of 28 groups (Table 3.1) were sown in controlled conditions of greenhouse ofInstitute of Horticultural Sciences (IHS), University of Agriculture Faisalabad (UAF), Pakistan. A mixture of sand, silt, clay and compost was used for appreciable germination, robust growth and high survival rate of citrus seedlings. For germination and raisingof seedling, greyish black polythene bags having a size of 15 inch length and 9 inch width were used (Figure 3.1). Single seed was sown in each polythene bag so as to avoid seedlings transplantation shock. Seedlings were irrigated with canal water whenever required. Fertilizer containing macro and micro nutrients (Table 3.2) was applied with

17 irrigation water during the entire period of experiment twice a month to prevent nutrition deficiency (Gottwald et al., 2012).

Figure 3.1 Greyish black polythene bags having a size of 15x9 inch used forcitrus germplasm seed sowing

Table 3.1 Citrus germplasm sown for the detection and expression profiling of huanglongbing

Sr.No. Group Group Cultivar Binomial Accession No. No. 1 1 Calamondin Calamondin Citrus madurensis PI 539349 2 2 Benton X citroncirus sp. PI 539819 3 Citrange C-35 X citoncirus sp. PI 539821 4 Citrange Carrizo X citroncirus sp. PI 150916 5 Citrange Savage Citrange X citroncirus sp. Pak 6 Citrange Troyer X citroncirus sp. PI 539810 7 Citrange Unnamed (Poncirus X citroncirus sp. PI 539818 trifoliata x S 8 3 Bengal Citrus medica PI 539417 9 Citron Corsican Citrus medica PI 539415 10 Citron Diamante Citrus medica PI 539424

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11 Citron Djerok Citrus medica PI 539416 12 Citron Dulcia Citrus medica PI 539436 13 Citron Hiawassie Citrus medica PI 539426 14 Citron Indian Citrus medica PI 539413 15 Citron Indian Citrus medica PI 53414 16 Citron Indian Citrus medica PI 31981 17 Citron Limau Matu Susu Citrus medica PI 539439 18 Citron Papuan Citrus medica PI 539430 19 Citron Philipine Citrus medica PI 539431 20 Citron S-1 Citrus medica PI 539441 21 Citron Sicily Citrus medica PI 539432 22 Citron Unnamed, ex-Morocco Citrus medica PI 230626 23 4 Sacaton X citroncirus sp. PI 539826 24 Citrumelo Swingle X citroncirus sp. PI 539828 25 5 Citrus relative Bergera Koenigii Bergera koenigii PI 539745 26 Citrus relative Clausena Excavata Clausena excavata PI 535419 27 Citrus relative Clausena Harmandiana Clausena PI 600640 harmandiana 28 Citrus relative Clausena Lansium Clausena lansium PI 539716 29 Citrus relative Eremocitrus Glauca Eremocitrus glauca PI 539717 30 Citrus relative Eremocitrus Glauca Eremocitrus glauca PI 654891 hybrid 31 Citrus relative Glycosmis Pentaphylla Glycosmis PI 127866 pentaphylla 32 Citrus relative Hawaiian Mock orange Murraya paniculata PI 539747 33 6 Grapefruit Duncan Citrus paradisi PI 539482 34 7 Fortunella Hindsii Fortunella hindsii PI 539723 35 Kumquat Fortunella Polyandra Fortunella PI 539731 polyandra 36 8 Lemon- Frost Eureka Citrus limon PI 539318 Eureka 37 9 Lemon hybrid Improved Meyer Citrus meyeri PI 539447 38 Lemon hybrid Kharna Citrus hybrid PI 213224 39 Lemon hybrid Perrine Citrus hybrid PI539205 40 Lemon hybrid Unnamed (Tavares Citrus limon hybrid PI 539804 ) 41 10 Mandarin Citrus Sunki Citrus sunki PI 539678 42 Mandarin Cleopatra PI539492 43 Mandarin Kinnow Citrus reticulata Pak Blanco

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44 Mandarin Kinokuni Citrus kinokuni PI 539270 45 Mandarin Parson‟s Special Citrus reticulata PI539497 46 Mandarin Sun Chu Sha Citrus reticulata PI 539544 47 Mandarin Nules Citrus clementina Pak hort.ex Tanaka 48 Mandarin Scarlet Emperor Citrus reticulata PI 539505 49 11 Mandarin Borneo Citrus limonia hybrid - RCRC 4132 Rangpur 50 12 Microcitrus Microcitrus Australsica Microcitrus PI 312872 australsica 51 Microcitrus Microcitrus Inodora Microcitrus inodora PI539742 52 Microcitrus Var. Sanguinea Microcitrus PI 312872 australasica 53 13 Microcitrus Faustrime X citroncirus sp. PI 539808 hybrid 54 14 Hanayu Citrus hanaju PI 539198 55 Papeda Honghe Citrus hongheensis PI 539672 56 Papeda Ichang Papeda PI 539253 57 15 Papeda hybrid Citrus celebica PI 539182 58 Papeda hybrid Citrus junos PI 45945 59 16 Pummelo Reinking Citrus maxima PI 539391 60 Pummelo Tahitian Citrus maxima PI 539392 61 17 Pummelo Kinkoji Citrus obovoidea PI 539458 hybrid 62 18 Rangpur Kirumakki Nucellar Citrus limonia RCRC 4131 Osbeck 63 Rangpur Knorr Nucellar Citrus limonia RCRC 4132 64 Rangpur Rangpur Poona Citrus limonia RCRC 4135 Nucellar 65 Rangpur Srirampur Nucellar Citrus limonia RCRC 4137 66 Rangpur Tuningmeng Nucellar Citrus limonia RCRC 4139 67 19 Florida Citrus jambhiri PI 539268 68 Rough lemon Schaub Citrus jambhiri PI 539266 69 20 Sour orange Bitter Sweet orange Citrus aurantium Pak 70 Sour orange Bouquet des Fleurs Citrus aurantium PI 539174 71 Sour orange Brazillian Sour orange Citrus aurantium L. Pak 72 Sour orange Gadadehi Citrus aurantium Pak 73 Sour orange Goutoucheng Citrus aurantium PI 539170 74 Sour orange Nansho Citrus taiwanica PI 539680 75 Sour orange Sour Orange Citrus aurantium L. Pak

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76 Sour orange Standard Citrus aurantium PI 539176 77 21 Sour orange Bergamot Citrus bergamia PI 539179 hybrid 78 Sour orange Gabbuchinee Citrus aurantium L. PI 539225 hybrid 79 Sour orange Keen Sour orange Citrus aurantium L. Pak hybrid 80 22 Sweet lime Palestine Citrus limetoides PI 539283 81 23 Sweet orange Atwood Citrus sinensis Pak 82 Sweet orange Lane Late Citrus sinensis L. Pak Osbeck 83 Sweet orange Madam Vinous Citrus sinensis PI 539625 84 Sweet orange Pineapple Citrus sinensis PI 539622 85 Sweet orange Succari Citrus sinensis Pak 86 Sweet orange Valencia Citrus sinensis L. Pak Osbeck 87 24 Orlando Citrus X tangelo PI 539711 88 25 Dweet Citrus noblis PI 539240 88 Tangor Tangor Citrus reticulata x Pak sinensis 89 26 Trifoliate Citrumelo 1452 X citroncirus sp. Pak 90 Trifoliate Flying Dragon Poncirus trifoliata PI 539768 91 Trifoliate Poncirus Trifoliate Citrus trifoliata Pak L.(previously Poncirus trifoliata (L.)Raf.) 92 Trifoliate Rich 16-6 Citrus trifoliata PI 539776 93 27 Trifoliate Cox Mandrin Hybrid Scarlet Pak hybrid mandarinxP. trifoliata 94 Trifoliate Fraser Hybrid Smooth SevillexP. Pak hybrid trifoliata 95 Trifoliate X-639 X citroncirus sp. PI 539847 hybrid 96 Trifoliate Yuma Citrange X citroncirus sp. Pak hybrid 97 28 Valencia Frost Citrus sinensis PI 539660 orange

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Table 3.2 Nutrientscomposition applied to the citrus germplasm

Macro nutrients Micro nutrients Sr.No. Nutrient Sr.No. Nutrient Sr.No. Nutrient 1 N 20% 1 Fe 400ppm 4 Zn 250ppm 2 P2O5 20% 2 B 15ppm 5 Cu 50ppm 3 K2O 20% 3 Mn 150ppm 6 MgO 1000ppm

3.2. EXPERIMENT-2

Asian citrus psyllid rearing

For expression profiling of huanglongbing disease in citrus germplasm, it was necessary to keep the germplasm free of any other graft transmissible disease except huanglongbing. For this purpose, Asian Citrus Psyllid (ACP) was reared in the growth room situated ina laboratory at the Center of Agricultural Biochemistry and Biotechnology (CABB), University of Agriculture Faisalabad (UAF), Pakistan, according to a modified protocol from Nava et al. (2007).Screenhouse raised healthy plants of Sweet orange (Citrus sinensis) and Kinnow (Citrus reticulata Blanco) were acclimatized in this growth room for ACP rearing. Pruning of those plants was done before starting the rearing experiment for the growth of new flushes and equal height of all plants.

Different citrus orchards of district Faisalabad were surveyed for the presence of ACP and HLB diagnosis. A sweet orange orchard with a huge population of ACP nymphs and adults with numerous symptoms of HLB on leaves was selected to achieve the target (Fig. 3.2).Symptomatic leaves for HLB diagnosis were collected from that sweet orange field and subjected to molecular studies for the detection of HLB bacterium Candidatus Liberibacter (Fig. 3.3). Positivity of the field sweet orange plants was confirmed by standard PCR using 16S ribosomal RNA gene primer pair OI1/OI2c(Jagoueix et al., 1996)and rplKAJL-rpoBC operon primer pair A2/J5(Hocquellet et al.,1999; Chohan et al., 2007)for Candidatus Liberibacter asiaticus.

After the detection of HLB pathogen, ACP was collected from those trees and released on Sweet orange and Kinnow plants for colonization in growth room (Fig.3.4)at the Center of Agricultural Biochemistry and Biotechnology (CABB), UAF, Pakistan, at 26±2 ᴼC temperatureand

22 photoperiod of 13:11(Light :Dark) (Nava et al., 2007).ACP was released on those plants fortnightly upto one year from November 2011 to October 2012. Establishment of ACP colonies and appearance of HLB symptoms were observed regularly. Macro and micro nutrients were provided to the plants in the growth room raised for ACP rearing for better growth and sufficient attractive food for adult and nymph ACP.

Figure 3.2(A&B)Sweet orange plants in field with a huge population of ACP adults

A

B

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Figure 3.3Sweet orange leaf samples with HLB symptoms from field trees for molecular detection of Candidatus Liberibacter asiaticus.A,C&D; Mottling and vein yellowing symptoms, B; Vein yellowing

B A

C D

Figure3.4 Sweet orange and kinnow plants in growth room

Figure3.5 Sweet orange leaf sample with blotchy mottle and vein yellowing symptom

24 after infestation of ACP in growth room conditions for HLB pathogen detection

3.3. EXPERIMENT-3

Huanglongbing diagnosis in ACP infested plantsand inoculation of citrus germplasm

3.3.1.Huanglongbingdiagnosis

Thesweet orange and kinnow plants were infested by Asian citrus psyllids (ACP) collected from HLB positive sweet orange field plants for their inoculation and colonization of ACP.After one year of ACP releaseon those plants, leaf samples with HLB symptoms (Fig. 3.5) were exised and their DNA were tested for the presence of HLB pathogen by conventional PCR.A primer pair OI1/OI2c was used for pathogen‟s 16S ribosomal RNA gene. Gene specific primer pair A2/J5was used to amplify rplKAJL-rpoBCbeta operon and for the amplification of 16S and 23S intergenic region, primer pair OI2/23S1 was used. After confirmation of the HLB bacterium presence, those plants were used for inoculation of 51 genotypes of citrus germplasm.

3.3.1.1 DNA extraction

DNA was extracted from midribs and petioles of mature leaf of inoculated and healthy plants by the CTAB (cetyltrimethylammoniumbromide) method (modified from Ruangwong and Akarapisan, 2006). About 0.5g leaf midribs and petioles were cut in to small pieces with sterile scissors. Chopped midribs and petioles pulverized in liquid nitrogen by using sterile pestle and mortar. To that powder, 4mL CTAB buffer (Table3.4) added and incubated at 55°C for 1 hour

25 and centrifuged at 4000rpm for 5 minutes. Mercapto ethanol (1µL/mL of CTAB) added to the CTAB buffer before starting the process of DNA extraction. To the supernatant, added 0.125 volume of 5M NaCl and 10% CTAB in 0.7M NaCl and incubated at 65°C for 10 minutes. Added equal volume of phenol, chloroform and isoamyl alcohol (25:24:1), shaked the mix gently and centrifuged at 12000rpm for 10 minutes. The supernatant taken carefully in properly labelled separate eppendorf tubes and added 1/10 volume of 3M sodium acetate. Then added equal volume of chilled isopropanol and placed at -20°Covernight. DNA pellet was formed by centrifugation at 13000 rpm for 15 minutes. Supernatant was removed carefully. Then DNA pellets were washed twice with freshly prepared 70% ethanol. Pellets were dried for 30 minutes at room temperature and dissolved in100µL of 1X TE buffer.

Table 3.3 Composition of CTAB buffer for DNA extraction

Chemical Final Concentration CTAB 2% Lauroyl sarcosine 1% Tris HCl 100mM NaCl 1.4mM EDTA (pH8) 20mM

3.3.1.2 DNA quantification by agarose gel electrophoresis

Genomic DNA extracted from suspected to be HLB positive leaf midribs of sweet orange field plants, screenhouse raised healthy sweet orange seedlings and growth room maintained ACP infested kinnow and sweet orange samples was quantified by gel electrophoresis technique. Ultra pure agarose, Invitogen by life technologies, USA was used for DNA quantification.Agarose gel (1%) prepared in 0.5X TBE buffer (Table 3.6). Gel was stained with 0.5mg/mL ethidium bromide. An electric current of 100 volt was applied for 90 minutes. Gel was visualized under UV light in the gel documentation system (BioRad) by using a software, quantity one.

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3.3.1.3 Polymerase chain reactions for HLB diagnosis

Singleplex and multiplex conventional PCR was performed after DNA extraction and quantification on peqSTAR thermal cycler. 16S rDNA primer OI1/OI2c (Jagoueix et al., 1996), ribosomal protein gene of the rplKAJL-rpoBC operon (β-operon) primer A2/J5 ( Hocquelletet al.,1999) and 16S/23S rDNA intergenicregionprimers OI2/23S1 were used for the detection of HLB bacterium in suspected positive, ACP infested samples and healthy controls. Sequences of primer pairs are given in table 3.4.Reaction mixtures used for singleplex, and multiplex conventional PCR are given in table 3.9. A total volume of 25 µL was used in the PCR reaction mix. Thin walled, flat capped, 0.2 mL, nuclease free, individual PCR tubes were used for PCR reaction mix. Thermal profile used for OI1/OI2c, A2/J5 and OI2/23S1 primers is given in table 3.10.

