AETIOLOGY, IMPORTANCE AND CONTROL OF A VINE-BROWNING AND DIE-
BACK DISEASE OF WATER YAM (DIOSCOREA ALATA L.) IN THE VOLTA REGION 
OF GHANA 
 
 
 
BY 
 
SHADRACK ASIEDU COFFIE 
(10363967) 
 
 
 
 
THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON IN 
PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF M. PHIL 
CROP SCIENCE (PLANT PATHOLOGY) DEGREE 
 
 
 
 
 
 
 
 
JULY, 2013  
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DECLARATION 
I do hereby solemnly declare that, this thesis is the original research of Shadrack Asiedu Coffie 
towards the award of the Master of Philosophy Crop Science (Plant Pathology) in the Department 
of Crop Science, College of Agriculture and Consumer Sciences (CACS), University of Ghana, 
Legon except for other people’s work which have been aptly cited and duly acknowledged and 
thus, neither part nor whole of this thesis has been presented elsewhere for another degree. 
 
 
....................................................... 
COFFIE, SHADRACK ASIEDU 
(STUDENT) 
 
DATE........................................... 
 
 
 
..........................................................      ................................................ 
PROF. SAMUEL K. OFFEI      DR. ERIC W. CORNELIUS 
(PRINCIPAL SUPERVISOR)                                                           (CO-SUPERVISOR)  
CROP SCI. DEPT.        CROP SCI. DEPT. 
UNIV. OF GHANA                                                                            UNIV. OF GHANA 
LEGON                                                                                               LEGON  
 
DATE...............................                  DATE..................................... 
 
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ACKNOWLEDGEMENTS 
To Him who gives victory, the Supreme being, the ancient Father, the good Shepherd, Lord God 
Almighty I owe him my life in totality for the grace, mercies, protection, wisdom and strength 
granted me throughout my two-year academic work in the University of Ghana, Legon.  
Indeed, I am indebted to a great number of individuals and corporate bodies whose immense 
contributions and/or supports have brought this research work thus far. The first and foremost 
earnest appreciation goes to my productive and energetic supervisors, Prof. Samuel K. Offei and 
Dr. Eric W. Cornelius who did not only spend sleepless nights in writing for the award of this 
project but also criticised constructively, advised fatherly, exercised patience, guided excellently 
and contributed immeasurably for the successes of this study. May the good Lord reward you 
accordingly! I undeniably owe you other thanks for introducing me to the world of molecular and 
bacteriological studies during my M.Phil Coursework. To Prof. Kwadwo A. Oduro, I say ‘Ayekoo’ 
for your paternal care, love, fortitude and impartation of Plant Pathology knowledge in my second 
degree programme. May you never grow weary!    
To my cherished parents, Mr. Charles K. Coffie and Mrs. Mary Coffie together with my lovely 
siblings, Mr. Stephen Coffie, Mrs. Victoria Tawiah, Miss Esther Coffie, Mrs. Margaret Mensah, 
and Mr. Daniel K. Gyasi I say a very big thanks to you all and seek God blessings for all. Indeed, 
you are fantabulous family and may you never lack! In fact, Mrs. Grace Coffie, Mr. George 
Mireku, Mr. Seth Tawiah, Miss Mary Appiah Kyeraa and George Nti’s support can never go 
unnoticed. May the loving Father expand your territory.  To the project sponsors, West Africa 
Agriculture and Productivity Programme (WAAPP) and Leventis Scholarship Foundation, I say 
well-done and may you never want.  
To Dr. and Dr. (Mrs.) Amoatey (Heads – Nuclear Agriculture and Crop Sci. Depts. respectively,  
Univ. of Ghana), Dr. Kenneth E. Danso (Director of BNARI, GAEC), Mr. Asumeng (Senior 
Technician, TBC-BNARI), Dr. R. Asiedu (Director of IITA, Ibadan,  Nigeria), Prof. (Mrs.) 
Antonio-Lopez (Senior Scientific Researcher, Univ. of Oxford, UK.), Dr. Emmanuel Otu, Dr. 
Asumadu and Mr. Mark Amankwah all of CSIR-CRI, Fumsua, Kumasi, Dr. Stephen T. Odonkor 
(RAMSRI, GAEC), Mr. S. Atsu and Mr. T. Akator (Directors of MoFA for Nkwanta-North and 
Krachi-West districts respectively), Mr. Emmanuel O. Ankrah (formally of the Dept. of Crop Sci., 
Univ. of Ghana) and Mr. S. Oppong (Research Farm Manager, PGGRI, Bunso) I am dumbfounded 
of your magnificent and diverse contributions made towards this research, either in cash or in kind. 
May God richly bless you all! To Mr. Rodney Darko, Mrs. Christolite Tagoe, Mr. Kwaku Acquah, 
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Mr. Enock Sarpeh and Mr. Benard Owusu all of the Biotechnology Centre – CACS, Mr. J. Otoo, 
Mr. E. Appiah, Mr. J. Honger, Mr. K. Agyekum, Mr. W. Asante, Mr. Christopher A. Arthur, Mr. 
X. Kanfra, Mr. K. Agnaka, Mr. A. Hador, Mr. K. Oyao, Atiwa, M. A. and Mr. B. Awudu all of the 
Dept. of Crop Sci., Univ. of Ghana, Legon, Mr. B. Joshua, Mr. F. Aboni, Mr. O. Abdallah, Mr. A. 
Isaac (AEAs, MoFA) as well as Mr. S. Owusu and Mr. K. Emmanuel all of the TBC, BNARI, I 
really appreciated your efforts rendered – ‘Me ma mo nyinaa amo!’  
To my spiritual team, Very Rev. and Mrs. Aidoo (Ex-Minister and wife in Charge, Maranatha 
Meth. Ch., Dansoman), the Singing Band Union, Bible Study Class Members, Mr. Kingsley 
Opuku, Miss Henrietta Maame-Esi Aidoo and Miss Cynthia Aidoo – all of The Methodist Ch. 
Ghana together with Mad. Hellen Aidoo (Assemblies of God), Miss Harriet Afari-Aikins and Mr. 
Seth K. Yobunt both of Treasure House Ministry, I say, thank a lot for your spiritual supports. May 
you never languish in life! To all my colleagues especially, Mr. S. Lamini, Mr. L. Lawson and 
Mr. A. Camara who showed love, care and concern in diverse ways, I say, may Allah open His 
heavenly blessings onto all your accomplishments in life. Certainly, you are colleagues! 
Ultimately, I offer my apology and sincere thanks to those that time and space did not permit me to 
mention their names. God richly bless you all! 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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DEDICATION 
 This thesis is wholly dedicated firstly to my good Shepherd, the Alpha and Omega for His 
spectacular love, care, strength, wisdom, protection and mercies He bequeathed upon me 
throughout my educational pursuits. Secondly, to my worthy supervisors, Prof. Samuel K. Offei 
and Dr. Eric W. Cornelius for their wonderful guidance and support towards my achievement. 
Next, to my entire family members especially, my lovely parents, Mr. and Mrs. Coffie and my 
compassionate siblings for their financial, social, moral and spiritual supports towards my 
upbringing. Last but not least, to my laudable educational counselor, Dr. Harry. M. Amoatey for 
encouraging me to pursue an M. Phil degree in Plant Pathology and finally, to my dearest friend, 
Miss Henrietta Aidoo for her prayers, love and encouragement towards my studies.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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TABLE OF CONTENTS 
 
DECLARATION .............................................................................................................................................. i 
ACKNOWLEDGEMENT ............................................................................................................................... ii 
DEDICATION ................................................................................................................................................ iv 
TABLE OF CONTENTS................................................................................................................................. v 
LIST OF TABLES .......................................................................................................................................... ix 
LIST OF FIGURES ......................................................................................................................................... x 
LIST OF PLATES .......................................................................................................................................... xi 
LIST OF ABREVIATIONS ......................................................................................................................... xiii 
ABSTRACT .................................................................................................................................................. xv 
 
CHAPTER ONE .............................................................................................................................................. 1 
1.0 INTRODUCTION ..................................................................................................................................... 1 
 
CHAPTER TWO ............................................................................................................................................. 6 
2.0 LITERATURE REVIEW .......................................................................................................................... 6 
2.1 Yam: Origin and distribution ..................................................................................................................... 6 
2.2 Botany of yam ............................................................................................................................................ 7 
2.3 Distinguishing characteristics of D. alata and D. rotundata ..................................................................... 9 
2.4 Economic importance and uses of yam ................................................................................................... 10 
2.5 World production and trade of yam ......................................................................................................... 13 
2.6 Yam production in Ghana ........................................................................................................................ 13 
2.7 Climatic, water and soil requirements of yam ......................................................................................... 15 
2.8 Husbandry practices/staking .................................................................................................................... 15 
2.9 Harvesting ................................................................................................................................................ 16 
2.10 Field diseases of yam ............................................................................................................................. 16 
     2.10.1 Foliar fungal diseases ..................................................................................................................... 17 
2.11 Economic importance of anthracnose disease of D. alata ............................................................. 18 
2.12 Prevalence, incidence and severity of yam anthracnose disease………………………………….…..  20 
2.13 Symptoms of anthracnose disease of D. alata ............................................................................... 21 
2.14 Etiology of anthracnose disease of D. alata .................................................................................. 22 
2.15 Colletotrichum species ................................................................................................................... 23 
2.15.1 Existence of Colletrotrichum species…….…………………………………………….…………….24 
2.16 Host relations and specificity in Colletotrichum gloeosporioides and C. truncatum…………..............27 
2.17 Characterization of the strains of Colletotrichum gloeosporioides and C. truncatum isolated ............. 28 
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2.18 Screening of D. alata cultivars for resistance to anthracnose disease using tissue culture-derived 
whole-plant assay ........................................................................................................................................... 30 
       2.18.1 Methods of screening of D. alata cultivars .......................................................................... 30 
       2.18.2 Methods of in-vitro propagation of D. alata ........................................................................ 32 
2.19 Development of IPM strategy for management of yam anthracnose disease ........................................ 33 
2.19.1 Stages of yam anthracnose disease development and its cycle ................................................... 33 
2.19.2 Epidemiology of yam anthracnose .............................................................................................. 34 
2.20 Management of anthracnose disease of D. alata in the field ......................................................... 35 
 
CHAPTER THREE ....................................................................................................................................... 37 
3.0 MATERIALS AND METHODS ............................................................................................................. 37 
3.1 Socio-economic background of the two important D. alata growing districts in the Volta region of 
Ghana ............................................................................................................................................................. 37 
3.1.1 Nkwanta-North District ................................................................................................................ 37 
3.1.2 Krachi-West District ..................................................................................................................... 38 
3.2 Assessment of farmers’ perception on importance and control of a vine-browning and die-back disease 
of D. alata in the Volta region........................................................................................................................ 39 
3.2.1 Selection of experimental sites within the two districts for the disease survey ............................ 39 
3.2.2 Survey to assess farmers’ knowledge, perception, experiences and economic importance of a 
vine-browning and die-back disease of D. alata in the Volta Region ................................................... 40 
3.2.3 Symptoms decription of vine-browning and die-back disease of water yam and samples 
collection ................................................................................................................................................ 41 
3.2.4 Determination of incidence and severity of a vine-browning and die-back disease of   D. alata in 
the Nkwanta-North and Krachi-West districts of the Volta Region ...................................................... 42 
3.3 Isolation and identification of the causal agent(s) of a vine-browning and die-back disease of D. alata in 
the Volta region ............................................................................................................................................. 45 
3.3.1 Pathogen Isolation ......................................................................................................................... 45 
3.3.2 Identification of micro-organisms associated with anthracnose disease of D. alata .................... 46 
3.4 Characterization of the strains of C. gloeosporioides and C. truncatum ................................................. 50 
3.4.1 Diversity study in C. gloeosporioides and C. truncatum infecting D. alata in Ghana ................. 50 
3.4.2 Genome analysis of C. gloeosporioides and C. truncatum strains ............................................... 51 
3.4.2.1 Rapid amplified polymorphic DNA (RAPD) analysis .............................................................. 51 
3.4.2.2 Restriction Enzyme Digestion of rDNA ITS regions ................................................................ 53 
3.5 Screening of D. alata cultivars for resistance to anthracnose disease using tissue culture-derived whole-
plants assay .................................................................................................................................................... 54 
3.5.1 Determination of appropriate hormones concentration for the tissue culture test ........................ 54 
3.5.2 Raising of D. alata explants for the screening trial ...................................................................... 55 
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3.5.3 Screening of 12 D. alata cultivars for resistance to anthracnose disease ..................................... 57 
3.6 Development of IPM strategies for management of anthracnose disease of D. alata ............................. 59 
3.6.2 Field management of anthracnose disease of D. alata in Ghana .................................................. 61 
 
CHAPTER FOUR ......................................................................................................................................... 64 
4.0 RESULTS ................................................................................................................................................ 64 
4.1 Farmers’ perception and practices on the cause, spread, control and importance of a vine-browning and 
die-back disease of D. alata in the Volta region ............................................................................................ 64 
4.1.1 General background of D. alata farmers ...................................................................................... 64 
4.1.2 Farmers’ knowledge and perception on disease symptoms, susceptibility of D. alata culitvars and 
prevalence of anthracnose diseaseof D. alata in the Volta Region ....................................................... 65 
4.1.4 Knowledge and experience of farmers on, yield and economic impact of a vine-browning and 
die-back disease of D. alata on livelihoods of farmers.......................................................................... 70 
4.1.5 Symptoms of a vine-browning and die-back disease of D. alata ................................................. 73 
4.1.6 Incidence and severity of a vine-browning and die-back disease of D. alata in the Nkwanta-North 
and Krachi-West districts ....................................................................................................................... 74 
4.2 Micro-organisms associated with a vine-browning and die-back disease of D. alata in the Volta Region
 ....................................................................................................................................................................... 75 
4.2.1 Identification using morpho-cultural characteristics of isoaltes ................................................... 75 
4.2.2 Molecular Identification of isolates .............................................................................................. 77 
4.2.3 Pathogenecity test of isolates from the Volta Region ................................................................... 78 
4.3 Characterization of the strains of C. gloeosporioides and C. truncatum causing anthracnose disease of 
D. alata in Ghana ........................................................................................................................................... 82 
4.3.1 Diversity in C. gloeosporioides and C. truncatum causing anthracnose disease of D. alata in 
Ghana ..................................................................................................................................................... 82 
4.4.2 Incidence of anthracnose disease on two-month old potted explants of 12 D. alata cultivars using 
six Colletotrichum isolates ................................................................................................................... 107 
4.5 Integrated management of anthracnose disease of D. alata ................................................................... 108 
 
CHAPTER FIVE ......................................................................................................................................... 113 
5.0 DISCUSSION ........................................................................................................................................ 113 
5.1 Assessment of farmers’ knowledge and perception of a vine-browning and die-back disease of D. alata 
in the Volta Region ...................................................................................................................................... 113 
5.4 Characterization of the strains of C. gloeosporioides and C. truncatum associated with anthracnose 
disease of D. alata in Ghana ........................................................................................................................ 120 
5.5 Screening of D. alata cultivars for resistance to anthracnose disease ................................................... 123 
5.6 Development of IMP strategy to manage anthracnose disease of D. alata ........................................... 126 
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CHAPTER SIX ............................................................................................................................................ 128 
6.0 CONCLUSION AND RECOMMENDATION ..................................................................................... 128 
6.1 Conclusion ............................................................................................................................................. 128 
6.2 Recommendation ................................................................................................................................... 129 
 
REFERENCES ............................................................................................................................................ 130 
 
APPENDICES ............................................................................................................................................. 151 
APPENDIX I: Questionnaire on etiology, importance and control of a vine-browning and die-back disease 
of water yam (Dioscorea alata L.) in the Volta Region of Ghana .............................................................. 151 
APPENDIX II: Surveyed Areas for the Diversity Study ............................................................................. 155 
APPENDIX IV: ANOVA for pathogenecity test for the Volta region strains ............................................ 157 
APPENDIX V: ANOVA for the performance of D. alata explants on different hormones combinations in 
the main experiment..................................................................................................................................... 157 
APPENDIX VI: ANOVA for Mean severity (AUDPCs) for a vine-browning and die-back disease on 12 
tissue-cultured D. alata cultivars raised in a screen house .......................................................................... 158 
APPENDIX VII: ANOVA for the mean disease incidence of a vine-browning and die-back disease of 12 
tissue-cultured D. alata cultivars raised in a screen house .......................................................................... 158 
APPENDIX VIII: ANOVA for fungicides inhibitory effect on radial mycelial growth and sporulation of 
three Colletotrichum spp. ............................................................................................................................. 159 
 
 
 
 
 
 
 
 
 
 
 
 
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LIST OF TABLES 
Table 3.1: Experimental sites and number of farmers interviewed per site ................................................... 40 
Table 3.3: Disease assessment key for severity of a vine-browning and die-back disease of D. alata ......... 44 
Table 4.1: Educational background of respondents ....................................................................................... 64 
Table 4.2: Symptoms used by farmers to identify a vine-browning and die-back disease of D. alata in the 
field. ............................................................................................................................................................... 66 
Table 4.5: Reasons for planting D. alata by farmers in the Nkwanta-North and Krachi-West districts. ...... 70 
Table 4.6: Expected and actual yield of D. alata harvested by farmers in the Nkwanta-North and Krachi-
West districts ................................................................................................................................................. 71 
Table 4.7: Farmers’ reasons for not achieving expected yield of D. alata in the Nkwanta-North and Krachi-
West districts ................................................................................................................................................. 72 
Table 4.8: Socio-economic impacts of a vine-browning and die-back disease of D. alata on the   livelihoods 
of farmers in the Nkwanta-North and Krachi-West districts. Number of farmers = 120. ............................. 73 
Table 4.9: Disease incidence and severity of anthracnose disease of D. alata in the selected communities of 
the Nkwanta-North and Krachi-West districts of the Volta Region. ............................................................. 75 
Table 4.11: Detection of DNA fragments of C. gloeosporioides and C. truncatum strains obtained from 
diseased D. alata plants in the Volta Region using ITS1/ITS4 primer pair and CgInt/ITS4 species-specific 
primer pair. .................................................................................................................................................... 78 
Table 4.12: Disease infection (%) by Colletotrichum isolates on two-month old potted tissue-cultured D. 
alata cultivars (TDa008 and TDa010). .......................................................................................................... 81 
Tabel 4.14 (86-96 pp): Distinguishable morphological and cultural characteristics of the 22 Colletotrichum 
spp. isolated from anthracnose disease of D. alata in Ghana. ....................................................................... 86 
Table 4.15: Detection of DNA fragments of C. gloeosporioides and C. truncatum strains obtained from 
diseased D. alata plants in Ghana using ITS1/ITS4 primer pair and CgInt/ITS4 species-specific primer pair.
 ....................................................................................................................................................................... 98 
Table 4.21: Severity (mean AUDPCs) scores and ranks (parenthesis) of anthracnose disease on two-month 
old potted explants of 12 D. alata cultivars using six Colletotrichum isolates. .......................................... 107 
Table 4.16: Incidence (%) of anthracnose disease on two-month old potted explants of 12 D. alata cultivars 
using six Colletotrichum isolates. ................................................................................................................ 108 
 
 
 
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LIST OF FIGURES 
Fig. 2.1: Origin and distribution of Dioscorea species worldwide. Source: Coursey (1975) and Degras 
(1993). .............................................................................................................................................................. 6 
Fig. 3.2: Inoculated (INO) and non-inoculated (C) two-month old potted tissue culture-derived whole-plants 
of D. alata cultivars enclosed in transparent latex bags to maintain high RH%  for disease infection. 
Pathogen used = C. gloeosporioides and C. truncatum with spore suspensions of 1.6 x 106 conidial/ml. .... 50 
Fig. 4.2: Perception of D. alata farmers on the cause of a vine-browning and die-back   disease of D. alata .
 ....................................................................................................................................................................... 68 
Fig. 4.3: Perception of D. alata farmers on the spread of a vine-browning and die-back   disease of D. alata 
within farms. .................................................................................................................................................. 69 
Fig. 4.4: Perception of D. alata farmers on the spread of a vine-browning and die-back   disease of D. alata 
among farms. ................................................................................................................................................. 69 
Fig 4.5: Perception of D. alata farmers on measures needed for successful of control of a vine-browning 
and die-back   disease of D. alata in the Volta Region. ................................................................................. 70 
Fig. 4.7: UPGMA dendrogram based on Jaccard’s coefficient, indicating relationships among the 16 strains 
of C. gloeosporioides obtained from six Regions of Ghana. Genetic distances were based on the combined 
data from the random amplified polymorphic DNA (RAPD) and the internal transcribed spacer-restriction 
fragment length polymorphism (ITS-RFLP) analyses. RAPD primers: OPA-11, OPA-18, OPA-05, OPC-07 
and OPC-16 and Restriction enzymes used were: HaeIII, HinfI, RsaI, MspI and HhaI. ............................. 103 
Fig. 4.8: UPGMA dendrogram based on Jaccard’s coefficient, indicating relationships among the four 
strains of C. truncatum obtained from three Regions of Ghana. Genetic distances were based on the 
combined data from the random amplified polymorphic DNA (RAPD) and the internal transcribed spacer-
restriction fragment length polymorphism (ITS-RFLP) analyses. RAPD primers: OPA-11, OPA-18, OPA-
05, OPC-07 and OPC-16 and Restriction enzymes used were: HaeIII, HinfI, RsaI, MspI and HhaI.. ........ 104 
Fig. 4.9: Fungistatic effects of four fungicides (Carbendazim, Mancozeb, Nordox and Shatiz F1) on radial 
mycelial growth and sporulation of two Colletotrichum isolates – C. truncatum (A) and C. gloeosporioides 
Cg111 (B). PDA amended cultures and controls (*NF) were incubated at 26±2 oC and 65-72% RH in the 
laboratory. Vertical bars indicate LSD5%. Percentage reduction in radial mycelial growth of test fungi (I) 
was calculated based on the formula of Kiran et al. (2010). ....................................................................... 109 
 
 
 
 
 
 
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LIST OF PLATES 
Plate 4.1: Stages of development of symptoms of a vine-browning and die-back disease of D. alata. Healthy 
plant (I); pin-point spots on lamina (II); pin-points spots surrounded by yellow halo on lamina (III); small 
irregular brown spots with yellow halo (IV); brown regular spots with gray centre bordered by dark brown 
ring (V); large brown spots bordered by yellow patches on leaves and die-back of vines (VI); and browning 
and/or blackening of vines and defoliation of leaves (VII). .......................................................................... 74 
Plate 4.6: Amplified DNA fragments of C. gloeosporioides strains (lanes – 2, 3, 4 and 6) and C. truncatum 
strains (lanes – 1 and 5) obtained from diseased D. alata in the Volta region using ITS1/ITS4 primer pair 
(A) and ITS4/CgInt species specific primer pair (B). M = Molecular size marker GeneRuler™ DNA Ladder 
Mix (100-10,000 bp); C = Negative controls (sterile distilled water). .......................................................... 78 
Plate 4.7: Two-month old potted tissue culture-derived whole-D. alata plants showing symptoms of 
anthracnose disease. Die-back (blue arrows) (A-C) and necrotic lesions on leaves (brown rings) (D-G) 13 
DAI with C. gloeosporioides isolate (Cg111) using spore suspension of 1.6 x 106 conidia/ml.. .................. 79 
Plate 4.8: Two-month old potted tissue culture-derived whole-D. alata plants without symptoms of 
anthracnose disease. Plants were sprayed with sterile distilled water amended with two drops of Tween 80 
(2% v/v) – as controls. ................................................................................................................................... 80 
Plate 4.10: Different growth patterns of C. truncatum strains on PDA at 25±2 oC, 7 days. Volta strains (I 
and II); Ashanti strains (E-G); Central strain (III); Eastern strain (IV)……………………..……………83  
 
Plate 4.11: Differnent formation of setae and acervuli of C. gloeosporioides strains obtained from diseased 
D. alata plants – setae (A) and acervuli (B). Black bars in panels = 60 µm.................................................. 83 
Plate 4.12: Differnent formation of setae and acervuli of C. truncatum strains obtained from diseased D. 
alata plants – setae (A-C) and acervuli (D and E). Black bars in panels = 60 µm……………………….84 
 
Plate 4.13: Amplified DNA fragments of C. gloeosporioides strains (lanes - 1, 2, 4, 5, 7, 8, 9, 11, 12, 13, 14, 
15, 17, 18, 19 and 20) and C. truncatum strains (lanes - 3, 6, 10 and 16) obtained from diseased D. alata in 
Ghana using ITS1/ITS4 primer pair (A) and ITS4/CgInt species specific primer pair (B). M = Molecular 
size marker GeneRuler™ DNA Ladder Mix (100-10,000 bp); C = Negative controls (sterile distilled water); 
Pc = Positive controls (C. gloeosporioides from mango). Lanes: 1 = Cg111, 2 = Cg114, 3 = Ct130, 4 = 
Cg117, 5 = Cg125, 6 = Ct132, 7 = Cg141, 8 = Cg147, 9 = Cg148, 10 = Ct142, 11 = Cg149, 12 = Cg150, 13 
= 151, 14 = Cg158 162, 15 = Cg162, 16 = Ct168, 17 = Cg165, 18 = Cg166, 19 = Cg167 and 20 = Cg169. ...... 97 
Plate 4.14: RAPD fingerprints obtained with three RAPD Primers OPA-18 (A), OPC-07 (B) and OPC-16) 
(C) for 20 Colletotrichum  isolates  (1-20) obtained from diseased D. alata in Ghana. M = Molecular size 
marker GeneRuler™ DNA Ladder Mix (100-10,000 bp); Pc = Positive controls (C. gloeosporioides from 
Mango); Lanes: 1 = Cg111, 2 = Cg114, 3 = Ct130, 4 = Cg117, 5 = Cg125, 6 = Ct132, 7 = Cg141, 8 = 
Cg147, 9 = Cg148, 10 = Ct142, 11 = Cg149, 12 = Cg150, 13 = 151, 14 = Cg158 162, 15 = Cg162, 16 = 
Ct168, 17 = Cg165, 18 = Cg166, 19 = Cg167 and 20 = Cg169. ................................................................. 100 
Plate 4.16: Three-week old in-vitro explants of D. alata incubated at 16-hour light/8-hour dark photoperiod 
(cool, white fluorescent light – 30 µmol m-2 S-1) and a temperature of 25 °C, 60-70% RH. ....................... 105 
Plate 4.17: Three-week old hardened explants of D. alata cultivars TDa019 (A), TDa007 (B) and TDa013 
(C) without symptoms of anthracnose disease. Medium used – 1:1 vermiculite: compost (peat: sand 9:1) 
(IITA, Nigeria) maintained under bioclimatic chamber. ............................................................................. 105 
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Plate 4. 18: Six-week old explants of D. alata cultivars TDa010 (A); TDa019 (B); TDa018 (C) and TDa007 
(D) with varying number of roots and shoots. Explants were incubated at 16-hour light/8-hour dark 
photoperiod (cool, white fluorescent light – 30 µmol m-2 S-1) and a temperature of 25 °C,  60-70% RH. .. 105 
 
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LIST OF ABREVIATIONS 
AEA   Agricultural Extension Agent 
ANOVA  Analysis of Variance 
AR   Ashanti Region 
BNARI  Biotechnology of Nuclear Agriculture Research Institute, Accra 
B/A-R   Brong-Ahafo Region 
C   Control 
CR   Central Region 
CRD   Completely Randomized Design 
CPDA   Carbendazim Potato Dextrose Agar 
CSIR-CRI Council for Scientific and Industrial Research-Crops Research Institute, 
Kumasi 
Ct   Colletotrichum truncatum 
Cg   Colletotrichum gloesoporioides 
CTAB   Cetyltrimethylammonium Bromide 
DAI   Days after Inoculation 
DSDW  Double Sterile Distilled Water 
DNA   Deoxyribonucleic Acid 
EDTA   Ethylene Diamino Tetra-acetic Acid 
e.g.   Example 
ELISA   Enzyme Linked Immunosorbent Assay 
ER   Eastern Region 
F   Fisher’s Test 
FAO   Food and Agricultural Organization of the United Nations 
FAOStat  Food and Agricultural Organization of the United Nations’ Statistics 
g   Gramme 
GAEC   Ghana Atomic Energy Commission, Accra 
GEPC   Ghana Exports Promotion Council 
i.e.   That is 
ISSER   Institute of Statistical, Social and Economic Research 
IITA   International Institute of Tropical Agriculture 
IPM   Integrated Pest Management 
JHS   Junior High School 
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L   Litre 
LSD   Least Significant Difference 
MAS   Months after sprouting 
MoFA   Ministry of Food and Agriculture, Ghana 
MoFA-SRID Ministry of Food and Agriculture – Statistics, Research and Information 
Directorate 
MoFA-ESD  Ministry of Food and Agriculture – Extension Service Directorate 
MPDA   Mancozeb Potato Dextrose Agar 
NPDA   Nordox Potato Dextrose Agar 
NAPDA  Non-amended Potato Dextrose Agar 
NR   Northern Region 
P   Probability 
RCBD   Randomized Complete Block Design  
PCR   Polymerase Chain Reaction 
PDA   Potato Dextrose Agar 
PGRRI  Plant Genetic Resource Research Institute, Bunso  
RAPD   Randomized Amplified Polymorphic DNA 
RFLP   Restriction Fragment Length Polymorhism  
RH   Relative Humidity 
RNA   Ribonucleic Acid 
RT-PCR  Reverse Transcription Polymerase Chain Reaction 
RPM   Revolution per Minute 
SPDA   Shafiz Potato Dextrose Agar 
SHS   Senior High School 
SPSS   Statistical Package for Social Sciences 
ssDNA  Single Stranded Deoxyribonucleic Acid 
SDW   Sterilized Distilled Water 
TAE   Tris-Acetic-Ethylene Diamino Tetra-acetic Acid 
TBC-BNARI Tissue-Culture and Biotecnology Centre of the Biotechnology and Nuclear 
Agriculture Research Institute, Accra 
TRIS Trizma Base 
TRIS-EDTA Trizma Base-Ethylene Diamino Tetra-acetic Acid 
UG University of Ghana 
UV Ultra Violet 
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ABSTRACT 
Vine-browning and die-back disease (anthracnose disease) is a major constraint and a predominant 
disease in all yam producing areas especially, in Africa. The disease reduces photosynthetic area of 
infected yam plants resulting in severe yield loss. Farmers and Agricultural Extension Agents in 
the Nkwanta-North and Krachi-West districts of the Volta Region have recently reported high 
incidence of the disease on their farms. A study was, therefore, conducted from January, 2012 to 
June, 2013 to assess farmers’ knowledge and perception of the disease and its economic impacts 
on their livelihoods, determine the incidence and severity of the disease, identify the causal 
organisms of the disease, characterize the strains of the pathogens inducing the disease, use tissue 
culture-derived whole-plant assay to screen water yam cultivars for anthracnose resistance and 
develop effective integrated pest management strategy for the disease in the field. Assessment of 
farmers’ knowledge and perception was ascertained through questionnaires and interviews of 120 
D. alata farmers in the Nkwanta-North and Krachi-West districts of the Volta Region. The 
findings indicated that, most D. alata farmers in both districts were aware of the presence of a 
vine-browning and die-back disease in their fields and could identify it by its symptoms but were 
ignorant of its causal organisms, spread and control. The incidence and severity of the disease in 
the Nkwanta-North district (75.30% and 1.80) were higher compared to Krachi-West district 
(55.70% and 1.60). The pathogens responsible for the disease in the Volta region and other yam 
growing regions (Ashanti, Brong-Ahafo, Central, Eastern and Northern regions) in Ghana were 
confirmed as Colletotrichum gloeosporioides Penz. and Sacc. and C. truncatum Andrus and Moore 
using symptoms of the disease, morpho-cultural characteristics and genome analysis of isolates, 
and pathogenicity test. The vine-browning and die-back disease was identified as anthracnose 
disease of D. alata. Morpho-cultural characters and Polymerase Chain Reaction (PCR) with five 
Random Amplified Poly-Dimorphism (RAPD) primers (OPA-11, OPA-18, OPC-5, OPC-7 and 
OPC-16) and five Inter-Spacers Restriction Fragment Length Polymorphism (ITS-RFLP) enzymes 
(HhaI, HaeIII, HinfI, MspI and RsaI) were used to characterize 16 strains of C. gloeosporioides 
and four strains of C. truncatum obtained from diseased D. alata samples in the Volta Region and 
other five yam growing regions in Ghana (Ashanti, Brong-Ahafo, Central, Eastern and Northern).  
The 16 C. gloeosporioides strains clustered into two major groups (with similarity of 52%) and six 
sub-groups while the C. truncatum strains were put into two groups (with similarity of 30%) using 
unweighted pair-group method with arithmetic average (UPGMA). Screening of 12 D. alata 
cultivars for resistance to the disease was performed using tissue culture-derived whole-plant 
assay. Of the 12 cultivars, six (TDa008, TDa010, TDa014, TDa023, TDa025 and TDa027) were 
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resistant to the disease but at varying levels whiles the six other cultivars (TDa007, TDa013, 
TDa015, TDa018, TDa019 and TDa021) were susceptible at varying levels. Cultivar TDa023 was 
highly resistant while TDa019 was highly susceptible. The use of integrated pest management 
strategy consisting of hot-water treated seed yam (45±1oC for 20 minutes and drying for 30 
minutes) of the highly resistant cultivar (TDa023) along with monthly weeding and Mancozeb 
sprays significantly (P < 0.05) reduced anthracnose severity (1.2) compared to the control (3.0) in 
the field. Although, the technology developed could effectively reduce the disease, it requires 
additional research at two or more locations to be sure of its sustainability before recommending to 
farmers. Moreover, benefit-cost analysis ought to be undertaken on the developed IPM strategy. 
.  
 
 
 
 
 
 
 
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CHAPTER ONE 
1.0 INTRODUCTION 
 
Yam (Dioscorea spp.) constitutes an economically important staple food for millions of people in 
the tropics and subtropics. West Africa accounts for about 95% of the total yam production area 
(FAO, 2002). The crop is one of Ghana’s major staples, being ranked second in importance (in 
tonnage terms) after cassava in staple food production but its relatively high unit price makes it 
the most important food crop in terms of value of production (Lebot, 2009). Along with cassava, 
it provides 31% of ‘national food security’ and supplies in excess of 50% of the calorie needs of 
the average Ghanaian (Dadzie, 2004). Yam is grown mainly by smallholders and serves as 
valuable income source for producers and retailers.  In addition, it generates foreign exchange 
earnings for the Country and plays a significant role in the socio-cultural lives (such as 
celebration of New Yam Festivals) of some producing regions such as the Ashanti, Volta and 
Brong-Ahafo Regions (Narula et al., 2007). The two most important cultivated edible yams in 
Ghana are white Guinea yam (D. rotundata Poir) and water yam (D. alata L.) (Ofori and Hahn, 
1991). 
 
D. alata is composed of food nutrients such as carbohydrates (starch, sugars, and fibers), proteins, 
minerals, a negligible amount of lipids and accounts for over 20% of the total dietary calorie 
intake in Africa and South Pacific (Opara, 2003; Bhandari and Kawabata 2004; Lebot, 2009). It is 
traditionally consumed as boiled, mashed, fried and roasted. However, some cultivars are well 
suited for pounding (Lebot et al., 1998). Industrially, certain D. alata cultivars are processed into 
flour, crisps, chips or cubes, flakes and ice-cream (Degras, 1993).  
 
In Ghana, D. alata is commercially produced in six major regions with Volta being the leading 
producer followed by the Eastern, Brong-Ahafo, Northern, Ashanti and Central regions (MoFA, 
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2010).  In comparison with D. rotundata, D. alata has superior characteristics for sustainable 
production, including high yield potential, ease of propagation, early vigour for weed suppression 
and storability of tubers (Asiedu et al., 2003). The limitation of D alata production in most areas 
in Ghana is the susceptibility of most cultivars to a leaf necrosis, vine-browning and die-back 
disease. Recently, prevalence of this disease has been reported by farmers and agricultural 
extension agents (AEAs) in the Volta region but it appears to be less significant in the other D. 
alata growing regions (MoFA-ESD, Personal Communication, 2012). The disease has, therefore, 
become a major constraint to D. alata production in the Volta region. Infections that commenced 
prior to or during tuber formation resulted in excessive yield loss of about 90% (Atsu and Akator, 
Personal Communication, 2012). A similar disease was reported to limit the production of the 
crop in the Caribbean (Degras et al. 1984; Green and Simons, 1994).  Abang et al. (2001a) 
reported 85% yield loss of D. alata due to the disease in Nigeria while 75-95% yield loss have 
been reported in the Caribbean (Mignucci et al., 1988; Green and Simons, 1994).  
 
Several important D. alata cultivars were lost in the Caribbean due to high incidence (85-100%) 
of a vine-browning and die-back disease (Degras et al., 1984; Mignucci et al., 1988; Green, 
1994). Recently, the Nkwanta-North and Krachi-West districts of the Volta Region has been 
encountering high incidence of the disease which has led to abandoning of D. alata production by 
many farmers with a negative impact on their livelihoods (Atsu and Akator, Personnal 
Communication, 2012). It was, therefore, imperative to assess the economic importance of the 
vine-browning and die-back disease affecting D. alata in the Volta Region of Ghana.  
 
The vine-browning and die-back disease symptoms described by the farmers and AEAs in the 
Volta Region were typical of Colletotrichum spp. infection as similar symptoms were reported on 
D. alata in Nigeria (Abang et al., 2001a) and the Caribbean (Green and Simons, 1994) were 
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associated with Colletotrichum gloeosporioides  and C. truncatum.  However, whether the same 
causal organisms were responsible for the disease in the Volta Region of Ghana was unknown. It 
was, therefore, necessary to identify the causal agents of the vine-browning and die-back disease 
of D. alata in the Volta region.  
 
