Field Evaluation And Virus Indexing Of Selected Hybrid White Yam (Dioscorea rotundata Poir) In Two Selected Locations Of South-Eastern Nigeria

ABSTRACT

Yam (Dioscorea spp.) is an important crop in tropical and subtropical regions. The presence of any virus within the vegetative planting material poses a significant risk. Obtaining virus-free plants are very important for both international and local germplasm exchange among farmers and breeders, hence the need to employ sensitive molecular diagnostic tool that will detect the presence of both DNA and RNA viruses if present in any part of the plant. The experiment was laid out in Randomized Complete Block Design (RCBD) with nine treatments of yam genotypes and three replications to assess the three (3) viral strains: Yam Mosaic Virus (YMV), Yam Mild Mosaic Virus (YMMV) and Badnavirus (BaDv). The experimental layout was carried out in two locations: the National Root Crops Research Institute (NRCRI) research farm Umudike, Abia State and Faculty of   Agriculture research farm Abakaliki, Ebonyi State University (EBSU).  The study employed the molecular diagnostic strategy of PCR-based detection tools targeting the three different yam-infecting viruses. CTAB techniques were   used to   extract the total nucleic acid from the leaves samples of the nine   genotypes from the two locations, the nucleic acid were quantified using the Nano Drop 2000 spectrophotometer. RT-PCR was employed for the analysis of YMV with primer pair YMV- F3x FP: (5ˈ-GACAATGATGGACG GTGCGG-3ˈ) and YMV-B3x RP:(5ˈCTTTGCCATC AAATCCAAACA T- 3ˈ), and YMMV with primer pair YMMV-FP: (5ˈ-GGCACACAT GCA AATGAARGC-3ˈ) and YMMV RP: (5ˈCACCAGTAGAGTGAACATAG- 3ˈ), while BaDv was subjected to the PCR with primer pair Badna –FP: (5ˈ–ATGCCITTYGGIIT IAARAAYGCICC-3ˈ) and Badna-RP: (5ˈATGCCITTY GGIITIAARAAYGCICC–3). The PCR product was electrophoresed, and then visualized under UV transilluminator. There were amplifications in the wells containing the positive controls, while there were no amplifications in the wells of the nine tested genotypes indicating that the plant tissues are healthy. The genotypes showed some variations within and across the locations; Genotypes grown in Abakliki were superior in the length of vines (cm), while those grown in Umudike were superior in vine girth. At harvest those in Abakaliki recorded total mean yield of 8 tubers and 3.17kg tuber weight, while those in Umudike recorded mean yield of 12 tubers and mean weight of 5.20kg. TDr1401785 recorded the highest yield performance with mean yield of 19 tubers while TDr1000021 recorded the least with mean yield of 4 tubers. TDr1400359 recorded the maximum tuber yield with mean weight of 6.8kg while TDr8902665 recorded the least with mean weight of 1.17kg. The study demonstrated that environment has a significant effect on performance of the 9 genotypes as seen across the locations. The non-detection of viruses among the genotypes screened could be due to absence of the three year viruses screened for in the two locations.

 TABLE OF CONTENTS

Title page                                                                                                                                i

Declaration                                                                                                                              ii

Certification                                                                                                                            iii

Dedication                                                                                                                              iv

Acknowledgements                                                                                                                v

Table of Contents                                                                                                                   vi

List of Tables                                                                                                                          x

List of Figures                                                                                                                         xi

List of Plates                                                                                                                           xii

Abstract                                                                                                                                  xiii

 

CHAPTER 1: INTRODUCTION

1.1              Background of the Study                                                                                           1

1.2              Statement of the Problem                                                                                           2

1.3              Aim and Objectives of the Study                                                                               3

1.4              Justification of the Study                                                                                           3

CHAPTER 2: LITERATURE REVIEW

2.1       Origin of Yam                                                                                                             5

2.2       Nutritional Value of Yam                                                                                           11

2.3       Yam as a Dietary Source of Energy                                                                           12

2.4       Yam as a Mineral Source                                                                                            17

2.5       Yam Bioactive Compounds                                                                                        19

2.6       Therapeutics Potential of Yam                                                                                   22

2.7       Antimicrobial Potential of Yam                                                                                  25

2.8       Yam Antioxidant Properties                                                                                       26

2.9       Yam Anti-Inflammatory Properties                                                                            27

2.10     Anticancer Activity of Yam                                                                                       28

2.11     Anti-Diabetic Properties of Yam                                                                                30

2.12     Yams Future Prospective                                                                                            32

2.13     Yam Infecting Viruses                                                                                                37

12.14   Genus Potyvirus, Family Potyviridae                                                                          40

12.14.1Yam mosaic virus (YMV)                                                                                           40

12.14.2 Dioscorea alata virus (DAV)                                                                        40

12.14.3 Other potyviridae                                                                                                       41