Table 3.4 The primerssequence for HLB diagnosis by conventional PCRstudies

Prime Sequences Target Orientatio Region of Comments r DNA n amplification OI1 GCGCGTATGCAA Las Forward 16s ribosomal Primer described TACGAGCGGCA RNA by Jagoueix et al., 1996 OI2c GCCTCGCGACTT Las Reverse 16s ribosomal Primer described CGCAACCCAT RNA by Jagoueix et al., 1996 A2 TATAAAGGTTGA Las Forward rplKAJL- Primer described CCTTTCGAGTTT rpoBC(β operon) by Hocquellet et al.,1999 J5 ACAAAAGCAGAA Las Reverse rplKAJL- Primer described ATAGCACGAACA rpoBC(β operon) by Hocquellet et A al.,1999 OI2 5′-ATGGGTTGCGA Las Forward 16S/23S rDNA Primer described AGTCGCGAGGC-3′ intergenic by Jagoueix et al., region 1997 23S1 5′-CGCCCTTCTCT Las Reverse 16S/23S rDNA Primer described CGCGCTTGA-3′ intergenic by Jagoueix et al., region 1997

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Table 3.5Composition of reaction mixture for conventional PCR

Final conc. of Reagent Stock conc. Per reaction Products by reagent (µL) Nuclease free water 16.8 Ambion ® 1x Taq buffer 10x 2.5 Fermentas 0.25mM dNTP 10 µM 0.625 Fermentas 0.25µM OI1 10 µM 0.625 IDT 0.25µM OI2c 10 µM 0.625 IDT 0.25µM A2 10 µM 0.625 IDT 0.25µM J5 10 µM 0.625 IDT 0.2u Taq polymerase 0.5 Fermentas 15-30ng Template DNA 2 µL Total 25

Table 3.6 Thermal profile for conventional PCR

Function Temperature Time No.of cycles Initial denaturation 95°C 10 minutes 1 Denaturation 94°C 30 seconds 35 Annealing 58°C 1 minute 35 Extension 72°C 1minute 35 Final extension 72°C 10 minutes 1 Hold 4°C forever

3.3.1.4 PCR product analysis

The PCR products were analyzed by gel electrophoresis using 1.2% agarose in 1x TAE buffer (Table 3.7). The gel was run for 90 minutes at 80 volts. Ethidium bromide was used for staining agarose gel. Gel was visualized in BioRad GelDoc imaging system using quantity one software.

Table 3.7 Composition of 5 X TBE buffer for one litre solution

Sr.No. Reagent Quantity 1 Tris base 54g 2 Boric acid 27.5g 3 0.5M EDTA (pH. 8) autoclaved 20mL

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Table 3.8 Composition of 50X TAE bufferfor one litre solution

Sr.no. Reagent Quantity 1 Tris base 242g 2 Glacial acetic acid 57.1 mL 3 0.5M EDTA (pH. 8) autoclaved 100mL

3.3.2 Inoculation of citrus germplasm

There were 51 genotypes of citrus including citrus relatives in replicates for the purpose of inoculation by the Candidatus Liberibacter asiaticus bacterium. There would be chances of like and dislike by ACP for a large number of citrus genotypes at the same place while, our objective was to inoculate all of the 51 genotypes and then see what happens with these genotypes. Therefore, only sweet orange and kinnow plants were provided to ACP for their colonization and infestation. Further, it was decided to use the above mentioned sweet orange and kinnow plants to inoculate the citrus germplasm by graft challenge because, the acquisition of grafts or buds or the inoculation material from HLB positive plants was easy, safe and suitable for the whole experimental citrus germplasm inoculation.

The grafts and leaf midribs used for citrus germplasm inoculation were obtained from HLB positive sweet orange (Citrus sinensis) plants that were used for ACP rearing and infestation in controlled conditions of growth room.Fifty one genotypesof citrus germplasm(Table.4.1) were inoculated in triplicate. Three plants for each genotype were separated as healthy controls. Each replicate of citrus genotypes was inoculated by budding one midrib andone bud to ensure inoculation (Fig. 3.6). The inoculated citrus germplasm including citrus and citrus relatives was maintained in controlled conditions of screenhouse (Fig.3.7 & 3.8 ). Application of nutrients was continued during the entire period of experiment to both treatments i.e. healthy control and inoculated plantsto prevent expression of nutrition deficiency symptoms.

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Figure 3.6Healthy control plants of citrus (A to F)in greenhouse

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Figure 3.7 Citrus germplasm replicates for inoculation of HLB bacterium (A through D)

A B

C D

Figure 3.8 Few genotypes of citrus out of 51 showing graft/midrib inoculation; (A); Sicily (B); Orlando (C); X639 (D); Benton (E); Unnamed (F) Carrizo (G); Tuningmeng Nucellar (H); Eremocitrus glauca hybrid (I); Flying Dragon (J); Rich 16-6

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3.4. EXPERIMENT-4

Detection of Candidatus Liberibacter asiaticus in citrus germplasm

Molecular studies were carried out for the detection of huanglongbing bacterium “Candidatus Liberibacter asiaticus” in citrus germplasm.Extraction of DNA and conventional PCR studies for HLB diagnosis from field and growth room plants infested by ACP were conducted in the Centre of Agricultural Biochemistry and Biotechnology (CABB), University of Agriculture, Faisalabad (UAF), Pakistan.

Detection of Candidatus Liberibacter asiaticus in citrus germplasmby real-time PCRwas accomplishedat theUniversity of California, Riverside (UCR), USA. The process of leaf sampling, DNA extraction and real-time PCR studies are as under:

3.4.1 Sample Collection

Mature leaf samples were collected from inoculated and healthy control plants of citrus germplasm maintained in greenhouse, Institute of Horticultural Sciences, University ofAgriculture, Faisalabad (UAF), Pakistan for molecular detection of Candidatus Liberibacter asiaticus. Samples were collected after 12 months of inoculation. Prominent symptoms of huanglongbingwere not found to be presentin all of the inoculated genotypes of citrus however, efforts were made to collect those leaves which had symptoms and exist within area of 12 inches of midrib/graft inoculation. From each replicate,3 to4 leaves were collected. Leaves were 33 excised, placed in zip lock bags, labelled properly, put in ice box immediately, shifted to the lab and stored in the refrigerator at 4°C until DNA extraction. Collected sapmles were further processed on the same day rather than to store them for a long time.

3.4.2Shipment of DNA to USA and Zymopurification

The DNA was extracted from midribs and petioles of mature leaf of inoculated and healthy plants by using 2X CTAB buffer (Table.3.3).The DNA extraction process was stopped upto the addition of equal volume of chilled isopropanollevel (Fig. 3.9) for shipment toUniversity of California, Riverside (UCR), USA. The eppendorff tubes containing plant DNA were properly labelled, sealed with parafilm and placed in ziplock plastic bags which were shipped to UCR in dry ice by FedEx. Upon receiving, all DNA samples were stored at -20°C till further purifications.

For further purification, stored DNA samples were centrifuged at 13000 rpm for 15 minutes.Discarded the supernatant carefullyand short spin the tubes for 30 second. Remaining isopropanol then removed carefully by using 10µL pipette tip.For checking the quality, DNA pellets from only few samples were washed with 70% ethanol, air dried for 15 minutes and dissolved in 200µL 1X TE (pH8). When those samples were quantified by nanodrop, there was sufficient quantity of DNA in each sample but the peaks at 260/280 nm were showing impurities in the DNA.

Table 3.9 DNA clean and concentrator contents ZR-96 DNA clean and D4024 Storage Temperature concentrator TM -5(Kit Size) (4×96 Preps.) DNA binding buffer 2×100 ml 25 ᴼC DNA wash buffer 48 mL 25 ᴼC DNA elution buffer 16 mL 25 ᴼC Zymo-SpinTM 1-96 plate 4 25 ᴼC Collection plate 4 25 ᴼC Elution plate 4 25 ᴼC

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Mature leaves contain higher quantities of polyphenols, tannins and polysaccharides (Porebski et al., 1997) which makes it very difficult to isolate pure DNA. As we used mature leaves, so pellets from remaining samples were dried for 5 minutes at room temperature and dissolved directly in200µL 1X TE (pH8) without ethanol washing. This dissolved DNA was further purified by using ZYMO RESEARCH DNA cleaning kit (ZR-96 DNA Clean & ConcentratorTM- 5. Catalog No.D4024) according to the manufacturer‟s instructions. Before starting purification, added 192 mL 100% ethanol to the 48 mL DNA wash Buffer concentrate. Added 2 volumes (400µL) of DNA Binding Buffer to 200µL of genomic DNA sample and mixed briefly by vortexing. Sample mixtures were transfered to the wells of a Zymo-Spin 1-96 plate mounted on a collection plate. Centrifuged for 25 minutes until sample mixtures had been completely filtered and discarded the flow- through. DNA wash buffer (300µL)then added to each well of the Zymo-Spin 1-96 plate. Centrifuged the plate for 15 minutes and discarded the flow- through. Again added 300µL DNA wash buffer to each well of the Zymo-Spin 1-96 plate. Sealed with the adhesive film and centrifuged for 15 minutes at 4000 rpm and discarded the flow- through. Theelution plate was placed below the Zymo-Spin 1-96 plate. Added 50µL DNA elution buffer in each well of the Zymo-Spin 1-96 plate and covered the plate with the adhesive film. Centrifuged for 20 minutes at 4000 rpm (37°C) to elute the DNA.The Zymo-Spin 1-96 plate was removed from the elution plate and shifted the eluted DNA from plate to the already labeled eppendorf tubes. Tube Labels were prepared by LabJet BLX-028 microtube label printer. The dilutions from purified DNAwere madefor real time quantitative PCR.

3.4.3 Citrus germplasm DNA quantification by spectrophotometry

After completion of inoculated and healthy citrus germplasm DNA purification by using zymo research DNA cleaning kit,it was quantified by spectrophotometric method using 1µL genomic DNA from the total with spectrophotometer NanoDrop ND 2000TM (Thermo Scientific) at the Institute for Integrative Genome Biology (IIGB), UCR. 1µL of DNA elution buffer was loaded on the nanodrop with the help of micro pipette and clicked on blank to measure the blank reading. Then 1µL of each sample was taken for DNA quantification and clicked on measure. For every new sample cleaned the lense of nanodrop with kim wipes. The concentration of DNA was obtained by absorbance at 260 nm. The ratio of nucleic acids to proteins in the sample was

35 evaluated by the ratio of the absorbance at 260 and 280 nm (A260/A280) (Sambrook and Russel, 2001). The quality of DNA was judged by the size and shape of peaks at A260/A280 nm.

3.4.4 Real-time PCR for Candidatus Liberibacter asiaticus detection in citrus germplasm

In the DNA samples of citrus germplasm,multiplex quantitative real time PCR was conducted for the detection of Candidatus Liberibacter asiaticus sequence. The DNA extracted and purified from 51 genotypes of citrus in triplicate was pooled into 51 DNA samples for inoculated as well as healthy controls each and used in PCR. Each PCR reaction was run in triplicate for each genotype for the detection of HLB bacterium. The measurement was made after each amplification cycle, and this is the reason why this method is called real time PCR. Quantitative TaqMan PCR was conducted using 16S rDNA based TaqMan primer-probe set specific to Candidatus Liberibacter asiaticus.Aprimer-probe set based on plant cytochrome oxidase (COX) gene was used as a positive internal control to assses the quality of the DNA extracts. This assay does not cross-react with other pathogens or endophytes commonly resident in citrus plants and is very sensitive (Li et al., 2006).

Sequence specific DNA probes consisting of oligonucleotides were labeled at 5´ end with fluorescent reporters 6- carboxyfluorescein (FAM) and JOE while black hole quencher 1(BHQ1) and black hole quencher 2 (BHQ2) at 3´ ends which permitted detection only after hybridization of the probe with its complementary sequence (Table 3.10).

3.4.4.1Taqman based qPCR protocol for Las detection in citrus germplasm

Before starting preparation of reaction mixture for qPCR, cleaned the bench with 70% ethanol or 1% bleach.After drying, placed a new bench top paper on the reservedarea for qPCR. Pippets were also cleaned with 70% ethanol. Only designated pippets and nuclease free water used for qPCR.Real-time quantitative PCR assays were carried out in 96 well plates (non-skirted TempPlate, std. depth, white cat.No.1402-9589, USA ScientificR). Barrier tips were used for adding DNA in the 96 well plate. Two µL of template DNA in 25 µL total reaction volume was used for PCR. Composition of reaction mixture for qPCR along with the names of primers and probes for HLB pathogen and plant cytochrome oxidase gene is given in the table 3.10.Each plate consisted of sets of controls including no template control (NTC) and positive control. Template DNA added first in 96 well plate. After adding master mix, sealed the plate firmly with 36 adhesive film (Microseal B Film BioRad MSB1001) and short spinnedfor 10 second. BIORAD iQ5 thermocycler was used for running qPCR.

Table 3.10 The primer/probe sequence for Las detection by qPCR studies

Primer Sequences Target Orientation Region of Comments /Probe DNA amplification HLB GTC GAG CGC GTA Las Forward 16s ribosomal Primer for16s as f TGC AAT AC RNA ribosomal RNA gene described by Li et al., 2006 HLB TGC GTT ATC CCG Las Reverse 16s ribosomal Primer for16s as r TAG AAA AAG GTA RNA ribosomal RNA G gene described by Li et al., 2006 cox f GTA TGC CAC GTC Citrus Forward Citrus cox-1 gene Primer for internal GCA TTC CAG A cox-1 control cytochrome gene oxidase gene from plant described by Li et al., 2006 cox r GCC AAA ACT GCT Citrus Reverse Citrus cox-1 gene Primer for internal AAG GGC ATT C cox-1 control cytochrome gene oxidase gene from plant described by Li et al., 2006 FAM- AGA CGG Las Forward 16s ribosomal Probe for16s HLB GTG AGT AAC GCG- probe RNA ribosomal RNA as p BHQ1 gene described by Li et al., 2006 cox p CY3-ATC CAG ATG Citrusco Forward Citrus cox-1gene Probe for internal CTT ACG CTG G- x-1 gene control cytochrome BHQ2 probe oxidase gene from plant described by Li et al., 2006 cox p JOE-ATC CAG ATG Citrus Forward Citrus cox-1 gene Probe for internal CTT ACG CTG G- cox-1 control cytochrome BHQ2 gene oxidase gene from probe plant described by Li et al., 2006

Cycling conditions of PCR consisted of an initial denaturation step at 95°C for 3 minutes followed by 40 cycles of denaturation at 95°C for 10 seconds and annealing at 58 °C for 20 seconds (Figure 3.10). The instrument records the cycle threshold (Ct) values for all samples. The Ct value is the number of PCR cycle at which the fluorescence increases above a set threshold value. Record of Ct values are exported to excel files for analysis.