 
Although, Colletotrichum spp. are widely accepted as the causal agents of a vine-browning and 
die-back disease of D. alata (Singh et al., 1966; Winch et al., 1984), identification and 
differentiation of species based on morphological and cultural characteristics have often been 
inadequate (Alonso-Gonzalez et al., 2006). C. gloeosporioides isolates from a vine-browning and 
die-back disease of D. alata, often appear similar in cultural and morphological characteristics but 
are highly variable in terms of virulence – from location to location (Abang et al., 2001). 
Diversity studies among Colletotrichum spp. using molecular techniques such as polymerase 
chain reaction (PCR) with species-specific primers (Adaskaveg and Hartin, 1997), inter-spacers 
restriction fragment length polymorphism (ITS RFLP), random amplified polymorphic DNA 
(RAPD) markers (Thottappilly et al. 1999; Alleyne, 2001) and inter-spacers (ITS) sequence 
analysis (Sreenivasaprasad et al., 1994; Sreenivasaprasad et al., 1996; Freire et al., 2001; Martín 
and García-Figueres, 1999)  are more efficient in resolving the systematic issues in 
Colletotrichum spp. Thus, the combined use of molecular diagnostic tools and traditional morpho-
cultural techniques would present an appropriate and good approach for studying Colletotrichum 
spp. complexes existing in the Volta region and/or Ghana at large.  
 
 
 
 
 
 
 
The deployment of durable host plant resistance in D. alata would contribute significantly to a 
high level of field performance. The highly spatial and temporal variations among Colletotrichum 
spp. coupled with long growth cycle of D. alata and the high cost of conducting multiple-location 
trials more often than not, limit field evaluation and generally makes it unproductive (Green et al., 
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2000). Previously, resistant cultivars in the Caribbean were reported to be susceptible as a result 
of new or previously unknown virulent strains of C. gloeosporioides (Ano et al., 2005) and 
selection of D. alata cultivars under sub-optimal conditions for disease development under natural 
field infection.  
 
 
Lack of standard screening procedures for yam anthracnose resistance delayed progress in 
resistance breeding until Onyeka et al. (2006) optimized tissue culture-derived whole-plant assay 
to evaluate D. alata cultivars for varying levels of resistance. This assay provides a probable rapid 
technique for evaluating breeding populations and studying the relationship between D. alata 
cultivars and pathogen isolates. Augmenting this method by taken into accounts, the disease 
development, rate of the disease progression and the final level of diseased area under the 
progress curve (AUDPC) (Walther et al. 1999; Jeger, 2004) would, therefore, not only provide a 
strong tool for identification of levels of resistance to the vine-browning and die-back disease but 
also the analysis of genetic basis for resistance in D. alata cultivars in the Volta region and/or 
Ghana at large. The study, then, proposed the use of tissue culture-deverived whole-plant assay to 
screen D. alata cultivars in the Volta region and/or Ghana – taking into consideration, the 
AUDPC.  
 
 
Currently, there are no available measures that ensure sustainable control of a vine-browning and 
die-back disease of D. alata in Ghana. Resistant D. alata cultivars seem to be the best means of 
controlling the disease in the Caribbean (Green and Simons, 1994) but cultivars which initially 
proved resistant to the disease later succumbed to it partly due to the high variability of the 
pathogen (Green et al., 2000). Furthermore, since resistance is relative, not all the D. alata 
cultivars in the Volta region or collections in Ghana would prove resistant to the disease. Hence, 
integrated pest management (IPM) measures which involve the use of healthy planting materials, 
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good agronomic practices and judicious use of effective fungicides would, therefore, form the 
basis of sustainable management strategies for the vine-browning and die-back disease in Ghana. 
The study, therefore, employed IPM strategy to manage the disease. 
The objectives of the study were to: 
• Assess the farmers’ knowledge, perception and practices on the cause, spread, control and 
importance of a vine-browning and die-back disease of D. alata in the Volta Region 
• Determine the incidence and severity of the disease in the Volta Region 
• Confirm the identity of the causal organisms as Colletotrichum spp. 
• Characterize the strains of the pathogen isolated  
• Utilize tissue culture-derived whole-plant assay to screen 12 D. alata cultivars for 
resistance to the disease. 
 
• Develop an integrated pest management (IPM) strategy to manage the disease.  
 
 
 
 
 
 
 
 
 
 
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CHAPTER TWO 
2.0 LITERATURE REVIEW 
2.1 Yam: Origin and distribution 
Yam (also called Ñamé -Spanish and Igname - French) belongs to the genus, Dioscorea and a 
family, Dioscoreaceae. Of the estimated 300-600 species available (Govaerts et al., 2007), only 
over half a dozen principal species are grown for consumption (Ofori and Hahn, 1991) while 
others are grown for medicinal purposes (Wu et al., 2005; Lebot, 2009). Yams originated in the 
Far East and spread westwards (Coursey, 1975) (Fig. 2.1). They have since evolved 
independently in the Eastern and Western Hemispheres and today yams are grown widely 
throughout the tropics (Asiedu et al., 2003). In the West African yam zone, which is the principal 
producer on a global basis, D. rotundata, D. alata and D. esculenta are the most common species 
(Degras, 1993). 
 
       D. alata;             D. dumetorum and D. hipsida;          D. trifida;          D. esculenta;        D. cayenensis 
and D. rotundata and           D. bulbifera 
 
 
 
 
 
Fig. 2.1: Origin and distribution of Dioscorea species worldwide. Source: Coursey (1975) and Degras 
(1993). 
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2.2 Botany of yam 
Yam (Dioscorea spp. L) is a polyploid and a vegetatively propagated tuber crop that is cultivated 
in the yam belt of West and Central Africa (IITA, 2009). It belongs to a group of crops labelled 
“orphaned crops”, which have not received research attention for a long period of time and, 
therefore, has attain very little improvement (Otoo, 2001b). The major edible species of yam of 
African origin are white Guinea yam (D. rotundata Poir.), yellow Guinea yam (D. 
cayenensis Lam.), and trifoliate or bitter yam (D. dumetorum Kunth). Edible species from Asia 
include water or greater yam (D. alata L.), and lesser yam (D. esculenta). Cush-cush yam (D. 
trifida L.) originated from the Americas (IITA, 2009). White Guinea yam and water yam are the 
most important in terms of cultivation and use in West Africa. Yam is grown in about 51 
countries in the tropics and subtropics with yields averaging about 11 million metric tons/hectare 
in the major producing countries of West Africa (Nigeria, Cote d’Ivoire, Ghana, and Benin) 
(Okwu and Ndu, 2006). 
Dioscorea is a genus of over 600 species with subterranean tubers or rhizomes (Govaerts et al., 
2007). The tubers are storage organs and often grown to a substantial size; they produce short, 
fibrous roots and annual shoots, which are winding (except in dwarf species), the direction of 
winding being specific. The genus Dioscorea is divided into a number of taxonomic sections; the 
important food yams are grouped in the following: Enantiophyllum- D. alata, D. rotundata/D. 
cayenensis complex, D. opposita and D. japonica; Lasiophyton- D. dumetorum and D. hispida; 
Combilium - D. esculenta and Macrogynodium- D. trifida; Opsophyton- D. bulbifera (Lebot, 
2009).  
Yam has an annual vegetative system composed of a root apparatus (some extend throughout the 
upper layers of the soil, others consist of root hairs), a stem apparatus, and a foliar apparatus. The 
adventitious roots arising from the base of the stem absorb mineral nutrients, and water (Orkwor 
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et al., 1998). The yam stem is usually a thin twining vine allowing the plants to climb. It is 
frequently winged and commonly spiny. Several species have deep striations in their stem, some 
contain anthocyanins whiles others have large thorns and generally, the direction of the twining is 
used as a taxonomic feature (Bourret, 1973). The leaves are petiolate (except for D. dumetorum, 
D. hispida and D. pentaphylla, which have trifoliate leaves and hairs on their stems), often 
cordate with strongly marked reticulate veining (unusual for a mono-cotyledon), sometimes 
lobed, occasionally palmately compound (Okwu and Ndu, 2006) and have an arrangement either 
opposite or alternate with axillary buds (Degras, 1993a).  
It has small flowers borne in elongated racemes, with male and female separate and usually borne 
on different plants. The female flowers form dehiscent casing, usually trilocular, with 6 seeds, 
generally winged and flat for wind dispersal, though many of the cultivated forms have become 
partially or highly sterile (Akoroda and Hahn, 1995). The seed usually goes through a dormancy 
period of three to four months before germination can occur. The genus Dioscorea is dioecious 
with an extremely irregular production of male and female flowers pollinated by insects (Bourret, 
1973). As flowering is rare, yams are vegetatively propagated using the basal nodal region, the 
tuber and the bulbils (Degras, 1993a). Plants usually produce a single annual tuber, which is 
usually 20 to 40 cm long and weighs from two to several dozen kilograms, depending on cultivar 
and growing conditions (Dadzie, 2004). The body can be elongated or spherical with a white, 
yellow or purple flesh. Bulbils, which are characteristics of D. alata, are produced in the axils of 
the leaves which have the morphology and appearances of condensed stems and in a few 
instances are relatively large and tuberous weighing about 20 to 100 g (Degras, 1993, 1993b). By 
contrast, D. bulbifera produces only aerial tubers, which can weigh up to 1 kg (Dadzie, 2004).  
 
Yam tuber does not sprout during the early part of storage, even under suitable growth conditions 
(Alexander and Coursey, 1969). The length of tuber dormancy is endogenously controlled and 
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conditions such as availability of soil moisture or cool temperature are ineffective triggers of 
sprouting (Passam and Noon, 1977). The length of dormancy is also influenced by the yam 
species (Otoo, 2001b). The environmental conditions affecting yam tuber dormancy are 
photoperiod, white and coloured lights, temperature, relative humidity and partial oxygen 
pressure (Akem and Asiedu, 1994). Physiological age of tubers affects their readiness to sprout, 
but by approximately 6 months after harvesting, dormancy disappears completely and bud-less 
setts planted after that period will require nearly the same time to sprout (Onwueme and Charles, 
1994).  
2.3 Distinguishing characteristics of D. alata and D. rotundata 
D. alata (water yam) and D. rotundata (white yam or white guinea yam) are the two most 
important yam species cultivated in Ghana and/or West Africa (Dadzie, 2004; IITA, 2009; 
Cornelius, 2011) with distinct characteristics. D. alata is a large climbing plant, which usually 
reaches 15 m in height, with quadrangular winged stems, twining anticlockwise (to the right) 
(Putnam and Gaskalla, 2010). Leaves are opposite, variable in size and shape, but usually ovoid to 
cordate with a deep basal sinus, acuminate. The male flowers are borne on panicles, up to 30 cm 
long whilst the female flowers are on small axillary spikes. Few cultivars produce fertile seed and 
most are completely sterile (Akoroda and Hahn, 1995). Bulbils are liberally formed in leaf axils 
of some cultivars (Degras, 1993b).  
The tubers are usually single except few cultivars and exhibit wide variation in shape, colour and 
size: they are generally cylindrical but may be long and bending to almost globular and are often 
pronged or lobed, or even flattened and fan-shaped (Alexander and Coursey, 1969). Their weight 
is usually 5-10 kg though special cultivation can yield giant tubers of 15 kg or more (Putnam and 
Gaskalla, 2010). The flesh of some cultivars can be deep reddish-purple or pink and these forms 
have been classified as D. purpurea Roxb. and D. afropurpurea Roxb. (Lebot, 2009).  Dioscorea 
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rotundata on the other hand, is a deciduous, herbaceous thorny vine, dying to the ground after 
physiological maturity (Bourett, 1973). The stems are twining, growing to 20 m or more, with 
four often purplish wings (cross-shaped or square in cross-section) (Alexander and Coursey, 
1969). Tubers formed in some cultivars are elongated, hairy with rough-textured surface which 
grow to as much as 15 cm long (Amusa et al., 2003). The leaves are opposite, but often alternate 
at the base of the vine. The blades are heart-shaped, with rounded basal lobes, or arrowhead-
shaped, with angular basal lobes; the five to seven arching, longitudinal veins are conspicuous 
(IITA, 2009). Leaf blades are as much as eight inches long and six inches broad; the petioles are 
often as long as the blades. Flowering is frequent and conspicuous and fruits usually produced are 
3-winged capsules borne in pendent racemes (Putnam and Gaskalla, 2010). 
2.4 Economic importance and uses of yam 
Yams are second to cassava as the most important tropical root crop (in terms of production or 
consumption) (Dadzie, 2004). Yams are a staple crop in many parts of Africa and Southeast Asia 
(Amusa et al., 2003). In the South Pacific, the yam is a significant food crop, accounting for over 
20%, 8.1%, and 4.6% of the total dietary calorie intake in the Kingdom of Tonga, Solomon 
Islands, and Papua New Guinea, respectively (Simons, 1993). Besides their importance as food 
source, yams also play a significant role in the socio-cultural lives of some producing regions like 
the celebrated New Yam Festival in West Africa, a practice that has also extended to overseas 
where significant population of tribes from yam growing areas observe it (Narula et al., 2007). In 
some parts of Southeastern Nigeria, the meals offered to gods and ancestors consist principally of 
mashed yam. Yams store relatively longer in comparison with other tropical fresh produce, and 
therefore stored yam represents stored wealth which can be sold all-year-round by the farmer or 
marketer.  
 
 
 
 
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In parts of Igbo land in Southeastern Nigeria, it is customary for the parents of a bride to offer her 
yams for planting as a resource to assist them in raising a family (Egesi et al., 2007). The Dioscorea 
species have long been cultivated for their medicinal properties (sapogenin steroids, used in 
production of cortisone and synthetic hormones) in the following order of importance: D. 
bulbifera, D. cayenensis, D. dumetorum, D. alata, D. trifida, D. laxiflora, and D. microbotrya 
(Karnick, 1969; Pedralli et al., 2002). The wide array of saponins and steroidal sapogenins from 
various cultivars provides the pharmaceutical industry with compounds for oral contraceptives 
and prevention of diseases such as malaria, yellow fever, skin rashes and dengue (Bhandari and 
Kawabata, 2004; Lebot, 2009).  Wu et al. (2005) analyzed the consumption of D. alata by post-
menopausal women and concluded that although the mechanisms are not yet fully understood, its 
consumption reduces the risk of breast cancer and cardiovascular disease. Nutritionists stress the 
importance of yams in treating anaemic patients given their wealth of nutrients, vitamins, and 
minerals (Okwu and Ndu, 2006). In Africa, it was found that the tuber is responsible for increased 
fertility in women who habitually consume it (Balbach and Boarim, 1993). Diosgenin can be 
extracted from D. bulbifera (Narula et al., 2007) and can also be used as an anti-tumor agent (Gao 
et al., 2007).   
 
According to FAOSTAT (2005), an estimated 48.7 million tons of yam were produced worldwide 
in 2005, with Sub-Saharan Africa accounting for 97 per cent of this figure. In Nigeria alone, 
export earnings from yams hit N56 bn in 2008, up from N37 bn realized in 2007 and about N70 
bn was realized from yam export in 2010. In 2008, the World production of yam was 51.4 million 
tons out of which Nigeria accounted for an average of 36.7 million tons, Ghana for 3.6 million 
tons and Cote d'Ivoire for 4.8 million tons (FAOSTAT, 2010). Farmers have constantly affirmed 
that yam cultivation is a veritable source of income. According to statistics, average daily 
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consumption per capita of yams is highest in Benin (364 kcal), Côte d‘Ivoire (342 kcal), Ghana 
(296 kcal), and Nigeria (258 kcal) (IITA, 2009). 
 
 
 
 
 
 
 
A typical analysis of the edible portion of the tubers is: water 65-73 per cent; protein 1.12-2.78 
per cent; fat 0.03-0.27 per cent; carbohydrate 22-29 per cent; fiber 0.65-1.40 per cent; ash 0.67-
2.06 per cent (IITA, 2000). The starch contains a high proportion of fairly large granules: sizes 
ranging from 5 to 50 microns have been reported. The gelatinization temperature ranges from 
69° to 88° and the viscosity from 100 to 200 Brabender units. Unlike most other yam species, 
starch from D. alata has high gel strength. Starch from white-fleshed and purple-fleshed cultivars 
have similar typical composition averaging: moisture 13.6 per cent; protein 0.14 per cent; ash 
0.22 per cent; amylose 21.1 per cent; reducing sugars 0.18 per cent; pH 7.1 (Tetteh and Saakwa, 
1991; Bailey and Jeger, 1992). Ascorbic acid contents ranging from 4.9 to 8.2 mg/100 g of edible 
portion have been reported, while certain cultivars in the South Pacific have been found to contain 
6 mg/100 g of carotene. Three anthocyanins have been isolated from D. alata var. atropurpurea 
and rubella and found to be cyanidin glycosides (Degras, 1993a).  
 
Yams are a source of food, a source of income to access food, leads to reduction in imported food 
and thus adds to local food self-sufficiency (FAO, 2000). This is even more significant when the 
untapped potential of yams is considered. Jamaica grows and exports several varieties of yams but 
the most popular and lucrative is yellow yams (D. cayenesis) which displaced Negro yam (D. 
rotundata) as the main yam variety by the mid-1970s. Yellow yams currently account for close to 
60 percent of total yam production and perhaps as much as 90 percent of all Jamaican yam 
exports. Jamaica is the world‘s leading yam exporter accounting for 16 percent of the global yam 
trade but has a monopoly of the yellow yam market (FAOSTAT, 2010). It serves a critical 
employment role with thousands of rural families making a living from yam cultivation.  
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2.5 World production and trade of yam 
Most of the world production of yam is from Africa (about 96%) with Nigeria alone accounting 
for nearly 75% of the total world production; World annual production was estimated to be 25 
million metric tons in 1974, and 24 million metric tons in 1992 (Moura-Costa et al., 1993). In 
1995, total world production increased from 32.7 million metric tons to 37.5 million metric tons 
in 2000 (IITA, 2009).  Also, in the period, export quantity declined slightly while export income 
remained fairly steady (FAOSTAT, 2010). During the period 1975-90, total yam cultivated area 
increased by about 38.8% globally, while the total production increased by 45.8%. However, the 
importance of yam in the economy of the main producing areas appears to be declining due partly 
to competition with other crops like cassava in Nigeria and taro in the South Pacific (Opara, 
1999). The major producing areas have also continued to experience high population growth 
rates. During the last four decades, the annual growth rate (%) of per capita production in the 
major yam zones in Africa has declined (Egesi et al., 2007). 
2.6 Yam production in Ghana 
In Ghana, yam is grown on free draining, sandy and fertile soil after clearing the first fallow. 
Land is prepared in the form of mound or ridge or heap of 1 meter (3 ft. 3 inch) height or on the 
flat. The yams recommended for such soil conditions are the white yam or white guinea yam (D. 
rotundata) and water yam or yellow yam (D. alata). Planting is done by seed yam or cut setts 
from ware tubers (CCPGS, 2011). The tubers are subjected to treatment with wood ash or a 
fungicide (thiabendazole) 24 hours before planting to prevent damage to the tubers. The setts are 
planted at an interval of 15–20 cm (5.9–7.9 inch) with the cut surface facing up. Mulching is 
essential during October–November with dry grass or plant debris weighed down with balls of 
mud. Dosage of fertilizer application, as essential, is decided after chemical analysis of the soil 
samples. Manual weeding by hoeing is done three or four times depending on rate of weed growth 
(Dadzie, 2004). Yam is currently the fourth most important tuber-root crop in the world, after 
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potato (Solanum tuberosum L.), cassava (Manihot esculenta Crantz), and sweet potato (Ipomoea 
batatas L.). In 2008, the estimated world production of yams was 51.7 million tons, with Africa 
leading the production. West Africa is the most important area for yam production in the world.  
 
 
 
 
 
 
Ghana is the second largest producer of yams in the world next to Nigeria (FAOSTAT, 2010). In 
Ghana, during 1997 and 1998, the crop was ranked second in importance (in terms of tonnage) 
after cassava and was the most important crop in terms of value (Dadzie, 2004). A survey by GTZ 
in the Northern Region of Ghana identified yams as the most important cash and food crop in that 
region, followed by groundnuts, cassava and maize. Also, the National Agricultural Research 
Strategy Plan (NARSP) identified yam, cassava and cocoyam as first priority commodities for 
research, with yam receiving the highest priority rating of all crops. However, there are a number 
of serious constraints to yam production. These include: pests and diseases, the low multiplication 
rate (and hence low availability of planting material), declining soil fertility and the high and 
expensive labour inputs required (Degras, 1993). Yams are almost entirely vegetatively 
propagated by planting pieces of tubers, or setts or whole small tubers. Each seed piece usually 
weighs 115 to 140 g. They can be planted 7 to 9 cm deep in 1.2 m rows with plants spaced 60 cm 
apart, or in hill plantings 1 m apart. 
 
Traditionally in Ghana, farmers obtain their planting materials either from their own farms, or by 
buying the surplus from neighboring farmers. This means that the planting material is often of 
low quality, being infected with fungal pathogens, virus and/or nematodes and may be relatively 
expensive (Tetteh and Saakwa, 1991; Latunde-Dada et al., 2001). In order to ensure the survival 
and growth of the planting materials, fairly large pieces of seed yam (220 g) are used which adds 
to the cost and results in a very low multiplication rate. Inter-cropping is the most common 
practice, with the distance between yam plants determined by the number and types of the other 
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crops in the field. However, where sole cropping is done, rows are 1 m apart, with intra-row 
spacing of 50-100 cm (Dadzie, 2004). 
2.7 Climatic, water and soil requirements of yam 
 
 
Yam is a plant of the sub-humid to humid tropics but also does well in semi-arid regions. 
Reported temperature range for growth is 14 - 40 °C with the optimum between 20-32 °C (Abang 
et al., 2003). Since it takes 7-10 months to mature in the field, a well-distributed annual rainfall 
(or water supply) ranging between 700-8000 mm with the optimum between 1200-4000 mm is 
required for yam production (Akem and Asiedu, 1994; Egesi et al., 2007). However, dry spells of 
up to 1 month during the growing season do not severely reduce yields. Yam is sensitive to 
aluminium toxicity in the soil. It thrives in deep, fertile, well drained, loose, loams and sandy-
loam soils with low levels of salinity and responds well to manuring. In comparison with other 
tropical tuber crops, yam requires soil of high fertility (Dadzie, 2004). Heavy clays tend to be 
waterlogged and result in tuber rots and difficult harvesting. Gravel or rocky soils tend to hinder 
tuber penetration. Required soil pH range for growth is 4.8 - 8.5 with the optimum between 5.5 - 
6.5 (IITA, 2009).  
2.8 Husbandry practices/staking 
Plants are usually staked soon after emergence. However, unstaked cultivation which suppresses 
weeds better but gives lower yields is also practiced (CCPGS, 2011). Weeding is done 3-4 times 
during the season, using hand tools. In general, yam is the first crop in the rotation after fallow. 
Some kind of support is usually required for good production of yams. D. rotundata and D. 
cayenensis are sometimes grown without support, but the sacrifice in yield is so great that only 
dire shortages of labour or staking material should prevent staking. The staking systems are 
usually built of local materials such as sticks or branches, slim bamboos, or bamboo with side 
branches. Sometimes dead materials from previous crops such as corn, sorghum, or tobacco are 
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used, but these usually do not last long enough to give support through the growing season. The 
use of living plants is also practiced but not recommended because they compete with the yams 
and severely reduce yields (Otoo, 2001b). However, because the staking of yams is so costly, 
further attempts to develop a living-plant system especially, with legumes such as Cajanus and 
Sesbaniay is being advocated (Dadzie, 2004). One of the best staking system currently designed 
in Nigeria by the IITA (2009) and being replicated in Ghana involves twelve-foot teak poles 
strung with 12-gage galvanized wire 6 to 8 feet off the ground and tied to stakes driven into the 
ground. Two rows of yams are then planted, one on each side and sufficiently developed vines are 
passed onto the wire with support of strings.  
 
2.9 Harvesting  
In many parts of West African yam zone, mature yams are harvested at the end of the rainy 
season or early part of the dry season, which coincides with the end of vegetative growth (Dadzie, 
2004). Yams for long-term storage (for marketing or seed) are usually harvested during the 
harmattan period (Dec-Jan) in many parts of southeastern Nigeria when the crops have attained 
maximum growth and maturity. Harvesting occurs 7-10 months after planting. Hand tools are 
used to dig up the tubers. Although the tubers can be kept quite well, they are easily damaged 
during and after harvest, and therefore need careful handling to avoid bruises.  
2.10 Field diseases of yam  
Yam diseases do not only cause severe yield losses but also lead to genetic erosion as well as 
restricting international germplasm movement and exchange (IITA, 2000). However, it has been 
estimated that an average of over 25% of the yield is lost annually to diseases and pests (Arene, 
1987; Ezeh, 1998; FAO, 1998). Anthracnose disease of yam is often referred to by a number of 
names including yam necrosis, yam scorch, yam chlorosis and vine-browning and die-back. 
Yam anthracnose disease caused by C. gloeosporioides has a wide distribution and is becoming a 
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major worldwide biotic constraint to yam (Dioscorea species) production especially, in the 
humid and sub-humid tropics (Emehute et al., 1998). Fournet et al. (1974) and Nwankiti and 
Arene (1976) also reported the destruction of the disease to D. alata species especially, in the 
Caribbeans and the yam zones of tropical Africa. On the same account, Nwankiti and Ene (1984) 
and Winch et al., (1984) reported that D. alata, the most widely distributed species are 
particularly susceptible to the disease whiles other yam species are affected to a much lesser 
extent (Mignucci et al., 1988).  
 
 
 
Despite the minor effects of pests on yam cultivars in comparison to other crops, phytosanitary 
problems represent the main difficulties for producers. The virus [Yam mosaic virus (YMV)], 
anthracnose disease of yam caused by the fungus (Glomerella cingulata), nematodes 
(Meloidogyne spp., Pratylenchus spp. and Scutellonema bradys) are the main diseases that 
account for the highest field losses in susceptible cultivars (Onayemi, 1983; Abang et al., 2003; 
Amusa et al., 2003). A vine-browning and die-back disease stands out as the principal 
phytosanitary problem in several areas of yam cultivation and production (IPGRI/IITA, 1997). 
Nematodes, which interact with fungi and bacteria, attack the tubers in the field and continue to 
cause damage in post-harvest. Yam meloidogynoses are diseases caused by nematodes of the 
genus Meloidogyne, which show high incidence and severity in production, causing heavy losses 
in yam production and marketing (Abang et al., 2002; Mignouna et al., 2003b). Yam mosaic virus 
caused by an aphid-transmitted potyvirus is another important field disease affecting yam (Egesi 
et al., 2007). 
2.10.1 Foliar fungal diseases  
Anthracnose disease is one of the major foliar diseases affecting yam and has had a considerable 
impact on water yam production world-wide (Nwakiti and Arene, 1976; Simons, 1993). It is 
caused mostly by the fungus C. gloeosporioides (Nwakiti et al., 1987). The disease attacks 
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several species of Dioscorea, particularly D. alata, D. cayenensis, D. rotundata and D. trifida. 
The symptoms observed in each host can be vein banding, curling, mottling, green-spotting and 
flecking (Mantell, 1980; IITA, 1993; Asiedu et al., 2003). Concentric leaf spot disease of yam 
caused by Sclerotium rolfsii has also been reported in south western Nigeria (Amusa, 2000). 
Symptoms of the disease include circular leaf spots of varying sizes that form concentric rings. At 
maturity, the center of the leaf spots contains sclerotia of the fungus. The lesion may merge, 
together with the center eventually falling out due to necrosis (Mantell, 1980; IITA, 1993). At 
nurseries, the pathogen causes complete blight of sprouting yam with sclerotia being produced at 
the base of the infected yam leaves. Soil, plant debris and several weeds such as Aclipha ciliata, 
Chromolaena odoranta, Euphobia heterophilla, Ipomoea triloba and Commelina erecta can 
harbour the pathogen (Amusa, 2000). The effect of the disease on yield has not been investigated, 
but it is anticipated that under severe attack yield loss of more than 50% could be obtained (IITA, 
2009).  
 
Zonate leaf spot caused by Curvulariae ragroistides and Pestalotia macro trichia is occasionally 
encountered on D. alata fields (PANS, 1984; Amusa et al., 1993). Also, foliar symptoms of 
nematode infections on yams are sporadically observed on the field. Early yellowing of leaf and 
termination of vine growths have been observed on D. rotundata infected with M. incognita, but 
infections rarely reduces total tuber yield (Adesiyan and Odihirin, 1978). Cercospora leaf spot 
caused by Cercospora spp. also affect yam species resulting in appreciable yield reduction (IITA, 
2000). 
2.11 Economic importance of anthracnose disease of D. alata 
Anthracnose disease has had a dramatic impact on D. alata cultivation in regions such as the 
Caribbean where mono-cultures with a single popular susceptible variety are common (Orkwor 
and Asiedu, 1995). In this region, susceptibility to anthracnose disease has made it virtually 
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impossible to grow the popular D. alata cultivars Pacala and White Lisbon. The disease is 
considered the single most important factor responsible for the decline of water yam production in 
the Carribean (Ano et al., 2002; McDonald et al., 1998). Nwankiti and Ene (1984) reported D. 
alata yield loss of about 80% in Nigeria as a result of attack by anthracnose disease. On-farm 
yield-loss studies in three agro-ecologies within the water yam production zone of West Africa 
showed that the impact of the disease on yield is highly dependent on time of disease onset and 
the prevalent environmental conditions (Green, 1994). C. gloeosporioides was recently found to 
be associated with deep-seated (systemic) infection of botanic seeds of water yam (Abang, 1997).  
 
Yam breeders do report instances where it has proved impossible to obtain viable seeds from 
parental genotypes because of the high flower abortion and low fruit set associated with the 
disease (Orkwor and Asiedu, 1995). Although the exact role of infected seed in the epidemiology 
of water yam vine-browning and die-back is yet to be ascertained, it poses a serious problem in 
the international distribution and exchange of water yam (seed) germplasm since materials have 
to be certified disease-free. Furthermore, the disease is implicated in the erosion of genetic 
diversity in large-scale field collections of water yam germplasm (Orkwor and Asadu, 1997). C. 
gloeosporioides is also known to cause ‘dead skin’ disease in the Caribbean, in which severely 
affected tubers fail to sprout and become rapidly infected with other pests and pathogens (Green 
and Simons, 1994). The pathogen is believed to play a role in tuber bio-deterioration in Nigeria; 
however, ‘dead skin’ symptoms have not been reported.  
 
Vine-browning and die-back disease occurs wherever water yam is grown but extreme 
temperatures in parts of northern Nigeria appear unfavorable for disease development (Green, 
1994; Nwankiti and Ene, 1984). In areas where vine-browning and die-back is not currently 
considered a constraint, there is a risk of higher yield losses in the future, as the trend towards 
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shorter fallows increases and as new hybrids replace a wide spectrum of local landraces. The 
disease causes leaf necrosis and die-back of D. alata vines, leading to a reduction in the effective 
photosynthetic surface area of the crop with a concomitant reduction in ability of the yam tuber to 
store food reserves (Abang et al., 2002). Green (1994) recorded losses in excess of 85% while 
Mignucci et al. (1988) reported losses exceeding 90%. Farmers in the Caribbean can no longer 
rely on the hitherto effective benzimidazole fungicides due to the development of fungicide-
resistant C. gloeosporioides strains (Bayart and Pallas, 1994). The presence of several pathogenic 
fungi on the yam phylloplane led Amusa et al. (2003) to suggest that anthracnose disease is a 
‘disease complex’, requiring the concerted action of a number of fungal pathogens for significant 
symptom development. In virulence studies under controlled environment conditions, however, 
Abang (1997) showed that a single slow-growing grey isolate of C. gloeosporioides is capable of 
causing 100% leaf abscission and premature death of up to 76% of inoculated plants. Earlier 
reports showed that the disease was severe only on D. alata but more recent investigations have 
revealed that the disease is prevalent and severe on most of the widely cultivated species of yam 
(Akem and Asiedu, 1994). 
2.12 Prevalence, incidence and severity of yam anthracnose disease 
Although, yam anthracnose prevalence is usually confused with and at times used interchangeably 
with the disease incidence, prevalence refers to the number of geographical units (field, farms, 
districts, regions or other defined populations) where the disease has been visually observed or 
detected, divided by the total of the geographical units sampled and evaluated (Nutter et al., 1991; 
Cooke et al., 2006; Nutter et al., 2006). The disease prevalence data is usually multiplied by 100 
to obtain a percentage of the geographical units diseased.  Hu et al. (1995) also stated that disease 
(yam anthracnose) prevalence can be obtained by sampling whole plant samples or plant parts 
from a population and using appropriate indexing methods to detect the presence of the pathogen. 
According to Nutter (1997), pathogen prevalence may provide quantitative information on the 
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presence of the pathogen (Colletotrichum spp.) in symptomatic plants. Generally, samples 
collected from a single geographical location are bulked and indexed to detect the presence of the 
pathogen. Disease incidence on the other hand, is the number or proportion of plant units that are 
visibly diseased as a result of a pathogen (Colletotrichum spp.) in relation to the total number of 
plant units sampled and assessed (Madden and Huges, 1995; González-Pérez et al., 2011).  
2.13 Symptoms of anthracnose disease of D. alata  
Anthracnose disease attack all parts of D. alata plant at all stages of development (Egesi et al., 
2009). Infections concentrated or expressed on the shoot tips generally result in vine and/or tip 
die-back (Abang et al., 2006). Other symptoms expressed generally, depend on the age of the yam 
plant, the environmental conditions and the species and/or cultivar grown (Ayodele et al., n.d). 
Typically, when infection is initiated on the leaves, symptoms first appear on lower older leaves 
close to the soil surface as pinpoint lesions that are dark brown, with or without pale brown or 
yellow chlorotic halos (Winch et al., 1984; Akem and Asiedu, 1994). The lesions then expand 
rapidly and may coalesce, encompassing the entire leaf and resulting in premature leaf abscission. 
Severe infection results in defoliation leaving naked, black and drying vines (Arene, 1987; 
Amusa, 2000; Prasath and Ponnuswami, 2007). The enlarged spots form yellow margins and the 
centre may fall out giving a ‘shot-hole’ effect.  
 
Infected leaves sometimes fall off reducing the effective photosynthetic surface of the plant and 
early infection may result in premature death of the plant. Shoot tip and stem die-back frequently 
occur under such severe infections (Abang, 2003). In some cases, there is a superficial bronzing 
and blackening of mature leaves on surfaces exposed to the sun, a condition often referred to as 
scorching.  According to Green and Simons (1994), the disease affects the leaves, petioles, stems 
and veins of the plant, causing leaf spots, leaf blotches, petiole blights, pre-mature abscission, 
dieback and eventual death of the entire plant. The disease usually has a dramatic effect on 
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infected plants, converting a field of initially healthy yam plants from ‘green’ to ‘black’ within a 
few weeks (Green and Simons, 1994).  
2.14 Etiology of anthracnose disease of D. alata 
The pathogen causing anthracnose disease of D. alata was first described as Gloeosporium pestis 
Massee from yam in Fiji (Winch et al., 1984). It was later reported from D. alata in India (Singh 
et al., 1966) and subsequently classified as C. gloeosporioides. There have been conflicting 
reports on the actual cause of anthracnose disease of D. alata. Previous work by Amusa (1997) 
indicates that yam anthracnose is a disease complex and has been associated with the activities of 
C. gloelsporioides, Curvularia pallescens, C. eragrostides, Pestalotia spp. and Rhizoctonia 
solani. Many researchers have referred to the disease by the common names of: yam chlorosis or 
yam scorch in reference to the symptoms observed on leaves (Ayodele et al., n.d). The confusion 
associated with naming of the disease is due to uncertainties of its actual cause and has 
necessitated studies to determine the pathogen of the disease.  
 
 
 
 
 
Although other pathogens are involved in the anthracnose disease complex, several researchers 
have confirmed that colletotrichum spp. is the causal organism of the disease (Winch et al., 1984; 
Singh et al., 1966). Extensive studies by Nwankiti and Ene (1984), Abang et al. (2002) and 
Aduramigba-Modupe et al. (2010) have confirmed C. gloelsporioides as the primary cause of 
anthracnose disease of D. alata. Akem (1999) argued that, due to the predominance of C. 
gloeosporioides from sample isolations and the reproduction of typical symptoms of anthracnose 
disease with the fungus, C. gloeosporioides is the causal agent of the disease on D. alata. Studies 
by McDonald et al. (1998) and Ano et al. (2002) revealed that C. gloeosporioides is the main 
cause of anthracnose disease of D. alata in the Carribean. Earlier reports in Ghana revealed that 
Colletotrichum spp. were the causal agents of anthracnose of D. alata (Oduro, 2000; Offei et al., 
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2008). In Nigeria, both C. gloeosporioides and C. truncatum have been reported as the cause of 
anthracnose disease of D. alata (Ayodele et al., n.d). 
2.15 Colletotrichum species 
The genus Colletotrichum includes a number of plant pathogens of major importance, causing 
diseases of a wide variety of woody and herbaceous plants (Abang et al., 2006). It has a primarily 
tropical and subtropical distribution, although there are some high-profile species affecting 
temperate crops. Fruit production is especially affected; both high value crops in temperate 
markets such as strawberry, mango, citrus and avocado, and staple crops such as banana (Waller 
et al., 2002). Colletotrichum species cause devastating disease of coffee berries in Africa, and 
seriously affect cereals including maize, sugar cane and sorghum. The genus was recently voted 
the eighth most important group of plant pathogenic fungi in the world, based on perceived 
scientific and economic importance (Yuan et al., 2011). As plant pathogens, Colletotrichum 
species are primarily described as causing anthracnose disease although other maladies are also 
reported such as red rot of sugar cane, coffee berry disease, crown rot of strawberry and banana, 
and brown blotch of cowpea (Rojas et al., 2010). Many species may be seed-borne and can 
survive well in soil by growing saprobically on dead plant fragments and may be spread through 
water-splash dispersal of conidia and air transmission of ascospores from the sexual morph 
(Nicholson and Moraes, 1980).  
 