12.14.4 Family caulimoviridae, genus badnavirus                                                                 41

12.14.5 Cucumber mosaic virus, genus cucumovirus                                                             42

12.14.6 Dioscorea, genus of latent viruses                                                                             43

12.14.7 Dioscorea mottle virus genus comovirus                                                                   43

CHAPTER 3: MATERIALS AND METHODS

3.1       Study Area                                                                                                                  44

3.2       Collection of Planting Material                                                                                   44

3.3       Experimental Design and Planting                                                                             45

3.4       Germination of the Seed Yam                                                                                                45

3.5       Virus Diseases Incidence                                                                                            45

3.6       Virus Disease Severity                                                                                                46

3.7       Tuber Yield at Harvest                                                                                               46

3.8       Statistical Analysis                                                                                                      46

3.9       Leaves Sampled for Virus Detections                                                                                    46

3.10     Molecular Studies for Detecting Viral Organisms                                                      47

3.10.1  Extraction of total nucleic acid method using hexadecyltrimethyl

           ammonium bromide (CTAB)                                                                                       47

3.10.2  Preparation of agarose gel nucleic acid electrophoresis and polymerase

            chain reaction                                                                                                              48

3.10.3  Loading samples and running the agarose gel                                                            49

3.11     Polymerase Chain Reaction                                                                                        49

3.11.1  PCR mix components                                                                                                 49

3.11.2  RT-PCR for YMV and YMMV multiplex                                                                51

3.11.3  PCR detection of badnavirus                                                                                     52

CHAPTER 4:            RESULTS AND DISCUSSION    

4.1 Results                                                                                                                              53

4.1.1 Sprouting percentage                                                                                                     53

4.1.2 Establishment count                                                                                                       56

4.1.3 Virus disease incidence                                                                                                  58

4.1.4 Virus disease severity                                                                                                    60

4.1.5 Vine girth                                                                                                                       62

4.1.6 Vine length                                                                                                                    65

4.1.7 Mean number of tubers                                                                                                  68

4.1.8 Tuber weight at harvest                                                                                                 70

4.1.9 Various parameters (variables) assessed across the locations                                        72

4.1.10 Nucleic acid quantification                                                                                          74

4.1.11 Molecular studies for virus detections                                                                         77

4.1.12 Soil Physiochemical Properties                                                                                    79

4.2       Discussion                                                                                                                   61

CHAPTER 5: CONCLUSION AND RECOMMENDATIONS

5.1       Conclusion                                                                                                                  84

5.2       Recommendations                                                                                                      84

References                                                                                                                  85

Appendices                                                                                                                 104

 

 

 

 

 

LIST OF TABLES

                                                                                                                                                Pages

2.1       The bioactive compound profile of yam (Dioscorea species)                         21

2.2       The medicinal applications of yam (Dioscorea species)                                             24

2.3       Virus species infecting yam (Dioscorea species)                                                        39

3.1       Primer sequences used for the analyses of the three infecting

yam viruses- YMV, YMMV and Badnavirus                                                            50

3.2       RT-PCR programme showing Thermal cycling conditions               

for YMV and YMMV multiplex                                                                                50

3.3       PCR programme showing thermal cycling conditions for Badnavirus                       51

4.1       Mean germination percentage of Umudike genotypes                                               54

4.2       Mean Germination Percentage of Abakaliki genotypes                                             55

4.3       Mean comparison of the various parameters (variables)

measured across the Locations                                                                                    

4.4       Nucleic acid quantification of the samples collected from the

nine (9) genotypes grown at the two locations with positive and

negative controls                                                                                                         78

4.5       Soil chemical properties                                                                                              83

4.6       Soil physical properties                                                                                               83

 

 

LIST OF FIGURES

                                                                                                                                     Pages

2.1       Global Distribution of Yam Production in 2018                                            7

4.1       Comparison of mean establishment counts at 4^th months of the

genotypes across the locations                                                                        57

4.2       Mean comparison of virus disease incidence of the genotypes

across the locations                                                                                         59

4.3       Comparison of mean virus disease severity of the genotypes

across the locations                                                                                         61

4.4       Mean comparison of vine girth of the genotypes across the locations

(mm) at 4^th month                                                                                           63

4.5       Mean comparison of vine girth of the genotypes across the locations          

(mm) at 5^th month                                                                                           64

4.6       Mean comparison of length of vine of the genotypes across the

locations at 4^th Month                                                                                     66

4.7       Mean comparison of length of vine of the genotypes across the

locations at 5^th Month                                                                                     67

4.8       Mean comparison of number of tubers of the genotypes across

the locations                                                                                                    69

4.9       Mean comparison of tuber weight of the genotypes across the locations      71

 

 

LIST OF PLATES

                                                                                                                                     Pages

4.1       Gel image of nucleic acid isolated from the nine (9) samples               76

4.2       Gel image PCR of YMVand YMMV Multiplex for the nine (9) samples       78

4.3       Gel image of Badnavirus for the nine (9) samples                                            79