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Table3.11Composition of reaction mixture for Taqman based qPCR Reagents Quantity per reaction Nuclease free water 16.125µL NEB Taq Buffer 2x dNTP mix 200 µM Primer HLBas f 200 nM PrimerHLBas r 200 nM PrimerCox f 200 nM PrimerCox r 200 nM Taq Polymerase (NEB) 2.5 units Probe HLB asp(FAM) 150 nM ProbeCox p (JOE) 150 nM Template DNA 20 ng Total volume 25 µL

Figure 3.9 DNA precipitated in isopropanol for storage and shipment to USA

Figure 3.10 Thermal profile for real-time PCR detection of HLB in plants using iQ5

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3.5 EXPERIMENT-5

Expression profiling of huanglongbing disease in citrus.

Molecular techniques were used for expression profiling of huanglongbing disease in citrus. From fifty one accessions/ genotypes of citrus, forty five were incorporated in the expression profiling experiment (Table 4.3). Three plants for each genotype of citrus germplasm with same size and health were inoculated and three plants for each genotype were isolated as healthy control with no inoculation.

After twelve weeks of inoculation, evaluation process for symptoms expression on the leaves of citrus germplasm was started for detection of HLB in the inoculated plants. Molecular studies for detection and expression profiling of HLB were carried out after one year of inoculation. Following steps were performed to accomplish the above mentioned parts of expression profiling of HLB in citrus germplasm experiments.

3.5.1 RNA extraction andshipment to USA

The materials used in citrus germplasm RNA extraction were as under:

Liquid nitrogen, nitrile gloves, autoclaveable pestles and mortars, single channel pipettes and pipett tips of 10, 100 and 1000 µL., ice buckets with ice, tissue papers, RNA zap for cleaning, RNA extraction buffer, water saturated RNA grade phenol, chloroform, 100% ethanol, sodium acetate, microfuge tubes (1.5mL), dry bath, refrigerated centrifuge machine and parafilm.

RNA extraction from whole leaf of 51 genotypes of citrus germplasm with three biological replicateswas carried out at Center of Agricultural Biochemistry and Biotechnology (CABB), University of Agriculture Faisalabad (UAF), Pakistan according to Sambrook and Russell, 2001. Before starting extraction, all equipments especially pipepettes and working bench were cleaned with RNA ZAP. Nuclease free eppendorff tubes were labeled with permanent marker and placed on ice in ice bucket.

About0.2g leaf sample pulverized in liquid nitrogen using sterile pestle and mortars. Shifted the powder to microfuge tube (1.5mL), added 300µLRNA extraction buffer (Table 3.12) followed by addition of 300µL phenol to the above mix.Vortexed the tubes for 1minute and incubated at

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65ºC for 5 minutes.Then plunged the tubes on wet ice for 2 minutes followed by centrifugation at 13000rpm for 10 minutes.To supernatant, added equal volume of RNA grade phenol chloroform (1:1) and centrifuged for 10 minutes.The supernatant was taken innew 1.5 mLnuclease free tube with pre added absolute ethanol and 3M sodium acetate. Those samples were incubated at -20 ºC overnight and centrifuged at 13000 rpm for 15 minutes. Removed the ethanol thoroughly.Then washed the RNA pellet with 70% ethanol.RNA pellet dissolved in 100µL ultra pure water and checked its quality on 2.5% agarose gel stained with ethedium bromide.

After checking the RNA quality for some citrus leaf samples, experimental citrus germplasm RNA was extracted according to the above mentioned protocol and wrapped the tubes with para film and stored at -80ºC freezer at Center of Agriculture Biochemistry and Biotechnology (CABB), University of Agriculture Faisalabad, Pakistan for shipment to USA.

Table 3.12 Composition of RNA extraction buffer

Chemical Final Concentration SDS 5% Tris HCl(pH 7.5) 200mM Sodium acetate (pH5.2) 50 mM EDTA (pH8) 10mM

For shipment, extraction of RNA from inoculated and healthy germplasm was stopped upto addition of 3M sodium acetate and absolute ethanol. Precipitated RNA samples inabsolute ethanol were shipped to University of California Riverside (UCR), USA in dry ice by FedEx.Upon receiving, all RNA samples were stored at -80 C till further purifications.

For further purification, initially twelve RNA samples precipitated in absolute ethanol were taken out from the -80 C freezer, unsealed the tubes and spin down for 1 minute at maximum speed. Removed the supernatant carefully and washed the pellets with 70 % ethanol. After removing 70 % ethanol used for washing, respinned the tubes for short time and removed all ethanol with 10 µL tip. Pellets were dried for 5-10 minutes and resuspended in 150 µL d3H2O. The RNA samples were quantified by NanoDrop ND 2000TM (Thermo Scientific). Nucleic acid concentration was good in almost all samples(Table 3.13) but peaks for RNA for few samples 40 were not upto the mark and absorbance at 260/280 nm ranged between 1.21-2.05 representing some impurities. However, those twelve samples were used to synthesize cDNA for further molecular assays.

Table3.13 Nucleic acid concentrations for initial evaluation of RNA by nanodrop

Nucleic Sample acid Sample ID conc. Unit A260 A280 260/280 260/230 type Factor R1 0.3992 µg/µl 9.981 4.846 2.06 1.92 RNA 40 R2 0.4074 µg/µl 10.186 4.909 2.07 1.96 RNA 40 R3 0.0952 µg/µl 2.38 1.158 2.05 2.1 RNA 40 R4 0.0604 µg/µl 1.51 0.943 1.6 0.87 RNA 40 R4 0.4581 µg/µl 11.453 5.506 2.08 2.17 RNA 40 R6 0.215 µg/µl 5.375 4.29 1.25 0.66 RNA 40 R7 0.1866 µg/µl 4.664 5.186 0.9 0.48 RNA 40 R8 0.0617 µg/µl 1.543 1.279 1.21 0.6 RNA 40 R9 0.1418 µg/µl 3.546 2.921 1.21 0.57 RNA 40 R10 0.0897 µg/µl 2.241 1.633 1.37 0.69 RNA 40 R11 0.1104 µg/µl 2.76 1.379 2 1.57 RNA 40 R12 0.1254 µg/µl 3.136 2.647 1.18 0.55 RNA 40

3.5.2Synthesis ofcDNA using NEB MMLV enzyme

Total RNA extracted from greenhouse raised citrus germplasm was converted to complimentary DNA using NEB MMVL enzyme. Twelve RNA samples initially used for cDNA synthesis. Invitrogen random primers (50-250ng) were used for cDNA synthesis. Each reaction was of 20µL. Reaction cocktails for cDNA synthesis are given below in tables 3.14 and 3.15.

Table 3.14 Primers and dNTPs cocktail for cDNA

chemicals 1x 20µL 13x for 20 reaction mix µL reaction mix Random primers 1 13 (Invitrogen;50-250ng) 10mM dNTPs 1 13 Cocktail for primers and dNTPs 26 Total RNA 10 10

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Table3.15 Reverse transcription (RT)reaction cocktail

RT reaction cocktail 1x (µL) 13x (µL) 5X MMLV RNase H- rxn buffer 4 52 0.1M DTT 2 26 Murine RNase inhibitor 1 13 MMLV reverse transcriptase 1 13 Total 8 104 8

Aliquotes of 2µLcocktail for primers and dNTPs prepared in 12 blank tubes.To those aliquots added 10µL RNA.Incubated that mix at 80 C for 5 minutes.After incubation the tubes immediately plunged into ice bucket containing ice and water for 5 minutes.Then spin the tubes for 30 seconds. Then 8µL RT reaction cocktail added to the above mix and spin for 30 second.Incubated the above mix at room temperature for 10 minutes followed by incubation at 42 C for 50 minutes.For inactivation of the above reaction, tubes were incubated at 80 C for 10 minutes. Spin the tubes and stored cDNA at -80 C for downstream processes.

3.5.3Conventional PCR for the detection of internal control gene malate dehydrogenase. In conventional PCR,cDNA was used to detect the housekeeping gene malate dehydrogenase (MDH) as internal control in few genotypes of citrus.Reaction mix volume used for PCR was 25 µL (Table 3.16). PCR condition in thermocycler are provided in table 3.17.Trifoliate cDNA positive control was made from RNA that was not treated with with DNAse 1 and negative control using water as template also ran with 12 cDNA samples. Analysis of PCR product was done on 1% agarose gel in TAE buffer in gel documentation machine BioRad using software quantity one. After observing the PCR product results on gel, it was decided to perform DNase treatment of same RNA samples before proceeding for expression profiling steps.

Table 3.16Composition of PCR reaction mix for MDH gene

PCR Reaction mix 25 µl / rxn(1X) Water 20.25 10X buffer (NEB) 2.5 10mM dNTP 0.5 Cit 534 10µM 0.25 Cit 535 10µM 0.25 Taq Polymerase 0.25 Template 1 42

Table 3.17 PCR Conditions for MDH gene

PCR Conditions Initial denaturation at 95 C 3 min denaturation at 95 C 30 sec Annealing at 48 C 45 sec Extension at 72 C 45 sec Go to 2 35 times Final extension at 72 C 10 min 10 C forever

3.5.4Treatment of RNA samples with DNase

For the elimination of DNA from RNA samples, above mentioned RNA samples were treated with DNase. From 150 µL total RNA dissolved in water, 50µL RNA was taken in separate 1.5mL ependorff tube. To that RNA added 3µL of RQ1 10x reaction buffer, 1µL of RQ1RNase free DNase (NEB) and 6µL RNase free water. Incubated the mixture at 37 Cfor one hour. Reprecipitated in 6µL of3M sodium acetate and 150 L 100 ethanol and placed at -80 C. For further use they were eluted in water.

Next 24 samples of RNA were processed, eluted in water and subjected for quantification by using nanodrop. Again the peaks of nucleic acid at 260/280 nm were presenting some impurities. Elution of RNA in water and nanodrop reading was performed in National Clonal Germplasm Repository for citrus and dates, Riverside, CA. There it was suggested to dissolve remaining RNA samples that were extracted from fifty one genotypes of citrus and citrus relatives directly in water skipping the washing step of RNA pellet in70% ethanol. This suggestion was for cleaning the RNA samples in purification columns.

Water dissolved RNA was then shifted to UCR carefully in dry ice and immediately stored at- 80 C.Before cleaning the RNA samples in purification colums, few samples from DNase treated samples and few from the same genotypes but not DNase treated were planned to check on 2% native agarose gel. Four RNA samples extracted fromCarrizo citrange, Corsican, Indian and Troyer citrange were checked for this purpose (Fig.3.11). Agarose gel was prepared in 1x TAE buffer stained with ethidium bromide.The gel was run at 70 volt for 45 minutes.

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3.5.5 Qubit quantification of RNA samples

As the results of native agarose gel for DNase treated and not treated samples were confusing, the need was felt for qubit quantification of those samples both for DNA and RNA concentration. The UV absorbance method use a spectrophotometer to measure the natural absorbance of light at 260 nm for DNA and RNA or 280 nm for proteins. Because so many molecules absorb light at 260 nm, this measurement may be inaccurate due to potential contamination of the sample with other molecules. In addition, using the absorbance method, it is not possible to distinguish between DNA, RNA, protein or free nucleotides or amino acids in the sample, leading to inaccurate measurements.

Qubit fluorometer is a small instrument used for quantification of DNA, RNA and protein. It use fluorescent dyes to determine the concentration of nucleic acids and proteins in the sample. The dyes used in qubit assay are specific for one type of molecule: DNA, RNA or protein. These dyes have extremely low fluorescence until they bind to their targets. Upon binding, they become intensely fluorescent. The advantage of using these specific dyes is that the researcher can tell precisely how much of biomolecule (DNA, RNA or protein) is in a specific sample even in the presence of other biomolecules.

Two samples from DNase treated and two from not treated were used for qubit quqntification for RNA as well as DNA concentrations. Two standards for each of the nucleic acid were also subjected for the assay resulting in total twelve samples.

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Figure 3.11TotalRNA samples extracted from Carrizo, Corsican, Indian andTroyer, withand without DNase treatment

3.5.6 Qubit assay for RNA Set up two assay tubes for the standards and one tube for each sample. Prepared the qubit working solution by diluting the qubit reagent 1:200 in qubit buffer.After that prepared 200µL of working solution for each standard and sample.The assay tubes were prepared according to the specifications given in table 3.18.

Table 3.18 Specification of Qubit assay for RNA and DNA

Standard assay tubes User sample assay tubes Volume of working solution 190 µL 180-199 µL (from step 2) to add Volume of standard (from kit) 10 µL - to add Volume of user sample to add - 1-20 µL Total volume in each assay 200 µL 200 µL tube

Took 1400 µL buffer for 4 samples and two standards (Qubit RNA buffer) in a falcon tube and then added 7µL dye (Qubit RNA Assay Reagent) and shake gently.Then took 198 µL mixture from master mix in each tube for 4 samples and 190 µL for two standards each. Then added10 µL Qubit RNA standard-1 in C1 where already added above 190 µL mix. Then added10 µL Qubit RNA standard-2 in C2 where already added above 190 µL mix.Then added 2 µL RNA sample in each 198 µL mix added already prepared tube.Thin walled, clear, 0.5 mL qubit assay

45 tubes (set of 500, cat.no.Q32856 or Axygen PCR-05-c tubes (WWR, Part no.10011-830) were used for assay.Vortexed all tubes for 2-3 seconds. Incubated the tubes for 2 minutes at room temperature. Inserted the tubes in the qubit 2.0 flourometer and taken readings. By using the dilution calculator feature of the qubit, determined the stock concentration of original sample.

3.5.7Qubit assay for DNA The procedure for qubit assay of DNA was same as for RNA. The only difference in the assay was the qubit assay kit used for DNA. Qubit double stranded DNA broad range assay was choosed. For the assay buffers and dyes used were:

1.Molecular probes by life technology =1 kit. 2.Qubit ds DNA BR assay kit. For untreated samples, only names of samples are written while for DNAse treated samples, Dt is written before the name of citrus RNA samples.