Infection occurs through an appressorium that develops from the germinating spore on the plant 
surface, followed by turgor driven penetration of the cuticle and in some cases, of epidermal cells 
by infective hyphae (Bailey and Jeger., 1992). Establishment within plant tissues is aided through 
the production of host-induced virulence effectors by the fungus (Kleeman et al., 2012; 
O’Connell et al., 2012). Nascent colonies in most cases then enter a biotrophic phase with 
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infected tissues remaining externally symptomless which may be short (O’Connell and Reed, 
2012) or extended and presumably involve dormancy (Prusky and Plumbley, 1992). Then, the 
fungus enters a necrotrophic phase that results in significant death of plant cells and the 
emergence of pathogenic lesions. This delayed onset of disease symptoms may lead to significant 
post-harvest losses, with apparently healthy crops degenerating in storage (Prusky and Plumbley, 
1992). The biotrophic life strategies adapted by Colletotrichum species may also contribute to 
their prominence as symptomless endophytes of living plant tissues (Lu et al., 2004; Joshee et al., 
2009; Rojas et al., 2010; Yuan et al., 2011).  
2.15.1 Existence of Colletotrichum spp.  
Colletotrichum species have been reported to exist as endophytes, epiphytes, saprobes, plant 
pathogens and even human pathogens (Sutton, 1992; TeBeest et al., 1997; Cano et al., 2004; 
Kumar and Hyde, 2004; Photita et al., 2004, 2005; Promputtha et al., 2007). Colletotrichum 
species that cause serious plant disease are also commonly isolated as endophytes from healthy 
plants and have been identified as saprobes on dead plant material (Photita et al., 2001a; Kumar 
and Hyde, 2004; Liu et al., 2007; Promputtha et al., 2007; Damm et al., 2009; Prihastuti et al., 
2009). In many cases, the same species have been recorded with several lifestyles although with 
the ambiguity of species identification, it is not always clear whether they are definitely the same 
species. Prihastuti et al. (2009) described C. fructicola and C. siamense from coffee berries, 
isolated as epiphytes, endophytes and pathogens and these species have since been shown to be 
widespread on several hosts (Yang et al., 2009). C. dematium also occurs as an endophyte, 
pathogen and saprobe (Latunde-Dada et al., 2001; Damm et al., 2009).  
 
Colletotrichum is one of the most economically important genera of fungi, causing anthracnose 
disease, affecting a wide host range, especially on tropical and subtropical crops as well as fruit 
trees (Sutton, 1992). Above ground plant parts can be affected by anthracnose diseases at all 
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stages on stems, leaves, flowers and fruits. An example of anthracnose disease caused by 
Colletotrichum spp. familiar to many is the blackening of tropical fruits (Tang et al., 2005; 
Crouch and Beirn, 2009), especially bananas and mangoes in fruit bowls. The disease often takes 
two forms, resulting in spots on leaves or the blackening of fruits, usually post-harvest. In the case 
of persimmon, Colletotrichum horii can infect fruits, twigs, cause dieback and even tree death 
(Zhang, 2008; Hyde et al., 2009). Colletotrichum acutatum, C. capsici and C. gloeosporioides 
have been reported causing anthracnose disease on chili fruits in Thailand (Than et al., 2008b). 
2.15.2 Colletotrichum as endophytes 
Colletotrichum species have been found as symptomless inhabitants (endophytes) in plant tissues 
(Liu et al., 2007; Damm et al., 2009; Prihastuti et al., 2009). For example, putative C. 
gloeosporioides and C. acutatum strains were isolated from healthy leaves and pseudo stems of 
banana (Musa acuminata), ginger (Alpinia malaccensis), Euphatorium thymifolia and wild ginger 
(Amomum siamense) in Thailand and in low frequencies from rhizomes of wild ginger (Bussaban 
et al., 2001; Photita et al., 2005). Lu et al. (2004) isolated C. gloeosporioides and C. boninense as 
endophytes from leaves of 12 different tree species in the Iwokrama Forest Reserve, Guyana. 
Hyde and Soytong (2008) reported that Colletotrichum spp. that exist as endophytes can become 
primary saprobic decomposers or as latent infections of pathogens that cause disease under 
specific conditions. Simmonds (1941) showed in field experiments that Gloeosporium musarum 
(= C. musae) can remain latent within the skin of green banana fruits for almost five months, and 
develop anthracnose disease of fruit rot as the fruit ripens. The development of the fungus in the 
latent phase is restricted due to a poor capacity for secreting macerating enzymes and pectin 
esterase (Simmonds, 1963). Quiescent infections of strawberry transplants by C. gloeosporioides 
are common and play an important role as inoculum sources of anthracnose disease of crown rot 
(Raman and Louws, 2008). 
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2.15.3 Colletotrichum gloeosporioides and C. truncatum 
2.15.4.1 Colletotrichum gloeosporioides 
Colletotrichum gloeosporioides (Penzig) Penzig and Sacc. is an asexual filamentous fungal 
pathogen belonging to the Phylum: Ascomycota and genus: Colletotrichum that causes 
anthracnose disease on various tropical, sub-tropical and temperate fruits across the world and 
also in Ghana (Akem and Asiedu, 1994). Mycelial colour ranges from gray to white, dark-brown 
to orange and rosy with colony elevation ranging from thick floccose aerial mycelia to flat-mat 
mycelia (IMI, 1995). C. gloeosporioides easily sporulate producing abundant, visible fruiting 
bodies with submerged or superficial acervuli (Ayodele et al., n.d). Some strains sporulate 
superficially with scattered acervuli while others produce both superficial and submerged 
sporulation. Conidia produced are usually cylindrical with obtuse ends, hyaline, aseptate, 
uninucleate formed in setose, globose and/or saucer-shaped acervuli. Setae produced by C. 
gloeosporioides ranges from dark brown to black, highly profused to sparse, slightly curved to 
straight, 1-4 septate, swollen at the base and tapering towards the apex measuring 50-240 µm long (IMI, 
1995; Onyeka et al., 2005). Acervuli produced by C. gloeosporioides vary from round to irregular and 
globose to saucer-shaped. The C. gloeosporioides species complex has been studied by Weir et al. 
(2012) and it is a well-supported clade on a very long branch and shows few differences in the 
gene loci. However it is a diverse clade in terms of morphology and includes a number of 
important plant pathogens. Weir et al. (2012) recognized two sub-clades within the species 
complex based on an eight-locus analysis, both of which were supported by Bayesian posterior 
probability values of 1.   
 
2.15.3.2 Colletotrichum truncatum 
Colletotrichum truncatum Andrus and Moore is a filamentous fungal pathogen belonging to the 
Phylum: Ascomycota and genus: Colletotrichum that causes anthracnose disease of on few 
selected crops such as yam (Ayodele et al., n.d; IMI, 1995). It has array of mycelial colour from 
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dark brown to salmon. Conidia produced are strongly curved, fusiform with acute apex and 
obtuse base, hyaline, aseptate and uninucleate usually produced in acervuli ranging from sparse to 
saucer-shaped. Setae are frequently produced ranging from sparse to profuse, black to dark 
brown, straight to slightly curved, 1-4 septate, swollen at the base and tapering towards the apex 
measuring 40-190 µm long (IMI, 1995). Acervuli formed ranges from setose to saucer-shaped.  
 
2.16 Host relations and specificity in Colletotrichum gloeosporioides and C. truncatum 
Colletotrichum gloeosporioides and C. truncatum are cosmopolitan with either multiple species 
occurring on a single host or a single species occurring on multiple hosts (Sander and Korsten, 
2003). Fungus-host relationships are broad, imprecise and often overlapping (Freemand and 
Shabi, 2000). Colletotrichum species can infect many hosts and may adapt to new environments 
(Sanders and Korsten, 2003; Photita et al., 2004), leading to serious cross infection problems in 
plant production. Earlier reports showed that the anthracnose disease caused by C. 
gloeosporioides and C. truncatum was severe only on D. alata but more recent investigations 
have revealed that the disease is prevalent and severe on most of the widely cultivated yam 
species (Akem and Asiedu, 1994). C. gloeosporioides has been reported as a pathogen on a wide 
range of host species throughout the world (Cannon et al., 2008). Weeds such as Spigella 
anthelmia, Calapogonium mucunoides and Commelina sp. and several other non-yam hosts 
harbour populations of C. gloeosporioides that can be highly virulent on yam (Abang et al., 
2006). Citrus spp. harboured populations of C. gloeosporioides that were highly virulent on yam 
(Green, 1994) and isolates from lime (Citrus aurantiifolia) were highly virulent on D. alata 
(Simons, 1993). 
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 2.17 Characterization of the strains of Colletotrichum gloeosporioides and C. 
truncatum isolated 
2.17.1 Polymerase chain reaction (PCR) 
PCR amplification is a rapid and sensitive method for detecting and characterizing members of 
pathogen groups. It can be used to confirm the results obtained from ELISA and electron 
microscopy. PCR was first used for the amplification of ß-globin genomic sequences for 
diagnosis of sickle cell anemia (Saiki et al., 1985). Since then, it has been employed extensively 
for the identification and differentiation of distinct strains of pathogens by amplification of 
sequences (Ward et al., 1992; Ward and Shukla, 1991; Shukla and Ward, 1989).  
 
PCR amplification requires Deoxynucleotide triphosphates (dNTPs) for the DNA extension, an 
optimized buffer containing magnesium, reverse and forward primers, which are complementary 
to the opposite strands of DNA and a thermostable DNA polymerase enzyme isolated from e.g. 
the thermophilic bacterial species, Thermus aquaticus (Taq) (Gelfand and White, 1990). Specific 
or degenerate primers are designed according to the available sequence data of viral genomic 
nucleic acids of the same group of viruses. Degenerate primers are a mixture of oligonucleotides 
varying in base sequence but with the same number of bases. Isolated DNA goes through 
temperature cycles. First DNA strands are disassociated at high temperature (94-96oC). The 
temperature is then lowered (50-55oC) to allow primers to anneal to the DNA strands. Finally, the 
DNA is subjected to the optimum extension temperature for the polymerase (usually 72oC). The 
three steps are repeated 30-45 times, allowing an exponential accumulation of DNA fragments. 
Amplified products are analyzed by agarose gel electrophoresis and DNA markers are included to 
allow the size of the PCR products to be estimated (Saiki et al., 1985). PCR works directly for the 
amplification of DNA templates and badnaviruses (Ward et al., 1992; Nie and Singh, 2000; 
Sharman et al., 2000). 
 
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2.17.2 Diversity study in Colletotrichum gloeosporioides and C. truncatum 
Traditionally, Colletotrichum gloeosporioides and C. truncatum have been identified and 
delimited on morphological characters; several features have been utilized by taxonomists 
including size and shape of conidia and appressoria; presence or absence of setae, sclerotia, 
acervuli and teleomorph state and cultural characters such as colony colour, growth rate and 
texture (Simmonds, 1965; Smith and Black, 1990; Sutton, 1992; Photita et al., 2005; TeBeest et 
al., 1997; Than et al., 2008a-c; Thaung, 2008). These criteria alone are not always adequate for 
reliable differentiation among these two species due to variation in morphology and phenotype 
among species under environmental influences (Abang et al., 2002).  
 
 
 
 
 
 
 
To overcome the inadequacies of these traditional schemes, several kinds of molecular techniques 
have been used to study genetic diversity, for example, labelled probes to detect restriction 
fragment length polymorphism (RFLP) (Hegedeus and Khachatourians, 1993; Sreenivasaprasad 
et al., 1996; Maurer et al., 1997; Abang et al., 2002; Peres et al., 2002; Moriwaki et al., 2002; 
Guerber et al., 2003; Du et al., 2005; Photita et al., 2005; Whitelaw-Weckert et al., 2007; Shenoy 
et al., 2007b; Peres et al., 2008; Than et al., 2008a-c) and methods based on the polymerase chain 
reaction (PCR) such as the use of random amplified polymorphic DNA (RAPD) (Fegan et al., 
1993; Bidochka et al., 1994; Piatti et al., 1998; Freire et al., 2001; Jensen et al., 2001; Gaitan et 
al., 2002), simple sequences repeat (SSR or microsatellites) analysis (Kretzner et al., 2000; 
Enkerli et al., 2001, 2005) and internal transcribed spacer (ITS)-rDNA sequence RFLP analysis 
(Rakotonirainy et al., 1994; Buscot et al., 1996; Coates et al., 2002a).   
 
 
 
 
Even though, they have different and complementary discriminatory power, RAPD and RFLP 
analyses have been very effective in detecting genetic diversity in several species (Couteaudier et 
al., 1998; Glare and Inwood, 1998; Coates et al., 2002b; Enkerli et al., 2005). PCR technique 
combined with RAPD and RFLP analyses have been extensively used to determine the genetic 
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diversity of various entomopathogenic fungi (Welsh and McClelland, 1990; Williams et al., 1990; 
Cobb and Clarkson, 1993; Bidochka et al., 1994; Neuvéglise and Brygoo, 1994; Neuvéglise et al., 
1994; Bidochka et al., 1995; Hodge et al., 1995; Fungaro et al., 1996; Maurer et al., 1997) 
including Metarhizium (Fegan et al., 1993; Leal et al., 1994) and to relate genetic diversity to 
pathogenicity index (Bridge et al., 1997) and to identify strains (Milner et al., 2002).  
 
The combined use of molecular diagnostic tools such as rapid amplified poly-dimorphism 
(RAPD) banding pattern (Thottappilly et al., 1999), inter-spacers restriction fragment length 
polymorphism (ITS RFLP), polymerase chain reaction (PCR) using species-specific primers 
(Adaskaveg and Hartin, 1997) alongside the traditional morpho-cultural techniques is at present 
an appropriate and good approach for studying Colletotrichum species complexes (Cannon et al., 
2000; Cai et al., 2009). This approach was employed in the characterization of the strains of 
Colletotrichum gloeosporioides and C. truncatum that existed in the Volta Region and the five 
other yam growing regions in Ghana – for accurate identification. Once a species is accurately 
named, it unlocks data that can be used for developing and implementing effective disease 
management strategies (Freeman et al., 1993).  
2.18 Screening of D. alata cultivars for resistance to anthracnose disease using tissue 
culture-derived whole-plant assay 
2.18.1 Methods of screening of D. alata cultivars  
D. alata, like any other crop, is often evaluated for disease incidence and severity in green house 
and the field using artificial or natural inoculation of the pathogen. Various in-vitro methods have 
been described for assessment of yam response to anthracnose disease based on the inoculation of 
in-vitro plantlets in glass tubes and purified mesophyll protoplasts (Moura-Costa et al., 1993) and 
inoculation of detached leaves (Green et al., 2000; Jacqua et al., 2005). These methods are useful 
for pathogenicity tests and characterization of C. gloeosporioides isolates and could be used for 
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pre-screening of varietal resistance (Abang et al., 2001); they are, nonetheless, unsuitable for 
analysis of the relationship between yam genotypes and pathogen isolates (Onyeka et al., 2006) 
because they involve inoculation of plant parts in the artificial states. Abang et al. (2001) 
proposed a tissue culture-derived whole plant technique for assessing resistance to yam 
anthracnose disease. This method involves spore inoculation of potted tissue culture-derived 
whole-plants and disease evaluation and has the advantage of using whole-plants in a controlled 
environment. It therefore, provides a probable rapid technique for evaluating breeding populations 
and studying the relationship between yam genotypes and pathogen isolates. This technique was 
adapted in screening of popularly grown D. alata cultivars in Ghana in the present study.  
 
 
 
 
 
 
 
 
Tissue culture-derived whole-plant method (Abang et al., 2001) provides a rapid technique for 
assessment of yam accessions and segregating populations for resistance to anthracnose disease. 
Due to the long time needed for field evaluation and the need sometimes, to screen genotypes for 
resistance to distinct pathogen isolates (Onyeka et al., 2006), breeding programs continue to rely 
on controlled environmental studies to achieve rapid progress towards development of resistant 
varieties. Besides, the high cost of conducting multiple-location trials coupled with the long 
growth cycle of water yam (Otoo, 2001b) and the highly spatial and temporal variations among C. 
gloeosporioides isolates limits field evaluation and often makes it unproductive (Green et al., 
2000).  
 
2.18.2 Disease assessment  
Disease assessment based on percentage whole plant area (WPA) scoring method proposed for 
field assessment of yam anthracnose disease (Simons and Green, 1994) and spray inoculation 
method (Onyeka et al., 2006) enhance the efficiency of the tissue culture-derived whole-plant 
technique. The critical stage for optimal single disease assessment of tissue culture-derived 
whole-plant is seven days after inoculation (7 DAI) (Onyeka et al., 2005b). Nevertheless, the use 
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of more than one assessment is a requirement to satisfactorily compare different yam genotypes. 
Thus, the use of AUDPC derived from assessment before and after the critical stage is the most 
suitable for quantitative resistance assessment (Onyeka et al., 2005a). This study then 
encompassed the assessment of area under the disease progress curve (AUDPC) for a defined 
period of crop growth, rather than the critical point model approach (Jeger and Viljanen-
Rollinson, 2001). 
 
2.18.3 Methods of in-vitro propagation of D. alata  
There are several methods of in-vitro propagation of D. alata (Moura-Costa et al., 1993; Abang et 
al., 2001). However, the most widely used methods are the plant tissue and meristem cultures. 
2.18.3.1 Plant tissue cultue 
The most important economic use of plant tissue culture to date is probably for the rapid 
propagation of plants, which facilitates the interchange of plant breeding material. In-vitro culture 
in combination with other techniques allows the elimination of viruses and other diseases from 
valuable clones (Mantell et al., 1978). Germplasm conservation in-vitro is also important for the 
preservation of the genetic resources. Micro-propagation is the in-vitro asexual reproduction of 
plant materials from very small pieces of plants in an artificial medium under aseptic conditions 
(Hartmann and Kester, 1975c). Tissue culture allows the propagation of a wide variety of plant 
materials such as foliage plants, woody ornamentals, vegetables, fruits and horticulture. Axillary 
shoot production is the most common form of propagation used by commercial propagators. The 
indirect formation of shoots can also be made from callus. However, callus is genetically unstable 
and new plants may not be true to type (Mantell et al., 1978). The production of in-vitro culture 
does not necessarily free plants from viruses. For safe germplasm movement, the plant materials 
need to be tested and only that material found virus-free used (Walkey, 1991).  
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2.18.3.2 Meristem cultures 
Morel and Martin (1952) were the first to demonstrate that virus-free plants can be recovered 
from infected plants through meristem culture. Plant virus particles are generally found in the 
conducting systems of plants and are rarely present in actively dividing meristematic cells (Sahin 
et al., 2003). Evans et al. (1983) demonstrated that the shoot and root apices of virus-infected 
plants are frequently devoid of viral particles or contain very low virus concentrations. Meristem-
tip culture is the most important and effective method in comparison with plant tissue culture 
since meristem-tips grow more quickly into plantlets than cultured tissues from other sources. In 
addition to that, the regenerated plantlets usually retain the genetic characteristics of the parent 
plants (Mantell et al., 1978). Meristem-tip culture was found to be efficient for the eradication of 
viruses and fungi in garlic (Verbeek et al., 1995), in sweet potato (Frison and Ng, 1981) and also 
in D. trifida (Saleil et al., 1990). This method was used to raise the D. alata cultivars used in the 
screening trial of the current study. 
2.19 Development of IPM strategy for management of yam anthracnose disease  
 
Fair knowledge of the stages of yam anthracnose disease development and its spread (i.e. 
epidemiology) aid in the disease management (Onyeka et al., 2006). Hence, extensive reviews of 
these areas were done in order to effectively devise appropriate strategy to manage the disease. 
2.19.1 Stages of yam anthracnose disease development and its cycle 
C. gloeosporioides was previously thought to survive in soil but Ekefan et al. (2000) showed that 
survival in soil is unlikely. Microbial antagonism appears to play a significant role in the apparent 
non-survival of C. gloeosporioides in soil. The pathogen over-seasons in diseased stems, leaves 
and tubers as spores. The ascospores produced by surviving mycelium produce the primary 
inoculum. Conidia can spread over relatively short distances by rain splash or overhead irrigation. 
Ascospores are airborne and important in long distance dispersal (Galvan et al. 1997; Ebenebe, 
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1980). Important source of C. gloeosporioides inoculum is infested crop debris (Green, 1994) 
hence older leaves  closer to the soil are first infested by the pathogen or young leaves and vines 
that have reached the tops of supporting poles and drops near to the soil. When the spores come in 
contact with the leaves and vines, it penetrates through natural openings such as stomata 
(Nwankiti and Okpala, 1981) and wounds, however, wounding do not increase symptom severity 
on D. alata (Kolattukudy et al., 2000; Abang et al., 2001).  
Mature leaves with well-developed cuticle are known to be considerably more resistant to attack 
by C. gloeosporioides than are younger leaves (Sweetmore et al., 1994; Abang, 1997). The stages 
of anthracnose disease development are enhanced by optimum temperature for growth and 
sporulation of the pathogen which is known to vary between 26 oC and 32 oC. Spore viability and 
germination are extremely sensitive to atmospheric humidity; such that, at 99% relative humidity 
(RH), germination was half that at 100% RH and was found to be negligible below 97% RH 
(Wharton, 1994). Lesion formation is, therefore, dependent on periods of 100% RH or leaf 
wetness of sufficient duration to allow for spore germination and host penetration.  
2.19.2 Epidemiology of yam anthracnose  
Studies investigating the epidemiology of yam anthracnose highlighted three major sources of 
infection: infected planting material (tubers) (Simons and Green, 1994); alternative hosts 
(Simons, 1993; Green, 1994); plant debris (Green, 1994). Infected soil was however, discounted 
as a source of inoculums (Ekefan et al., 2000; Arene, 1980). However, the relative importance of 
these sources of inoculum varies depending on geographical location. Survival of the pathogen 
from one season to the next may occur, but is unlikely to be important where growers practice 
crop rotation and plough-in crop debris. The fungus, C. gloeosporioides, infects many crops and 
weeds and is likely that spores from these plants also affect the yam crop (Morse et al., 2000). If 
tuber rots occur in the Pacific as they do in the Caribbean, then infected planting material is likely 
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to be the most important way that new crops become diseased (Osai, 1993). Small immature 
tubers, derived from early shoot death, may be a major source of infection of the shoots as they 
develop (Amusa and Ayinla, 1997). Once infection is established in the crop, development of 
management measures against the disease depends on rainfall and host variety.  
 
Severe outbreaks develop on susceptible varieties following rainstorms or cyclones (Amusa and 
Ayinla, 1997). Spores are formed in large numbers on the leaf spots and are splashed in rain and 
or carried by dripping dew to adjacent and lower leaves and stems (Akinnusi et al., 1987; Ogali et 
al., 1991). Abang (2003) obtained different strains of C. gloeosporioides (FGS and SGG) from 
dissected botanic seeds that were surface sterilized and plated on culture media but the incidence 
of infection in seed lots and the efficiency of C. gloeosporioides transmission through seed 
remains to be established. The importance of infected tubers as a source of inoculum was 
established in epidemiological studies, which found a correlation between infected tubers sown 
and the amount of foliar disease subsequently observed. However, long periods of rain favour 
epidemics of the disease because conidia are spread by rain splash (Galvan et al. 1997; Ebenebe, 
1980; Schwartz and Mohan, 1995).  
2.20 Management of anthracnose disease of D. alata in the field 
Management of anthracnose disease of D. alata in the field has always been challenging although 
management measures which include chemical and cultural methods give appreciable results 
(Akem and Asiedu, 1994). Measures for the management of yam anthracnose disease in the field 
include cultural practices, use of chemicals, and resistant varieties as planting materials that 
provide partial control by reducing the rate of disease development (Nwakiti and Arene, 1976; 
Ogundana, 1981; Nwakiti, 1982; Walther et al., 1999). Chemical sprays (Benlate) reduced 
anthrancnose disease of D. alata to some extent in the Carribean however, regular application of 
chemicals led to the development of fungicide resistance besides being inappropriate for resource-
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poor farmers (Green and Simons, 1994; Bayart and Pallas, 1994). Use of host plant resistance is 
one of the sustainable management strategies for controlling yam anthracnose.  
 
The development of yam populations with multiple resistances to the disease presents a cheaper 
and more effective approach to its control, necessitating the screening of local and introduced 
germplasm for reaction to anthracnose disease in different agro-ecological zones (Nwakiti and 
Ene, 1984; Nwakiti et al., 1987). The use of anthracnose-resistant cultivars bred and released by 
the International Institute of Tropical Agriculture (IITA) has been advocated (IITA, 1993). 
However, it is reported that D. alata cultivars which were initially resistant to anthracnose disease 
later succumbed to the disease due to the emergence of different strains of C. gloeosporioides in 
the Carribean (Green and Simons, 1994; Ano et al., 2005). It is, therefore, argued that, integrated 
disease management strategy which combines cultural methods (the use of crop rotation, 
fallowing, planting of healthy materials, staking, good farm sanitation and the destruction of 
infected crop cultivars), use of host resistant cultivars and chemical sprays (effective fungicides) 
proves most appropriate in managing anthracnose disease on the field (IITA, 2000; Onyeka et al., 
2005a; Offei et al., 2008). The study, then, used the integrated disease management approach in 
managing anthracnose disease of D. alata established on the field.   
 
 
 
 
 
 
 
 
 
 
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CHAPTER THREE 
3.0 MATERIALS AND METHODS 
3.1 Socio-economic background of the two important D. alata growing districts in the 
Volta region of Ghana 
Nkwanta-North and Krachi-West are the two major districts in the Volta Region of Ghana where 
D. alata is commercially produced (Dadzie, 2004). These two districts were the areas where the 
study was specifically conducted. 
3.1.1 Nkwanta-North District 
The Nkwanta-North (red arrowed) (Fig. 3.1) district is one of the 18 districts in the Volta region 
and located between Latitude 7° 30’ North and 8° 45’ North and Longitude 0° 10’ West and 0o 
45’ East. The district capital, Kpassa is located 270 km to the north of Ho (the Regional Capital) 
(Anonymous, 2006).  It has a surface area of approximately 11,510 km2 thus making it one of the 
smallest districts in the region. The area experiences wet and dry seasons with the dry season 
occuring between November and March.  Mean annual rainfall ranges from 922 mm to 1,874 mm 
and temperatures between 11 °C and 39 °C (Anonymous, 2006). The district lies in the 
transitional vegetation zone and is covered by Sanannah woodland and grassland with occasional 
pockets of semi-deciduous forest. The soil in the district is mainly of the ferric acrisols and dystric 
fluvisols type. The ferric acrisols covers about 70% of the entire area and supports the cultivation 
of a wide variety of crops including root crops (especially, yam), cereals, legumes, and oil palm. 
The structure of the local economy is skewed towards agriculture, which employs about 70% of 
the working population (MoFA-SRID, 2010). Unlike in other parts of the country, where farming 
is left in the hands of the elderly, people of all ages in the district are involved in farming due to 
the high returns derived from yam production. The district is one of the leading producers of yam 
in the country. About 80% of the working population is involved in its production as either farm 
owners, tenants or farm labourers.   
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3.1.2 Krachi-West District 
Krachi-West district (black arrowed) (Fig. 3.1) lies in between the northern part of the central 
uplands of the Volta Region dominated by highlands ranging from 850 m to 1000 m above sea 
level (Anonymous, 2006).  However, the northern part is flat with slopes rising from 85 m to 300 
m above sea level. The entire district forms part of the Voltaian Basin and the district capital is 
Kete-Krachi. The population distribution in the district shows that 51.7 percent are males while 
48.3 percent are a female which is different from the national distribution where there are more 
females (50.68) than males (49.32 percent) (Ghana Statistical Service, 2010). The district covers 
an area of 4169 sq. km out of which 37% is covered by water. The northern part of the district is 
part of the northwest savannah zone in the North and the forest zone in the south. About 80% of 
the district is covered with savannah grassland. However, forests and woodlands can be found 
along the lake, streams, rivers, foothills and the southern portion of the Oti zones. The major soils 
in the District include the Techiman Association (mainly sandy soils) in the north, the 
Kpelesawgu association (sandy clay soils) in the mid-portions and southern-most tip, the 
Dormabin-Dentesso association (silty sand) in the south and the Ejura-Amantim association 
(sandy loam) in the west. The district has about 70% of the population actively engaged in 
agricultural related ventures (MoFA-SRID, 2010). The crops cultivated include yam, cassava, 
cowpea, maize and rice. The district is one of the leading producers of yam in the country and, 
therefore, faces the problem of glut in good harvests (Dadzie, 2004).  Some farmers rear animals 
such as goats, sheep, cattle and poultry. 
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Fig. 3.1: Map of Volta Region indicating the study area. Nkwanta-North (red arrowed) and 
Krachi-West (black arrowed).  Source: Anonymous (2006). 
 
3.2 Assessment of farmers’ perception on importance and control of a vine-browning 
and die-back disease of D. alata in the Volta region 
3.2.1 Selection of experimental sites within the two districts for the disease survey  
 Two districts (Nkwanta-North and Krachie-West) where anthracnose disease of D. alata had 
been reported (Atsu and Akator, Personal Communication, 2012) were selected and three 
communities were randomly selected in each district for the study. The selection was done 
stepwisely by first obtaining a list of all D. alata producers from the Agricultural Extension 
Agents (AEAs) working in the two districts. Initial focus group discussions were then held with 
the AEAs of each district to determine the communities in their respective districts where the 
disease was most predominant. Fifteen D. alata farmers per community with disease hot-sopt 
were randomly selected from the lists of D. alata farmers provided by the AEAs. Experimental 
sites and number of farmers selected for the survey are presented in Table 3.1. 
 
 
 
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Table 3.1: Experimental sites and number of farmers interviewed per site. 
Districts   Communities                              Number of farmers interviewed 
    Kpasa-Tindani    23 
Nkwanta-North   Kpasa-Abunyanya    18 
Bajimpoa-Akura    19    
 
 
Gyidiasu     17 
Krachi-West   Osramae     23 
Ohiamankyene    20 
 
3.2.2 Survey to assess farmers’ knowledge, perception, experiences and economic 
importance of a vine-browning and die-back disease of D. alata in the Volta Region 
A field survey was conducted between February and June, 2012 to obtain baseline data on 
farmers’ knowledge, perception, experiences and economic importance of anthracnose disease of 
D. alata in the Nkwanta-North and Krachi-West districts of Volta Region of Ghana. One hundred 
and twenty D. alata farmers (from both smallholders with ≤ 1 ha of D. alata and plantation 
growers with ≥ 10 ha of D. alata) were randomly chosen from the experimental sites in the two 
districts. Pre-tested semi-structured questionnaires designed were read and interpreted to the 
farmers and their responses documented. The survey was aimed at establishing farmers’ 
knowledge, perception and experiences concerning prevalence, spread, control and economic 
importance of the disease especially its impact on the livelihood of the farmers. The data collected 
using the questionnaire included the demographical data of the farmers; knowledge, perception 
and experience of farmers concerning cause of the disease, spread and control and the socio-
economic impact of the disease including the livelihood of farmers   (Appendix 1). All data 
collected through the questionnaires were analyzed using Statistical Package for Social Sciences 
(SPSS) version 18 (SPSS Inc., 2009). 
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3.2.3 Symptoms decription of vine-browning and die-back disease of water yam and samples 
collection 
Symptoms of a vine-browning and die-back disease consistently observed on D. alata plants in all 
the farms visited in the Nkwanta-North and Krachi-West districts of the Volta Region were 
described by the stages of the diseases. This was done by examining diseased leaves and vines of 
selected D. alata cultivars [using systematic sampling technique with ‘W-shaped’ sampling path 
(Lin et al., 1979)] and symptoms compared with the description and/or pictures provided by Egesi 
et al. (2007); Abang et al. (2006); Akem and Asiedu (1994); Ayodele et al. (n.d.). During the 
field survey, other D. alata diseases such as leaf curl, witches broom and leaf spots were observed 
but not considered important in the study. Photographic records were taken on diseased D. alata 
cultivars, agronomic practices and mode of storage of planting materials with DSC–S750 Sony 
Digital Camera.  
 
 
 
Samples (leaves and vines) of D. alata cultivars exhibiting a vine-browning and die-back disease 
symptom together with healthy samples were collected, sealed in sterile transparent latex bags – 
each of size 15 cm x 20 cm, well-labelled, packaged in an ice chest container and transported to 
the Plant Pathology laboratory of the Department of Crop Science, University of Ghana – Legon. 
The samples were kept in a Lec laboratory refrigerator model BK 700 at approximately 4 oC and 
used for the isolation and identification of the causal organism. Besides, healthy matured tubers of 
nine D. alata cultivars grown in the Volta region (Table 3.2) were collected and transported along 
with the diseased samples. In addition, three D. alata cultivars were collected from the Plant 
Genetic Resources Research Institute (PGGRI) – Bunso, Eastern region (Table 3.2). These 
cultivars were planted in both the Experimental Garden of the Biotechnology and Nuclear 
Agriculture Research Institute (BNARI) of the Ghana Atomic Energy Commission (GAEC), 
Accra and Sinna’s Garden of the Department of Crop Science, University of Ghana – for nodal 
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cuttings to be used in the tissue culture experiment, personnal observation of their growth and 
yield characters (Table 3.2), germplasm maintenance and for subsequent studies.  
Table 3.2: General characteristics of the 12 D. alata cultivars used in the study. 
Cultivar 
code 
Local name/ 
source no. 
Characteristics Original 
Source 
TDa007  Niboltutu Multiple tubering, low yielding and oval-
cylindrical doted tubers  
 
 
 
 
 
 
Volta region 
TDa008 TDa008-12 Oval-cylindrical tuber shape, high yielding PGRRI-Bunso 
TDa010 Sum ne hyen Irregular tuber shape, pinkish-white 
tubers, high yielding, purple vines  
 
 
 
 
 
 
Volta region 
TDa013 TDa013-12 Irregular tuber shape, high yielding  PGRRI-Bunso 
 TDa014 Chachribon Cylindrical tuber shape, high yielding, 
light-purple vines with less-thick leaves      
Volta region 
 
 
 
TDa015 
 
 
 
TDa015-12 
 
 
 
Irregular tuber shape, high yielding  
 
 
 
PGRRI-Bunso 
TDa018 Akomea Cylindrical tuber shape, moderate yielding     Volta region 
TDa019 Matches (short tubered) Multiple tubering, oval tuber shape, thin-
leaves 
Volta region 
 
 
 
 
TDa021 
 
 
Matches (long tubered) 
 
 
Cylindrical tuber shape, multiple tubering     
 
 
Volta region 
TDa023 Akaba (thorn-less) Oval-cylindrical tuber shape, high 
yielding, thick-leaves  
Volta region 
TDa025 Poka     Cylindrical tuber shape, low yielding Volta region 
TDa027 Akaba (thorned vine) Oval-cylindrical tuber shape, high 
yielding, thick-leaves.  
Volta region 
 
3.2.4 Determination of incidence and severity of a vine-browning and die-back disease of   
D. alata in the Nkwanta-North and Krachi-West districts of the Volta Region 
 
 
 
 
 
The incidence and severity of a vine-browning and die-back disease of D. alata in the two 
districts were determined to assess the extent of spread in farms and to effect appropriate 
management of the disease. In each district, a total of three communities and fifteen D. alata 
farms per community were randomly selected and in every farm, a minimum of 20 and maximum 
of 50 plants (depending on farm size) were selected (using systematic sampling technique with 
‘W-shaped’ sampling path (Lin et al., 1979) and assessed for disease incidence and severity.  In 
order to get rid of selection bias, every eighth D. alata plant was selected, assessed and scored for 
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both incidence and severity (IMI, 1995). Severity of a vine-browning and die-back disease of D. 
alata in the six communities of the two districts were determined using a disease assessment key 
with a subjective score-scale of 0-5 (modified after Akem and Asiedu, 1994; Prasath and 
Ponnuswami, 2007) (Table 3.3). Percentage whole-plant area scoring method (Simons and Green, 
1994; Abang et al., 2006) was employed in the assessment of the selected plants. The proportion 
of diseased tissues’ (leaves and vines) area of each selected plant (i.e. percent whole-plant lesion 
area) against non-diseased tissues’ area was scored on a scale of 0-5. The scores were then 
converted to disease severity index (Kim et al., 2000) as: 
Disease severity index (I) =                  Sum of all numerical ratings x 100  
           Total number of observations x maximum disease score  
 
Disease incidence was determined using +/- scoring method (Abang et al., 2006) whereby (+) 
represented presence of the disease whereas (-) represented absence of the disease.  The number 
of both diseased and healthy inspected plants in each farm was counted respectively and the 
incidence value calculated by the formulae: 
Disease incidence (Farm)             =       Number of diseased plants  x     100% 
              Total number of plants inspected per farm 
 
 
 
 
 
 
 
 
 
Disease incidence (Community)       =     ∑ Disease incidence in each farm x         x     100% 
                    Total number of farms inspected 
 
 
 
 
 
 
Disease incidence (District)              =  ∑ Disease incidence in each Community   x    100% 
                   Total number of Communities inspected 
 
 
 
 
 
The data obtained from the two districts were then compared using student t-test and GenStat 
Statistical Software version 9.0 was used to perform the analysis. Due to the importance and 
greater economic impacts of the anthrancnose disease on the livelihoods of farmers in the 
Nkwanta-North and Krachi-West in the Volta Region, there was the need therefore, to confirm the 
identity of the pathogens causing the disease in order to devise appropriate IPM strategy to 
manage it.  
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Table 3.3: Disease assessment key for severity of a vine-browning and die-back disease of D. 
alata 
Index   Qualitative Rating              Pictorial Rating 
0.0             No symptoms on leaves and vines 
 
 
 
1.0  1 – 10 per cent of leaves and vines showing disease  
                        symptoms 
 
 
 
 
2.0  11 – 25 per cent of leaves showing disease symptoms  
                         with browning of vines  
 
                                                                                                                      
 
 
3.0  26 – 40 per cent of leaves showing disease symptoms 
  with browning of vines and some tip die-back  
 
 
 
 
4.0  41 -70 per cent of leaves showing disease symptoms    
  with heavy browning of vines and tip die-back 
 
 
 
5.0   71 – 100 per cent of leaves defoliated and whole-   
plant dying with naked black drying 
 
 
 
 
 
 
 
 
 
 
 
Source: Modified after Akem and Asiedu (1994) and Prasath and Ponnuswami (2007).  
 
