Identification and characterization of colletotrichum species associated with mango and citrus diseases in the Ashanti region of Ghana BY Kidist Fekadu Wolde (M.Sc. PLANT PATHOLOGY) AUGUST, 2015 1 IDENTIFICATION AND CHARACTERIZATION OF COLLETOTRICHUM SPECIES ASSOCIATED WITH MANGO AND CITRUS DISEASES IN THE ASHANTI REGION OF GHANA A Thesis submitted to the Department of Crop and Soil Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana in partial fulfilment of the requirements for the Degree of DOCTOR OF PHILOSOPHY in Plant Pathology KIDIST FEKADU WOLDE (M.Sc. PLANT PATHOLOGY) AUGUST, 2015 2 DECLARATION I, hereby, declare that this submission is my own work towards the PhD degree and that, to the best of my knowledge, it contains no material previously published by another person nor material which has been accepted for the award of any other degree of the University, except where due acknowledgement has been made in the text. KIDIST FEKADU WOLDE ................................ ............................. Student No. 20069724 Signature Date DR CHARLES KWOSEH ............................ .............................. (Supervisor) Signature Date PROF. RICHARD AKROMA ........................ ............................... (Co- Supervisor) Signature Date Certified by: DR E. A.OSEKRE ........................ ............................... (Head of Department) Signature Date ii ABSTRACT The purposes of the study were to identify Colletotrichum species, detect their variability, to investigate their cross infectivity potential and determine farmers' awareness about the diseases that are associated with mango and citrus. Culture characteristics, size and shape of conidia and mycelial growth rates were used to characterize the isolates into different morphological groups. Those grouped isolates were further studied for their genetic similarity and differences, using multigene loci primers and restriction enzymes. ITS region, β- tub gene and GPDH primers were also used to characterize the isolates. Farmers were aware of mango anthracnose but did not know any such disease on citrus. Colletotrichum species isolated from mango and citrus had overlap in their cultural characteristics. All of the isolates were assigned into ten morphogroups. Seven of the morphogroups were characterized by their cylindrical conidia and three of the morphogroups (G1, G3 and G6) had curved conidia. Apart from the conidial shape, most of the morphogroups had unique characters to stand as a group on their own. However, G2, G5 and G9 showed overlap in many of their characters. The results from molecular analysis were different, depending on the gene/region targeted. The ITS region amplification showed high genetic similarity among the ten morphogroups, irrespective of their conidial or other cultural characteristic variations. Further investigation on the same ITS region, using Colletotrichum species-specific primer, CgInt, confirmed the similarity for six of the morphogroups such as (G1, G2, G4, G5, G8 and G9) but the other four morphogroups were not picked. The overall results of the ITS region were very helpful in understanding the close relationship of the different iii morphogroups. Further, analysis of the same group of isolates, using β-tubulin primer, clearly showed existence of genetic variability in between the groups. The variability expressed on β-tub gene analysis was in line with the characterization made, based on conidial shape. It gave two distinct groups; one group having cylindrical conidia and the other having curved conidia. This result is an indicator that conidial shape is governed by a genetic factor. The third gene, GPDH analysis gave high genetic variability results. It amplified nine distinct groups from the ten morphogroups studied. Eight of them were in line with eight of the morphogroups. Therefore, GPDH results were more detailed than the other studied regions of Colletotrichum species in showing genetic variability. However, the combined results of the morphological characterization and the multigene analyses played their own role for a better understanding of species variability. The pathogenicity tests on mango and citrus fruits confirmed most of the morphogroups as pathogenic. However, all the curved morphogroups isolated from diseased mango tested negative on mango. The cross-infectivity potential of citrus and mango isolates of Colletotrichum were confirmed. The cross tests on papaya also showed that most of the morphogroups were pathogenic. From the study, it is understood that critical investigation of Colletotrichum spp., using the traditional techniques of morphological characterization, can assist in determining the genetic variability among isolates but it is tedious, time consuming and wrong conclusions could be drawn if it is first time study. Once the organisms are sequenced and named, the conventional investigation could play a great role to detect the variability. Therefore, it is recommended iv that the identified groups of isolates be sequenced for the purpose of accurately naming them to the species level. v TABLE OF CONTENT CONTENT PAGE DECLARATION....................................................................................................................... ii ABSTRACT .............................................................................................................................. iii TABLE OF CONTENT ........................................................................................................... vi LIST OF TABLES ................................................................................................................... xi LIST OF PLATES ................................................................................................................. xiii LIST OF FIGURES ............................................................................................................... xvi ABBREVIATIONS ............................................................................................................... xvii AKNOWLEDGEMENTS ..................................................................................................... xix DEDICATION........................................................................................................................ xxi CHAPTER ONE ....................................................................................................................... 1 1.0 GENERAL INTRODUCTION .......................................................................................... 1 1.1 Background of the study ................................................................................................... 1 1.2 Objectives of the Study ..................................................................................................... 4 1.3 References ......................................................................................................................... 5 CHAPTER TWO ...................................................................................................................... 8 2.0 LITERATURE REVIEW .................................................................................................. 8 2.1 Mango and citrus ............................................................................................................... 8 2.1.1 Climatic and ecological requirements of mango and citrus ...................................... 9 2.1.2 Production, export and market potential .................................................................... 9 2.1.3 Challenges of citrus and mango production ............................................................ 10 2.2 Agricultural science and farmers' role ............................................................................ 11 2.2.1 Farmers' indigenous knowledge and success of farming ......................................... 11 2.2.2 Farmers' perception and its importance for plant protection .................................... 12 2.3 Anthracnose disease ........................................................................................................ 13 2.3.1 Anthracnose disease cycle ....................................................................................... 14 2.3.2 Anthracnose disease symptoms ............................................................................... 15 vi 2.3.3 Causal agent (s) of anthracnose disease, Colletotrichum species ........................... 17 2.4 Identification of Colletotrichum species ......................................................................... 19 2.4.1 Conventional identification techniques .................................................................... 20 2.4.2 Advanced techniques ............................................................................................... 21 2.4.2.1 PCR- based identification techniques ............................................................... 22 2.5 References ....................................................................................................................... 23 CHAPTER THREE ................................................................................................................ 38 3.0 FARMERS' PERCEPTION AND PREVALENCE OF ANTHRACNOSE DISEASE ON MANGO AND CITRUS IN the ASHANTI REGION OF GHANA ............................................................................................................................ 38 3.1 Summary ........................................................................................................................