Table 3.19 Qubit assay values for RNA and DNA

Qubit assay result for RNA Qubit assay result for DNA Sr. No. Samples Result (ng/µL) Sr. No. Samples Result (ng/µL) 1 Corsican >100 1 Corsican 209 2 Indian >100 2 Indian 243 3 Dt corsican 82 3 Dt corsican < 100 4 Dt indian >100 4 Dt indian < 100

The results of qubit assay in the given samples represented a potential amount of DNA contamination. By keeping in mind the impurities in RNA, it was decided to purify and concentrate the RNA by using purification columns. Before purification by columns, it was thought important to know the integrity of RNA for downstream processes of expression profiling in citrus. Bioanalyzer fluorometric analysis of already DNase treated and not treated RNA samples was performed to assess RNA integrity (Fig.3.12). Four genotypes of citrus including: Var. sanguinea, Flying dragon, Indian and Hawaiian mock orange were sent for bioanalyzer analysis to genomic core facility IIGB, Dept. of Botany and Plant Sciences, Bachelor Hall UCR, USA.

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Quantification of RNA concentration in samples by nanodrop was done before sample submission for bioanalyzer. Comparison of RNA concentrations by nanodrop and bioanalyzer along with RNA integrity number was done for downstream processes (Table 3.20).

Figure 3.12Total RNA electrophoresis file run summary produced on Agilent 2100 bioanalyzerof already DNase treated and not treated samples

3.5.8 Purification of citrus germplasm RNA

After quantificationof few samples of RNA by nanodrop spectrophotometery and qubit fluorometry and RNA integrity determination by bioanalyzer, it was felt necessary to purify the RNA samples using purification columns. For that purpose, ZR-96 RNA clean and concentrator kit (Catalog No. R1080) by Zymo Research corp. was used according to manufacturer‟s instructions.

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Table 3.20 Comparison of RNA concentrations checked by nanodrop and bioanalyzer along with RIN values Sr. No. Sample ID/ name Nanodrop Total vol. RIN value RNA conc. in concentration submit for bio bioanalyzer (ng/ µL) analyzer (µL) (ng/ µL) 1 R3/ Var sanguinea 78.4 3 7.3 88 2 R4/ Flying dragon 50.1 3 1.3 32 3 R5/ Indian 97.1 3 5.6 124 4 R11/ Hawaiian 111.6 3 2.5 99 mock orange 5 R3dt/ Var sanguinea 36 3 N/A 7 6 R4dt/ Flying dragon 46.4 3 1.6 17 7 R5dt/ Indian 55.3 3 2.6 35 8 R11dt/ Hawaiian 63.4 3 2 16 mock orange

Table 3.21RNA clean and concentrator contents ZR-96 RNA clean and R1080 Storage concentrator TM (2×96) Preps Temperature (Kit Size) RNA binding buffer 100 ml 25ᴼC RNA prep buffer 4x25 ml 25ᴼC RNA wash buffer (concentrate) 2x24 mL 25ᴼC DNase/RNase free water 6 mL 25ᴼC Zymo-SpinTM 1-96 plate 2 25ᴼC Collection plate 2 25ᴼC Elution plate 2 25ᴼC Cover foil 4

First of all RNA wash buffer was prepared by adding 96 mL of 100% ethanol to the 24 mL RNA wash buffer concentrate present in the kit. Then added 2 volumes of RNA binding buffer to each RNA sample and mixed well. Equal volume of 95-100% ethanol was added to the mixture of

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RNA binding buffer and RNA sample and mixed well. Transferred the above mix to the wells of Zymo-Spin l-96 plate mounted on a collection plate and centrifuged at 3000 x g for 5 minutes. Discarded the flow through and mounted the Zymo-Spin l-96 plate back onto the collection plate. For RNA purification with in-plate DNase digestion, added 30 L DNase 1 cocktail (3 L of RNase free DNase1,3 L of 10x reaction buffer and 24 L of RNA wash buffer) directly to the matrix in each well.Incubated the plate at 37 C for 30 minutes.Centrifuged theZymo-Spin l- 96 plateat 3000 x g for 5 minutes.RNA prep buffer (400 µL) was added to each well of Zymo- Spin l-96 plate, centrifuged at 3000 x g for 5 minutes and discarded the flow through. Zymo-Spin l-96 plate was centrifuged at 3000 x g for 5 minutes after adding 800µL RNA wash buffer to each well and discarded the flow through. This step was repeated with 400µL RNA wash buffer.Centrifugedthe Zymo-Spin l-96 plateat 3000 x g for 5 minutes on the emptied collection plate to ensure complete removal of the wash buffer.Then transferred the Zymo-Spin l-96 plate onto the elution plate.Added 50µL DNase/RNase-free water including RNase inhibitor (ambion) at a final concentration of 1 U/µLdirectly to the column matrix in each well and let stand for 1 minute at room temperature. RNA was eluted by centrifugation at 3000 x g for 10 minutes. The eluted RNA was stored at -80 C after making aliqoutes.

The aliquots of RNA were used for quantification by nanodrop spectrophotometer, bioanalyzer analysis, cDNA synthesis for qPCR and RNA-seq libraries prepration from few samples of RNA. Concentrations of RNA with their absorbance ratio values at 260/280 nm after purification and DNase treatment are shown in table 3.22.

Table 3.22 Nanodrop concentrations of RNA after purification and DNase treatment

Nucleic Acid Sample # Conc.ng/µl A260 A280 260/280 260/230 Type Factor 1 148.4 3.711 1.849 2.01 1.47 RNA 40 2 341.8 8.545 4.1 2.08 2.09 RNA 40 3 322.1 8.052 3.98 2.02 1.77 RNA 40 4 224.2 5.606 2.838 1.98 1.58 RNA 40 5 482.4 12.059 6.012 2.01 1.67 RNA 40 6 470.5 11.763 5.704 2.06 2.04 RNA 40 7 131.1 3.279 1.663 1.97 1.7 RNA 40 8 215.6 5.389 2.788 1.93 1.49 RNA 40 9 163.7 4.093 2.069 1.98 1.73 RNA 40 10 367.9 9.197 4.604 2 1.61 RNA 40

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11 360.9 9.024 4.374 2.06 1.91 RNA 40 12 289.7 7.243 3.584 2.02 1.79 RNA 40 13 479.9 11.998 5.803 2.07 1.98 RNA 40 14 97.5 2.438 1.337 1.82 1.24 RNA 40 15 588.6 14.714 7.094 2.07 2.1 RNA 40 16 411.2 10.281 5.043 2.04 1.84 RNA 40 17 323.1 8.078 3.908 2.07 1.96 RNA 40 18 242.7 6.069 3.118 1.95 1.51 RNA 40 19 628.5 15.713 7.655 2.05 1.99 RNA 40 20 180.3 4.509 2.152 2.09 2.3 RNA 40 21 828.2 20.705 9.839 2.1 2.22 RNA 40 22 57 1.425 0.805 1.77 1.25 RNA 40 23 500.2 12.506 5.916 2.11 2.45 RNA 40 24 387.1 9.677 4.638 2.09 2.02 RNA 40 25 238.3 5.958 2.924 2.04 1.93 RNA 40 26 418.9 10.471 4.965 2.11 2.16 RNA 40 27 236.6 5.916 3.037 1.95 0.87 RNA 40 28 588.6 14.715 7.089 2.08 1.98 RNA 40 29 153.1 3.827 2.001 1.91 1.39 RNA 40 30 209.8 5.244 2.87 1.83 1.13 RNA 40 31 187.3 4.684 2.458 1.91 1.29 RNA 40 32 187.3 4.683 2.336 2 1.84 RNA 40 33 156.1 3.903 1.99 1.96 1.56 RNA 40 34 374.3 9.357 4.468 2.09 2.21 RNA 40 35 275.3 6.883 3.346 2.06 1.96 RNA 40 36 208.9 5.221 2.565 2.04 1.81 RNA 40 37 177.9 4.448 2.17 2.05 1.9 RNA 40 38 91 2.276 1.208 1.88 1.35 RNA 40 39 356.8 8.92 4.356 2.05 1.91 RNA 40 40 379.9 9.496 4.575 2.08 2.13 RNA 40 41 195.4 4.884 2.364 2.07 1.96 RNA 40 42 174.5 4.362 2.163 2.02 1.88 RNA 40 43 365.7 9.142 4.661 1.96 1.57 RNA 40 44 234.3 5.858 2.986 1.96 1.53 RNA 40 45 433.7 10.843 5.19 2.09 2.22 RNA 40 46 284.7 7.119 3.381 2.11 2.13 RNA 40 47 93 2.324 1.277 1.82 1.24 RNA 40 48 833.8 20.844 9.785 2.13 2.29 RNA 40 49 1352 33.8 15.861 2.13 2.26 RNA 40 50 234.6 5.865 2.817 2.08 1.8 RNA 40 51 115.1 2.877 1.626 1.77 1.11 RNA 40 52 183.7 4.591 2.181 2.1 2.07 RNA 40 53 171.4 4.285 2.048 2.09 1.87 RNA 40 50

54 283.9 7.097 3.601 1.97 1.64 RNA 40 55 220.3 5.507 2.534 2.17 2.07 RNA 40 56 234.6 5.866 2.946 1.99 1.65 RNA 40 57 83.7 2.092 0.983 2.13 1.69 RNA 40 58 101 2.526 1.168 2.16 1.79 RNA 40 59 132.5 3.313 1.586 2.09 1.86 RNA 40 60 302.4 7.559 3.657 2.07 1.87 RNA 40 61 154.4 3.859 1.883 2.05 1.61 RNA 40 62 171.7 4.291 2.127 2.02 1.5 RNA 40 63 694.7 17.368 8.335 2.08 2.23 RNA 40 64 1294.1 32.352 15.301 2.11 2.29 RNA 40 65 152.6 3.815 1.817 2.1 2.04 RNA 40 66 118.8 2.969 1.392 2.13 1.65 RNA 40 67 119.8 2.995 1.405 2.13 1.85 RNA 40 68 132.2 3.306 1.553 2.13 1.74 RNA 40 69 191.7 4.792 2.251 2.13 1.9 RNA 40 70 526 13.151 6.38 2.06 2.15 RNA 40 71 695.2 17.379 8.299 2.09 2.12 RNA 40 72 609.2 15.23 7.38 2.06 2.16 RNA 40 73 102.4 2.559 1.287 1.99 1.33 RNA 40 74 142 3.55 1.944 1.83 0.53 RNA 40 75 548.2 13.706 6.638 2.06 2.12 RNA 40 76 146.9 3.673 2.169 1.69 0.81 RNA 40 77 145.2 3.631 1.782 2.04 1.68 RNA 40 78 205.4 5.135 2.458 2.09 1.65 RNA 40 79 476 11.9 5.859 2.03 1.79 RNA 40 80 490.3 12.259 6.086 2.01 1.89 RNA 40 81 959.6 23.989 11.51 2.08 2.14 RNA 40 82 685.3 17.131 8.195 2.09 2.12 RNA 40 83 131.4 3.284 1.539 2.13 1.9 RNA 40 84 151.9 3.799 1.856 2.05 1.82 RNA 40 85 485.1 12.128 5.842 2.08 2.12 RNA 40 86 213.5 5.336 2.517 2.12 2.06 RNA 40 87 149.6 3.74 1.744 2.14 1.9 RNA 40 88 280.3 7.008 3.354 2.09 1.78 RNA 40 89 212.1 5.302 2.641 2.01 1.82 RNA 40 90 211.9 5.297 2.54 2.09 1.65 RNA 40 91 224.9 5.623 3.45 1.63 0.97 RNA 40 92 375.3 9.383 4.466 2.1 2.08 RNA 40 93 375.7 9.393 4.471 2.1 2.09 RNA 40 94 200 5.001 2.462 2.03 1.84 RNA 40 95 225.9 5.648 2.871 1.97 0.82 RNA 40 96 72.1 1.802 0.952 1.89 0.54 RNA 40 51

97 176.6 4.414 2.121 2.08 1.96 RNA 40 98 293.3 7.334 3.489 2.1 1.79 RNA 40 99 178.1 4.452 2.199 2.02 1.18 RNA 40 100 534.2 13.354 6.222 2.15 2.18 RNA 40 101 453.5 11.338 5.378 2.11 2.03 RNA 40 102 277.2 6.931 3.297 2.1 1.95 RNA 40 103 237.1 5.927 2.836 2.09 1.92 RNA 40 104 357 8.925 4.209 2.12 2.22 RNA 40 105 188.1 4.703 2.252 2.09 2.12 RNA 40 106 227.2 5.68 2.778 2.05 1.98 RNA 40 107 206.9 5.172 2.45 2.11 1.99 RNA 40 108 273.1 6.829 3.228 2.12 2.14 RNA 40 109 171 4.275 2.076 2.06 1.73 RNA 40 110 294.1 7.352 3.442 2.14 2.17 RNA 40 111 298.6 7.464 3.515 2.12 2.21 RNA 40 112 331.1 8.278 3.891 2.13 2.15 RNA 40 113 405.9 10.147 4.821 2.1 2.15 RNA 40 114 234.5 5.863 2.786 2.1 1.91 RNA 40 115 289.3 7.232 3.421 2.11 2.2 RNA 40 116 138.6 3.465 1.698 2.04 1.7 RNA 40 117 172.9 4.321 2.365 1.83 1.33 RNA 40 118 211.6 5.29 2.518 2.1 2.2 RNA 40 119 268.4 6.711 3.195 2.1 2.23 RNA 40 120 164.4 4.109 2.168 1.9 1.49 RNA 40 121 241 6.026 2.87 2.1 1.95 RNA 40 122 151.1 3.776 1.815 2.08 2.11 RNA 40 123 240.4 6.009 2.854 2.11 2.18 RNA 40 124 249.3 6.233 2.934 2.12 2.08 RNA 40 125 234.1 5.853 2.92 2 1.77 RNA 40 126 113.4 2.835 1.414 2.01 1.91 RNA 40 127 474.1 11.852 5.639 2.1 2.07 RNA 40 128 191 4.776 2.357 2.03 1.72 RNA 40 129 293.8 7.346 3.495 2.1 2.15 RNA 40 130 93.9 2.347 1.146 2.05 2.08 RNA 40 131 298.6 7.466 3.584 2.08 1.93 RNA 40 132 127.3 3.182 1.935 1.64 1.03 RNA 40 133 224 5.599 2.711 2.07 1.93 RNA 40 134 86.5 2.163 1.077 2.01 1.57 RNA 40 135 137.6 3.441 1.671 2.06 1.4 RNA 40 136 205.9 5.148 2.474 2.08 2.03 RNA 40 137 198.2 4.956 2.446 2.03 1.55 RNA 40 138 228 5.699 2.802 2.03 1.71 RNA 40 139 260.4 6.51 3.119 2.09 2.07 RNA 40 52

140 121.9 3.046 1.47 2.07 1.93 RNA 40 141 91.1 2.277 1.113 2.05 1.43 RNA 40 142 152.4 3.81 1.872 2.04 1.42 RNA 40 143 206.3 5.157 2.523 2.04 1.84 RNA 40 144 113.9 2.848 1.426 2 1.38 RNA 40 145 128.3 3.207 1.642 1.95 1.74 RNA 40 146 114.8 2.871 1.392 2.06 2.04 RNA 40 147 248.9 6.221 2.956 2.1 1.88 RNA 40 148 224.9 5.623 2.684 2.1 2.11 RNA 40 149 163.7 4.092 2.115 1.94 1.47 RNA 40 150 125 3.124 1.547 2.02 1.73 RNA 40 151 121.9 3.047 1.474 2.07 1.77 RNA 40 152 187.1 4.678 2.239 2.09 1.76 RNA 40

3.5.9 Expression profiling of huanglongbing disease in citrus by realtime quantitative PCR To see the response of citrus and citrus relatives to HLB infection, one of the very sensitive molecular techniques, real time PCR was used. Total RNA was extracted from ≈ 200mg leaf samples of HLB infectedas well as healthy genotypes of citrus and citrus relatives. After purification and concentration, total RNA samples were transformed into complementary DNA by using protoscript II reverse transcriptaseenzyme (NEB) and oligo (dT)20 primer (invitrogen). To make cDNA, 1µg of total RNA was used.The procedure for cDNA synthesis has already been described in section 3.5.2 of experiment 5. Complementary DNA synthesized from fifty one genotypes of citrus was quantified by nanodrop spectrophotometer. The cDNA, synthesized from 51 genotypes of citrus in triplicate, was pooled into 51 cDNA samples. For use in PCR reactions, cDNA aliqoutes were diluted to ten times i.e. 1:10. 3.5.9.1 SYBR green based quantitative real time PCR

For expression profiling of huanglongbing disease in citrus, a SYBR green based reaction was utilized. A 2X SYBR green ready to use mix, SensiMixTM SYBR & Flourescein master mix was obtained from BIOLINE, USA.