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3.3 Isolation and identification of the causal agent(s) of a vine-browning and die-back 
disease of D. alata in the Volta region 
3.3.1 Pathogen Isolation 
 
Isolation of pathogens associated with the disease was first done on water agar (WA) from Agar 
agar (OXOID) and then sub-cultured onto potato dextrose agar (PDA). The WA was prepared by 
dissolving 15 g of Agar powder in 250 ml of sterilized distilled water (SDW) in a 1 L conical 
flask. The flask was shaken vigorously to get a uniform mixture and then topped to the 500 ml 
mark. The flask was plugged with cotton wool and covered with Aluminium foil and autoclaved 
at 121 oC for 15 minutes at a pressure of 15 psi. On the other hand, to prepare 500 ml PDA, 19.5 g 
of the PDA powder (OXOID) was dissolved in 250 ml of SDW in a 1 L conical flask. The flask 
was shaken vigorously to get a uniform mixture and then topped to the 500 ml mark. The flask 
was plugged with cotton wool and covered with Aluminium foil and autoclaved at 121 oC for 15 
minutes at a pressure of 15 psi.   
 
 
 
 
 
 
The infected tissues (leaves and vines) of D. alata showing typical symptoms of anthracnose 
disease were selected and washed thoroughly in several changes of SDW and blotched with 
sterile cotton wool. Portions of advancing necrotic margins on both leaves and vines were then 
cut out into small bits (measuring about 2 mm) and surface sterilized in 1% sodium hypochlorite 
solution for one minute. Sterilized tissues were severally rinsed in double sterile distilled water to 
remove the traces of sodium hypochlorite and blotted with sterile cotton wool. Blotted tissues 
were transferred into Petri plates containing WA under aseptic conditions of standard tissue 
isolation (Rangaswami, 1958) and incubated at 26±2 oC in the laboratory for seven days under 12-
h artificial light/darkness cycle. Healthy D. alata tissues (leaves and vines) were used as controls. 
Incubated plates were examined macroscopically after 72 hours for pathogen growth 
(Thangamani et al., 2011). Where there signs of pathogen growth on five-day old WA plates, 
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were sub-culturing was done by transfering 2 mm diameter mycelial plugs (taken from the 
advancing disease margins) onto freshly prepared PDA plates and incubated at 26±2 oC for 7-10 
days. Mono-conidial cultures were then prepared for each pure isolate after identification and 
maintained on both PDA plates and slants at 4°C in a refrigerator for later use.  
3.3.2 Identification of micro-organisms associated with anthracnose disease of D. alata 
Isolated pathogens from the diseased D. alata samples were identified using morphological, 
cultural and molecular methods and confirmed with pathogenecity or infectivity test.  
3.3.2.1 Identification of isolates using culture characteristics 
Cultural characteristics such as conidial masses, colony growth and colour were observed on PDA 
at 26±2 oC; 10 replicates were prepared for each isolate. The mean colony growth for each isolate 
from 48 to 168 hours (i.e. 2 – 7 days) was calculated. The colour of each colony and conidial 
masses were also recorded on the seventh day. The description and naming of the Colletotrichum 
species was done according to Chaube and Pundhir (2005); IMI (1995); Barnett and Hunter 
(1998); Mordue (1971); Sutton and Waterston (1970); Thom and Raper (1945).  
3.3.2.2 Identification of isolates using morphological characteristics 
In the morphological identification, the shapes and sizes of conidia, setae and acervuli of each 
isolate were examined under a light microscope after incubation at 26±2 oC for seven days on 
PDA. The mean size (length and width) of conidia per isolate was measured with the aid of 
electron scanning microscope.   
 
3.3.2.3 Genome analysis of isolates 
Molecular techniques such as polymerase chain reaction (PCR) with Oligonucleotide and species-
specific primers (Adaskaveg and Hartin, 1997) were used to confirm the identity of isolates and 
discriminate pathogens (C. gloeosporioides from C. truncatum) involved in the disease.  
 
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3.3.2.3.1 Culturing of isolates for DNA extraction  
Fungal isolates obtained from D. alata cultivars in the different geographic districts and 
maintained in the Plant Pathology Laboratory of the Department of Crop Science, University of 
Ghana – Legon were used for the analysis. Mono-sporic cultures of each fungal isolate were 
obtained by plating multi-sporic cultures stored in the refrigerator onto Petri plates containing 
freshly prepared PDA and incubating the plates at 26±2 °C for 7 days and then cultivating single 
spore colonies (from the 7-day old culture) on Sabouraud media: containing (gL-1) Peptone, 10; 
Dextrose, 40; agar, 20; supplemented with 1% (w/v) of yeast extract (Difco, France) to produce 
mycelium for DNA extraction.  
3.3.2.3.2 DNA extraction 
A modified cetyltrimethylammonium bromide (CTAB) method (McGarvey and Kaper, 1991) was 
used for DNA extraction. Aerial mycelia-conidia were scraped off 7-day old cultures (about 50 
mg) from each isolate and ground in liquid nitrogen to a fine powder. Samples were thawed in 2 
mL homogenization buffer (0·1m Tris–HCl, 1·4 m NaCl, 20 mm EDTA pH 8·0) and incubated at 
37 oC for 15 min. Extraction buffer (1 mL) (7% CTAB, 1% polyvinylpyrrolidone, 0·1 m Tris–
HCl, 1·4 m NaCl, 20 mm EDTA pH 8·0), 300 µL 20% sodium dodecyl sulphate and 0·5% 
mercaptoethanol (v/v) were added and mixed by vortexing. After incubation at 65 oC for 1 hour, 
the homogenate was extracted twice with an equal volume of phenol/chloroform/ isoamylalcohol 
(25:24:1, v/v/v). The aqueous phase was then separated by centrifugation and incubated with 
RNase A (100 mg mL–1) and proteinase K (10 mg mL–1) for 15 min at 37 °C. After a final 
chloroform/isoamylalcohol (24: 1, v/v) extraction, DNA was precipitated by adding 0·6 vol 
isopropanol and incubating on ice for 30 min. After centrifugation (30 min at 10 000 revs/min) to 
collect DNA, nucleic acid was further purified and re-precipitated by adding 2·5 vol ice-cold 70% 
ethanol and  0·5 vol 7·5 m ammonium acetate pH 7.7, standing at 23 °C for 30 min and then 
centrifuging for 20 min at 10 000 revs/min. The final pellet was washed twice in 70% ethanol, air-
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dried and re-suspended in 100 µL Tris-EDTA (TE) (10 mM Tris-Cl/1 mM EDTA) buffer pH 7·4 
(Sahin et al., 2003). 
3.3.2.3.3 PCR amplification using Oligonucleotide and species-specific primers 
Oligonucleotide primer pair, ITS1 (5'-TCCGTAGGTGAACCTCCG-3') and ITS4 (5'-
TCCTCCGCTTATTGATATGC-3') with rDNA genes flanking the entire ITS1-5.8S-ITS4 region 
(about 500-600 bp) was used for ITS PCR-amplification (White et al., 1990). A species specific 
primer pair, ITS4 (5'-TCCTCCGCTTATTGATATGC-3') and CgInt (5'-
GGCCTCCCGCCTCCGGGCGG-3') (Mills et al., 1992) was used in the species-specific PCR 
amplification. For each of the twenty isolates, PCR of the extracted DNA was performed in a 
BIO-RAD PTC-220 thermocycler (Dyad MJ Research) for 30 cycles of 35 s at 94 °C, 35 s at 55 
°C and 1 min at 72 °C and a final additional 10 mins cycle at 72 °C. Reactions were carried out in 
a total volume of 25 µL containing 0.2 µM of dNTPs, 0.5 µM of the primer, 2.5 µM of 1x PCR 
amplification buffer, 0.5 mM of MgCl2, 0.4 U Taq DNA polymerase (Invitrogen) and 0.4 µM of 
DNA template and 50 ng ultrapure water. Reactions were done twice to evaluate the consistency 
of the banding patterns for all isolates studied. Products were separated on a 2% (w/v) agarose gel 
in 1x TAE buffer (40 mM Tris acetate, 1 mM EDTA, pH 8.0) at 125 V for 3 hours. The gels were 
stained with ethidium bromide and visualized with UV light using a Cole Palmer FLUO-LINK 
FLX apparatus and photographed for later assessment.  
 
3.3.2.4 Pathogenecity test of Colletotrichum isolates from the Volta region  
Pathogenecity test was done to confirm the identity of the pathogen isolated from D. alata on a 
randomly selected tissue culture-derived whole-D. alata cultivar TDa 008. 
3.3.2.4.1 Inoculum preparation  
To prepare inoculum for spray-inoculations, 2 mm mycelia plugs of three Colletotrichum isolates 
(Cg111, Cg125 and Ct132) – which represented genetic variations in the characterization study of 
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the Volta Region isolates, were taken from the sub-cultures established from single spores and 
stored in the fridge and transferred onto freshly prepared PDA plates; and left to sporulate under 
continuous light for 7-10 days at 26±2 oC. Inoculum suspensions were then prepared by flooding 
each culture plate with 50 ml double sterile distilled water (DSDW) with two drops of Tween 80 
(2% v/v). The surface of each culture plate was gently brushed with a fine paintbrush (Egesi et al, 
2009) to dislodge spores. The mycelia-spore suspensions were then filtered through double-
layered cheesecloth to remove mycelia. The resulting spore suspension per isolate was adjusted to 
1.6 x 106 conidia/ml using Eubauer haemocytometer and a tally counter. 
3.3.2.4.2 Inoculation of D. alata cultivars 
 
 
 
A day to the experimental inoculation, two-month old potted tissue culture-derived whole-D. 
alata cultivars (TDa 008 and TDa 013) supported with 1.5 m wooden stakes were sprayed with 
carborandum using a fine De-vilbis atomizer. Before inoculation was done, all D. alata plants 
were enclosed in transparent latex bags, each of size 0.5 m x 1.5 m to prevent cross-inoculation 
by the different isolates. Plants were then either spray-inoculated with the prepared inoculum of 
each isolate or sterile distilled water amended with two drops of Tween 80 (2% v/v) (as controls) 
with a 1 litre hand-held sprayer until run-off. Inoculums were allowed to adhere onto the leaves 
and vines for about 30 minutes (Abang et al., 2006), after which each plant was covered again 
with the transparent latex bag misted with sterile distilled water to maintain a moist environment 
(conditions: natural 12-h daylight/night cycle, 26±2 °C and 98–100% RH) for 48 hours – for 
infection to occur (Fig. 3.2). The experiment was replicated 15 times, using 6 x 12 factorial 
treatments in completely randomized design (CRD).  
 
  
 
 
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Fig. 3.2: Inoculated (INO) and non-inoculated (C) two-month old potted tissue culture-derived 
whole-D. alata cultivars enclosed in transparent latex bags to maintain high RH% for disease 
infection. Pathogen used = C. gloeosporioides and C. truncatum with spore suspensions of 1.6 x 
106 conidial/ml. 
3.3.2.4.3 Re-isolation of pathogen 
Infected leaves and vines of D. alata cultivars were collected, labelled and packaged in an ice 
chest and sent to the Plant Pathology laboratory, Department of Crop Science, University of 
Ghana, Legon for re-isolation of the suspected causal agents. The isolation was done first on 
water agar (WA) and incubated at 26±2 °C, about 70-80% RH for five days and sub-cultured onto 
PDA and maintained at the same incubation conditions for seven days. The pathogen was then 
observed and identified as described in sections under 3.3.2. 
3.4 Characterization of the strains of C. gloeosporioides and C. truncatum 
3.4.1 Diversity study in C. gloeosporioides and C. truncatum infecting D. alata in Ghana 
A survey was carried out between July and December, 2012 to obtain baseline knowledge on the 
diversity of the pathogen associated with vine-browning and die-back disease of D. alata in 
Ghana. Prior to the survey, a list of all the major D. alata growing districts in each of the five 
regions (Ashanti, Brong-Ahafo, Central, Eastern and Northern) was obtained from the Extension 
Service Department of the Ministry of Food and Agriculture, Ghana. Two major D. alata growing 
C INO C C C INO INO INO INO INO C 
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districts per region were then randomly selected for the study. Initial focus group discussions 
were held with the AEAs of each of the selected districts to determine the communities within 
their respective districts where the disease was most predominant. A maximum of three 
communities per district were randomly selected and fifteen D. alata farms per community were 
further selected randomly from the list of D. alata farms provided by the respective AEAs.  
 
 
 
 
 
 
 
 
A total of 12 districts, 27 communities and 405 farms (Appendix II) were surveyed for the study. 
During the survey, several samples of D. alata leaves and vines exhibiting varying symptoms of a 
vine-browning and die-back disease were collected using systematic sampling technique with ‘W-
shaped’ sampling path (Lin et al., 1979) from different locations and cultivars. The samples were 
then sealed in sterile transparent latex bags of size 0.5 m x 1.5 m, well-labelled, packaged in ice 
chest container and transported to the Plant Pathology Laboratory for isolation (as discussed in 
section 3.3.1), identification (as discussed in section 3.3.2) and characterization (as discussed in 
sections 3.4.2 to 3.4.3) of the micro-organisms associated with the diseased D. alata tissues. 
Photographic records were taken on the diseased D. alata plants and agronomic practices 
undertaken by farmers with DSC–S750 Sony Digital Camera. Pathogenecity test was carried out 
as described in section 3.3.2.4 using six selected Colletotrichum isolates (Cg146, Cg147, Cg149, 
Cg151, Cg165 and Ct168) which were diverse from the other isolates in the genome diversity 
study conducted using five Random Amplified Poly-Dimorphism (RAPD) primers (OPA-11, 
OPA-18, OPC-5, OPC-7 and OPC-16) and five Inter-Spacers Restriction Fragment Length 
Polymorphism (ITS-RFLP) enzymes (HhaI, HaeIII, HinfI, MspI and RsaI). 
3.4.2 Genome analysis of C. gloeosporioides and C. truncatum strains  
3.4.2.1 Rapid amplified polymorphic DNA (RAPD) analysis  
In the RAPD analysis of isolates, PCR-RAPD of the extracted genomic DNA was performed in a 
BIO-RAD PTC-220 thermocycler (Dyad MJ Research) for 45 cycles of 35 s at 94 °C, 35 s at 55 
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°C and 1 min at 72 °C and a final additional 10 mins cycle at 72 °C. Reactions were carried out in 
a total volume of 25 µL containing 0.2 µM of dNTPs, 0.5 µM of the primer, 2.5 µM of 1x PCR 
amplification buffer, 0.5 mM of MgCl2, 0.4 U Taq DNA polymerase (Invitrogen) and 0.4 µM of 
DNA template and 20.5 µM ultrapure water. Reactions were done twice to evaluate the 
consistency of the banding patterns for all isolates studied. Products were separated on a 2% (w/v) 
agarose gel in 1x TAE buffer (40 mM Tris acetate, 1 mM EDTA, pH 8.0) at 125 V for 3 hours. 
The gels were stained with ethidium bromide and visualized with UV light using a Cole Palmer 
FLUO-LINK FLX apparatus and photographed for later evaluation.  
 
A pre-screening of twenty Colletotrichum isolates with ten primers from the OPA, OPC and OPD 
series (Operon Technologies, California, USA) was performed to select the most informative 
primers. For the final evaluation of isolates, five primers: OPA-11 (5’-GGGTAACGCC-3’); 
OPA-18 (5’-AGCCAGCGAA-3’); OPC-05 (5’-GATGACCGCC-3’); OPC-07 (5’-
GTCCCGACGA-3’) and OPC-16 (5’-TTCCGAACCC-3’) were used. Computer analysis of 
RAPD patterns were performed as described by Halmschlager et al. (1994), in which the band 
pattern obtained from agarose gel electrophoresis was digitalized by hand to a two discrete-
character-matrix (0 and 1 for absence and presence of RAPD-bands, respectively). For each 
enzyme, polymorphic bands were considered as binary characters and scored as present (1) or 
absent (0). Genetic similarity between pairs was estimated using Jaccard’s similarity coefficient 
with the SIMQUAL option (Jaccard, 1908). The similarity matrix was run on sequential, 
agglomerative, hierarchical and nested clustering (SAHN) (Sneath and Sokal, 1973) using the 
unweighted pair-group method with arithmetic average (UPGMA) clustering algorithm to 
generate a dendrogram. All computations were undertaken using the NTSYSpc 2.1 program 
(Rohlf, 2000).   
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3.4.2.2 Restriction Enzyme Digestion of rDNA ITS regions 
All fungal isolates obtained from the six regions were used for PCR restriction fragment analysis. 
Oligonucleotide primers, ITS1 (5'-TCCGTAGGTGAACCTCCG-3') and ITS4 (5'-
TCCTCCGCTTATTGATATGC-3') with rDNA genes flanking the entire ITS1-5.8S-ITS4 region 
(about 500-600 bp) were used for ITS PCR-amplification (White et al., 1990). The PCR reactions 
were carried out in a BIO-RAD PTC-220 thermocycler using a total volume of 25 µL containing 
0.2 µM of dNTPs, 0.2 µM of the ITS forward and reverse primers, 1 x PCR amplification buffer, 
0.25 mM of MgCl2, 1 U Taq DNA Polymerase (Invitrogen) and 25 mg of DNA template and an 
initial amplification step of 2 min at 94 °C, followed by 45 cycles of 35 s at 94 °C, 35 s at 55 °C 
and 10 min at 72 °C. Restriction of the PCR products was carried out according to the 
manufacturer’s instructions using five endonucleases: HhaI, HaeIII, RsaI, HinfI and MspI to study 
polymorphisms in the ITS regions of the isolates. Enzyme treatments were made by taken a 3 µL 
aliquot of the PCR reaction and incubating it with 1 unit of the respective enzyme for 1–2 hours at 
37 oC using the digestion buffer specified by the manufacturer.  
 
Restriction fragments generated were analyzed by electrophoresis on 2% polyacrylamide gel (20 
x 20) in 1 x Tris- Borate-EDTA (TBE) pH 8.0 buffer, at 125 V for 3 hours. Gels were stained 
with ethidium bromide and thereafter, DNA fragments were visualized under UV light using a 
Cole Palmer FLUO-LINK FLX apparatus and the molecular weights of the fragments determined 
by comparison with a 100 bp DNA ladder.  
 
For each marker, polymorphic bands were considered as binary characters and scored as present 
(1) or absent (0). Genetic similarity between pairs was estimated using Jaccard’s similarity 
coefficient with the SIMQUAL option (Jaccard, 1908). The similarity matrix was run on 
sequential, agglomerative, hierarchical and nested clustering (SAHN) (Sneath and Sokal, 1973) 
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using the unweighted pair-group method with arithmetic average (UPGMA) clustering algorithm 
to generate a dendrogram. All computations were undertaken using the NTSYSpc 2.1 program 
(Rohlf, 2000).   
3.5 Screening of D. alata cultivars for resistance to anthracnose disease using tissue 
culture-derived whole-plants assay 
Nine D. alata cultivars grown in the Volta Region and and three cultivars collected from PGGRI, 
Bunso – Eastern Region of Ghana were screened for their resistance to the Colletotrichum spp. 
isolated from the diseased D. alata obtained in the various regions. The methods (adapted to 
accomplish this objective are elaborated in sections 3.5.1 to 3.5.3 below. 
3.5.1 Determination of appropriate hormones concentration for the tissue culture test 
In order to maximize reagents and/or hormones use (Svabova and Lebeda, 2009; Sebastiani et al., 
1994), preliminary experiment was conducted at the Tissue Culture and Biotechnology Centre 
(TCBC) of the Biotechnology and Nuclear Agricultue Research Institute (BNARI) in Ghana 
Atomic Energy Commission (GAEC) in April, 2012. The sole aim of this trial was to arrive at the 
choice of appropriate treatment combination (i.e. hormones combination) that could give best 
performance of explants to be used in the tissue culture experiment. Six randomly selected D. 
alata cultivars: TDa008, TDa013, TDa018, TDa019, TDa025 and TDa027 and six-combination of 
hormones (BAP + NAA + Kinetin: T1 = [0.25 + 0.25 + 0.25 mg/l], T2 = [0.25 + 0.5 + 0.5 mg/l], T3 = 
[0.25 + 1.0 + 1.0 mg/l], T4 = [0.25 + 1.5 + 1.5mg/l], T5 = [0.25 + 2.0 + 2.0 mg/l] and T6 = [0.25 + 2.5 + 2.5 
mg/l]) were used in the trial. A 6 x 7 factorial arrangement in CRD with 25 replications (i.e. 
culture tubes) per cultivar per hormones combination was used and each replication consisted of a 
single tube. Auxin-free basal medium (BAP + NAA + Kinetin: 0.0 + 0.0 + 0.0 mg/l) was used as the 
control. All activities that were carried out were done as described in the sections of the main 
experiment (section 3.5.3) with the exception of the design used together with the number of 
culture tubes and/or replications.  
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Data on root-shoot responses (i.e. days required to root and shoot initiation, mean number of roots 
and shoot per cultivar and mean shoot growth over a period of six weeks) of each cultivar were 
recorded. The main tissue culture experiment was then performed after the appropriate hormones 
combination and/or concentration had been selected from the preceding trial. Photographic 
records were taken on every stage of the experiment with Sony Digital Camera model DSC-S750.  
3.5.2 Raising of D. alata explants for the screening trial 
Explants of the 12 D. alata cultivars were raised using tissue culture-derived whole-plant assay 
with the detailed methods described in sections 3.5.2.1 to 3.5.2.3 below. 
3.5.2.1 Plantlets source and preparation 
Nodal cuttings (about 2.5-3.0 cm long) of healthy vines with active buds (i.e. meristematic buds) 
were made from the 12 D. alata cultivars maintained in the Experimental Garden,  BNARI – 
GAEC  and Sinna’s Garden, Department of Crop Science, University of Ghana – Legon. Samples 
collected were well packaged, properly labelled and transported to the Tissue Culture and 
Biotechnology Centre, BNARI during August and September, 2012. The vines were then cut into 
1.5 cm to 2.0 cm length with single node intact. These nodal vine cuttings were washed with 5% 
(v/v) detergent solution (Teepol, Qualigen, Mumbai, India) for 10 minutes and rinsed several 
times with running tap water. These nodal cuttings were surface sterilized with bavistin 0.3 % 
(w/v) and streptomycin 0.2% (w/v) for 10 minutes each and then washed with sterile distilled 
water and transferred to laminar air flow cabinet. In the laminar chamber, nodal segments were 
again treated with 70% ethyl alcohol for 30 seconds to one minute followed by another treatment 
in 0.1% (w/v) mercuric chloride (HgCl2) for another 5 minutes. Finally, the nodal cut vines were 
washed thoroughly in 3 to 4 changes of double sterile distilled water and soaked on sterile 
blotting paper. Sterilized blotted explants were then implanted on a fortified solid MS medium 
(Murashige and Skoog, 1962) prepared according to Maura-Costa and Mantell (1993).  
 
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All cultures were incubated at 16-hour light/8-hour dark photoperiod (cool, white fluorescent light 
– 30 µmol m-2 S-1) and a temperature of 25/29 °C (night/day) growth room, in 2.5 cm diameter x 
10 cm long tubes under 60-70% relative humidity (RH). Fifty replicate tubes per cultivar were 
used in the experiment with each replication comprising of a single culture tube.   
3.5.2.2 Plantlets multiplication 
Plantlets cultures were maintained by regular sub-culturing at 2-week intervals on freshly 
prepared MS medium (Flow Laboratories), 2% sucrose, 0.8% agar, pH 5.8, amended with root-
shoot hormones (Mantell et al. 1978). In-vitro raised shoots measuring about 4-5 cm length 
grown in multiplication medium were excised from the shoot clumps and transferred onto a half-
strength MS basal medium supplemented with 2.0 mg/l α-Naphthalene acetic acid (NAA), 2.0 
mg/l Kinetin and 0.25 mg/l BAP (which proved best amongst the six hormones-combinations in 
the preliminary experiment) for root and shoot formation. In addition to the aforementioned root-
shoot hormone supplements, the medium was amended with ascorbic acid 100 mg/l (as an 
antioxidant) and artificial charcoal (making the medium assume the natural darkness condition in 
the root-region of the soil). The root-shoot responses of each cultivar in-vitro which included: 
rooting and shooting percentage (i.e. % explants response), days required to root and shoot 
initiation, mean number of roots and shoot per cultivar and mean shoot growth over a period of 
six weeks were recorded. Auxin-free basal medium was used as the control.   
3.5.2.3 Acclimatization and establishment of plantlets  
Six-week old rooted micro-propagules were removed from culture tubes and the roots were 
washed under running tap water to remove the agar. Plantlets were then transferred into sterile 
poly pots (small plastic cups) of size 22 x 35 x 18 cm containing pre-soaked sterile 1:1 
vermiculite: compost (peat: sand, 9:1) (IITA, Nigeria) and maintained under moist condition by 
covering plantlets with sterile poly pots misted with double sterile distilled water for three days at 
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28/24 °C day/night temperatures, 70-80% RH and 16-h photoperiod. Mean weaning survival per 
cultivar was tabulated. After the three days, the coverings were removed and the plantlets were 
kept under bioclimatic chamber for further three weeks for advanced hardening. Following that, 
plantlets were transplanted into black plastic pots measuring 40 cm wide x 50 cm long containing 
a mixture of soil + sand + compost in 1:1:1 ratio and were still kept under the bioclimatic 
chamber for a period of one week after which they were transferred to 70% luminous screen 
house and remained there for the pathogen-screening experiment. Each plant was supported with 
a wooden stake of height, 1.5 m. No pesticide was applied during the course of the trial. Rather, 
hand-pickings of weeds were done when necessary and potted plants were irrigated thrice a week.   
 
3.5.3 Screening of 12 D. alata cultivars for resistance to anthracnose disease 
Six Colletotrichum isolates obtained from the Volta region and the other five regions (Ashanti, 
Brong-Ahafo, Central, Eastern and Northern regions) were used in the screening tests. Inoculums 
were prepared as described in section 3.3.2.4.1. A day to the experimental inoculation, two-month 
old potted tissue culture- derived whole-D. alata cultivars (TDa007, TDa008, TDa010, TDa013, 
TDa014, TDa015, TDa018, TDa019, TDa021, TDa023, TDa025 and TDa027) supported with 1.5 
m wooden stakes were sprayed with carborandum using a fine De-vilbis atomizer. Before 
inoculation was done, each D. alata plant was covered with a transparent latex bag of size 0.5 m x 
1.5 m to prevent cross-inoculation by the different isolates. Fifteen potted D. alata plants per 
cultivar received a spray-inoculum treatment of an isolate at a concentration of 1.6 x 106 
conidia/ml amended with two drops of Tween 80 (2% v/v) with a 1 litre hand-held sprayer until 
run-off. The control plants (15 per cultivar), however, were sprayed with sterile distilled water 
amended with two drops of Tween 80 (2% v/v). 
 
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Inoculums were allowed to adhere onto the leaves and vines for about 30 minutes, after which 
each plant was covered again with the transparent latex bag misted with sterile distilled water to 
maintain a moist environment (conditions: natural 12-h daylight/night cycle, 26±2 °C and 98–
100% RH) for 48 hours – for infection to occur (Fig. 3.3). The layout of this experiment was a 6 x 
12 factorial arranged in CRD with 20 replicates and each replication consisting of a single plant. 
The height and number of leaves per plant at the time of inoculation were recorded. Disease 
severity for each cultivar was evaluated using the percentage whole plant area scoring method 
(Egesi et al., 2007), in which percentage of whole plant area affected by a vine-browning and die-
back disease was scored based on a scale of 0-5 modified after Akem and Asiedu (1994) and 
Prasath and Ponnuswami (2007). 0 = no symptoms (highly resistant); 1 = 1-10 per cent of plant 
area showing disease symptoms (resistant); 2 = 11-25 per cent of plant area showing disease 
symptoms (moderately resistance); 3 = 26-40 per cent of plant area showing disease symptoms 
(susceptible); 4 = 41-70 and 5 = 71-100 per cent of plant area showing disease symptoms were 
ranked highly susceptible.  
 
 
 
 
 
 
 
The disease severity was scored at 5, 9, 12 and 14 days after inoculation (DAI) (modified after 
Onyeka et al, 2006) whilst disease incidence (presence or absence of the disease) was recorded on 
14 DAI – for all strains. Variations in resistance of D. alata cultivars were evaluated based on the 
ideal method for assessment of partial or rate reducing resistance (Onyeka et al., 2006) by taking 
into account, the beginning of the disease development, rate of the disease progression and final 
level of the diseased cumulative area under the progress curve (AUDPC) before and after the 
critical stage after inoculation and calculated using Shaner and Finney (1977) formula as 
indicated below. The disease progress rate (Rd) was then calculated as the regression slope 
coefficient of disease severity over time (Onyeka et al., 2006). Data were pooled and subjected to 
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analysis of variance (ANOVA) and the differences between individual means were tested using 
Fisher’s Least Significant Difference Test (LSD Test) at 5% (Steel et al., 1997).  
 
AUDPC =          [(Xi+1 + Xi)/2] [ti+1 – ti] 
Where,  Xi = the proportion of the host tissue damaged at ith day 
  ti = the time in days after appearance of the disease at ith day 
  n = the total number of observations 
 
The highly resistant (TDa023) and highly susceptible (TDa019) (but farmers’ preference) D. alata 
cultivars obtained from the screening test were purposively selected and used in the integrated 
management strategies for the disease.  
3.6 Development of IPM strategies for management of anthracnose disease of D. 
alata 
Integrated method of disease management which involved selection of healthy planting materials, 
hot-water treatment (45±1 oC for 20 minutes and drying for 30 minutes), frequent weeding 
(monthly) and chemical (fungicides sprays) was employed in the study to manage anthracnose 
disease of D. alata. 
 
3.6.1. in-vitro evaluation of fungistatic effect of four fungicides on radial mycelial growth 
and sporulation of C. gloeosporioides and C. truncatum 
The efficacies of four fungicides readily available on the local market were assessed for the 
management of anthracnose disease of D. alata. In-vitro fungicides screening experiment was 
conducted in the Plant Pathology Laboratory of the Department of Crop Science, University of 
Ghana, Legon to assess the efficacies of four fungicides on the radial mycelial growth and 
sporulation of the selected C. gloeosporioides (Cg111) and C. truncatum (Ct132) strains – which 
proved highly pathogenic in the screening trial and showed distinct diversities in the genome 
n 
i = 1 
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analyses). Poisoned food technique (Kiran et al., 2010) was employed in the study for the 
measurement of the radial mycelial growth and sporulation of the isolates on both amended and 
non-amended potato dextrose agar (PDA). Efficacies of systemic (Carbendazim) and three 
protectant (Mancozeb, Shafiz F1 and Nordox) fungicides’ efficacies were assessed in the Plant 
Pathology Laboratory by plating each isolate on fungicide-amended PDA (modified after 
Maldonado et al., 2005); Carbendazim potato dextrose agar (CZPDA), Mancozeb potato dextrose 
agar (MPDA), Shafiz potato dextrose agar (SPDA) and Nordox potato dextrose agar (NPDA) 
with treatment levels shown in Table 3.4.  
Tabel 3.4: Types of fungicides and application rates used in the in-vitro screening of fungicides 
Fungicide       Recommended concentration 
Carbendazim (Bendazim® 50WP)     1.3 g/litre 
Shafiz F1 ® 71.5 WP       3.1 g/litre 
Mancozeb (Agrithane® 80 WP)     2.7 g/litre 
Nordox (Cuprous oxide (Cu2O) ® 86 WP)      5.0 g/litre 
 
 
The control used in the experiment was non-amended potato dextrose agar (NAPDA). Each of the 
media was centrally inoculated with a 2 mm diameter fungus plug taken from the advancing 
margin of a five-day old single colony culture of each isolate and incubated at 26±2 oC and 65-
72% RH in the laboratory. The design used in this experiment was 4 x 4 factorial experiments in 
completely randomized design (CRD) layout with ten replications per treatment. Daily 
observation of plates was made up to the seventh day and colony diameter measured by taking the 
average of the two perpendicular distances across the centre of the colony using a meter rule. 
Kiran et al. (2010) formula for calculating percentage reduction in radial growth (I) of test fungus 
over control (C) was used for the percentage radial growth reduction.  
I = C – T   x 100 
         C  
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Where, I = Percent reduction in growth of test fungi. 
 C = Radial growth (mm) in control.  
T = Radial growth (mm) in treatment. 
 
A sterile cork borer of diameter 2 mm was used to take mycelial plugs from relative positions 
equidistant to the center of Petri dish with mycelial growth after the eighth day of incubation. 
Each detached plug was put into 10 ml sterile distilled water with two drops of Tween 80 solution 
in a McCartney bottle and capped (Maldonado et al., 2005). The spores of each plug were then 
detached by electronic shaking for about 20 seconds and counted under a compound microscope 
with the aid of eubauer haemocytometer and a tally counter. Kiran et al. (2010) formula for 
determining the percentage reduction in sporulation (I) over control (C) was employed in 
calculating the percentage reduction in sporulation of the C. gloeosporioides isolates.  
 
 
 
 
 
 
 
I = C – T   x 100 
       C  
Where, I = Percent reduction in sporulation of test fungi. 
 C = Number of spores in control.  
T = Number of spores in treatment. 
 
3.6.2 Field management of anthracnose disease of D. alata in Ghana  
 
The experiment was conducted at the University of Ghana Farm, Legon (5 o 58’ N, 0 o 8’ W; 153 
m asl) in the Greater Accra Region of Ghana which is located within the Coastal Savannah Zone, 
with annual mean rainfall of 750 mm and average temperature of 26 oC. The soil belongs to the 
Adenta series, ferric Acrisol which is light in texture, well drained and easy to work with. The 
topsoil profile is sandy loam and consists of approximately 20 cm of pale-brown to brown sand 
with loose, friable consistence and a weak, fine granular structure (FAO/UNESCO, 1990). 
 
 
 
Clean seed-yam (i.e. setts) of the two purposively selected D. alata cultivars TDa023 (highly 
resistant) and TDa019 (highly susceptible but farmers’ preference) were established for integrated 
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management of the disease from November, 2012 to May, 2013. Prior to planting, setts of both 
cultivars (each of weight, 200 g) were dipped into hot-water (45 oC) for 20 minutes and allowed 
to dry for 30 minutes (Small, 1988). The non-treated setts (controls) were, however, not treated 
with the hot-water. The total land area was 45 x 30 m2 prepared by ploughing and harrowing with 
a tractor after which ridges of height 50 cm and length 12 m separated at 1 m were raised 
manually with hoe. The whole plot was divided into three main blocks and each block was further 
divided into two sub-plots (i.e. plots A and B) separated at 2 m apart where the treated and control 
setts were randomly assigned (to either of the plots). Allocation of seed-pieces per sub-plot was 
done randomly. The design used was 2 x 4 factorial in randomized complete block design 
(RCBD) with three replications. Setts were placed 8 cm deep and 1m apart within the 50 cm high 
ridges giving a population of 10,000 plants per hectare. Each plant was supported with a wooden 
stake of height 2.5 m. Each block contained 20 ridges with each treatment appearing 25 times in a 
block. The sub-plots assigned with treated setts were weeded once every month whilst the control 
sub-plots were left unweeded. 
 