Primer pairs for fifteen genes of interest (Liao and Burns, 2012) and a housekeeping gene actin were ordered for synthesis to integrated DNA technologies (IDT), Coralville, Iowa, USA (table 3.24). The primer pairs were dissolved in TE buffer (10: 0.1mM, pH 7.5). Recipe for TE buffer preparation is given in table 3.23. The pH of tris base solution was adjusted at 7.5 by adding few 53 drops of concentrated hydrochloric acid. Similarly, pH of EDTA stock solution was adjusted at 8 by adding and dissolving few pellets of sodium hydroxide.

Table.3.23Composition of TE buffer for primer dissolving

Chemical Stock solution Vol. needed for Working 50 mL TE buffer soln. conc. Tris base 100mM (pH 7.5) 5 mL 10mM EDTA 500mM (pH 8) 10 µL 0.1 mM Millipore 45 mL water

The primers were optimized for their annealing temperatures using gradient PCR and melt curve analysis. It was performed to find out the right temperature for the primers to anneal efficiently to their targets, while preventing non specific annealing and primer dimer formation. A melt curve analysis was performed for each primer at the end of the PCR cycles to confirm the specificity of primer annealing. A triplicate no template control (NTC) reaction also included in every run for each primer pair to test buffers and solutions for cDNA contamination and to assess for primer dimmers.

To evaluate PCR efficiency, cDNA samples from three genotypes of citrus including: Flying dragon, Fortunella hindsii and Clausena harmandia were initially diluted ten fold and from that tenfold dilution, produced two fold dilution series over eight points. For each dilution, a standard qPCR protocol performed in triplicate for all the primer pairs to be used in the experiment.

Sequence from actin gene was used as reference gene for gene expression analysis (Table 3.25). Actin is an extremely abundant protein that comprises a dynamic polymeric network present in all eukaryotic cells, known as actin cytoskeleton (Staigers et al., 2000; Lloyd, 1998).The most notable genes among the above mentioned fifteen genes used in the experiment included for the processes of cell defense, transport, photosynthesis and carbohydrate metabolism to see the gene expression in citrus following infection with the bacterial pathogen Candidatus Liberibacter asiaticus causing huanglongbing.

After optimizations, PCR reactions consisted of 12.5µL of 2X SYBR green ready to use mix, 200 nMof forward and reverse primers and 2µL of diluted cDNA 1:10 in water. Total volume of

54 the PCR reaction mix was adjusted to 20 µL by adding qPCR grade water (ambion). The qPCR reactions were carried out using Bio-Rad CFX96 thermocycler at the department of Botany and Plant Sciences, Bachelor Hall, UCR, USA. Low profile plates with 96 wells (Bio-Rad, MLL9601) were used forPCR.qPCR reactions for all samples were carried out in triplicate. To each well of 96-well plate, added 2µL template first.After the addition of template cDNA, added 18 µL of the mix to each well and sealed with BioRad optical sealer (MSB1001) and mixed briefly. After completion of all wells, briefly spinned the plate at 50xg to eliminate air bubbles and loaded into CFX96 for qPCR.Thermal conditions for SYBR green based PCR reactions are given in table 3.24.

Table 3.24Thermal conditions for SYBR green based PCR reactions

Cycles Conditions Temperature Time 1 Initial denaturation 95 C 10 minutes denaturation 95 C 15 second 39 annealing 60 C 15 second extension 72 C 15 second 95 C 10 second Melt curve 65 C 5 second 95 C end

Table 3.25List of primers used for expression profiling of HLB in citrus by qPCR.

Citrus Primer Accession Primer Primer sequences (5ʹ – 3ʹ) Reference gene ID/ TM number sets CsSUL RC1-55.7 forward ACAGGAATTGCCGCATTTAGAG Liao and F RC2- 55.4 JN793452 reverse AAACCGAATCCTTCCCAATGAC Burns. 2012 CsSB1 RC3- 56.6 forward CCTCCTTCTAAGATGCTTGATGCT Liao and RC4- 55.9 JN793454 reverse GCACCTTCTGATATGCAAGATCG Burns. 2012 CsSB2 RC5- 57.4 forward CAGTAGATGTGGATGCAGTGTCC Liao and RC6- 56.6 JN793455 reverse GCCGTCAATTCCAGGTTCAC Burns. 2012 CsSD1 RC7- 57.8 forward GGTATCCTCCAAGCTGCTGTG Liao and RC8- 55.0 JN793456 reverse ACTTTATCCGATGGGAATGGC Burns. 2012 CsSD2 RC9- 55.1 forward AAGGAATAAAATCCACTGCCGTAG Liao and RC10- JN793457 reverse CTTGGAGGTTCATAATGACCTGGT Burns. 2012 56.6 CsSD3 RC11- forward AAGAATTTTGCGAGAGCTTTAAGTC Liao and 55.2 JN793458 T Burns. 2012 RC12- reverse CCAACTCCAGGGATTTTGCTAC 55.9

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CsSD4 RC13- forward CAGGTCTACTCAGATTACATGAGGA Liao and 58.0 JN793459 GC Burns. 2012 RC14- reverse TTTCTGGATAAGCTGGGTATCTCA 55.3 CsSUR RC15- forward GCGGCG ACTATGGTTTGG Liao and 2 56.7 JN792547 Burns. 2012 RC16- reverse CCT TTTTCATCACTCTAGGATGCA 54.9 CsNCE RC17- forward TCTCTTCAAACACCTTCCATTCTTC Liao and D 55.1 JN794601 Burns. 2012 RC18- reverse TGCAGGTGATGG AGGGTATTTT 56.5 CsACS RC19- forward TTCGAATCCACTAGGCACAACTT Liao and 1 56.5 AJ011095 Burns. 2012 RC20- reverse CAACGCTCGTGAACTTAGGAGA 56.7 CsACO RC21- forward AAGATGGCCAGTGGATTGATG Liao and 55.3 AF321533 Burns. 2012 RC22- reverse TCACCGAGGTTGACAACAATG 55.4 CsLHC RC23- forward TGGAGGGTTCATCTTATCTTGGC Liao and B 56.7 JN793451 Burns. 2012 RC24- reverse CACTGGATCAAGCTCTGCATTT 55.7 CsELIP RC25- forward TCAACCAACCATACCGAAAGC Liao and 55.5 JN793450 Burns. 2012 RC26- reverse ATCGCAAGCCTCCCGTTT AT 56.9 CsPG RC27- forward ATGGAACATCGGCAACAGAAGT Liao and 57.0 EF121320 Burns. 2012 RC28- reverse GTAACTGAGCTTCACATCCTCCAA 56.7 CsATC RC29- forward GCTAATGAAGTCCAGAGTTGCCA Liao and 57.2 JN793453 Burns. 2012 RC30- reverse GCTGCGGTAAAATGCACCAC 57.6 Actin RC31 forward TCACAGCACTTGCTCCAAGCA RC32 reverse TGCTGGAAGGTGCTGAGGGA

Although primer pairs for fifteen genes were optimized for gene expression experiment by qPCR, but among those genes, starch synthesis, carbohydrate metabolism,cell defense and transporterwere selected. From fifty one genotypes those were inoculated with bacterial pathogen Candidatus Liberibacter asiaticus, forty five (Table 3.26) were tested with seven genes of

56 interest including: sulfate transferase(CsSULF), glucose-1-phosphate adenyl transferase(CsSB1), granule bound starch synthase(CsSB2), alpha amylase (CsSD1), alpha amylase 3 (CsSD2), beta amylase 9(CsSD3) and cytochrome P450 mono oxygenase 83B1 (CsSUR2). Relative expression of the said genes were calculated using the software, CFX manager version 3.0.1224.1015.Calculations for the relative quantity, accurate normalization and fold change of gene expression were done according to Pfaffl, (2001) and Vandesompele et al. (2002) using formulas: For relative quantity, ∆Ct = GOI- HKG Where: GOI= average Ct values of gene of interest and HKG= average Ct values of housekeeping gene. For normalization, ∆ ∆Ct = ∆Ct experimental samples - ∆Ct controls Fold change = 2(-∆ ∆Ct)

Table 3.26 Citrus germplasm tested for expression profiling of HLB Sr. Group Group Cultivar Binomial Accession No. No. No. 1 1 Calamondin Calamondin Citrus madurensis PI 539349 2 2 Citrange Benton X citroncirus sp. PI 539819 3 Citrange Troyer X citroncirus sp. PI 539810 4 3 Citron Bengal Citrus medica PI 539417 5 Citron Indian Citrus medica PI 539413 6 Citron Papuan Citrus medica PI 539430 7 Citron Philipine Citrus medica PI 539431 8 Citron S-1 Citrus medica PI 539441 9 Citron Sicily Citrus medica PI 539432 10 4 Citrumelo Sacaton X citroncirus sp. PI 539826 11 5 Citrus relative Clausena Harmandiana Clausena PI 600640 harmandiana 12 Citrus relative Eremocitrus Glauca Eremocitrus glauca PI 654891 hybrid 13 Citrus relative Glycosmis Pentaphylla Glycosmis PI 127866 pentaphylla 14 Citrus relative Hawaiian Mock orange Murraya paniculata PI 539747 15 6 Grapefruit Duncan Citrus paradisi PI 539482 16 7 Kumquat Fortunella Hindsii Fortunella hindsii PI 539723

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17 8 Lemon- Eureka Frost Eureka Citrus limon PI 539318 18 10 Mandarin Citrus Sunki Citrus sunki PI 539678 19 Mandarin Kinnow Citrus reticulata Pak Blanco 20 Mandarin Parson‟s Special Citrus reticulata PI539497 21 Mandarin Sun Chu Sha Citrus reticulata PI 539544 22 11 Mandarin Borneo Rangpur Citrus limonia hybrid - RCRC 4132 Rangpur 23 12 Microcitrus Var. Sanguinea Microcitrus PI 312872 australasica 24 13 Papeda Honghe Citrus hongheensis PI 539672 25 14 Papeda hybrid Yuzu Citrus junos PI 45945 26 15 Pummelo Reinking Citrus maxima PI 539391 27 Pummelo Tahitian Citrus maxima PI 539392 28 16 Rangpur Srirampur Nucellar Citrus limonia RCRC 4137 29 Rangpur Tuningmeng Nucellar Citrus limonia RCRC 4139 30 17 Rough lemon Schaub Citrus jambhiri PI 539266 31 18 Sour orange Brazillian Sour orange Citrus aurantium L. Pak 32 Sour orange Gadadehi Citrus aurantium Pak 33 Sour orange Goutoucheng Citrus aurantium PI 539170 34 Sour orange Nansho Daidai Citrus taiwanica PI 539680 35 Sour orange Sour Orange Citrus aurantium L. Pak 36 19 Sour orange Keen Sour orange Citrus aurantium L. Pak hybrid 37 20 Sweet orange Madam Vinous Citrus sinensis PI 539625 38 Sweet orange Pineapple Citrus sinensis PI 539622 39 Sweet orange Succari Citrus sinensis Pak 40 21 Tangelo Orlando Citrus X tangelo PI 539711 41 22 Trifoliate Citromelo 1452 X citroncirus sp. Pak 42 Trifoliate Flying Dragon Poncirus trifoliata PI 539768 43 Trifoliate Rich 16-6 Citrus trifoliata PI 539776 44 23 Trifoliate X-639 X citroncirus sp. PI 539847 hybrid 45 24 Valencia orange Frost Citrus sinensis PI 539660

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3.5.10 RNA-Seqlibrary preparation for Illumina

For learning of advance technique for expression profiling, RNA-Seq libraries for Illumina were also practiced to prepare. Six pairs of citrus genotypes comprising of healthy and inoculated were selected for RNA-seq libraries preparation. Those six genotypes were: Benton, Clausena Harmandiana, Glycosmis Pentaphylla, Kinnow (Citrus reticulata Blanco), Sweet orange (Citrus sinensis) and Keen sour orange. For RNA-seq library preparation, kits manufactured by NEW ENGLAND BioLabs were used.Three different types of kits used were as follows:

1- NEB Next Ultra RNA Library Prep Kit for Illumina. NEB # E7530S/L

2- NEB Next Poly(A) mRNA Magnetic Isolation Module (NEB # E7490)

3- NEB Next Multiplex (NEB #7335) Oligos for Illumina

Other materials required for library preparations were:

1- Magnetic rack

2- AgencourtR AMPureRXP beads (Beckman Coulter, Inc. # A63881)

3- Nitrile, powder free examination gloves. Kimberly-Clark 500.

4- Freshly prepared 80% ethanol.

In total, 12 libraries were prepared from 6 genotypes of citrusc.