Two months after sprouting (MAS) of setts, all D. alata plants on the treated sub-plots (except the 
bordered ones) were covered with transparent latex bags each of size 0.5 m x 2.5 m – a day to the 
fungicides treatment in order to prevent fungicides-cross effect. Each of the three fungicides: 
Carbendazim, Mancozeb and Nordox (Cu2O) (which significantly inhibited the radial mycelial 
growth and sporulation of the Colletotrichum isolates Cg111 and Ct132 in in-vitro assay) with 
preparations of 2.7, 5.0 and 1.3 g/l of water respectively was sprayed onto seven randomly 
selected D. alata plants per cultivar per sub-plot using a 15 l Knapsack sprayer at 30 psi after 
carefully removing the latex bags (one at a time). The control plants were, however, sprayed with 
SDW. Fungicides sprays were either repeated on every 14 days (Carbendazim and Mancozeb) or 
28 days (Nordox) for two months.  
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A day after the first fungicides application, all D. alata plants (i.e. both treated and controls) were 
sprayed with carborandum using a fine De-vilbis atomizer. Next day after the carborandum 
application, all the D. alata plants (except the bordered plants) were spray-inoculated with mixed 
spore-suspension of Colletotrichum isolates (Cg111 + Ct132) with concentration, 1.6 x 106 
conidia/ml. The inoculum concentration was amended with two drops of Tween 80 (2% v/v) 
before spraying until run-off with a 1 litre hand-held sprayer. Inoculum was allowed to adhere 
onto the leaves and vines of D. alata for about 30 minutes, after which each plant was covered 
with new latex bag misted with SDW – to maintain high humidity for infection to occur. The 
experiment was conducted under natural irrigation condition, however, when necessary, artificial 
irrigation was done. No fertilizer was applied in the course of the trial. 
3.6.2.1 Disease assessment 
Disease ratings per cultivar per plot were undertaken a week after inoculation (WAI) for severity 
of anthracnose disease using a subjective score-scale of 0-5 (modified after Prasath and 
Ponnuswami, (2007); Akem and Asiedu, 1994) (Table 3.3). The scoring was repeated weekly for 
two months (Mignucci et al., 1988) to estimate foliage and vine necrosis caused by 
Colletotrichum spp. Variation of treatments in managing and/or controlling the disease were 
evaluated based on ideal method for assessment of disease D. alata plant (Onyeka et al., 2006) by 
taking into consideration, the beginning of the disease development, rate of the disease 
progression and final levels of the diseased cumulative area under the progress curve (AUDPC) 
before and after the critical stage of the disease. The disease progress rate was then taken as the 
slope of regression line for disease severity against time. Data collected were statistically 
analyzed with GenStats software version 9.0 and the differences between individual means were 
separated using Fisher’s Least Significant Difference (LSD) test at 5% (Steel et al., 1997). 
 
 
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CHAPTER FOUR 
4.0 RESULTS 
4.1 Farmers’ perception and practices on the cause, spread, control and importance 
of a vine-browning and die-back disease of D. alata in the Volta region  
 
4.1.1 General background of D. alata farmers  
The survey revealed that majority (93.3%) of D. alata farmers in the Nkwanta-North and Krachie- 
West districts of the Volta region were males and 6.7% were females. Majority of the respondents 
were aged 41-50 years (56.7%). The rest were aged 21-30 years (4.2 %), 31-40 years (33.3%) or 
51 years and above (5.8%).  
 
Majority (31.0%) of the D. alata farmers in the two districts had no formal education (Table 4.1). 
The rest had formal education up to the Junior High School (JHS) level (27.0%), Senior High 
School (SHS) (22%) and Primary (15%). However, a small population of the farmers (5%) had 
received Tertiary education.  
Table 4.1: Educational background of respondents 
     Education Level Frequency Percent Cumulative Percent 
  No formal education 37 31.0 31.0 
Primary 18 27.0 58.0 
JHS 32 22.0 80.0 
SHS 
Tertiary 
27 
6 
15.0 
5.0 
               95.0 
  100.0 
Total 120 100.0  
 
Majority (62.0%) of the farmers had between 1-5 years farming experiences in D. alata 
production. The rest had 6-10 years (30.0%) or below 1 year (8.0%) farming experience.   
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Fig. 4.1: Years of farming of D. alata by farmers in the Nkwanta-North and Krachi-West districts 
of the Volta Region  
 
4.1.2 Farmers’ knowledge and perception on disease symptoms, susceptibility of D. alata 
culitvars and prevalence of anthracnose disease of D. alata in the Volta Region 
 
 
 
 
A greater proportion (58%) of the farmers in the two districts indicated that in the presence of 
vine-browning and die-back disease, D. alata plants exhibit black spots with dark brown borders 
merging to form lesions and defoliation while 37% described large brown spots on the leaves 
bordered by yellow patches and die-back (Table 4.2). Some farmers (18%), however, described 
brown regular spots on leaves surrounded by dark brown rings with gray centres whilst a few 
(7%) indicated that small regular brown spots with yellow halos were seen on leaves. A handful 
of the farmers (2%) indicated that the disease appears as pin-point spots scattered on the lamina 
surrounded by yellow halos whilst 1% described the disease as pin-point spots scattered on the 
lamina without yellow halos (Table 4.2).  
 
 
 
 
 
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Table 4.2: Symptoms used by farmers to identify a vine-browning and die-back disease of D. 
alata in the field. 
Disease symptoms  Frequency 
Percent of  
respondetns 
Cumulative 
percent 
  Pin-point spots scattered on the lamina without 
yellow halos  
 
Pin-point spots scattered on the lamina 
surrounded by yellow halo  
 
Small irregular brown spot with yellow halo    
 
Brown regular spots surrounded by a  dark 
brown ring and having a gray center 
 
Large brown spots bordered with yellow patches 
and die-back 
 
Black spots with dark brown border merging to 
form lesions and causing defoliation                                                            
 
1 
 
 
2 
 
7 
 
 
18 
          
          
37 
 
 
55
 
0.8 
 
 
2.5 
  
5.8 
 
 
15.1 
 
 
30.8 
 
 
45.0
 
0.8 
 
 
3.3 
 
9.1 
 
 
24.2 
 
 
55.0 
 
         
 
100.0 
Total 120 100.0  
  
D. alata cultivars, ‘Matches’ (long and short tubered), ‘Akaba’ (thorned and thorn-less), 
‘Akomea’, ‘Nibultutu’, ‘Chachribon’, ‘Poka’ and ‘Sum ne hyen’ were the popularly grown 
cultivars in both districts. Farmers (100%) indicated that all the cultivars grown were susceptible 
to the vine-browning and die-back disease but to different levels. Majority (40.8%) of the farmers 
perceived Matches ‘short tubered’ as the highly susceptible cultivar, followed by Matches ‘long 
tubered’ (30.0%) and ‘Chachribon’ (15.1%). However, ‘Akomea’ (5.8%) and ‘Poka’ (4.2%) were 
perceived to be moderately susceptible whiles ‘Sum ne hyen’ (2.5%), ‘Nibultutu’ (1.6) and 
‘Akaba’(thorned and thorn-less) (0.8%) were the least susceptible (Table 4.3).  
 
 
 
 
 
 
 
 
 
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Table 4.3 Farmers knowledge on susceptibility of D. alata cultivars to a vine-browning and die-
back disease of D. alata in the Volta Region. 
 
 
            Cultivar Frequency 
Percent of 
respondents 
    Cumulative        Host  
       Percent         Responses* 
  Matches (short tubered) 
Matches (long tubered) 
Akaba (thorned) 
Akaba (thorn-less) 
Akomea 
Poka 
Sum ne hyen 
Nibultutu 
Chachribon 
48 
36 
1 
1 
6 
5  
3 
2 
18 
40.0 
30.0 
0.8 
0.8 
5.0 
4.2 
2.5 
1.6 
15.1 
40.0 
70.0 
70.8 
71.6 
76.6 
80.8 
83.3 
84.9 
100.0 
HS 
 
 
S 
 
 
 
LS 
 
 
 
LS 
MS 
 
 
MS 
 
 
LS 
 
 
 
 
LS 
S 
Total 120 100.0   
*Host responses: HS = Highly susceptible; S = Susceptible; LS = Less susceptible; MS = Moderately susceptible. 
 
Majority (55.8%) of the farmers perceived to have encountered disease incidence of 50-70% 
while 27.5% of the farmers recorded 25-50% dsease incidence. Seventeen percent of the farmers 
perceived an incidence of less than 25% whereas 3.0% had between 75 and 100% of their farms 
affected by the disease (Table 4.4).  
 
 
 
 
 
 
 
Table 4.4: Percentage of D. alata plants in farms affected by a vine-browning and die-back 
disease of D. alata in Nkwanta-North and Krachi-West districts. 
Incidence (%) of disease on farms 
Frequency 
Percent of 
respondetns 
Cumulative 
Percent 
  Less than 25 
25 - 50 
50 - 75 
75 - 100                                                                            
17 
33 
67 
3 
14.2 
27.5 
55.8 
2.5 
14.2 
         41.7 
  97.5 
        100.0 
Total         120       100.0  
 
4.1.3 Farmers’ perception, knowledge and experience on the cause, spread and control of a 
vine-browning and die-back disease of D. alata  
 
 
 
 
A wide range of causes of a vine-browning and die-back disease of D. alata were perceived by 
the farmers in the two districts (Fig. 4.2). Most of the farmers (73.3%) had no idea of the cause of 
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the disease while 12.5% it to excessive rainfall. Poor management practices by farmers (6.7%), 
excessive sunlight (5.0%), superstition and belief (2%) and fungus (0.8%) were also perceived as 
causes of the disease.  
                          
 
 
 
 
 
 
 
 
 
Fig. 4.2: Perception of farmers on the cause of a vine-browning and die-back   disease of D. alata.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
D. alata farmers in both districts had different perceptions on the mode of spread of a vine-
browning and die-back disease of D. alata within and among farms (Fig. 4.3). A high proportion 
of the farmers (63.3%) had no idea as to how the disease spread within a given farm, while 20.0% 
attributed the spread to planting of infected materials (i.e. seed-yam). On the other hand, 7.5% 
perceived the disease spread to be by contact between healthy and diseased plants while 5.0% and 
4.2% ascribed it to rainfall and wind respectively  
Causal agent 
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Fig. 4.3: Perception of farmers on the spread of a vine-browning and die-back disease of D. alata 
within farms.  
 
Farmers’ perception on the spread of the disease between and within farms was the same (Fig. 
4.4). Majority (69.2%) of the farmers did not have any idea as to how the disease is spread from 
one farm to another, however, some (15.8%) believed the disease spread was due to exchange of 
infected planting materials among farmers while others (9.2%) attributed the spread to wind. The 
rest credited the spread of the disease among farms to rainfall (6.0%) and contaminated tools 
(0.8%) respectively.  
 
 
 
 
 
 
 
 
 
 
 
 
Fig. 4.4: Perception of farmers on the spread of a vine-browning and die-back disease of D. alata 
among farms.  
 
 
 
 
 
 
  
 
 
 
 
 
 
 
Majority (63.30%) of farmers in both districts were of the view that the disease could be 
controlled. Nonetheless, 36.67% of them believed the disease was uncontrollable. Out of the 
Agents of spread 
Agents of spread 
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63.3% farmers who perceived that the disease could be controlled, 50.0% indicated that 
application of chemicals was likely to control the disease while 27.6% considered regular 
weeding to be the best control measure. About 11.8% of the respondents had no idea of any 
control measure, though they believed the disease could be controlled whereas 10.5% percieved 
use of healthy planting materials as the most effective disease control measure (Fig. 4.5).  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Fig 4.5: Perception of farmers on measures needed for successful of control of a vine-browning 
and die-back disease of D. alata in the Volta Region. 
 
 
 
 
4.1.4 Knowledge and experience of farmers on, yield and economic impact of a vine-
browning and die-back disease of D. alata on livelihoods of farmers 
 
 
 
 
 
 
Majority (55.8%) of the farmers in both districts produced D. alata production mainly for food 
security. Some (40.8%) produced it exclusively for income generation while the rest (3.4%) 
produced the crop in order to buy assets such as houses, lands and bullock (Table 4.5).  
Table 4.5: Reasons for planting D. alata by farmers in the Nkwanta-North and Krachi-West districts. 
Reasons for planting D. alata by farmers  Frequency 
Percent of 
respondents 
Cumulative 
Percent 
  For food security 
For income generation 
Purchase of assets 
67 
49 
4 
55.8 
40.8 
3.4 
55.5 
         96.6 
        100.0 
Total 120      100.0  
 
 
 
    Chemical control 
    Regular weeding 
    No idea 
    Use of healthy planting materials 
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The yields expected by farmers in both districts were between 4.0 and 21.0 tons per 2 ha and 15 
ha farm size respectively but the actual harvest of most farmers (51) was between 4 and 6 tons 
(Table 4.6). Out of 27.5% who cultivated 2 ha farmlands, only 5.8% realized their expectations in 
terms of yield (i.e. between 4-6 tons/2 ha farm size) while the majority (26) did not reach their 
harvest target and had a yield between 1-3 tons/2 ha. None of the farmers who cultivated 
farmlands ranging from 5 to 15 ha achieved his/her expected yield; implying that, 72.5% of the 
farmers did not reach their anticipated yields (Table 4.6).  
Table 4.6: Expected and actual yield of D. alata harvested by farmers in the Nkwanta-North and 
Krachi-West districts. 
                Expected  
         yield (tons)  
Farm       of D. alata  
size/ha     per annum  
Actual yield (tons) per annum 
Freq. %   1-3         4-6         7-9     10-12       13-15     16–18    19-21   
2 
5 
7 
10 
13 
15 
 
        4.0 – 6.0  
        7.0 – 9.0 
        10.0 – 12.0 
        13.0 – 15.0 
        16.0 – 18.0 
        19.0 – 21.0                                        
  26           7            0            0              0            0           0    
  4            19           0            0              0            0           0   
  2            11           5            0              0            0           0 
  0            14           6            9              0            0           0 
  0             0            0            5              7            0           0 
0 0 0            1              3            1           0 
      33 
      23 
      18 
      29 
      12 
       5 
27.5 
19.2 
15.0 
24.2 
   10.0 
   100 
        Total  32           51          11           15           10           1           0         120  
 
Majority (63.7%) of the respondents attributed their yield losses to D. alata diseases. The rest 
ascribed their losses to excessive drought (15.8%), inadequate labour (8.0%) and excessive 
rainfall (6.2%) (Table 4.7). Some farmers attributed low yield to non-staking (2.7%) of plants,   
flooding (2.7%) during heavy down-pour of rain and assigned their yield losses to poor land 
preparation (0.9%).  
 
 
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Table 4.7: Farmers’ reasons for not achieving expected yield of D. alata in the Nkwanta-North 
and Krachi-West districts. 
 
 
Reasons for not achieving 
expected yield Frequency 
 
Percent of 
respondents            
                                                     63.7 
    6.2                      
  15.8 
           0.9 
           2.7 
           2.7 
           8.0 
  
Cumulative     
percent 
 
 
             63.7                                                           
 
 
 
                                                                  69.9                                               
 
 
                                                                 85.7       
 
 
                                                                86.6 
 
 
 
 
                                                               89.3 
 
 
                                                            92.0 
 
 
 
 
                                               100.0 
                                        
  Diseases of D. alata 
Excess rainfall 
Extensive drought 
Poor land preparation 
Non-staking 
Flooding 
Inadequate labour  
      72 
        7 
      18 
1 
3 
3 
9 
Total       113  100.0 
 
Responses of D. alata farmers in Nkwanta-North and Krachi-West districts concerning the socio-
economic impacts of a vine-browning and die-back disease on their livelihoods are presented in 
Table 4.8. The diseases impacted negatively on the farmers’ livelihoods as majority were unable 
to pay: their school fees (80.8%), service loans (90.8%) and medical bills (69.2%) and purchase 
of assets (88.4%). Nearly all the farmers (95.8%) reported that poor yield resulting from the 
disease really impacted negatively on their social relations with their spouses and in some cases, 
even extends to the extended family body. Most of them (76.7%) were shy to contact extension 
agents in times of poor yield. However, few respondents (7.6%) stated that poor yields due to the 
disease did not negatively influence their relationships with extension agents. 
 
 
 
 
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Table 4.8: Socio-economic impacts of a vine-browning and die-back disease of D. alata on the   
livelihoods of farmers in the Nkwanta-North and Krachi-West districts. N = 120.  
Economic and social responsibilities 
of farmers   
Level of Influence* 
        1   2        3  4       5 
 Payment of school fees 
Payment of service loans 
Payment of medical bills 
Purchase of assets 
Relationship with spouse and neighbours 
Relationship with Extension Officers 
80.8** 
90.8 
69.2 
88.4 
95.8 
76.7 
10.0 
9.2 
18.3 
5.8 
3.4 
13.3 
7.5 
 
0.0 
12.5 
5.8 
0.8 
3.4 
1.7 
 
0.0 
0.0 
0.0 
0.0 
 
5.0 
 0.0 
 
 0.0 
 0.0 
 0.0 
 0.0 
 1.6 
*1 = Highly negatively influenced; 2 = Negatively influenced; 3 = Neutral; 4= Positively influenced; 5 = Highly 
positively influenced; ** Values represent proportion of farmers. 
 
4.1.5 Symptoms of a vine-browning and die-back disease of D. alata 
When D. alata cultivars infected by a vine-browning and die-back disease were examined at the 
various farms in the Nkwanta-North and Krachi-West districts during the field survey, different 
symptoms of the disease were observed. The most common field symptoms were: 
• Pin-point spots scattered on the lamina (stage 1);  
• Pin-point spots scattered on the lamina and surrounded by yellow halo (stage 2);  
• Small brown spot with yellow halo (stage 3);  
• Brown regular spots surrounded by a dark brown ring and having a gray center (stage 4);  
• Large brown spots bordered by yellow patches and die-back of vines (stage 5); and  
• Black spots with dark brown border merging to form lesions and causing defoliation as 
well as browning of vines (stage 6).  
 
In the advanced stage of the disease, some plants die before maturity leaving the tuber 
mummified and/or shriveled; harvested tubers decay shortly after storage. In some cases, mycelial 
mat and acervuli of Colletotrichum sp. were found on well defined lesions on leaves, petioles and 
vines. It was also noted that, some local D. alata cultivars, especially ‘Nibultutu’ exhibited black 
coatings on upper leaf surfaces exposed to the sun (named of ‘yam scorch syndrome’) (Plate 4.1). 
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Generally, most cultivars in the surveyed areas exhibited the typical characteristic symptoms that 
start with circular black spots randomly distributed on leaf surfaces, expanding to leaf edge 
necrosis and then progressing to vine blackening and tip die-back.   
                                
                                                     
Plate 4.1: Stages of development of symptoms of a vine-browning and die-back disease of D. alata. 
Healthy plant (I); pin-point spots on lamina (II); pin-points spots surrounded by yellow halo on lamina 
(III); small irregular brown spots with yellow halo (IV); brown regular spots with gray centre bordered by 
dark brown ring (V); large brown spots bordered by yellow patches on leaves and die-back of vines (VI); 
and browning and/or blackening of vines and defoliation of leaves (VII).  
 
4.1.6 Incidence and severity of a vine-browning and die-back disease of D. alata in the 
Nkwanta-North and Krachi-West districts 
The mean incidence of a vine-browning and die-back disease of D. alata recorded in the 
Nkwanta-North (75.3%) was significantly higher (P < 0.05) than that of the Krachi-West (55.7%) 
IV I II III 
VI VII V 
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(Appendix III) (Table 4.9). However, the mean severity of the disease in the two districts were not 
significantly different (P > 0.05) (Table 4.9).  
Table 4.9: Disease incidence and severity of anthracnose disease of D. alata in the selected 
communities of the Nkwanta-North and Krachi-West districts of the Volta Region. 
District Community Disease Incidence (%) Disease Severity 
 Kpasa-Tindani 73.01 1.81 
Nkwanta-North Kpasa-Abunyanga 88.17 2.01 
 Bajimpoa-Akura 65.12 1.57 
 Mean  75.30 1.80 
  
Osramae 
 
67.14 
 
1.65 
Krachi-West Gyidaesu 49.11 1.52 
 Ohiamankyene 51.13 1.63 
 
 
Mean  
 
LSD (P < 0.05) 
55.75 
 
 
 
 
 
 
 
 
   6.52 
                 1.60 
                 1.46 
       
 
4.2 Micro-organisms associated with a vine-browning and die-back disease of D. 
alata in the Volta Region 
4.2.1 Identification using morpho-cultural characteristics of isoaltes 
When D. alata leaves and vines exhibiting typical symptoms of yam anthracnose disease 
collected from the Volta Region of Ghana were cultured on PDA and incubated at 26±2 oC, 70-
85% RH for seven days, C. gloeosporioides Penz. and Sacc. and C. truncatum Andrus and Moore 
were consistently isolated.  
4.2.1.1 Colletotrichum gloeosporioides  
C. gloeosporioides was the micro-organism consistently isolated from lesions of the D. alata  
samples collected from the Volta Region. The morpho-cultural studies revealed different growth 
patterns, mycelial colours and other distinguishable morphological characters in PDA culture 
(Plate 4.2). Colony growths of isolates were visible 48 hours after sub-culturing onto freshly 
preprared PDA. A total of four different growth patterns ranging from concentric with distinct 
rings to growth with less pronounced rings were observed among isolates (Plate 4.2). Mycelial 
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colour of isolates ranged from gray to white to dark brown and orange. Colony elevation also 
ranged from thick floccose aerial mycelia (Cg111) to flat-mat mycelia (Cg114) (Plate 4.2). The 
isolates sporulated producing abundant and visible fruiting bodies with submerged acervuli. Some 
isolates sporulated superficially with scattered acervuli while others produced both superficial and 
submerged sporulation. Mean radial growth of colony on PDA incubated at 26±2 oC and 70-85% 
RH for seven days was 4.2 cm. Conidia of isolates were cylindrical with obtuse ends, hyaline, aseptate, 
uninucleate (Plate 4. 3) formed in globose to sparse acervuli.  
               
               Cg111                                  Cg114                                      Cg117                                    Cg125                    
 
 
 
 
 
Plate 4.2: Culture characteristic of C. gloeosporioides isolates (obtained from diseased D. alata in the 
Volta Region) on PDA incubated at 26±2 oC and 70-85% RH for seven days. Whitish thick floccose aerial 
mycelial growth with light yellow ring at the border (Cg111); Light-yellowish flat-mat mycelial growth 
(Cg114); Dark-brown mycelial growth with white pustule at the centre (Cg117); and whitish fluffy 
mycelia growth with less pronounced rings (Cg125).  
 
      
Plate 4.3: Morphological characteristics of C. gloeosporioides on PDA incubated at 26±2 oC and 70-85% 
RH for seven days. Conidia – cylindrical with obtuse ends, hyaline, aseptate and uninucleate; Black bar 
in each panel = 20 µm long. 
4.2.1.2 Colletotrichum truncatum  
C. truncatum isolates produced different growth pattern and distinct morphological characters in 
PDA culture. Colony growths were visible 48 hours after sub-culturing onto PDA. Isolates 
produced dark/blackish mycelial growth on culture surface with colony margin ranging from uniform 
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(Ct131) to distinct profuse branching of mycelia (Ct130) (Plate 4.4).  The isolates produced abundant 
superficial sporulation with peculiar black acervuli. Two distinct colony growth patterns ranging from 
concentric with distinct rings to growth with profused branching were observed among isolates 
(Plate 4.4). The mean radial mycelial growth of colony on PDA incubated at 26±2 oC and 70-85% 
RH for seven days was 3.5 cm. Conidia of isotaes were strongly curved, fusiform with acute apex 
and obtuse base, hyaline, aseptate and uninucleate (Plate 4.5) formed in acervuli ranging from 
saucer-shaped to sparse type.  
                                                                                   
Plate 4.4: Morphological characteristics of C. truncatum isolates (obtained from diseased D. alata in the 
Volta Region) on PDA incubated at 26±2 oC and 70-85% RH for seven days. Dark mycelial growth 
with profuse branching of mycelia (Ct130) and concentric growth with distinct rings and uniform 
margin (Ct132).   
 
Plate 4.5: Cultural characteristics of C. truncatum on PDA incubated at 26±2 oC and 70-85% RH for 
seven days. Conidia – strongly curved, fusiform with acute apex and obtuse base, hyaline, aseptate and 
uninucleate; Black bar in each panel = 20 µm long. 
4.2.2 Molecular identification of isolates 
Amplification of the ITS1-5.8s-ITS4 locus with the Oligonucleotide primer pair (ITS1/ITS4) 
generated PCR products of approximately 600-bp for both the C. gloeosporioides (2, 3, 4 and 6) 
Ct130 Ct132 
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and C. truncatum strains (1 and 5) (Plate 4.6A). Using the ITS4/CgInt species-specific primer 
pair, PCR products of about 530-bp was generated for all the C. gloeosporioides strains (2, 3, 4 
and 6) but no amplicon was obtained in C. truncatum strains (1 and 5) (Plate 4.6B). The negative 
controls (C) did not produce any PCR product (Table 4.11). 
                      
Plate 4.6: Amplified DNA fragments of C. gloeosporioides strains (lanes – 2, 3, 4 and 6) and C. truncatum 
strains (lanes – 1 and 5) obtained from diseased D. alata in the Volta region using ITS1/ITS4 primer pair 
(A) and ITS4/CgInt species specific primer pair (B). M = Molecular size marker GeneRuler™ DNA 
Ladder Mix (100-10,000 bp); C = Negative controls (sterile distilled water).   
 
Table 4.11: Detection of DNA fragments of C. gloeosporioides and C. truncatum strains obtained 
from diseased D. alata plants in the Volta Region using ITS1/ITS4 primer pair and CgInt/ITS4 
species-specific primer pair.  
Isolate Code               Lab ID     ITS1/ITS4 ITS4/CgInt 
*Ct130                             1                  +      - 
aCg111                             2           + + 
aCg114                             3        + + 
aCg117                             4          + + 
*Ct132                             5          
aCg125                             6           
+ 
+ 
- 
+ 
 aCg = C. gloeosporioides; *Ct = C. truncatum; (+) = presence of amplified DNA fragment and (-) = Absence of 
DNA fragment. 
 
4.2.3 Pathogenecity test of isolates from the Volta Region 
Pathogenecity test of Colletotrichum isolates on two-month old potted tissue culture-derived D. 
alata plants with inoculum suspension of 1.6 x 106 condia/ml carried out in the screenhouse 
B A 
600-bp 
C 6 5 4 3 2 1 M C 6 5 4 3 2 1 M 
530-bp 
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revealed all isolates to be pathogenic and thus, incited disease lesions true to type (i.e. 
anthracnose disease symptoms) on the D. alata plants (Plate 4.7). The control plants did not show 
any symptoms of the disease (Plate 4.8). Infection caused by the various isolates appeared similar 
but re-isolation yielded only the isolate with which the particular D. alata cultivar was inoculated. 
Small, irregular, brown spots with yellow halo symptoms were more often observed on younger 
than older leaves. Brown, regular spots bordered by dark brown ring with gray center, however, 
dominated the older leaves with sporadic die-back symptoms.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Plate 4.7: Two-month old potted tissue culture-derived whole-D. alata plants showing symptoms of 
anthracnose disease. Die-back (blue arrows) (A-C) and necrotic lesions on leaves (brown rings) (D-G) 13 
DAI with C. gloeosporioides isolate (Cg111) using spore suspension of 1.6 x 106 conidia/ml. 
A C B 
D E F G 
H I J K 
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Plate 4.8: Two-month old potted tissue culture-derived whole-D. alata plants without symptoms of 
anthracnose disease. Plants were sprayed with sterile distilled water amended with two drops of Tween 80 
(2% v/v) – as controls. 
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The first evidence of infection was noted on the 3rd day after incubation (DAI) in cultivar TDa 
008 whilst cultivar TDa 010 exhibited disease symptoms on the 4th DAI. The symptoms caused 
by the two strains of C. gloeosporioides and the C. truncatum isolate were undistinguishable at 
the initial stage of infection. The lesion first appeared as pin-point spots scattered on the lamina 
and later marked as brown, regular spots bordered by dark brown rings and gray center. However, 
as the infection advanced, three clearly discernible foliage symptoms were detected: (1) brown 
regular spots surrounded by dark brown ring with gray center; (2) large brown spots bordered by 
yellow patches; and (3) die-back. In D. alata cultivar TDa008, Ct132 isolate caused significantly 
higher (P < 0.005) disease with mean whole-plant area infection of 17.3% than isolate Cg125 with 
a mean whole-plant area infection of 10.8% (Table 4.12). However, between isolates Cg111 and 
Cg125, there was no difference. In cultivar TDa010, Ct132 caused significantly higher (P < 
0.005) disease with mean whole-plant area infection of 11.3% than isolate Cg125 with a mean 
whole-plant area infection of 8.9%. Isolate Cg111 recorded significantly higher (P < 0.005) mean 
whole-plant area infection of 13.0% in TDa 008 than in TDa010 (9.1%). Nonetheless, Cg111 was 
not significantly (P > 0.005) virulent from Cg125 in both cultivars (TDa 008 than in TDa010) 
(Appendix IV).    
Table 4.12: Disease infection (%) by Colletotrichum isolates on two-month old potted tissue-cultured D. 
alata cultivars (TDa008 and TDa010). 
 
    Colletotrichum Isolate 
  
D. alata Cultivar   Cg111           Cg125           Ct132  Control (SWD*)       LSD (P < 0.05) 
 
 
 
 
 
TDa008                13.0  10.80             17.30             0.00                  2.90  
TDa010                  9.1    8.91                11.32  0.00       1.30 
LSD (P < 0.005)         1.80    1.41    2.62  0.00 
 
 
 
 
*SDW = Sterile distilled water.  
 
 
 
 
 
 
 
 
 
The varied strains of C. gloeosporioides and C. truncatum revealed in the culture studies 
necessitated the need to genetically characterize them in order to resolve the systematic issues that 
existed among them. 
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4.3 Characterization of the strains of C. gloeosporioides and C. truncatum causing 
anthracnose disease of D. alata in Ghana  
 
4.3.1 Diversity in C. gloeosporioides and C. truncatum causing anthracnose disease of D. 
alata in Ghana  
C. gloeosporioides and C. truncatum were consistently isolated from the D. alata samples which 
exhibited typical symptoms of anthracnose disease collected from the six regions of Ghana 
(Volta, Ashanti, Brong-Ahafo, Central, Eastern and Northern Regions). A total of 18 different 
strains of C. gloeosporioides and four of C. truncatum were obtained which exhibited varied 
growth patterns in PDA culture. Out of the 18 strains of C. gloeosporioides found, a total of four 
were obtained from the Volta, three from Ashanti, three from Brong-Ahafo, two from Central two 
from Eastern and four from Northern Region. In the case of C. truncatum, two strains were 
isolated from the Volta, one from Central and one from the Eastern Region. 
 
 
 
 
 
 
4.3.1.1 Diveristy in growth patterns of C. gloeosporioides and C. truncatum strains 
 
 
 
 
 
4.3.1.1.1 Colletrichum gloeosporioides 
A total of 18 different growth patterns of C. gloeosporioides ranging from concentric growth with 
distinct rings to growth with less pronounced rings were observed on PDA (Plate 4.9). Mycelial 
colour ranged from white to gray to dark brown and orange. Colony elevation also ranged from 
thick floccose aerial mycelia to fluffy and flat. All isolates sporulated within 3 to 10 days with either 
copious or scarce conidia. Some isolates sporulated superficially with scattered acervuli while 
some too exhibited both superficial and submerged sporulations. 
         
                                                  
 
 
 
 
 
 
 
              
 
 
 
 
 
 
 
 
  
 
                                                                       
  
 
 
 
 
Plate 4.9: Different growth patterns of C. gloeosporioides strains on PDA at 25±2 oC, 7 days. Volta strains (A-D); 
Ashanti strains (E-G); Central strains (H and I); Eastern strains (J and L); Brong-Ahafo strains (M-O); and Northern 
strains (P-R). 
M N O P R Q 
G H I J K 
F 
L 
E D C B A 
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4.3.1.1.2 C. truncatum                                         
 
Four different colony growth patterns of C. truncatum ranging from concentric growth with 
distinct rings to growth with profused branching were observed (Plate 4.10). The strains produced 
either dark/blackish or salmon mycelial growth on culture surface with colony margin ranging from 
uniform to distinct profuse branching of mycelia. There was production of abundant superficial 
sporulation with peculiar black acervuli (Plate 4.10).  
 
 
 
 
 
Plate 4.10: Different growth patterns of C. truncatum strains on PDA at 25±2 oC, 7 days. Volta strains (I and II); 
Ashanti strains (E-G); Central strain (III); Eastern strain (IV).  
 
 
 4.3.1.2 Diveristy in setae and acervuli formation of C. gloeosporioides and C. truncatum strains 
 
 4.3.1.2.1 C. gloeosporioides  
 
 
 
 
Setae formed in some selected strains of C. gloeosporioides ranged from highly profused to 
sparse, dark brown to black, slightly curved to straight, 1-4 septate, swollen at the base and tapering 
towards the apex measuring 55-174 µm long (Plate 4.11A).  However, in some strains there was no setae 
formation. Acervuli formed also ranged from round to irregular, globose to saucer-shaped measuring 50-
260 µm in diameter (Plate 4.11B).  
               
 
 
                
 
 
 
 
Plate 4.11: Different formation of setae and acervuli of C. gloeosporioides strains obtained from 
diseased D. alata plants – setae (A) and acervuli (B). Black bars in panels = 60 µm.  
I II III IV 
(A) 
(B) 
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4.3.1.2.2 C. truncatum 
Setae formed in C. truncatum strains ranged from sparse to profuse, black to dark brown, straight to 
slightly curved, 1-4 septate, swollen at the base and tapering towards the apex measuring 50-190 µm long 
(Plate 4.12a-c). Acervuli formed ranged from setose to saucer-shaped measureing 70-190 µm in diameter 
(Plate 4.12d-e). 
     
 
 
 
 
 
 
 
 
Plate 4.12: Differnent formation of setae and acervuli of C. truncatum strains obtained from diseased 
D. alata plants – setae (A-C) and acervuli (D and E). Black bars in panels = 60 µm. 
 
 
 
 
 
 
 
 
 
 
 
4.3.1.3 Diveristy in radial mycelial growth and pathogenecity test of some selected strains of C. 
gloeosporioides  
 
 
 
 
 
 
 
 
Radial mycelial growth of the 10 selected strains of C. gloeosporioides on PDA incubated for 
seven days at 26±2oC and 70-85% RH in 12/12 h light/dark cycle increased progressively with 
time (i.e. duration of incubation) (Fig. 4.6). The growth ranged from 2.3 cm (Cg169) to 4.4 cm 
(Cg141 and Cg162) on day 7. At day 2, there were no significant differences (P > 0.05) in 
mycelial growths among the 10 strains However, at day 3, 4, 5, 6 and 7, strain Cg141 produced 
significantly higher (P < 0.05) mycelial growth of 2.5, 3.4, 4.0, 4.2 and 4.4 cm respectively 
compared with strains Cg169 (0.9, 1.1, 1.4, 1.6 and 1.8 cm) and Cg151 (1.0, 1.2, 1.5, 1.7 and 1.9 
cm) (Fig 4.6). Strain Cg162 initially grew slowly but at day 7, it produced the highest radial 
mycelial growth (4.4 cm).  
A B C D E 
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Fig. 4.6: Radial mycelial growth of 10 selected C. gloeosporioides strains on PDA incubated at 26±2 oC, 
70-85% RH for seven days in 12/12 h light/dark cycle. Vertical bars indicate LSD at 5%. 
 
All the Colletotrichum isolates (Cg146, Cg147, Cg149, Cg151, Cg165 and Ct168) used in the 
pathogenicity test proved pathogenic but to varying levels. Isolate Ct168 produced significant (P 
< 0.05) higher disease severity (2.1) compared with Cg146 (1.3) and Cg147 (1.4). However, there 
were no significant (P > 0.05) differences among the isolates Cg149, Cg151, Cg165 and Ct168.  
4.3.1.4 Distinguishable morphological and cultural characteristics of the 22 Colletotrichum isolates 
from anthracnose disease of D. alata in Ghana 
 
 
The macro and microscopic studies of each of the 22 Colletotrichum isolates revealed varied 
growth patterns, mycelial colours and other distinguishable morphological and cultural characters 
in PDA culture (Table 4.14; 92-101 pp). Conidial measurements ranged from 2.0-13 µm x 1-4 µm 
in isolate Cg111 to 15.5-24 µm x 3.5-4 µm (Ct130). Average radial growth of 7-day old PDA 
cultures of isolates incubated at 26±2oC and 70-85% RH ranged between 2.2 to 4.5 cm. Setae and 
acervuli were present in some isolates with varying characters. 
Isolates 
code 
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86
 
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University of Ghana          http://ugspace.ug.edu.gh
87
 
 
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bo
di
es
 
             
  
 
    
    
    
 
   
    
    
    
    
    
    
    
    
    
    
    
  Co
nid
ia:
 x 
40
0  
    
    
    
    
    
    
    
Ro
un
d a
cer
vu
lus
: x
 40
0 
   
Se
ta
e: 
Ab
sen
t. C
on
id
ia
: C
yli
nd
ric
al 
wi
th 
ob
tus
e e
nd
s, h
ya
lin
e, 
ase
pta
te,
 u
nin
uc
lea
te,
 2
-13
 µ
m 
x 
1-4
 µ
m,
 fo
rm
ed
 in
 ro
un
d 
ac
erv
uli
 w
ith
 di
am
ete
r r
an
gin
g f
rom
 80
-26
0 µ
m.
   
   
  
 
    
    
    
    
  S
eta
e: 
x 4
00
    
    
    
    
    
    
    
    
  C
on
idi
a: 
x 4
00
 
 
Se
ta
e: 
Pre
sen
t –
 pr
ofu
sed
, b
lac
k, 
str
aig
ht 
to 
sli
gh
tly
 cu
rve
d, 
1-
4 s
ep
tat
e, 
sw
oll
en
 at
 th
e b
ase
 an
d t
ap
eri
ng
 to
wa
rds
 th
e a
pe
x 
me
asu
rin
g 
50
-12
0 
µm
 l
on
g. 
C
on
id
ia
: 
Str
on
gly
 c
urv
ed
, 
fus
ifo
rm
 w
ith
 ac
ute
 ap
ex
 an
d 
ob
tus
e b
ase
, h
ya
lin
e, 
ase
pta
te,
 
un
inu
cle
ate
, 1
5-2
4 µ
m 
x 3
.5-
4 µ
m,
 fo
rm
ed
 in
 se
tos
e a
ce
rvu
li 
of 
65
-17
0 µ
m 
in 
dia
me
ter
. 
  