Preparation of libraries was started from total RNA. About 1µg total RNA quantified by bioanalyzer for each genotype was used as starting material. Procedures for library preparation were followed according to manuals of kit manufacturers. The process was laborious and intricate. A little bit negligence could result in the loss of precious RNA samples as well as costly kit contents. Only three samples per day were possible to reach the step where the library preparation could be stopped and samples could be stored at -20 C.For completion, another complete working day was required. Following steps were involved in the library preparation for RNA-seq analysis:

1- Messenger RNA isolation

2-Fragmentation and priming

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3-First strand cDNA synthesis

4-Second strand cDNA synthesis

5-Purification of double-stranded cDNA using 1.8X Agencourt AMPure XP beads

6-End repair/dA-tail of cDNA library

7-Adaptor ligation

8-Purification of ligation reaction using AMPure XP beads

9-Library enrichment by PCR

10-Purification of PCR reaction using Agencourt AMPure XP bead

11-Assessment of library quality on bioanalyzer.

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CHAPTER-4

RESULTS AND DISCUSSION

Citrus is one of the most important fruit crops contributing in the revenue of Pakistan. Bahrain, Dubai, Indonesia, Kuwait, Malaysia, Netherlands, Oman, Qatar, Russia ,Saudi Arabia,Singapore, and UK are the major market places of Pakistan‟s kinnow. Different diseases and pests affect the genus citrus, resulting in reduced quality and taste of fresh fruit, low production and ultimately less profit for the growers (Martinelli et al.,2015). Today, huanglongbing is the major problem for citrus producing countries including Pakistan all over the world.Different strategies are being used for the management of HLB, but still there is no report of complete cure of the disease. Present study is an effort to raise the citrus germplasm in greenhouse conditions and study the expression of some selected genes in response of HLB infection by molecular techniques.

4.1. EXPERIMENT-1 Rearing of citrus germplasm in greenhouse. Citrus germplasm seed (PI numbers) was acquired from the USDA National Clonal Germplasm Repository for Citrus and Dates, Riverside, California, USA and University of Agriculture, Faisalabad, Pakistan source trees (Table 3.1). Out of 97 genotypes of citrus seeds sown in controlled conditions of greenhouse ofInstitute of Horticultural Sciences (IHS), University of Agriculture Faisalabad (UAF), Pakistan, 51 genotypes were survived in sufficient number to meet the requirement of replicates for inoculation purpose as well as healthy controls (Table 4.1).

From 97 genotypes of citrus sown for the experiment, 8 more types along with 51 also germinated and survived. Those 8 varieties were: Citrumelo 1452, C35, Savage citrange, Improved Meyer, Kharna, Nules, Scarlet Emperor and Palestine belonging from Trifoliate, Citrange, Lemon hybrid, Mandarin and Sweet lime groups. The reason for not including those genotypes in the list of 51 survived was, that some seedlings died before inoculation, and from the remaining plants, inoculated replicates were died. The remaining healthy number of plants was not enough to inoculate them in triplicate. Apart from that, the time period of inoculation

61 would be different for those 8 varieteis if inoculated later and the results for the detection of bacterial pathogen and gene expression could be biased.

There was not hundred percent result of seedlings survival from all goups of citrus genotypes. Althoug, the number of cultivars survived from the groups of genus citrus including: Calamondin, Grapefruit, Lemon eureka, Mandarin hybrid Rangpur, Tangelo and Valencia orange was found to be 100% (Fig.4.1).

Table 4.1.Survivedcitrus germplasm from 97 sown genotypes of citrus

Sr.No. Group Group Cultivar Binomial Accession No. No. 1 1 Calamondin Calamondin Citrus madurensis PI 539349 2 2 Citrange Benton X citroncirus sp. PI 539819 3 Citrange Carrizo X citroncirus sp. PI 150916 4 Citrange Troyer X citroncirus sp. PI 539810 5 3 Citron Bengal Citrus medica PI 539417 6 Citron Corsican Citrus medica PI 539415 7 Citron Indian Citrus medica PI 539413 8 Citron Papuan Citrus medica PI 539430 9 Citron Philipine Citrus medica PI 539431 10 Citron S-1 Citrus medica PI 539441 11 Citron Sicily Citrus medica PI 539432 12 4 Citrumelo Sacaton X citroncirus sp. PI 539826 13 5 Citrus relative Clausena Harmandiana Clausena PI 600640 harmandiana 14 Citrus relative Eremocitrus Glauca Eremocitrus glauca PI 654891 hybrid 15 Citrus relative Glycosmis Pentaphylla Glycosmis PI 127866 pentaphylla 16 Citrus relative Hawaiian Mock orange Murraya paniculata PI 539747 17 6 Grapefruit Duncan Citrus paradisi PI 539482 18 7 Kumquat Fortunella Hindsii Fortunella hindsii PI 539723 19 8 Lemon- Frost Eureka Citrus limon PI 539318 Eureka 20 9 Lemon hybrid Unnamed (Tavares Citrus limon hybrid PI 539804 limequat) 21 10 Mandarin Citrus Sunki Citrus sunki PI 539678 22 Mandarin Kinnow Citrus Pak

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reticulataBlanco 23 Mandarin Parson‟s Special Citrus reticulata PI539497 24 Mandarin Sun Chu Sha Citrus reticulata PI 539544 25 11 Mandarin Borneo Rangpur Citrus limonia hybrid - RCRC 4132 Rangpur 26 12 Microcitrus Var. Sanguinea Microcitrus PI 312872 australasica 27 13 Papeda Honghe Citrus hongheensis PI 539672 28 14 Papeda hybrid Yuzu Citrus junos PI 45945 29 15 Pummelo Reinking Citrus maxima PI 539391 30 Pummelo Tahitian Citrus maxima PI 539392 31 16 Rangpur Knorr Nucellar Citrus limonia RCRC 4132 32 Rangpur Rangpur Poona Citrus limonia RCRC 4135 Nucellar 33 Rangpur Srirampur Nucellar Citrus limonia RCRC 4137 34 Rangpur Tuningmeng Nucellar Citrus limonia RCRC 4139 35 17 Rough lemon Florida Citrus jambhiri PI 539268 36 Rough lemon Schaub Citrus jambhiri PI 539266 37 18 Sour orange Brazillian Sour orange Citrus aurantium L. Pak 38 Sour orange Gadadehi Citrus aurantium Pak 39 Sour orange Goutoucheng Citrus aurantium PI 539170 40 Sour orange Nansho Daidai Citrus taiwanica PI 539680 41 Sour orange Sour Orange Citrus aurantium L. Pak 42 19 Sour orange Keen Sour orange Citrus aurantium L. Pak hybrid 43 20 Sweet orange Madam Vinous Citrus sinensis PI 539625 44 Sweet orange Pineapple Citrus sinensis PI 539622 45 Sweet orange Succari Citrus sinensis Pak 46 21 Tangelo Orlando Citrus X tangelo PI 539711 47 22 Trifoliate Citrumelo 1452 X citroncirus sp. Pak 48 Trifoliate Flying Dragon Poncirus trifoliata PI 539768 49 Trifoliate Rich 16-6 Citrus trifoliata PI 539776 50 23 Trifoliate X-639 X citroncirus sp. PI 539847 hybrid 51 24 Valencia Frost Citrus sinensis PI 539660 orange

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Figure 4.1.Groups of citrus presenting total number of cultivars sown and survived

16 14 12 10 8 6 4 No. of cultivars sown 2

0 No. of cultivars survived

Citron

Tangor

Kumquat

Calamondin

Roughlemon

Sweet orange

Lemonhybrid

Citrusrelative

Papeda hybrid

Trifoliate hybrid

Pummelohybrid Microcitrus hybrid Sour orange hybrid

Figure 4.2 (A&B)Germination of citrus germplasm seedlings

A

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B

4.2. EXPERIMENT- 2 Asian citrus psyllid rearing For expression profiling of huanglongbing disease in citrus germplasm, it was necessary to keep the germplasm free of any other graft transmissible disease except huanglongbing. For this purpose, Asian citrus psyllid (ACP) Diaphorina citri (D. citri) were captured from HLB positive sweet orange trees tested by conventional PCR and released for rearing in the growth room situated in a laboratory at the Center of Agricultural Biochemistry and Biotechnology (CABB), University of Agriculture Faisalabad (UAF), Pakistan. Screenhouse raised healthy plants of sweet orange (Citrus sinensis) and kinnow (Citrus reticulata Blanco) were used for ACP release and rearing. Pruning of those plants was done to induce the growth of new flushes and equal height of all plants. The same work has been reported by Hall and Albano (2014) with different genotypes of citrus in a way that the selection of a rearing plant for ACP may be greatly influenced by the flushing properties of a plant species, especially if the goal is to produce large numbers of ACP. This is because ACP is dependent on flush for reproduction. Plants can be trimmed to stimulate flush growth. For ACP rearing,Murraya exotica (Skelley and Hoy, 2004;

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Hall et al., 2007) and Citrus macrophylla(Hall and Richardson, 2013)has been used. Westbrook et al. (2011) have studied eighty seven genotypes of citrus and its relatives in field for colonization of ACP. As the temperature of the growth room was optimum for the growth of plants and ACP, very soon after the release, we were able to see the colonies of nymphs on the new flushes of both varieties. According to Liu and Tsai (2000), the optimum range of temperatures for the growth of ACP population was recorded as 25-28ᴼC.

The white waxy excretions called honeydew of the nymphs are the evidence of the presence of D. citri(Fig. 4.3A & B; Fig. 4.5A). The adults can also be seen in the figures on citrus leaves or branches sitting at an angle of 45ᴼ as mentioned by some other researchers (Wenninger et al., 2009; Bove, 2006).

As far as the expression of HLB symptoms in the infested plants of sweet orange and kinnow are concerned, typical symptoms of HLB in leaves expressed in both types. New flushes on both varieties arose with twisted leaves. Very clear blotchy mottled leaves as well as vein yellowing symptoms were also expressed on the infested plants (Fig.4.4A & B; Fig. 4.5B).

Figure 4.3. Colonies of ACP on Sweet orange and Kinnow plants in growthroom conditions

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Figure 4.4 Appearance of blotchy mottle, vein yellowing and leaf curling symptoms in sweet orange and kinnow plants after acquisition of HLB bacterium from natural vector in growthroom conditions

Figure 4.5 Successful rearing of ACP resulting in establishment of ACP colonies and expression of HLB symptoms on the source plants

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4.3 EXPERIMENT- 3 Huanglongbing diagnosisin ACP infested plants and inoculation of citrus germplasm 4.3.1 Huanglongbing diagnosis The sweet orange and kinnow plants were used for the colonization and infestation by Asian citrus psyllids (ACP) in controlled conditions of growth room at the University of Agriculture, Faisalabad, Pakistan. The ACP were collected from HLB positive sweet orange field plants of Faisalabad. As the insect vector of HLB pathogen was captured from HLB positive plants and released on the growth room plants for one year, those plants were kept under observation for HLB symptom appearance. Blotchy mottle and vein yellowing symptoms along with twisted small sized leaves were found to be present in almost all of the infested pants (Fig. 4.4 A& B; Fig.4.5B). After one year of infestation, they were tested for the presence of HLB pathogen by conventional PCR. The primer pairs OI1/OI2c and A2/J5 were used for the amplification of pathogen‟s 16S ribosomal RNA gene and rplKAJL-rpoBC beta operon respectively. A product of 1160 nucleotides (Fig. 4.8) and ~700 nucleotides (Fig. 4.9) was observed for OI1/ OI2c and A2/J5 primer pairs respectively on agarose gel as also reported by other researchers (Ruanguang and Akarapisan, 2006; Chohan et al., 2007; Razi et al., 2014). For the amplification of 16S/23S rDNA intergenic region, primer pair OI2/23S1 was used, resulting in an amplicon of about 800 bp (Fig. 4.7) indicating the presence of Candidatus Liberibacter asiaticus in the sweet orange and kinnow plants infested by ACP as reported by the founder of the primer OI2/23S1 (Jaguoeix et al., 1997) and other researchers (Subandiyah et al., 2000; Ding et al., 2009).

4.3.2 Inoculation of citrus germplasm

After the detection of HLB pathogen in ACP infested sweet orange plants, fifty one genotypes of citrus germplasm (Table 4.1) were inoculated by grafts and leaf midribs for the transmission of HLB bacterium: Candidatus Liberibacter asiaticus. Several workers reported inoculation by grafting. For grafting they used budsticks of 2 to 4 cm size from field trees, infected with huanglongbing bacteria and grafted on half to one year old plants of citrus (Lopes and Frare, 2008).

Reports of several experiments showed that higher number of Ca.L.asiaticus were transmitted as compare to Ca. L. Americanus. It was assumed on the basis of qPCR results that higher

68 transmission efficiency is due to higher multiplication of Ca. L. asiaticus in the phloem of citrus (Lopes et al., 2009a).

To see the success and response of inoculation, observation of HLB symptom expression was started after one month of inoculation. Inspite of nutrients application, the leaf color of inoculated plants was found to be light green as compared to dark green for the healthy control plants. Of 51 genotypes of citrus and citrus relatives, only 21 genotypes (41 %) expressed HLB symptoms in leaves including: blotchy mottle, vein yellowing, vein corking and leaf yellowing (Fig.4.10). whereas, 30 genotypes (59 %) were found to have no observable symptoms (table 4.2). Vein yellowing symptom of HLB was observed in highest number of citrus genotypes (76%) while, blotchy mottle, vein corking and leaf yellowing was observed in 66.6%, 33.3% and 19% of citrus genotypes respectively (Fig. 4.11).