 
University of Ghana          http://ugspace.ug.edu.gh
88
 
 
So
ur
ce
 
   
Gy
ida
esu
, 
V/
R                           
Oh
iam
an
ky
en
e, 
V/
R           
Is
ol
at
e 
C
od
e    
Cg
 11
4                    
Ct
 13
0            
C
ul
tu
re
 c
ha
ra
ct
er
is
ti
cs
 
         
  
   
    
    
    
    
To
p  
    
    
    
    
    
    
  U
nd
ers
ide
  
   Lig
ht 
ye
llo
wi
sh 
gro
wt
h 
at 
the
 ce
ntr
e s
urr
ou
nd
ed
 
wi
th 
wh
itis
h 
rin
g 
of 
my
ce
lia
, s
low
 g
row
th,
 le
ss 
dis
tin
ct 
co
nc
en
tric
 
rin
gs 
on
 
sur
fac
e 
wh
ils
t 
un
de
rsi
de
 w
ith
 y
ell
ow
ish
-gr
ee
n 
co
lou
r b
ord
ere
d 
wi
th 
wh
ite
 r
ing
 a
nd
 p
rod
uc
tio
n 
of 
sub
me
rge
d 
dis
tin
ct 
da
rk-
bro
wn
 c
on
idi
al 
pu
stu
les
 a
t 
the
 
ce
ntr
al 
reg
ion
. R
ad
ia
l g
ro
w
th
: 2
.5 
cm
. 
  
  
 
    
    
    
    
To
p  
    
    
    
    
    
    
  U
nd
ers
ide
  
 Da
rk/
bla
ck
ish
 m
yc
eli
al 
gro
wt
h 
on
 b
oth
 c
ult
ure
 
sur
fac
e 
an
d 
un
de
rsi
de
, 
slo
w 
gro
wt
h, 
dis
tin
ct 
pro
fus
e 
bra
nc
hin
g 
of 
my
ce
lia
, 
ab
un
da
nt 
sup
erf
ici
al 
spo
rul
ati
on
 of
 pe
cu
lia
r b
lac
k a
ce
rvu
li. 
R
ad
ia
l g
ro
w
th
: 3
.5 
cm
. 
  
M
or
ph
ol
og
y 
of
 c
on
id
ia
, m
yc
el
ia
 a
nd
 f
ru
it
in
g 
bo
di
es
 
                  
  
 
    
    
    
    
    
  C
on
idi
a: 
x 4
00
    
    
    
    
    
    
    
    
   A
cer
vu
lus
: x
 40
0 
 
Se
ta
e:
 A
bse
nt.
 C
on
id
ia
: 
Cy
lin
dri
cal
 w
ith
 r
ou
nd
ed
 e
nd
s, 
hy
ali
ne
, a
sep
tat
e, 
un
inu
cle
ate
, 4
-20
 µ
m 
x 2
-5 
µm
, f
orm
ed
 in
 
bro
wn
ish
 sa
uc
er-
sha
pe
d a
cer
vu
li o
f d
iam
ete
r, 6
0-2
35
 µm
.  
                          
  
 
   
   
   
  S
eta
e: 
x 4
00
    
    
    
    
    
    
Co
nid
ia:
 x 
40
0 
 
Se
ta
e: 
Pre
sen
t –
 p
rof
use
, d
ark
 b
row
n, 
str
aig
ht,
 1
-4 
sep
tat
e, 
sw
oll
en
 at
 th
e b
ase
 an
d t
ap
eri
ng
 to
wa
rds
 th
e a
pe
x m
ea
sur
ing
 
65
-19
0 µ
m 
lon
g. 
C
on
id
ia
: S
tro
ng
ly 
cu
rve
d, 
fus
ifo
rm
 (ta
pe
rin
g 
tow
ard
s b
oth
 en
ds)
 w
ith
 ac
ute
 ap
ex
 an
d o
btu
se 
ba
se,
 hy
ali
ne
, 
ase
pta
te,
 un
inu
cle
ate
, 1
5.5
-24
 µ
m 
x 3
.5-
4 µ
m,
 fo
rm
ed
 in
 da
rk 
sau
cer
-sh
ap
ed
 ac
erv
uli
 m
ea
sur
ein
g 7
0-1
90
 µm
 in
 di
am
ete
r. 
 
University of Ghana          http://ugspace.ug.edu.gh
89
 
 
So
ur
ce
 
    
Ne
w 
Ed
ub
ias
e, 
A/
R*
 
               
CS
IR
-C
RI
, 
Fu
me
sua
, A
/R
 
          
Is
ol
at
e 
C
od
e    
Cg
 15
1                 
Cg
 15
8            
C
ul
tu
re
 c
ha
ra
ct
er
is
ti
cs
 
     
  
    
    
  
To
p  
    
    
    
    
    
    
    
 U
nd
ers
ide
 
 Gr
ay
ish
 ye
llo
w 
gro
wt
h w
ith
 th
ick
 fl
oc
co
se 
ae
ria
l 
my
ce
lia
 an
d 
da
rk 
co
nid
ial
 p
ust
ule
s a
t t
he
 ce
ntr
e 
an
d 
sup
erf
ici
al 
spo
rul
ati
on
. U
nd
ers
ide
 o
f c
olo
ny
 
wi
th 
les
s 
pro
no
un
ce
d 
lig
ht 
bro
wn
 
rin
gs 
int
ers
pe
rse
d 
wi
th 
da
rk 
co
nid
ial
 p
ust
ule
s 
an
d 
pro
no
un
ce
d 
da
rk 
ce
ntr
e 
an
d 
sub
me
rge
d 
spo
rul
ati
on
. R
ad
ia
l g
ro
w
th
: 4
.5 
cm
.  
        
  
 
    
    
    
    
 To
p  
    
    
    
    
    
    
   U
nd
ers
ide
 
 W
hit
ish
 co
tto
ny
 gr
ow
th 
wi
th 
thi
ck
 fl
oc
co
se 
aer
ial
 
my
ce
lia
, le
ss 
pro
no
un
ce
d g
row
th 
rin
gs 
on
 su
rfa
ce 
an
d 
ae
ria
l s
po
rul
ati
on
 w
ith
in 
co
tto
ny
 m
yc
eli
al 
gro
wt
h. 
Un
de
rsi
de
 of
 co
lon
y w
ith
 di
sti
nc
t c
en
tra
l 
ros
y b
uff
 zo
na
tio
ns 
sur
rou
nd
ed
 w
ith
 w
hit
ish
 ri
ng
 
of 
my
ce
lia
. R
ad
ia
l g
ro
w
th
: 
2.6
 cm
. 
    
  M
or
ph
ol
og
y 
of
 c
on
id
ia
, m
yc
el
ia
 a
nd
 f
ru
it
in
g 
bo
di
es
  
  
 
  
 
    
    
   I
rre
gu
lar
 A
cer
vu
lus
: x
 40
0  
    
    
    
    
    
    
    
    
  C
on
idi
a: 
x 4
00
 
 
Se
ta
e:
 A
bse
nt.
 C
on
id
ia
: c
yli
nd
ric
al 
wi
th 
ob
tus
e e
nd
s, 
hy
ali
ne
, 
ase
pta
te,
 tr
inu
cle
ate
, 5
-16
 µ
m 
x 
3.0
-4 
µm
, f
orm
ed
 in
 se
tos
e 
ac
erv
uli
. A
cer
vu
li i
rre
gu
lar
, 7
0-2
50
 µm
 in
 di
am
ete
r. 
        
 
  
 
    
    
    
    
   C
on
idi
a: 
x 4
00
    
    
    
    
Ae
ria
l m
yc
eli
a w
ith
 Sp
oru
lat
ion
: 
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
   (
arr
ow
) x
 40
0 
     
Se
ta
e:
 A
bse
nt.
 C
on
id
ia
: c
yli
nd
ric
al 
wi
th 
ob
tus
e e
nd
s, 
hy
ali
ne
, 
ase
pta
te,
 un
inu
cle
ate
, 4
-23
 µ
m 
x 3
.5-
4 µ
m,
 fo
rm
ed
 in
 se
tos
e 
ac
erv
uli
. A
cer
vu
li i
rre
gu
lar
, 5
0-1
80
 µm
 in
 di
am
ete
r. 
   
University of Ghana          http://ugspace.ug.edu.gh
90
 
 
So
ur
ce
 
    
Br
on
iku
rom
,  
A/
R                
Ba
wj
ias
e, 
 
C/
R*
 
          
Is
ol
at
e 
C
od
e    
Cg
 14
7                 
Cg
 14
1            
C
ul
tu
re
 c
ha
ra
ct
er
is
ti
cs
 
  
  
 
    
    
    
    
   T
op
    
    
    
    
    
    
    
    
   U
nd
ers
ide
 
 W
hit
ish
-gr
ay
 c
ott
on
y 
fas
t 
gro
wt
h 
wi
th 
les
s 
pro
no
un
ce
d 
rin
gs 
on
 s
urf
ac
e 
ha
vin
g 
da
rk-
gra
y 
pu
stu
le 
at 
the
 c
en
tre
. U
nd
ers
ide
 o
f c
olo
ny
 w
ith
 
lig
ht 
gra
y 
to 
bro
wn
 p
ron
ou
nc
ed
 g
row
th 
rin
gs 
bo
rde
red
 w
ith
 w
hit
ish
 re
gio
n. 
R
ad
ia
l g
ro
w
th
: 4
.5 
cm
. 
  
 
    
    
    
    
To
p  
    
    
    
    
    
    
    
    
 U
nd
ers
ide
 
 Gr
ay
ish
 f
luf
fy 
gro
wt
h 
wi
th 
les
s 
pro
no
un
ced
 
co
nc
en
tric
 r
ing
s 
on
 t
he
 s
urf
ace
, 
sub
me
rge
d 
spo
rul
ati
on
, b
lac
k 
ma
ss 
at 
the
 ce
ntr
e. 
Un
de
rsi
de
 
wi
th 
lig
ht 
gra
y t
o w
hit
e p
ron
ou
nc
ed
 gr
ow
th 
wi
th 
les
s d
ist
inc
t d
ark
 ri
ng
s a
nd
 bl
ac
k c
on
idi
al 
pu
stu
le 
at 
the
 ce
ntr
e. 
R
ad
ia
l g
ro
w
th
: 4
.4 
cm
. 
  
M
or
ph
ol
og
y 
of
 c
on
id
ia
, m
yc
el
ia
 a
nd
 f
ru
it
in
g 
bo
di
es
  
   
  
 
    
    
    
   C
on
idi
a: 
x 4
00
    
    
    
    
    
    
    
 A
ce
rvu
lus
: x
 40
0 
 
Se
ta
e:
 A
bse
nt.
 C
on
id
ia
: C
yli
nd
ric
al 
wi
th 
rou
nd
 en
ds,
 hy
ali
ne
, 
ase
pta
te,
 bi
nu
cle
ate
, 4
-23
 µm
 x 
2-5
 µm
, fo
rm
ed
 in
 da
rk 
sau
ce
r-
sha
pe
d a
cer
vu
li. 
Ac
erv
ulu
s e
lon
ga
ted
 m
ea
sur
ing
 60
-18
0 µ
m 
in 
dia
me
ter
. 
                     
  
 
    
    
    
 A
ce
rvu
lus
: x
 40
0  
    
    
    
    
Co
nid
ia:
 x 
40
0 
   
Se
ta
e:
 A
bse
nt.
 C
on
id
ia
: C
yli
nd
ric
al 
wi
th 
ob
tus
e e
nd
s, h
ya
lin
e, 
ase
pta
te,
 un
inu
cle
ate
, 4
.5-
24
 µm
 x 
3.5
-4 
µm
, fo
rm
ed
 in
 br
ow
n 
to 
da
rk 
set
ose
 ac
erv
uli
. A
cer
vu
lus
 ir
reg
ula
r m
ea
sur
ing
 55
-17
0 
µm
 in
 di
am
ete
r. 
     
University of Ghana          http://ugspace.ug.edu.gh
91
 
 
So
ur
ce
 
    
Od
um
asi
,  
C/
R               
As
sin
-E
du
bia
se,
  
C/
R            
Is
ol
at
e 
C
od
e    
Ct
 14
2                
Cg
 14
3             
C
ul
tu
re
 c
ha
ra
ct
er
is
ti
cs
 
          
  
 
    
    
    
   T
op
    
    
    
    
    
    
    
 U
nd
ers
ide
 
 Sa
lm
on
 gr
ow
th 
wi
th 
sca
nty
 m
yc
eli
a, 
pro
no
un
ced
 
rin
g 
gro
wt
hs 
on
 b
oth
 su
rfa
ce 
an
d 
un
de
rsi
de
 o
f 
co
lon
y. 
Ab
un
da
nt 
sup
erf
ici
al 
spo
rul
ati
on
 
ap
pe
ari
ng
 as
 da
rk 
rin
g a
t t
he
 ce
ntr
e f
or 
bo
th 
the
 
sur
fac
e a
nd
 th
e r
ev
ers
e o
f c
olo
ny
. R
ad
ia
l g
ro
w
th
: 
2.7
 cm
. 
  
 
    
    
    
  T
op
    
    
    
    
    
    
    
Un
de
rsi
de
    
    
 
 Sm
ok
y 
gra
y 
co
lon
y 
wi
th 
thi
ck
 f
loc
co
se 
aer
ial
 
my
ce
lia
 an
d d
ark
 co
nid
ial
 pu
stu
les
 at
 th
e c
en
tre
. 
Un
de
rsi
de
 of
 co
lon
y w
ith
 di
sti
nc
t o
liv
ac
eo
us 
gra
y 
zo
na
tio
n 
alt
ern
ate
d 
wi
th 
da
rk 
bu
ff 
zo
na
tio
n 
an
d 
pro
no
un
ce
d 
sup
erf
ici
al 
an
d 
sub
me
rge
d 
spo
rul
ati
on
. R
ad
ia
l g
ro
w
th
: 4
.4 
cm
.  
 
M
or
ph
ol
og
y 
of
 c
on
id
ia
, m
yc
el
ia
 a
nd
 f
ru
it
in
g 
bo
di
es
  
          
  
 
    
    
    
   S
eta
e: 
x 4
00
    
    
    
    
    
    
    
    
    
Co
nid
ia:
 x 
40
0 
Se
ta
e:
 P
res
en
t –
 sp
ars
e 
to 
pro
fus
e, 
da
rk 
bro
wn
, s
tra
igh
t t
o 
sli
gh
tly
 cu
rve
d, 
1-3
 se
pta
te,
 sw
oll
en
 at
 th
e b
ase
 an
d t
ap
eri
ng
 
tow
ard
s t
he
 ap
ex
, 4
0-7
0 
µm
 lo
ng
. C
on
id
ia
: W
ea
kly
 cu
rve
d, 
fus
ifo
rm
 (t
ap
eri
ng
 to
wa
rds
 b
oth
 e
nd
s) 
wi
th 
acu
te 
ap
ex
 a
nd
 
ob
tus
e 
ba
se,
 h
ya
lin
e, 
ase
pta
te,
 u
nin
uc
lea
te,
 1
6-2
5 
µm
 x
 3
-4 
µm
, f
orm
ed
 in
 br
ow
n s
eto
se 
ace
rvu
li 
me
asu
rei
ng
 70
-19
0 µ
m 
in 
dia
me
ter
. 
           
  
 
    
    
    
   S
eta
e: 
x 4
00
    
    
    
    
    
    
    
    
    
  C
on
idi
a: 
x 4
00
 
 Se
ta
e:
 P
res
en
t –
 p
rof
use
, b
lac
k, 
sli
gh
tly
 cu
rve
d, 
1-4
 se
pta
te,
 
sw
oll
en
 at
 ba
se 
an
d t
ap
eri
ng
 to
wa
rds
 th
e t
ip,
 50
-15
0 µ
m 
lon
g. 
C
on
id
ia
: 
Cy
lin
dri
cal
 w
ith
 o
btu
se 
en
ds,
 h
ya
lin
e, 
ase
pta
te,
 
un
inu
cle
ate
, 4
-20
 µ
m 
x 
2.5
-4 
µm
, f
orm
ed
 in
 d
ark
 s
eto
se 
ac
erv
uli
. 
Ac
erv
ulu
s 
irr
eg
ula
r 
me
asu
rin
g 
65
-17
0 
µm
 i
n 
dia
me
ter
.  
 
University of Ghana          http://ugspace.ug.edu.gh
92
 
 
So
ur
ce
 
     
PG
RR
I-B
un
so,
  
E/R
*             
As
ese
wa
,  
E/R
             
Is
ol
at
e 
C
od
e     
Cg
 16
7              
Ct
 16
8              
C
ul
tu
re
 c
ha
ra
ct
er
is
ti
cs
 
       
  
 
    
    
    
    
  T
op
    
    
    
    
    
    
    
    
    
Un
de
rsi
de
 
 Da
rk 
gra
y 
my
ce
lia
l 
gro
wt
h 
wi
th 
ser
rat
ed
-en
d 
gro
wt
h 
an
d 
int
ers
pe
rse
d 
wi
th 
wh
ite
 p
ust
ule
s o
n 
the
 su
rfa
ce
 an
d s
ub
me
rge
d s
po
rul
ati
on
. U
nd
ers
ide
 
of 
co
lon
y 
wi
th 
rin
g 
of 
da
rk 
bro
wn
 g
row
th 
sep
ara
tin
g 
rin
gs 
of 
tw
o 
lig
ht 
bro
wn
 g
row
ths
. 
R
ad
ia
l g
ro
w
th
: 3
.7 
cm
. 
 
   
 
    
    
    
    
To
p  
    
    
    
    
    
    
   U
nd
ers
ide
 
 Lig
ht 
ye
llo
wi
sh 
bro
wn
 m
yc
eli
a, 
fas
t 
gro
wt
h, 
dis
tin
ct 
co
nc
en
tric
 r
ing
s, 
ab
un
da
nt 
spo
rul
ati
on
, 
sub
me
rge
d 
an
d 
sup
erf
ici
al 
pro
du
cti
on
 o
f d
ist
inc
t 
bro
wn
 a
cer
vu
li 
on
 b
oth
 s
urf
ace
 a
nd
 u
nd
ers
ide
. 
R
ad
ia
l g
ro
w
th
: 4
.3 
cm
.  
  
M
or
ph
ol
og
y 
of
 c
on
id
ia
, m
yc
el
ia
 a
nd
 f
ru
it
in
g 
bo
di
es
  
  
  
 
    
    
    
    
Se
tae
: x
 40
0  
    
    
    
    
    
    
    
    
 C
on
idi
a: 
x 4
00
 
 Se
ta
e:
 P
res
en
t 
– 
spa
rse
, d
ark
 b
row
n, 
str
aig
ht 
to 
sli
gh
tly
 
cu
rve
d, 
1-4
 se
pta
te,
 sw
oll
en
 at
 ba
se 
an
d t
ap
eri
ng
 to
wa
rds
 th
e 
ap
ex
, 6
5-1
75
 µm
 lo
ng
. C
on
id
ia
: C
yli
nd
ric
al 
wi
th 
ob
tus
e e
nd
s, 
hy
ali
ne
, a
sep
tat
e, 
un
inu
cle
ate
, 1
5-2
2 µ
m 
x 3
.5-
5 µ
m,
 fo
rm
ed
 
in 
bro
wn
 irr
eg
ula
r a
cer
vu
li m
ea
sur
ing
 60
-18
0 µ
m 
in 
dia
me
ter
. 
                          
  
 
   
   
   
   
  S
eta
e: 
x 4
00
    
    
    
    
    
    
 C
on
idi
a: 
x 4
00
 
 Se
ta
e:
 P
res
en
t –
 pr
ofu
se,
 bl
ac
k, 
str
aig
ht,
 1-
4 s
ep
tat
e, 
sw
oll
en
 
at 
ba
se 
an
d 
tap
eri
ng
 t
ow
ard
s 
the
 e
nd
, 5
0-1
45
 µ
m 
lon
g. 
C
on
id
ia
: 
Str
on
gly
 c
urv
ed
, f
usi
for
m 
wi
th 
ac
ute
 a
pe
x 
an
d 
ob
tus
e b
ase
, h
ya
lin
e, 
ase
pta
te,
 un
inu
cle
ate
, 1
3.5
-23
 µm
 x 
2.5
-4 
µm
, f
orm
ed
 in
 da
rk 
set
ose
 ac
erv
uli
 m
ea
sur
ein
g 6
0-1
70
 µ
m 
in 
dia
me
ter
. 
University of Ghana          http://ugspace.ug.edu.gh
93
 
 So
ur
ce
 
    
Se
ke
soa
,  
E/R
               
Ta
no
so,
  
B-
A/
R*
 
           
Is
ol
at
e 
C
od
e    
Cg
 16
9                
Cg
 16
2             
C
ul
tu
re
 c
ha
ra
ct
er
is
ti
cs
 
  
  
 
    
    
    
   T
op
    
    
    
    
    
    
  U
nd
ers
ide
 
 Da
rk 
bro
wn
 m
yc
eli
a, 
slo
w 
gro
wt
h w
ith
 ab
un
da
nt 
sup
erf
ici
al 
spo
rul
ati
on
 o
n 
the
 su
rfa
ce
. U
nd
ers
ide
 
of 
co
lon
y 
wi
th 
da
rk 
bro
wn
 w
ith
 s
ub
me
rge
d 
spo
rul
ati
on
. R
ad
ia
l g
ro
w
th
: 2
.2 
cm
. 
     
  
 
   
   
   
   
   
To
p  
    
    
    
    
    
    
 U
nd
ers
ide
 
 Lig
ht 
ye
llo
wi
sh 
gra
y 
gro
wt
h 
wi
th 
lig
ht 
flo
cco
se 
ae
ria
l 
my
ce
lia
 i
nte
rsp
ers
ed
 w
ith
 d
ark
 c
on
idi
al 
pu
stu
les
 an
d a
 lig
ht 
bro
wn
 ri
ng
 at
 th
e c
en
tre
 w
ith
 
sup
erf
ici
al 
spo
rul
ati
on
. U
nd
ers
ide
 of
 co
lon
y w
ith
 
lig
ht 
ye
llo
w 
wi
th 
gra
yis
h 
bro
wn
 
ce
ntr
e 
int
ers
pe
rse
d 
wi
th 
da
rk 
pu
stu
les
 o
f c
on
idi
a 
an
d 
sub
me
rge
d s
po
rul
ait
on
. R
ad
ia
l g
ro
w
th
: 4
.5 
cm
.  
 
M
or
ph
ol
og
y 
of
 c
on
id
ia
, m
yc
el
ia
 a
nd
 f
ru
it
in
g 
bo
di
es
  
           
  
 
    
    
    
   S
eta
e: 
x 4
00
    
    
    
    
    
   C
on
idi
a: 
x 4
00
 
 Se
ta
e:
 P
res
en
t –
 h
igh
ly 
spa
rse
, d
ark
 b
row
n, 
str
aig
ht,
 1
-4 
sep
tat
e, 
sw
oll
en
 at
 ba
se 
an
d t
ap
eri
ng
 to
wa
rds
 th
e a
pe
x, 
70
-19
0 
µm
 l
on
g. 
C
on
id
ia
: 
Sh
ort
, 
cy
lin
dri
cal
 w
ith
 o
btu
se 
en
ds,
 
hy
ali
ne
, a
sep
tat
e, 
un
inu
cle
ate
, 3
-15
 µ
m 
x 3
-4 
µm
, f
orm
ed
 in
 
bro
wn
 se
tos
e a
ce
rvu
li m
ea
sur
ing
 65
-17
5 µ
m 
in 
dia
me
ter
. 
  
  
   
   
 
Co
nid
ial
 pu
stu
les
 of
    
    
    
    
    
    
    
    
  C
on
idi
a: 
x 4
00
 
    
   c
olo
ny
: x
 40
0  
    
    
  
Se
ta
e:
 A
bse
nt.
 C
on
id
ia
: c
yli
nd
ric
al 
wi
th 
ob
tus
e e
nd
s, 
hy
ali
ne
, 
ase
pta
te,
 u
nin
uc
lea
te,
 1
3-2
2 
µm
 x
 3
.5-
5 
µm
, f
orm
ed
 in
 d
ark
 
irr
eg
ula
r a
cer
vu
li m
ea
sur
ing
, 6
0-1
80
 µm
 in
 di
am
ete
r. 
     
University of Ghana          http://ugspace.ug.edu.gh
94
 
 
So
ur
ce
 
     
Tw
um
ia,
  
B-
A/
R            
Su
nk
un
de
i, 
B-
A/
R              
Is
ol
at
e 
C
od
e     
Cg
 16
5             
Cg
 16
6               
C
ul
tu
re
 c
ha
ra
ct
er
is
ti
cs
 
         
  
   
    
    
    
    
To
p  
    
    
    
    
    
    
 U
nd
ers
ide
 
 W
hit
e 
gra
y, 
slo
w 
gro
wt
h 
wi
th 
sub
me
rge
d 
da
rk 
rou
nd
 to
 ir
reg
ula
r a
cer
vu
li 
an
d 
no
 d
ist
inc
t r
ing
 
gro
wt
h 
on
 b
oth
 s
urf
ace
 a
nd
 u
nd
ers
ide
. R
ad
ia
l 
gr
ow
th
: 2
.2 
cm
. 
 
  
 
    
    
    
    
 To
p  
    
    
    
    
    
    
    
    
 U
nd
ers
ide
 
 Da
rk 
gra
y 
my
ce
lia
l 
gro
wt
h, 
les
s 
pro
no
un
ce
d 
gro
wt
h 
rin
gs 
wi
th 
sub
me
rge
d 
spo
rul
ati
on
. D
ark
 
bro
wn
 g
row
th 
on
 u
nd
ers
ide
 i
nte
rsp
ers
ed
 w
ith
 
sm
all
-si
zed
 co
nid
ial
 pu
stu
les
.  R
ad
ia
l g
ro
w
th
: 3
.3 
cm
. 
     
M
or
ph
ol
og
y 
of
 c
on
id
ia
, m
yc
el
ia
 a
nd
 f
ru
it
in
g 
bo
di
es
  
         
  
 
   
   
 A
ce
rvu
lus
: x
40
0  
    
    
    
    
    
  C
on
idi
a: 
x 4
00
 
 Se
ta
e:
 A
bse
nt.
 C
on
id
ia
: c
yli
nd
ric
al 
wi
th 
ob
tus
e e
nd
s, 
hy
ali
ne
, 
ase
pta
te,
 u
nin
uc
lea
te,
 4
-20
 µ
m 
x 
3-4
 µ
m,
 fo
rm
ed
 in
 d
ark
 
glo
bo
se 
ace
rvu
li m
ea
sur
ing
, 7
0-2
30
 µm
 in
 di
am
ete
r. 
        
        
  
 
   
  C
on
idi
al 
pu
stu
les
: x
 40
0  
    
    
    
    
    
   C
on
idi
a: 
x 4
00
 
 Se
ta
e: 
Ab
sen
t. 
C
on
id
ia
: c
yli
nd
ric
al 
wi
th 
ob
tus
e 
en
ds,
 
hy
ali
ne
, a
sep
tat
e, 
un
inu
cle
ate
, 2
-19
 µm
 x 
2-4
 µm
, fo
rm
ed
 
in 
set
ose
 or
 gl
ob
ose
 ac
erv
uli
 or
 on
 so
lita
ry 
ph
ial
ide
s o
n 
my
cel
ium
. A
ve
rvu
lus
 ro
un
d t
o e
lon
ga
ted
 to
 ir
reg
ula
r 6
5-
19
0 µ
m 
in 
dia
me
ter
. 
    
University of Ghana          http://ugspace.ug.edu.gh
95
 
 
So
ur
ce
 
     
SA
RI
,  
N/
R*
 
             
Ny
ari
ga
,  
N/
R            
Is
ol
at
e 
C
od
e     
Cg
 14
6               
Cg
 14
8             
C
ul
tu
re
 ch
ar
ac
te
ri
st
ic
s 
         
  
 
    
    
    
    
 To
p  
    
    
    
    
    
  U
nd
ers
ide
 
 Sm
ok
y 
gra
y 
co
lon
y 
wi
th 
lig
ht 
flo
cco
se 
aer
ial
 
my
ce
lia
 an
d w
hit
e p
ust
ule
 at
 th
e c
en
tre
. U
nd
ers
ide
 
of 
co
lon
y 
wi
th 
dis
tin
ct 
ros
y-c
olo
ure
d 
my
ce
lia
. 
R
ad
ia
l g
ro
w
th
: 4
.2 
cm
.  
                   
 
 
    
    
    
    
To
p  
    
    
    
    
    
  U
nd
ers
ide
 
 W
hit
ish
 g
ray
 f
luf
fy 
my
ce
lia
 g
row
th 
wi
th 
les
s 
pro
no
un
ce
d 
rin
gs 
gro
wt
h 
on
 
the
 
sur
fac
e. 
Un
de
rsi
de
 w
ith
 w
hit
e g
ray
 gr
ow
th 
wi
th 
lig
ht 
ros
y 
ce
ntr
e. 
R
ad
ia
l g
ro
w
th
: 3
.7 
cm
. 
   
M
or
ph
ol
og
y 
of
 c
on
id
ia
, m
yc
el
ia
 a
nd
 f
ru
it
in
g 
bo
di
es
  
                   
  
 
    
    
    
    
 Se
tae
: x
 40
0  
    
    
    
    
    
    
Co
nid
ia:
 x 
40
0 
 Se
ta
e:
 P
res
en
t –
 h
igh
ly 
pro
fus
ed
, d
ark
 b
row
n, 
str
aig
ht 
to 
sli
gh
tly
 cu
rve
d, 
1-4
 se
pta
te,
 sw
oll
en
 at
 th
e b
ase
 an
d t
ap
eri
ng
 
tow
ard
s t
he
 ap
ex
, 6
0-1
80
 µ
m 
lon
g. 
C
on
id
ia
: c
yli
nd
ric
al 
wi
th 
ob
tus
e e
nd
s, 
hy
ali
ne
, a
sep
tat
e, 
un
inu
cle
ate
, 4
-21
 µ
m 
x 
3-4
.5 
µm
, 
for
me
d 
in 
set
ose
 a
ce
rvu
li 
me
asu
rin
g 
60
-19
0 
µm
 i
n 
dia
me
ter
.  
          
  
 
    
    
    
  C
on
idi
a: 
x 4
00
    
    
    
    
    
    
Co
nid
ia:
 x 
40
0 
 Se
ta
e:
 A
bse
nt.
 C
on
id
ia
: c
yli
nd
ric
al 
wi
th 
ob
tus
e e
nd
s, 
hy
ali
ne
, 
ase
pta
te,
 un
inu
cle
ate
, 2
.5-
18
 µ
m 
x 3
-5 
µm
, f
orm
ed
 in
 se
tos
e 
ac
erv
uli
 m
ea
sur
ing
 60
-17
0 µ
m 
in 
dia
me
ter
. 
     
University of Ghana          http://ugspace.ug.edu.gh
96
 
 
So
ur
ce
 
     
To
lon
,  
N/
R                         
Bo
le,
 N
/R
 
 
Is
ol
at
e 
C
od
e     
Cg
 14
9                    
Cg
 15
0  
C
ul
tu
re
 c
ha
ra
ct
er
is
ti
cs
 
          
  
 
    
    
    
    
 To
p  
    
    
    
    
    
    
    
  U
nd
ers
ide
 
 Da
rk 
gra
y c
olo
ny
 w
ith
 bl
ack
 pu
stu
le 
at 
the
 ce
ntr
e 
an
d 
sup
erf
ici
al 
spo
rul
ati
on
 
at 
the
 
sur
fac
e. 
Un
de
rsi
de
 o
f 
co
lon
y 
wi
th 
dis
tin
ct 
bro
wn
ish
-
co
lou
r 
an
d 
sub
me
rge
d 
spo
rul
ati
on
 w
ith
 d
ark
 
ce
ntr
e. 
R
ad
ia
l g
ro
w
th
: 3
.8 
cm
.  
 
  
    
    
    
    
   T
op
    
    
    
    
    
    
    
    
   U
nd
ers
ide
 
      W
hit
ish
 li
gh
t m
ass
 m
yc
eli
a, 
bo
rde
red
 w
ith
 li
gh
t 
ye
llo
w 
to 
gra
y r
ing
 to
wa
rds
 th
e e
nd
 on
 th
e s
urf
ace
 
wi
th 
sub
me
rge
d s
po
rul
ati
on
. U
nd
ers
ide
 of
 co
lon
y 
wi
th 
lig
ht 
ros
y 
int
ers
pe
rse
d 
wi
th 
lig
ht 
da
rk 
pu
stu
les
 of
 co
nid
ia.
 R
ad
ia
l g
ro
w
th
: 3
.6 
cm
 
  
M
or
ph
ol
og
y 
of
 c
on
id
ia
, m
yc
el
ia
 a
nd
 f
ru
it
in
g 
bo
di
es
  
          
 
 
   
   
  S
eto
se 
ac
erv
ulu
s: 
x 4
00
    
    
    
    
    
    
  C
on
idi
a: 
x 4
00
 
 Se
ta
e:
 A
bse
nt.
 C
on
id
ia
: c
yli
nd
ric
al 
wi
th 
ob
tus
e 
en
ds,
 
hy
ali
ne
, 
ase
pta
te,
 u
nin
uc
lea
te,
 6
-23
 µ
m 
x 
3.5
-5 
µm
, 
for
me
d 
in 
set
ose
 a
cer
vu
li 
me
asu
rin
g 
70
-23
0 
µm
 i
n 
dia
me
ter
.  
          
  
 
    
    
    
    
    
Se
tae
: x
 40
0  
    
    
    
    
    
    
    
    
Co
nid
ia:
 x 
40
0 
Se
ta
e:
 P
res
en
t –
 h
igh
ly 
pro
fus
ed
, d
ark
 b
row
n, 
str
aig
ht 
to 
sli
gh
tly
 cu
rve
d, 
1-4
 se
pta
te,
 sw
oll
en
 at
 th
e b
ase
 an
d t
ap
eri
ng
 
tow
ard
s t
he
 ap
ex
, 5
0-1
70
 µ
m 
lon
g. 
C
on
id
ia
: c
yli
nd
ric
al 
wi
th 
ob
tus
e 
en
ds,
 h
ya
lin
e, 
ase
pta
te,
 u
nin
uc
lea
te,
 1
3-2
3 
µm
 x
 3
-5 
µm
, f
orm
ed
 in
 ir
reg
ula
r a
ce
rvu
li 
me
asu
rin
g 
60
-18
0 
µm
 in
 
dia
me
ter
.  
      
    
* V
/R
 = 
Vo
lta
 re
gio
n, 
A/
R 
= A
sha
nti
 re
gio
n, 
C/
R 
= C
en
tra
l re
gio
n, 
E/
R 
= E
ast
ern
 re
gio
n, 
B-
A/
R 
= B
ron
g-A
ha
fo 
reg
ion
 an
d N
/R
 = 
No
rth
ern
 re
gio
n. 
Bl
ac
k b
ar 
in 
ea
ch
 
pa
ne
l o
f c
on
idi
a m
ea
su
res
 20
 µm
 w
hil
st 
tha
t o
f s
eta
e a
nd
 ac
erv
uli
 m
ea
su
res
 60
 µm
. R
ad
ial
 gr
ow
th 
of 
iso
lat
es 
wa
s r
ec
ord
ed
 on
 th
e 7
th  d
ay
 of
 in
cu
ba
tio
n a
t 2
6±
2o C
, 7
0-
85
% 
RH
. 
University of Ghana          http://ugspace.ug.edu.gh
97 
 
4.3.1.5 Molecular characterization of Colletotrichum spp. obtained from anthracnose disease of D. 
alata in the six regions of Ghana  
Of the 22 isolates obtained from the six regions (Ashanti, Brong-Ahafo, Central, Eastern, Northern 
and Volta Regions), 20 were used in the genome analysis since two of the C. gloeosporioides 
strains exhibited similar morpho-cultural characters. In the ITS1/ITS4 primer pair analysis, 
amplification of the ITS1-5.8s-ITS4 locus of all the 20 Colletotrichum isolates generated PCR 
products of approximately 600-bp on agarose gel (Plate 4.13a) as was indicated in section 4.2.2. 
However, ITS4/CgInt species specific primer pair produced PCR products of about 530-bp for all 
strains of C. gloeosporioides (lanes: 1, 2, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 17, 18 and 19) (Plate 
4.13b). No amplified products were recorded in C. truncatum strains (lanes: 3, 10, 16 and 20) 
(Plate 4.13b). This indicates that the C. truncatum strains identified among the isolates in the 
culture studies were actually confirmed. The negative controls (C) in both analyses did not produce 
any PCR product.  All of the profiles generated were examined visually and bands scored (as 
present or absent) (Table 4.15).  
 
 
 
 
 
 
                                                                                            (A) 
 
 
 
 
 
 
(B) 
Plate 4.13: Amplified DNA fragments of C. gloeosporioides strains (lanes - 1, 2, 4, 5, 7, 8, 9, 11, 12, 13, 14, 15, 17, 
18, 19 and 20) and C. truncatum strains (lanes - 3, 6, 10 and 16) obtained from diseased D. alata in Ghana using 
ITS1/ITS4 primer pair (A) and ITS4/CgInt species specific primer pair (B). M = Molecular size marker 
GeneRuler™ DNA Ladder Mix (100-10,000 bp); C = Negative controls (sterile distilled water); Pc = Positive 
controls (C. gloeosporioides from mango). Lanes: 1 = Cg111, 2 = Cg114, 3 = Ct130, 4 = Cg117, 5 = Cg125, 6 = 
Ct132, 7 = Cg141, 8 = Cg147, 9 = Cg148, 10 = Ct142, 11 = Cg149, 12 = Cg150, 13 = 151, 14 = Cg158 162, 15 = 
Cg162, 16 = Ct168, 17 = Cg165, 18 = Cg166, 19 = Cg167 and 20 = Cg169.    
600-bp 
13 15 M 1 5 7 9 8 6 2 C 3 4 10 11 12 14 17 16 18 19 20 Pc 
530-bp 
13 15 M 1 5 7 9 8 6 2 C 3 4 10 11 12 14 17 16 18 19 20 Pc 
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98 
 
Table 4.15: Detection of DNA fragments of C. gloeosporioides and C. truncatum strains obtained 
from diseased D. alata plants in Ghana using ITS1/ITS4 primer pair and CgInt/ITS4 species-
specific primer pair. 
Source of Isolate 
     (Region) 
Isolate Code Lab ID  ITS1/ITS4 ITS4/CgInt 
Volta #Cg111 1       +      + 
Volta Cg114 2  + + 
Volta *Ct130   3  + - 
Volta  Cg117 4 + + 
Volta 
Volta  
Cg125 
*Ct132 
5 
6 
 
 
+ 
+ 
+ 
- 
Central Cg141 7      +      + 
Central *Ct142 10 + - 
Northern Cg148 9 +   + 
Northern Cg149 11  + + 
Northern 
Ashanti 
Ashanti 
Ashanti 
Brong-Ahafo 
Brong-Ahafo 
Brong-Ahafo 
Eastern 
Eastern 
Eastern 
Cg150 
Cg151 
Cg158 
Cg147 
Cg162 
Cg165 
Cg166 
*Ct168 
Cg167 
Cg169 
12 
13 
14 
8 
15 
17 
18 
16 
19 
20 
 
 
 
 
 
 
 
 
 
 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
- 
+ 
+ 
#Cg = C. gloeosporioides; *Ct = C. truncatum; (+) = presence of amplified DNA fragment; (-) = Absence of DNA 
fragment. 
 
 
In the analyses of the RAPD primers (OPA-11, OPA-18, OPC-5, OPC-07 and OPC-16) tested on the 
genome of the 20 Colletotrichum isolates collected from diseased D. alata in the Volta Region and 
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99 
 
the other five Regions (Ashanti, Brong-Ahafo, Central, Eastern and Northern), DNA fingerprints 
were generated and evaluated for overall clearness of banding patterns (Plate 4.14 a-c). All the 
primers used generated short amplicons located between: 100 and 400-bp (OPC-16) (Plate 4.14c), 
100 and 700-bp (OPA-18) (Plate 4.14a), 100 and 750-bp (OPC-07) (Plate 4.14b), 150 and 650-bp 
(OPC-05) (data not shown) and 130 and 730 (OPA-11) (data not shown). Sets of major amplicons 
were generated in each primer and were shared by two or more isolates. In OPA-18, amplicon of 
approximately 100-bp was shared by almost all isolates whiles 250 and 530-bp were shared by 
three C. gloeosporioides strains (Cg125, Cg150 and Cg158: Lanes 5, 12 and 14) and a C. 
truncatum strain (Ct130: Lane – 3) (Plate 4.14a).  
 
In using primer OPC-07, amplicon of approximately 120-bp was shared by almost all isolates 
(both C. gloeosporioides and C. truncatum isolates); 400 and 680-bp were shared by isolates 
(Ct130, Cg125 and Cg150: Lanes 3, 12 and 14 respectively); 750-bp amplicon was shared by 
seven C. gloeosporioides strains (Cg117, Cg148, Cg150, Cg158, Cg162, Cg166 and Cg169: Lanes 
4, 9, 12, 14, 15, 18 and 20 respectively) (Plate 4.14b). Using OPC-16 primer, discernible amplicons 
of about 100 and 130-bp were shared by almost all the isolates (both C. gloeosporioides and C. 
truncatum isolates) whilst amplicons of approximately 280 and 400-bp were shared by two C. 
gloeosporioides strains (Cg147 and Cg166: Lanes – 8 and 18 respectively) and two C. truncatum 
strains (Ct142 and Ct168: Lanes – 10 and 16 respectively). Similar results were demonstrated with 
primers OPA-11 and OPC-05 (data not shown). 
 
 
 
 
 
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100 
 
 
. 
 
 
 
 
 
   (A) RAPD Primer OPA-18  
  
 
 
 
 
 
 
 
 
    (B) RAPD Primer OPC-07  
 
 
 
 
 
 
 
    (C) RAPD Primer OPC-16 
Plate 4.14: RAPD fingerprints obtained with three RAPD Primers OPA-18 (A), OPC-07 (B) and OPC-16 (C) for 20 
Colletotrichum  isolates  (1-20) obtained from diseased D. alata in Ghana. M = Molecular size marker 
GeneRuler™ DNA Ladder Mix (100-10,000 bp); Pc = Positive controls (C. gloeosporioides from Mango); Lanes: 1 
= Cg111, 2 = Cg114, 3 = Ct130, 4 = Cg117, 5 = Cg125, 6 = Ct132, 7 = Cg141, 8 = Cg147, 9 = Cg148, 10 = Ct142, 
11 = Cg149, 12 = Cg150, 13 = 151, 14 = Cg158 162, 15 = Cg162, 16 = Ct168, 17 = Cg165, 18 = Cg166, 19 = Cg167 
and 20 = Cg169.    
  
In the analyses of the restriction fragments of the amplification products with five endonucleases (HaeIII, 
HinfI, RsaI, MspI and HhaI), HaeIII enzyme produced the most fingerprints ranging from 100 to 
130-bp 100-bp 
400-bp 
210-bp 280-bp 
Pc 13 15 M 1 5 7 9 8 6 2 3 4 10 11 12 14    17 16 18 19 20 c 13 15  1 7 9 8 6  10 11 12      18   
700-bp 
100-bp 
250-bp 
180-bp 
530-bp 
Pc 13 15 M 1 5 7 9 8 6 2 3 4 10 11 12 14    17 16 18 19 20 
750-bp 
680-bp 
320-bp 
120-bp 
530-bp 
400-bp 
Pc 13 15 M 1 5 7 9 8 6 2 3 4 10 11 12 14    17 16 18 19 20 
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1500-bp (Plate 4.15) whilst enzyme MspI gave the simplest fingerprints ranging between 100 to 
800-bp (data not shown). HaeIII enzyme produced discernible DNA fragment amplified at about 100-
bp band was shared by almost all isolates whilst 250-bp band was shared by nine isolates (Cg117, 
Cg125, Cg149, Cg141, Cg148, Cg149, Cg162, Cg166 and Cg169: Lanes – 4, 5, 6, 7, 9, 11, 15, 18 
and 20 respectively) (Plate 4.15). The positive control (Pc – reference isolate) also produced an 
amplicon at this band region (250-bp). However, none of the C. truncatum strains (Ct130, Ct132, 
Ct142 and Ct168: Lanes – 3, 6, 11 and 16 respectively) produced fingerprints that matched with 
that of the reference isolate (C. gloeosporioides from mango) (Plate 4.15).  
 
Using HhaI digestion, C. gloeosporioides strains (Cg111 and Cg118: Lanes – 1 and 18) produced marked 
DNA fragment of about 150-bp; Cg162: Lane – 15 (200 and 300-bp); and Cg149: Lane – 10 (330 and 490-
bp) (Plate 4.16).  Among the C. truncatum strains, Ct130: Lane – 3 produced discernible fragment of 
approximately 200-bp; Ct132: Lane – 6 (200-bp) and Ct168: Lane – 16 (150, 200 and 250-bp) (Plate 4.16). 
MspI digestion yielded 400-bp fragments for seven C. gloeosporioides strains (Cg111, Cg141, Cg147, 
Cg148, Cg151, Cg165 and Cg169: Lanes – 1, 7, 8, 9, 13, 17 and 20 respectively); 350-bp fragments for four 
isolates (Cg125, Cg149, Cg158 and Cg166: Lanes – 5, 10, 14 and 18 respectively); 150 and 300-bp for 
isolates (Ct130 and Ct132: Lanes – 3 and 6 respectively); and 400-bp for isolates (Ct142 and Ct168: Lanes 
– 11 and 16 respectively) (data not shown). HinfI and RsaI produced major bands ranging between 100 – 
450-bp and 120 – 750-bp respectively (data not shown). 
 
 
 
 
 
 
 
 
Plate 4.15: RFLP fingerprints obtained with HaeIII primer for  16  C. gloeosporioides strains  (Lanes – 1, 2, 
4, 5, 7, 8, 9, 10, 12, 13, 14, 15, 17, 18,19 and 20) and four C. truncatum strains (Lanes – 3, 6, 11 and 16) 
obtained from  diseased D. alata in Ghana. M = Molecular size marker GeneRuler™ DNA Ladder Mix 
(100-10,000 bp); Pc = Positive control (C. gloeosporioides from mango); Lanes – 1 = Cg111, 2 = Cg114, 3 = 
Ct130, 4 = Cg117, 5 = Cg125, 6 = Ct132, 7 = Cg141, 8 = Cg147, 9 = Cg148, 10 = Cg149, 11 = Ct142, 12 = Cg150, 
13 = 151, 14 = Cg158 162, 15 = Cg162, 16 = Ct168, 17 = Cg165, 18 = Cg166, 19 = Cg167 and 20 = Cg169.    
 
850-bp 
100-
350-bp 530-bp 
1500-bp 
Pc 13 15 M 1 5 7 9 8 6 2 3 4 10 11 12 14    17 16 18 19 20 
250-bp 
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Plate 4.16: RFLP fingerprints obtained with HhaI primer for  16  C. gloeosporioides strains  (Lanes – 1, 2, 
4, 5, 7, 8, 9, 10, 12, 13, 14, 15, 17, 18,19 and 20) and four C. truncatum strains (Lanes – 3, 6, 11 and 16) 
obtained from  diseased D. alata in Ghana. M = Molecular size marker GeneRuler™ DNA Ladder Mix 
(100-10,000 bp); Pc = Positive control (C. gloeosporioides from mango); Lanes – 1 = Cg111, 2 = Cg114, 3 = 
Ct130, 4 = Cg117, 5 = Cg125, 6 = Ct132, 7 = Cg141, 8 = Cg147, 9 = Cg148, 10 = Cg149, 11 = Ct142, 12 = Cg150, 
13 = 151, 14 = Cg158 162, 15 = Cg162, 16 = Ct168, 17 = Cg165, 18 = Cg166, 19 = Cg167 and 20 = Cg169.    
 
4.3.1.5 .1 Cluster analyses of the 20 Colletotrichum isolates 
 
 
 
4.3.1.5 .1.1 C. gloeosporioides strains 
 
 
 
The dendogram of genetic relatedness generated by scoring of the presence (1) or absence (0) of 
reproducible bands of the five RAPD primers OPA-11, OPA-18, OPC-05, OPC-07 and OPC-16 
(Operon Technologies, California, USA) and the five RFLP endonucleases HaeIII, HinfI, RsaI, 
MspI and HhaI clearly showed the genetic variability between the 16 C. gloeosporioides strains 
isolated from diseased D. alata in Ghana (Fig. 4.7). The 16 C. gloeosporioides strains identified in 
the culture study (i.e. traditional approach) were summarized by the cluster analysis into two main 
groups: Group A (Cg111 and Cg117 – both from Volta Region) and group B (Cg114, Cg125, 
Cg141, Cg146, Cg147, Cg149, Cg150, Cg152, Cg151, Cg158, Cg162, Cg165, Cg166 and Cg169 – 
from Volta and the other five Regions) (Fig. 4.7). However, there were five main sub-groupings 
(B1-B5) among the strains found in group B. Strains in group A shared 52% similarity with group 
B.  The four strains from the Volta Region (Cg111, Cg114, Cg117 and Cg125) were basically 
grouped into two – Cg111 and Cg117 (group A) and Cg114 and Cg125 (group B) with Cg111 
recording 100% similarity with Cg117 and Cg114 (78%) with Cg125 (Fig. 4.7).  
 
330-bp 
100-bp 
200-bp 250-bp 
490-bp 
650-bp 
150-bp 
Pc 13 15 M 1 5 7 9 8 6 2 3 4 10 11 12 14    17 16 18 19 20 
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Strains obtained from the Ashanti Region were grouped into three: Cg147 (group B2), Cg151 
(group B1) and Cg158 (group B5); Brong-Ahafo Region into three: Cg162 (group B1), Cg165 
(group B3) and Cg166 (group B5); Central Region into two: Cg141 (group B5) and Cg146 (group 
B2); Eastern into two: Cg167 (group B2) and Cg169 (group B5); and Northern Region into two: 
Cg149 (group B5) and Cg150 (group B2). Strains Cg114 and Cg117 from the Volta Region were 
similar whilst Cg111 and Cg125 were 80% similar. Again, Cg125 and Cg149 from Volta and 
Northern Regions respectively were 100% similar. Three isolates Cg158, Cg166 and Cg169 from 
Ashanti, Brong-Ahafo and Eastern Regions respectively also revealed 100% similarity (Fig. 4.7).  
 
 
 
 
 
 
 
 
 
 
 
Fig. 4.7: UPGMA dendrogram based on Jaccard’s coefficient, indicating relationships among the 16 strains 
of C. gloeosporioides obtained from six Regions of Ghana. Genetic distances were based on the combined 
data from the random amplified polymorphic DNA (RAPD) and the internal transcribed spacer-restriction 
fragment length polymorphism (ITS-RFLP) analyses. RAPD primers: OPA-11, OPA-18, OPA-05, OPC-07 
and OPC-16 and Restriction enzymes used were: HaeIII, HinfI, RsaI, MspI and HhaI. 
 
4.3.1.5 .1.2 C. truncatum strains 
 
 
 
 
Cluster analysis of the restriction fragments from the amplification products of the rDNA genes flanking 
the entire ITS1-5.8S-ITS4 region of the isolates using the five RFLP and the five RAPD primers 
showed the existence of two distinct groups (group A and B) (Fig. 4.8). However, sub-groupings occurred 
in the group B strains (i.e. group B1 and B2) which resulted in three groupings. The first group comprised 
A 
B1 
B2 
B4 
B5 
B3 
                             Volta Region: Cg114 
                             Volta Region: Cg125 
                       Northern Region: Cg149 
                Brong-Ahafo Region: Cg166 
                        Ashanti Region: Cg158 
                          Eastern Region: Cg169 
                         Central Region: Cg141 
                 Brong-Ahafo Region: Cg165                           
                                                                                                                                                                                                                                                                 Ashanti Region: Cg147                            
 
 
                                                                                                                                                                                                                                                                     Central Region: Cg146 
                         Eastern Region: Cg167 
                        Northern Region: Cg150 
                         Ashanti Region: Cg151 
                 Brong-Ahafo Region: Cg162 
                             Volta Region: Cg111 
                                                                                                                                                                                                                                                                                          Volta Region: Cg117 
Similarity Coefficient (%) 
0.51.0 0.9 0.8 0.7 0.6
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104 
 
two identical strains Ct130 and Ct168 (group B2) from the Volta and Eeastern Regions respectively and 
were 100% similar (Fig. 4.8). The second group (B1) was strain from the Central Region (Ct142) which was 
65% similar to Ct130 and Ct168 obtained from the Volta and Eastern Regions respectively. The strain 
Ct132 which proved highly virulent in the pathogenecity test was grouped separately (i.e. put into group A) 
from the other strains and recorded 30% similarity to the first and second groups of isolates. The Volta 
Region strains (Ct130 and Ct132) were put into two different groups, B2 and A respectively. 
 
 
 
 
 
 
 
 
Fig. 4.8: UPGMA dendrogram based on Jaccard’s coefficient, indicating relationships among the four 
strains of C. truncatum obtained from three Regions of Ghana. Genetic distances were based on the 
combined data from the random amplified polymorphic DNA (RAPD) and the internal transcribed spacer-
restriction fragment length polymorphism (ITS-RFLP) analyses. RAPD primers: OPA-11, OPA-18, OPA-
05, OPC-07 and OPC-16 and Restriction enzymes used were: HaeIII, HinfI, RsaI, MspI and HhaI. 
 
 
4.4 Screening of D. alata cultivars for resistance to anthracnose disease of D. alata 
The 12 D. alata tissue culture-derived whole-plants used for the screening trial gave higher  (70-
90%) explants response (i.e. root-shoot). However, the rate of explants growth in-vitro (Plat 4.16) 
was slow in comparison with the hardened explants on 1:1 vermiculite: compost (peat: sand 9:1). 
No anthracnose disease symptoms were observed on hardened D. alata explants (Plate 4.17). Days 
to root initiation and bud break, number of root and shoot per explants (Plate 4.18), root and shoot 
length and nodes per shoot of explants were significantly different (P < 0.05) among the cultivars 
(Appendix V).  
 
 
Similarity Coefficient (%) 
0.21.0 0.8 0.6 0.4
Central Region 
 
Volta Region 
 
Ct132 
 
Ct142 
3 
2 
 
Eastern Region 
   Volta Region 
 
 
Ct168 
Ct130 
 
1 
B 
A 
B1 
B2 
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105 
 
 
 
 
 
 
 
 
 
  Plate 4.16: Three-week old in-vitro explants of D. alata incubated at 16-hour light/8-hour dark photope    
riod (cool, white fluorescent light – 30 µmol m-2 S-1) and a temperature of 25 °C, 60-70% RH. 
  
 
 
 
 
 
 
 
 
Plate 4.17: Three-week old hardened explants of D. alata cultivars TDa019 (A), TDa007 (B) and TDa013 
(C) without symptoms of anthracnose disease. Medium used – 1:1 vermiculite: compost (peat: sand 9:1) 
(IITA, Nigeria) maintained under bioclimatic chamber.  
 
 
 
 
 
 
 
 
Plate 4. 18: Six-week old explants of D. alata cultivars TDa010 (A); TDa019 (B); TDa018 (C) and TDa007 
(D) with varying number of roots and shoots. Explants were incubated at 16-hour light/8-hour dark 
photoperiod (cool, white fluorescent light – 30 µmol m-2 S-1) and a temperature of 25 °C, 60-70% RH. 
A D C B 
A B C 
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106 
 
 
4.4.1 Mean severity (AUDPCs) of anthracnose disease of the 12 D. alata cultivars  
 
 
 
 
The AUDPC scores of anthracnose disease in the two-month old potted tissue culture-derived 
whole-D. alata cultivars (12) revealed wide range of resistance among the cultivars. The cultivars 
were grouped into two main categories: resistant – TDa008, TDa010, TDa014, TDa023, TDa025 
and TDa027 and susceptible – TDa007, TDa013, TDa015, TDa018, TDa019 and TDa021. 
However, the levels of resistance and susceptibility varied among cultivars (Table 4.16). D. alata 
cultivar TDa023 had AUDPC value of 29.67 which was significantly lower (P < 0.005) than each 
of the remaining five resistant cultivars TDa027 (36.53), TDa025 (45.02), TDa008 (47.48), 
TDa014 (48.49) and TDa010 (49.40) (Table 4.16) but there were no significant (P > 0.005) 
difference among the cultivars TDa008, TDa010, TDa014, TDa025 and TDa027.  
 
Among the six susceptible cultivars, TDa018 had a significantly lower (P < 0.05) AUDPC value 
(54.37) compared with TDa007 (66.61), TDa021 (74.31) and TDa019 (97.90). There was 
significant (P < 0.05) interaction between the 12 D. alata cultivars and six Colletotrichum isolates 
(Table 4.16) (Appendix VI). The ranking of cultivars varied among isolates (Table 4.21). 
Nonetheless, cultivar TDa023 had the lowest rank (1) in almost all the isolates (Cg111, Cg151, 
Ct132 and Ct168) whilst cultivar TDa019 recorded the highest rank (12) in nearly all isolates 
(Cg111, Cg151, Ct132 and Ct168) used (Table 4.16) 
University of Ghana          http://ugspace.ug.edu.gh
10
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LS
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 L
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 su
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; S
 =
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 H
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C
ul
ti
va
r 
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108 
 
 
TDa023 had a mean disease incidence value of 71.95% which was relatively lower than cultivar 
TDa019 (89.53%) (Table 4.16). Among the isolates, Ct132 recorded the highest mean disease 
incidence (86.76%) while Cg151 recorded the least (75.97%). Although, there were no significant 
(P > 0.005) interactions among the isolates and cultivars, interaction between isolate Ct132 and 
cultivar TDa019 recorded the highest disease incidence (96.72%) whilst isolate Cg151 and cultivar 
TDa023 produced the lowest mean disease incidence (65.94%).  
Table 4.16: Incidence (%) of anthracnose disease on two-month old potted explants of 12 D. alata 
cultivars using six Colletotrichum isolates. 
 
*SDW = Sterile distilled water; Disease incidence was scored on the 14th DAI using a score of (+) for disease presence 
and (-) for disease absence and percent disease incidence calculated using the formula below: 
 
Disease incidence (I)         =   Number of diseased plants  x     100 
                          Total number of plants inspected 
 
4.5 Integrated management of anthracnose disease of D. alata  
 
When the strains of C. gloeosporioides (Cg111) and C. truncatum (Ct132) which proved highly 
virulent in the cultivar screening trial were selected and treated with four fungicides (Carbendazim, 
Mancozeb, Nordox and Shafiz F1), there was significant (P < 0.005) fungistatic effect on radial 
      Disease Incidence 
 
 
 
 
 
Colletotrichum Isolates 
                      
Cultivar Cg111 Ct132 Cg150 Cg151 Cg162 Ct168 *SDW     Mean 
TDa007  80.81 90.3 78.40 81.87 82.73 79.30 0.00 82.24 
TDa008 85.21 86.4 81.93 79.24 81.40 80.12 0.00 82.38 
TDa010 76.94 79.93 80.01 75.01 79.21 76.35 0.00 77.92 
TDa013 88.12 91.62 78.99 76.03 83.17 77.03 0.00 82.49 
 TDa014 75.97 80.53 74.75 69.56 77.92 70.17 0.00 74.83 
TDa015 80.68 86.41 79.89 78.16 84.26 79.16 0.00 81.45 
TDa018 84.45 88.87 81.12 79.92 83.19 80.24 0.00 82.97 
TDa019 93.69 96.72 85.03 81.08 91.09 89.58 0.00 89.53 
TDa021 79.64 91.44 83.56 76.82 81.21 78.98 0.00 81.94 
TDa023 73.33 79.10 67.23 65.94. 70.18 69.91 0.00 71.95 
TDa025 84.03 86.09 74.92 70.25 75.24 73.18 0.00 77.29 
TDa027 79.12 83.76 75.33 67.74 74.02 72.51 0.00 75.42 
Mean  
 
81.83 
 
86.76 
 
78.43 
 
75.97 
 
80.30 
 
77.21 
 
0.00 80.09 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
LSD (P > 0.001): Isolate = ns, Cultivar =   ns, Isolate x cultivar = ns 
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mycelial growth and sporulation of isolates in-vitro (Appendix VIII).  Radial mycelial growth and 
sporulation inhibition ranged from 100% in Carbendazim, Mancozeb and Nordox to 0% in the 
control (PDA without fungicide) (Fig. 4.9). Shafiz F1 had significantly lower (P < 0.05) fungistatic 
effect (80%) on radial mycelial growth and sporulation of isolate Ct132 compared with 
Carbendazim (100%), Nordox (100%) and Mancozeb (100%) (Fig. 4.9a). Similarly, in isolate 
Cg111, Shafiz F1 produced significantly lower (P < 0.05) fungistatic effect (86%) on radial 
mycelial growth and sporulation of isolate Cg111 compared to Carbendazim (100%), Mancozeb 
(100%) and Nordox (100%) (Fig. 4.9b).  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Fig. 4.9: Fungistatic effects of four fungicides (Carbendazim, Mancozeb, Nordox and Shatiz F1) on 
radial mycelial growth and sporulation of two Colletotrichum isolates – C. truncatum (A) and C. 
gloeosporioides Cg111 (B). PDA amended cultures and controls (*NF) were incubated at 26±2 oC 
and 65-72% RH in the laboratory. Vertical bars indicate LSD5%. Percentage reduction in radial 
mycelial growth of test fungi (I) was calculated based on the formula of Kiran et al. (2010).  
The information on mean rainfall, relative humidity and temperature of the study area obtained 
from the Ghana Metrological Station, ‘Mpehuasem’ during the period of study (November, 2012 to 
May, 2013) revealed that there was positive correlation between mean rainfall and mean relative 
humidity. However, there was no positive correlation between the mean temperatures recorded and 
mean rainfall or relative humidity (Table 4.17). The study area had a maximum mean rainfall in 
Period of Incubation Period of Incubation 
Pathogen: Cg111 Pathogen: Ct132 
A B (Days) (Days) 
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May, 2013 (245.3 mm) with 33.5 mm as the minimum in December, 2012 (Table 4.17). The 
maximum and minimum mean temperatures were 34.3oC and 19.3oC respectively recorded in 
December, 2012. The mean relative humidity ranged from 64 % in December, 2012 to 79% in May, 
2013.  
Table 4.17: Mean rainfall, relative humidity and temperature distributions of the study area during 
the period of the experiment (November, 2012 to May, 2013). 
Month 
(2012/13) 
Mean 
Rainfall 
(mm) 
Mean Relative 
Humidity     
(%) 
 
           Mean Temperature (oC) 
              Max.                      Min. 
November  
 
December 
 
January 
 
February 
 
March 
 
April 
 
May 
36.1 
 
33.5 
 
42.1 
 
70.8 
 
96.9 
 
98.3 
 
245.3 
66 
 
64 
 
67 
 
69 
 
72 
 
74 
 
79 
              32.1                       20.2 
 
              34.3                       19.3 
 
29.1 23.3 
 
29.6 23.5 
 
31.4 24.0 
 
29.4 23.9 
 
28.7 22.4 
 Source: Ghana metrological station (2013). 
 
 
The integrated disease management trial using artificial inoculation of mixed Colletotrichum 
isolates (Cg111 and Ct132 – highly virulent strains) with spore suspension of 1.6 x 106 conidial/ml 
revealed that, D. alata cultivars which received hot-water treatments, fungicides sprays and 
weeding in an integration (i.e. cultivar + hot-water treatment (H) + weeding (W) + fungicide 
sprays) gave significantly lower (P < 0.05)  disease severities compared to cultivars that did not 
receive hot-water treatment, fungicide sprays and weeding (controls) (i.e. cultivar, no hot-water 
treatment (NH), no weeding (NW) and no fungicide sprays) (Fig. 4.10). The disease severity of 
both the treated cultivars and the controls increased with time (weeks) such that mean disease 
severities of 0.1 and 1.3 in week 1 and 8 respectively were recorded in the treated cultivars whilst 
the controls recorded mean disease severities of 0.6 in week 1 and 3.4 in week 8. In cultivar 
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TDa019, treatment combination (TDa019 + H + W + Mancozeb sprays) had a significantly lower (P 
< 0.05) disease severity (1.1) in week 8 compared with the treatment combination (TDa019 + H + 
W + Nordox sprays) (2.0). However, there was no significant difference (P > 0.05) between the 
treatment combinations (TDa019 + H + W + Mancozeb sprays) and (TDa019 + H + W + 
Carbendazim sprays). In cultivar TDa023, there were no significant differences (P > 0.05) among the 
treatment combinations (TDa023 + H + W + Mancozeb sprays), (TDa023 + H + W + Carbendazim 
sprays) and (TDa023 + H + W + Nordox sprays) (Fig. 4.10). However, TDa023 + H + W + 
Mancozeb sprays had the least disease severity value (1.2). Among the controls, cultivar TDa023 
had significantly lower (P < 0.05) disease severity (3.0) in week 8 in comparison with TDa019 (3.7).   
 
Fig. 4.10: Progress of anthracnose disease of D. alata cultivars (TDa019 and TDa023) on the field 
following artificial inoculation of mixed Colletotrichum isolates (Cg111 and Ct132) with spore 
suspension 1.6 x 106 conidial/ml.  *Keys: TDa019 and TDa023 = D. alata cultivars; H = Hot-water 
treatment; W = Weeding; NH = No hot-water treatment; NW = No weeding. Vertical bars indicate 
LSD at 5%. 
 
4.5.1 Mean AUDPC scores associated with each IPM strategy for the management of 
anthracnose disease of D. alata on the field 
 
 
 
 
The mean AUDPC of the mixed isolates on the two D. alata cultivars (TDa019 and TDa023) 
revealed significance (P < 0.05) (Table 4.18). In cultivar TDa019, the mean AUDPC scores ranged 
KEYS* 
 
, H, W. 
, H, W. 
, H, W. 
, H, W. 
, H, W. 
, H, W. 
, NH, NW. 
, NH, NW. 
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from 24.06 to 102.05. The treatment combination (TDa019 + H + W + Mancozeb sprays) had 
significantly lower (P < 0.05) mean AUDPC score (24.06) compared with (TDa019 + H + W + 
Nordox sprays) (52.27) and the control (102.05). However, there was no significant difference (P > 
0.05) between treatment combinations (TDa019 + H + W + Mancozeb sprays) and (TDa019 + H + 
W + Carbendazim sprays). In cultivar TDa023, the mean AUDPC scores ranged from 16.54 to 
91.76 (Table 4.18). Treatment combination (TDa023 + H + W + Mancozeb sprays) again recorded 
significantly lower (P < 0.05) mean AUDPC score (17.01) compared with (TDa023 + H + W + 
Nordox sprays) (30.99). There was no significant difference (P > 0.05) between the treatment 
combinations (TDa023 + H + W + Mancozeb sprays) and (TDa023 + H + W + Carbendazim 
sprays) (Table 4.18).  
 
Table 4.18: Mean AUDPC scores associated with each IPM strategy for the management of anthracnose 
disease of D. alata on the field and ranks (parenthesis). Isolates used = (Cg111 + Ct132). 
 
 
* TDa019 and TDa023 = D. alata cultivars; H = Hot-water treatment (45oC for 20 minutes); W = Weeding 
(monthly); NH = No hot-water treatment; NW = No weeding. Fungicides sprays were initiated 2 months after 
sprouting (MAS) and terminated at 4 MAS. Total Carbendazim application (fortnightly) = 4 at 500 g a.i/ha; Total 
Mancozeb applicaton (fortnightly) = 4 at 800 g a.i/ha; and total Nordox application (tri-weekly) = 3 at 800 g a.i/ha.  
**AUDPC = Area under disease progress curves for anthracnose disease of D. alata using 0-5 scale and calculated 
using the formula of Shaner and Finney (1977). 
#Lower scores in parenthesis indicate higher disease reduction (AUDPC). 
Treatment Combination* Mean AUDPC** 
Mancozeb + TDa019 + H + W  24.06 (1)# 
Carbendazim + TDa019 + H + W 25.18 (2) 
Nordox + TDa019 + H + W 52.27 (3) 
No-fungicide + TDa019 + NH + NW 102.05 (4) 
Mancozeb + TDa023 + H + W     16.54 (1)# 
Carbendazim + TDa023 + H + W   17.01 (2) 
Nordox + TDa023 + H + W    40.99 (3) 
No-fungicide + TDa023 + NH + NW    91.76 (4) 
LSD (P < 0.05)                                   4.78 
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CHAPTER FIVE 
5.0 DISCUSSION 
5.1 Assessment of farmers’ knowledge and perception of a vine-browning and die-back 
disease of D. alata in the Volta Region   
Analyses of the data collected from the field survey to assess the knowledge and perception of D. 
alata farmers in the Nkwanta-North (60) and Krachi-West (60) districts of the Volta Region 
revealed that most of the farmers have little or no formal education. Probably, most people with 
secondary or tertiary education seek for jobs in the cities and thus, leave the farming activities in the 
hands of those who have had little or no formal education. This finding corroborates Sesay et al. 
(2013) report that farming in Sierra Leone is left in the hands of the little or no educated people 
while the highly educated ones seek for jobs in the cities. The involvement of people with little or 
no formal education in water yam production in both districts might have influenced the disease 
spread since they perceived diseased D. alata plants as early maturing cultivars. Strategic programs 
to educate farmers on novel advancements and improved technologies in farming for business could 
make farming lucrative to attract educated people in the districts into D. alata production. 
 
Most farmers in the two districts could identify the vine-browning and die-back disease of D. alata 
by its symptoms and referred to it as “brown brown” which connotes browning of leaves and vines 
of the D. alata cultivars. Majority (88.0%) of the respondents were, however, ignorant of the cause 
of the disease and rather attributed it to heavy rainstorm after a prolonged drought and superstition. 
The causal organisms have been identified as the fungi Colletotrichum gloeosporioides and 
Colletotrichum truncatum by earlier workers (Ayodele, et al., n.d; Singh et al., 1966; Nwankiti and 
Ene, 1984; Akem, 1999; Abang et al., 2002; Aduramigba-Modupe et al., 2010). Since the real 
cause of the disease was unknown to most of the farmers, diseased D. alata plants were left intact 
on the field and farm sanitation and other suitable cultural practices to minimize the spread of the 
disease were not carried out. The association of heavy rainfall with the occurrence of the disease by 
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114 
 
the farmers (15.0%) supported the report that fungal infection of plant hosts is influenced by 
adequate moisture (Schwartz and Mohan, 1995; Agrios, 2005). The ignorance of farmers in both 
districts about the spread of the disease within and among farms might have contributed to the high 
incidence of the disease as the sources of inoculum (diseased plant parts) were left on farms after 
harvest. Nevertheless, a few farmers (24.0%) ascribed the disease spread within farms to planting of 
infected seed yam and among farms to exchange of infected seed yam (19.0%). This agrees with the 
findings of Orkwor and Asadu (1997) and Kambaska et al. (2009) that 30% of previous harvests of 
farmers’ seed yam reserved for the next cropping season in Nigeria served as primary sources of 
inoculum on farms especially, when exchanged among farmers. Moreover, infected planting 
materials have been identified as triggering epidemics of the disease as fungi survive beneath the 
periderm of setts (Simons and Green 1994).  
The few farmers (9.0%) who attributed the spread of the disease within farms to contact between 
diseased and healthy plant were in agreement with Schwartz and Mohan (1995), Agrios (2005) and 
Chaube and Pundhir (2005) who related the spreading of the disease to plants contact, insects, 
irrigation water, infected tools and debris. Wind (11.0%) and rainfall (6.0%) were perceived by 
some farmers as modes of the disease spread among farms. This agrees with findings of Ebenebe 
(1980), Schwartz and Mohan (1995) and Galvan et al. (1997) who reported that C. gloeosporioides 
infection on leaves is mainly caused by air-borne conidia. Hence, the disease spread in the Volta 
Region might have been influenced by both wind and water.  
 
 
 
 
 
 
 
 
D. alata cultivars commonly grown by farmers in the districts were ‘Akaba’ (thorned and thorn-
less), ‘Matches’ (long and short tubers), ‘Akomea’, ‘Poka’, ‘Nibultutu’, ‘Chachribon’ and ‘Sum ne 
hyen’ in decreasing order of popularity. This may have been influenced by ‘Akaba’ cultivars being 
less prone to anthracnose and high yielding with better quality compared to the other cultivars. 
Cultivar resistance to diseases in yam greatly influences farmers’ selection for cultivation (Sesay et 
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115 
 
al., 2013). High dry matter content and low percent peel loss (i.e. yield and food quality) are also 
important selection criteria for yam genotype especially, if it is to be processed into secondary 
products (Egesi et al., 2007).Although, ‘Matches’ was highly susceptible to the vine-browning and 
die-back disease, it was the second prime cultivar to ‘Akaba’ commercially grown in both districts. 
This may partly be due to its ability to sprout from small seed or sett sizes (even, as small as 
matches-box size) and multiple tuber formation. The susceptibility of ‘Matches’ cultivar to the 
disease may probably be due to a lower concentration of phenolics (antifungal compounds) and 
other resistance factors to prevent or reduce disease infection. The strains of the pathogen in the 
region might have also produced excessive phenolases (tyrosinase) in the cultivars to breakdown 
their resistance (Plumbley and Sweetmore, 1994).  
 
Furthermore, D. alata has a longer maturity period of about 9 to 12 months which increases its 
exposure to the strains of the pathogen in the field and continuous high production of a given D. 
alata cultivar (especially, ‘Matches’) on the same piece of land year after year, might have also 
contributed to the high losses (50 to 75%) encountered by the farmers. It was, however, observed 
during the field survey that ‘Nibultutu’, ‘sum ne hyen’ and ‘Akomea’ which were resistant to the 
anthracnose disease were cultivated on a small-scale; usually, for consumption (Coffie, Personnal 
Observation, 2012). The pathogen strains might have developed resistant-breakdown gene(s) to 
match the resistant gene(s) in the cultivars as supported by the concept of ‘gene for gene matching’ 
(Maloy, 1993). McDonald et al. (1998) and Ano et al (2002a) reported 90-100% loss of D. alata 
cultivars to C. gloeosporioides strains in the Caribbean where monocultures with a single popular 
susceptible cultivar were common.  
 