Figure 4.6 DNA extracted from healthy controls and HLB infected genotypesofcitrus.Lane1-6= healthy controls, lane7-12 = inoculated

Figure 4.7 Amplification of16S&23S intergenic region showing 800bpamplicon specific for Candidatus Liberibacter asiaticus

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Figure 4.8Candidatus Liberibacter asiaticus detection in sweet orange plants infested by ACP.Amplicons of 1160bp obtained in plants 4&5 by using16S rDNA primer OI1/OI2C. M1= 1Kb DNA ladder, lane 1= no template control, 2&3= healthy controls,4&5 HLB infected samples

Figure 4.9Candidatus Liberibacter asiaticus detection in sweet orange plants infested by ACP.Amplicons of 703 bp obtained in plants 2-4 by usingLas specific primer A2/J5. M=1Kb ladder, 1=healthy control, 2= positive control,3, 4 &5= inoculatedplants

Table 4.2 Citrus genotypesexpressing huanglongbing symptoms Sr.No. Group Cultivar Blotchy Vein Vein Leaf mottle yellowing corking yellowing 1 Mandarin Citrus Sunki - -  - 2 Mandarin Kinnow  - - - 3 Mandarin Parson‟s Special     4 Mandarin Sun Chu Sha     5 Tangelo Orlando  -

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6 Citron Papuan  - - - 7 Citron Corsican   8 Citron Indian  -  - 9 Citron Sicily  - - - 10 Sour orange Keen Sour orange   - - hybrid 11 Sour orange Nansho dai dai - -   12 Sour orange Brazillian Sour   - - orange 13 Citrus relative Hawaiian Mock   - - orange 14 Sweet orange Pineapple -   - 15 Sweet orange Succari   - - 16 Trifoliate X-639 -   - hybrid 17 Citrange Carrizo -  - - 18 Lemon hybrid Tavares limequat   - - 19 Mandarin Borneo Rangpur   - - hybrid Rangpur 20 Rangpur Rangpur Poona -  - - nucellar 21 Rangpur Tuningmeng   - - Nucellar

Figure 4.10 Bars showinghuanglongbing symptoms expressionin citrus germplasm

HLB symptoms in citrus germplasm 35 30 25 20 15 10 No. of citrus 5 genotypes expressing 0 No. of citrus citrus of No. genotypes symptoms

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Figure 4.11Inoculated citrus plants in screenhouse showing blotchy mottle and vein yellowing symptoms of HLB (A); Hawaiian Mock orange (B); Brazillian Sour orange (C); Corsican (D); Tuningmeng Nucellar (E); Keen Sour orange (F); Indian (G); Rangpur Poona nucellar (H); Papuan (I); Sicily and (J); Orlando

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Experiment4

4.4 Detection of Candidatus Liberibacter asiaticus in citrus germplasm

A totalof 306 DNA samples were extracted from 51 genotypes of citrus including inoculated and healthy replicates. The replicates of DNA were pooled into 102 samples comprising of 51 for inoculated and 51 for healthy controls.Candidatus Liberibacter asiaticus was detected in citrus germplasmDNA by usingmultiplex real time quantitative PCR. Each PCR reaction was run in triplicate for each genotype for the detection of HLB bacterium. The measurement was made after each amplification cycle, and this is the reason why this method is called real time PCR. Quantitative TaqMan PCR was conducted using 16S rDNA based TaqMan primer-probe set specific to Candidatus Liberibacter asiaticus.Aprimer-probe set based on plant cytochrome oxidase (COX) gene was used as a positive internal control to assses the quality of the DNA extracts. This assay does not cross-react with other pathogens or endophytes commonly resident in citrus plants and is very sensitive (Li et al., 2006).

The DNA from field trees of sweet orange, cultivar Succari, was used as a positive control for HLB bacterium detection. Keeping in view the results of some other researchers, we considered the DNA samples positive, for HLB bacterium,with Ct values up to or less than 36.9 (Fig.4.10 A-D). The DNA samples with Ct values above 36.9 or no amplification (NA) were considered negative for the presence of HLB pathogen (Hoffman et al., 2013). No amplification was observed in healthy controls of citrus germplasm. No amplification signals in inoculated genotypes of citrus germplasm indicate that there was no detectable titre in them, while, in the healthy controls mean that the samples were not infected.

Real time qPCRamplification results for Candidatus Liberibacter asiaticus detection in citrus germplasm revealed no amplification or zero amplification in Clausena harmandiana. The mean Ct value of 37.70 was obtained for Glycosmis pentaphylla indicating these plants negative for HLB(Hoffman et al., 2013). Of 51 genotypes of citrus, a large number of citrus genotypes were found to have Ct values between 36.9 and 35 including: Schuab, Florida, Sacaton, Sour orange, Gou tou Cheng, Flying Dragon, S-1, Hawaiian Mock orange, Keen Sour orange, Benton and Var. Sanguinea. Citru genotypes having Ct values between 35 and 30 indicate medium titre of the bacterial pathogen in citrus host. The varieties included in the range of 35 to 30 were:

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Bengal, Eremocitrus Glauca hybrid, Philipine, Reinking, Rich 16-6, Papuan and Troyer. Citrus genotypes with Ct values between 30 and 20 indicate a high titer of the bacteria causing HLB disease in citrus host. All of the remaining genotypes, except those presented in the range of Ct values of 36.9 to 30, fall in the category of highly susceptible varieties for HLB also called .From 51 genotypes of citrus, the proportion of HLB positive and negative genotypes came at 96.07% and 3.92% respectively. From this HLB positive, 96.07% part of citrus genotypes, 11 varieties (22.45% ) fall in the category, having low titer of Ca. Las in a range of 36.9 to 35 mean Ct values while, only 7 varieties (14.28%) contained medium titer of Ca. Las showing a range of 35 to 30 mean Ct values. Remaining 31citrus genotypes (63.3%) were found to have a high titer of Ca. Las with Ct values ranging from 30 to 20 (Fig. 4.12).

All of the inoculated varieties of citrus were found infected upon testing for HLB pathogen with a difference of percentage of infection except Clausena harmandiana and Glycosmis pentaphylla. Among 24 groups of citrus in this study, Mandarins and Sweet oranges were found to have lowest mean Ct values indicating higher rates of HLB infection (Fig 4.13 A-D; Table 4.3).The highest rate of HLB infection was observed in Sweet orange cultivar Succari with a minimum mean Ct value of 20.0. Citrus relative, Glycosmis Pentaphylla produced lowest signals for HLB pathogen detection in real time qPCR with a mean Ct value of 37.70 (Table 4.3).Amplification plots for Candidatus Liberibacter asiaticus 16S rDNAdetection and plant‟s cytochrome oxidase gene detection in citrus plants are shown in figure 4.14 and 4.15 respectively.

4.4.1 Correlation between HLB symptoms and Ct values There were 21genotypes of citrus that expressed huanglongbing symptoms.The number and type of symptoms expressed by each genotypewere mixed. The correlation coefficientwas calculated statistically by using Pearson‟s correlation formula in MS excell between HLB symptoms and cycle threshold values (Fig. 4.16).A negative correlation between symptoms and ct values was obtained with a value of -0.253321. It is concluded from the result that the intensity of HLB infection in citrus host is related to some extent with the expression of symptoms because there are some genotypes of citrus that do not express HLB symptoms but their Ct values show them susceptible e.g., Eremocitrus Glauca hybrid, Honghe and Bengal.

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Figure 4.12 HLB positive citrus genotypes with a range of Ct values from 36.9-20 and their number in that range presenting the titer of CandidatusLiberibacter asiaticus on the basis of Ct values

No. of citrus genotypes 35

30

25

20

15 No. of citrus genotypes

10

5

0 Mean Ct values 36.9-35 35-30 30-20

Figure 4.13 A-D.Ct values of 51 genotypes of graft inoculated citrus germplasm

40 35 30 25 20 15 10 5 0

A Citrus genotypes

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40 35 30 25 20 15 10 5 0

B

40 35 30 25 20 15 10 5 Series1 0

C

40 35 30 25 20 15 10 Series1 5 0

D 77

Figure 4.14Candidatus Liberibacter asiaticus amplification plot showing Las detection in citrus plants

Pos ctrl

Reinking Tahitian Corsican Yuzu Fortunella Hindsii

Figure 4.15 Plant’s cytochrome oxidase gene amplification plot in citrus plants

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Table 4.3.Mean cycle threshold values in citrus germplasm DNA for the detection of Candidatus Liberibacter asiaticus

Sr.No. Group Cultivar Fluor FAM Ct Mean 1 Calamondin Calamondin 24.10 2 Citrange Benton 35.08 3 Carrizo 28.71 4 Troyer 33 5 Citron Bengal 33.92 6 Corsican 28.47 7 Indian 24.21 8 Papuan 34.18 9 Philipine 32.29 10 S-1 35.92 11 Sicily 29.60 12 Citrumelo Sacaton 36.55 13 Citrus relative Clausena Harmandiana NA 14 Eremocitrus Glauca 33.97 hybrid 15 Glycosmis Pentaphylla 37.70 16 Hawaiian Mock orange 35.68 17 Grapefruit Duncan 29.81 18 Kumquat Fortunella Hindsii 24.05 19 Lemon- Eureka Frost Eureka 26.09 20 Lemon hybrid Unnamed (Tavares 24.35 limequat) 21 Mandarin Citrus Sunki 23.95 22 Kinnow 25.06 23 Parson‟s Special 24.30 24 Sun Chu Sha 22 25 Mandarin Borneo Rangpur 28.55 hybrid -Rangpur 26 Microcitrus Var. Sanguinea 35.74 27 Papeda Honghe 24.01 28 Papeda hybrid Yuzu 26.0 29 Pummelo Reinking 30.78 30 Tahitian 27.95

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31 Rangpur Knorr Nucellar 26.30 32 Rangpur Poona Nucellar 27.30 33 Srirampur Nucellar 25.71 34 Tuningmeng Nucellar 27.75 35 Rough lemon Florida 36.30 36 Schaub 35.68 37 Sour orange Brazillian Sour orange 24.02 38 Gadadehi 28.90 39 Goutoucheng 35.92 40 Nansho Daidai 25.06 41 Sour Orange 35.29 42 Sour orange Keen Sour orange 36.21 hybrid 43 Sweet orange Madam Vinous 24.18 44 Pineapple 25.69 45 Succari 20.0 46 Tangelo Orlando 24.31 47 Trifoliate Citromelo 1452 29.10 48 Flying Dragon 35.36 49 Rich 16-6 33.67 50 Trifoliate hybrid X-639 26.0 51 Valencia orange Frost 24.81

Figure 4.16 Correlation between HLB symptoms and Ct values of citrus germplasm

40 35 30

25 Ct values 20 Symptoms

Ct values Ct 15 Linear (Ct values) 10 Linear (Symptoms) 5 0 0 5 10 15 20 25 Symptoms

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Experiment5 4.5. Expressionprofiling of huanglongbing disease in citrus germplasmby realtime quantitative PCR To study the response of citrus and citrus relatives to HLB infection compared with healthy plants, real time quantitative PCR was used. Forty five genotypes of citrusinoculated with bacterial pathogen,Candidatus Liberibacter asiaticus, were tested with seven selected genes from Liao and Burns (2012). These genes were selected because of: 1- Gene sequences were identical to the functional homologues of already published research (Howarth et al., 2003; Li et al., 2003; Krinke et al., 2009; Liao and burns, 2012). 2- Selected genes were confirmed for their response to pathogen infection as indicated by published research. For expression profiling of HLB in citrus, primer pairs for 7 genes were selected based on their functions. Those 7 genes with their putative functions were: Sulfate transferase (CsSULF), a transporter gene for sulfate transport.Five genes involved in the regulation of starch metabolism were; glucose-1-phosphate adenyl transferase (CsSB1), granule bound starch synthase (CsSB2), alpha amylase (CsSD1), alpha amylase 3 (CsSD2)and beta amylase 9(CsSD3). Cytochrome P450 mono oxygenase 83B1 (CsSUR2)involved in defense response is a phytohormone related gene. Relative expression of the said genes were calculated using the software, CFX manager version 3.0.1224.1015. Gene expression data analysis results for relative quantity and fold change for all of the seven genes in forty five genotypes of citrus in response of HLB infection are given. Relative quantities of the target genes were determined with respect to healthy control of each genotype. For normalization, actin gene was used as reference or housekeeping. Upregulated and down regulated gene expression values with fold change compared to healthy controls are presented in table 4.4.The negative values are representing down regulation whereas, positive values indicate up regulation of the respective gene in a given genotype of citrus.

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Table 4.4 Comparison of expression of 7 genes in 45 genotypes of HLB infected citrus using qRT PCR analysis

Sr. Group Cultivar Fold change No. CsSU CsSB CsSB CsSD CsSD CsSD CsSU LF 1 2 1 2 3 R 1 Calamondin Calamondin 2 10 NA = NA 2 Citrange Benton 4 6.9 16 23 NA 5 3 Citrange Troyer 4 -2.5 -9 -16 = -9 4 Citron Bengal -7 -5.5 -1.7 NA NA -60 5 Citron Indian = 2 -47 -25 NA 6 Citron Papuan -2 -2 = NA -3 7 Citron Philipine = NA -68 -10 NA NA 8 Citron S-1 -3 23 11 4 5 2 9 Citron Sicily 12 -147 = = 2 10 Citrumelo Sacaton 4 -9 = 5 10 11 Citrus Clausena -2 -177 NA NA NA -55 relative Harmandiana 12 Citrus Eremocitrus = -6 = = -2 -2 -2 relative Glauca hybrid 13 Citrus Glycosmis = NA NA NA NA NA relative Pentaphylla 14 Citrus Hawaiian 3 -57 = NA 26 relative Mock orange 15 Grapefruit Duncan = -3.3 -3 -2 = = 16 Kumquat Fortunella -2 NA -9 5 2 8 Hindsii 17 Lemon- Frost Eureka = -2 -26 -7 -8 Eureka 18 Mandarin Citrus Sunki -8 = -22 -3 NA 19 Mandarin Kinnow -24 NA 25 9 = 20 Mandarin Parson‟s -20 -454 2 5 -2 Special 21 Mandarin Sun Chu Sha 44 -2 -36 NA 3 22 Mandarin Borneo = 1.3 -4 3 3 hybrid - Rangpur Rangpur 23 Microcitrus Var. Sanguinea 20 3 -10 -19 = 24 Papeda Honghe -8 NA NA 2 26 25 Papeda Yuzu -3 -2 -1.8 -7 -2 -4 hybrid

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26 Pummelo Reinking 6 -73 -1.4 NA 3 27 Pummelo Tahitian -45 NA NA NA NA NA 28 Rangpur Srirampur -4 -173 -21 -56 NA NA Nucellar 29 Rangpur Tuningmeng = -5 -1.5 = NA Nucellar 30 Rough Schaub -44 NA NA NA NA NA lemon 31 Sour orange Brazillian Sour = -12.7 -2 -3 -4 orange 32 Sour orange Gadadehi -3 -17 -225 NA -58 -1.6 33 Sour orange Goutoucheng 1025 -7 NA NA NA 34 Sour orange Nansho Daidai 2 NA -6 4 NA = 35 Sour orange Sour Orange -2 -3.5 -16 NA -10 -23 36 Sour orange Keen Sour -8 2 -54 NA -15 -5 hybrid orange 37 Sweet Madam Vinous 52 = = 21 56 13 orange 38 Sweet Pineapple = -2.7 = = 2 2.7 = orange 39 Sweet Succari 97 -7 -2 = 2 orange 40 Tangelo Orlando = -487 -8 2 2 41 Trifoliate Citromelo 16 -2 -2 = NA NA 1452 42 Trifoliate Flying Dragon 12 -205 5 3 23 43 Trifoliate Rich 16-6 5.3 -2.6 = 3 9 44 Trifoliate X-639 = -3 -13 -4 hybrid 45 Valencia Frost -24 NA -479 NA NA NA orange

4.5.1 Sulfate transferase (CsSULF)

From 51 genotypes of citrus, only 2 were found to be negative for the presence of Candidatus Liberibacter asiaticus using Taqman based qPCR. These two genotypes may be placed in tolerant category against HLB as the causal bacterium was not detected in them. Transporter gene,CsSULF was expressed at equal level in healthy and diseased Glycosmis pentaphyllawhile, it was 2 fold down regulated in Clausena harmandian. Remaining genes did not express in

83 inoculated cDNA samples of both of these varieties. When the citrus genotypes with Ct values ranging from 20 to 25 for Las detection by qRT PCR were compared for the gene expression changes withClausena harmandianaand Glycosmis pentaphylla following results were found: Sulfate transferase (CsSULF) gene was upregulated in Calamondin, Sun chu sha, Madam vinous and Succari sweet orange. Overall picture for upregulation of CsSULF gene among 45 genotypes of citrus indicate 16 genotypes with 2 to 1025 fold change (Table 4.5).These results suggest that upregulation of CsSULF was due to sulfur deficiency,indicating week root systemin susceptible genotypes(Kataoka et al., 2004; Liao and Burns, 2012).In Tahitian and Schuab no gene was amplified except CsSULF gene that was down regulated indicating strong root system for plant survival. Relative expression of CsSULF is presented in figure (4.17 A&B).