Most farmers perceived that the disease could be controlled by the use of chemicals but chemical 
control was not practiced. The farmers rather resorted to cultural control practice such as regular 
weeding (27.63%) and use of healthy planting materials (10.53%). It is, however, suggested that 
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116 
 
integrated disease management measures involving the use of resistant cultivars, judicious use of 
chemicals and good phytosanitory measures such as weeding, burning or burying of debris may 
give desirable results in controlling the disease (Ogundana et al., 1970; Nwakiti and Arene, 1976; 
Nwakiti and Okpala, 1981; IITA, 1993). Anthracnose disease of D. alata was identified as the most 
important factor affecting farmers’ income as they were unable to realize their expected yield. 
Many farmers have, therefore, abandoned the production of D. alata in both districts for other food 
crops. The disease had been estimated to cause excessive yield loss of about 85 to 100% in several 
fields in the Carribeans especially, when the disease epidemics commenced prior to or during tuber 
formation (Degras et al., 1984; Mignucci et al., 1988; Green, 1994). Some farmers, however, 
suggested extensive drought, excessive rainfall and poor land preparation as major causes of yield 
loss in D. alata. Germination of conidia of Colletotrichum spp. under high humidity and long 
period of drought significantly influence tuber formation of yam (Abang, 1997).   
 
Farmers in the Nkwanta-North and Krachi-West districts produce D. alata principally for food 
consumption (food security) followed by income generation and then, for purchasing assets.  
Failure of farmers to realize their expected yield poses a threat not only to their economic 
responsibilities but also, their social relations as farmers in both districts were unable to settle 
school fees, medical bills, loans and acquire assets due to low or no returns from farms. The social 
relation of farmers with family members especially, spouse and Extension Officers were also 
negatively affected.  
 
5.2 Incidence and severity of the disease in the Volta Region 
 
 
 
 
The incidence of vine-browning and die-back disease in Nkwanta-North (75.30%) and Krachi-
West (55.80%) were very high. During the survey, it was observed that diseased D. alata debris 
from previous harvests were gathered and left on the field and on top of mounds which might have 
retained the inoculum (Akem and Asiedu, 1994; Abang et al., 2006; Egesi et al., 2007) and 
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117 
 
contributed to the high disease incidence. D. alata plants were also left unstaked resulting in inter-
twinning of vines (both healthy and diseased) which may have facilitated the spread of the disease. 
The practice of liberal exchange of germplasm (planting materials exchange) among farmers may 
account for the high disease incidence. The higher disease incidence at Nkwanta-North district may 
have resulted from the higher rainfall recorded during the production season (1700 mm) compared 
to Krachi-West district (900 mm) (Baddim and Aboni, Personnal Communication, 2012).  
 
Disease severity was similar in the two districts [Nkwanta-North (1.80) and Krachi-West (1.60)]. 
During the survey, it was observed that initiation of tubers in D. alata cultivars in the Nkwanta-
North district had initiate tubers which coincided with the peak of the disease in the district around 
June/July when rainfall was very high (Atsu, Personnal Communication, 2012). Translocation of 
resources for tuber formation at the expense of defensive mechanisms in leaves and vines might 
have contributed to the high disease severity (Onyeka et al., 2005). 
 
5.3 Identification of the causal agents of anthracnose disease of D. alata in the Volta 
Region 
 
 
 
 
The symptoms on diseased D. alata plants, morpho-cultural characteristics of the isolates, genome 
analysis, and pathogenicity test confirmed Colletotrichum gloeosporioides Penz. and Sacc. and C. 
truncatum Andrus and Moore as the causal agents of vine-browning and die-back disease of D. 
alata in the two districts of the Volta region. Colletotrichum spp. have been reported as the cause 
of anthracnose disease of D. alata in Ghana, but the actual species were not confirmed (Oduro, 
2000; Offei et al., 2008). This work, therefore, is the first confirmation of the actual species of 
Colletotrichum causing anthracnose disease of D. alata in Ghana and the foremost to report of a 
new species (C. truncatum). The same fungi were also implicated in the disease in the Central and 
Eastern regions. Colletotrichum spp. have been reported to be the pathogen causing anthracnose 
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118 
 
disease of D. alata (Singh et al., 1966; Winch et al., 1984). C. gloeopsorioides and C. truncatum 
have also been reported as the pathogens responsible for the disease in Nigeria (Ayodele, et al., 
n.d). However, in the Ashanti, Brong-Ahafo and Northern Regions, only C. gloeosporioides was 
confirmed as the prime cause of the disease. This finding corroborates reports of Nwankiti and Ene 
(1984), McDonald et al. (1998), Akem (1999), Abang et al. (2002), Ano et al. (2002) and 
Aduramigba-Modupe et al. (2010) who found C. gloeosporioides as the causal agent of 
anthracnose disease of water yam. 
 
 
 
 
 
 
 
 
 
 
 
 
 
The yam breeding program at IITA had lost valuable D. alata germplasm which could have been 
protected by fungicide sprays due to the erroneous concept that the disease was of complex 
etiology involving a virus until Colletotrichum gloesoporioides was confirmed as the principal 
cause of yam anthracnose (Akem, 1999). The presence of C. truncatum strains in the three regions 
of Ghana (Central, Eastern and Volta) could partly be due to the introduction of the strain from 
Nigeria (where it is well noted) through the importation of D. alata germplasm to the Southern-
most part of Ghana (possibly, Plant Genetic Resource Research Institute (PGRRI), Bunso) and 
might have been spreading to the major D. alata growing areas. This then calls for internal 
quarantine to restrict the spread of this strain into other regions because, the strain is more virulent 
and thus, has the potential of degrading the D. alata industry in Ghana as was reported in the 
Carribean (Degras et al., 1984; Mignucci et al., 1988; Green, 1994; Green and Simons, 1994).   
  
The varying levels of pathogenicity in the D. alata cultivars could probably be due to different 
levels of phenolases (tyrosinase) produced by strains of C. gloeosporioides and C. truncatum 
(Plumbley and Sweetmore, 1994). Studies by Abang et al. (2001) confirms that variations exist 
among C. gloeosporioides responsible for anthracnose disease of D. alata and described four forms 
associated with anthracnose of yam in Nigeria: the slow-growing grey, the fast-growing salmon, 
the fast-growing grey and the fast-growing olive forms. These variations have wide-ranging effect 
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on disease severity as they posses’ different levels of virulence (Abang, 1997). The present study 
revealed that anthracnose disease attacks mainly leaves and vines of D. alata which confirms 
earlier works (Simons, 1993; Akem and Asiedu, 1994; Onyeka et al., 2005; Abang et al., 2006; 
Egesi et al., 2009). In the initial stages of the disease development, small regular, brown necrotic 
spots with yellow halo symptoms were more frequently observed on the younger leaves than the 
older. However, as the disease progressed, brown regular spots surrounded by dark brown rings 
with gray centres or shot-holes dominated on the older leaves with occasional symptoms of die-
back. This corroborates the findings of Offei et al. (2008). The rate of progression of the symptoms 
of anthracnose disease of D. alata was affected by the age of leaf and vine, stage of the epidemic, 
cultivar susceptibility, rainfall and agronomic practices such as fungicide application (Sweetmore 
et al., 1994; Wharton, 1994).  
 
The earlier expression of the disease symptoms on the younger leaves may probably be due to the 
tenderness of the cuticle membrane of the younger leaves than the older (Chaube and Pundhir, 
2005). Again, the restriction of disease development on the younger leaves could partly be due to 
higher accumulation of phenols (antifangal compounds) (Plumbley and Sweetmore, 1994). Shot-
holes observed later in the leaves may possibly be due to the activities of secondary microbes 
(Akem, 1999). Fusarium sp., Rhizopus stolonifer, Curvularia pallescens and C. eragrostides were 
isolated from anthracnose diseased yam samples (Amusa, 1997).  
 
Currently, no work has been done on the actual causes of the vines die-back and the browning of 
leaves. Perhaps, the plant might have been induced to release excessive phenols as counter attack 
to the pathogen phenolases (tyrosinase) (Green, 1994; Plumbley and Sweetmore, 1994; Agrios, 
2005) which might have caused reduction in chlorophyll contents at the infected portions; and 
subsequently, led to browning of leaves and dying back of vines – a necrotic structural defense 
reaction (Winch et al., 1993). Even though, Fusarium sp., Cercospora sp. and Colletotrichum spp. 
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were isolated from diseased D. alata samples collected in the present stuty – making it a disease 
complex (Amusa, 1997), spray-inoculation of healthy potted tissue culture-derived whole-plants of 
D. alata cultivars with the Colletotrichum isolates alone proved pathogenicity with typical 
symptoms of a vine-browning and die-back disease on both leaves and vines similar to that 
ascribed by Winch et al. (1984). Akem (1999) also argued that, the predominance of 
Colletotrichum spp. from sample isolations and the reproduction of typical symptoms with C. 
gloeosporoides were proves that C. gloeosporoides is the main causal agent of anthracnose disease 
of D. alata. It is, therefore, suggested that Colletotrichum isolates which have been identified as the 
primary causal agent of the disease be used to screen D. alata cultivars for resistance to the disease. 
 
5.4 Characterization of the strains of C. gloeosporioides and C. truncatum associated 
with anthracnose disease of D. alata in Ghana 
 
 
 
 
 
Colletotrichum isolates from diseased D. alata in the Volta region together with the countrywide 
collections exhibited variable cultural and morphological characters such that, even, isolates with 
similar conidial measurements differed in some morphological characters, sporulation and growth 
patterns – from one location which is similar to observations made in Nigeria (Ayodele et al., n.d). 
Traditionally, Colletotrichum spp. have been identified and classified based on morphological 
characters (Simmonds, 1965; Smith and Black, 1990; Sutton, 1992) and several features have been 
utilized by taxonomists including size and shape of conidia and appressoria; presence or absence of 
setae, sclerotia, acervuli and teleomorph state and cultural characters such as colony colour, growth 
rate and texture (Photita et al., 2005; TeBeest et al., 1997; Than et al., 2008a-c; Thaung, 2008).  
 
 
 
 
 
 
 
 
The high morpho-cultural variations that existed among the isolates could be due to collections of 
diseased samples from a wide area with varied environmental conditions as samples collected too 
closely did not show any or much variations. Colletotrichum spp. easily become varied, both in 
morphology and phenotype under environmental influences (Abang et al., 2002; Moriwaki et al., 
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2002; Whitelaw-Weckert et al., 2007). Cannon et al. (2000) also suggested nucleic acid analyses 
should provide the most reliable framework for classifying Colletotrichum spp. as DNA characters 
are not directly influenced by environmental factors. Based on the morphological and cultural 
characters obtained in the present study, rapid identification and/or differentiation of Colletotrichum 
spp. from D. alata could be done.  
 
The 16 C. gloeosporioides and four C. truncatum strains identified in the culture studies were 
clustered into two major groups (Groups: A and B) after the genome analyses with the RAPD 
primers and RFLP enzymes. This finding corroborates that of Xiao et al. (2004) who noted 
clustering of 15 isolates of C. gloeosporioides from strawberry into two separate groups with 
RAPD primers. The two major groupings observed could probably be due to long period of 
independent evolution from a single Colletotrichum strain that might have gained ability to infect a 
particular D. alata cultivar (Peres et al., 2002). Asexual reproduction by conidia of isolates which 
results in occurrences of identical genotypes and linkages between independent sets of genetic 
markers might have also accounted for the observation made (Milgroom et al., 1996).  
 
The close relatedness of the isolates could lead to incorrect identification using morpho-cultural 
characteristics. Once the species are inaccurately named and/or identified, it is probable that vital 
information needed for effective management of the disease would not be provided (Freeman et al., 
1993). Colletotrichum isolates Cg111 and Ct132 (from the Volta Region) which were highly 
pathogenic to the potted tissue culture-derived whole-plant D. alata cultivars in the screening trial 
were grouped separately from the less pathogenic; indicating that pathogenicity was decisive in 
group formation. This finding agrees with that of Ratanacherdchai et al. (2010) who reported that 
RAPD markers and RFLP enzymes are quick and reliable alternatives for differentiating isolates of 
Colletotrichum spp. into their respective pathogenic group. It is possible that these two isolates 
possessed distinct genetic make-ups from the others. As both isolates were found in the Volta 
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Region, effective internal quarantine measures have to be implemented to prevent the spread of 
these isolates (especially, the most virulent (Ct132) from the Volta Region to other yam growing 
regions in Ghana. Moreover, sustainable management measures have to be deployed to manage the 
spread of the pathogens within the region.  
 
The high genetic relatedness of the isolates affirms the argument that identification and 
differentiation of Colletotrichum spp. based on morphological and cultural characteristics have 
often been inadequate (Brown et al., 1996). The combined usage of molecular diagnostic tools 
along with traditional morpho-cultural techniques in this study, therefore, present an appropriate 
approach for studying Colletotrichum spp. complexes (Cannon et al., 2000). Accurate identification 
of Colletotrichum spp. can be achieved by combining multigene analysis and morpholo-cultural 
characters (Cai et al., 2009).  
 
Although, the isolates were closely related, high genetic diversity existed among isolates within a 
specified region especially, Volta which may probably be due to the high evolutionary and genetic 
recombination rates of the isolates (Abang et al., 2006). Analyzing C. gloeosporioides from 
diseased D. alata in the Caribbean using RAPD markers demonstrated a high level of genetic 
variability among isolates (Thottappilly et al., 1999; Alleyne, 2001). Extensive studies have also 
confirmed high genetic diversity in Colletotrichum spp. (Freeman et al., 1998; Sahin et al., 2003). It 
is more probable that age (i.e. period of colonization), higher mutation rates and varied 
environmental conditions at sampling locations/sites have accounted for the wider genetic diversity 
found among the isolates (Hartl et al., 1989; Hodson et al., 1993). The wider variations among 
strains within regions especially, Volta affirms the high incidence of anthracnose disease in the 
region in comparison to the other five regions (Coffie, Personnal Observation, 2012). There is, 
therefore, the need to develop sustainable management measures for managing the disease in the 
region, however, same could be replicated in the other regions of Ghana. 
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5.5 Screening of D. alata cultivars for resistance to anthracnose disease  
 
 
 
 
 
 
The general performance of explants of the 12 D. alata cultivars which were further used for 
screening was very good. This may be due to the ratio of hormones (BAP + NAA + Kinetin: 0.25 + 
2.0 + 2.0 mg/l respectively) used. The same ratio gave significantly higher (P < 0.05) root-shoot 
number and nodes per explants of sweet potato (Maura-Costa and Mantell, 1993). The marked 
differences observed among explants of the cultivars in the present study regarding days to bud 
break, mean root, mean shoot and shoot length per explant as well as nodes per shoot indicated 
genetic variability in responding to growth regulators, traumatic or osmotic shock, mechanical 
perturbation and unusual culture and environmental conditions (Gaspar et al., 2004).  
 
 
The screening of explants of the 12 D. alata cultivars in Ghana for resistance to anthracnose disease 
revealed six cultivars (TDa008, TDa010, TDa014, TDa023, TDa025 and TDa027) resistant to the 
anthracnose disease but to varying degrees. TDa023 was the highly resistant cultivar while TDa025 
and TDa027 were resistant with TDa008, TDa010 and TDa014 being the least resistant. The 
presence of varying levels of antifungal compounds (including phenols) and leaf thickness among 
others may have accounted for the relative disease resistance exhibited by the cultivars to the 
disease. The production of phenolases (tyrosinase) is a likely mechanism by which host resistance 
is overcome by some C. gloeosporioides strains (Plumbley and Sweetmore, 1994). Structural 
defensive mechanisms such as cell wall thickness, formation of cork layers and tyloses are also 
used by plants against pathogens (Agrios, 2005).     
 
The least resistant cultivars TDa008, TDa010 and TDa014 are amongst the early-breeding lines in 
Ghana (Oppong, Personal Communication, 2012). Hence, they might have lost their resistance to 
the pathogens due to the development of severe strains over several years of exposure to varying 
environmental conditions. Field resistance of D. alata cultivars to yam anthracnose disease have 
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been observed to vary between locations resulting in differences in performance (Aduramigba-
Modupe et al., 2008). It is worth noting, that TDa027 which recorded lower disease severity (in 
comparison to the other resistant cultivar (TDa025) is a parent of TDa023 – which was highly 
resistant to the disease (Oppong, Personal Communication, 2012). Plant breeders should, therefore, 
breed for novel resistant D. alata cultivars periodically as strains of Colletotrichum spp. change 
their infective mechanisms (‘pathogen race shift’) (Maloy, 1993) 
 
The results also revealed six D. alata cultivars (TDa007, TDa013, TDa015, TDa018, TDa019 and 
TDa021) susceptible to the disease but to varying levels such that TDa019 was highly susceptible 
whereas TDa021 was susceptible and TDa007, TDa13, TDa15 and TDa018 were the least 
susceptible. Although, cultivars TDa019 and TDa021 were bred and/or released not too long (Atsu, 
Personal Communication, 2012), their extensive cultivation (monocropping) might have enabled 
the pathogen adapt to them as substrates and developed resistant-breakdown gene(s) to overcome 
the resistant gene(s) in these cultivars. Several studies (Ayodele et al., n.d; Akem and Asiedu, 1994; 
Onyeka et al., 2006) have confirmed that pathogen and/or pests’ damage is prevalent and severe on 
widely cultivated crop species – of which yam was emphatically stressed (Ayodele, et al., n.d; 
Akem and Asiedu, 1994; Onyeka et al., 2006).  
 
The high susceptibility of TDa019 to the pathogen could also mean that phenols (antifungal 
compounds) in the cultivar are expressed in low quantity and thus, unable to overcome phenolases 
(tyrosinase) released by the pathogen (Plumbley and Sweetmore (1994). In addition, the thin-leaf of 
TDa019 in comparison with the highly resistant cultivar TDa023 (Coffie, Personal observation, 
2013) might have augmented the pathogen infection since there is the likelihood of easy penetration 
of infection pegs (haustoria) of the pathogen. Cultivars TDa013 and TDa018 which were the best 
performers among the least susceptible in the screening trial, has not been extensively cultivated in 
comparison with the highly susceptible cultivar (TDa019) (Klomega and Aboni, Personnal 
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Communication, 2012). The results corroborate finding of Svabova and Lebeda (2009) who 
inoculated tissue cultured plants with spore and mycelial filtrate as inocula. The pathogen might not 
have adapted to them as substrates. Differences in phenolic contents of the cultivars may also 
account for the difference in susceptibility observed. The biochemistry of the D. alata cultivars 
screened, therefore, needs to be studied so that the true nature of resistance of the test D. alata 
cultivars to anthracnose disease could be identified. The possible chemical resistant factors 
produced by the pathogen should be comprehensively researched and potential pathotoxins 
identified used for screening cell suspensions from which anthracnose disease tolerant cell lines 
could be obtained. 
 
The C. gloeosoporioides isolate (Cg111) and C. truncatum (Ct132) found in the Volta Region were 
more virulent compared to isolates from the other five regions. These isolates (Cg111 and Ct132) 
may possess complex disease-infection mechanisms (Abang et al., 2006) than the others which 
might have contributed to the sparate groupings in the cluster analyses. This affirms the high 
occurrence and severity of anthracnose disease of D. alata in the region compared to the other 
regions (Atsu and Akator, Personnal Communication, 2012; Abdallah and Isaac, Personnal 
Communication, 2012; Coffie, Personnal Obsrvation, 2012; Charles, Personnal Communication, 
2013). The resistant cultivars identified in this study have to be subjected to field-screening trials in 
two or more other regions of Ghana to ascertain their true resistance before recommending them to 
farmers. However, at present, efficient management of the disease in the Volta region would require 
the planting of the resistant cultivars (TDa008, TDa010, TDa014, TDa023, TDa025 and TDa027) 
alongside judicious use of fungicides and farm sanitation in an integrated disease management 
strategy.  
 
 
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5.6 Development of IMP strategy to manage anthracnose disease of D. alata 
 
The in-vitro screening of fungicides for control of C. gloeosporioides (Cg111) and C. truncatum 
(Ct132) isolates revealed Carbendazim (systemic fungicide), Mancozeb (protectant fungicide) and 
Cuprous oxide (Nordox) as 100% effective in inhibiting radial mycelial growth and sporulation of 
of the isolates while Shafiz F1 was the least effective. This confirms the findings of Naphtali (2012) 
that Carbendazim and Mancozeb are effective fungicides inhibiting radial mycelial growth and 
sporulation of C. gloeosporioides from diseased onion seedlings. The poor performance of Shafiz 
F1 could partly be owed to the inability of its active ingredient to degrade the cell-wall of the 
isolates (Gullino et al., 2000). 
 
The integrated disease management studies on the field revealed that integration of the highly 
resistant D. alata cultivar (TDa023), hot-water treatment of seed yam (45±1 oC for 20 minutes), 
monthly weeding and Mancozeb sprays was an active strategy for reducing the disease severity 
followed by the selection of the highly resistant cultivar, hot-water treatment, weeding and 
Carbendazim sprays with the least being the integration of the highly resistant cultivar, hot-water 
treatment, weeding and Nordox sprays. Integrated pest management measure that combines 
selection of healthy  planting materials, hot-water treatment, good sanitation, crop rotation, 
destruction of infected crop residues, chemical application and proper time of harvest has often 
been the best approach in controlling crop diseases and/or pests (Ogundana, 1971; Nwakiti and 
Arene, 1976; Nwakiti, 1982; Small, 1988; Egesi et al., 2007; Obeng-Ofori, 2007). Selection of 
resistant cultivars, use of clean pathogen-free planting setts, crop rotation practices, spraying of 
recommended fungicides, removing weeds that may be alternative hosts, early staking and planting 
of barrier crops like maize in integration is effective in the management of anthracnose disease of 
yam (Offei et al., 2008). 
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The better performce of the disease management package containing Mancozeb sprays partly agrees 
with the finding of Schwartz and Mohan (1995) who reported that Mancozeb spray is effective in 
controlling diseases in many crops. Mancozeb sprays significantly reduce anthracnose disease of 
yam Akem and Asiedu (1994) and onion Naphtali (2012). Mancozeb is multi-site in its mode of 
action and hence, could delay resistance of the pathogen (Gullino et al., 2000). It is therefore, 
recommended that a disease management measure which integrates selection of healthy planting 
materials (setts) of the resistant cultivar (TDa023) alongside hot-water treatment, regular weeding 
and Mancozeb sprays be used to manage anthracnose disease of D. alata in the Volta Region and/or 
Ghana. However, Mancozeb sprays could be rotated with Carbendazim sprays to delay build-up of 
pathogen resistance.  
 
The performance of the fungicides on the field contrasted the in-vitro findings (where all recorded 
100% inhibition of radial mycelial growth and sporulation of the test fungi). This could partly be 
attributed to reaction of the respective active ingredients (a.i) to the varied envirionmental 
conditions (rainfall, temperature and relative humidity) that existed on the field at the period of the 
trial (Gullino et al., 2000). Although, the cultivar TDa019 (farmers’ preference) could not reduce 
the disease severity as did cultivar TDa023, it is recommended that, if possible, the two cultivars be 
planted as a ‘mixed-crop’ to reduce the disease build-up and/or spread (Maloy, 1993; Akem and 
Asiedu, 2004; Egesi et al., 2007) alongside the use of Mancozeb-Carbendazim sprays in rotation 
and proper farm sanitation in an integrated disease management in the Volata Region. Currently, 
the development of D. alata populations with multiple resistances to antracnose disease by the 
International Institute of Tropical Agriculture (IITA) has been advocated (IITA, 1993) in Nigeria 
which could be replicated in Ghana to manage the disease.  
 
 
 
 
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CHAPTER SIX 
6.0 CONCLUSIONS AND RECOMMENDATIONS 
6.1 Conclusions 
• The results of the study indicated that most D. alata farmers in the Nkwanta-North and 
Krachi-West districts of the Volta Region were aware of the presence of anthracnose disease 
of water yam on their fields and could identify it by its symptoms. They were, however, 
ignorant of the causal organisms and spread of the disease within and among farms. 
Although, the disease was not controlled, some farmers perceived use of chemicals as the 
right option for control of the disease. The disease impacted negatively on the livelihoods of 
the farmers.  
• The Nkwanta-North district had higher disease incidence (75.30%) than Krachi-West 
district (55.70%) but disease severity was similar in both districts – Nkwanta-North (1.80) 
and Krachi-West (1.60). 
• The vine-browning and die-back disease was idenfied as anthracnose disease of water yam. 
The pathogens responsible for the disease of D. alata in the Volta region and other yam 
growing regions (Ashanti, Brong-Ahafo, Central, Eastern and Northern regions) in Ghana 
are strains of Colletotrichum gloeosporioides and C. truncatum.   
• A total of four strains of Colletotrichum gloeosporioides and two C. truncatum were found 
in the Volta region. However, in the six regions (Volta, Ashanti, Brong-Ahafo, Central, 
Eastern and Northern), a total of 18 strains of C. gloeosporioides and four of C. truncatum 
were found.   
• D. alata cultivars TDa008, TDa010, TDa014, TDa023, TDa025 and TDa027 were resistant 
to anthracnose disease but at varying levels. Cultivar TDa023 was highly resistant while 
TDa025 and TDa027 were resistant and TDa008, TDa010 and TDa014 were the least 
resistant. Six other cultivars (TDa007, TDa013, TDa015, TDa018, TDa019 and TDa021) 
were susceptible to the disease but also, at varying levels. Cultivar TDa019 was highly 
susceptible whereas TDa021 was susceptible and TDa007, TDa13, TDa15 and TDa018 
were the least susceptible. 
• IPM strategy involving the use of highly resistant cultivar (TDa023) alongside hot-water 
treatment of seed yam (45oC for 20 minutes and drying for 30 minutes), monthly weeding 
and Mancozeb sprays was effective for managing anthracnose disease of D. alata in the 
field.  
 
 
 
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6.2 Recommendations 
• Education of D. alata farmers in the Nkwanta-North and Krachi-West districts on the causes 
and mode of spread of the pathogens could change their perception and contribute to 
effective management of the disease. 
• Effective internal quarantine measures should be implemented to prevent and/or reduce the 
spread of the C. truncatum, the most virulent strain from Volta Region to other yam 
growing regions in Ghana.   
• Identification of disease resistance factors in D. alata should be done to aid Plant breeders in 
breeding for novel resistant cultivars to manage anthracnose disease. 
• RAPD and ITS-RFLP techniques using different primers could be used to further 
characterize Colletotrichum strains in the country. SSR primers could be employed 
alongside the RAPD and ITS-RFLP primers for characterization of wide number of strains. 
• Benefit-cost analysis of the IPM strategy developed for the management of anthracnose 
disease of water yam should be undertaken.  
• Screening of the resistant D. alata cultivars identified should be repeated on the field at 
different locations to assess their performance and yield. Resistant D. alata cultivars bred in 
IITA, Nigeria could be sourced and screened for resistance to the Colletotrichum isolates 
found in Ghana. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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Fungal Ribosomal RNA Genes for Phylogenetics. Phytopathology 9(2): 123-145. 
 
Whitelaw-Weckert, M. A., Curtin, S. J., Huang, R., Steel, C. C., Blanchard, C. L. and Roffey, P. E. 
(2007). Phylogenetic Relationships and Pathogenicity of Colletotrichum acutatum Isolates 
from Grape in Subtropical Australia. Plant Pathology 56(3), 448– 463. 
 
Williams, J. G. K., Kubelik, A. R., Livak, K. J., Rafalki, J. A. and Tingy, S. V. (1990). DNA 
Polymorphisms Amplified by Arbitrary Primers are Useful as Genetic Markers. Nucleic 
Acid Research 18: 6531- 6535. 
 
Winch, J. E., Newhook., F. J. G., Jackson, V. H. and Cole, J. S. (1984). Studies of Colletotrichum 
gloeosporioides Disease on Yam (Dioscorea alata L.) in Solomon Islands. Plant 
Patholology 33: 467–477. 
 
Winch, J. E., Jackson, G. V. H., Newhook, F. J. and Cole, J. S. (1993). Blackening on Yam in 
Response to Colletotrichum gloeosporioides. Plant Pathology 42: 187–194. 
 
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Wu, W. H., Liu, L. Y., Chung, C. J., Jou, H. J. and Wang, T. A. (2005). Estrogenic Effect of Yam 
Ingestion in Healthy Postmenopausal Women. Journal of the American College of 
Nutrition 24: 235-243. 
 
Xiao, C., Tuthill, T., Bator, J., Kelly, C. M., Challinor, L. J. and Chipman, P. R. (2004). 
Discrimination Among Rhinovirus Serotypes for a Variant ICAM-1 Receptor Molecule. 
Journal of Virology 78: 10034–10044. 
 
Yang, Y. L., Liu, Z. Y., Cai, L., Hyde, K. D., Yu, Z. N. and Mckenzie, E. H. C. (2009). 
Colletotrichum Anthracnose of Amaryllidaceae. Fungal Diversity 39: 123-146. 
 
Yuan, K., Qin, W., Dong, M., Liu, J. and Sun, J. (2011). Gray Matter Deficits and Resting-State 
Abnormalities in Abstinent Heroin-Dependent Individuals. Neuroscience Letters 48(2): 
101–105.  
 
Zhang, J. Z. (2008). Anthracnose of Persimmon Caused by Colletotrichum gloeosporioides in 
China. Asian and Australasian Journal of Plant Science and Biotechnology 2: 50-54. 
 
 
 
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APPENDICES 
APPENDIX I: Questionnaire on aetiology, importance and control of a vine-browning and 
die-back disease of water yam (Dioscorea alata L.) in the Volta Region of Ghana 
Documentation of farmers’ knowledge, perception and experiences concerning prevalence, spread, 
control and economic importance of a vine-browning and die-back disease of D. alata.  
Questionnaire No……… 
A. Background 
 
1. Name of farmer………………………………………………….Male/female………….. 
2. Level of education……………………………………………………………………….. 
3. Name of District…………………………………………………………………………. 
4. Name of village…………………………………………………………………………... 
 
5. Size of yam farms, year of cultivation, sources of planting materials and expenditure 
 
Year 
 
Size 
Varieties Source of 
planting materials 
Expenditure 
 land labour Planting materials others 
 
2008 
       
 
2009 
       
 
2010 
       
 
2011 
       
 
B. Farmers Knowledge, perception and experiences concerning disease prevalence 
 
6. Please mention the diseases that affect the following parts of your yam 
I. Leaves…………………………………………………………………………………………
…………………………………………………………………………………………… 
II. Vines…………………………………………………………………………………………
…………………………………………………………………………………………………
………………………………………………………………………………………… 
III. Tubers…………………………………………………………………………………………
…………………………………………………………………………………………… 
 
7. What is the size of the farm affected?…………………………………………………………. 
 
8. What percentage of your yam farm has been affected by anthracnose?   A) Less than 25%      B) 
Between 25% and  50%   C) Between 50% and 75% ,     D) Between 75% and 100%,      E)  
100%  
 
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9. How do the yam tubers look like when they are affected by anthracnose disease? 
……………………………………………………………………………………………………
…………………………………………………………………………………………………… 
10. How do the leaves and vines look like when the yam is affected by anthracnose disease? 
……………………………………………………………………………………………………
…………………………………………………………………………………………………… 
11. Please describe the stages of the disease? 
……………………………………………………………………………………………………
…………………………………………………………………………………………………… 
12. What periods of the year do your yams suffer from anthracnose disease?  
……………………………………………………………………………………………………
…………………………………………………………………………………………………… 
13. At what age from date of planting do the yams suffer from anthracnose?  
……………………………………………………………………………………………………
…………………………………………………………………………………………………… 
14. Which varieties of yams are usually affected by the disease?  
……………………………………………………………………………………………………
…………………………………………………………………………………………………… 
15. What percentage of the yam farms in this town/village do you think are affected by yam 
anthracnose?  A) less than 25%,       B)between 25% and  50%,   C)between 50% and 75% ,     
D) between 75% and 100%,      E)100%  
 
C.  Knowledge, perception and experiences concerning cause and spread of the disease 
 
16. What do you think are the causes of yam anthracnose disease?  
……………………………………………………………………………………………………
…………………………………………………………………………………………………… 
 
17. Which year and month was the first time your yam was affected by yam anthracnose disease?  
Year…………………………………………Month……………………………………………. 
 
18. What condition was the farm before your yam first suffered the disease?   
A) Weedy   [    ]      B)  Flooded  [    ]          C) Drought    [    ]        D) Normal condition  [    ] 
 
19. What did you do when you noticed your yams were affected with the disease?  
……………………………………………………………………………………………………
…………………………………………………………………………………………………… 
20. Do your yams still suffer from yam anthracnose?  
……………………………………………………………………………………………………
…………………………………………………………………………………………………… 
21. How does yam anthracnose disease spread on the same farm? 
……………………………………………………………………………………………………
…………………………………………………………………………………………………… 
22. How does the yam anthracnose spread from one farm to another?  
……………………………………………………………………………………………………
……………………………………………………………………………………………………
………… 
University of Ghana          http://ugspace.ug.edu.gh
153 
 
23. If your yam fails due to anthracnose disease what crop will you plant and why? 
……………………………………………………………………………………………………
…………………………………………………………………………………………………… 
 
D. Knowledge, perception and experiences concerning control 
 
24. Can yam anthracnose be controlled?    Yes………….. No…………… 
 
25. If yes, what do you think can be done to control the spread of yam anthracnose disease? 
What can be done Who should do it Why 
   
   
   
   
 
26. What have you done to your yam to control anthracnose disease?  
……………………………………………………………………………………………………
……………………………………………………………………………………………………
………… 
27. If no to question 26, give reasons 
……………………………………………………………………………………………………
…………………………………………………………………………………………………… 
 
E. Knowledge, perception and experiences concerning economic importance of yam 
anthracnose disease 
 
28. Expected yield, volume harvested and reasons for yield difference 
 
Year  Farm size Expected yield Volume 
harvested 
Reasons for difference 
2008     
2009     
2010     
2011     
 
29. Reasons for planting yam and what was done when yam was diseased 
Year  Reasons for 
planting yam 
% of yam 
affected 
What did you do when was diseased? 
2008    
2009    
2010    
2011    
 
University of Ghana          http://ugspace.ug.edu.gh
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30. Please explain the effect of yam anthracnose disease on the following: 
 
I. Money to pay your children’s school fees 
…………………………………………………………………………………………………
………………………………………………………………………………………………… 
II. Money to repay loans 
…………………………………………………………………………………………………
………………………………………………………………………………………………… 
III. Money to pay medical bills 
…………………………………………………………………………………………………
………………………………………………………………………………………………… 
IV. Money to buy assets 
…………………………………………………………………………………………………
………………………………………………………………………………………………… 
V. Your relationship with your spouse and neighbours 
…………………………………………………………………………………………………
………………………………………………………………………………………………… 
VI. Your relationship with extension officers 
…………………………………………………………………………………………………
………………………………………………………………………………………………… 
 
Farmer’s contact number……………………………… 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
University of Ghana          http://ugspace.ug.edu.gh
155 
 
APPENDIX II: Surveyed Areas for the Diversity Study 
Table of Regions, Districts, Communities and number of farms selected for the Diversity Study. 
 
Regions  Districts   Communities  Number of farms 
                  Fumesua             15 
 Ejisu-Juabeng             Kentinkrono      15 
                             Kwamo   15 
Ashanti                      
 
 
 
New Edubiase   15 
 
Adansi-South   Bronikurom                 15 
                          Odumasi   15 
              
       Twumia No. 1   15 
                                     Tanoso-Central Twumia No. 2   15                     
Sunkundei                         15 
Brong-Ahafo                       
        
   Tanoso-South                         Tanoso                      15 
                                                       
 
 
 
     
          
 
 Awutu Senya District                                                  Bawjiase Central            15 
 
 
 
 
 
 
 
Central       Odumase   15 
 
  Assin-Central   Assin-Fosu   15 
     Assin-Edubiase            15 
 
                                    Nkurakan      Sekesoa   15 
Eastern         Asesewa   15 
               
   Akim-Tafo   Bunso     15 
 
       SARI    15 
Tolon Kunbung  Nyankpala-Central  15 
       Nyanriga       15 
Northern  
 
 
 
 
 
 
Bole    Bole Central   15 
        
Kpasa-Tindani  15 
   Nkwanta-North  Kpasa-Abunyanya  15 
Volta       Bajimpoa-Akura  15 
       Osramae   15 
Krachi-West   Ehiamankyene  15 
       Gyidiasu   15 
405 
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6 
 A
P
P
E
N
D
IX
 I
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: 
A
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 f
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 t
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 19
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mm
ary
  
 
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7 
 A
P
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IX
 I
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<.0
01
 
University of Ghana          http://ugspace.ug.edu.gh
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APPENDIX VI: ANOVA for Mean severity (AUDPCs) for a vine-browning and die-
back disease on 12 tissue-cultured D. alata cultivars raised in a screen house  
 
Variate: Screening sum_AUDPC      
Source of variation d.f. s.s. m.s. v.r. F pr. 
Pathogen 5    8263.75    1652.55    20.98 <.001 
Cultivar 11 22189.76  2017.25  25.61 <.001 
Pathogen.Cultivar   55  45274.33  823.17  10.45  <.001 
Residual 289  22764.23  78.77     
Total       360    98492.07 
 
APPENDIX VII: ANOVA for the mean disease incidence of a vine-browning and die-
back disease of 12 tissue-cultured D. alata cultivars raised in a screen house 
 
Variate: Screening mean_incidence      
Source of variation d.f. s.s. m.s. v.r. F pr. 
Pathogen 5        37983.65    7596.73    1.82 .035 
Cultivar 11       52189.76  4744.52  1.14 .107 
Pathogen.Cultivar   55       105274.33  1914.08  0.46  .429 
Residual                                      289    1203044.37      4162.78    
Total        360   1398492.11 
   
 
 
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9 
 A
P
P
E
N
D
IX
 V
II
I:
 A
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eat
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29
 
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. 
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s.s
. 
89
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s.s
. 
32
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