Figure 4.17(A,B) Relative expression of sulfate transferase (CsSULF) in healthy and inoculated citrus germplasm

A

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B

4.5.2 Glucose-1-Phosphate adenyl transferase (CsSB1)

Glucose-1-Phosphate adenyl transferase is also known as ADP glucose pyrophosphorylase. This enzyme takes part in starch and sucrose metabolism.Relative expression of CsSB1in healthy and inoculated citrus germplasmis shown in figure 4.18 (A&B).qPCR results for gene expression in HLB infected citrus germplasm reveal upregulation of this enzyme in 6 genotypes including: Calamondin, Benton, Indian, Keen sour orange, S-1 and Var Sanguinea (Table 4.5). These results resemble with the qRT PCR results of Albrect and Bowman (2008). They reported 8.33 fold upregulation of CsSB1in HLB affected sweet orange.A reaction of sweet orange to Ca. L. asiaticus attack with microarray analysis indicated the up-regulation of ADP-glucose pyrophosphorylase(Kim et al., 2009). Up regulation as well as down regulation in HLB infected plants relate to the starch synthesis. Equal expression or no amplification of this gene was found in Madam Vinous, Glycosmis Pentaphylla, Citrus Sunki, Honghe, Philipine, Schuab, Nansho daidai and Tahitian suggesting no starch accumulation in the said varieteis. CsSB1 was down regulated in 27 genotypes of HLB infected citrus. This result is in agreement with the results of Liao and Burns (2012) and Martinelli et al. (2015).

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Figure 4.18A-B: Relative expression of glusose 1 phosphate adenyl transferase(CsSB1) in healthy and inoculated citrus germplasm

A

B

4.5.3 Starchsynthase (CsSB2)

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CsSB2 is responsible for starch synthesis. Equal expression or no amplification of this gene was found in: Calamondin, Frost, Hawaiian Mock orange, M.Vinous, Rich16-6, Sacaton, Clausena Harmandiana, Honghe, Pineapple, Schuab, Glycosmis Pentaphylla,Papuan and Tahitian suggesting no starch accumulation in the said varieties. In present study, this gene was upregulated in Flying Dragon, Benton, S-1, Kinnow and Parson‟s Special. The upregulation of the same gene was described in response to HLB infection by Martinelli et al. (2015). A reaction of sweet orange to Ca. L. asiaticus attack with microarray analysis indicated the up-regulation of granule bound starch synthase and starch debranching enzyme. These genes are responsible for starch accumulation in HLB infected leaves (Kim et al., 2009). CsSB2 was down regulated in 26 genotypes of citrus infected with HLB pathogen (Table 4.5; Fig.4.19).Majority of citrus genotypes vary in susceptibility for HLB pathogen, lemons are found less susceptible. A study on response of lemon towards Ca. L. asiaticus infection resulted in increased quantity of starch synthase(Nwugo et al., 2013).

Figure 4.19 Relative expression of granule bound starch synthase (CsSB2) in healthy and inoculated citrus germplasm

4.5.4 Alpha amylase (CsSD1)

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Alpha amylase act on starch at any place and break it down into maltose and glucose etc. qRT PCR results for CsSD1 gene expression revealed upregulation in 8 genotypes of HLB infected citrus including: Benton, Sacaton, S-1, Honghe, Madam Vinous, Orlando, Flying Dragon and Rich 16-6. CsSD1 was down regulated in 8 genotypes including: Troyer, Indian, Philipine, Frost Eureka, Citrus Sunki, Yuzu, Srirampur Nucellar and X-639. In the remaining genotypes, gene was either not amplified or equally amplified as in the healthy samples (Table 4.5; Figure 4.20 A&B). These results are in agreement with the results of HLB infected and girdled fruit tissues from Liao and Burns (2012). When the gene expression results of CsSD1 were compared with the Ct values for HLB pathogen detection and symptom appearance, the genotypes were found to be highly susceptible for HLB having Ct values in a range from 23 to 35. Upregulation of this gene indicates tolerance for phloem plugging in the above mentioned 8 varieties, whereas down regulation of CsSD1 indicates HLB symptom epression.

Figure 4.20(A&B) Relative expression of alpha amylase-1(CsSD1) in healthy and inoculated citrus germplasm

A

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B

4.5.5 Alpha amylase3 (CsSD2)

In this study, qRTPCR to differentiate expression of genes in HLBinfected leaves from that in healthy, CsSD2 was upregulated in 7 genotypes including: S-1, Fortunella Hindsii, Parson‟s Special, Borneo Rangpur, Nansho DaiDai and Pineapple. This gene was down regulated in Eremocitrus Glauca hybrid, Var. sanguine, Duncan and Yuzu (Figure 4.21 AtoC). Downregulation of this gene may be correlated with the non appearance of HLB symptoms.According to Liao and Burns (2012) shared genes whose expression changed in a similar way included alpha-amylase 3 (CsSD2) and beta-amylase 9 (CsSD3). 4.5.6 Beta amylase9 (CsSD3) During fruit ripening, beta amylase degrades starch into maltose, causing sweetness in ripe fruit (Grennan, 2006). qPCR results for gene expression changes in HLB infected leaf samples of citrus germplasm revealed upregulation of CsSD3 in Benton, S-1, Sicily, Sacaton, Hawaiian Mock orange, Fortunella Hindsii, Sun Chu Sha, Borneo Rangpur, Honghe, Reinking, Madam Vinous, Pineapple, Succari, Orlando, Flying Dragon and Rich 16-6. This gene was found to be down regulated in Keen Sour orange, Gadadehi, Brazillian Sour orange, Yuzu, Parson‟s Special, Frost Eureka, Eremocitrus Glauca hybrid, Clausena Harmandiana, Papuan and Bengal (Figure 4.22).

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Figure 4.21A-C Relative expression of alpha amylase-3(CsSD2)

A

B

90

C

Figure 4.22Relative expression of beta amylase 9 (CsSD3)

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4.5.7 Cytochrome P450 mono oxygenase83B1(CsSUR2)

Phytohormone metabolism related gene cytochrome P450 mono oxygenase83B1(CsSUR2) was upregulated in Fortunella hindsii, Madam vinous and Succari sweet orange (Table 4.5; fig. 4.23) pointing towards synthesis and breakdown of hormones responsible for leaf formation and shedding, fruit development and ripening while this gene was not expressed in Clausena harmandianaand Glycosmis pentaphylla.Genes for cytochrome P450 family were upregulated upto 6 fold in sweet orange (Albrect and Bowman, 2008).A study on response of lemon towards Ca. L. asiaticus infection resulted in decrease in production of defense associated proteins and increase in Zinc concentration (Nwugo et al., 2013).

Figure 4.23Relative expression of cytochrome P450 monooxygenase (CsSUR2)

Thisstudy was performed to differentiate expression of genes in HLB infected and healthy leaf samples of forty five genotypes of citrus and its relatives. Up till now, gene expression studieshave been done mostly on Citrus sinensis cultivars only (Albrecht and Bowman, 2008;

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Albrecht and Bowman, 2012; Liao and Burns, 2012; Parra, 2012; Nwugo et al., 2013; Du et al.,2015). Figure 4.24 (A through I) Gene expression of seven genes presenting fold change in forty five genotypes of citrus. Upward bars indicating upregulation and downward bars represent down regulation of genes

30 A 20

10 Calamondin 0 Benton -10

Troyer

CsSB1 CsSB2

CsSD1 CsSD2 CsSD3 CsSUR -20 CsSULF Bengal -30 Fold change Indian -40

-50

-60

50 Clausena B Harmandiana 0 Eremocitrus Glauca hybrid -50 Glycosmis Pentaphylla -100 Hawaiian Mock orange -150 Duncan

-200

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50 C 0 -50 -100 Fortunella Hindsii -150 Frost Eureka -200 Citrus Sunki -250 Kinnow -300 -350 Parson‟s Special -400 -450 -500

40 D 20 0 -20 Papuan -40 Philipine -60 S-1 -80 Sicily -100 Sacaton -120 -140 -160

50 E 40 30 Sun Chu Sha 20 Borneo Rangpur 10 Var. Sanguinea 0 Honghe -10 Yuzu -20 -30 -40

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20 F 0 -20 Reinking -40 -60 Tahitian -80 Srirampur Nucellar -100 Tuningmeng Nucellar -120 Schaub -140 -160 -180

1200 G Brazillian Sour 1000 orange 800 Gadadehi 600 Goutoucheng 400 200 Nansho Daidai 0 Sour Orange -200 -400

100 H

0 Keen Sour orange -100 Madam Vinous -200 Pineapple Succari -300 Orlando -400

-500

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100 I

0 Citromelo 1452 -100 Flying Dragon -200 Rich 16-6 X-639 -300 Frost -400

-500

4.6Assessment ofRNA-Seq library quality on bioanalyzer For learning of advance technique for expression profiling, RNA-Seq libraries were preparedsuccessfully. It was done to use the knowledge of libraries preparation in future research. After completion of the process of libraries preparation, quality of the RNA-seq libraries were assessed. After quantification of the cDNA libraries using nanodrop spectrophotometer, 1.5 µL of each sample was assessed for quality by bioanalyzerat genomic core facility, IIGB, UCR. Concentrations of cDNA in each library were quantified by nanodrop after the attachment of expected index primer sequences (table 4.5) Table 4.5 Sample information for cDNA library concentrations by nanodrop and index primer sequences

Sample Sample Name Conc. Total Index information (ng/µl) Vol.(µl) primer sequences 1 Clasena harmandiana (H) 8.8 2 CAGATC 2 Clasena harmandiana (I) 10.2 2 CTTGTA 3 Keen sour orange (H) 23.3 2 TGACCA 4 Keen sour orange (I) 12.4 2 ACAGTG 5 Kinnow (H) 10.1 2 TGACCA 6 Kinnow (I) 12.3 2 ACAGTG 7 Sweet orange (H) 8.2 2 GATCAG 8 Sweet orange (I) 7.2 2 ACTTGA 9 Benton (H) 8.8 2 CGATGT 10 Benton (I) 7.7 2 GCCAAT 11 Glycosmis pentaphylla (H) 39 2 GATCAG 12 Glycosmis pentaphylla (I) 25 2 TAGCTT

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Figure 4.25 Bioanalyzer results of RNA-seq libraries

There were low molar concentrations of few libraries as shown in the electropherograms of libraries (fig. 4.25) from glycosmis pentaphylla (inoculated) and kinnow (healthy).No library had any adapter dimer which would be at~ 120 to 128 bp, so this was good.There was need to calculate the molar concentration, in nano mole (nM), of each library. So, it was calculated by taking the Molarity value from the region table value, multiplying by the dilution factor, and dividing by 1000.For Illumina next-generation libraries, experts prefer to have molar concentrations ranging from >1 nM to 2 nM.

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CHAPTER-5

SUMMARY

In Pakistan, HLB has been ignored unintentionally. Due to ignorance, uprooting of citrus trees has been a major practice in Pakistan without knowing the actual cause of decline. In these situations, a detailed study of HLB and its natural vector was truly needed so as to assess the citrus germplasm for gene expression changes in response of HLB to manage the disease. Further, the present research was optimistically designed to seek knowledge of modern and sophisticated techniques for HLB diagnosis and expression profiling. Moreover, ACP rearing in controlled conditions of growthroom was aimed for HLB transmission in sweet orange and kinnow plants and use of those plants for inoculation of citrus germplasm. Citrus germplasm was inoculated by the leaf midribs and budsticks of HLB positive sweet orange plants used for ACP rearing because, it was necessary to transmit only HLB pathogen in the citrus germplasm instead of any other graft transmissible pathogen. There were pretty much chances of transmission of some other pathogens, if the grafts would be taken from HLB positive field trees, that could give wrong results of gene expression in citrus. The fundamental goal of this research is to help protect citrus plants. Use of latest technology for disease detection is required to manage HLB and save the citrus in Pakistan.

In this study,following effortswere made to achieve the objectives.

1- Citrus germplasm was reared in the greenhouse of Institute of Horticultural Sciences, University of Agricultue, Faisalabad, Pakistan for HLB detection and expression profiling.

2- Citrus orchards of Faisalabad district of Punjab, Pakistan were surveyed for HLB detection and acquisition of ACP from HLB positive citrus trees for ACP rearing and inoculation of Kinnow and sweet orange plants used for ACP rearing in contolled conditions of growth room.

3- ACP was successfully reared and Candidatus Liberibacter asiaticus was detected by conventional PCR in the plants used for ACP rearing.These HLB positive sweet orange plants were used for the inoculation of citrus germplasm.

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4- Fifty one genotypes of citrus (citrus and its relatives) were inoculated by using midribs. Of 51 genotypes, 49 were found HLB positive by real time PCR with Ct values ranging from 36 to 20. Only two genotypes i.e. Clausena Harmandiana and Glycosmis Pentaphylla were negative for Candidatus Liberibacter asiaticus.

5- From 51, twenty onegenotypes(41%) of citrusexpressed huanglongbing symptoms.

6- Expression profiling results for seven selected genes revealed tolerance against HLB in two genotypes of citrus relatives including: Clausena Harmandiana and Glycosmis Pentaphylla.

Expression profiling of huanglongbing disease in citrus through molecular techniques was done first time on 51 genotypes in Pakistan and this work has opened here a new era by the use of advanced techniques for HLB management research and will prove best method for fighting against HLB.

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RECOMMENDATIONS FOR FUTURE WORK

1- From the seven tested genes in response of HLB infection, all were important, but CsSB1 and CsSB2 were proved to be novel. As these genes are involved in carbohydrate metabolism regarding starch synthesis which is the major problem in response of HLB infection, silencing of these genes in susceptible genotypes of citrus against HLB may help in the development of tolerance.

2- For HLB management, there is need to test and identify further genes that may incorporate tolerance in citrus against HLB. Those identified genes may be used to make transgenic citrus, that will be tolerant against HLB.

3- Control of natural vector of HLB bacterium is very important. There is need to find some ways to control this vector that would be environment friendly.

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